Univers

ity of

Cap

e Tow

n

Repositioning fusidic acid for tuberculosis: semi-

synthesis of analogues and impact of mycobacterial

biotransformation on antibiotic activity

Antonina Wasuna

Supervisor: Professor Kelly Chibale

Department of Chemistry, University of Cape Town

Co-Supervisor: Associate Professor Digby F. Warner

Department of Pathology & Institute of Infectious Disease and Molecular Medicine, University

of Cape Town

A thesis presented for the degree of Doctor of Philosophy in the Department of Chemistry,

University of Cape Town

March 2017

Univers

ity of

Cap

e Tow

n

The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non-commercial research purposes only.

Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author.

i

Declaration

I, Antonina Wasuna, hereby declare that:

i. The above thesis is my own unaided work, both in concept and execution, and that apart from

the normal guidance of my supervisors, I have received no assistance apart from that

acknowledged.

ii. Neither the substance nor any part of this thesis has been in the past, or is being, or is to be

submitted for a degree in the University of Cape Town or any other University.

Signed 22nd March 2017

ii

Acknowledgements

To my supervisors, Professor Kelly Chibale and Associate Professor Digby F. Warner, thank you for taking

a chance on me and for the mentorship you have provided, beyond the enormous scientific knowledge

you have imparted upon me. You have redefined hard work for me, and your patience and guidance

through the constant challenges during this project have left an indelible mark.

I extend my gratitude to the Carnegie Corporation of New York and the Schlumberger Faculty for the

Future Foundation for financial support. I am grateful to Mrs. Elaine-Rutherfoord-Jones, Mrs. Deirdre

Brooks and Ms Saroja Naicker, whose formidable administrative capability made this journey smooth. I

would also like to thank Mr. Pete Roberts for the NMR training, support and recording of spectra. In

addition, I am grateful for the Mass Spectrometry analysis performed by Mr. Gianpiero Benincasa. I

thank Dr. Gurminder Kaur, Dr. Mathew Njoroge, Dr. Nigel Makoah and Ms Natasha Strydom for the

synthesis of several analogues evaluated in this project and provision of pharmacokinetic data. I also

thank Dr. Vinayak Singh for construction of the knockdown mutant strain, and Professors Tom Ioerger

and James Sacchettini (Texas A & M University, USA) for whole genome sequencing of my samples.

To the Medicinal chemistry research group and the MMRU, your generosity, problem-solving skills and

humour made the labs my home away from home. Thank you for the technical, scientific and moral

support, from bench to hospital bedside. Dr. Peter Njogu, Dr. Dennis Ongarora, Dr. Atica Moosa and Dr.

Krupa Naran, and Mr. Charles Omollo, thank you for your unrelenting patience in helping me to settle

into the labs without recourse to the first aid kit. To my friends scattered across the globe, including

many whom I have not mentioned here, your patience and generosity through my long silences and

absence over the years is truly humbling. Tamara Sutila, Gladwell Ng’ang’a, Miriam Maina, Claire Le

Manach, Christel Brunschwig, Teboho Nchaba, Hellen Mahlase, Karabo Kgoleng, Dorothy Semenya,

Tatenda Dinha, Nkulumo Zinyengere, Taigh Anderson, Rachel Piontak, Tewa Oduro, various members of

St. Michael’s Rondebosch community, Wiebke Ringel, Julia Baum, Arlindo Meque, and Natasha Strydom,

thank you for your encouragement to push on. Hvala, ahsante sana, merci, kea leboha, ndinotenda,

thank you, danke schön, obrigada, baie dankie. To the Kenyan “mafia”, ahsanteni sana, for making the

journey more bearable and improving my chapati-making skills. To my Joburg family, wat imedo gi osiep.

Ero kamano ahinya.

Mum and Dad, thank you for your support. I am because you are. Tony, Brian and Nikky Wasuna, you

can finally have “that your sister” back. To the Almighty God be all praise and honour.

iii

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the leading causes of death

globally, especially in low and middle-income countries. TB is primarily a curable disease, with

chemotherapy predicated on a combination of four drugs. The increase in multiple forms of drug-

resistant TB is a major cause for concern, underpinning the importance of a continuous pipeline of new

anti-TB agents. Drug repositioning – that is, the optimization of existing drugs for new therapeutic

indications – has shown promise in expanding the therapeutic options for TB chemotherapy. Fusidic acid

(FA), a natural product-derived antibiotic, has modest in vitro antimycobacterial activity. Through a

multi-disciplinary approach combining aspects of chemistry and biology, this study investigated the

pharmacological and physicochemical properties of FA that might be exploited for optimization of FA as

a lead compound for TB drug discovery.

FA is a weak carboxylic acid, and it was hypothesised that the carboxylic acid moiety limits its

permeation of the complex mycobacterial cell wall. Therefore, this study aimed to identify novel FA

analogues with improved permeation properties and designed to act as potential prodrugs. By

modifying the C-3 hydroxyl and the carboxylic acid moiety, alkyl and aminoquinoline derivatives were

covalently fused to FA through ester and amide coupling reactions to generate hybrids and/or potential

prodrugs (Figure 1).

Figure 1: Fusidic acid and representative ester and amide derivatives.

iv

Turbidimetric solubility assays revealed that most of the hybrids were 5- to 10-fold less soluble than FA.

Moreover, the resultant amides and esters were found to be less potent than FA. Through the testing of

1:1 physical molar mixtures of the constituent pharmacophores, the contribution of the individual

scaffolds to the antimycobacterial activity of the hybrids was evaluated. The aminoquinoline derivatives

were found to reduce the overall antimycobacterial activity of the FA-aminoquinoline hybrids. The poor

aqueous solubility of these compounds may have precluded the accurate determination of their

antimycobacterial activity, as some of the compounds precipitated out of the aqueous media used for

the drug susceptibility tests. The FA hybrids were also evaluated against two mammalian cell lines and

found to be cytotoxic at very low concentrations. 1:1 physical molar mixtures of the selected

aminoquinoline derivatives and FA revealed that the aminoquinoline scaffold contributed to the

cytotoxicity of the hybrids. This unsatisfactory cytotoxic profile was a basis for the discontinuation of

these hybrids and only the C-3 prodrugs were progressed to subsequent assays.

Mtb is an intracellular pathogen, and in vitro macrophage models of Mtb infection that attempt to

simulate host infection conditions are widely used to assess the efficacy of antimycobacterial

compounds in disease-relevant conditions. FA and selected analogues were evaluated for their

intracellular efficacy, and found to reduce bacterial burden in infected THP-1 cells at concentrations two

or five times higher than their inhibitory concentrations in broth cultures. FA and three ester analogues,

GKFA16, GKFA17 and GKFA61, as well as two amides, AW23 and AW25, were investigated for the role

of mycobacterial metabolism in their efficacy. In Mtb cultures, the two esters were hydrolysed to FA to

achieve a higher concentration of FA than that seen in incubations with FA, suggesting that they are

prodrugs. In contrast, GKFA61 and the two C-21 amides were not hydrolysed to FA in Mtb cultures.

Although the mode of action of FA in other organisms is well-established, there are no published reports

on its antimycobacterial target and mechanism(s) of resistance (MoR). Through the generation of

spontaneous resistant mutants, the target of FA and the corresponding MoR in Mtb were confirmed. A

substitution mutation (H462Y) in the fusA1-encoded Elongation Factor G (EF-G) conferred resistance to

FA and several derivatives. No cross-resistance was observed between FA and other standard anti-TB

drugs in vitro. Notably, the fusA1 mutation conferred slight hypersensitivity to streptomycin, another

v

protein synthesis inhibitor, indicating its potential exploitation as a synergistic partner in TB drug

combination therapy. Target-based whole cell screening was conducted using an anhydrotetracycline-

responsive knockdown strain of FA underexpressing EF-G, with consequent hypersensitivity observed for

FA and three analogues. This provided further confirmation of EF-G as the target of FA in Mtb.

In conclusion, these findings demonstrate that FA can be used as a template for repositioning in TB drug

discovery.

vi

List of abbreviations

ACN Acetonitrile

ADMET Absorption, Distribution, Metabolism, Excretion and Toxicity

ATc Anhydrotetracycline

BCG Bacille Calmette-Guérin

BDQ Bedaquiline

bp Base pair

CD3OD deuteromethanol

CEM Cryo-electron microscopy

CET Cryo-electron tomography

CFU Colony forming unit

CH2Cl2 dichloromethane

CHO Chinese hamster ovarian

CIP Ciprofloxacin

cKD conditional knockdown

CTAB N-cetyl-N, N, N-trimethyl ammonium bromide

DCC Dicyclohexyl carbodiimide

DCS D-cycloserine

DIPEA N, N-Diisopropylethylamine

DMF N, N-Dimethylformamide

vii

DMSO Dimethylsulphoxide

DNA Deoxyribonucleic acid

dNTP Deoxynucleotide triphosphate

DOTS Directly Observed Therapy – Short Course

EDCI 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide

EDTA Ethylenediaminetetraacetic acid

EF-G Elongation Factor G

EMB Ethambutol

EPI Efflux pump inhibitor

ERY Erythromycin

ETH Ethionamide

EtOAc Ethyl acetate

FA Fusidic acid

FBS Fetal bovine serum

FDA US Food and Drug Administration

g Gravitational force

GAST/Fe Glycerol Alanine Salts Tween80

GAT Gatifloxacin

GDP Guanosine 5’ diphosphate

viii

GEN Gentamycin

GTP Guanosine triphosphate

HCl Hydrochloric acid

HIV Human Immunodeficiency Virus

HLM Human liver microsomes

HOBt 1-Hydroxybenzotriazole hydrate

HPLC High Pressure Liquid Chromatography

HTS High throughput screening

Hz Hertz

Hyg Hygromycin

IC Inhibitory concentration

INH Isoniazid

KAN Kanamycin

LC-MS Liquid chromatography coupled mass spectrometry

LEV Levofloxacin

LRMS Low Resolution Mass Spectrometry

LTBI Latent tuberculosis infection

MABA Microplate alamar blue assay

MDR Multidrug resistant

ix

MeOH Methanol

MIC90 Minimum Inhibitory Concentration

MLM Mouse liver microsomes

MmpL3 Mycobacterial membrane protein Large 3

MoA Mechanism of action

MOX Moxifloxacin

M.p. Melting point

MS Mass Spectrometry

Msm Mycobacterium smegmatis

Mtb Mycobacterium tuberculosis

MTT 3-(4, 5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

NaCl Sodium chloride

NH4OAc Ammonium acetate

Nm Nano metre

NMR Nuclear Magnetic Resonance

OADC Oleic acid-albumin-dextrose-catalase

OFX Ofloxacin

PAS para-aminosalicylic acid

PBS Phosphate buffered saline

x

PBP Penicillin binding proteins

PCR Polymerase Chain Reaction

PD Pharmacodynamics

PK Pharmacokinetics

PMA Phorbol myristate acetate

Ppm parts per million

PZA Pyrazinamide

Rf Retardation factor

RLM Rat liver microsomes

RIF Rifampicin

RNS Reactive nitrogen species

ROS Reactive oxygen species

rpm rotations per minute

SAR Structure-activity relationship

sdH2O Sterile distilled water

SI Selectivity index

SNPs Single nucleotide polymorphisms

SPEC Spectinomycin

STM Streptomycin

xi

Tet Tetracycline

TB Tuberculosis

TLC Thin Layer Chromatography

TB-WCS Target-based whole-cell screening

TE Tris-EDTA

TMS Tetramethylsilane

UCT University of Cape Town

UV Ultraviolet

v/v Volume per volume

v/w Weight per volume

VER Verapamil

WGS Whole-genome sequencing

WHO World Health Organization

WT Wild-type

XDR Extensively Drug Resistant

xii

Contents Declaration ..................................................................................................................................................... i

Acknowledgements ....................................................................................................................................... ii

Abstract ........................................................................................................................................................ iii

List of abbreviations ..................................................................................................................................... vi

List of Appendices ....................................................................................................................................... xix

List of Figures ............................................................................................................................................... xx

List of Graphs ............................................................................................................................................. xxii

List of Schemes.......................................................................................................................................... xxiii

List of Tables ............................................................................................................................................. xxiv

List of publications arising from this thesis and conferences attended .................................................... xxv

Chapter 1 ...........................................................................................................................................1

Introduction and Literature Review ....................................................................................................1

1.1 Tuberculosis: Epidemiological burden, disease aetiology and management ..................................... 1

1.2 Antimycobacterial chemotherapy ...................................................................................................... 2

1.2.1 Drug-susceptible TB Therapy ....................................................................................................... 2

1.2.2 Management of Drug-resistant TB .............................................................................................. 5

1.3 Limitations of current chemotherapy ................................................................................................. 9

1.4 Advances in TB drug discovery: Opportunities and challenges .......................................................... 9

1.5 Drug repurposing and repositioning: recent additions to TB drug regimens ................................... 10

1.6 Current drug discovery paradigms .................................................................................................... 13

1.6.1 The challenges ............................................................................................................................ 14

1.7 Relevance of Natural products to drug discovery ............................................................................. 14

1.7.1 Nature’s toolbox: The role of natural products in drug discovery ............................................. 14

1.7.2 Nature’s suitability: The concept of privileged structures ......................................................... 17

xiii

1.7.3 Declining natural product-based drug discovery ....................................................................... 17

1.7.4 Saving graces: Advances from technology platforms and genomics ......................................... 18

1.7.5 Chemistry: Synthesis and semi-synthesis as a strategy in drug discovery ................................. 19

1.7.6 Semi-synthesis in TB drug discovery .......................................................................................... 20

1.8 Target identification and determination of mechanisms of resistance ............................................ 23

1.8.1 Target identification and Mechanism of Action determination ................................................ 23

1.8.2 The value of re-discovery: Polypharmacology ........................................................................... 26

1.8.3 Mechanisms of drug resistance ................................................................................................. 27

1.9 Mycobacterium tuberculosis: An intracellular pathogen ................................................................. 30

1.10 Absorption, Distribution, Metabolism and Excretion (ADME) ........................................................ 34

1.10.1 Drug Metabolism ..................................................................................................................... 35

1.11 Rationale ......................................................................................................................................... 41

1.12 Hypothesis....................................................................................................................................... 41

1.13 Aim .................................................................................................................................................. 42

1.14 Specific objectives: .......................................................................................................................... 42

1.15 References ...................................................................................................................................... 43

Chapter 2 ......................................................................................................................................... 62

Design, semi-synthesis and in vitro evaluation of pharmacological and physicochemical properties of

fusidic acid-aminoquinoline hybrids ................................................................................................. 62

Chapter overview ........................................................................................................................................ 62

2.1 Fusidic acid ........................................................................................................................................ 62

2.1.1 Unique structure of FA ............................................................................................................... 63

2.1.2 Antibacterial SAR........................................................................................................................ 63

2.2 Quinolines in drug discovery ............................................................................................................. 64

2.3 Hybrid drugs ...................................................................................................................................... 65

xiv

2.3.1 Classification of hybrid drugs ..................................................................................................... 65

2.4 Prodrugs: design and application in drug discovery ......................................................................... 66

2.4.1 Classification of prodrugs ........................................................................................................... 67

2.5 Rationale for fusidic acid-aminoquinoline hybrids and C-3 ester prodrugs ..................................... 70

2.6 Aims and objectives .......................................................................................................................... 71

2.7 Semi-synthesis of FA hybrids ............................................................................................................ 71

2.7.1 Retrosynthetic analysis of the FA amide and ester prodrugs based on aminoquinolines ........ 72

2.7.2 Synthesis of 4-aminoquinoline diamines and alcohols .............................................................. 73

2.7.3 Synthesis of FA amides............................................................................................................... 75

2.7.4 Synthesis of FA esters ................................................................................................................ 75

2.7.5 1H NMR characterisation: Key spectroscopic indicators of selected intermediates and target

compounds ......................................................................................................................................... 78

2.8 Physicochemical evaluation of the FA hybrids.................................................................................. 80

2.8.1 Experimental determination of solubility .................................................................................. 80

2.8.2 Results of solubility testing of the compounds .......................................................................... 80

2.9 Pharmacological evaluation of the FA analogues ............................................................................. 82

2.9.1 Antimycobacterial evaluation of the FA analogues ................................................................... 82

2.9.2 Results of antimycobacterial evaluation of FA-aminoquinoline hybrids ................................... 82

2.9.3 Evaluation of the contribution of the pharmacophoric units to the pharmacological activity of

the hybrids .......................................................................................................................................... 85

2.10 Summary ......................................................................................................................................... 87

2.11 References ...................................................................................................................................... 88

Chapter 3 ......................................................................................................................................... 92

Evaluation of cytotoxicity, intracellular efficacy and mycobacterial metabolism of Fusidic acid and

selected analogues ........................................................................................................................... 92

3.1 Introduction ...................................................................................................................................... 92

xv

3.2 Aims and objectives .......................................................................................................................... 93

3.3 Evaluation of cytotoxicity .................................................................................................................. 93

3.3.1 Evaluation of cytotoxicity of the FA-aminoquinoline hybrids in CHO cells................................ 93

3.3.2 Evaluation of cytotoxicity of the FA-aminoquinoline hybrids in THP-1 cells ............................. 94

3.3.3 Results: Cytotoxicity in CHO cells ............................................................................................... 94

3.3.4 Discussion ................................................................................................................................... 98

3.4 Evaluation of selected FA analogues for intracellular antimycobacterial efficacy in THP-1 ............. 98

3.4.1 Compound selection .................................................................................................................. 98

3.4.2 Intracellular infection ................................................................................................................. 99

3.4.3 Results of CFU log reduction for Mtb-infected THP-1 treated with FA ................................... 100

3.4.4 Results of CFU log reduction for Mtb-infected THP-1 treated with GKFA16 and GKFA17 ...... 100

3.4.5 Results of CFU log reduction for Mtb-infected THP-1 treated with GKFA37 ........................... 101

3.4.6 Discussion ................................................................................................................................. 102

3.5 In vitro Metabolism of FA and selected analogues ......................................................................... 104

3.5.1 Introduction ............................................................................................................................. 104

3.5.2 Metabolism of FA ..................................................................................................................... 104

3.5.3 Metabolism of selected FA analogues ..................................................................................... 107

3.6 Role of mycobacterial metabolism in bioactivation and biodegradation of drugs ........................ 108

3.7 Rationale ......................................................................................................................................... 108

3.8 Aim .................................................................................................................................................. 109

3.8.1 Incubation of compounds in Mtb ............................................................................................ 110

3.8.2 Results ...................................................................................................................................... 110

3.8.3 Discussion ................................................................................................................................. 113

3.9 Summary ......................................................................................................................................... 115

3.10 References .................................................................................................................................... 117

xvi

Chapter 4 ....................................................................................................................................... 120

Target Identification and Mechanism of Resistance of Fusidic Acid .................................................. 120

4.1 The identification of distinct modes of action for the same molecule in different organisms. ..... 120

4.2 Mechanism of action of fusidic acid (FA) ........................................................................................ 121

4.2.1 Antiprotozoal mode of action of FA ......................................................................................... 124

4.3 Elongation Factor G (EF-G): Function and structure ....................................................................... 125

4.3.1 Function ................................................................................................................................... 125

4.3.2 Structure .................................................................................................................................. 125

4.4 Resistance to FA .............................................................................................................................. 129

4.4.1 In vitro resistance ..................................................................................................................... 129

4.4.2 In vivo resistance ...................................................................................................................... 130

4.4.3 Evaluation of the fitness cost of FAR ........................................................................................ 132

4.5 Validation of targets using target-based whole cell screening ....................................................... 133

4.6 Aims and objectives ........................................................................................................................ 134

4.7 Identification of spontaneous resistant mutants and determination of frequency of mutation. .. 134

4.7.1 Results of MIC90 screening of putative FAR Msm mutants ....................................................... 134

4.8 Fitness cost of the FA resistance-conferring mutation in Mtb ....................................................... 136

4.9 Determination of the susceptibility of the GKFA isolate to selected FA analogues and

antituberculosis agents ......................................................................................................................... 137

4.10 Validation of EF-G as the target of FA in Mtb: Evaluation of the effect of transcriptional silencing

of fusA on susceptibility of Mtb to FA and selected analogues ............................................................ 138

4.10.1 Results of the effect of transcriptional silencing of fusA on susceptibility of Mtb to FA and

selected analogues ............................................................................................................................ 139

4.11 Discussion...................................................................................................................................... 140

4.11.1 Msm results ............................................................................................................................ 140

4.11.2 Mtb result .............................................................................................................................. 142

xvii

4.12 Summary ....................................................................................................................................... 143

4.13 References .................................................................................................................................... 144

Chapter 5 ....................................................................................................................................... 150

Conclusions and Recommendations for Future Work ...................................................................... 150

5.1 Conclusions ..................................................................................................................................... 150

5.2 Recommendations for future work ................................................................................................ 153

Chapter 6 ....................................................................................................................................... 155

Experimental ................................................................................................................................. 155

6.1 Chemistry ........................................................................................................................................ 155

6.1.1 Reagents and solvents ............................................................................................................. 155

6.1.2 Chromatography ...................................................................................................................... 155

6.1.3 Physical and spectroscopic characterization ........................................................................... 155

6.1.4 General synthetic procedure for 4-aminoquinoline derivatives .............................................. 158

6.1.5 General synthetic procedure for fusidic acid amides .............................................................. 160

6.1.6 General synthetic procedure for fusidic acid esters ................................................................ 165

6.2 Biology experimental section .......................................................................................................... 168

6.2.1 Turbidimetric Solubility ............................................................................................................ 168

6.2.2 Bacterial strains and growth conditions .................................................................................. 170

6.3 Assessment of the effect of transcriptional silencing of fusA1 on the susceptibility of Mtb to FA

and selected analogues using the checkerboard assay ....................................................................... 173

6.4 Generation of spontaneous resistant mutants ............................................................................... 174

6.4.1 Generation of FA-resistant mutant strains of Msm ................................................................. 174

6.4.2 Generation of FAR Mtb mutants .............................................................................................. 174

6.4.3 DNA extraction, purification and amplification ....................................................................... 175

6.4.4 PCR amplification of genomic DNA .......................................................................................... 177

xviii

6.4.5 Sequencing ............................................................................................................................... 177

6.4.6 Targeted sequencing of MsmfusA1 and fusA2 ........................................................................ 177

6.4.7 Targeted sequencing of Mtb fusA1 .......................................................................................... 178

6.5 Competition assay ........................................................................................................................... 178

6.5.1 Calculation of relative fitness ................................................................................................... 179

6.6 Cytotoxicity assays .......................................................................................................................... 179

6.6.1 Cytotoxicity assay in CHO ......................................................................................................... 179

6.6.2 Cytotoxicity in THP-1 cells ........................................................................................................ 180

6.6.3 Macrophage cytotoxicity assay ................................................................................................ 181

6.7 Macrophage efficacy assay ............................................................................................................. 181

6.7.1 Infection of THP-1 with Mtb .................................................................................................... 181

6.8 Evaluation of mycobacterial metabolism of FA and selected FA analogues .................................. 182

6.9 References ...................................................................................................................................... 184

Appendix 1: genomic DNA amplified from the FAR Mtb mutant strain GKFA .......................................... 185

Appendix 2: Primers used for targeted sequencing of PCR amplicons ..................................................... 186

Appendix 3: Checkerboard assay plate of FA tested against the Mtb fusA knockdown strain ................ 188

Appendix 4: COSY NMR spectrum of AW23 ............................................................................................. 189

xix

List of Appendices

Appendix 1: genomic DNA amplified from the FAR Mtb mutant strain GKFA 185

Appendix 2: Primers used for targeted sequencing of PCR amplicons 186

Appendix 3: Checkerboard assay plate of FA tested against the Mtb fusA knockdown

strain 188

Appendix 4: COSY NMR spectrum of AW23 189

xx

List of Figures

Figure 1.1: First-line anti-TB drugs 3

Figure 1.2: Examples of second-line anti-TB drugs 7

Figure 1.3: Examples of anti-TB agents developed by repurposing and repositioning 11

Figure 1.4: Examples of natural product-derived clinical drugs 14

Figure 1.5: The percentage of natural product-derived small molecule approved

drugs in the years 1981-2010 15

Figure 1.6: Proportion of drugs developed from natural products 16

Figure 1.7: Examples clinical drugs containing privileged scaffolds 18

Figure 1.8: Semi-synthesis of rifampicin 21

Figure 1.9: Spectinomycin and representative spectinamides 22

Figure 1.10: Causes of compound attrition in drug discovery 35

Figure 1.11: Examples of active metabolites developed into clinical drugs 39

Figure 1.12: Morphine and its metabolites in man 40

Figure 2.1: Fusidanes with their respective carbon atom numbering indicated 62

Figure 2.2: Cyclopentanoperhydrophenanthrene with a trans-syn-trans arrangement 63

Figure 2.3: A summary of FA antibacterial SAR 64

Figure 2.4: Examples of quinolines with antimycobacterial activity 65

Figure 2.5: Classification of hybrids, adapted from Srivastava and Lee 66

Figure 2.6: Illustration of the prodrug concept 67

Figure 2.7: Fusidic acid amide and ester aminoquinoline hybrids 71

Figure 2.8a: Canonical structures for the inductive and mesomeric effects of the

quinoline nitrogen 74

Figure 2.8b: Mechanism of formation of 7-chloroquoniline-4-amine derivatives 74

Figure 2.9: Spectrum of AW21 illustrating the aliphatic protons of the C-4 side chain 78

Figure 2.10: Spectrum of AW23 illustrating the aromatic protons of the quinoline

ring and identifiable FA protons 79

Figure 3.1: FA and analogues selected for evaluation of intracellular efficacy in THP- 99

xxi

1

Figure 3.2: FA metabolites in man 104

Figure 3.3: Postulated conversion of FA to 3-epiFA 105

Figure 3.4: C-16 hydrolysis of FA 105

Figure 3.5: Examples of aryl and alkyl esters evaluated for metabolic stability in

microsomes 106

Figure 3.6: Structures of FA analogues for evaluation of mycobacterial metabolism 108

Figure 4.1: Structure of cyclomarin A 120

Figure 4.2: Antibiotic target sites during bacterial protein synthesis 122

Figure 4.3: Crystal structure of T. thermophilus EF-G from Margus et al. 125

Figure 4.4: Translocation on the ribosome and the inhibitory effect of FA 126

Figure 4.5: Schematic of the proposed mechanism of FA resistance mediated by

FusB-type proteins 130

Figure 4.6: Structures of selected FA analogues tested against putative Mtb FAR

mutants 137

Figure 4.7: Sequence alignment of EF-G and EF-G2 from Mtb and Msm 140

Figure 4.8: Domain conservation comparison between EF-G and EF-G2 141

Figure 6.1: Layout of turbidimetric solubility assay compound pre-dilution plate 167

Figure 6.2: Turbidimetric solubility assay plate layout 168

Figure 6.3: Layout of checkerboard assay 96-well microtitre plate 172

Figure 6.4: Structure of ME-29 181

xxii

List of Graphs

Graph 3.1: Log reduction of CFU in THP-1 infected Mtb treated with FA 100

Graph 3.2: Log reduction of CFU in THP-1 infected Mtb treated with GKFA16 101

Graph 3.3: Log reduction of CFU in THP-1 infected Mtb treated with GKFA17 101

Graph 3.4: Log reduction of CFU in THP-1 infected Mtb treated with GKFA37 102

Graph 3.5: Ratio of FA to internal standard in live and heat-killed Mtb cultures over

time 109

Graph 3.6: Ratio of FA and GKFA16 to internal standard in live and heat-killed Mtb

cultures over time 110

Graph 3.7: Ratio of FA and GKFA17 to internal standard in live and heat-killed Mtb

cultures over time 110

Graph 3.8: Ratio of GKFA61 to internal standard in live and heat-killed Mtb

cultures over time 111

Graph 3.9: Ratio of AW23 to internal standard in live and heat-killed Mtb cultures

over time 111

Graph 3.10: Ratio of AW25 to internal standard in live and heat-killed Mtb cultures

over time 112

Graph 4.1: Growth curve of wild-type Mtb and FA-resistant mutant strain Mtb

GKFA 135

xxiii

List of Schemes

Scheme 2.1: Retrosynthetic analysis of FA amide and ester derivatives 72

Scheme 2.2: Synthesis of 4-aminoquinoline diamines and alcohols 73

Scheme 2.3: Synthesis of FA amide derivatives 75

Scheme 2.4: Synthesis of FA ester derivatives 76

xxiv

List of Tables

Table 1.1: Advantages and disadvantages of natural products in drug discovery 19

Table 1.2: Examples of non-CYP450 enzymes involved in metabolism of xenobiotics 37

Table 2.1: Examples of prodrugs with improved lipophilicity or permeability 69

Table 2.2: Yields of 4-aminoquinoline alcohols and diamines 74

Table 2.3: Yields of FA amides and esters 76

Table 2.4: Results of turbidimetric solubility evaluation of the FA-aminoquinoline

hybrids 81

Table 2.5: Results of antimycobacterial testing of FA–aminoquinoline hybrids 83

Table 2.6: Results of MIC of selected individual aminoquinoline derivatives and their

mixtures with FA 86

Table 3.1: Results of IC90 determinations of individual aminoquinoline derivatives

and their mixtures with FA in CHO 95

Table 3.2: Results of evaluation of cytotoxicity of the FA-aminoquinoline hybrids 96

Table 3.3: FA and analogues evaluated for intracellular efficacy in THP-1 99

Table 4.1: Sequence homology between EF-G and EF-G2 among several species 127

Table 4.2: Results of MIC90 screening of putative FAR Mtb mutants 134

Table 4.3: Results of selected MIC90 screening of FA against colonies isolated during

resistant mutant generation 136

Table 4.4: MIC90 of FA, selected analogues and anti-TB drugs in fusA conditional

knockdown strain 138

Table 6.1: Gradients used for investigation of the purity of compounds with

preparatory HPLC 155

Table 6.2: Gradient used for investigation of the purity and mass of compounds

using LC-MS 155

Table 6.3: Bacterial strains used in this study 169

xxv

List of publications arising from this thesis and conferences attended

Publications

Review:

Kigondu E. M., Wasuna A., Warner, D. F., Chibale K. Pharmacologically active metabolites, combination

screening and target identification-driven drug repositioning in antituberculosis drug discovery.

Bioorganic and Medicinal Chemistry, 2014, 15; 22(16):4453-61.

Manuscripts in preparation:

Kaur G., Makoah N., Njoroge M., Strydom S., Wasuna A., Warner, D. F., Wiesner L., Chibale K. Synthesis

and biological characterization of fusidic acid C-3 esters.

Wasuna A., Moosa A., Kaur G., Singh, V., Ioerger T. R., Chibale K., Warner D. F. Fusidic acid inhibits

Elongation Factor G in Mycobacterium tuberculosis.

Conferences and Training courses attended

Keystone conference, Novel Therapeutic Approaches to Tuberculosis, Keystone, Colorado, 30th March-

4th April 2014. Poster presentation: Fusidic acid is bacteriostatic against Mycobacterium smegmatis.

H3D symposium, Innovative Approaches to Tuberculosis therapy, Victoria Falls, Zambia, 27th - 30th

August 2014. Poster presentation: Mutation in fusA1 causes in vitro resistance of Mycobacterium

tuberculosis to Fusidic acid.

Novartis Next Generation Scientist Program (June - August 2015), Novartis Pharma AG, Basel,

Switzerland.

1

Chapter 1

Introduction and Literature Review

Chapter overview

This chapter provides an overview of the current Tuberculosis (TB) chemotherapy and its limitations.

Additions to the existing anti-TB agents are highlighted in the context of current drug discovery

paradigms, with a focus on the example of several old drugs that have been repositioned in recent

efforts to expand the TB drug pipeline. The relevance of natural products in drug discovery is discussed,

arguing its continued relevance using current examples of antimycobacterial compounds derived from

natural products. The importance of drug target identification earlier in the discovery process is

discussed, with highlights of the widely used methods. The major classes of drug metabolising enzymes

and the systems used in the study of metabolism are also outlined, highlighting the value of

incorporating drug metabolism studies in the optimization of compounds at various stages of discovery

and development. Finally, the justification and aim of this work are provided.

1.1 Tuberculosis: Epidemiological burden, disease aetiology and management

It is estimated that up to one third of the world’s population is infected with Mycobacterium

tuberculosis (Mtb).1 Of these, about 10% progress to active tuberculosis (TB) disease in their lifetime.2

Consequently, TB is a major cause of morbidity and mortality. The World Health Organization (WHO)

estimates that, in 2015, there were 10.4 million new cases of infection and that the disease claimed the

lives of 1.4 million people. Of major concern is the heavy burden of the disease in sub-Saharan Africa

and parts of Asia.3 The alarming rise in the emergence of drug-resistant forms of TB has compounded

global efforts to control this scourge. In sub-Saharan Africa, this is exacerbated by the Human

Immunodeficiency Virus (HIV) pandemic, which synergizes with TB in debilitating the host immune

response. HIV has been shown to increase the risk of activating latent TB infection (LTBI) to active

disease, and to accelerate progression to active TB.4,5 South Africa provides a stark example of this co-

epidemic: almost 70% of TB-related deaths are HIV-associated.1

Mtb is a Gram positive, intracellular pathogen infecting humans as obligate host. Transmission of Mtb

occurs via aerosol and, upon inhalation, the innate immune response is activated. Bacilli are

phagocytosed by immune cells, chiefly alveolar macrophages. Phagocytosis in turn stimulates a pro-

2

inflammatory response, and other immune cells such as lymphocytes, monocytes and fibroblasts are

recruited, culminating in the formation of a granuloma, the hallmark of TB infection.6 Infection is

contained through the activation of innate and adaptive immune responses that create a hostile

environment for Mtb. However, through several mechanisms, the bacilli evade host elimination,

resulting in productive infection.7 These include inhibition of phagosome-lysosome maturation,

cytokine-mediated host defences and resistance to host bactericidal mechanisms.6,8,9 The bacilli not only

succeed in evading elimination, but have been shown to replicate within the host.10 TB is primarily a

pulmonary disease, although dissemination to other body organs occurs in some cases6, especially in HIV

co-infected individuals.

The existing measures for TB prophylaxis and cure are vaccination and chemotherapy respectively. In

most countries, the public health sector has implemented vaccination programmes as well as the

detection and treatment of confirmed TB cases. The only available vaccine, Bacille Calmette-Guérin

(BCG), is administered to infants at birth. However, it confers protection against disseminated TB only in

infancy, with highly variable protection against pulmonary disease in adolescents and adults.11 To date,

efforts to create an alternative, more effective TB vaccine have been unsuccessful. Recently, MVA85A, a

vaccine candidate that reached phase IIb trials in South Africa, demonstrated no protection against TB in

infants.12 There are, however, several vaccine candidates at various stages of clinical trials which offer

hope for a TB vaccine in the future.11 Although prevention is ultimately cheaper and better than cure,

chemotherapy remains the mainstay in the management of TB.………………………………………………….

1.2 Antimycobacterial chemotherapy

In 1882, Robert Koch identified Mtb as the causative agent of TB. However, streptomycin (STM), the first

anti-TB antibiotic, was only developed in 1944 by Waksman and co-workers.13–15 Three decades

thereafter, during the “Golden era” of antibiotic discovery, TB chemotherapy evolved to the current

first-line multidrug regimen. The past 5 years have seen the addition of several new drugs to the

therapeutic options, but these are reserved for the management of drug-resistant forms of TB.16

1.2.1 Drug-susceptible TB Therapy

Pulmonary TB is a curable disease, with figures varying between 80-95% of the cases reported classified

as drug-susceptible.17,18 At programmatic level, this is implemented through the Directly Observed

Therapy, Short course (DOTS), using a combination of 4 drugs administered in 2 phases. The initial phase

3

of 4 months involves treatment with isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA) and

ethambutol (EMB), followed by a 2-month continuation phase with INH and RIF. However, the 6-month

multidrug regimen required to achieve cure has certain limitations: (1) toxicity and intolerance, which

necessitate treatment interruption and regimen changes, (2) drug-drug interactions, especially with

antiretrovirals in HIV co-infected patients, (3) and thus poor patient adherence, which have contributed

to the development of drug resistance.17 The first line drugs used in the management of drug-

susceptible TB are discussed briefly below.

Rifamycins

The rifamycins (Figure 1.1) are a class of antibiotics derived as a biosynthetic metabolite of the

bacterium Amycolaptosis mediterranei and consist of drugs such as rifampicin (RIF), rifabutin, and

rifapentine. Introduced in 1963, RIF is the most commonly used member of this class, while rifabutin

and rifapentine are newer generation derivatives. Rifamycins have a unique mechanism of action

amongst the clinically used antibiotics; they inhibit RNA synthesis through the formation of a stable

complex with RNA polymerase binding with the -subunit (encoded by rpoB). This leads to alterations in

RNA transcription and, consequently, nucleic acid metabolism, resulting in cell death. Widespread

resistance to RIF has been reported, known to be the result of single point mutations in rpoB, which

inspired the development of Gene Xpert, a diagnostic tool for the detection of RIF-resistant TB.16,19–21

Figure 1.1: First-line anti-TB drugs

4

Isoniazid

Isonicotinylhydrazine, more commonly known as isoniazid (INH), is a prodrug that was introduced in

1952. INH causes cell death through inhibition of enoyl-[acyl-carrier-protein] reductase, an enzyme

involved in the synthesis of mycolic acids that are a critical component of the mycobacterial cell wall.22,23

Its bioactivation from a prodrug relies on KatG, a mycobacterial catalase-peroxidase enzyme that

couples INH to NADH to achieve inhibition. Most cases of clinical resistance to INH have been mapped to

mutations in the katG gene, which reduces the catalytic activity of the peroxidase and thus markedly

reduces activity of the drug. In a few cases, mutations occur in either inhA or the promoter region of the

mabA-inhA operon; the former results in decreased affinity of the catalase enzyme for NADH, while the

latter causes over-expression of the wild-type enzyme and thus effective titration of the drug, resulting

in defective INH activation.24,25

Pyrazinamide

Like INH, pyrazinamide (PZA) is a prodrug. It requires bioactivation by mycobacterial pyrazinamidase

(encoded by pncA) to yield pyrazinoic acid (POA) which is pharmacologically active.26,27 Its mode of

action has been elusive, although it has been implicated in disruption of protein translation by targeting

a ribosomal protein, RpsA.28–30 PZA is characterized by differential kill kinetics, possessing bactericidal

activity against slow and non-replicating bacilli, whereas against fast-replicating bacteria, it is

bacteriostatic.31 Clinical resistance to PZA is associated with mutations in the pncA gene.26 Initially

thought to be activated exclusively by mycobacterial deamidase, Via and co-workers have recently

established that host amidases contribute to its hydrolysis and overall efficacy.32 PZA is unique in that its

development followed an unconventional path; it lacks activity against most strains of mycobacteria in

the conventional in vitro drug susceptibility assays. At acidic pH (often 5.5), however, it is active against

Mtb in vitro. A likely reason for its efficacy in vivo is that environment inside the macrophage is acidic.

PZA contributes to treatment shortening when added to drug regimens.33

Ethambutol

Ethambutol (EMB) was introduced into clinical use in 1961, and has several attractive features. It is

cheap, with low toxicity and consequently better patient tolerability than the other anti-TB drugs. Along

with PZA, it is used as adjunct therapy in treatment of MDR-TB.33 EMB inhibits arabinosyl transferases

5

essential for mycobacterial cell wall biosynthesis, resulting in cell death.34–36 Resistance to EMB is most

commonly associated with point mutations in the embBC operon while, in some cases, multi-step

mutations in genes encoding cell wall precursors precede embBC mutations, with varying degrees of

EMB resistance.37

1.2.2 Management of Drug-resistant TB

Resistance to STM was reported within 2 years of its initial use in TB treatment. Over the years, in the

estimated 5-20% of drug-resistant cases of TB patients, the emergence of drug resistance has taken on

multiple forms.15,16 These are classified as follows: (1) mono-resistance (resistance to a single agent); (2)

multi-drug resistant TB (MDR-TB), that is, resistance to INH and RIF; (3) extensively drug-resistant TB

(XDR-TB), that is, MDR plus resistance to two classes of the second-line drugs, fluoroquinolones and the

injectable drugs, and (4) totally drug resistant TB (TDR-TB) which is not officially defined but is ascribed

to forms of TB that are resistant to all first and second line drugs18. Drug-resistant forms of TB pose

additional challenges to drug-susceptible TB: therapy is longer, ranging from 18-24 months. In addition,

the drugs are costlier and more toxic. Some of the drugs are injectable and, together, these factors

further reduce patient adherence. Some of the second-line drugs are briefly discussed below.

Aminoglycosides

Aminoglycosides form a critical component of MDR and XDR TB management. They include STM,

kanamycin (KAN) and amikacin (AMK). The first of these, STM, was isolated from an actinobacterium,

Streptomyces griseus, and introduced clinically in 1944.13 Aminoglycosides are protein synthesis

inhibitors that act by binding irreversibly to the bacterial 30S ribosomal subunit. Resistance to

aminoglycosides is mostly associated with changes in the ribosomal drug binding site, specifically,

mutations in rpsL, the gene which encodes S12, an essential ribosomal protein. Mutations in this gene

account for approximately half of all STM-resistant clinical isolates reported. Fewer cases of resistance

are the result of mutations in the rrs gene.38,39 In addition, enzyme-mediated drug modification is also

associated with resistance to KAN. A mycobacterial acetyltransferase, enhanced intracellular survival

(Eis), has been shown to carry out multiple acetylations on KAN, rendering Mtb resistant to the drug.

Supporting evidence is the upregulation of mycobacterial Eis observed in KAN-resistant clinical

isolates.40,41 Aminoglycosides are formulated as injectables, requiring daily administration of painful

6

injections to patients. Another major drawback is their side-effect profile, with ototoxicity and

nephrotoxicity reported in a proportion of patients.42,43

Fluoroquinolones

These are an established class of broad spectrum antibiotics that have been developed from the first

member, nalidixic acid, that was used to treat urinary tract infections.44 The second generation agents

include levofloxacin and sparfloxacin, while the third generation consists of moxifloxacin (MOX) and

gatifloxacin (GAT). The latter two are a critical component of MDR-TB treatment. Their molecular target

is DNA gyrase, an enzyme that causes relaxation of supercoiled DNA strands. They inhibit DNA

replication by intercalating into DNA to form a drug-enzyme-DNA complex, which causes breaks in the

DNA strand.45 Recently, three landmark phase III studies investigated treatment shortening in drug-

susceptible TB using modified regimens incorporating the fluoroquinolones, MOX and GAT. Despite

evidence of treatment shortening in mouse efficacy studies, all three regimens failed to show superiority

over the existing first-line regimen.46–48

Macrolides

This class of antibiotics includes erythromycin, clarithromycin and azithromycin. They inhibit protein

synthesis by binding to 23S rRNA and thus prevent peptide bond formation. Mechanisms implicated in

the resistance to macrolides include drug efflux and mutations in the ribosomal drug binding site.44

7

Figure 1.2: Examples of second-line anti-TB drugs

Capreomycin

Capreomycin, an antibiotic isolated from Streptomyces capreolus, is a cyclic polypeptide used to treat

MDR-TB. It targets the mycobacterial ribosome at the interface of the small and large subunits and

requires ribosomal rRNA methylation for optimal binding, which in turn inhibits ribosome function.

Resistance in Mtb arises from mutations in tlyA, which encodes a methyltransferase which is also known

to be a virulence factor in Mtb.49 Mtb resistance is also attributed to mutations in the rrs gene that

encodes 16S rRNA.19,50

8

Thioamides

The main members of this class are ethionamide and protionamide, with the former more commonly

used to treat MDR-TB. As structural analogues of INH, they are also prodrugs (activated by EthA) and, in

addition, share a molecular target with INH. Thus, cross-resistance with INH has been reported.51–55

Thioamides also inhibit mycolic acid biosynthesis, resulting in cell death. In addition to mutations in

inhA, mutations in ethA have been associated with resistance to ethionamide.52 As might be expected,

resistance to these two drugs develops rapidly due to their cross-resistance.33 Based on a study of of

inhA and ethA drug resistance mutation patterns, Müller et al proposed alternative guidelines for the

individualised treatment of TB patients, which, if implemented, could potentially reduce the loss of INH

and ethionamide as chemotherapeutics for MDR-TB.56

para-Aminosalicylic acid (PAS)

An analogue of aspirin, the popular anti-inflammatory drug, PAS is a prodrug that was developed in

1948. It is activated by dihydropteroate synthase (DHPS) and dihydrofolate synthase (DHFS) to the active

moiety, a hydroxyl dihydrofolate which inhibits folic acid biosynthesis.57 Resistance is associated with

mutations in DHPS and DHFS, preventing its activation to the active dihydroxyl metabolite.57,58 It is a last-

choice drug among the second-line agents, due to its lower efficacy, poor gastric tolerability, and

requirement of cold chain storage (the less efficacious sodium salt does not, however, require this).33

D-cycloserine

Introduced in 1955, it is a D-alanine analogue that is a cornerstone of MDR-TB treatment whose major

drawback is its short shelf-life of 2 years.33 It is bacteriostatic, inhibiting D-alanine racemase and ligase,

enzymes essential for the synthesis of the peptidoglycan precursor UDP-muramyl pentapeptide.59 A

recent study by Desjardins et al in which whole genome sequence data from a diverse collection of

clinical isolates was analysed, revealed that resistance to D-cycloserine was associated with loss of

function mutations in ald and alr which encode D-alanine ligase and racemase respectively. Amongst the

anti-TB drugs, a remarkable feature of D-cycloserine is its central nervous system (CNS) effects which

make it ideal for TB management in psychiatric patients.60

9

1.3 Limitations of current chemotherapy

As highlighted above, the lengthy duration of treatment, toxicity and relatively poor efficacy of some of

the drugs demonstrate the need for new and better drugs for TB management. Inherent properties of

the bacillus are a huge contributor to these outcomes, and cannot be overlooked. In the face of host-

induced stresses to Mtb infection, mainly nitrosative, acidic, hypoxic and nutritional, the bacillus is able

to alter and down-regulate its metabolism. This metabolic shift allows it to enter a state of non-

replication and, consequently, emergence of sub- populations of cells. These include persister cells,

which are refractive to anti-TB drugs, necessitating alternative and longer periods of chemotherapy.61,62

The microenvironments in the caseous lesions in which Mtb reside during infection also require

differential drug pharmacokinetic considerations to achieve sterilization.9,63–65

The spectrum of Mtb disease, from latent to active disease and cases of relapse, has spurred a re-

thinking in the diagnosis and monitoring of TB in high-burden settings, especially in low- and middle-

income countries. Logistical challenges in drug access, diagnosis and drug susceptibility testing and

administration at the point of care also play a role16. Cases of short supply of some anti-TB drugs, and

the associated toxicities which necessitate drug interruption or withdrawal of specific agents, create

windows of monotherapy which, in turn, accelerate progression of resistance to some of the drugs66,67.

In all countries surveyed, resistance has emerged against all the clinically used drugs, attesting to the

survival capability of Mtb68. For most of the drugs discussed above, chromosomal mutations (single or

step-wise), cause changes in the molecular drug targets or auxiliary changes in the bacillus which inhibit

or greatly reduce pharmacological activity of the drugs69. The challenges posed by drug-resistant forms

of TB cannot be underestimated, with subsequently grave increases in morbidity and mortality

worldwide. In 2006, an outbreak of drug-resistant TB in Tugela Ferry, a small town in rural South Africa,

resulted in the death of 52 out of 53 patients with MDR-TB68.

1.4 Advances in TB drug discovery: Opportunities and challenges

As highlighted in section 1.4 above, the shortcomings of the current chemotherapy necessitate the

expansion of chemotherapeutic agents to treat both drug-susceptible and drug-resistant forms of TB.

Some of the goals include identification of: (1) Agents with novel mechanisms of action to overcome the

resistance observed in the current drugs; (2) Sterilizing drugs that eradicate the different sub-

populations of bacilli; (3) Treatment shortening agents, which can be incorporated into the existing drug

10

regimens; (4) The optimal pharmacokinetic parameters required to eliminate bacilli in macrophages and

caseous lesions that have variable susceptibility to the current drugs.70

Global efforts to these ends are variably structured, with private-public partnerships, and partnerships

between pharmaceutical companies and academia as some of the existing models. These include,

among others, the TB Alliance, Stop TB Partnership Working Group on New TB Drugs, and the TB Drug

Accelerator (TBDA) programme.71 The approval in 2012 of bedaquiline (BDQ), a diarylquinoline (Figure

1.3), was a milestone in TB drug discovery as this was the first novel drug with a novel mechanism of

action in over 4 decades.72 Subsequent trials have, however, elicited safety concerns owing to its

cardiotoxicity. In addition, soon after these trials, cases of resistance to BDQ emerged.

1.5 Drug repurposing and repositioning: recent additions to TB drug regimens

An approach that is increasingly applied to accelerate the drug development process is called drug

repurposing or repositioning.73,74 Previously, these two terms were used interchangeably; however, it is

now acknowledged that their individual meanings are different. Drug repurposing refers to the

application of an existing drug for a new indication, without any chemical modification. Such a drug only

requires dose optimization during preclinical and clinical development, since prior knowledge of its

safety and pharmacokinetic profiles are available. Consequently, this reduces the cost and shortens the

duration of drug development. On the contrary, drug repositioning is ascribed to the chemical

modification of a pre-existing drug for a new indication. This term can also be applied to drugs which

have been rescued from phase III or IV clinical stages, often due to poor safety or efficacy. Repurposed

or repositioned drugs may have similar, or, in some cases, additional targets in the new indications. In

the search for new TB drugs, drug repurposing and drug repositioning have seen several drugs progress

to various stages of clinical development. Drug repurposing and repositioning have expanded the

therapeutic options for TB, ameliorating morbidity and mortality associated especially with MDR- and

XDR-TB. A few examples are discussed below.

Nitroimidazoles

These drugs were based on the modest antimycobacterial activity of metronidazole, a nitroimidazole

used to treat protozoal and anaerobic bacterial infections.75,76 Structural modifications led to the

11

nitroimidazo-oxazines, with PA-824 and OPC-67683, now known as pretomanid and delamanid,

respectively.70,75,77 They are active against both replicating and hypoxic, non-replicating bacilli, and

possess a complex mechanism of action resulting in inhibition of cell wall mycolic acid biosynthesis and

respiratory poisoning.76 They are prodrugs, requiring bio-activation by the enzyme deazaflavin (cofactor

F420) -dependent nitroreductase (Ddn).78,79 Reports of resistance to delamanid have already emerged,

with most mutations mapped to fgd1, the gene encoding the nitroreductase responsible for its

bioactivation. Mutations have also been mapped to fbiA, which encodes an enzyme required for

generation of the co-factor F420, with depletion of this co-factor resulting in decreased bioactivation of

delamanid.80,81

Figure 1.3: Examples of anti-TB agents developed by repurposing and repositioning

12

Oxazolidinones

The oxazolidinones include linezolid (Lzd), the first member of this class, with sutezolid and tedizolid as

newer generation agents. A landmark study by Barry et al, in which Lzd was added to a failing regimen,

showed efficacy in 35% of XDR patients within 2 months, and 87% within 6 months.82,83 Oxazolidinones

act by binding to the 50S subunit of 23S rRNA, blocking the translation step of protein synthesis. A major

drawback of Lzd is its toxicity which, unfortunately, is linked to its mechanism of action. In addition to

inhibiting bacterial protein synthesis, it also inhibits mammalian mitochondrial protein synthesis,

resulting in adverse events such as myelosuppression and neuropathy.70,84 Because the toxicity is dose-

dependent, ongoing studies to determine efficacious but less toxic dose ranges are underway.

Derivatives tedizolid, sutezolid and AZD-5847 with better safety profiles have advanced to phase II

clinical trials. Tedizolid is clinically approved, while sutezolid is currently in phase II clinical trials. 70,85,86

Mutations in rplC, which encodes the 50S ribosomal protein, were identified in vitro in spontaneous

resistance, while clinical Lzd-resistant isolates with no corresponding mutations led Richter and co-

workers to the postulation of efflux as a mechanism of resistance.87,88

Riminophenazines

Clofazimine is the first-in-class among the riminophenazines, and was developed as an anti-TB drug, but

repurposed for treatment of leprosy.89 In cases of MDR and XDR-TB, clofazimine has shown efficacy and

relatively low toxicity.90–92 Its major drawbacks, the discolouration of skin, along with central nervous

system effects, have been a deterrent to its second entry as a repurposed anti-TB agent. It has shown

activity against slow-replicating bacilli, which are implicated in the formation of biofilm in vitro (in the

streptomycin–resistant strain SS18B) model of non-replication and in granuloma in vivo, and is thus an

attractive chemotherapeutic option for non-replicating forms of Mtb. Although there is contention on

the exact mechanisms of action of clofazimine, it has been shown to inhibit Mtb growth by intracellular

redox recycling and membrane disruption; consequently, there is a low frequency of resistance to

clofazimine.89 Less lipophilic analogues with improved efficacy and lower side effect profiles are in the

early stage of pre-clinical development.93,94

13

Ethylenediamines

These are structural analogues of EMB, with SQ109 as the front-runner in phase II clinical trials. Despite

their structural similarity to EMB, they are cell wall inhibitors with a different mechanism of action, with

polypharmacological effects on fungi and bacteria that lack the mycolic acids found in Mtb. They target a

mycobacterial cell wall protein, Mycobacterial membrane protein Large 3, (MmpL3), which has garnered

interest as a novel target of several new antimycobacterial agents.70,95–97 In addition, SQ109 inhibits

menaquinone synthesis, cellular respiration and ATP synthesis. These multiple mechanisms of action

suggest limited development of resistance, making it suitable for MDR-TB treatment.70

Drug repurposing has lent a new lease on life to TB drug discovery, with other established drug classes

such as the -lactams, rifamycins, carbapenems, macrolides and sulfonamides in ongoing pre-clinical

and clinical development.47,70,84,98,99

1.6 Current drug discovery paradigms

The most common approaches to drug discovery are either phenotypic or target based. The former

involves the use of whole cells to determine the minimum inhibitory concentration (MIC) of compounds.

Compound libraries are screened for in vitro activity against Mtb or, in some cases, such as in that of the

initial identification of BDQ, Mycobacterium smegmatis (Msm), a non-pathogenic surrogate of Mtb.

With the technological advancements over the last few decades, this is done at a high-throughput scale,

allowing thousands of compounds to be tested rapidly. Once hits are identified, their mechanisms of

action and resistance are investigated, typically through the identification of spontaneous resistant

mutants. Chemical synthesis is usually employed to generate structural analogues to develop Structure-

Activity Relationships (SAR) to allow optimization of pharmacological and pharmacokinetic properties of

the molecules that can be progressed to in vivo animal studies as a prelude to pre-clinical development.

Following the publication of the Mtb genome in 1998, a landmark study by Sassetti et al identifying a list

of essential genes has provided a useful tool for the unravelling of novel drug targets.100,101 Target-based

approaches begin with structural information on a target, which enables the high-throughput screening

(HTS) of compounds using computational (in silico) tools to identify small molecule inhibitors. The drug

targets, often enzymes, are then heterologously expressed and, using in vitro assays, enzyme inhibitors

are identified. A major drawback of this approach is often the lack of correlation between (often active)

enzyme inhibition in biochemical assays in vitro and lack of potency in whole-cell phenotypic screening.

14

This is attributed to factors such as compound permeability and, in the case of Mtb, the unusually thick

mycobacterial cell wall acts as a barrier to drug penetration.102,103

1.6.1 The challenges

Current drug discovery paradigms are hampered by several challenges: (1) in vitro to in vivo transition of

identified leads; (2) mitigation of off-target pharmacology, since most of these drugs have a background

in other disease indications; (3) repeated discovery of compounds that target the same enzymes, mostly

cell wall inhibitors102; (4) frequent discovery of prodrug-activated electrophiles, which raise concerns if

targeted by the human drug metabolism systems, and (5) the length and complexity associated with

target identification for the hits identified through phenotypic screening.70,102

1.7 Relevance of Natural products to drug discovery

1.7.1 Nature’s toolbox: The role of natural products in drug discovery

Figure 1.4: Examples of natural product-derived clinical drugs

15

Throughout history, natural products and their derivatives have played a key role as a source of

therapeutic agents. Chemical substances derived from plants, animals and microbes have been used to

treat disease since the dawn of medicine.104–106 Figure 1.4 above provides examples of some

pharmaceutical drugs that were derived from nature, spanning different therapeutic areas: artemisinin

(antimalarial), digoxin (heart failure), paclitaxel (anticancer), STM (antibiotic).

Natural products continue to play a key role in the drug discovery and development process, as

demonstrated in a review of all new chemical entities (NCEs) by Newman and Cragg covering the years

1981-2010, which showed that natural products contributed up to 50% of all small molecule approved

drugs in 2010. Figure 1.5 below demonstrates this.105

Their usefulness as pharmaceuticals comes in several forms: as natural products, as semi-synthetic

natural product analogues or as synthetic compounds based on natural product pharmacophores. A

pharmacophore is described as the ensemble of steric and electronic features that is necessary to

ensure optimal interactions with a specific biological target structure and to trigger (or to block) its

biological response.104

Figure 1.5: The percentage of natural product-derived small molecule approved drugs in the years

1981-2010 105

25.6

38.5

23.6

34.9

46.7

32.7

46.4

36.7

31.6

28.2

20

41.7 40 39.5

28.9

21.1

12.2

27.3

38.9

35.1

45.5

35.3

33.3

38.9 39.4

23.8

42.3

37.5

20.8

50

0

10

20

30

40

50

60

1980 1985 1990 1995 2000 2005 2010

Per

cen

tage

Year

16

Salient points in this comprehensive review conducted by Newman and Cragg include:

As shown in Figure 1.6 below, a considerable proportion of NCEs that are considered synthetic have

their origins in natural products. This may be in several forms: natural product mimetics, that is,

molecules that mimic the biological activity of natural products [synthetic drug (SD) mimicking a natural

product (NP), SD mimicking NP]; those that mimic natural product pharmacophores, and those based on

natural product pharmacophores.

Notably, of all the therapeutic areas that have benefited from natural product-based drug discovery in

the last thirty years, anti-infectives took the lion’s share, with antiviral vaccines topping the list. Thus,

infectious diseases continue to depend heavily on natural products and their structures for

replenishment of the drug discovery pipeline.

A novel designation, natural product botanicals (NB), has emerged to cover botanical defined mixtures,

which have gained recognition by the Food and Drug Administration (FDA) and other regulatory

authorities. This is a welcome change to the single drug-single target paradigm of modern day drug

discovery, and acknowledges Nature’s approach where a desirable phenotype may be the result of the

interaction of multiple targets, be they receptors or enzymes. This also implies that these desirable

phenotypes can be targeted by mixtures of natural products, as opposed to single or pure chemicals.

Figure 1.6: Proportion of drugs developed from natural products105

17

1.7.2 Nature’s suitability: The concept of privileged structures

Nature has been shown to provide natural ligands that have an affinity for a wide variety of receptors.

The concept of privileged structures was introduced by Evans et al in 1988, when they deduced that

certain “privileged structures” are capable of providing useful ligands for more than one receptor, and

that judicious modification of such structures could be a viable alternative in the search for new

receptor agonists and antagonists.107 These privileged structures are presumed to have undergone

evolutionary selection over time to allow optimal bioactivities and bioavailabilities. The exploitation of

these scaffolds to treat disease offers the strategic advantage of Nature targeting Nature, causing

natural products to be touted as keys whose locks remain to be uncovered in biological systems.104 As a

consequence, natural products have been extensively used to elucidate complex cellular mechanisms,

including signal transduction and cell cycle regulation leading to the identification of important targets

for therapeutic intervention.108

Structurally, privileged scaffolds are characterized as typically rigid, polycyclic heteroaromatic systems

capable of orienting varied substituent patterns in a well-defined 3-D space.108 In the quest for novel

drugs, medicinal chemistry has applied several strategies to incorporate these scaffolds of biological

relevance to small-molecule libraries. Figure 1.7 illustrates examples of privileged scaffolds found in

nature alongside clinical drugs that contain them. Among these, the quinoline and steroid scaffolds are

relevant in TB drug discovery and to the work reported in this project. Other scaffolds that present

opportunities for the field of TB include the chalcones, flavonoids, polyketides, steroids and peptides.109

As the field of natural product research is inexhaustible, no comprehensive list of privileged scaffolds is

achievable.

1.7.3 Declining natural product-based drug discovery

Despite this, in the 50 odd years that have been widely regarded as the ‘Golden era’ of antibiotic drug

discovery, there has been a decline in natural products-based drug discovery globally, attributable to

several reasons including but not limited to104:

1. The emergence of high throughput screening, resulting in a shift to chemical libraries of smaller,

‘screen-friendly’ molecules with defined molecular targets.

18

2. The evolution in the 1990’s of combinatorial chemistry, which promised chemically diverse, drug-like

screening libraries.

3. Major advances in molecular biology, cellular biology and genomics that have elucidated molecular

targets, presenting the tempting option of shorter drug discovery timelines.

4. A general decline in research on infectious diseases which has, until recently, relied heavily on natural

products as the source of new scaffolds.

Figure 1.7: Examples clinical drugs containing privileged scaffolds

1.7.4 Saving graces: Advances from technology platforms and genomics

Two major obstacles to advancing a natural product medicinal chemistry program are compound supply

and the ease of diversification strategies. Most natural products are sourced from plants, or from the

fermentation of microorganisms. Over the years, advances in chromatography that enable rapid, high

resolution refining steps to obtain pure products, coupled with powerful spectroscopic techniques that

allow characterization of the most complex natural products, have made what was a daunting

endeavour, a routine task in chemistry laboratories across the world. Grzelak et al have demonstrated

this by developing a platform consisting of microbiological, chromatographic and spectrometric

techniques, allowing the early identification of antimycobacterial compounds early in the isolation

process.110 Coupled to this, genomic tools have revolutionized this field of research by enabling

19

scientists to provide alternative ways of efficient compound supply. An example of this is the successful

probing of biosynthetic pathways to create strains that yield single components of interest. Doramectin,

a superior analogue of avermectin, has been successfully produced after gene knockout of aveC in

Streptomyces avermitilis, affecting the ratio of avermectin to doramectin.111 Another example is that of

genetic engineering of natural biosynthetic pathways to produce ‘unnatural’ natural products, especially

in the field of polyketide synthesis.111–113 These, among other numerous examples of bioorganic

chemistry and chemical biology, provide evidence that natural products remain a viable option as a

sustainable source of new molecules for the drug discovery pipeline.

Table 1.1: Advantages and disadvantages of natural products in drug discovery105,106

Natural products Synthetic molecules

Great structural diversity. Less structural diversity.

“Privileged” structures for optimal biological

interaction.

Structures have lower optimal suitability for

biological activity.

Tendency to possess multiple modes of action/

biological targets.

Less diversity in modes of action/biological

targets.

Multidisciplinary expertise is required for processing

of samples from the source.

Comparatively less multidisciplinary expertise

required.

Complex mixtures that require lengthy isolation,

purification and characterization.

Shorter timelines for isolation, purification and

characterization.

Low concentrations of compound and difficulty in

scale-up and limited supply.

Large quantities can be synthesised and supply

issues avoided.

1.7.5 Chemistry: Synthesis and semi-synthesis as a strategy in drug discovery

Natural products identified in screening campaigns can either be used as new molecules in their own

right, or can serve as the starting point for medicinal chemistry programs aimed at enhancing their

biological and other profiles.108 Thus, apart from ensuring a sustainable supply, diversification of natural

products is a key component of any natural products-based drug discovery programme. Organic

synthesis is a useful tool for both of these ends; de novo (total) synthesis of a compound aims to address

20

the drawback of supply. It is, however, labour- intensive and time-consuming, thus hardly pragmatic for

large-scale, pharmaceutical manufacturing of drugs. The other approach, chemical semi-synthesis,

involves the modification of an existing scaffold, and usually aims at improving biological properties such

as potency, or altering other physicochemical properties such as solubility, lipophilicity, or to improve

the toxicity profile by making a molecule more selective for the intended drug target.

The effort to incorporate privileged scaffolds from Nature in small-molecule libraries as starting points

for medicinal chemistry programs inspired advancements in synthetic chemistry. These include

combinatorial chemistry, diversity-oriented synthesis (DOS) and privileged substructure-based diversity-

oriented synthesis (pDOS), which resulted in the generation of molecularly diverse, large, natural

product-like libraries efficiently, expediently and at a fraction of the cost of conventional synthetic

methods.82,108,114,115

1.7.6 Semi-synthesis in TB drug discovery

The existing TB regimens are composed of drugs that fall into three categories: totally synthetic

molecules, unaltered natural products and semi-synthetic derivatives of natural products. INH and PZA

are purely synthetic, small molecules in the first line TB chemotherapy regimen. Unaltered natural

products include STM, while a pertinent example of a semi-synthetic derivative of a natural product is

RIF, the 3-(4-methylpiperazinoiminomethyl) derivative of 3-formylrifamycin SV, a member of the

rifamycins.116 As illustrated in Figure 1.8 below, through several enzymatic modifications, rifamycin B,

the primary product of fermentation of Nocardia mediterranei, yields precursors of the key intermediate

rifamycin SV, which is subsequently chemically derivatized to RIF.

21

Figure 1.8: Semi-synthesis of rifampicin

22

Recently, Lee and co-workers synthesised spectinamides (Figure 1.9), amide derivatives of the old

antibiotic, spectinomycin (SPEC), a protein synthesis inhibitor. The spectinamides are a promising new

class of amide derivatives of SPEC with potent antimycobacterial activity. They were designed to

overcome the poor antimycobacterial activity of SPEC.117 These examples illustrate the continued

relevance of semi-synthetic modification of natural products in the expanding the panel of

antimycobacterial agents for TB drug discovery.

Figure 1.9: Spectinomycin and representative spectinamides

Other examples include griselimycin, a cyclic peptide isolated from Streptomyces species in the 1960s

that has garnered renewed interest recently. It has a novel mechanism of action, and is active in vitro

and in vivo against both drug-sensitive and drug-resistant forms of Mtb. Its excellent pharmacological

profile has been followed up with medicinal chemistry efforts which have identified derivatives with

improved pharmacokinetic properties, with one progressing to late pre-clinical studies.118–120

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1.8 Target identification and determination of mechanisms of resistance

Target identification and mechanism of action studies are useful in the drug discovery process, as they

provide valuable information that enables faster, optimal design of inhibitors by guiding SAR studies.

Furthermore, they provide an understanding of off-target effects that may be responsible for drug

toxicity, thus facilitating the optimisation of selectivity through appropriate chemical modification

earlier in the drug discovery/development cascade.103 An additional outcome of target identification is

the potential for drug repurposing, as exemplified in section 1.1.4 above by the antibacterial agents that

have been repurposed for Mtb based on their mode of action. In some cases, drugs have been

repurposed as a result of the discovery of novel modes of action applicable to other indications

Following recent insights into host-pathogen interactions, pathogenesis, inflammatory pathways, and

the host’s innate and acquired immune responses to TB infection, a wide range of host-directed

therapies (HDTs) with different mechanisms of action have been identified. Some of the HDTs identified

are drugs that have been repurposed from the treatment of non-communicable diseases. Statins

(cholesterol lowering agents), metformin (an oral hypoglycaemic agent), valproic acid (an anti-epileptic),

and imatinib (an anticancer drug), have demonstrated a role in the induction of autophagy and

phagosome maturation. The use of HDTs as adjuncts to existing TB treatment regimens could potentially

shorten the duration of treatment or improve clinical outcomes in patients infected with both drug-

susceptible and drug-resistant TB.121,122 Ongoing preclinical and clinical studies to evaluate the efficacy of

several repurposed drugs are underway. There are ongoing pre-clinical and clinical studies evaluating

the efficacy of several repurposed drugs as HDTs.121,123,124

1.8.1 Target identification and Mechanism of Action determination

Target identification is an upstream process independent of drugs that involves identifying pathways or

components thereof that are critical to survival of an organism or pathogenesis. Subsequent

characterization of these essential enzymes or proteins can facilitate drug discovery efforts to identify

suitable chemical inhibitors. Part of the challenge of identifying new classes of anti-TB drugs is the

paucity of novel chemically and biologically validated targets. The genome of Mtb H37Rv was

sequenced in 1998 and reannotated in 2002, providing the sequences of all genes and the function of

most.125,126The accompanying challenge is the identification of ‘druggable’ targets in the genome;

24

properties such as essentiality, susceptibility to inhibition, lack of human homologues or the lowest

possible homology to human counterparts are difficult to achieve in a single molecule or class of

molecules.77

Mechanism of action, on the other hand, is a drug-dependent downstream process that involves the

elucidation of the specific way or ways in which a drug elicits the desired inhibitory biological effect.

Many of the drug discovery tools available today were not in existence in the ‘golden era’ of antibiotic

discovery, and thus most of the anti-TB drugs in use were developed through phenotypic screening.

Moreover, very few clinical trials were conducted, therefore very few of the drugs entered clinical use

with a priori knowledge of their mechanisms of action; RIF and PZA had their mechanisms of action

established almost a decade after the onset of clinical use127 while that of INH was explained until >30

years after introduction.128

Approaches to target identification can be broadly classified into genomic, biochemical and

computational methods. Due to their complementarity, often more than one approach is employed to

uncover the targets of successful hit compounds from phenotypic screens.

1.8.1.1 Biochemical approach: proteomics

The classical biochemical approach is based on the interaction between a small molecule or natural

product and a protein target, which is broadly referred to as proteomics.129 It relies largely on the use of

drug-affinity chromatography techniques - more commonly, the use of a labelled ligand or small

molecule suspected to have affinity for a particular protein target.130–132 This is referred to as chemical

proteomics, and is useful for obtaining powerful and precise information on the modes, affinity and

kinetics of the binding of molecules to their targets103,133–136. Proteomics has been successfully used to

complement the phenotypic approach in identification of targets of several drugs.137–139

1.8.1.2 Genomic approach

The genomic approach has been the mainstay of target identification for new drugs.140,141 Present-day

technology has enabled the relative ease of small and large-scale modifications of DNA and RNA, and

advancements in parallel sequencing technology have made it relatively inexpensive and fast.141 Genes

25

with previously unknown and essential functions have been uncovered in this way, as demonstrated by

the seminal work of Sassetti et al who used Transposon site hybridization (TraSH) mutagenesis to

comprehensively identify genes required for optimal growth, the so-called “survivasome” of Mtb. This

was a milestone in unraveling the functions of Mtb genes, consequently expanding the panel of

potential drug targets.100,101

In one study, Boshoff et al characterised the gene expression profiles of Mtb when subjected to 75

different growth and environmental conditions. These included growth on alternative carbon sources,

starvation states, and treatment with different classes of anti-TB drugs.142 In addition, the ability to

modulate pathways using gene knockdowns, knock-ins and knockouts has facilitated drug target

identification at a high-throughput scale. A landmark study by Abrahams et al demonstrated the power

of target-based whole cell screening (TB-WCS) as a tool for drug discovery. These researchers designed

and generated conditional mutants that underexpress essential genes, consequently identifying

inhibitors of pantothenate synthetase (PanC), an enzyme considered essential in Mtb.143 A recent review

by Evans and Mizrahi highlighted the strengths and limitations of this approach in subsequent validation

of identified targets.144

1.8.1.3 Computational tools

It is not feasible to identify the targets and mechanisms of action of each compound or class in drug

discovery; the financial and time costs are massive, with high attrition rates for individual compounds.145

Computational methods offer an expedient complementary tool for drug target identification and,

subsequently, can be exploited in improving the efficiency of their binding to the target, reducing

potential on or off-target toxicity, and even by designing multi-targeting compounds. Various

computational tools have been used with varying levels of success in target identification, and the

success of these methods depends on the choice of screening tools and their implementation in each

phase of the drug discovery process.146,147

Computer-aided drug design uses chemistry to predict whether molecules bind to a target and, if so,

how strongly. Several in silico methods such as homology modelling, molecular docking, similarity

searches, and others are used in computational approaches to target identification.146–148 They can be

broadly categorised into structure-based and ligand-based methods. A third method, the use of

26

bioinformatics tools, is based on genomic, proteomic and statistical data and has gained traction in

recent years with the development of powerful computational systems. Much like in vitro experiments,

these are screening platforms that allow the reduction of enormous libraries of compounds to more

manageable numbers, to filter the number of drug candidates that can be progressed to each stage of

the process.

1.8.2 The value of re-discovery: Polypharmacology

The single drug-single target paradigm that was widely held in drug discovery has been challenged in

recent years as further research on established drugs with apparently singular known modes of action

has revealed additional targets. This has led drug discovery scientists to the knowledge that efficacy and

toxicity (on- and off-target effects) are often the result of modulation of multiple targets.149 This

phenomenon, known as polypharmacology, can be exploited in several ways. Multiple drug targets can

be harnessed for potential synergistic effects, enhancing the desired phenotype. Secondly, knowledge of

off-target pharmacological effects can be used to mitigate off-target toxicity through the design of more

selective drug molecules. Thirdly, uncovering additional targets of drugs or compounds can lead to the

repositioning of de-prioritised hit or lead compounds for different disease indications. In addition,

polypharmacological prediction can unravel new molecular targets for existing drugs, expanding their

scope of clinical benefit at a lower cost for the preclinical phase in comparison to a de novo drug

discovery project.150

A greater challenge in polypharmacology is the deliberate design of multi-targeted compounds or drugs,

demonstrated by Apsel and co-workers who rationally designed small molecule inhibitors targeting two

different protein families that are popular targets in cancer chemotherapy.151 A plausible benefit of this

approach is decreased likelihood of resistance to the drug by compensatory mechanisms, as two

different targets are inhibited simultaneously: the development of resistance by compensatory

mechanisms is therefore theoretically more difficult.

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1.8.3 Mechanisms of drug resistance

Exposure to a drug induces mycobacterial stress responses that favour both genetic and physiological

survival mechanisms.70 Mechanisms of drug resistance can be broadly classified as intrinsic and

acquired.

1.8.3.1 Intrinsic drug resistance

Intrinsic resistance refers to the innate ability of a bacterium to resist the activity of a particular

antimicrobial agent through its inherent structural or functional characteristics.19 Intrinsic factors

include the cell wall as a permeability barrier, transcriptional regulation of drug resistance, drug

modification, mycothiol-mediated protection, and drug efflux. Efflux is responsible for both intrinsic and

acquired drug resistance, and will be discussed under acquired drug resistance mechanisms.

The mycobacterial cell wall as a permeability barrier

The architecture, molecular composition and structural complexity of the mycobacterial cell wall

distinguish it from that of all other microorganisms. Rich in mycolic acids, these characteristics enable it

to present a barrier to permeability to both hydrophobic and hydrophilic molecules, antibiotics included.

The cell envelope of mycobacteria is notorious for being several-fold less permeable to

chemotherapeutic agents when compared to functionally similar cell walls of other bacteria.152 Apart

from impeding entry to antibiotics, this multi-layered barrier enables the organism to survive under

harsh conditions by resisting chemical injury and dehydration through the various roles played by each

of the components of the cell envelope.153–155 Thus, it is not surprising that since the advent of TB

chemotherapy, inhibition of mycolic acid biosynthesis has been one of the most widely exploited and

successful drug targets. Disruption of the mycolic acid biosynthetic pathway is a well proven and

effective therapeutic target of several anti-TB drugs such as INH and EMB. Interestingly, these two anti-

TB agents were developed without prior knowledge of their ultimate molecular targets.

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Transcriptional regulation of drug resistance

During infection and consequent antimicrobial treatment, Mtb undergoes various environmental

stresses such as hypoxia, genotoxic stress, and nutrient starvation. Mtb generates a coordinated,

specific response to antimicrobial treatment. A number of drug-induced transcriptional changes are

activated that lead to antimicrobial tolerance or intrinsic drug resistance. For example, Colangeli et al

demonstrated that antibiotic-induced expression of an MDR efflux pump operon was associated with

resistance to INH and EMB.156 Understanding the essentiality of the roles of these transcriptional factors

in mycobacteria would reap the benefit of expanding the potential drug targets for TB drug discovery.

Drug modification/degradation

Mycobacteria are able to inactivate xenobiotics such as antimicrobials through enzyme-mediated

chemical modification, resulting in drug resistance. In addition to the example of KAN in section Error!

Reference source not found. above, there are several other examples of antimicrobials degraded by

Mtb. Intrinsic resistance of Mtb to -lactams arises through two major mechanisms: poor outer

membrane permeability and drug modification.157 Mtb is resistant to -lactams through the constitutive

expression of -lactamase (encoded by blac), which inactivates -lactams through hydrolysis of the -

lactam ring.158 There are two widely-used approaches to circumvent this; co-formulation of -lactams

with -lactamase inhibitors such as clavulanic acid, and the development of carbapenems, which are

resistant to -lactamase.159 In a recent study, Warrier et al reported N-methylation as a mechanism of

Mtb resistance in vitro. These researchers demonstrated that a novel bactericidal compound was

inactivated through methylation by a previously uncharacterized methyltransferase, Rv0560c.160

Mycothiol-mediated protection

In the case of pathogenic organisms like Mtb, toxic oxidants from the host phagocytic response impose

oxidative stress, resulting in the alteration and corruption of lipids, carbohydrates and nucleic acids in

the mycobacterial cell wall and consequent death. Bacterial thiols play several roles in microorganisms.

They act as redox co-factors, redox buffers (important in the management of oxidative stress) and in the

detoxification of xenobiotics. Mycothiol (MSH), the predominant thiol in mycobacteria, has analogous

functions to Glutathione (GSH), an antioxidant in other bacteria, and functions to mop up free radicals

29

such as reactive oxygen species (ROS) and reactive nitrogen species (RNS).161,162 Decreased levels of MSH

have been associated with increased susceptibility of Mtb to RIF, STR and erythromycin, and even Mtb

resistance to the pro-drugs INH and ETH have been associated with decreased levels of MSH. The role of

xenobiotic detoxification is dependent on a number of enzymes, which have been discussed in a review

by Jothivasan and Hamilton.163

1.8.3.2 Acquired drug resistance

Acquired drug resistance occurs when a microorganism obtains the ability to resist the activity of a

particular antimicrobial agent to which it was previously susceptible. Acquired drug resistance in Mtb is

caused mainly by spontaneous mutations in chromosomal genes, and the selective growth of such drug-

resistant mutants may be promoted during suboptimal drug therapy.19

Spontaneous mutations

In Mtb, the evolution of resistant strains is driven mainly by the sequential acquisition and accumulation

of spontaneous mutations in chromosomal genes, with several physiological consequences that

manifest phenotypically as drug resistance. These mutations result in the alteration of the drug targets

in several ways such as: (1) over-expression of the drug target, as in the case of INH; (2) alteration of the

drug-binding site, consequently decreasing its affinity for the drug in the case of RIF, the

aminoglycosides and macrolides; and (3) partial or complete inhibition of prodrug activation in the case

of INH and PZA, among others.69,164 Spontaneous mutations may arise in genes that encode drug-

modifying enzymes, as is the case for the aminoglycoside KAN. Mtb evolves to MDR and XDR through a

step-wise accumulation of chromosomal mutations, each of which confers resistance to individual

drugs.165

In addition to mutations within single genes, drug resistance is also influenced by phenomena such as

epistasis – the interaction of additional genetic mutations, where the phenotypic effect of one mutation

is determined by the presence of one or more other mutations – and bacterial fitness – that is, where

mutation(s) may impact the level of resistance, growth rate, virulence, and transmissibility of a resistant

strain of Mtb.20,69

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Drug Efflux

Efflux is a ubiquitous mechanism responsible for intrinsic and acquired drug resistance in prokaryotic

and eukaryotic organisms. Mtb presents one of the largest numbers of putative efflux pumps (EPs)

compared with its genome size. Consequently, active drug efflux systems have been shown to be

present in mycobacteria, extruding structurally and functionally unrelated compounds, including drugs.

This results in drug tolerance, as reported by Adams et al, who demonstrated the acquisition of drug-

tolerance during bacterial residence in macrophage as a result of efflux.166 Efflux pumps may select for a

single substrate or may be non-selective, conferring a multidrug resistant (MDR) phenotype.167

There are several clinical implications of the MDR efflux pumps; they have been implicated in the efflux

of a wide variety of substrates, among them -lactam antibiotics, which are being explored as a

therapeutic option for MDR-TB, ethionamide, the aminoglycosides, fluoroquinolones and macrolides, all

of which are part of the MDR-TB treatment regimen.167,168

Several strategies to counter the clinical impact of EPs are being explored. These include the use of

efflux pump inhibitors (EPIs) or efflux inhibitors (EI) such as thioridazine, verapamil, reserpine, and

omeprazole, all of which are clinically used drugs, albeit for non-infectious diseases.15,169–171 Medicinal

chemistry approaches have been employed to improve the design of existing EPIs and EIs and also to

design novel antimicrobials that are not substrates of the efflux pumps.117,172 These different strategies

have had varying levels of success at the in vitro and in vivo stages of testing and will hopefully form the

basis of adjunctive therapy or offer novel anti-TB drugs with less potential for efflux-mediated

resistance.173

1.9 Mycobacterium tuberculosis: An intracellular pathogen

As mentioned in section Error! Reference source not found. above, following inhalation of bacilli,

activation of innate and adaptive immune responses in the host contains the infection.174 Phagocytosis

by alveolar macrophages is followed by the recruitment of other immune cells such as neutrophils to the

site of infection, forming the granuloma, the primary structure that contains Mtb in the host. Within the

granuloma, several stress responses are activated during host infection, briefly summarized as:(1)

reactive intermediates, which cause oxidative and nitrosative stress; (2) acidification of the

phagolysosome; (3) nutritional starvation and (4) hypoxia.8,175

31

Through a range of host and bacterial-mediated mechanisms, the bacilli are able not only to survive, but

to replicate within the intracellular environment, consequently bursting out of the macrophages and

causing necrosis of the granulomatous lesions. Some of the mechanisms employed are: (1) inhibition of

phagosomal maturation; (2) production of radical scavengers that can process/convert reactive oxygen

species; (3) use of pH as an environmental cue, releasing chloride ions to counter the proton influx in the

phagosome; (4) synthesis of mycothiol, which serves as an antioxidant, maintaining intrabacterial

homeostasis; (5) induction of a ‘foamy macrophage’ phenotype in which lipid bodies may shelter

bacteria. The bacilli within, having undergone a metabolic shift, scavenge lipids such as triacylglycerides

(TAG) and cholesterol from the host as a nutrient source.8,9,176

These lesions lead to the destruction of surrounding tissue, encasing the lesion in a fibrotic capsule

consisting of a collagen rim. The resultant structure, the caseum, has been shown to have distinct

microenvironments within, adding heterogeneity to the outcome of infection and variability in the

response to drug therapy. In some cases, the caseum liquefies, resulting in cavitary disease, which is

associated with poor clinical outcomes, the development of drug resistance, and relapse. The widely-

held paradigm of TB as a binary disease (either active or latent) was first challenged by Barry et al, who

proposed a spectrum of disease; from those who have cleared infection with no future risk of active

disease, to those who incubate actively replicating bacilli without clinical symptoms177. This was

corroborated by Lenaerts and colleagues, who demonstrated the dynamic nature of TB lesions in human

infection, contrary to the assumption that they are stable and unchanging, due to the long duration of

time between infection and symptomatic disease.9 Furthermore, these researchers found that,

compared to individuals who have cleared infection, asymptomatic individuals infected with actively

replicating bacilli are at a higher risk of developing active disease.177

The outcome of lesions is dependent on the interplay of bacterial and host factors. In some cases, the

failure of the adaptive arm of immunity leads to liquefaction and necrosis. Moreover, recent studies

have shown the involvement of lipid mediators such as eicosanoids in the regulation of pro- and anti-

inflammatory responses associated with cytokines such as IL-1, leukotrienes and Tumour Necrosis

Factor (TNF).9,178 The mouse model has been most commonly used in studies of host infection, with the

limitation that mice do not form granulomas upon Mtb infection. These fail to capture several features

highly relevant to human Mtb pathology: the development of caseous necrosis and subsequent fibrosis,

which may limit drug penetration; hypoxia, which affects metabolic state of the bacilli; intra and extra

32

cellular bacterial populations, which cause variability in drug efficacy; and liquefaction and cavity

formation, which affect clinical outcome, drug resistance, and relapse. The recently developed Kramnik

mouse model is promising since it is characterized by granuloma formation in some mice, and so has

gained popularity in in vivo studies.179 However, non-human primates best simulate the full spectrum of

human infection.9

The relevance of macrophages in the overall course of infection has been challenged in recent years.

Although bacilli survive and disseminate from the phagolysosome of activated macrophages, sputum

and bronchoalveolar lavage (BAL) samples from infected patients have revealed a larger proportion of

bacilli contained within neutrophils than in macrophages.180 The “life cycle” of Mtb has also been

disputed; some researchers argue that the bacillus spends most of its life extracellularly, with the

intracellular stage as a brief interlude following infection. Nevertheless, intracellular bacilli create an

additional hurdle on the path of TB drugs from the blood compartment to their molecular targets. A

drug must permeate the granuloma to reach the bacilli. Using MOX, Prideaux et al demonstrated that

the lack of correlation between its excellent in vitro activity and poor efficacy in eradicating bacilli in

caseum was due to its failure to reach cidal concentrations.64 Furthermore, the microenvironment of the

phagolysosome contributes to decreased drug action, leading to heterogeneity in response to drug

treatment.9,64

Macrophages are among the first cells that Mtb encounters, and play a continued role in the outcome of

infection8. Furthermore, in a recent review, Tan and Russell argue that, in contrast to neutrophils, which

elicit a highly aggressive but short-lived response to Mtb, macrophages generate a longer, more

regulated antimicrobial response.8 It is therefore important to understand how Mtb survives under the

hostile intracellular environment. In vitro models of macrophage infection represent a powerful tool for

studying the host-pathogen interaction.181–183 Moreover, given the limitations of both target-based and

in vitro phenotypic screens, ex vivo screens employing Mtb-infected macrophages have been integrated

into the screening cascade in most drug discovery programs.184 These offer several advantages: they

provide information about uptake and cellular activity of compounds, and allow the identification of

cytotoxic compounds earlier in the screening cascade.184 In addition, these studies offer insights into the

bacterial mechanisms essential for intracellular survival that can be explored as part of host-directed

therapies.184 In a landmark study by VanderVen and colleagues, 340 000 compounds were screened and

found to have differential antimycobacterial activity, with a subset that were only active

33

intracellularly.185 These researchers further delineated the activity of these compounds, revealing a

smaller subset that inhibited intracellular Mtb growth by blocking Mtb catabolism of cholesterol, which

the bacilli scavenge from the host as a source of carbon due to the nutritional starvation induced by host

immune responses. In addition, these researchers developed fluorescent reporter strains of Mtb with

wide applicability in studying host-pathogen interactions at individual bacterium level when coupled

with confocal microscopy and other techniques; the heterogeneity in intracellular bacterial populations

in response to the intracellular environment, and its consequent impact on the outcome of infection and

treatment.185

In another landmark study, Brodin and colleagues used high-content imaging to screen a library of small

molecules, identifying inhibitors of a well-established drug target, decaprenylphosphoribosyl-ß-D-ribose

2’-epimerase 1 (DprE1), and reported that RIF and INH mitigate the cytopathogenic effects of Mtb

infection on macrophages.186 This study also reported 100-fold less activity of RIF intracellularly,

corroborating previous claims by other researchers.187 In recent years, macrophage models of infection

have evolved, and have even been coupled with imaging. This is exemplified by Stanley and co-workers,

who used high-content, high-throughput imaging to screen a library of clinical drugs, with the aim of

identifying those that target host pathways and consequently inhibit intracellular growth of Mtb.188

This study identified two broad categories; inhibitors of host pathways exploited by Mtb for virulence;

and those that activate immune responses targeting intracellular bacteria. Fluoxetine, an

antidepressant, was found to induce autophagy, while gefitinib, an anticancer drug, demonstrated ex

vivo and in vivo antimycobacterial efficacy in vitro and in mice infected with Mtb.188

Studies by Christophe et al showed that unstimulated human macrophages can restrict Mtb growth and

elicit both slow and fast replication phenotypes187. These authors demonstrated that bacterial

replication rates are dependent on bacterial burden, supporting the findings of Welin and co-workers,

who showed that, in low bacterial burden in macrophages, bacterial death is due to phagosomal

acidification, while in higher bacterial burden, necrosis is the main cause of bacterial death.189,190 In

addition, the findings of Christophe and colleagues challenged the widely held notion that bacterial

replication rates determine antibiotic susceptibility. This was corroborated by Raffetseder et al, who also

showed that drug susceptibility is not affected by the multiplicity of infection (MOI). MOI of 1 bacilli: 1

macrophage and 10 bacilli: 1 macrophage showed no differences in MIC values for INH, RIF and EMB.191

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1.10 Absorption, Distribution, Metabolism and Excretion (ADME)

The activity of a drug in vivo is dependent not only on its action at the intended target, but also on the

concentration and duration of action of the drug at the target site.192 When administered orally, a drug

dissolves in the gastrointestinal tract, is absorbed through the walls of the intestine either passively or

actively, before its distribution to various compartments within the body, after which it undergoes

metabolism and is eventually excreted. These aspects are measured by the parameters, absorption,

distribution, metabolism and excretion, which collectively form the acronym ADME. The interplay of

these parameters affects the overall fate of a drug in the body, commonly referred to as the

pharmacokinetics (PK). Toxicity, which is considered along with ADME properties, has in recent years

extended the acronym to ADMET193. Due to its implications for the safety and efficacy of a drug, PK

optimisation is a key component of the drug discovery and development process.193 As can be seen from

Figure 1.10, after the year 2000, compound attrition rates dropped significantly after the integration of

PK earlier in the drug discovery and development cascade194.

Figure 1.10: Causes of compound attrition in drug discovery adapted from Kola and Landis, 2004194

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1.10.1 Drug Metabolism

As part of their survival mechanism, living organisms eliminate foreign compounds (xenobiotics) that

pose a threat to their survival.195 Xenobiotics include environmental pollutants and toxins, food and the

chemicals present therein. Chemical modification facilitates the elimination process by making

xenobiotics amenable to excretion. Drugs are treated as xenobiotics, therefore the primary function of

drug metabolism is to make a lipophilic molecule more hydrophilic to enable conjugation and/or

excretion from the body.195–197

Metabolism involves a wide array of chemical modifications carried out by a variety of enzymes. In

addition to metabolising xenobiotics, these enzymes are also involved in the transformation of

endogenous substrates such as hormones, steroids and neurotransmitters.197,198 The liver is the main

organ involved in drug metabolism, although the skin, lungs, blood cells and plasma also contribute to

the biotransformation of certain drugs.

1.10.1.1 Drug metabolising enzymes

An array of enzymes is involved in the metabolism of drugs, and a review by Testa et al of more than

1000 xenobiotics found that cytochrome P450 (CYP450) enzymes account for up to 40% of the resultant

metabolites.199 CYP450 enzymes are encoded by 57 genes and 58 pseudogenes in man, pointing to their

significance in drug metabolism.200 Specifically, five major isoforms, namely CYP1A2, 2C9, 2C19, 2D6 and

3A4, are responsible for the metabolism of about 75% of clinically used drugs.201 This superfamily of

haem-containing enzymes is a class of oxidoreductases, specifically monooxygenases, which catalyse the

chemical addition of a single oxygen atom to substrates in a general reaction:

R-H + O2 + NADPH + H+ R-OH + NADP+ + H2O

Highest concentrations of CYP450 enzymes are expressed in the liver, with lower concentrations of

specific isoforms occurring in almost all body tissues, significantly in the respiratory tract, the kidneys,

gastrointestinal tract, the heart and skin.202

In addition to the CYP450 superfamily, other enzymes such as uridine diphosphate-glucuronyl

transferases (UGTs), esterases, dehydrogenases, among others also metabolise drugs, albeit to a lesser

extent.199 Table 1.2 below summarises some of the more common examples of non-CYP450 enzymes.199

36

Table 1.2: Examples of non-CYP450 enzymes involved in metabolism of xenobiotics

Enzyme class Function

Phase I

Flavin-containing monooxygenases

Oxygenation of highly polarisable

nucleophilic heteroatom-containing

drugs203

Monoamine oxidases Metabolism of catecholamines 204–

206

Esterases Activation of amide, ester and

thioesters prodrugs

Alcohol and aldehyde dehydrogenases Oxidative metabolism of alcohols

and aldehydes207

Aldo-keto reductases

Detoxification of reactive aldehydes

and unsaturated carbonyl

compounds208–210

Phase II

Glutathione-S-transferases Conjugative enzymes that couple

xenobiotics or phase I metabolites

to endogenous substrates to

facilitate excretion211–221

Uridine glucuronosyl transferases

Sulfotransferases

Methyl transferases

N-acetyl transferases

1.10.1.2 Classification of drug metabolism

In the past, drug metabolism was traditionally categorised into phase I and phase II. Phase I reactions

involve oxidoreductive and hydrolytic reactions that unmask reactive functional groups such as thiols,

amines, and carboxylic acids among others. These reactions include hydroxylation (aliphatic and

aromatic), epoxidation, dealkylation (N-, S- , and O) and oxidation (N-, S- , P-). Given the prevalence of

oxidoreductases, between 60 and 75% of phase I reactions are catalysed by CYP450 enzymes.199,222

Other enzymes involved in phase I metabolism include the flavin-containing monooxygenases (FMOs)

and monoamine oxidase (MAO) which metabolise endogenously produced neurotransmitters known as

catecholamines.203,223 In some cases, phase I metabolism products are marginally more hydrophilic.

37

Phase II reactions, in turn, typically involve conjugation of the phase I metabolites to more polar

endogenous molecules which facilitate excretion. In certain cases, they play a role in inactivation and

detoxification and, in even fewer, bioactivation of drugs.224 The enzymes involved include, among

others, uridine glucuronosyl transferases, sulfotransferases and methyl transferases.211 In a few cases,

phase I and II metabolism do not occur sequentially, as the substrates of conjugation are not always

products of phase I metabolism. This, in part, prompted Testa et al, who challenged this classification

through an extensive review of reactions and enzymes involved in metabolism of xenobiotics. These

researchers pointed out the importance of conjugation reactions before the phase I-associated redox

reactions.199,225

1.10.1.3 Classification of metabolites

Irrespective of the routes of metabolism, the resultant metabolites can be broadly classified into stable

and unstable, as discussed in the following sections.

Biologically inactive stable metabolites

The chemical modifications that occur during metabolism alter the chemical properties and,

consequently, the interaction between drugs and their target sites. Thus, in many instances, metabolism

leads to the significant or total loss of pharmacological activity. Knowledge of drug metabolism has been

exploited in the design of new drugs, with metabolic stability as one of the criteria for advancing a

compound or series of compounds. This is done with the aim of ensuring predictable optimal dosing

after metabolism, which has implications on efficacy. In some cases, a drug can be intentionally

designed to undergo metabolism within a time frame that allows it to achieve pharmacological activity

at the site of action. This approach is known as “soft drug” design, and has been used as a strategy to

mitigate on-target or, in the case of multiple metabolic products, off-target toxicity elicited by certain

metabolites.226

Biologically stable active metabolites

In some cases, the products of metabolism can result in metabolites with pharmacological activity that is

less, comparable to, or greater than, the parent molecule. This may be because some of the chemical

38

modifications due to metabolism do not alter the pharmacophore, and thus the drugs or compounds

retain activity. In such instances, other physicochemical properties such as aqueous solubility,

permeability, and chemical stability are altered, either to the benefit or detriment of overall drug

efficacy.227–230 Such metabolites are termed metabolic products of biotransformation, and examples

include marketed drugs such as acetaminophen, cetirizine and desloratidine. Figure 1.11.228,231 With

regards to definition, a minor contention exists, as they can be strictly defined as metabolites that share

a pharmacological target with the parent molecule, as described by Fura et al228. In contrast, other

scientists have widened the term to include prodrugs, which are devoid of activity by design, requiring

enzymatic modification (usually hydrolytic biotransformation) to elicit pharmacological activity.228,232,233

Figure 1.11: Examples of active metabolites developed into clinical drugs

Although most active metabolites stem from phase I metabolism, some phase II metabolites are known

to be active. Morphine is one such example, with two products of phase II metabolism, one active and

the other inactive (Figure 1.12). Morphine-6-glucuronide has superior analgesic activity to its parent

39

morphine, whereas morphine-3-glucuronide has been shown to be a potent opioid antagonist in animal

models and, paradoxically, has been reported to enhance pain through unknown mechanisms.234,235

Figure 1.12: Morphine and its metabolites in man

Reactive metabolites

Although metabolism is primarily a detoxification process, exceptions exist. In certain cases, drug

metabolism results in the formation of unstable and chemically reactive metabolites which result in

adverse drug reactions. Such metabolites are termed reactive metabolites. Their reactivity often occurs

through covalent binding to endogenous macromolecules in their immediate environment, which may

even involve the enzymes responsible for their formation. Other macromolecules they commonly bind

to are DNA, RNA and membrane proteins, resulting in long term carcinogenic or teratogenic effects.236–

241 Certain chemical functional groups are implicated in these reactions, and thus the term ‘structural

alerts’ was coined as a result of the existing evidence supporting their involvement in reactive

metabolite formation.242

40

1.10.1.4 Systems for drug metabolism studies

Various in vivo and in vitro systems have been developed to study drug metabolism for drug discovery

purposes. Mice and rats are the most commonly used animal models while, in certain cases, depending

on cost, throughput and the relevance to the disease under investigation, larger animals such as guinea

pigs and dogs are used. While animal models undoubtedly offer the most optimal biological complexity

and relevance to human disease, the cost and ethical implications have necessitated in vitro models as a

first step in screening compounds for later stages. In vitro systems are used in the early stages as a

preliminary means not only to prioritise the large number of compounds in the hit confirmation stage,

but also as a predictive tool for suitable dosage determination for pre-clinical in vivo studies.

Due to its central role as the major drug metabolising organ, the frequently used in vitro models are

hepatic. In decreasing order of biological complexity, the systems include: (1) whole perfused liver (2)

liver slices (3) hepatocytes (4) s9 fractions and (5) microsomes. S9 are sub-cellular liver fractions that

contain a complement of most phase I and II enzymes and are, as far as enzyme activity is concerned,

therefore the most complete sub-cellular fraction.243 Microsomes are the most commonly used model,

since they contain a complement of most CYP450 enzymes which, in turn, are of great relevance in the

metabolism of clinically used drugs.244 In the last two decades, recombinant expression of specific CYP

450 isoforms have become a household tool in biotransformation studies.201,222,237

1.10.1.5 Relevance of metabolism and metabolites in drug discovery and development

As mentioned in section 1.9 above, ADME properties have implications on the safety and efficacy of

drugs. Regulatory requirements for drug approval include characterisation of the metabolites of drug

candidates. It is therefore important to understand the contribution of metabolites to the activity of

compounds early in order to:

(1) Anticipate and incorporate strategies to mitigate toxicity of compounds in pre-clinical drug

discovery in order to minimise compound attrition rates downstream.

(2) Optimise PK properties of compounds, through for instance, the design of prodrugs.

Exploit potentially beneficial pharmacological effects of active metabolites in the design of

novel compounds.

41

1.11 Rationale

The literature reviewed above highlights the need for novel anti-TB agents with improved safety and

efficacy profiles; more potent agents with novel mechanisms of action, and activity against both drug-

sensitive and drug-resistant strains of Mtb. The past decade has seen massive gains in TB drug discovery,

with several new drugs approved and others in various stages of clinical development. However, the

sobering reality of ever emerging resistance even to the new drugs, coupled with the high attrition rates

along the chain, underscore the need to constantly replenish the pipeline. Notably, drug repurposing

and repositioning have provided a significant proportion of the novel agents in clinical use. Furthermore,

several of these molecules are derivatives of natural products, subsequently sparking renewed interest

in old drugs and natural products as starting points for new leads.

Evidence from the foregoing literature also emphasizes the value of in vitro assays such as target

identification, intracellular efficacy evaluation and drug metabolism; if conducted upstream, they

provide valuable insights into the pharmacokinetic and pharmacological properties of compounds. In

turn, these can be exploited to streamline the drug discovery process in two ways: optimization or,

alternatively, de-prioritization of compounds earlier in the process.

1.12 Hypothesis

Natural products can serve as a template for new molecules with potential as antimycobacterial agents.

This project explores the repositioning of fusidic acid, a natural product-derived antibiotic for

antituberculosis drug discovery. Through a combination of chemistry and biology, this study entails the

in vitro characterization of pharmacological and pharmacokinetic properties of fusidic acid and several

derivatives.

42

1.13 Aim

The overall aim of this study was two-fold:

a) To identify novel analogues of fusidic acid with improved potency and pharmacokinetic

properties.

b) To carry out in vitro characterization of the pharmacological and pharmacokinetic properties of

fusidic acid and several derivatives.

1.14 Specific objectives:

i. Design and perform chemical semi-synthesis of fusidic acid derivatives.

ii. Conduct in vitro pharmacological evaluation of the fusidic acid derivatives.

iii. Determine the solubility of the derivatives.

iv. Evaluate the cytotoxicity of fusidic acid and derivatives.

v. Evaluate the intracellular efficacy of fusidic acid and selected derivatives in THP-1 cells.

vi. Perform in vitro pharmacokinetic evaluation of fusidic acid and selected derivatives.

vii. Identify the target of, and mechanisms of resistance to, fusidic acid in Mycobacterium

tuberculosis.

43

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62

Chapter 2

Design, semi-synthesis and in vitro evaluation of pharmacological and physicochemical properties

of fusidic acid-aminoquinoline hybrids

Chapter overview

In this chapter, a brief overview of hybrid drugs and prodrugs is outlined after which the design and

semi-synthesis of fusidic acid hybrids is then presented. The hybrids are subsequently evaluated for

antimycobacterial activity, and the contribution of the constituent pharmacophores to the activity of

the hybrids is investigated. In addition, the solubility of these hybrids is determined experimentally.

2.1 Fusidic acid

Fusidic acid (FA; Figure 2.1) is a natural product derived from Fusidium coccineum that was first

isolated in 1960. It possesses activity against gram positive bacteria and was developed by Leo

Laboratories as an antibiotic for the treatment of Methicillin-resistant Staphylococcus aureus (MRSA)

infections, especially in combination with rifampicin (RIF).1–4 It has shown moderate in vitro activity

against drug-susceptible Mtb and clinical isolates resistant to the first line anti-TB agents.5,6 Despite

this, it has shown in vivo inefficacy in a well-established mouse model of infection, with no decrease

in bacterial load in mice treated with FA up to 200 mg/kg (Lenaerts et al, unpublished results). Its

status as an established clinical drug attests to its optimal clinical pharmacokinetic profile,

motivating further studies into its optimization for anti-TB drug discovery.

Figure 2.1: Fusidanes with their respective carbon atom numbering indicated

63

2.1.1 Unique structure of FA

FA is the most potent of the fusidanes, a class of naturally occurring antibiotics, which include

helvolic acid and cephalosporin P1 (Figure 2.1). Fusidanes resemble steroids structurally, but are

devoid of any hormonal or anti-inflammatory activity due to a difference in stereochemistry that

distinguishes them from other tetracyclic triterpenes and sterols.7 FA is unique due to the unusual

stereochemistry of its tetracyclic ring system, where rings A, B and C are arranged in a trans-syn-

trans manner in contrast to the usual trans-anti-trans arrangement seen in tetracyclic triterpenes

and sterols. This forces ring B into a boat conformation, resulting in a chair-boat-chair conformation

of the cyclopentanoperhydrophenanthrene ring system (Figure 2.2). In addition, these compounds

have a common carboxylic acid-bearing side chain linked to the ring system at C-17 via a double

bond and an acetate group at C-16.8 It is weakly acidic (pKa of 5.7) and is mostly ionized in plasma

and tissue at the physiological pH of 7.4.

Figure 2.2: Cyclopentanoperhydrophenanthrene with a trans-syn-trans arrangement

2.1.2 Antibacterial SAR

The main aims of semi-synthetic modification of FA have been to improve potency and spectrum of

antibacterial activity, as well as improving physicochemical parameters such as solubility, due to its

highly hydrophobic nature. Extensive Structure-Activity Relationship (SAR) studies of FA have been

carried out with respect to its antibacterial activity outlined in Figure 2.3.8

64

Figure 2.3: A summary of FA antibacterial SAR adapted from Duvold et al, 20018

However, only a few of these analogues showed activities comparable to that of FA, while all have

shown a similar antibacterial spectrum to FA.9–11 In the search for analogues with improved aqueous

solubility, Koreeda et al synthesised C-3, C-24 and C-25 O-glycosylated analogues using various five-

and six-membered cyclic sugar moieties (glycals). The details of the activity and solubility of the

analogues are not discussed in the published patent.12 The antibacterial SAR can serve as a starting

point to guide the repositioning of FA through semi-synthesis for TB drug discovery.

2.2 Quinolines in drug discovery

As mentioned in Chapter 1, the quinoline scaffold is considered a privileged structure in drug

discovery. Compounds derived from nature containing this scaffold have been reported to have anti-

infective properties including antimalarial, antiviral, antileishmanial and antitubercular activity, as

well as anticancer and cardiovascular properties among others.13 Quinolines have a long-standing

history as antimalarial agents.14 Purely synthetic molecules based on quinolines have also been

developed successfully, as evidenced by bedaquiline (BDQ) and others (Figure 2.4), a novel drug

reserved for the treatment of MDR-TB. It is a diarylquinoline that inhibits mycobacterial Adenosine

Triphosphate (ATP) synthase.15,16

Among these sub-classes is primaquine, an 8-aminoquinoline used in the treatment of malaria

caused by Plasmodium vivax (P. vivax) and Plasmodium ovale (P. ovale) as well as transmission-

blocking of malaria caused by Plasmodium falciparum (P. falciparum). It was reported to have an

MIC of 5 M at 90% inhibition against Mtb H37Rv.17 Another example is mefloquine, a 4-substituted

quinoline also used in the prophylaxis and treatment of malaria, that showed moderate

antimycobacterial activity in the same assay.17 Due to its relevance in TB drug discovery, the

quinoline scaffold was chosen for the synthesis of hybrids in this project.

65

Figure 2.4: Examples of quinolines with antimycobacterial activity

2.3 Hybrid drugs

A hybrid compound is a chemical modification created by combining two or more chemical scaffolds,

either directly, or via a linker.18,19 Hybrids potentially offer several advantages over single molecules

and combination therapy. They have the potential to overcome the development of resistance to

therapy by combining two pharmacophores with different modes of action into a single entity. In

addition, this may lead to synergistic effects of the pharmacophores, and thus increased potency.20,21

Physicochemical parameters such as solubility and permeability can be improved by incorporating

moieties with desired properties. Paclitaxel, a highly hydrophobic anticancer drug, has been

successfully improved using this strategy.22 It is also possible to design selectivity for a drug target by

covalent attachment of moieties with affinity for specific receptors with proximity to the target

organ or tissue.23 Overcoming the additive toxic effects of the individual drugs may thus be avoided,

with the overall added convenience of a single formulation, improving overall patient adherence at a

potentially lower cost.

2.3.1 Classification of hybrid drugs

Hybrid molecules can be classified as cleavable, non-cleavable and merged (Figure 2.5).22 Cleavable

hybrids are covalently linked directly or indirectly through a spacer designed to be hydrolysed,

usually enzymatically. Esters, amides and carbamates are the most commonly used linkers. Upon

hydrolysis, the individual molecules are expected to elicit their pharmacological effects. Non-

cleavable hybrids are linked covalently, and are designed to retain the biological activities as a single

molecule. Merged or overlapping hybrids are obtained by overlapping common structural motifs of

two or more pharmacophores. The hybrid may retain the functional properties of the source

molecules or elicit a pharmacological effect distinct from the source molecules.

66

Figure 2.5: Classification of hybrids, adapted from Srivastava and Lee.22

2.4 Prodrugs: design and application in drug discovery

Prodrugs are bioreversible derivatives of drug molecules designed to undergo an enzymatic and/or

chemical transformation in vivo to release the parent drug, which then exerts the desired

pharmacological effect.24–27 Prodrugs have become a widely-used strategy for improving

physicochemical, pharmacokinetic or biopharmaceutical properties of pharmacologically active

agents in both drug discovery and development. In the last decade, between 5–7% of known clinical

drugs were classified as prodrugs, and the use of this approach in the early stages of drug discovery

is a growing trend; 15 % of all new drugs approved between 2001 and 2002 were prodrugs. Some of

the drawbacks which have been successfully addressed by prodrug design include challenges in

formulation and drug delivery such as aqueous solubility, poor oral absorption, rapid metabolism

and toxicity.28

67

2.4.1 Classification of prodrugs

In most cases, prodrugs only require one or two chemical steps (more commonly enzymatic

transformation) to yield the pharmacologically active compound as illustrated in Figure 2.628

Figure 2.6: Illustration of the prodrug concept, adapted from Rautio et al 200828

Prodrugs can be broadly classified into two categories: co-drugs or bioprecursor drugs. A third

category, referred to as soft drugs, does not conform to the classical definition of a prodrug. Co-

drugs refer to the case of two pharmacologically active drugs that are covalently fused into a single

molecule, in which each drug acts as a promoeity for the other.29 Bioprecursor drugs result from

modification of the active drug itself, resulting in the generation of a novel compound, which is

subsequently chemically or enzymatically converted to the active metabolite.28 Soft drugs, which are

designed to undergo predictable deactivation or metabolism in a controlled manner after achieving

therapeutic effect, are the opposite of prodrugs. They are typically used in the detoxification of

drugs with narrow therapeutic indices.30–32 A major factor to be considered in prodrug design is the

presence of functional groups in a molecule that are amenable to chemical prodrug derivatization.

Commonly used functional groups include hydroxyl, carboxylic acid, amine, and phosphonate

groups.28 Modification of these groups typically produces, among others, ester, amide, carbonate,

and phosphate group prodrugs.28

68

The use of amides as prodrugs is less common, due to their relative enzymatic stability in vivo.

Notwithstanding, a few examples exist. As highlighted in Chapter 1, the anti-TB agent pyrazinamide

(PZA) is an amide prodrug that undergoes enzymatic hydrolysis; it is a substrate of mycobacterial

and host amidases, which hydrolyse it to pyrazinoic acid, the pharmacologically active moiety.33

Esters are the most common prodrugs, and are estimated to constitute 49% of all marketed

prodrugs.25,34 By masking charged groups such as carboxylic acids and phosphates, they are often

used to enhance lipophilicity and consequently membrane permeability of hydrophilic drugs.28,35

Once administered, the ester bond is hydrolysed by ubiquitous esterases found in the blood, liver as

well as other organs and tissues. These include carboxylesterases, acetylcholinesterases,

arylesterases and paraoxonases.28 Examples of clinically used ester prodrugs are Angiotensin

converting enzyme (ACE) inhibitors and -lactam antibiotics, both of which improve bioavailability.28

Another successful example of the application of the prodrug strategy is that of the experimental

compound MGS0210, an alkyl ester of MGS0039- a glutamate receptor antagonist used for the

treatment of psychiatric disorders such as schizophrenia36,37( Table 2.1). MGS0039 has a carboxylic

acid group which limits its intestinal permeability, consequently reducing its bioavailability in rats

and monkeys. In separate clinical studies, administration of several ester prodrugs, among them

MGS0210, achieved a 31% increase in oral bioavailability of MGS0039 in monkeys following

esterase-mediated bioconversion by esterases.36,37 Table 2.1 illustrates examples of ester prodrugs.

69

Table 2.1: Examples of prodrugs with improved lipophilicity or permeability28

Prodrug name

(therapeutic

area)

Functional group Structure Pharmacologically active

metabolite/moiety

Enalapril

(Angiotensin-

converting

enzyme

inhibitor

Monoethyl ester of

enalaprilat

Pivampicillin

(-lactam

antibiotic)

Pivaloylmethylester

of ampicillin

MGS0210

(glutamate

receptor

(MGLUR2)

antagonist

n- heptyl ester of

MGS0039

70

2.5 Rationale for fusidic acid-aminoquinoline hybrids and C-3 ester prodrugs

Physicochemical parameters have a significant effect on drug efficacy, for instance, implications on

the extent and rate of drug absorption. These in turn, are dependent on the ability of a molecule to

permeate across the cell membrane of microorganisms. Lipophilicity and solubility are the two key

physicochemical factors that affect both the extent and rate of drug absorption.38 Increasing

lipophilicity generally enhances membrane permeability. In order for absorption to occur, orally

administered drugs must dissolve in the aqueous environment of the stomach and intestines before

they can be absorbed.38 In addition to crossing the pathogen’s membrane, these properties may

affect drug permeation across host membranes to the target organs and tissues. TB pathology is

encumbered with the added barrier of the caseum, a necrotic, hydrophobic ring that consists chiefly

of immune cells such as macrophages and neutrophils encasing bacilli.39,40 The added challenge of

drug penetration into the necrotic lesions where persistent bacilli reside requires a

hydrophobic/hydrophilic balance to achieve an effective concentration for antimycobacterial

activity.41–44

The carboxylic acid functional group is an important pharmacophore in certain classes of

compounds. It is also, however, responsible for certain drawbacks such as toxicity, limited passive

diffusion across membranes among others.45 It was hypothesised that in addition to the hydroxyl

groups at positions 3 and 11 of FA, the carboxylic side chain may limit its permeation of the thick

mycobacterial cell membrane, with consequent modest antimycobacterial activity in comparison to

other Gram positive organisms. As illustrated in Figure 2.3 above, the C-21 carboxylic acid group has

been shown to be essential for in vitro antibacterial activity. As such, the use of esters and amides

as potential prodrugs to mask the carboxylic acid group would retain its interaction with the

biological target while potentially improving its lipophilicity.

As discussed in Chapter 1, esters and amides have been shown to improve the antimycobacterial

activity of spectinomycin.46 We hypothesized that derivatization of the C-21 carboxylic acid group in

this manner through hybridization to quinoline moieties would enhance the permeability properties

of the hybrids required for improved activity against Mtb. Additionally, potential enzyme-mediated

hydrolysis would deliver the quinoline pharmacophore, which in itself is plausibly endowed with

antimycobacterial activity. The FA-aminoquinoline hybrids thus synthesised would fall under the

classification of cleavable hybrids (Figure 2.5). We also hypothesised that upon enzyme-mediated

hydrolysis, C-3 ester prodrugs of FA would increase the concentration of FA at the site of action as a

result of improved permeability. The C-3 ester prodrugs evaluated in this project were synthesised

by Dr. Gurminder Kaur at the University of Cape Town.

71

2.6 Aims and objectives The main aim of this chapter was to identify FA analogues with improved physicochemical and

pharmacodynamic properties through chemical semi-synthesis. The specific objectives were:

(1) Semi- synthesis of ester and amide FA-aminoquinoline hybrids and their characterisation

using spectroscopic techniques.

(2) Pharmacological evaluation of the synthesised hybrids.

(3) Evaluation of the contribution of the pharmacophoric units to the pharmacological

activity of the hybrids by screening appropriate intermediates as well as 1:1 mixtures of

the same intermediates.

(4) In vitro physicochemical profiling (solubility and permeability) of the FA-aminoquinoline

hybrids.

2.7 Semi-synthesis of FA hybrids

This work focuses on modification of the carboxylic acid group, exploring the effect of hydrophobic

substituents at this position on potency. To this end, aminoquinoline ester and amide hybrids of FA

shown in Figure 2.6 were synthesised. Selected 4-aminoquinolines and primaquine, an 8-

aminoquinoline, were selected due to their relevance in TB drug discovery as discussed in section

2.2.

Figure 2.7: Fusidic acid amide and ester aminoquinoline hybrids

72

2.7.1 Retrosynthetic analysis of the FA amide and ester prodrugs based on aminoquinolines

The amides were envisaged from coupling between the C-21 carboxylic acid of FA and the primary

amines shown in Figure 2.7 above. The esters were to be obtained by coupling of the acid to the

primary alcohols shown in Figure 2.7. A C-N disconnection was expected to furnish the

aminoquinoline alcohols and diamines, leading to the starting material, commercially available 4, 7-

dichloroquinoline, the corresponding diamines and amino alcohols (Scheme 2.1)

Scheme 2.1: Retrosynthetic analysis of FA amide and ester derivatives.

73

2.7.2 Synthesis of 4-aminoquinoline diamines and alcohols

The 1-(4’-amino-7’-chloroquinoline)- diamines and alcohols were synthesised using a modification of

the literature procedure described by de Souza et al.47 The quinoline diamine with the 2 and 4-

carbon methylene spacers as well as the N-methyl 3-carbon methylene spacer were readily

available, hence the only ones synthesised during this project were those containing the 3, 4 and 5-

carbon methylene spacers. All the quinoline amino alcohol analogues were synthesised. The

quinoline analogues were furnished by a nucleophilic substitution reaction between 4, 7-

dichloroquinoline and an excess (5 equivalents) of the appropriate diamines and alcohols. Since the

diamines and alcohols were liquid, the reactions were carried out by reflux in the neat, affording the

desired products in good yields (90–98%) without using column chromatography for purification.

They were characterized by NMR and Mass spectroscopy, and in their melting points compared with

values previously reported.48 The synthesis is depicted in the Scheme 2.2.

Scheme 2.2: Synthesis of 4-aminoquinoline diamines and alcohols

Reagents and conditions: (i) corresponding diamine, 80 oC 1 hr then 135oC, N2, 3hrs, 90–98%.

The nucleophilic substitution reaction is enabled by the reactivity of the quinoline ring (I), which is a

fused benzo[b]pyridine, therefore conferring upon it the capacity for both electrophilic and

nucleophilic reactivity. Through mesomeric and inductive effects, the nitrogen atom causes π-

electron deficiency at the neighbouring carbon atoms at position 2 and 4. The presence of the

electron-withdrawing chloro group at position 8 further enhances this electron deficiency, rendering

C-2 and C-4 prone to nucleophilic attack (Figure 2.8a). The regioselectivity observed is driven by the

ability of the nitrogen atom to act as an electron ‘sink’ through resonance, facilitating nucleophilic

attack by the amine as illustrated in Figure 2.8b.48,49

74

Figure 2.8a: Canonical structures for the inductive and mesomeric effects of the quinoline nitrogen

Figure 2.8b: Mechanism of formation of 7-chloroquoniline-4-amine derivatives

2.7.2.1 Results: Yields of aminoquinoline diamines and alcohols

Table 2.2: Yields of 4-aminoquinoline alcohols and diamines

Compound

code X n % yield

AW21 O 2 98

AW26 O 3 95

AW28 O 4 96

AW29 O 5 90

AW27 NH 3 90

AW18 NH 4 96

AW43 NH 5 98

75

2.7.3 Synthesis of FA amides

The synthesis of the amides employed carbodiimide chemistry, using a procedure described by

Gonzalez Cabrera et al.50 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) was used in favour

of Dicyclohexyl carbodiimide (DCC) to activate the carboxylic acid, as it is a salt and therefore easily

removed by washing the crude reaction mixture with water or aqueous salts during work up.

Furthermore, attempts at coupling using DCC returned very low yields, prompting the use of an

alternative coupling method. Scheme 2.3 below summarizes the amide coupling procedure.

Racemic primaquine was sourced commercially as a diphosphate salt, therefore the first step

involved neutralization using 2 equivalents of N, N-Diisopropylethylamine (DIPEA). This is in contrast

to the coupling reactions involving the other amines, in which 1.2 equivalents of DIPEA was used.

The compounds were obtained in poor to moderate yields after purification using column

chromatography.

Scheme 2.3: Synthesis of FA amide derivatives.

Reagents and conditions: (i) appropriate amine, EDCI, HOBt, DIPEA, DMF, 25 oC, 16h, 25-90%.

2.7.4 Synthesis of FA esters

Similar carbodiimide chemistry as in amide coupling was employed, with N, N-dimethyl-4-

aminopyridine (DMAP) used as a nucleophilic base instead of (DIPEA). Optimal yields were obtained

when 2.5 equivalents were used, as opposed to the more common use of a catalytic amount.

Scheme 2.4 illustrates the procedure for the synthesis of the ester hybrids of FA.

76

Scheme 2.4: Synthesis of FA ester derivatives

Reagents and conditions: (i) appropriate alcohol, EDCI, DMAP, DMF, 25 oC, 16h, 69-85%.

2.7.4.1 Results: Yields of FA amide and ester aminoquinoline hybrids

Table 2.3: Yields of FA amides and esters

R Compound

code X n R1 % Yield

AW32 O 2 H 69

AW42 O 3 H 74

AW34 O 4 H 82

AW35 O 5 H 85

AW23 NH 2 H 80

AW31 NH 3 H 55

AW25 NH 4 H 50

AW45 NH 5 H 90

AW24 NH 3 CH3 25

77

R Compound

code X n R1 % Yield

AW33 NH 3 - 25

Except for AW45 with a 5-carbon methylene spacer, the longer chain amides were obtained in lower

yields than the amides with shorter methylene spacers. Moreover, the loss of material after

purification by column chromatography led to even lower yields of the final products. HPLC was

used to profile the compounds for purity, with all the target compounds showing ≥ 95% purity. The

FA-primaquine hybrid was obtained as a diastereomeric mixture, since FA is chirally pure while

primaquine was commercially sourced as a racemic mixture. This was evidenced by the overlapping

signals observed in the proton NMR spectrum. Separation of the diastereomers was not attempted

in this study.

In the case of the esters, good yields were obtained for the compounds, with column

chromatography affording the purified compounds in higher yields compared to the amides. This

was primarily due to relatively minimal material lost during purification by column chromatography.

Plausibly, unlike the amides the esters were less prone to the “tailing” effect caused by interaction

of the amines with silanol groups on the surface of the silica stationary phase during column

chromatography purification. In addition, higher yields were obtained for the esters with longer

chain methylene spacers. HPLC was used to profile the compounds for purity, with all the target

compounds showing ≥95% purity.

78

2.7.5 1H NMR characterisation: Key spectroscopic indicators of selected intermediates and target compounds

2.7.5.1 Substituted 7-chloroquoniline-4-amine derivatives

In the case of the 4-aminoquinoline derivatives, the success of the substitution reactions was

confirmed by the appearance of proton peaks upfield in the aliphatic region, which was consonant

with reports from the literature.51 Figure 2.9 indicates 1H NMR spectrum of the amino alcohol

derivative containing the 2-carbon methyelene spacer, AW21.

Figure 2.9: Spectrum of AW21 illustrating the aromatic ring and C-4 aliphatic side chain protons

2.7.5.2 FA amides and esters

FA is a relatively large molecule with many aliphatic protons and therefore very few assigned NMR

data have been published in the literature. This has been achieved through advanced 1D and 2D

spectroscopic techniques that have not been carried out so far in this project. However, several

identifiable proton signals were observed, and their chemical shift values in each of the target

molecules have been highlighted. The assignment of the remaining signals proved challenging and

the chemical shift values reported here correspond to various literature reports of FA NMR data,

79

corroborated by the proton-proton coupling observed in a 2D COSY NMR experiment of compound

AW23 (Appendix 4). Nevertheless, the success of the amide coupling and esterification with the

quinoline amides and esters respectively was confirmed by the well-resolved signals of the quinoline

ring system, with downfield proton peaks observed between 6.38 parts per million (ppm) and 8.40

ppm as indicated on the spectrum of amide AW23 shown in Figure 2.10.

Figure 2.10: Spectrum of AW23 illustrating the aromatic protons of the quinoline ring and

identifiable FA protons

Furthermore, evidence from mass spectroscopy was used to confirm the expected molecular mass

for each of the target compounds. For all target compounds, both low-resolution mass spectroscopy

(LRMS) and liquid chromatography-coupled mass spectroscopy (LC-MS) confirmed the expected

molecular mass in positive mode [M + H]. The NMR and MS spectroscopy data for the target

compounds are provided in Chapter 6.

80

2.8 Physicochemical evaluation of the FA hybrids

2.8.1 Experimental determination of solubility

The aqueous solubility was determined using a plate-based turbidimetric solubility assay previously

described by Bevan and Lloyd.52 This method measures the onset of precipitation of any given

compound by a change in absorbance. All the compounds synthesised were dissolved in DMSO to

prepare stock solutions for all downstream assays, while phosphate buffer saline (PBS) is the

aqueous medium used for solubility assays. Since most organic compounds are not freely soluble in

aqueous media, as the concentration of compound in PBS increases, precipitation occurs, resulting

in increased absorbance in the PBS-containing wells relative to the DMSO-containing wells. As the

maximum aqueous solubility level is reached, precipitation occurs when solute particles fail to

dissolve in the PBS. The absorbance due to the occlusion of light by the particles is measured using

UV spectrophotometry at 620 nm. The concentration at which the onset of increased absorbance is

observed is considered the upper limit of solubility.

10 mM stock solutions of test compounds were prepared in 100% DMSO, followed by dilution with

phosphate buffered saline (PBS) at pH 7.4, over a concentration range of 0 to 200 µM. The dilutions

were effected to a final DMSO concentration of 2.2% v/v. The assay plates were incubated for two

hours at ambient temperature before absorbance readings were taken. Reserpine and

hydrocortisone were used as the insoluble and highly soluble controls respectively.

2.8.2 Results of solubility testing of the compounds

As envisioned, all the analogues were significantly less soluble in comparison to FA, which is

regarded as highly soluble. There was no observable trend with respect to chain length, or

comparison between the ester and amide analogues, with low and partial solubility observed across

both series. AW34, the ester with a 4-carbon methylene spacer, was the least soluble at 5 µM, while

AW32, the ester with a 2-carbon methylene spacer was the most soluble compound, with maximum

solubility at 80 µM. Among the amides, AW45 and AW23 with a 5-and 2-carbon methylene spacer

respectively, were the least soluble at 10 µM.

The addition of the non-polar lipophilic aminoquinoline derivatives increased the molecular weight

of the FA analogues by a range of 204 to 247 atomic mass units from the least to the most bulky.

Since quinolines are aromatic in nature, π-stacking interactions may have played a role in the

average 10-fold lower solubility of the hybrids. Since the heteroatoms oxygen and nitrogen are

present in all the compounds, hydrogen bonding may have contributed to mitigating their overall

81

hydrophobicity, as in the case of AW32 whose maximum solubility was on the higher end of partial

solubility. The results are summarized in Table 2.4. The data are representative of two independent

experiments performed on different days.

Table 2.4: Results of turbidimetric solubility evaluation of the FA-aminoquinoline hybrids

R Compound

code X n R1

Solubility (µM)

AW32 O 2 H 80

AW42 O 3 H 10

AW34 O 4 H 5

AW35 O 5 H 20

AW23 NH 2 H 10

AW31 NH 3 H 10

AW25 NH 4 H 40

AW45 NH 5 H 10

AW24 NH 3 CH3 20

AW33 NH 3 - 40

The solubility of each compound was ranked according to the following criteria: <10 µM insoluble; 10-100

partially soluble; 100 µM = highly soluble. FA solubility: > 200 µM.

82

2.9 Pharmacological evaluation of the FA analogues

2.9.1 Antimycobacterial evaluation of the FA analogues

Evaluation of the antimycobacterial activity of the intermediates and FA analogues was conducted

by broth microdilution method, using the Microplate Alamar Blue Assay (MABA). The strain of Mtb

used was Mycobacterium tuberculosis H37Rv (MA), a virulent strain adapted for laboratory use.53

The broth microdilution method enables the determination of the minimum inhibitory

concentration (MIC) of compounds over a range of concentrations serially diluted (two-fold) on a

single 96-well microtitre plate.54,55 In the MABA assay, metabolic activity of the cell correlates with

the reduction of resazurin to resorufin, and the resulting change in colour from blue to pink and

fluorescent, allowing visual detection and fluorometric or colorimetric quantification. Wells with

non-viable cells thus remain blue while those with viable cells exhibit pink colouration. The results

reported herein relied on visual detection to assess growth and inhibition.

The assays were conducted in the routinely used enriched 7H9 medium supplemented with oleic

acid dextrose catalase (OADC), as well as glycine alanine salts supplemented with iron (GAST/Fe), a

relatively minimal medium. 10 mM stocks of each compound dissolved in DMSO were diluted to a

final concentration not exceeding 160 μM at the highest concentration at the onset of two-fold

serial dilution on the assay plates. The 14-day assay was conducted in duplicate for each compound,

with 3 biological replicates performed on different days. At day 7, the plates were inspected for the

formation of pellets, as an indicator of growth. Rifampicin (RIF), a potent first-line anti-TB drug, was

used as a control. Detailed experimental procedures of the assay are provided in Chapter 6.

2.9.2 Results of antimycobacterial evaluation of FA-aminoquinoline hybrids

Overall, the FA-aminoquinoline hybrids were less potent than FA (Table 2.5). This is not surprising

since these hybrids were hypothesized to undergo enzyme-mediated hydrolysis (plausibly by

mycobacterial esterases and amidases) to release FA and the quinoline component. There was no

observable trend in comparing the amide and the ester analogues. Among the esters, increase in

chain length of the methylene spacer correlated with decreased activity, although not to the extent

of the inactivity observed for the corresponding amide with a 5-carbon methylene spacer AW45.

Among the amides, the most potent compound, AW25 with a 4-carbon methylene spacer, had a MIC

2- and 4-fold lower than FA in the two types of medium used for the assay. With the exception of

this compound, activity decreased as the chain length of the methylene spacer increased, with very

poor activity observed for AW45 with a 5-carbon methylene spacer.

83

Table 2.5: Results of antimycobacterial testing of FA–aminoquinoline hybrids

R Compound

code X n R1

MIC90 7H9/OADC (µM) MIC90 GAST/Fe (µM)

Day 7 Day 14 Day 7 Day 14

AW32 O 2 H 10 10 5 10

AW42 O 3 H 40 40 20 40

AW34 O 4 H 40 40 20 40

AW35 O 5 H 40 40 20 40

AW23 NH 2 H 20 20 10 20

AW31 NH 3 H 20 20 20 20

AW25 NH 4 H 10 20 10 10

AW45 NH 5 H 160 160 40 160

AW24 NH 3 Me 20 20 10 20

84

R Compound

code X n R1

MIC90 7H9/OADC (µM) MIC90 GAST/Fe (µM)

Day 7 Day 14 Day 7 Day 14

AW33 NH 3 - 160 160 >160 >160

RIF MIC90 against Mtb was 0.01 µM. FA MIC90 against Mtb was 5 µM. Data are representative of 3 independent experiments.

.

85

The poor activity observed for AW45 may be partially attributed to its poor solubility. Evidence of

this was the significant difference between the MIC at day 7 and day 14. It was noted that in the

time frame between day 7 and 14, the compound precipitated in the growth medium at 40 µM. On

repeat experiments, precipitation was observed upon addition of the compound to the growth

medium, even before inoculation of the assay plates with Mtb culture. With respect to the growth

medium used, it was observed that FA had an 8-fold difference lower MIC in GAST/Fe compared to

7H9/OADC.

This may be attributed to protein binding by the albumin in OADC. Albumin is a weak base known to

bind to acidic and neutral drugs, and thus may bind FA, reducing the effective concentration

available to achieve maximum potency.56

2.9.3 Evaluation of the contribution of the pharmacophoric units to the pharmacological activity of the hybrids

Selected aminoquinoline derivatives were evaluated for antimycobacterial activity, as well as a 1:1

(molar ratio) mixture of FA and each intermediate. This was carried out in order to establish the

contribution of the aminoquinoline derivatives to the activity of the FA-aminoquinoline hybrids.

Moreover, this would also determine if there is benefit in covalent linkage of the two

pharmacophores as opposed to co-administration of the individual compounds. The latter is

beneficial in a clinical setting, as the added convenience of a single formulation of two compounds

or drugs improves patient compliance. Along with primaquine, the amine and aminoalcohol

derivatives with 2- and 5-carbon methylene spacer were selected for evaluation. The data are from

two independent assays performed on different days.

86

2.9.3.1 Results of evaluation of the contribution of the pharmacophoric units to the pharmacological activity of the hybrids

Table 2.6: Results of MIC of selected individual aminoquinoline derivatives and their mixtures with

FA

Compound

code n X

MIC90 in 7H9/OADC at day

14 (µM), singly

MIC90 in 7H9/OADC at

day 14 (µM), in

combination

AW21 2 O >160 5

AW28 4 O >160 5

AQ1 2 NH >160 5

AW18 4 NH >160 5

MIC90 of primaquine and FA singly were 160 µM and 5 µM respectively

The selected aminoquinoline derivatives all had poor activity, with MIC values greater than 160 µM.

These data correspond to reports published by De Souza et al, who reported similarly poor activity

of aminoquinoline derivatives.57 In 1:1 molar ratio mixture with FA, the MIC value observed

corresponded to that of FA on its own. It can be inferred from these data that the aminoquinoline

pharmacophore did not contribute to activity, but instead reduced the overall activity of the hybrids.

Surprisingly, primaquine, previously reported to have modest antimycobacterial activity (MIC90 5

µM) by Lougheed et al, was inactive in this study, with an MIC90 of 160 µM. In the study reported by

these authors, the drug susceptibility assays were conducted using a different growth medium

(Dubos medium supplemented with albumin and glycerol, with no addition of Tween80). This may

account for the significant variation in the MIC of this compound in the two assays.17

87

2.10 Summary

Several C-21 ester and amide hybrids of FA fused to aminoquinolines were successfully synthesised

in moderate to good yields. NMR and LC-MS spectroscopic data confirmed the structures of the

expected compounds.

Semi-quantitative determination of the aqueous solubility of the target compounds in aqueous

medium was conducted using an established turbidimetric assay as a means of anticipating potential

challenges in conducting subsequent in vitro assays in aqueous solutions. All the hybrids scored

poorly in the solubility scale, with the overall average solubility 10-fold lower than that of FA.

The compounds were evaluated for their in vitro activity against Mtb. Compared to FA, all the esters

and amides synthesised were less active, with MIC values ranging from 2- to 8-fold lower than that

of FA. The exception to this was AW45, the amide analogue with a 5-carbon methylene spacer, the

longest in the series. As a consequence of its poor aqueous solubility, its MIC value could not be

established, as the compound precipitated in the growth medium at concentrations below the

average MIC values observed in the series.

In order to establish the contribution of each pharmacophore to the activity of the hybrids, selected

constituents of the hybrid molecules were tested in a 1:1 molar ratio. In addition, the

aminoquinoline derivatives were also evaluated for their MIC. The MIC values of all the 1:1 mixtures

were similar that of FA, while the aminoquinoline derivatives proved inactive on their own. The

aminoquinoline scaffold was therefore not beneficial to the overall activity of the hybrids.

Conversely, covalent attachment of FA improved their antimycobacterial activity. These data

demonstrate that the use of aminoquinoline-based esters and amides to mask the carboxylic acid

was not tolerated. This result corroborates antibacterial SAR reported8–10, which showed that bulky

hydrophobic substituents at this position were not tolerated.

It is critical to note that the poor solubility of these hybrids was taken into consideration in the

selection of FA analogues to be evaluated in subsequent in vitro assays, which would be conducted

in aqueous media. Moreover, given their low potency, the series could not be progressed further,

save for evaluation of cytotoxicity and evaluation of mycobacterial-mediated hydrolysis, which are

presented in Chapter 3. Therefore, the FA-aminoquinoline hybrids were not assessed for membrane

permeability. The detailed experimental procedures for the synthesis, spectroscopic

characterization, biological evaluation and solubility assay are presented in Chapter 6.

88

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4

Chapter 3

Evaluation of cytotoxicity, intracellular efficacy and mycobacterial metabolism of Fusidic acid and

selected analogues

Chapter overview

In this chapter, the fusidic acid hybrids and several other analogues are evaluated for their

cytotoxicity. Subsequently, selected analogues are evaluated for their intracellular efficacy in THP-1

cells. Finally, the mycobacterial metabolism of several of the hybrids and analogues is investigated.

3.1 Introduction

A combination of bacterial and host-mediated factors enables Mtb not only to survive, but to

replicate, within macrophages.1 For this reason, macrophage models of infection have been useful

preclinically in evaluating the intracellular activities of compounds under different conditions.2

Efficacy against intracellular bacilli reflects physicochemical and pharmacodynamic properties of

compounds: permeability across the host membrane, avoidance of or activation by host metabolism

and additionally, the ability to inhibit an essential bacillary function despite the survival mechanisms

enlisted intracellularly. Therefore, the evaluation of intracellular efficacy of compounds has been

incorporated into the panel of most TB drug discovery assays. As a result, in vitro models of infection

have been developed to enable medium and high-throughput screening of compounds. An

additional consideration is that activity in macrophages is considered a prerequisite for mouse

efficacy studies, particularly given that TB in the mouse model is primarily macrophage driven.3 THP-

1 and J774 are the two most common cell lines used for the in vitro assessment of intracellular

efficacy of compounds owing to their ease of propagation and extensive literature describing the

activity of different anti-TB drugs in these models. THP-1 is a human monocytic cell line adapted to

mature into macrophages under laboratory conditions, thus enabling infection with Mtb and

consequently the evaluation of compound efficacy; these cells were used here to evaluate FA and

selected analogues.

In the absence of information on a compound’s mechanisms of action, general toxicity in an

intracellular infection model such as Mtb-infected macrophages cannot be ruled out as the cause of

inhibition. Since the ultimate goal of drug discovery is to develop safe and efficacious medicines, it is

equally important to establish the selectivity of compounds for microorganisms, and rule out their

inhibition of mammalian cells. As part of the broader goal of safety evaluation in any drug discovery

4

program, compounds are evaluated for in vitro cytotoxicity during the pre-clinical phase. A large

panel of cell lines, both animal and human, has been adapted for in vitro evaluation of cytotoxicity at

high-throughput scale. For this study, two mammalian cell lines were selected for use: Chinese

Hamster ovarian (CHO) cells, an epithelial cell line derived from the ovary of the Chinese hamster,

and THP-1 cells, the same human monocytic cell line used for intracellular infections, which is

derived from an acute monocytic leukaemia patient. The former is well-established for cytotoxicity

studies, while the latter was of relevance because of its use downstream in intracellular

antimycobacterial efficacy studies.

3.2 Aims and objectives

The aim in this first part of the chapter was to identify cytotoxic compounds as a prelude to

investigating the intracellular efficacy of FA and selected analogues. The specific objectives were:

(1) Evaluate the cytotoxicity of FA-aminoquinoline hybrids in CHO cells.

(2) Determine the contribution of the pharmacophoric units to the cytotoxicity of the FA-

aminoquinoline hybrids in CHO cells.

(3) Investigate the cytotoxicity of FA-aminoquinoline hybrids, selected C-3 FA esters and keto-

fusidic acid in THP-1 cells.

(4) Evaluate the intracellular activity of FA and selected analogues in THP-1 cells.

3.3 Evaluation of cytotoxicity

3.3.1 Evaluation of cytotoxicity of the FA-aminoquinoline hybrids in CHO cells

The compounds were screened for in vitro cytotoxicity using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-

diphenyltetrazolium bromide (MTT) assay, which compares well with other available assays.4,5 It is a

colorimetric assay routinely used to assess cellular growth and survival in the presence of

compounds over a range of concentrations and expressed as the concentration that inhibits growth

and survival by 50% (IC50). FA, selected aminoquinolines, and the FA-aminoquinoline hybrids were

screened. In addition, in order to establish the contribution of each pharmacophore to the

cytotoxicity of the hybrids, 1:1 molar mixtures of selected aminoquinoline derivatives and FA were

tested in combination. For this purpose, representative aminoquinoline alcohol and diamine

derivatives were selected, specifically: the 2-carbon methylene spacer-linked aminoalcohol and

diamine, alongside the 5 methylene carbon spacer-linked counterparts from the two series. The

assays were conducted at the Department of Pharmacology, University of Cape Town. The

experimental details are provided in Chapter 6.

4

3.3.2 Evaluation of cytotoxicity of the FA-aminoquinoline hybrids in THP-1 cells

Prior to carrying out antimycobacterial efficacy in THP-1 cells, the FA-aminoquinoline hybrids were

evaluated for cytotoxicity. For this assay, the 96-well plate format of the broth microdilution and

MABA assays were applied, allowing 2-fold serial dilutions of compound ranging from 1.56 µM to

200 µM. The THP-1 cells were incubated in the presence of the compounds for 3 days at 37oC with

5% CO2, after which alamar blue was added. Visual assessment was used to determine growth and

inhibition, expressed as IC90, defined as the lowest concentration that resulted in 90% inhibition of

cells.

3.3.3 Results: Cytotoxicity in CHO cells

3.3.3.1 Evaluation of the contribution of the pharmacophoric units to the cytotoxicity of the

hybrids

Table 3.1: Results of IC90 determinations of individual aminoquinoline derivatives and their mixtures

with FA in CHO

Compound

code n X

MIC90 in

7H9/OADC

at day 14 (µM),

singly

MIC90 in

7H9/OADC

at day 14 (µM),

in combination

IC90 in CHO

(µM), singly

IC90 in CHO

(µM), in

combination

AW21 2 O >160 5 47.1 >194

AW28 4 O >160 5 12.5 >194

AQ1 2 NH >160 5 >200 >194

AW18 4 NH >160 5 200 >194

FA - - 0.625 >194 -

primaquine - - >160 5 ND ND

ND- Not determined; Values for MIC90 represent screening against Mtb H37Rv, while those for IC90 represent

screening against the CHO cell-line.

At concentrations up to 200 µM, the aminoalcohol derivatives were not cytotoxic to the cells. In

contrast, the diamine derivatives were cytotoxic at very low concentrations. Furthermore, the

4

diamine with a 5 carbon methylene spacer was roughly 4-fold more cytotoxic than its 2 carbon

methylene-spaced counterpart. Primaquine, as expected of a clinically approved drug, was also

relatively non-cytotoxic. Calculation of selectivity indices would not be reflective of their cytotoxicity

in Mtb-infected cells, since the MIC values for antimycobacterial activity were based on 90 % and not

50% inhibition of growth. In all cases, the 1:1 molar mixtures matched that of FA.

3.3.3.2 Evaluation of cytotoxicity of the FA-aminoquinoline hybrids in CHO and THP-1

The FA hybrids were evaluated for cytotoxicity in both CHO and THP-1 cells. Relative to FA, all the

FA hybrids were cytotoxic, and at concentrations below 10 µM for the amides AW31 and AW25,

which had 3- and 4- carbon methylene spacers respectively.

It is noteworthy that the amides with the longest and shortest alkyl spacer - AW45 and AW23,

respectively - were equally cytotoxic. This suggests that factors other than increased molecular

weight or length of the alkyl spacer contributed to increased cytotoxicity. The primaquine hybrid,

AW33, was the least cytotoxic compound, and this correlates with the lack of cytotoxicity seen in

both pharmacophores.

Similarly, among the esters, there was no correlation between increased chain length and

cytotoxicity despite the non-cytotoxicity observed in the aminoalcohol derivatives that constitute

them. Calculation of selectivity indices would not be reflective of their cytotoxicity in Mtb, since the

MIC values for antimycobacterial activity were based on 90 % and not 50% inhibition of growth.

Table 3.2 summarises the results.

96

Table 3.2: Results of evaluation of cytotoxicity of the FA-aminoquinoline hybrids

R Compound

code X n R1

IC50 in CHO

(µM)

IC90 in

THP-1 (µM)

MIC90 in

7H9/OADC

(µM)

Selectivity

index

AW32 O 2 H 19.4 12.5 10 1.25

AW42 O 3 H 10.0 6.25 40 0.16

AW34 O 4 H ND* 6.25 40 0.16

AW35 O 5 H 13.1 6.25 40 0.16

AW23 NH 2 H 10.3 12.5 20 0.63

AW31 NH 3 H 2.75 6.25 20 0.31

AW25 NH 4 H 4.98 6.25 20

AW45 NH 5 H 10.0 ND* 160 0.31

AW24 NH 3 Me 17.5 6.25 20 0.31

97

R Compound

code X n R1

IC50 in CHO

(µM)

IC90 in

THP-1 (µM)

MIC90 in

7H9/OADC

(µM)

Selectivity

index

AW33 NH 3 - >220 12.5 >160 0.31

- FA - - - >194 200 5 40

- primaquine - - - 126.7 ND >160 -

- emetine - - - 0.104 - - -

ND- Not Determined (in the case of AW34, due to insufficient material, while AW45 precipitated in the cell culture medium at the lowest limit of concentration for testing.

Values for MIC90 represent screening against Mtb H37Rv, while IC50 and IC90 represent screening against CHO and THP-1 cells respectively. Selectivity index is expressed

relative to IC90 in THP-1.

98

3.3.4 Discussion

Relative to FA, the FA-aminoquinoline hybrids were cytotoxic to both CHO and THP-1 cells; although

by virtue of the different definitions of cytotoxicity (50% inhibition for CHO and 90% for THP-1), no

direct comparison of the absolute values could be made. Variations in the cytotoxicity profile of

compounds across different cell lines are an inherent limitation of these widely used in vitro

cytotoxicity assays. Differences in function and structure of the cell lines may have implications for

their sensitivities to compounds. In addition, as these cell lines are immortalised and adapted for in

vitro lab use, there is a limit to their correlation in vivo.

There was no clear trend with relation to the length of the alkyl spacer across the amide and ester

hybrids. In the THP-1 assay, the primaquine hybrid AW33 was the least cytotoxic of the hybrids.

The cytotoxicity observed for the diamine aminoquinoline derivatives in the MTT assay suggests that

the quinoline scaffold contributes to the cytotoxicity of the hybrids synthesised in this project. The

non-cytotoxicity observed for FA supports this and, in addition, the aminoquinoline scaffold has

been associated with cytotoxicity. For example, Andayi et al reported cytotoxic activity of

aminoquinoline hybrids with similar alkyl spacers at the 4-position.6

Given their low aqueous solubility, as can be seen in the case of AW45 that precipitated in the

culture medium used for the assay, this may have precluded the detection of the in vitro cytotoxicity

in the case of several of the hybrids which were found to be insoluble at 10 µM in the solubility

assay discussed in Chapter 2.

By virtue of their poor solubility in aqueous media, in vitro cytotoxicity and poor antimycobacterial

activity in comparison to FA, these compounds were not progressed to macrophage efficacy studies.

3.4 Evaluation of selected FA analogues for intracellular antimycobacterial efficacy in THP-1

3.4.1 Compound selection

As part of ongoing work aimed at the repositioning of FA for TB drug discovery, structure-activity

relationship (SAR) studies on this molecule have resulted in the synthesis of a wider panel of

analogues with variable antimycobacterial activity. Along with FA, two C-3 ester and 3-ketofusidic

acid (keto-FA) were selected based on their antimycobacterial activities, relatively low cytotoxicity to

THP-1 cells and their promising PK profiles from preliminary in vivo mouse studies. 3-ketoFA

(designated as GKFA37), has been reported as a metabolite in man with lower antibacterial activity

compared to the parent drug.7

The compounds were diluted to achieve 1X, 2X, and 5X MIC90 concentrations, with the exception of

GKFA16 which was used at 1X and 2X MIC90 due its lower selectivity index (Table 3.3). Isoniazid

(INH), a first-line anti-TB agent with in vitro intracellular efficacy, was used at 2X its MIC90 value.8

99

Figure 3.1: FA and analogues selected for evaluation of intracellular efficacy in THP-1

3.4.2 Intracellular infection

To assess the intracellular efficacy of the compounds, THP-1 cells were infected with approximately

1 x 105 colony forming units (CFU) per ml of Mtb H37Rv MA at a multiplicity of infection (MOI) of 10

bacilli: 1 macrophage. At 4 hours post-infection, extracellular bacteria were washed off and the cells

were treated with the compounds in triplicate at a range of concentrations (Table 3.3).The cells

were incubated for 6 days as the viability of THP-1 cells is known to decrease after this period.

Table 3.3: FA and analogues evaluated for intracellular efficacy in THP-1

Compound 7H9/OADC MIC90 in

(µM)

IC90 in THP-1 in

(µM)

Selectivity

index

Concentrations for

assay (µM)

FA 5 >200 >40 1x, 2x, and 5x MIC

GKFA16 20 100 5 1x and 2x MIC;

GKFA17 5 100 10 1x, 2x, and 5x MIC

GKFA37 2.5 >200 >80 1x, 2x, and 5x MIC;

The culture medium (with and without compound for the treated and untreated cells, respectively)

was replenished every two days (adding fresh medium to the untreated controls).

Concomitantly, cells in representative wells were lysed using distilled water with 0.05% Tween80

and the lysates serially diluted in standard 7H9 medium. Serial dilutions of the lysates were plated

on 7H10 agar plates and CFU were enumerated after incubation for 21-28 days. The post-

phagocytosis inoculum (untreated cells at the onset of infection) was used as a baseline to

determine the increase in CFU. Details of the experimental procedure are described in Chapter 6.

100

3.4.3 Results of CFU log reduction for Mtb-infected THP-1 treated with FA

At 1X MIC, there was no reduction in CFU, while at 2X and 5X MIC there was a 2-log reduction in CFU

by day 6. The 2-log reduction in CFU was more rapidly achieved at 5X its MIC. However, this is lower

than the activity observed for the control drug, INH at 2X MIC, which caused a 2-log reduction in CFU

by day 2 (Graph 3.1).

Graph 3.1: Log reduction of CFU in THP-1 treated with FA

3.4.4 Results of CFU log reduction for Mtb-infected THP-1 treated with GKFA16 and GKFA17

In comparison to FA, a more modest reduction in CFU was observed for GKFA16, with a 1-log

reduction achieved by day 6 at both 1X and 2X MIC (Graph 3.2). However, at 5X MIC, a 2-log

reduction was observed for GKFA17 over the 6 days, with earlier onset of log reduction in CFU by

day 2 (Graph 3.3).

101

Graph 3.2: Log reduction of CFU in THP-1 treated with GKFA16

Graph 3.3: Log reduction of CFU in THP-1 treated with GKFA17

3.4.5 Results of CFU log reduction for Mtb-infected THP-1 treated with GKFA37

At 1X and 2X MIC, a 1-log reduction in MIC was observed, while, at 5X MIC, a more rapid decline in

CFU similar to that of the control INH was achieved (Graph 3.4).

102

Graph 3.4: Log reduction of CFU in THP-1 treated with GKFA37

3.4.6 Discussion

FA, GKFA17 and GKFA37 were active against intracellular Mtb, effecting a 2-log reduction in CFU

over the course of 6 days. This was more rapidly achieved at the highest concentration for each of

these compounds. GKFA16 only caused a 1-log reduction, correlating with its lower potency in Mtb

broth cultures compared to FA and the other two analogues. In this macrophage model of infection,

GKFA37, which has been reported as a metabolite of FA in man with lower antibacterial activity than

the parent drug, showed a 1-fold higher difference in potency compared to FA. This was a similar

result to that observed in broth culture, where it showed a 1-fold higher difference in potency

compared to FA.

The intracellular efficacy observed for FA and the analogues in this study point to their ability to

accumulate within phagocytic cells, consistent with the findings of Lemaire et al who evaluated FA

for intracellular efficacy against several strains of Staphylococcus aureus. As part of their study,

Lemaire and co-workers evaluated FA accumulation in THP-1 cells, and found that increased

accumulation was not associated with better activity in broth. This led these authors to speculate

the involvement of other resistance mechanisms in the relatively lower intracellular efficacy of FA

compared to its activity in broth.9

As discussed in section 3.1, macrophage models of infection have proven to be useful in evaluating

the intracellular efficacy of compounds under different conditions.2 In a study by Vanderven et al,

from a high-throughput screen of 1,359 compounds with antimycobacterial activity ≤ 5 µM, only

10% inhibited growth of Mtb intracellularly.2 The results obtained for GKFA16, GKFA17 and GKFA37

103

give some measure of confidence in the antimycobacterial efficacy of these compounds, albeit

tempered by several factors: macrophages are part of a wider context of the granuloma in host

infection, thus excluding other mechanisms that play a role in the outcome of infection in host

disease. Cell lines adapted for in vitro lab use have limited predictive value for in vivo disease, and in

addition, different results are sometimes observed across various cell lines.

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3.5 In vitro Metabolism of FA and selected analogues

3.5.1 Introduction

As discussed in Chapter 1, drug metabolism has implications for the efficacy and safety of a drug as a

consequence of the following: (1) enzymatic inactivation (detoxification); (2) biotransformation into

active metabolites; (3) enzymatic modification into reactive metabolites that cause toxicity. Because

of its status as an old drug, the in vivo metabolism of FA has been studied extensively, and more

recently by various members of our lab, using a variety of in vitro systems.

3.5.2 Metabolism of FA

3.5.2.1 Metabolism of FA in humans

In humans, FA is mainly cleared through hepatic metabolism, and to a lesser extent, through biliary

excretion. The C-21 glucuronide (2) is the major metabolite, while 3-keto FA (GKFA37), 27-carboxy

FA (4) and a ring hydroxylation (5) product are minor metabolites. (Figure 3.2 below).7,10,11

Interestingly, at high doses, FA has been reported to inhibit its own clearance; although the

mechanisms for this auto-inhibition are as yet unknown.7,12,13

105

Figure 3.2: FA metabolites in man

3.5.2.2 Metabolism in microsomes

Human, mouse and rat liver microsomes (denoted as HLM, MLM and RLM respectively) were used

for the evaluation of metabolic stability and metabolite identification. Overall, comparable

microsomal stability of 65% ± 5% was observed across the three species. With regards to specific

metabolites, differential metabolism was observed across the rodent and human species. FA was

shown to have a shorter half-life in rodent microsomes. In addition, one of the metabolites reported

in FA metabolism in man, carboxyfusidic acid, was absent in all the in vitro incubations.

106

The major metabolite detected in RLM and MLM was 3-epifusidic acid (3-epi-FA), which was

hypothesised to result from reversible oxidation at C-3, wherein the reverse reduction occurs in a

non-stereo selective manner, resulting in both FA and its C-3 epimer, 3-epiFA. In addition, 3-keto

fusidic acid (3-ketoFA) and a ring hydroxylation product were detected as minor metabolites (Figure

3.3). This epimerisation has also been reported for another steroid, cinobufagin.14, 15 Interestingly, in

cinobufagin, this reaction is also species-specific, reported only for in vitro and in vivo rat

metabolism, but not in mouse or humans. Conversely, in HLM, the major metabolite reported was 3-

ketoFA, with no 3-epiFA detected. Negligible traces of the ring hydroxylation metabolite were also

detected.

Figure 3.3: Postulated conversion of FA to 3-epiFA

3.5.2.3 Metabolism in mycobacterial lysates

FA was found to be metabolically stable in incubations of Mtb lysate, while in that of Msm, it was

unstable, with a half-life of 3 hours. This was found to be as a result of hydrolysis of the acetate at C-

16, with an unstable hydroxyl intermediate. The resultant metabolite, fusidic acid lactone, has been

reported as a metabolite with lower antibacterial activity compared to FA.10 Figure 3.4 below

illustrates the hydrolysis.

Figure 3.4 C-16 hydrolysis of FA

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3.5.2.4 Metabolism in plasma

The stability in plasma varied as follows: mouse>>rat>man, which corresponds to the literature

reports of the hierarchy of hydrolase activity across these species.16 In rat and mouse plasma, the C-

16 hydrolysis reported in Msm lysate also occurred, resulting in formation of the lactone. In human

plasma, however, this modification did not take place.17

3.5.3 Metabolism of selected FA analogues

Previously, several aryl and alkyl esters of FA were evaluated for their metabolism in vitro in our lab,

and these are discussed briefly below due to their relevance to this project.17

3.5.3.1 C-3 esters

Metabolism in microsomes

In RLM, both aryl and alky esters exemplified by ME166, ME175, GKFA16 and GKFA17 (Figure 3.5)

were extensively metabolised to FA. Among the aryl esters, a hydroxylated metabolite was also

produced.

Figure 3.5: Examples of FA aryl and alkyl esters metabolized to FA in rat liver microsomes

Metabolism in plasma

Metabolism in rat plasma also produced FA, albeit at a slower rate in comparison to RLM

incubations. One compound in particular, GKFA17 (Figure 3.5) was evaluated for metabolism in

human, rat and mouse plasma, and its stability decreased in the order: human>>rat>>mouse. In

mouse plasma, GKFA17 had a short half-life of 30 minutes, exhibiting rapid hydrolysis to FA, with

less than 10% of the parent compound detected at the end of the incubation period. Conversely, in

human plasma, it was very stable, with >99% remaining 3 hours after incubation.

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3.6 Role of mycobacterial metabolism in bioactivation and biodegradation of drugs

Mycobacteria possess hydrolytic enzymes that are known to metabolise xenobiotics. One such

example is pyrazinamide (PZA), a first-line anti-TB drug. PZA is well-established as a prodrug which

undergoes hydrolysis by mycobacterial deamidase to yield the weakly acidic pyrazinoic acid (POA),

which is responsible for the pharmacological activity observed.18,19 To support the assertion of

intrabacillary hydrolysis, it has been shown that clinical resistance to PZA is associated with

mutations in the pncA gene which encodes the mycobacterial gene.19 Via and co-workers, however,

reported host-mediated hydrolysis of PZA, demonstrating the contribution of host bioactivation to

the clinical efficacy of PZA.20 Another example is that of the aminoglycosides, a class of second-line

anti-TB agents used in the treatment of MDR-TB. As highlighted in Chapter 1, the inactivation of the

aminoglycoside kanamycin through enzymatic modification has been reported. A mycobacterial

acetyltransferase enhanced intracellular survival (Eis) has been shown to carry out multiple

acetylations on kanamycin (KAN), rendering Mtb resistant to several aminoglycosides. To support

this, DNA sequencing of KAN-resistant clinical isolates revealed the upregulation of mycobacterial

eis.21,22 Also highlighted in Chapter 1, recently, Warrier et al reported N-methylation of an

antimycobacterial pyridobenzimidazole by Rv0560c, a previously uncharacterized methyltransferase

in Mtb.23

As discussed in chapter 1, a common mechanism of resistance to anti-TB drugs that are prodrugs is

the emergence of resistance mutations in genes that encode the enzymes required for their

bioactivation. Ethionamide, a second-line anti-TB agent, is one such example, with mutations

reported in ethA, which encodes the monooxygenase required for its bioactivation. A recent study

published by Blondiaux and co-workers reported that a novel molecule, Small Molecule Aborting

Resistance (SMARt-420) fully reversed ethionamide resistance in Mtb-infected mice, and, in

addition, increased the basal sensitivity of bacteria to ethionamide.24

3.7 Rationale

As mentioned in section 3.5.2.3, data from our lab demonstrated that, in Msm, FA is metabolised by

mycobacterial hydrolases to its lactone, which has significantly less antibacterial activity than FA.17

Since these experiments were carried out using lysates of Msm and Mtb, the hydrolysis observed

could only be attributed to cytosolic hydrolases. This excludes the contribution of enzymes

expressed in the mycobacterial cell membrane and/or enzymes that may be inactivated during cell

109

lysis, which may play a role in the in vitro metabolism of xenobiotics. In the prokaryotic world, Mtb is

unrivalled in the range of enzymes involved in lipid metabolism, including a variety of hydrolases,

some of which are expressed in the cell membrane.25–27 Although their substrates are typically

bacterial, their substrate specificity may be variable and some of them may hydrolyse xenobiotics.

The example of the prodrug PZA attests to the contribution of mycobacterial metabolism to

pharmacological activity. Moreover, some species of Streptomyces and Nocardia have been reported

to hydrolyse the C-16 acetyl group in FA as a mechanism of resistance to FA.28–31 As mentioned in

section 3.5.3.1, the C-3 esters were hydrolysed to FA in vitro. Among the FA analogues evaluated,

GKFA16 and GKFA17 showed good in vitro antimycobacterial activity in broth cultures and in Mtb-

infected THP-1 cells. In addition, relative to several other analogues, they had good selectivity

indices. GKFA61, a C-3 silicate ester, was selected for evaluation due to its promising in vivo PK data

from our lab, which revealed that, when administered to C57BL/6 mice at equivalent doses, it had a

longer half-life than FA. On this basis, they were selected for evaluation of in vitro mycobacterial

metabolism. In addition, two C-21 amide FA-aminoquinoline hybrids, AW23 and AW25, were also

selected for similar evaluation. As discussed in Chapter 2, these two hybrids were 4-fold less potent

than FA against Mtb. We hypothesised that this may be due to their stability in broth cultures; the

failure of hydrolysis to release FA and the aminoquinoline pharmacophore is therefore a plausible

explanation for their poor antimycobacterial activity relative to FA. Figure 3.6 illustrates their

chemical structures, the MIC90 (in Mtb H37Rv).

Figure 3.6: Structures of FA analogues for evaluation of mycobacterial metabolism

3.8 Aim

We hypothesised that there may be hydrolases in Mtb that may contribute to bioactivation or

biodegradation of FA and selected analogues. The aim of this work was therefore to investigate the

110

role of mycobacterial metabolism in the bioactivation and or biodegradation of FA and selected

ester and amide analogues in an in vitro system. The specific objective was to investigate the in vitro

metabolism of FA and selected C-3 ester and C-21 amide analogues in broth cultures of Mtb.

3.8.1 Incubation of compounds in Mtb

The compounds were added at 0.5X MIC to Mtb in PBS (0.05% Tween80) pH 7.4 and incubated in a

shaking incubator at 37oC over with sampling at various time-points over 48 hours. In parallel, the

compounds were added to heat-killed Mtb as a control to rule out degradation or bioactivation in

the absence of live bacteria. At each time-point, acetonitrile containing an internal standard was

used to deactivate the bacterial metabolism process. The samples were centrifuged and analysed by

LC-MS. The ratio of the analyte to internal standard over time was then plotted to give a measure of

the stability of the compound over the 48 hours.

3.8.2 Results

3.8.2.1 Stability of FA in Mtb cultures

Over 48 hours, FA was stable in both the live and heat-killed Mtb cultures (Graph 3.5). This suggests

that FA is not metabolised by the live bacilli and that any instability is due to non-specific

mechanisms such as aqueous degradation. The data shown are of technical triplicate samples,

representative of 2 experiments carried out on separate days.

Graph 3.5: Ratio of FA to internal standard in live and heat-killed Mtb cultures over time

3.8.2.2 Stability of GKFA16 and GKFA17 in Mtb cultures

In contrast, there was a gradual decline in the concentration of GKFA16 and GKFA17 in the live Mtb

culture (Graphs 3.6 and 3.7). Moreover, FA was detected in these cultures, with a concomitant

0

1

2

3

4

5

0 10 20 30 40

An

alyt

e/I

S ra

tio

Time (Hrs)

GKFA_HK

GKFA_LIVE

FA HK

FA live

111

increase in concentration over the 48 hours. This suggests that GKFA16 and GKFA17 are hydrolysed

to FA by the live bacilli. Furthermore, in both cases, when compared to the heat-killed Mtb cultures

containing FA where the concentration of FA remained constant over time, GKFA16 and GKFA17

were hydrolysed to higher concentrations of FA, a further testament to the mycobacterial hydrolysis

occuring. In the heat-killed cultures, this hydrolysis was marginal, and occurred at a much lower rate.

Graph 3.6: Ratio of FA and GKFA16 to internal standard in live and heat-killed Mtb cultures over

time

Graph 3.7: Ratio of FA and GKFA17 to internal standard in live and heat-killed Mtb over time

3.8.2.3 Stability of GKFA61 in Mtb cultures

Over 48 hours, GKFA61 was stable in both the live and heat-killed Mtb cultures, and no FA was

detected in the incubations (Graph 3.8). This suggests that it is not metabolised by the live bacilli

and that any instability is due to non-specific mechanisms such as aqueous degradation. The data

0

2

4

6

0 10 20 30 40

An

alyt

e/I

S ra

tio

Time (Hrs)

GKFA16_HK

GKFA16_LIVE

FA_HK

FA_LIVE

-2

0

2

4

6

8

10

12

0 10 20 30 40

An

alyt

e/I

S ra

tio

Time (Hrs)

GKFA17_HK

GKFA17_LIVE

FA_HK

FA_LIVE

112

shown are of technical triplicate samples, representative of 2 experiments carried out on separate

days.

Graph 3.8: Ratio of GKFA61 to internal standard in live and heat-killed Mtb over time

3.8.2.4 Stability of AW23 and AW25 in Mtb cultures

Similarly, the C-21 amides AW23 and AW25 were stable over the course of the 48 hours in both the

live and heat-killed Mtb cultures, and no FA was detected in the incubations (Graph 3.9).

Graph 3.9: Ratio of AW23 to internal standard in live and heat-killed Mtb over time

0

2

4

6

8

10

0 10 20 30 40

An

alyt

e/I

S

Time (Hrs)

GKFA61_HK

GKFA61_LIVE

0

0.4

0.8

1.2

1.6

2

0 10 20 30 40

An

alyt

e:I

S ra

tio

Time (Hrs)

AW23 HK

AW23 LIVE

113

Graph 3.10: Ratio of AW25 to internal standard in live and heat-killed Mtb over time

3.8.3 Discussion

FA remained stable in live Mtb cultures, a similar result to that previously observed in Mtb lysate.

This rules out the involvement of mycobacterial cell membrane-associated hydrolases in the

biodegradation of FA. Conversely, the C-3 esters GKFA16 and GKFA17 were hydrolysed by live bacilli

to achieve higher concentrations of FA compared to the heat-killed Mtb cultures, where the

concentration of FA remained relatively unchanged. These data are corroborated by preliminary in

vivo mouse PK (unpublished data from our lab), which have shown that these two compounds are

hydrolysed to achieve higher (lung) concentrations of FA when compared to the mice administered

with FA. Given the extensive hydrolysis of GKFA17 previously observed in mouse plasma and in rat

liver microsomes, it is plausible that mycobacterial and host metabolism could synergize to achieve

higher concentrations of FA in mice, increasing its bioavailability. In addition, both these compounds

were observed to be very stable in human plasma. Bacterial hydrolysis could therefore hydrolyse

them to achieve higher concentrations of FA in man.

The C-3 silicate ester GKFA61 and the C-21 amides AW23 and AW25 were stable in both live and

heat-killed Mtb cultures over 48 hours. The lack of hydrolysis of these compounds to FA in broth

cultures may contribute to their lower potency compared to FA. In the case of GKFA61, the isopropyl

groups provide steric hindrance around the C-3 position, improving metabolic stability, and,

consequently a longer half-life seen for this compound in mice.

Strictly speaking, prodrugs should be devoid of pharmacological activity, and require enzymatic

hydrolysis to release the pharmacologically active metabolite.32,33 The C-3 FA esters, GKFA16 and

GKFA17, in themselves possess modest in vitro and ex vivo activity against Mtb, albeit lower than

that of FA. Notwithstanding, these compounds are hydrolysed by mycobacterial and host esterases

0

2

4

6

8

0 10 20 30 40

An

alyt

e:I

S ra

tio

Time(Hrs)

AW 25 HK

AW25 LIVE

114

to the parent drug. Moreover, unpublished data from our lab demonstrated that, in an in vivo PK

mouse model, in comparison to mice that were administered with FA, higher concentrations of FA

were detected in the lungs of mice that were administered with GKFA16 and GKFA17. Potentially,

higher bioavailability in the lung- the organ of interest in the context of pulmonary TB, could result in

improved efficacy of these compounds. Since they enhance the bioavailability of FA, they fit the

broader definition of prodrugs, which includes drugs that improve properties of the

pharmacologically active metabolite, such as PK. Mtb-associated hydrolysis of these esters shows

that mycobacterial metabolism may facilitate compound efficacy, which can be incorporated into

the design of future analogues.

Apart from their relevance in PK, bioactivation or biodegradation may have implications for drug

efficacy, as certain Nocardia and Streptomyces species exploit biodegradation as a mechanism of

resistance to FA and other antibiotics.28–38 Mycobacteria are known to possess hydrolases, including

esterases. Although the substrates are typically endogenous, their activity against xenobiotics has

not been studied, and it would be worthwhile to characterise their specificity, since FA and

analogues shares the steroid nucleus with cholesterol and other lipid metabolites critical to Mtb

survival.25,27,39–41 The example of these two compounds illustrates the value of establishing the role

of mycobacterial metabolism in drug PK, for its overall implications on efficacy.

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3.9 Summary

Relative to FA, the FA-aminoquinoline hybrids were cytotoxic to both CHO and THP-1 cells.

There was no clear trend with relation to the length of the methylene spacer across the amide and

ester hybrids. In the THP-1 assay, the primaquine hybrid AW33 was the least cytotoxic of the

hybrids. Evaluation of selected aminoquinoline derivatives singly and in combination with FA in a 1:1

ratio in CHO cells suggests that the quinoline scaffold contributes to the cytotoxicity of these

hybrids. This is supported by the observation that FA was not cytotoxic in the same assay, and a

previous report in the literature, of compounds with a similar aminoquinoline scaffold, that were

found to be cytotoxic against this cell line.

The low aqueous solubility observed for these hybrids in Chapter 2 was further confirmed in the

cytotoxicity assays; AW45, the amide hybrid with a 5-carbon methylene spacer, precipitated in the

culture medium used for these assays. This may have precluded the accurate determination of the

cytotoxicity of some of these hybrids. By virtue of their poor solubility in aqueous media, in vitro

cytotoxicity and poor antimycobacterial activity in comparison to FA, these compounds were not

progressed to macrophage efficacy studies.

FA and three analogues, GKFA16, GKFA17 and GKFA37 (3-ketoFA), were evaluated for intracellular

efficacy in THP-1 cells. FA, GKFA17 and GKFA37 were active against intracellular Mtb, effecting a 2-

log reduction in CFU over the course of 6 days. This was more rapidly achieved at the highest

concentration for each of these compounds, which was 5x the MIC in broth cultures. GKFA16 only

caused a 1-log reduction, correlating with its lower potency in Mtb broth cultures compared to FA

and the other two analogues. In this macrophage model of infection, GKFA37, which has been

reported as a metabolite of FA in man with lower antibacterial activity than the parent drug, showed

a 1-fold higher difference in potency compared to FA. This was a similar result to that observed in

broth culture, where it showed a 1-fold higher difference in potency compared to FA. The

intracellular efficacy observed for FA and the analogues in this study point to their ability to

accumulate within phagocytic cells. This is consistent with existing literature reports of the

intracellular efficacy of FA against several strains of Staphylococcus aureus.

Three C-3 ester analogues, GKFA16, GKFA17 and GKFA61, as well as two C-21 FA amides, the FA-

aminoquinoline hybrids AW23 and AW25, were evaluated for in vitro mycobacterial metabolism. In

live Mtb cultures, GKFA16 and GKFA17 were extensively hydrolysed to FA, while, in heat-killed

cultures, their concentrations remained relatively unchanged. This bioconversion was not observed

for GKFA61, AW23 and AW25, whose concentrations remained relatively constant in both live and

heat-killed cultures. These findings corroborate in vivo PK data (unpublished), which showed higher

concentrations of FA in mice administered with GKFA16 compared to mice administered with

116

equivalent doses of FA. The lack of mycobacterial hydrolysis of GKFA61 by live bacilli correlates with

the longer half-life observed for this compound in mice, plausibly due to steric hindrance around the

C-3 position, which may confer metabolic stability.

117

3.10 References

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(17) Njoroge, M. PhD Thesis, University of Cape Town, 2014.

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M.; Delorme, V.; Djaout, K.; Trebosc, V.; Kemmer, C.; Wintjens, R.; Wohlkönig, A.; Antoine, R.;

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Chapter 4

Target Identification and Mechanism of Resistance of Fusidic Acid

Chapter overview

This chapter provides a summary of the target identification and mechanism of action studies of

fusidic acid in several organisms, with a brief overview of the function and structure of the known

target. The investigation of its target and subsequent validation are presented, in addition to

determination of its in vitro cross-resistance with other anti-TB agents.

4.1 The identification of distinct modes of action for the same molecule in different organisms.

TB chemotherapy only began in earnest in the 20th century, and majority of the first line anti-TB

chemotherapeutic agents were in clinical use decades before their modes of action were

established. For example, the mode of action of streptomycin (STM), the first drug that was used to

successfully cure a TB patient, was only established in the late 1950s despite being in use from the

early 1930s. Rifampicin (RIF) and isoniazid (INH), the mainstays of TB treatment since the 1960s, had

their mechanisms of action elucidated decades later. For instance, the bactericidal activity of INH

was only attributed to its inhibition of mycolic acid biosynthesis in the 1970s , and even then, the

true mode of action was only definitively elucidated much later.1,2 These discoveries were made

before the revolutions in genomics and molecular sciences, which enabled further (and often more

rapid) advances in drug target identification over the past 20 years.3,4

As highlighted in the previous chapter, with the growing awareness of polypharmacology, it is also

possible for a compound or drug to possess different molecular targets in different organisms. This is

the case with cyclomarin A (Figure 4.1), a naturally occurring cyclic peptide that inhibits the growth

of Plasmodium falciparum by inhibiting diadenosine triphosphate hydrolase (PfAp3Aase) whereas, in

Mtb, it targets the essential protease, ClpP.

121

Figure 4.1: Structure of cyclomarin A

This could be due to the fact that these two organisms are taxonomically unrelated, and

furthermore, the molecular targets of cyclomarin A are not essential for the viability of majority of

other organisms, due to redundancy of the genes that encode for these enzymes.5–7 Thus,

repositioning of even well-characterized anti-infectives should include identification of their target

or targets in the intended organism.

4.2 Mechanism of action of fusidic acid (FA)

FA is known to be a protein synthesis inhibitor in Gram-positive bacteria, inhibiting the translation

machinery, specifically the penultimate step in the elongation cycle, as well as ribosomal recycling.8

A recent study by Borg et al has uncovered deeper mechanistic understanding of its inhibitory

action.9 Protein synthesis can be divided into four steps: initiation, elongation, termination and

recycling. As illustrated in Figure 4.2 below, Elongation factor G (EF-G) is critical to the penultimate

elongation step, and is usually bound to Guanosine Triphosphate (GTP) in order to catalyse

translocation of the transfer RNA (tRNA) from the ribosomal A-site to the P-site.10

FA inhibits translocation of the tRNA from the A-site to the P-site by stabilizing the EF-G: Guanosine

Diphosphate (GDP) complex, preventing its release from the ribosome.8,10 It is presumed that the EF-

G:GTP complex binds to a defined site on the ribosome in the pre-translocation state. After

hydrolysis of GTP to GDP and inorganic phosphate (Pi), and consequent translocation, the EF-G:GDP

loses its affinity for the ribosome.11,12 Agrawal and colleagues used cryo-electron microscopy to

reconstruct fusidic acid-stalled EF-G:70S ribosome complexes, which delineate the binding site and

interaction mode of EF-G with the ribosome.13 These studies demonstrated that FA does not bind to

122

free EF-G, but rather to EF-G:GTP in complex with the ribosome, which implies that the antibiotic

requires a specific conformation of EF-G for binding.8 Thus, FA binding locks the ribosome in this

ternary complex, preventing the post-translocational conformation necessary for the next cycle of

elongation. Concomitantly, EF-G occupies the space on the ribosome, which overlaps with the

binding site of the elongation factor EF-Tu that is required for the amino acyl-tRNA delivery step.14

Several of the protein synthesis inhibitors listed in Figure 4.2 have antibacterial activity and are used

in the clinical management of a wide spectrum of infections, but lack activity against Mtb. These

include clindamycin, the macrolides erythromycin, neomycin and telithromycin, as well as

tetracyclines and chloramphenicol.15 Apart from their clinical or veterinary use in the treatment of a

wide spectrum of organisms, several of the inhibitors listed are relevant in TB drug discovery.

Interestingly, chloramphenicol (Cam) and tetracycline (Tet), which are used in the treatment of a

range of organisms, were shown to inhibit mycobacterial protein synthesis, but not growth, by Shaila

et al.16 This was attributed to poor permeability of the molecules across the cell wall. On the other

hand, paromomycin (Par) has been shown to possess in vitro and in vivo activity against Mtb,

including MDR-TB.17 The results of a clinical study by Donald and colleagues reported similar anti-TB

activity to STM.18

123

Figure 4.2: Antibiotic target sites during bacterial protein synthesis. Adapted from Wilson D. N.10

Initiation of protein synthesis involves the formation of a 70S ribosome (composed of a 30S and a 50S subunit) with the

initiator tRNA and start codon of the mRNA positioned at the P-site. This process is inhibited by the antibiotics edeine

(Ede), kasugamycin (Ksg), pactamycin (Pct) and thermorubin (Thb) on the 30S subunit, and by the orthosomycins,

avilamycin (Avn) and evernimicin (Evn), as well as thiostrepton (Ths) on the 50S subunit. The elongation cycle involves the

delivery of the aminoacylated-tRNA (aa-tRNA) to the A-site of the ribosome by elongation factor Tu (EF-Tu), which is

inhibited by Streptomycin (STM), tetracyclines (Tet) and glycylcyclines (tigecycline (Tig)). Peptide-bond formation between

the A- and P-site tRNAs is inhibited by blasticidin S (Bls), chloramphenicol (Cam), lincosamides (e.g., clindamycin (Cln)),

oxazolidinones (e.g., linezolid (Lzd)), pleuromutilins (Plu), puromycin (Pmn), streptogramin A (SA) and sparsomycin (Spr).

Translocation of the tRNAs is catalysed by EF-G and inhibited by the tuberactinomycins, capreomycin (Cap) and viomycin

(Vio); the aminoglycosides, hygromycin B (Hyg), neomycin (Neo) and paromomycin (Par); as well as fusidic acid (FA),

Spectinomycin (SPEC) and Ths. Elongation of the nascent chain is inhibited by the macrolides (e.g., erythromycin (Ert)),

streptogramin B (SB) and ketolides (e.g., telithromycin (Tel)). The final phases of termination and recycling lead to release

of the polypeptide chain and subsequent dissociation of the 70S ribosome, followed by recycling of the components for the

next round of initiation. Termination is inhibited by peptidyl-transferase inhibitors, such as Bls, Cam, Pmn and Spr, whereas

recycling is inhibited by translocation inhibitors, especially FA.

124

The tuberactinomycins, viomycin (Vio) and capreomycin (Cap) were identified as active against

multidrug-resistant tuberculosis in the 1950s.19–21 Streptomycin (STM) and Cap were advanced to

clinical use, and form part of the WHO standard regimens for MDR-TB.22

Following the successful treatment of XDR-TB in a small cohort of patients in a study by Lee et al,

linezolid (Lzd), a bacteriostatic antimicrobial, has garnered interest in TB drug discovery in recent

years.23,24 It is regarded as a “drug of last resort” in the treatment of Gram positive infections, mainly

vancomycin-resistant Enterococci (VRE) and Methicilllin-resistant Staphylococcus aureus (MRSA).25

The repurposing of Lzd underpins not only the continued relevance of drug repositioning in drug

discovery for drug-resistant TB, but also the usefulness of bacteriostatic antimicrobials. SPEC has

moderate in vitro activity against Mtb, and the encouraging development of novel spectinamide

analogues with potent in vitro activity and excellent in vivo profile in a mouse model of infection

show promise for drug-resistant TB.26

In addition to translocation inhibition, FA has been shown to inhibit ribosome recycling, which is

required for the release of the nascent polypeptide and therefore for the next round of initiation. In

fact, earlier reports reported that along with blasticidin S, another antibiotic, it has been shown to

be a more potent recycling inhibitor, with an IC50

in the ranges of ~0.1–15 μM for inhibition of

subunit dissociation, whereas its IC50 for inhibition of translocation is in the range of 10 –200 μM.8,10,

27,28 More recent studies by Borg et al have shown that FA’s inhibitory action is more nuanced,

ultimately dominated by the inhibition of elongation, but that its inhibition of ribosome recycling

may play a role in encoding FA resistance.9,29

4.2.1 Antiprotozoal mode of action of FA

FA has been shown to inhibit growth of the blood stage of the protozoan Plasmodium falciparum

which causes malaria.30 P. falciparum has two genome-containing organelles, the mitochondrion and

the relic plastid (apicoplast), the latter being a non-photosynthetic organelle unique to organisms

belonging to the phylum Apicomplexa. Several apicoplast pathways are critical for the blood and

liver stages of the parasite’s life cycle in the human host, and so have been studied as potential drug

targets.

As in bacteria, FA is a known inhibitor of the plasmodial translation machinery, targeting both

mitochondrial and apicoplast EF-G, albeit differentially, showing higher sensitivity in apicoplast EF-G.

This is partly attributable to a conserved GVG motif found in the critical GTP-binding domain (I) of

most other pro and eukaryotic EF-G that is absent in plastid/apicoplast EF-Gs. Additionally, FA has

also been found to inhibit ribosomal recycling in both the mitochondrion and the apicoplast.31,32

125

4.3 Elongation Factor G (EF-G): Function and structure

4.3.1 Function

EF-G is a member of the translational GTPase (trGTPase) superfamily whose bacterial members are

associated with diverse biological roles. It performs ribosome-dependent hydrolysis of GTP and is

involved in two distinct steps of protein synthesis: elongation and ribosome recycling. During the

elongation step, EF-G binds the ribosome and promotes the movement of tRNA and mRNA relative

to the ribosome. The relative shift of the mRNA is by a distance of one codon and the peptidyl- and

deacylated-tRNAs are shifted from the pre-translocational to the post-translocational site. Unlike

other GTPases, EF-G is active in the GDP-bound form and not in the GTP-bound form; that is, in a

kinetically stable ribosome–EF-G:GDP complex formed by GTP hydrolysis on the ribosome. During

the recycling step, EF-G acts in concert with the ribosome recycling factor (RRF) to effect the

dissociation of the ribosome into its individual subunits.33

Most of the functional characterization of this essential factor has been carried out by heterologous

expression in E. coli, whereas the majority of crystal structure elucidation studies have been

performed on EF-G from Thermus thermpohilus;34 even fewer studies have reported detailed

structures of E. coli EF-G in complex with FA.35–38

4.3.2 Structure

Early biochemical evidence indicated that EF-G is a multi-domain GTPase, an observation which has

been confirmed by crystallographic data.39–43 EF-G consists of five structurally well-defined domains

(Figure 4.3). The first G or GTPase domain binds and hydrolyses GTP and is common to all P-loop

GTPases (a structural class to which EF-G belongs), and so is highly conserved across different

species. It contains five typical motifs for GTP recognition – G1 to G5 – which form the GTP-binding

pocket. Within the G domain is a region termed the G’ insert, speculated to be an internal guanine

nucleotide exchange factor. Little information exists on the specific function of domain II. Domains

III, IV and V mimic amino acyl-tRNA when it is bound to EF-Tu:GTP in the ternary complex. Domain III

has been shown to be essential for the induction of GTP hydrolysis and subsequent translocation,

while domains IV and V are required for translocation but not for GTP hydrolysis.

126

Figure 4.3: Crystal structure of T. thermophilus EF-G from Margus et al. 41 The left panel indicates the

ribosome side of EF-G while the right column shows the opposite side, following a 180o rotation

about the y-axis. The numbers indicate the highly conserved amino acid residues, relative to

T.thermophilus.

Several studies have demonstrated that FA permits ribosome-stimulated GTP hydrolysis by EF-G, but

prevents the associated conformational changes in EF-G, and so inhibits EF-G turnover by stabilizing

EF-G:GDP on the ribosome.42 By binding at the interface of domains I and III, FA could restrict the

movement of these domains relative to each other, preventing EF-G from adopting the low affinity

GDP-bound conformation required for dissociation of the complex from the ribosome. Figure 4.4

illustrates the inhibitory effect of FA on the translocation process.43 In addition, two recent studies ,

one by Li and colleagues44, and another by Koripella et al45, have further delineated the activity of

EF-G by demonstrating that several mutations in a highly conserved amino acid residue, Histidine 91,

do not cause defects in intrinsic GTP hydrolysis, but lead to varying levels of defective ribosome-

associated GTP hydrolysis by EF-G due to conformational changes.

127

Figure 4.4: Translocation on the ribosome and the inhibitory effect of FA. Adapted from Frank et al.36

4.3.2.1 EF-G Homologue: EF-G2

EF-G was believed to exist exclusively in a single form as a bi-functional protein until recently, when

two genes (hEFG1 and hEFG2) encoding two different forms of EF-G were discovered in mammalian

mitochondria.46,47 An analysis of 191 bacterial genomes in a study of ribosomal associated GTPases

resulted in the identification of a second copy of EF-G in up to 30% of the bacterial strains

analysed.48 The second form of EF-G (EF-G2) has been isolated from several organisms and analysed

for activity. EF-G2 from T. thermophilus shows ribosome-dependent GTPase activity, with little

GTPase activity in the absence of ribosomes. It has a low level of activity in poly(U)-dependent

protein synthesis but its role in ribosome recycling is yet to be elucidated. EF-G2 isolated from M.

smegmatis was assayed for ribosome-dependent GTPase activity and ribosome recycling; neither

was observed, indicating a lack of ability to function in either of the roles of EF-G under the

conditions studied. Only in Borrelia burgdorferi (B. burgdorferi) and Pseudomonas aeruginosa (P.

aeruginosa) has the activity of both forms of EF-G been studied in depth.33,49 B. burgdorferi EF-G1

was found to act exclusively in translocation while EF-G2 was shown to function solely in ribosome

128

recycling, as is the case in human mitochondrial EF-G1 and EF-G2.49 In P. aeruginosa, EFG-1B, a form

of EF-G1, was found to be the sole translocase, while EFG1A (also a form of EF-G1) was surmised to

be responsible for ribosome recycling.33

Multiple sequence alignment of E-FG and EF-G2 from representative organisms by several groups

have revealed a wide variation in sequence homology; a high degree of homology between the EF-

G1 sequences, a slightly less uniform degree of homology between EF-G2 across these organisms

and lowest homology between EF-G and EF-G2 within the same organism. Importantly, regions of

sequence involved in GTP binding and hydrolysis are most strictly conserved between the two forms

of EF-G.50 Table 4.1 summarises the homology levels across several organisms.

Table 4.1: Sequence homology between EF-G and EF-G2 among several species50

Species comparison % homology

EF-G: Msm vs Mtb 83

EF-G: E. coli vs Tth 60

EF-G: Msm/Mtb vs E.coli/Tth 57-60

EF-G2: Msm vs Mtb 78

EF-G2: Msm vs Tth 33

EF-G2: Mtb vs Tth 36

Msm/Mtb EF-G vs EF-G2 30

Tth EF-G vs EF-G2 36

EF-G is encoded by the fus gene, which is located in the str operon that also contains genes encoding

two other ribosomal proteins, S7 and S12, along with a tuf gene, which encodes Elongation Factor

Tu (EF-Tu), a protein also involved in the elongation phase of protein synthesis.33 In the case of Msm,

earlier annotations in the NCBI database showed sequences corresponding to MSMEG_1400 and

MSMEG_6535 as EF-G and EF-G2 respectively. In current listing (also in the smegmalist database),

MSMEG_1400 is annotated as a pseudogene while MSMEG_6535 is annotated as the main

functional EF-G. Experimentally, however, Seshadri and co-workers observed that MSMEG_1400

encodes a functional EF-G, while MSMEG_6535 encodes EF-G2. In this study, we retained the earlier

annotations for these genes.50

129

4.3.2.2 Mtb EF-G

Genetic analysis of the Mtb genome predicted the presence of two forms of EF-G: EF-G1 (fusA1) and

EF-G2 (fusA2).51,52 Mtb fusA1 has previously been expressed in E. coli to study its interaction with

Mtb ribosome recycling factor (RRF)53, but, to date, there are no published reports of its GTPase

activity. Furthermore, the putative protein synthesis activity has not been evaluated in a biochemical

assay. The M. smegmatis homologues MsmfusA1 and MsmfusA2 have been expressed, purified and

evaluated in vitro, validating an earlier report by Martemyanov and co-workers that fusA2 does not

possess GTPase activity, but is capable of binding GTP.43,50

Although it might be inferred from these data that FA inhibits Mtb in a similar manner, there are no

published reports to support this. As already demonstrated, drugs may have distinct modes of action

in different organisms and, moreover, possibly more than one mode of action. In addition, as

observed for FA, mechanisms of resistance to the same drug may vary depending on the pathogen

and the nature of infection.54

4.4 Resistance to FA

4.4.1 In vitro resistance

In vitro resistance to FA in S. aureus strains has been selected for by the application of drug pressure

and mutations identified in fusA, the gene which encodes EF-G.43,55,56 Both low- and high-level

resistance have been achieved, with MIC50 and MIC90 values ranging from 1 g/ml to 132 g/ml.

57Analysis of mutations from the numerous resistant mutants revealed that high-level resistance

mutations cluster in two main regions of EF-G, at the interface between domains I and III. In most

cases reported, these were single amino acid substitutions. It was also noted that these mutations

did not confer a fitness cost on the bacteria, as assessed by doubling times in vitro.

As mentioned above, FA binding results in a conformation that locks the EFG:GDP complex on the

ribosome; therefore, mutations within this region possibly either facilitate the conformational

changes in EF-G required for dissociation from the ribosome (despite the presence of the drug), or

may simply prevent the drug from binding to EF-G.43 Evidence supporting this can be inferred from

studies such as that by Martemyanov and co-workers, who reported that EF-G lacking domain III

resulted in a 103-fold decrease in GTPase activity, but was able to bind to the ribosome in the

presence of the GDP or GTP and FA.43,58

130

In addition to mutations within fusA, other resistance-conferring determinants have been identified

in recent years. These are described in the context of in vivo resistance to FA, owing to their clinical

relevance.

4.4.2 In vivo resistance

Resistance to FA was reported as early as the 1970s, but the frequency of reports remained low,

with rates approximately 1-2 % in the early 1990s even in countries which employed FA extensively.

However, in the past two decades, there has been an increase in the prevalence of FA-resistant (FAR)

clinical isolates of staphylococcal species, including methicillin-resistant S. aureus (MRSA). Evidence

of this is found in numerous published reports of surveillance studies that have been conducted in

hospitals across various European countries, Malaysia, and the United States of America.59–64 For

example, one study of MRSA isolates collected over a 2-year period in Denmark reported that 17.6%

were FA-resistant.59,61 FA monotherapy has been strongly associated with the emergence of

resistance among both methicillin-resistant and susceptible S. aureus.

Previously, FA resistance was thought to be exclusively due to spontaneous chromosomal mutations

in fusA. However, in recent years, there have been published reports from extensive studies that

examined the relative prevalence of different FAR mechanisms, the genetic elements that carry

them, and the relative importance of clonal expansion of FAR strains versus horizontal dissemination

of these determinants. These additional resistance determinants, which have been designated as

fusB-type resistance determinants, include fusB-E. In fact, the predominant mechanism of clinical

FAR in S. aureus is the horizontal acquisition of determinants that encode FusB-type proteins.57,59 The

spread of FAR MRSA in particular regions of Europe has been attributed in part to clonal expansion of

community-acquired clones.60,61

Members of the FusB family, the best studied of which is FusB itself, are small (~25 kDa) proteins

that bind EF-G with a 1:1 stoichiometry and protect it from the inhibitory effect of FA. X-ray

crystallographic studies have determined the 3D structures of two representatives of the FusB

family, revealing a two-domain protein with an unusual zinc-binding fold in the C-terminal domain.

Using NMR to elaborate the structure of the EF-G:FusB complex, Tomlinson and co-workers

demonstrated that the C-terminal domain is the primary site of interaction of FusB with EF-G.62,63

Moreover, they showed that through direct interaction with EF-G, FusB promotes disassembly of the

FA-stalled post-translocation complex, thus enabling the elongation cycle to proceed. Guo et al

postulated that FusB rescues FA inhibition by facilitating dissociation of the FA-EF-G-ribosome

complex, in a study where they demonstrated that FusB can rescue both FA inhibition of

131

translocation and ribosome recycling.28 The ability of FusB to drive EF-G release explains why it

mediates resistance to FA. How FusB might achieve this effect has not been conclusively

established.62 A model by Cox and co-workers (Figure 4.5) illustrates the hypothesised mechanism of

FusB-type mediated resistance to FA.64

Figure 4.5: Schematic of the proposed mechanism of FA resistance mediated by FusB-type proteins,

Adapted from Cox et al.64 (A) EF-G catalyzes translocation in a reaction driven by the hydrolysis of GTP. (B) In the

presence of FA, the drug immobilizes EF-G·GDP complexes on the ribosome, sterically blocking the next stage of translation

and stalling protein synthesis. (C) FusB-type proteins compete with the ribosome for binding to EF-G, thereby destabilizing

the ribosome:EF-G:GDP:FA complex and prompting its dissociation; FA cannot bind to EF-G when the latter is not resident

on the ribosome, and will spontaneously dissociate once the complex has been dislodged from the ribosome. (D)

Dissociation of the EF-G·GDP·FA complex clears the ribosomal A site, allowing entry of the next incoming aminoacyl-tRNA

molecule, and translation resumes.

FusC has been detected in clinical strains of S. aureus that lack a mutation in fusA and fusB, while

FusD is the Staphylococcus saprophyticus homologue of FusB, conferring intrinsic resistance of this

organism to FA. Given that S. saprophyticus is unlikely to encounter antibiotic, and that the genes

encoding this protein are chromosomally located, it is speculated that the FusB homologues play an

as yet unknown role in housekeeping.59 It is plausible that the recruitment of one or more FusB

132

homologues from intrinsically resistant organisms into S. aureus may have occurred over time due to

selective pressure exerted by use of FA.

Interestingly, to date, no clinical strains harbouring mutations in both fusA and fusB have been

detected, implying that a combination of the two resistance genotypes is unlikely to confer high

level resistance to FA, and that emergence of resistant clinical strains bearing both resistance

determinants is an unlikely scenario. O’Neill and colleagues demonstrated that introduction of fusB

into S. aureus strains expressing low-level FAR did not elevate the level of FAR .57

4.4.3 Evaluation of the fitness cost of FAR

Resistance mutations in bacteria are associated with pleiotropic effects, which may include reduced

fitness of the resultant strains. It is important to consider the extent to which antibiotic resistance

determinants impose a fitness cost on the bacterium. A strain’s fitness reflects its robustness in the

face of adaptation to mutations, environmental and internal changes, allowing it to survive,

reproduce, and, in the case of pathogenic bacteria, retain virulence.65 From a mechanistic

perspective, fitness deficit can be attributed to the fact that these mutations target vital biological

processes, or alternatively, that the energy cost of resisting antimicrobial attack depletes resources

normally devoted to important cellular functions.66 Although many studies have demonstrated

associated fitness cost for the acquisition of antimicrobial resistance, some resistance-conferring

mutations have been shown to have low or no fitness deficit.67 Antibiotic resistance can result in

secondary mutations to abrogate such costs, as has been reported for in vitro strains of FA-resistant

S. aureus.8,59 It would be beneficial to identify such fitness costs in Mtb, as they may have

implications on in vivo growth rate, virulence and transmissibility.

Factors that affect fitness either independently or in combination include: selective drug pressure,

environmental changes, genotype of the strain and stress induced by competing strains.65 Due to the

multiple correlates and effects, fitness cost can be assessed in several ways, and there has been no

resolution on what method is best. Relative fitness is defined as the ability of a genotype or

individual to survive and reproduce in the presence of a second genotype or individual.66 It is often

determined by competition experiments between isogenic antibiotic-susceptible (wild-type) and

antibiotic-resistant (mutant) bacteria in culture. Competition experiments are based on the

assumption that competing strains don’t affect each other and compete only by intrinsic growth rate

and efficiency in utilizing nutrients.65 In this study, we applied two routinely used methods of

assessment: growth rate in liquid culture (comparing the doubling times of the wild-type and mutant

strain) and relative fitness.

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4.5 Validation of targets using target-based whole cell screening

As demonstrated by the case of RIF and INH in section 4.1, identification of the target of a

compound or chemical scaffold with antimycobacterial activity is not essential for subsequent stages

of drug discovery and development. However, knowledge of the target can facilitate the

optimization of pharmacological and pharmacokinetic properties of such compounds or compound

classes.68 In the case of drug repositioning, since there is putative knowledge of the drug target,

often, a reverse engineering approach to in vitro target identification and validation is employed.

Typically, this is achieved through the use of chemical genomics, often a combination of techniques,

including the isolation and sequencing of resistant mutants, whole genome transcriptional profiling,

and assays of macromolecular synthesis such as RNA, protein, DNA, and fatty acids. Subsequently,

the target(s) identified is/are validated through targeted gene modulation.69 Target-based whole cell

screening (TB-WCS) is applicable in such instances, since analogues of the parent drug can serve as

chemical probes to validate the target of interest.

A well-established strategy in TB-WCS involves the use of tetracycline (Tet) – regulatable promoter

elements to generate mutants that conditionally express the target or targets of interest. This can be

achieved through two configurations- Tet-ON, where addition of Tet induces gene expression, and,

consequently, increased levels of the target; or Tet-OFF, in which addition of Tet represses gene

expression, resulting in depletion of the target.70,71 TB-WCS using conditional knockdown (cKD)

mutants of Mtb hypersensitised to target- and pathway-specific inhibitors have been used in the

search for novel antimycobacterial chemical scaffolds for TB drug discovery, and also in the

validation of targets of interest.72–74 A recent example is the validation of CoaBC as a cidal target in

the Coenzyme A pathway of Mtb by Evans and co-workers.75

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4.6 Aims and objectives

Against this background of information on FA with respect to S. aureus, and, given that Mtb lacks

plasmids, it is likely that resistance to FA is driven by mutations in the chromosomal genes. The aim

of this study was to identify the mechanism of action (MoA) and plausible mechanisms of resistance

(MoR) to FA in Msm and Mtb. The specific objectives were:

1. To generate spontaneous resistant mutants by selective drug pressure, with subsequent

whole-genome sequencing (WGS) to identify causal mutations within the genomes of the

resistant isolates.

2. To validate the target of FA in Mtb by means of TB-WCS, using FA and selected analogues.

3. To determine potential fitness costs associated with the mutations identified. This was to be

carried out by growth comparison of the doubling time of FA-resistant strains against wild-

type strains and by competition assay.

4. To determine in vitro cross-resistance of FA with selected anti-TB agents and compounds in

development by determining the susceptibility of FA-resistant mutant strains to a panel of

approved and experimental compounds.

4.7 Identification of spontaneous resistant mutants and determination of frequency of mutation.

The frequency of FAR mutants of Msm and Mtb was determined by plating approximately 108 to 109

CFU/ml of logarithmic phase on standard solid medium (7H10 agar plates) containing various

concentrations of FA above the MIC in liquid medium. In the case of Msm, colonies were scored

after five to seven days, while for Mtb, four weeks. The spontaneous mutants arose at a frequency

of 1.0 x 10-8 in both cases. The colonies isolated underwent re-growth and exposure to FA to

establish a shift in MIC. For those that exhibited a significant MIC shift, genomic DNA was extracted,

amplified by Polymerase Chain Reaction (PCR) and subjected to targeted sequencing and whole

genome sequencing (only the Mtb isolate).

4.7.1 Results of MIC90 screening of putative FAR Msm mutants

An 8-fold shift in the MIC of FA (from 80.8 μM to 646.4 μM) was observed for one of the four

colonies isolated, while the remaining three colonies showed a 2-fold change in MIC, and were thus

not considered truly resistant isolates.

135

4.7.1.1 Sequencing results for Msm isolate

Genomic DNA was extracted from all four colonies isolated, and amplification of fusA1 (EF-G1) and

fusA2 (EF-G2) carried out by PCR. The amplicons were purified and subjected to targeted

sequencing, with reference to wild-type Msm mc2155. As no SNPs were identified, repeated

unsuccessful attempts at resistant mutant generation were made under varying conditions. Details

of the experimental conditions are described in Chapter 6.

4.7.1.2 Results of MIC90 screening of putative FAR Mtb mutants

Colonies arose at each of the various concentrations of FA at a frequency of 1 x10-8. The colonies

were screened against FA to determine their MIC90, with shifts observed in the range 2- to 32-fold

(Table 4.2). One of the colonies that arose at 20X MIC exhibited a 32-fold increase in MIC. This

isolate, designated GKFA, was selected for genomic analysis by targeted sequencing of MtufusA1,

the gene encoding Mtb EF-G, as well as WGS with H37Rv MA as the reference strain. The remaining

pseudoresistant isolates did not yield reproducible resistance after re-growth and re-exposure to FA.

Table 4.2: Results of MIC90 screening of putative FAR Mtb mutants

Colony ID and concentration of FA used for selection

MIC90 (M) in 7H9/OADC*

Fold shift in MIC*

Mtb H37Rv MA (wild-type) 5 -

GKFA (20X MIC) 160 32

SRM 2 (10X MIC) 10 2

SRM 3 (15X MIC) 10 2

SRM 4 (20X MIC) 10 2

SRM 5 (25X MIC) 20 4

SRM 6.1 (5X MIC) 5 1

SRM 6.2 (5X MIC) 5 1

*Data are representative of 2 biological replicates

136

4.7.1.3 Sequencing results for Mtb GKFA isolate

Whole genome sequencing of the FA-resistant Mtb isolate GKFA revealed a single nonsynonymous

SNP, which resulted in a H462Y substitution in fusA, similar to the result found in the targeted

sequencing of MtufusA1. This suggests that the mechanism of action of FA is by inhibition of EF-G,

and that mutations in the gene encoding for EF-G play a role in the in vitro resistance of Mtb to FA.

4.8 Fitness cost of the FA resistance-conferring mutation in Mtb

In order to determine the possible fitness cost associated with the H462Y mutation in fusA1, growth

kinetics of the mutant strain and wild-type were assessed during separate culture in liquid 7H9

medium as well as in competition. The growth kinetics of the two strains was comparable over 7

days (Graph 4.1). In the competition assay adapted from Bhatter et al, a value of 0.9 was obtained,

where values less than 1 indicate fitness cost in the absence of the antibiotic, 1 indicates no cost,

and values greater than 1 indicate benefit in the absence of the antibiotic.65,66 Thus, relative to the

wild-type, the FAR showed little cost.

Day

OD

60

0

Graph 4.1: Growth curve of wild-type Mtb and FA-resistant mutant strain Mtb GKFA. Data

are representative of 3 biological replicates. The strains were measured in duplicate; the

errors represent values of the mean standard deviation.

137

4.9 Determination of the susceptibility of the GKFA isolate to selected FA analogues and

antituberculosis agents

Table 4.3: Results of selected MIC90 screening of FA against colonies isolated during resistant

mutant generation*

14-day 7H9/OADC MIC in µM against selected isolated colonies screened

Drug or

compound

H37Rv

MA GKFA SRM 2 SRM 3 SRM 4 SRM 5

SRM 6.1

SRM 6.2

FA 5 160 10 10 10 20 5 5

RIF 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

KAN 3.12 3.13 1.56 1.56 1.56 1.56 1.56 1.56

EMB 1.56 0.78 1.56 3.13 1.56 1.56 1.56 1.56

Oflox 0.31 0.31 0.31 0.31 0.31 0.31 0.31 0.31

STM 20 5 2.5 20 20 20 2.5 2.5

INH 0.01 0.01 0.01 0.01 <0.01 0.01 0.01 0.01

GKFA54 80 160 80 80 80 160 80 80

GKFA17 5 >160 >160 >160 >160 >160 160 160

GKFA37 2.5 80 5 10 5 10 2.5 2.5

GKFA24 80 160 80 80 40 80 80 80

GKFA51 80 >160 160 160 80 160 80 80

KSFA44 >160 >160 160 >160 >160 >160 160 160

KSFA16 40 160 80 80 80 80 80 80

GKFA59 10 160 20 20 20 20 10 10

GKFA16 20 160 80 80 80 80 40 40

In addition to FA, selected analogues of FA, as well as a panel of standard antituberculosis drugs and

compounds in development were tested against wild-type and the GKFA strain of Mtb to identify

hyper susceptibility or hyper-resistance of this strain to selected compounds. Table 4.3 summarises

the 14-day assay results, carried out in standard 7H9 growth medium. This was carried out in order

to identify potential in vitro cross-resistance of FA with these compounds, due to the combinatorial

nature of anti-TB chemotherapy.

With the exception of STM, there were no differences in MIC of the compounds between wild-type

Mtb and the resistant mutant strain. This indicates a lack of in vitro cross-resistance between FA and

*Data are representative of 3 biological replicates

138

the standard first line anti-TB agents. The GKFA isolate was hyper resistant to the FA analogues

tested, most notably GKFA16, GKFA37 and GKFA59 (Figure 4.6), the three most potent compounds

against wild-type Mtb. STM showed a slight hypersensitivity, with 4 to 8-fold decrease in MIC against

4 of the 7 colonies screened.

Figure 4.6: Structures of selected FA analogues tested against putative Mtb FAR mutants

4.10 Validation of EF-G as the target of FA in Mtb: Evaluation of the effect of transcriptional

silencing of fusA on susceptibility of Mtb to FA and selected analogues

TB-WCS was used to validate EF-G as the target of FA in Mtb, using a Tet-OFF cKD mutant strain of

Mtb constructed by Dr. Vinayak Singh at the University of Cape Town. In this mutant strain,

supplementation of standard 7H9 medium with Tet caused transcriptional silencing of fusA,

subsequently depleting EF-G. This mutant strain was then tested for hypersensitivity to FA and

selected analogues (GKFA16, GKFA17, GKFA59; Figure 4.6) using an adaptation of the checkerboard

synergy assay reported by Ramón-García et al.76 This assay applies the broth microdilution method

to test combinations of two compounds across a range of concentrations in a 96-well format. The

susceptibility of the fusA cKD strain to FA and selected analogues was evaluated in 7H9 medium in

the absence (-ATc) or presence (+ATc) of a range of concentrations of ATc. RIF, whose MoA differs

from that postulated for FA, was also tested in order to rule out general hypersensitivity of the cKD

strain. In addition, the susceptibility of the cKD strain to STM, which had a 4-8 fold decrease in MIC

in the FAR Mtb strain, was also assessed. Bacterial viability was measured using the Microplate

Alamar Blue Assay (MABA), which is described in detail in Chapter 6.

139

4.10.1 Results of the effect of transcriptional silencing of fusA on susceptibility of Mtb to FA and

selected analogues

Using the cherckerboard assay, 2.5 ng/ml was determined as the lowest concentration of

anhydrotetracycline (ATc) that was sufficient for transcriptional silencing of fusA without inhibiting

Mtb growth. (Appendix 3). At this concentration, the fusA cKD strain exhibited strong

hypersensitivity to FA and the analogues; their MIC90 decreased by 32- and 64- fold shifts

respectively. There was no shift in the MIC of RIF, both in the presence and absence of ATc.

Interestingly, for STM, a 4-fold decrease in MIC of STM was observed. This was a similar result to

that obtained in the FAR Mtb strain GKFA.

Even in the absence of ATc, relative to wild-type (WT) Mtb, the fusA cKD strain showed

hypersensitivity to the FA analogues. The greatest hypersensitivity was observed for GKFA59, which

exhibited 8-fold higher potency in the cKD strain. RIF, on the other hand, showed no difference in

MIC between the two strains. Table 4.4 summarises the MIC90 of FA and selected analogues in the

fusA cKD strain in the absence (-ATc) or presence (+ATc, 2.5 ng/ml).

Table 4.4: MIC90 of FA, selected analogues and anti-TB drugs in fusA conditional knockdown strain

Drug/compound MIC in WT Mtb

µM

MIC in Tet-OFF

(-ATc) µM

MIC in Tet-OFF

(+ATc) µM

Fold shift in

MIC

FA 5 5 0.16 32

GKFA16 10 5 <0.16 >64

GKFA17 5 5 <0.16 >32

GKFA59 10 1.25 <0.16 >64

RIF 0.01 0.01 0.01 -

STM 20 5 5 4

140

4.11 Discussion

4.11.1 Msm results

Seshadri and co-workers generated fusA2 deletion mutants of Msm by allelic exchange in an

attempt to understand its functional relevance in mycobacteria, with heterologous expression of EF-

G2 in E.coli.50 Several pertinent findings arose from that study: EF-G2 is not essential for Msm

survival (under laboratory conditions); EF-G2 binds guanine nucleotides but does not possess EF-G-

like function: GTPase, ribosome recycling (in conjunction with ribosome recycling factor) and

translocation activities in Msm; Msm EF-G2 is upregulated in late stationary phase, similar to the

predicted expression pattern of Mtb EF-G2 from a micro-array based study, which also reported

upregulation of EF-G2 during starvation conditions.50,77 However, these authors were unable to

establish its exact biological significance in vitro or in vivo. In this study, the use of a heterologous

expression system for EFG-2 may have excluded the role of mycobacterium-specific factors or a

specific ribosomal or post-translational conformation required for its activity. Moreover, its

sequence homology with EF-G and EF-G2 with respect to the conserved G domain indicates that it

may play a role in protein synthesis.

Recent studies have shown that EF-G2 proteins are highly divergent in primary sequence, and

thought to be a duplication of EF-G1 early in prokaryotic evolution. This greater divergence in EF-G2

was illustrated by Margus and colleagues (Figure 4.8), who compared the domain and motif

conservation of EF-G1 and EF-G2.41 The highest conservation disparity between the two proteins is

observed in domains I, II and III, inviting supposition of difference in functionality of EF-G2. There

have been examples where gene duplication and a subsequent acquisition of new function have

been shown to be the most plausible explanation for the appearance of additional families.78 It is

tempting to speculate that, in the case of Msm, fusA1 and fusA2 may be paralogous genes, with the

gene duplication releasing EF-G from the constraints inherent to its dual functionality, enabling each

to become more specialized in distinct singular functions. In Msm, the two roles of EF-G, that is,

elongation and ribosome recycling, may each have been ascribed exclusively to one form following

gene duplication. Alternatively, the additional EF-G families may carry out auxiliary functions

required in specific phases of life or under certain environmental conditions. Under selective drug

pressure, EF-G2 may perform EF-G or “EF-G-like function”, rescuing protein synthesis in M.

smegmatis.

141

G2 G3

G4

G5

MsmEFG1_ 1 MA----------QKDVLTDLNKVRNIGIMAHIDAGKTTTTERILYYTGVNYKIGETHDGASTTDWMEQEQ 60

MtbEFG1_ 1 MA----------QKDVLTDLSRVRNFGIMAHIDAGKTTTTERILYYTGINYKIGEVHDGAATMDWMEQEQ 60

MsmEFG2_ 1 MADRTHSPAGNGSVPTAERPAAIRNVALVGPSGGGKTTLVEALLVAGGVLTRPGSVADGSTVCDFDEAEI 70

MtbEFG2_ 1 MADRVNASQGAAAAPTANGPGGVRNVVLVGPSGGGKTTLIEALLVAAKVLSRPGSVTEGTTVCDFDEAEI 70

Consensus_aa: MA............shhp..s.lRNhslht..stGKTThhE.lLhhs.l..+.Gphh-GsshhD@.E.Eb

Consensus_ss: hhhhhh hh eeeeee hhhhhhhhhhhhhhhhhh eee hhhhh

MsmEFG1_ 61 ERGITITSAAVTCFWNNNQINIIDTPGHVDFTVEVERSLRVLDGAVAVFDGKEGVEPQSEQVWRQADKYD 130

MtbEFG1_ 61 ERGITITSAATTTFWKDNQLNIIDTPGHVDFTVEVERNLRVLDGAVAVFDGKEGVEPQSEQVWRQADKYD 130

MsmEFG2_ 71 AQQRSVSLALASLSHNGIKVNLIDTPGYADFVGELRAGLRAADCALFVIAANETIDEPTKTLWQECNEVQ 140

MtbEFG2_ 71 RQQRSVGLAVASLAYDGIKVNLVDTPGYADFVGELRAGLRAADCALFVIAANEGVDEPTKSLWQECSQVG 140

Consensus_aa: .p..ols.Ahhoh.@ps.plNllDTPG@hDFhsElc.sLRhhDtAlhVhstpEsl-..ocplWpptsph.

Consensus_ss: h eeeeeeeeeee eeeeeee hhhhhhhhhhhh eeeeeee hhhhhhhhhhhh

MsmEFG1_ 131 VPRICFVNKMDKLGADFYFTVRTIEERLGAKPLVIQLPIGAENDFIGIIDLVEMKAKVWRGETALGEKYE 200

MtbEFG1_ 131 VPRICFVNKMDKIGADFYFSVRTMGERLGANAVPIQLPVGAEADFEGVVDLVEMNAKVWRGETKLGETYD 200

MsmEFG2_ 141 MPRAVVITKLDHPRANYDAALTAAQEAFGDKVAPLYFPVGDGESCKGVVGLLTRTYYDYSG-----GTHT 205

MtbEFG2_ 141 MPRAVVITKLDHARANYREALTAAQDAFGDKVLPLYLPSGD-----GLIGLLSQALYEYAD-----GKRT 200

Consensus_aa: hPRhhhlsKhD+..As@..slphh.-.hGspshslbhP.Gs.....GllsLlp..hb.@.s......p.p

Consensus_ss: eeeeee hhhhhhhhhhhh eeeeeeeee eeee hhhhh ee

MsmEFG1_ 201 VEDIPADLADKAEEYRTKLLETVAESD--EALLEKYFGGEELSVDEIKGAIRKLTVNSELYPVLCGSAFK 268

MtbEFG1_ 201 TVEIPADLAEQAEEYRTKLLEVVAESD--EHLLEKYLGGEELTVDEIKGAIRKLTIASEIYPVLCGSAFK 268

MsmEFG2_ 206 ARPPDGSHDAAIAELRGSLIEGVIEESEDETLMERYLGGEEIDEAVLIADLEKAVARASFFPVIPVCSQS 275

MtbEFG2_ 201 TRTPAESDTERIEEARGALIEGIIEESEDESLMERYLGGETIDESVLIQDLEKAVARGSFFPVIPVCSST 270

Consensus_aa: h...s.s.s..h.EhRs.LlEslhEps..E.LhE+YhGGEpls.s.lb.slcKhhh.tph@PVlsstt.p

Consensus_ss: hhhhhhhhhhhhhhhhhhh hhhhhhhh hhhhhhhhhhhhh eeeeeee

MsmEFG1_ 269 NKGVQPMLDAVIDYLPSPLDVESVQGHVPGKEDEVISRKPSVDEPFSALAFKIAVHPFFGKLTYVRVYSG 338

MtbEFG1_ 269 NKGVQPMLDAVVDYLPSPLDVPPAIGHAPAKEDEEVVRKATTDEPFAALAFKIATHPFFGKLTYIRVYSG 338

MsmEFG2_ 276 GVGTLELLDIISRGFPAPPEHQLPEVFTPQG-APRKPLSCDPDGPLLAEVVKTTSDPYVGRVSLVRVFSG 344

MtbEFG2_ 271 GVGTLELLEVATRGFPSPMEHPLPEVFTPQG-VPHAELACDNDAPLLAEVVKTTSDPYVGRVSLVRVFSG 339

Consensus_aa: s.Ghb.hL-hh.c.hPtP.-h..sbs@hP...s......sssD.Ph.AbhhKhhscP@hG+lohlRV@SG

Consensus_ss: hhhhhhhhhhhh ee ee eeeeeeeeee eeeeeeeeee

MsmEFG1_ 339 VVESGSQVVNSTKG---------------------------KKERLGKLFQMHANKENPVERASAGHIYA 381

MtbEFG1_ 339 TVESGSQVINATKG---------------------------KKERLGKLFQMHSNKENPVDRASAGHIYA 381

MsmEFG2_ 345 TIRPDATVHVSGHFSAFTDTSGSHGAAAAADGSTHSHADHDEDERIGTLSFPLGKQQRPAPTVVAGDICA 414

MtbEFG2_ 340 TIRPDTTVHVSGHFSSF-----------FGGGTSNTHPDHDEDERIGVLSFPLGKQQRPAAAVVAGDICA 398

Consensus_aa: hlcssspVhsts+............................ccERlG.L.b.htppppPh..hsAGcIhA

Consensus_ss: ee eeeee eeeeeeeeeee eeee ee eee

MsmEFG1_ 382 VIGLKDTTTGDTLCDPNEQIVLESMTFPDPVIEVAIEPKTKSDQEKLGTAIQKLAEEDPTFKVHLDQETG 451

MtbEFG1_ 382 VIGLKDTTTGDTLSDPNQQIVLESMTFPDPVIEVAIEPKTKSDQEKLSLSIQKLAEEDPTFKVHLDSETG 451

MsmEFG2_ 415 IGRLSRAETGDTLSDKTDPLVLRPWTMPDPLLPIAVAPRAKTDEDKLSVGLQRLAAEDPTLRIEQNPETH 484

MtbEFG2_ 399 IGKLSRAETGDTLSDKAEPLVLKPWTMPEPLLPIAIAAHAKTDEDKLSVGLGRLAAEDPTLRIEQNQETH 468

Consensus_aa: l..LpchpTGDTLtD.sp.lVLcshThP-Pll.lAl.s+hKoDp-KLthtl.+LA.EDPTh+lcbs.ET.

Consensus_ss: ee eeeeeee eeeeeeee hhhhhhhhhhhhhhhhh eeeeeee

MsmEFG1_ 452 QTVIGGMGELHLDILVDRMRREFKVEANVGKPQVAYRETIKRKVEKVEYTHKKQTGGSGQFAKVLIDLEP 521

MtbEFG1_ 452 QTVIGGMGELHLDILVDRMRREFKVEANVGKPQVAYKETIKRLVQNVEYTHKKQTGGSGQFAKVIINLEP 521

MsmEFG2_ 485 QIVLWCMGEAHAAVVLDALSRRYGVSVDTVELRVPLRETFAGKA-TGHGRHVKQSGGHGQYAVCDIEVEP 553

MtbEFG2_ 469 QVVLWCMGEAHAGVVLDTLANRYGVSVDTIELRVPLRETFAGNA-KGHGRHIKQSGGHGQYGVCDIEVEP 537

Consensus_aa: QhVl.tMGEhHhslllD.h.pc@.Vphsh.c.pVsh+ETh...h.psc.pH.KQoGGpGQ@t.h.IplEP

Consensus_ss: eeeeeee hhhhhhhhhhhhhh eeee eeeeeeee eeeee eeeeeeee

MsmEFG1_ 522 FVGEDGATYEFENKVTGGRIPREYIPSVDAGAQDAMQYGVL--AGYPLVNLKVTLLDGAYHEVDSSEMAF 589

MtbEFG1_ 522 FTGEEGATYEFESKVTGGRIPREYIPSVDAGAQDAMQYGVL--AGYPLVNLKVTLLDGAYHEVDSSEMAF 589

MsmEFG2_ 554 LPE--GSGFEFVDKVVGGAVPRQFIPSVEKGVRAQMEKGVTGESGYPVVDIRVTLFDGKAHSVDSSDFAF 621

MtbEFG2_ 538 LPE--GSGFEFLDKVVGGAVPRQFIPNVEKGVRAQMDKGV--HAGYPVVDIRVTLLDGKAHSVDSSDFAF 603

Consensus_aa: hs...Gts@EF.sKVhGG.lPRp@IPsV-.Ghps.MpbGV...tGYPlVsl+VTLhDG.hHpVDSS-hAF

Consensus_ss: eeeeeeeee hhhhhhhhhhhhhhh eee eeeeeeeeeeeeee hhh

MsmEFG1_ 590 KVAGSQALKKAAQAAQPVILEPIMAVEVTTPEDYMGEVIGDLNSRRGQIQAMEERS-GARVVKAQVPLSE 658

MtbEFG1_ 590 KIAGSQVLKKAAALAQPVILEPIMAVEVTTPEDYMGDVIGDLNSRRGQIQAMEERA-GARVVRAHVPLSE 658

MsmEFG2_ 622 QMAGALALRDAAAATKINLLEPVDDVTIVVPDDLVGTIMGDLSGRRGRVLGTDKHDADRTVIKAEIPEVE 691

MtbEFG2_ 604 QMAGALALREAAAATKVILLEPIDEISVLVPDDFVGAVLGDLSSRRGRVLGTETAGHDRTVIKAEVPQVE 673

Consensus_aa: phAGtbhL+cAA.hhp..lLEPl..lplhhP-DhhG.lhGDLstRRGplbth-p.s.s.pVl+AplPbsE

Consensus_ss: hhhhhhhhhhhhhh eeeeeeeeeeeee hhhhhhhhhhhhh eeeeeeee eeeeeeee hhh

MsmEFG1_ 659 MFGYVGDLRSKTQGRANYSMVFDSYAEVPANVSKEIIAKATGQ 701

MtbEFG1_ 659 MFGYVGDLRSKTQGRANYSMVFDSYSEVPANVSKEIIAKATGE 701

MsmEFG2_ 692 LVRYAIDLRSMSHGAGQFRRSFARYEPMPESAAARLRTSA--- 731

MtbEFG2_ 674 LTRYAIDLRSLAHGAASFTRSFARYEPMPESAAARVKAGAG-- 714

Consensus_aa: hh.Yh.DLRSbspG.tp@p.sFspY..hP.sht.cl.h.A...

Consensus_ss: h hhhhhhhh eeeeeeee eee hhhhhhhhhh

Figure 4.7: Sequence alignment of EF-G and EF-G2 from Mtb and Msm (generated using PROMALS3D)

Accession numbers for the sequences: MsmEFG1: AFP37835 NCBI; MsmEFG2:YP_890748.1 NCBI;

MtbEFG1: CCP43427 NCBI; MtbEFG2: CCP4285 NCBI

I II

III II

III IV

I

IV V

V IV

B

A

142

Figure 4.8: Domain conservation comparison between EF-G and EF-G2. Adapted from Margus et al.41

4.11.2 Mtb result

Mutations conferring in vitro and in vivo (from clinical isolates) resistance to FA have been mapped

in EF-G from several organisms: T. thermophilus, S. typhimurium and S. aureus and Pseudomonas

aeruginosa.33,43,55,56,79–83 Chen and co-workers analysed and classified all fusA-based mutations as

affecting: (i) FA binding: modification of the drug binding site with resultant decreased affinity for FA

(ii) EF-G conformation (iii) EF-G stability: altered interaction between EF-G and the ribosome.

Reiterated by Johansson et al in an earlier study, these factors contribute directly or indirectly to FA

resistance.83,84 FA binds to ribosome-bound EF-G in a pocket between domains II and III and the

switch II region of the G domain40, which are the most highly conserved regions across several

organisms (Figure 4.9). Several amino acid residues in T. thermophilus EF-G identified as interacting

with FA in crystallographic studies by Gao and co-workers are largely conserved across EF-Gs

examined, and substitutions in these residues have been associated with FA resistance.40 Further

corroboration of this was provided by Besier and co-workers who established a causal relationship

between several amino acid substitutions and FA resistance by introducing previously reported

amino acid substitutions into FA-susceptible S. aureus, which subsequently exhibited corresponding

levels of FA resistance.55 One of these mutations, H457Y, reported previously, both in vitro and in

vivo, putatively alters the surface in the FA-binding pocket, leading to alterations in drug binding.55 A

study by Ramakrishnan et al also identified His 458 in T. thermophilus as a corresponding conserved

residue to the His 462 in Mtb EF-G.85These findings corroborate the result in this study, that the

H462Y substitution leads to FA resistance. Thus, the FA resistance observed in the GKFA Mtb isolate

may be due to an altered drug binding pocket within the target.

143

Salient insights from the studies into the molecular basis of FA resistance include: a single amino

acid substitution mutation is necessary and sufficient to confer resistance to FA, and that different

amino acid exchanges at the same site may lead to different levels of FA resistance. It is imperative

that we generate more FA-resistant mutant Mtb strains, in order to study the scope of possible sites

of mutation within the gene and to determine potential fitness cost-mediating mutations.

The low-level increase in sensitivity of the FA-resistant strain to STM may be attributed to synergistic

inhibition of protein synthesis. Like FA, strep is a translation inhibitor, acting at the initiation step,

inhibiting Elongation Factor Tu (EF-Tu) which delivers the amino-acylated tRNA to the A-site of the

ribosome (Figure 4.2). The mutation harboured by the GKFA isolate may, in addition to altering drug

binding, also alter the function of EF-G, resulting in reduced elongation cycles compared to wild-type

(inferred from the absence of other potential fitness cost-mediating mutations within fusA).

Subsequent inhibition of initiation by STM may lead to further repression of protein synthesis,

manifested as hypersensitivity to the drug. This is supported by evidence from our lab (unpublished

data) where in vitro synergistic screening of FA with known drugs has shown synergistic effect with

other translation inhibitors (unpublished). This is not surprising, as Johanson and Hughes reported

that, compared to wild-type S. typhimurium, FA-resistant strains exhibited low-level sensitivity and

resistance to SPEC and KAN respectively.86 In addition, Macvanin and Hughes reported increased

susceptibility of a FAR strain of Salmonella enterica serovar Typhimurium to RIF and chloramphenicol

as well as selected antibiotics under the following classes: -lactams, aminoglycosides and

fluoroquinolones.87 Like FA, they are both translation inhibitors, with SPEC acting as a translocation

inhibitor, while KAN causes mistranslation in addition to inhibiting translocation.

The growth rates of the two strains in separate liquid cultures were comparable, and in the

competition assay, a relative fitness of 0.9 indicated that H462Y is a no-cost FAR mutation. This is

consistent with the finding of Melnyk et al, who reported a mean relative fitness of 0.8 for several

FAR strains of S. typhimurium, and concluded that resistance to fusidanes are no-cost mutations.66

Tet repression of fusA in a mutant strain led to strong hypersensitivity to FA and selected analogues,

thus confirming that these compounds inhibit Mtb growth by targeting EF-G. This effect was specific

to this class of compounds, as RIF, which inhibits RNA transcription, did not elicit a similar phenotype

in the knockdown strain. The 4-fold increase in potency of STM in the FAR strain was mirrored in the

knockdown, providing further evidence of its synergistic activity in inhibiting Mtb translation

machinery.

4.12 Summary

144

Repeated attempts at generating FAR strains of M. smegmatis proved unsuccessful. The inability to

raise spontaneous FA-resistant mutants in this study may be attributed to the unknown biological

role of EF-G2. EF-G was validated as the target of FA in Mtb, via resistant mutant selection and

whole genome sequencing. The resistant mutant strains arose at a frequency of 10-8, corresponding

to the frequency observed in naturally-occurring FA-resistant S. aureus. A substitution mutation in

the genome encoding gene fusA1 resulted in high-level in vitro resistance to FA, but did not affect

growth kinetics. The resulting mutated residue, located in a region in domain III conserved across EF-

G and EF-G2 in both Msm and Mtb, is conserved across several organisms of clinical relevance. To

date, there are no published reports of FA-resistant mutants of Mtb, although a corresponding

substitution mutation has been reported in both in vitro and clinical strains of S. aureus, H457Y, that

exhibited high level FA resistance. This mutation putatively alters the drug-binding pocket of EF-G.

Furthermore, the mutation is selective for FA and selected FA analogues, as no cross-resistance was

observed when tested against a panel of first and second-line anti-TB agents and compounds in

development.

A slight increase in susceptibility (4 to 8-fold decrease in MIC) of the FA-resistant strain to STM was

observed, which may be due to synergistic inhibition of protein synthesis because, like FA, STM is a

translational inhibitor.

A knockdown strain underexpressing EF-G was used to conduct target-based whole cell screening of

FA and three analogues, with consequent hypersensitivity observed for these compounds. This

effect was not observed with RIF, a transcriptional inhibitor, providing further confirmation of EF-G

as the target of FA in Mtb.

It would be useful to isolate more resistant mutants in order to identify other causal mutations of

FAR within the Mtb genome, as the evidence from clinical isolates of other organisms suggests a wide

scope of the mutations that lead to FAR. This information in turn, can be used to map mutational

“hotspots” as has been done for the standard anti-TB drugs such as RIF and INH.88–92 From the

existing knowledge of the effects of mutations on FA binding, coupled with structural information,

this could potentially guide structure-based design of FA analogues that can overcome the potential

inhibitory effects of the known resistance mutations.

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Chapter 5

Conclusions and Recommendations for Future Work

5.1 Conclusions

The overall objective of this study was to identify novel analogues of fusidic acid (FA) with improved

antimycobacterial activity through semi-synthesis. This was motivated by the hypothesis that FA’s

modest antimycobacterial activity is partly due to the presence of functional groups such as the C-21

carboxylic acid which may limit its permeation across Mtb’s hydrophobic cell wall.

Through ester and amide couplings, the C-21 carboxylic acid was functionalized to yield ester and

amide aminoquinoline hybrids. This was motivated in part by the recent report of potent

antitubercular amide derivatives of spectinomycin, an old antibiotic with weak antimycobacterial

activity, and the fact that bedaquiline, the first anti-TB drug licensed in over 40 years, bears the

quinoline scaffold.

The FA hybrids were 2- to 8-fold less potent than FA against Mtb, and were 2 to 10-fold less soluble

in aqueous media compared to FA. The poor solubility of these compounds in aqueous media

prevaricates their true antimycobacterial activity, because the antimycobacterial assays are

conducted in aqueous media. Furthermore, from a clinical perspective, this highlights the

importance of ensuring an optimum balance between aqueous solubility and permeability of

compounds. Aqueous solubility has implications on drug efficacy and safety. This is because the flux

of a drug across the intestinal membrane is proportional to the concentration gradient between the

intestinal lumen and the blood. Thus, even for drugs with high permeability, poor aqueous solubility

leads to decreased absorption. Similarly, in the case of injectable drugs, low aqueous solubility

results in decreased absorption into the bloodstream. In addition, high concentrations of poorly

soluble injectable drugs may crystallize in tissues and organs, resulting in acute toxicity.

The aminoquinoline pharmacophore lowered the antimycobacterial activity of the hybrids, as 1:1

molar mixtures of FA with selected aminoquinoline derivatives exhibited the MIC of FA, while the

aminoquinoline derivatives were all separately inactive. In comparing the length of the alkyl linker,

in both the amide and ester series, there was no correlation between chain length and

antimycobacterial activity. However, AW45, the amide with the longest alkyl linker, precipitated in

the aqueous media for the turbidimetric solubility assay at 10 M, distorting its antimycobacterial

assay result. The synthesis and antimycobacterial evaluation of these C-21 analogues contributes to

the expansion of FA Structure-Activity-Relationship (SAR) profile. The low potency of these C-21

151

amides and esters resembles its antibacterial SAR in other organisms, where the carboxylic acid

group has been shown to be essential for activity.

The FA hybrids were evaluated for cytotoxicity against two cell lines; Chinese Hamster Ovarian (CHO)

and THP-1, and found to be more cytotoxic than FA. Consistent with its in vivo safety profile, FA was

not cytotoxic in both cell lines, while the aminoquinoline derivatives on their own were cytotoxic.

These data suggest that the aminoquinoline moiety contributed to the overall cytotoxicity observed

in the hybrids. Like in the case of the antimycobacterial activity, there was no correlation between

the length of the alkyl linker and cytotoxicity. It is, however, important to note that these in vitro

cytotoxicity assays are conducted in laboratory-adapted cell lines whose predictive value is limited,

and, as observed for a few compounds in this study, the cytotoxicity profile of compounds may vary

across different cell lines. Nevertheless, due to their poor solubility, cytotoxicity and low

antimycobacterial activity, the FA-aminoquinoline hybrids were not progressed further.

As part of the broader goal of repositioning FA for antituberculosis drug discovery, other FA

analogues have been synthesised in our laboratory. The second objective of this study was to

evaluate the intracellular efficacy of FA and three C-3 analogues, GKFA16, GKFA17 and GKFA37 (3-

ketoFA) were evaluated for their intracellular efficacy in THP-1 cells. FA and all the analogues

evaluated were active against intracellular Mtb. Notably, in the case of GKFA37- a metabolite of FA

in humans, this was consistent with previous reports of its antibacterial activity. The intracellular

efficacy of the analogues studied in this project are an encouraging result for antitubercular drug

repositioning; it has been shown that activity in broth cultures does not always translate to

intracellular activity, since Mtb is an intracellular pathogen. This positive result notwithstanding,

several limitations are to be considered; the predictive value of data obtained from in vitro cell lines

is limited tempered by the inherent variations across different cell lines. Moreover, in the context of

in vivo disease, macrophages are part of the granuloma, which results from the interplay of several

other mechanisms that play an important role in the outcome of infection.

A third objective of this project was to evaluate the role of mycobacterial biotransformation in the

antimycobacterial activity of selected FA analogues. FA and five analogues were evaluated for their

stability in Mtb cultures. Three C-3 esters: GKFA16, GKFA17, GKFA61, as well as two C-21 amides,

AW23 and AW25. In live Mtb cultures, GKFA16 and GKFA17 were hydrolysed extensively to FA. In

heat-killed cultures, the concentrations of these compounds remained relatively unchanged,

suggesting that the metabolism was specifically associated with Mtb. Similar incubation of FA

revealed negligible hydrolysis, consistent with data from our laboratory, which showed that FA is

relatively stable in Mtb lysate incubations. Recently, preliminary in vivo studies in our laboratory

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revealed that these two compounds were hydrolysed to achieve higher concentrations of FA in the

lungs of healthy C57BL/6 mice that received GKFA16 and GKFA17 when compared to those that

received FA intravenously. Taken together, these data suggest that these two esters can be

exploited as prodrugs to enhance fusidic acid PK in the lungs, the primary site of TB infection.

Furthermore, from an in vivo PK perspective, the fact that the esters themselves have

antimycobacterial activity is plausibly beneficial; an initial (loading dose) of the ester can be

administered to achieve modest efficacy, while subsequent hydrolysis ensures a longer duration of

action due to increasing concentration of FA over time.

GKFA61, a C-3 silicate ester of FA, was relatively stable in both heat-killed and live cultures, with no

FA detected in these incubations. This result is consistent with its in vivo PK profile in mice, where it

has been shown to have a longer half-life. Its metabolic stability may be due to the presence of

isopropyl groups around the C-3 position, which cause steric hindrance, thus blocking this site of

metabolism. Similarly, the concentrations of the C-21 amides AW23 and AW25 remained relatively

unchanged in both heat-killed and live cultures.

A fourth objective, the identification and validation of the target of FA in Mtb, was achieved through

the generation of spontaneous resistant mutants and target-based whole cell screening (TB-WCS).

Spontaneous resistant mutant generation in Mtb revealed a point mutation (H462Y) in fusA1, which

encodes Elongation Factor G (EF-G), its known bacterial target. EF-G is a ribosomal GTPase, and this

mutation occurred in a highly conserved domain of EF-G associated with catalytic activity of the

enzyme. It has been demonstrated that similar mutations in other bacteria result in conformational

changes within EF-G that decrease drug affinity. In addition, this mutation was not associated with a

fitness cost, and no in vitro cross-resistance was observed with a panel of anti-TB drugs. This is an

encouraging result, since none of the existing ribosome-targeting anti-TB agents inhibits EF-G. A

knockdown strain underexpressing EF-G was used to conduct TB-WCS of FA and three analogues,

with hypersensitivity observed for these compounds. This effect was not observed with rifampicin, a

transcriptional inhibitor, thus providing further confirmation of EF-G as the target of FA in Mtb.

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5.2 Recommendations for future work

Since the carboxylic acid is essential for antimycobacterial activity, bioisosteres should be considered

in further expansion of fusidic acid SAR. Bioisosteres are functional groups or substituents that have

similar chemical or physicochemical properties and whose replacement retains biological activity.

Bioisosteric replacement is exploited in medicinal chemistry to address aspects associated with the

design and development of drug candidates, such as enhancing physicochemical properties,

improving potency or attenuating toxicity. Studies involving bioisosteric replacement of the

carboxylic acid are underway in our laboratories.

In addition, since FA is a high molecular weight compound, subsequent analogues synthesised may

benefit from supramolecular derivatization with biocompatible partner molecules such as inclusion

complexes, co-crystals and solvates or hydrates. These can be useful in improving its

physicochemical properties, specifically solubility.

Since the C-21 amides synthesised in this project were stable in Mtb incubations, the C-21 esters

should also be evaluated for stability in Mtb cultures subject to successful LC-MS method

development.

The evaluation of intracellular efficacy carried out in this study relied on CFU enumeration, which is

low-throughput, due to its labour-intensive and time-consuming nature. Studies are underway in our

lab to delineate the factors that affect drug permeation in an in vitro macrophage infection model. In

addition, a macrophage model of infection using fluorescent reporter strains of Mtb is being

developed, which would enable rapid, convenient, medium-throughput evaluation of intracellular

efficacy, in a 96-well format that allows more accurate dose-response curves for compounds over a

wider range of drug concentrations than CFU enumeration. However, as is applicable to all

laboratory-adapted cell lines, the relevance of macrophage assays to in vivo host infection

conditions is arguable. These cell lines exclude the interplay of host-associated factors with these

cells, which ultimately play a role in the outcome of infection. Moreover, in the case of Mtb

infection, the granuloma, and in a wider context, caseating granulomas, account for heterogeneity in

responses to drug treatment. To mitigating this limitation, bone-marrow derived macrophages or if

possible, primary human macrophages should be considered in testing selected derivatives further.

For target identification and mechanism of resistance determination, more spontaneous resistant

mutants should be isolated to study the spectrum of causal mutations of FAR. In conjunction with

structural information on Mtb EF-G, coupled with in silico tools, this can be useful in the design of

future derivatives that overcome resistance mechanisms associated with the drug target. In

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addition, efflux has been reported as a bacterial mechanism of FAR. Mtb has a variety of efflux

pumps with a wide range of substrates, including antimicrobials. Efflux pumps are associated with

Mtb resistance to fluoroquinolones and RIF. The role of efflux in Mtb FAR should be interrogated,

and if established, efflux inhibitors can be pursued as a strategy to overcome this.

The knockdown strain can be exploited as a tool to identify more potent antimycobacterial

compounds targeting EF-G with superior physicochemical and pharmacokinetic properties to FA; this

might be especially useful in screening recently discovered natural products.

Finally, FA is an old antibiotic that was approved for clinical use before the widespread availability

and use of animal models for in vivo pre-clinical work. Anecdotal reports from a group of researchers

in the 1960s reported higher clearance of FA in rodent species compared to the data generated from

PK studies in humans. This species-specific difference in metabolism may account for its poor in vivo

efficacy in mice (unpublished data), and thus, a more appropriate model of TB infection such as the

marmoset monkey should be considered for in vivo pre-clinical PK studies.

155

Chapter 6

Experimental

6.1 Chemistry

6.1.1 Reagents and solvents

All commercially available chemicals were purchased from Sigma-Aldrich South Africa or Merck

South Africa except for fusidic acid, which was purchased from AvaChem Scientific, Texas, USA.

N, N-Dimethylformamide (DMF) and methanol (MeOH) were purchased as anhydrous solvents,

while all other commonly used solvents were purchased from Kimix Chemicals, South Africa or

Protea Chemicals as Analytical Reagent (AR) grade then purified and dried using an SP-1 solvent

drying system from LC Technology. Ammonium acetate (NH4OAc) and MeOH, the High Performance

Liquid Chromatography (HPLC) grade solvents, were bought from Sigma Aldrich for chromatography

and Mass Spectrometry.

6.1.2 Chromatography

Reactions were monitored by Thin Layer Chromatography (TLC) using Merck F254 aluminum-backed

pre-coated silica gel plates purchased from Merck. Spots were visualized using ultraviolet (UV) light

(254/366 nm) or the following spraying reagents: cerric ammonium nitrate, anisaldehyde or

bromocresol green. Silica gel chromatography was performed using Merck kieselgel 60: 70-230 mesh

for gravity columns and 230-400 mesh for flash chromatography on Biotage® Isolera Prime™.

6.1.3 Physical and spectroscopic characterization

Melting points were determined on a Reichert-Jung Thermovar hot-stage microscope, adapted from

methods by Kofler and Kofler and are uncorrected.1,2

Low-resolution Mass Spectrometry (LRMS) was performed on a JEOL GC Mate II or an AB Sciex 4000

QTrap.

HPLC: Compound peak purity of the final compounds was determined by HPLC using a thermo

separation system comprising of a Spectra series P200 pump, AS100 automated sampler and UV 100

variable wavelength detector. For UV detection, a photo diode array (PDA) detector was set to

monitor the wavelength at 254 nm. The stationary phase used for Waters preparatory HPLC was a

Waters® X-bridge C18 5.0 μm column (4.6 x 150 mm) (Phenomenex, Torrance, CA) fitted with a

Supelguard TM Ascentis® guard cartridge C18 (2cm × 40 mm, 3 um) (Supelco Analytical, Bellefonte,

PA).

156

The mobile phase consisted of 0.4% acetic acid in 10 mM NH4OAc in HPLC grade (Type 1) water; with

flow rate = 1.20 ml/min; and acetonitrile (B) delivered at a flow rate 1.20 ml/min. All compounds

were confirmed to have ≥ 95% purity.

Table 6.1: Gradients used for investigation of the purity of compounds with preparatory HPLC

Time % A % A

Initial 75 25

9.00 0 100

14.00 0 100

14.10 75 25

20.00 75 25

Peak purity was reported as the integrated area of the compound peak as a percentage of total peak

areas observed.

LC-MS: Liquid chromatograph with mass spectrometer (LC-MS) analysis was performed using an

Agilent® 1260 Infinity Binary Pump, Agilent® 1260 Infinity Diode Array Detector (DAD), Agilent® 1290

Infinity Column Compartment, Agilent® 1260 Infinity Standard Autosampler, and a Agilent® 6120

Quadrupole (Single) mass spectrometer, equipped with an atmospheric pressure chemical ionization

(APCI) and electrospray ionization (ESI) multimode ionisation source. Purities were determined by

Agilent® LC-MS using a Kinetex Core C18 2.6 μm column (50 x 3 mm); organic phase (Mobile Phase

B): 0.4% acetic acid, 10 mM NH4OAc in a 9:1 ratio of HPLC grade methanol and Type 1 water,

aqueous phase (Mobile Phase A): 0.4% acetic acid in 10 mM NH4OAc in HPLC grade (Type 1) water;

with flow rate = 0.9 ml/min; detector: diode array (DAD) and all compounds were confirmed to have

≥ 95% purity.

Table 6.2: Gradient used for investigation of the purity and mass of compounds using LCMS

Time % A % B

Initial 75 25

1.00 75 25

3.00 0 100

4.50 0 100

5.20 75 25

6.00 75 25

157

NMR: All 1H NMR spectra were recorded in deuterochloroform (CDCl3) or deuteromethanol (CD3OD)

on a Bruker 300 Mega Hertz (MHz) or Varian Unity Spectrometer (400 MHz) with tetramethylsilane

(TMS) as an internal standard. All chemical shifts (δ) are given in parts per million (ppm), while

coupling constants (J) are expressed in Hertz (Hz) and are rounded off to one decimal place, resulting

in exactly the same coupling constants for coupling partners. 13C NMR spectra were recorded on the

same instruments at 75 MHz and in the same deuterated solvents, with TMS as an internal standard.

The 13C chemical shift values are listed without specific assignment to carbon atoms.

158

6.1.4 General synthetic procedure for 4-aminoquinoline derivatives3,4

A mixture of 4, 7-dichloroquinoline (1 g, 10.1 mmol), Et3N (2.1 ml, 15 mmol) and the appropriate

diamine or alcohol (2 mmol) was stirred under nitrogen at 80 oC for 1 hour without stirring. The

temperature was then raised to 140 oC and maintained for 3 hours with stirring, after which the

reaction mixture was left to cool to ambient temperature. Ice was then added, resulting in the

formation of a precipitate, which was stored in the refrigerator for 1 hour. The precipitate was

filtered and the residue washed with cold water (2 x 5 ml) followed by cold diethyl ether (2 x 5 ml) to

afford the substituted aminoquinoline diamines and amino alcohols in excellent yields (90-98%).

N-(7-chloroquinolin-4-yl)-propane-1, 3-diamine (AW27)

White powder (1.07 g, 90%) ; Rf 0.21 (1:9 MeOH:CH2Cl2); melting

point (m.p.) 125-127 oC ; literature m.p. (lit. m.p. 124-127 oC)4; δH

(400 MHz, CD3OD): 8.35 (1H, d, J 6.0 Hz, H2), 8.05 (1H, d, J 9.0 Hz,

H5), 7.75 (1H, d, J 3.0 Hz, H8), 7.38 (1H, dd, J 3.0 Hz, 9.0 Hz, H6),

6.54 (1H, d, J 6.0 Hz, H3), 3.59 (2H, t, J 6.3 Hz, H9), 1.37 (2H, m,

H10), 3.02 (2H, t, J 6.3 Hz, H9); δC (75 MHz, CD3OD): 154.0, 151.2,

146.9, 135.1, 130.3, 127.2, 122.5, 118.5, 112.4, 44.3, 41.6, 32.8. LRMS (EI) m/z 236.09 [M+H+]; HPLC

purity: > 95%.

N-(7-chloroquinolin-4-yl)-butane-1, 4-diamine (AW18)

Brown powder (1.21 g, 96%); Rf 0.21 (1:9 MeOH:CH2Cl2); m.p.

123-125 oC (lit. m.p. 124-127 oC)4; δH (400 MHz, CD3OD): 8.5

(1H, d, J 5.2 Hz, H2), 7.99 (1H, d, J 9.0 Hz, H5), 7.64 (1H, d, J 2.0

Hz, H8), 7.31(1H, dd, J 2.0 Hz, 9.0 Hz, H6), 6.4 (1H, d, J 5.2 Hz,

H3) 3.2 (2H, t, J 5.9 Hz, H9), 2.99 (2H, t, J 5.9 Hz, H12), 1.77 (2H,

m, H11), 1.61(2H, m, H10); δC (75 MHz, CD3OD): 153.0, 150.0,

147.0, 135.0, 129.1, 127.0, 124.0, 118.1, 113.6, 45.4, 41.4, 30.1, 26.0. LRMS (EI) m/z 250.10 [M+H+];

HPLC purity: > 95%.

159

N-(7-chloroquinoline-4-yl)-pentane-1, 5-diamine (AW43)

Light brown powder (1.30 g, 98%); Rf 0.22 (1:9

MeOH:CH2Cl2); m.p. 133-135 oC (lit. m.p. 136-138 oC)4; δH

(400 MHz, CD3OD): 8.41(1H, d, J 5.8 Hz, H2), 8.0 (1H, d, J 8.9

Hz, H5), 7.49 (1H, d, J 2.3 Hz, H8), 7.38 (1H, dd, J 2.3 Hz, 8.9

Hz, H6), 6.58 (1H, d, J 5.8 Hz, H3), 3.4 (2H, t, J 6.2 Hz, H9),

2.71 (2H, t, J 6.2 Hz, H13), 1.54 (2H, m, H12), 1.49 (2H, m,

H10), 1.38 (2H, m, H11); δC (75 MHz, CD3OD): 154.1, 150.6, 133.4, 130.0, 128.8, 126.5, 123.2, 119.1,

114.0, 44.6, 42.3, 32.3, 30.2, 26.1. LRMS (EI) m/z 264.12 [M+H+]; HPLC purity: > 95%.

2-(7-chloroquinolin-4-ylamino) ethanol (AW21)

Off-white powder (1.1 g, 98%); Rf 0.20 (1:9 MeOH:CH2Cl2); m.p 216-

218 oC ; δH (400 MHz, CD3OD): 8.35 (1H, d, J 5.7 Hz,H2), 8.10 (1H, d, J

9.4 Hz, H5), 7.77 (1H, d, J 2.1 Hz, H8), 7.40 (1H, dd, J 2.1 Hz, 9.4 Hz, H6),

6.58 (1H, d, J 5.7 Hz,H3), 3.83 (2H, t, J 6.3 Hz, H10), 3.68 (1H, t, J 6.3 Hz,

NH), 3.50 (2H, t, J 6.3 Hz, H9), 2.91 (1H, t, J 6.4 Hz, OH); δC (75 MHz,

CD3OD): 153.6, 149.9, 146.4, 136.1, 128.3, 126.7, 124.0, 117.9, 112.1,

60.3, 42.4. LRMS (EI) m/z 223.06 [M+H+]; HPLC purity: > 95%.

3-(7-chloroquinolin-4-ylamino) propan-1-ol (AW26)

Light brown powder (1.13 g, 95%); Rf 0.23 (1:9 MeOH:CH2Cl2); m.p.

221-223 oC; δH (400 MHz, CD3OD): 8.14 (1H, d, J 6.2, H2), 7.86 (1H,

d, J 8.9, H5), 7.57 (1H, d, J 2.4 Hz, H8), 7.20 ( 1H, dd, J 2.4 Hz, 8.9 Hz,

H6), 6.34 (1H, d, J 6.2 Hz, H3), 3.22 (2H, t, J 6.1 Hz, H11), 2.62 ( 2H, t,

J 6.1 Hz, H9), 2.56 (1H, m, NH), 1.71 ( 2H, m, H10 ), 1.47 (1H, m, OH);

δC (75 MHz, CD3OD): 153.2, 150.4, 148.7, 135.6, 128.7, 126.9,

123.1, 118.5, 114.2, 61.2, 44.9, 32.3. LRMS (EI) m/z 237.07 [M+H+]; HPLC purity: > 95%.

160

4-(7-chloroquinoline-4-ylamino) butan-1-ol (AW28)

Light green crystals (1.08 g, 96%); Rf 0.23 (1:9 MeOH:CH2Cl2);

m.p 216-218 oC; δH (400 MHz, CD3OD): 8.32 (1H, d, J 6.2 Hz, H2),

8.07 (1H, d, J 8.6 Hz, H5), 7.76 (1H, d, J 2.9 Hz, H8), 7.39 (1H, dd,

J 2.9 Hz, 8.6 Hz, H6), 6.51 (1H, d, J 6.2 Hz, H3), 3.63 (2H, t, J 6.1

Hz, H12), 3.38 (2H, t, J 6.1 Hz, H9), 1.81 (2H, m, H11), 1.69 (2H, m,

H10); δC (75 MHz, CD3OD): 154.0, 150.6, 148.1, 135.4, 130.2,

128.1, 122.2, 119.8, 113.6, 63, 45.9,30.6, 27.2. LRMS (EI) m/z 251.07 [M+H+]; HPLC purity: > 95%.

5-(7-chloroquinolin-4-ylamino) pentan-1-ol (AW29)

Light yellow powder (1.2 g, 90%); Rf 0.24 (1:9 MeOH:CH2Cl2);

m.p. 212-214 oC; δH (400 MHz, CD3OD): 8.32 (1H, d, J 6.3 Hz,

H2), 8.13 (1H, d, J 9.0 Hz, H5), 7.76 (1H, d, J 2.8 Hz, H8), 7.43

(1H, dd, J 2.8 Hz, 9.0 Hz, H6), 6.54 (1H, d, J 6.3 Hz, H3), 3.59

(2H, t, J 5.9 Hz, H13), 3.41 (t, J 5.9 Hz, H9), 1.77 (2H, m, H10),

1.51-1.61 (4H, m, H11 + H12); δC (75 MHz, CD3OD): 155.1,

151.3, 148.6, 136.7, 130.4, 128.2, 124.0, 120.1, 114.7, 64.0, 45.5, 33.2, 30.7, 25.1. LRMS (EI) m/z

265.10 [M+H+]; HPLC purity: > 95%.

6.1.5 General synthetic procedure for fusidic acid amides5

HOBt (130 mg, 0.97 mmol) was added to a solution of fusidic acid (200 mg, 0.39 mmol) and EDCI

(150 mg, 0.97 mmol) in DMF (8 ml) and the resulting reaction mixture was stirred for 15 minutes at

25 oC. In a separate round bottom flask, DIPEA (0.8 ml, 0.5 mmol) was added to the amine (0.42

mmol) in DMF (2 ml) and the resulting mixture was stirred for 10 minutes. In the case of primaquine

which was commercially available as a diphosphate salt, 0.78 mmol of DIPEA was used in order to

release the free base. The solution containing the amine was then added dropwise to the flask

containing the acid and the resulting reaction mixture was stirred at 25 oC for 16 hours. After

completion of the reaction (monitored by TLC), 15 ml of 20% MeOH/CH2Cl2 was added to the

reaction mixture, which was then washed with saturated aqueous sodium hydrogen carbonate

NaHCO3 (2 x 15 ml) followed by saturated aqueous sodium chloride (2 x 15 ml) and dried over

MgSO4. The solvent was evaporated under reduced pressure and the crude residue was purified

using column chromatography (10% MeOH/CH2Cl2) to furnish the amides in moderate to excellent

yields (25-90%).

161

N-(7-chloroquinolin-4-yl)-ethanoyl fusidic acid amide (AW23)

Bright yellow crystals (223 mg, 80%)

Rf 0.23 (1:9 MeOH:CH2Cl2); m.p. 148-

150 oC; δH (300 MHz, CDCl3): 8.40

(1H,d, J 6.0 Hz, H2’), 8.13 (1H, d, J 9.0

Hz, H5’), 8.01 (1H, d, J 2.3 Hz, H8’), 7.41

(1H, dd, J 2.3 Hz, 9.0 Hz, H6’), 6.38 (1H,

d, J 6.0 Hz, H3’), 5.88 (1H, d, J 8.2 Hz,

H16), 5.11 (1H, t, J 7.3 Hz, H24), 4.42

(1H, m, H11), 3.81 (1H, m, H3), 3.25-

3.55 (4H, m, H9’+ H10’) 3.09 (1H, m,

H13), 2.46 (2H, m, H22), 2.11 (1H, m, H5), 2.07-2.17 (2H, m, H23), 1.85-2.33 (2H, m, H12), 1.30-2.19 (2H,

m, H15), 1.96 (3H, s, acetyl), 1.51-2.17 (2H, m, H1), 1.67 (3H, s, H27), 1.60 (3H, s, H26), 1.58 (1H, m, H4),

1.57 (1H, m, H9), 1.12-1.74 (2H, m, H7), 1.13-1.59 (2H, m, H6), 1.38 (3H, s, H30), 0.98 (3H, s, H19), 0.92

(3H, d, J 6.2 Hz, H28), 0.91 (3H, s, H18); δC (75 MHz, CDCl3): 174.2, 171.2, 153.4, 151.1, 150.5, 148.7,

135.6, 132.9, 130.3, 128. 7, 126.9, 124.2, 123.7, 118.5, 114.3, 74.9, 72.3, 69.0, 50.3, 49.9, 48.1, 45.1,

42.1, 40.3, 39.2, 37.8, 36.9, 36.0, 34.9, 32.9, 30.5, 30.0, 29.2, 28.0, 26.1, 24.9, 23.1, 20.8, 19.9, 18.2,

17.3, 15.4; LRMS (ESI) m/z 721.41 [M+H+]; HPLC purity: > 95% (tr = 5.42 min).

N-(7-chloroquinolin-4-yl)-N-methylpropanoyl fusidic acid amide (AW24)

Light yellow crystals (73 mg, 25%); Rf

0.29 (1:9 MeOH:CH2Cl2); m.p. 137-140

oC; δH (300 MHz, CDCl3): 8.35 (1H,d, J

5.8 Hz, H2’), 7.96 (1H, d, J 8.9 Hz, H5’),

7.43(1H, d, J 3.0 Hz, H8’) 7.27 (1H, dd, J

3.0 Hz, 8.9, H6’), 6.33 (1H, d, J 5.8 Hz,

H3’), 5.57 (1H, d, J 8.2 Hz, H16), 5.0 (1H,

t, J 7.3 Hz, H24), 4.30 (1H, m, H11), 3.65

(1H, m, H3), 2.97 (1H, m, H13), 2.84 (2H,

m, H9’), 2.51( 2H, m, H11’), 2.38-2.46 (2H, m, H22), 2.19 (1H, m, H5), 2.03-2.11 (2H, m, H23), 1.85-2.33

(2H, m, H12), 1.27-2.14 (2H, m, H15), 1.86 (3H, s, acetyl), 1.72(2H, m, H10’), 1.51-2.17 (2H, m, H1), 1.69

(3H, s, H27), 1.55 (3H, s, H26), 1.58 (1H, m, H4), 1.57 (1H, m, H9), 1.12-1.74 (2H, m, H7), 1.13-1.59 (2H,

162

m, H6), 1.33 (3H, s, H30), 0.84-0.92 (9H, m, H18+19+28); δC (75 MHz, CDCl3): 173.4, 170.8, 153.2, 151.7,

149.9, 148.3, 136.1, 133.0, 130.6, 129. 1, 127.2, 124.0, 124.9, 117.9, 114.1, 75.0, 72.7, 69.7, 50.8,

49.2, 47.9, 46.3, 42.8, 41.3, 40.5, 39.4, 38.1, 37.6, 36.9, 35.2, 34.6, 33.5, 30.2, 31.3, 29.6, 28.1, 25.8,

24.9, 22.8, 20.4, 19.1, 18.0, 16.9, 14.8; LRMS (ESI) m/z 749.48 [M+H+]; HPLC purity: > 95% (tr = 5.29

min).

N-(7-chloroquinolin-4-yl)-butanoyl fusidic acid amide (AW25)

Yellow crystals (107 mg, 50%) Rf

0.29 (1:9 MeOH:CH2Cl2); m.p.

164-167 oC; δH (400 MHz, CDCl3):

8.24 (1H,d, J 5.9 Hz, H2’), 8.17

(1H, d, J 9.0 Hz, H5’), 7.91 (1H, d, J

3.2 Hz, H8’) 7.20 (1H, dd, J 3.2 Hz,

9.0, H6’), 6.33 (1H, d, J 5.9 Hz,

H3’), 5.73 (1H, d, J 8.2 Hz, H16),

4.98 (1H, t, J 7.3 Hz, H24), 4.27

(1H, m, H11), 3.75 (2H, m, H12’),

3.66 (1H, m, H3), 3.23 (2H, m, H9’), 2.88 (1H, m, H13), 2.36-2.45 (2H, m, H22), 2.11 (1H, m, H5), 2.11-

2.19 (2H, m, H23), 1.85-2.33 (2H, m, H12), 1.30-2.19 (2H, m, H15), 1.92 (3H, s, acetyl), 1.51-2.17 (6H, m,

H1+H10’+H11’), 1.55 (3H, s, H27), 1.46 (3H, s, H26), 1.58 (1H, m, H4), 1.57 (1H, m, H9), 1.21-1.68 (2H, m,

H7), 1.13-1.59 (2H, m, H6), 1.36 (3H, s, H30), 0.90 (3H, s, H19), 0.85 (3H, d, J 6.2 Hz, H28), 0.81 (3H, s,

H18); δC (75 MHz, CDCl3): 172.8, 171.0, 153.6, 150.4, 149.3, 148.0, 137.3, 133.8, 131.2, 129. 6, 128.1,

125.1, 124.5, 118.0, 114.7, 74.2, 73.1, 70.7, 51.1, 49.0, 48.0, 46.1, 42.7, 41.0, 40.3, 39.1, 38.0, 37.5,

37.0, 36.2, 34.0, 33.1, 30.8, 31.6, 30.2, 28.5, 26.1, 25.7, 23.2, 20.9, 19.9, 17.8, 17.0, 15.0; LRMS (ESI)

m/z 749.48 [M+H+]; HPLC purity: > 95% (tr = 4.99 min).

163

N-(7-chloroquinolin-4-yl)-propanoyl fusidic acid amide (AW31)

Yellow crystals (155 mg, 55%) Rf 0.30

(1:9 MeOH:CH2Cl2); m.p. 151-154 oC; δH

(400 MHz, CDCl3) 8.32 (1H,d, J 6.2 Hz,

H2’), 8.02 (1H, d, J 9.1 Hz, H5’), 7.78(1H,

d, J 3.2 Hz, H8’) 7.21 (1H, dd, J 3.2 Hz,

9.1 Hz, H6’), 6.39 (1H, d, J 6.2 Hz, H3’)

5.81 (1H, d, J 8.2 Hz, CH16), 5.0 (1H, t, J

7.3 Hz, H24), 4.29 (1H, m, H11), 3.62

(2H,m, H11’), 3.1 (2H, m, H9’), 3.39 (1H,

m, H3), 2.9 (1H, m, H13), 2.32-43 (2H, m, H22), 2.11 (1H, m, H5), 2.03-2.09 (2H, m, H23), 1.85-2.33 (2H,

m, H12), 1.30-2.19 (2H, m, H15), 1.97 (3H, s, acetyl), 1.51-2.17 (2H, m, H1+H10’), 1.62 (3H, s, H27), 1.6

(3H, s, H26), 1.58 (1H, m, H4), 1.57 (1H, m, H9), 1.14-1.73 (2H, m, H7), 1.13-1.59 (2H, m, H6), 1.34 (3H,

s, H30), 0.93 (3H, s, H19), 0.89 (3H, d, J 6.2 Hz, H28), 0.8 (3H, s, H18); δC (75 MHz, CDCl3): 174.6, 170.3,

154.0, 151.6, 149.9, 146.7, 135.7, 133.9, 130.5, 131.4, 128.0, 124.4, 122.8, 118.9, 113.0, 75.2, 71.9,

69.3, 49.3, 48.3, 45.3, 44.2, 41.0, 39.2, 38.5, 37.3, 36.1, 35.8, 34.8, 32.7, 31.1, 30.6, 29.6, 28.2, 27.6,

25.0, 24.6, 23.1, 21.2, 20.6, 18.1, 17.3, 15.0; LRMS (ESI) m/z 735.42 [M+H+]; HPLC purity: > 95% (tr =

5.42 min).

N-(6-methoxyquinolin-8-yl)-pentanoyl fusidic acid amide (AW33)

Bright yellow crystals (73 mg, 25%)

Rf 0.57 (1:9 MeOH:CH2Cl2); m.p. 107-

110 oC: δH (400 MHz, CDCl3): 8.53

(1H, dd, J 4.1 Hz, 1.8 Hz, H2’), 7.91

(1H, d, J 8.0 Hz, H4’ ), 7.39 (1H, m,

H3’), 6.33 (2H, d, J 4.9 Hz, H5’+7’),

5.75 (1H, d, J 8.2 Hz, H16), 5.05 (1H,

t, J 7.0 Hz, H24), 4.30 (1H, m, H11),

3.89 (3H, s, OMe) 3.60 (1H, m, H3),

2.84 (1H, m, H13), 3.4(2H, m, H13’),

3.2 (1H, m, H11’), 2.46 (2H, m, H22),

2.11 (1H, m, H5), 2.05-2.18 (2H, m, H23), 1.88-2.36 (2H, m, H12), 1.30-2.19 (2H, m, H15), 1.95 (3H, s,

164

acetyl), 1.51-2.17 (2H, m, H1), 1.64 (3H, s, H27), 1.60 (3H, s, H26), 1.58 (1H, m, H4), 1.57 (1H, m, H9),

1.35 (3H, s, H30), 1.53 (2H, m, H12’), 1.48 (2H, m, H9’), 1.27 (3H, s, H10’), 1.21-1.74 (2H, m, H7), 1.13-

1.59 (2H, m, H6), 0.95 (3H, s, H19), 0.90 (3H,s, H28), 0.88 (3H, s, H18); δC (75 MHz, CDCl3): 173.6, 171.1,

159.7, 151.3, 147.3, 146.3, 137.2, 135.0, 134.7, 133.2, 130.4, 124.2, 123.1, 101.3, 100.4, 75.7, 72.0,

69.2,56.9, 51.8, 50.9, 49.8, 45.3, 41.6, 40.5, 39.3, 37.4, 36.9, 36.0, 35.8, 34.6, 32.0, 30.9, 29.3, 28.1,

27.6, 26.3, 25.0, 24.2, 23.9, 23.1, 21.0, 20.5, 18.2, 17.1, 15.5; LRMS (ESI) m/z 759.50 [M+H+]; HPLC

purity: > 95% (tr = 8.44 min).

N-(7-chloroquinoline-4-yl)-pentanoyl fusidic acid amide (AW45)

Pale yellow crystals (265 mg, 90%)

Rf 0. 29 (1:9 MeOH:CH2Cl2); m.p.

143-146 oC; δH (400 MHz, CDCl3):

8.31 (1H,d, J 5.8 Hz, H2’), 8.01 (1H,

d, J 9.2 Hz, H5’), 7.87 (1H, d, J 3.0

Hz, H8’) 7.19 (1H, dd, J 3.0 Hz, 9.2,

H6’), 6.26 (1H, d, J 5.8 Hz, H3’), 5.65

(1H, d, J 8.2 Hz, H16), 4.97 (1H, t, J

7.1 Hz, H24), 4.26 (1H, m, H11), 3.66

(1H, m, H3), 3.27 (2H, m, H9’), 2.87

(1H, m, H13), 2.81 (2H, m, H12’), 2.68 (2H, m, H13’), 2.37-41 (2H, m, H22), 2.11 (1H, m, H5), 2.03-2.15

(2H, m, H23), 1.25-2.34 (2H, m, H12), 1.32-2.21 (2H, m, H15), 1.95 (3H, s, acetyl), 1.54-2.24 (6H, m,

H1+10’+11’), 1.59 (3H, s, H27), 1.47 (3H, s, H26), 1.57 (1H, m, H4+H9), 1.14-1.76 (2H, m, H7), 1.15-1.61 (2H,

m, H6), 1.26 (3H, s, H30), 1.06 (3H, s, H19), 0.90 (3H, d, J 6.2 Hz, H28), 0.85 (3H, s, H18); δC (75 MHz,

CDCl3): 174.3, 171.1, 154.3, 151.3, 150.7, 133.6, 132.6, 130.4, 129.8, 129.0, 127.0, 124.2, 123.1,

119.7, 115.2, 74.5, 71.5, 68.3, 49.3, 48.8, 45.1, 44.3, 42.6, 39.5, 39.0, 37.0, 36.3, 36.2, 35.6, 33.0,

32.3, 31.1, 30.3, 29.9, 28.8, 28.4, 26.4, 25.2, 23.8, 23.0, 21.1, 20.5, 18.3, 17.5, 16.2; LRMS (ESI) m/z

763.45 [M+H+]; HPLC purity: > 95% (tr = 5.32 min).

165

6.1.6 General synthetic procedure for fusidic acid esters

A solution of fusidic acid (200 mg, 0.39 mmol) and EDCI (150 mg, 0.97 mmol) was stirred in DMF (8

ml) at 25 oC and the solution was left to stir for 15 minutes. In a separate round bottom flask, DMAP

was added (118 mg, 0.97 mmol) to the alcohol (0.42 mmol) dissolved in DMF (2 ml) and stirred for

10 minutes. The solution containing the alcohol was then added dropwise to the flask containing the

acid, and the resulting reaction mixture was stirred at 25 oC for 15 hours. The reaction was

monitored by TLC and after completion, 15 ml of 20% MeOH/CH2Cl2 was added to the reaction

mixture which was then was washed with saturated aqueous NaHCO3 (2 x 15 ml) followed by

saturated aqueous NaCl (2 x 15 ml) and dried over MgSO4. The solvent was evaporated in vacuo and

purification of the resulting crude residue using column chromatography (10% MeOH/CH2Cl2)

afforded the esters in moderate yields (69-85%).

2-(7-chloroquinolin-4-ylamino)-ethanoyl fusidic acid ester (AW32)

White crystals (192 mg, 69%) Rf 0.29

(1:9 MeOH:CH2Cl2); m.p. 87-90 oC; δH

(400 MHz, CDCl3): 8.40 (1H,d, J 5.8

Hz, H2’), 8.15 (1H, d, J 8.9 Hz, H5’), 7.89

(1H, d, J 3.1 Hz, H8’), 7.60 (1H, dd, J 3.1

Hz, 8.9 Hz, H6’), 6.46 (1H, d, J 6.0 Hz,

H3’), 5.79 (1H, d, J 8.4 Hz, H16), 5.07

(1H, t, J 7.0 Hz, H24), 4.30 (1H, m, H11),

3.69 (1H, m, H3), 3.21-3.61 (4H, m,

H9’+ H10’) 3.16 (1H, m, H13), 2.31-2.39 (2H, m, H22), 2.11 (1H, m, H5), 2.07-2.17 (2H, m, H23), 1.84-2.32

(2H, m, H12), 1.33-2.21 (2H, m, H15), 1.94 (3H, s, acetyl), 1.51-2.16 (2H, m, H1), 1.65 (3H, s, H27), 1.61

(3H, s, H26), 1.58 (1H, m, H4), 1.57 (1H, m, H9), 1.09-1.69 (2H, m, H7), 1.13-1.59 (2H, m, H6), 1.31 (3H,

s, H30), 0.93 (3H, s, H19), 0.90 (3H, d, J 6.2 Hz, H28), 0.88 (3H, s, H18); (API) δC (75 MHz, CDCl3): 174.0,

170.6, 153.5, 150.6, 149.5, 148.9, 136.1, 133.4, 131.2, 128. 0, 127.2, 125.7, 124.9, 119.5, 113.0,

75.3, 72.7, 70.1, 50.8, 50.2, 47.5, 45.9, 43.1, 40.7, 40.0, 37.6, 36.1, 35.3, 34.9, 33.6, 31.8, 30.4, 32.2,

28.6, 26.3, 25.2, 23.1, 20.6, 19.0, 17.9, 16.3, 14.6; LRMS (ESI) m/z 722.39 [M+H+]; HPLC purity: > 95%

(tr = 6.73 min).

166

3-(7-chloroquinolin-4-ylamino)-propanoyl fusidic acid ester (AW42)

Pale white crystals (211 mg, 74%) Rf

0.28 (1:9 MeOH:CH2Cl2); m.p. 101-104

oC; δH (400 MHz, CDCl3): 8.39 (1H,d, J

6.1 Hz, H2’), 8.19 (1H, d, J 9.2 Hz, H5’),

7.88 (1H, d, J 2.0 Hz, H8’), 7.59 (1H, dd,

J 2.0 Hz, 9.2 Hz, H6’), 6.37 (1H, d, J 6.1

Hz, H3’), 5.78 (1H, d, J 8.4 Hz, H16), 5.04

(1H, t, J 7.2 Hz, H24), 4.28 (1H, m, H11),

3.63 (1H, m, H3), 3.35-3.57 (4H, m, H11’)

3.03 (1H, m, H13), 2.98 (1H, m, H9’), 2.39-2.43 (2H, m, H22), 2.11 (1H, m, H5), 2.12-2.17 (2H, m, H23),

1.88-2.37 (2H, m, H12), 1.34-2.19 (2H, m, H15), 1.93 (3H, s, acetyl), 1.51-2.17 (4H, m, H1+H10’), 1.60

(3H, s, H27), 1.58 (1H, m, H4), 1.57 (1H, m, H9), 1.52 (3H, s, H26), 1.14-1.75 (2H, m, H7), 1.5-1.62 (2H, m,

H6), 1.49 (3H, s, H30), 1.31 (3H, s, H19), 0.90 (3H, d, J 6.2 Hz, H28), 0.84 (3H, s, H18); δC (75 MHz, CDCl3):

173.9, 170.0, 154.3, 150.6, 149.7, 147.4, 136.1, 134.8, 131.9, 132.4, 129.3, 124.0, 123.5, 119.3,

113.7, 75.0, 73.8, 71.0., 50.2, 48.1, 46.5, 44.0, 41.9, 40.8, 38.1, 37.6, 36.3, 35.0, 34.1, 33.6, 31.9,

30.0, 29.3, 28.2, 26.5, 25.4, 24.0, 23.4, 21.0, 19.3, 18.7, 16.3, 14.9 ; LRMS (ESI) m/z 736.38 [M+H+];

HPLC purity: > 95% (tr = 6.99 min).

4-(7-chloroquinoline-4-ylamino)-butanoyl fusidic acid ester (AW34)

White crystals (238 mg, 82%) Rf

0.28 (1:9 MeOH:CH2Cl2); .m.p.

80-84 oC; δH (400 MHz, CDCl3):

8.37(1H,d, J 6.0 Hz, H2’), 8.09

(1H, d, J 8.9 Hz, H5’), 7.83 (1H, d,

J 3.3 Hz, H8’) 7.18 (1H, dd, J 3.3

Hz, 9.2 Hz, H6’), 6.40 (1H, d, J 6.0

Hz, H3’) 5.85 (1H, d, J 7.9 Hz H16),

5.15 (1H, t, J 7.0 Hz, H24), 4.34

(1H, m, H11), 3.71 (2H,m, H12’),

3.40 (1H, m, H3), 3.14 (2H, m, H9’), 2.97 (1H, m, H13), 2.39-2.44 (2H, m, H22), 2.11 (1H, m, H5), 2.09-

2.15 (2H, m, H23), 1.83-2.31 (2H, m, H12), 1.35-2.22 (2H, m, H15), 2.02 (3H, s, acetyl), 1.53-2.19 (6H, m,

H1+H10’+ H11’), 1.65 (3H, s, H27), 1.59 (3H, s, H26), 1.58 (1H, m, CH4), 1.57 (1H, m, H9), 1.13-1.71 (2H, m,

167

H7), 1.13-1.59 (2H, m, H6), 1.35 (3H, s, H30), 1.06 (3H, s, H19), 0.93 (3H, d, J 6.2 Hz, H28), 0.86 (3H, s,

H18); δC (75 MHz, CDCl3): 173.8, 171.6, 153.3, 151.4, 150.2, 148.6, 136.3, 134.1, 132.2, 130. 5, 129.3,

126.1, 124.3, 119.3, 115.7, 75.9, 74.2, 70.3, 50.7, 49.0, 47.9, 46.4, 43.1, 40.7, 40.5, 39.3, 38.2, 37.6,

37.0, 35.1, 34.6, 33.8, 32.7, 31.5, 30.4, 29.3, 28.5, 26.3, 24.2, 20.1, 19.4, 18.2, 17.6, 15.3; LRMS (ESI)

m/z 750.42 [M+H+]; HPLC purity: > 95% (tr = 7.02 min).

5-(7-chloroquinolin-4-ylamino)-pentanoyl fusidic acid ester (AW35)

Yellow crystals (253 mg, 85%) Rf

0.43(1:9 MeOH:CH2Cl2); m.p. 77-

80 oC; δH (400 MHz, CDCl3): 8.33

(1H,d, J 6.0 Hz, H2’), 8.11 (1H, d, J

9.1 Hz, H5’), 7.90 (1H, d, J 3.2 Hz,

H8’) 7.24 (1H, dd, J 3.2 Hz, 9.1 Hz,

H6’), 6.39 (1H, d, J 6.0 Hz, H3’), 5.77

(1H, d, J 8.4 Hz, H16), 5.02 (1H, t, J

7.3 Hz, H24), 4.28 (1H, m, H11), 3.78

(2H, m, H13’), 3.58 (1H, m, H3),

3.36 (2H, m, H9’), 2.98 (1H, m, H13), 2.81 (2H, m, H12’), 2.41-2.53 (2H, m, H22), 2.11 (1H, m, H5), 2.09-

2.15 (2H, m, H23), 1.85-2.33 (2H, m, H12), 1.31-2.17 (2H, m, H15), 1.91 (3H, s, acetyl), 1.51-2.26 (8H, m,

H1+H10’+H11’+H12’), 1.60 (3H, s, H27), 1.59 (1H, m, H4+H9), 1.51 (3H, s, H26), 1.12-1.74 (2H, m, H7), 1.13-

1.59 (2H, m, H6), 1.21 (3H, s, H30), 1.06 (3H, s, H19), 0.90 (3H, d, J 6.2, H28), 0.86 (3H, s, H18); δC (75

MHz, CDCl3): 173.6, 170.3, 153.5, 150.7, 148.2, 135.4, 133.1, 132.4, 130.2, 129.1, 128.7, 123.8, 122.7,

118.1, 116.0, 75.3, 72.6, 69.7, 53.3, 49.1, 46.3, 45.0, 43.6, 41.3, 40.0, 38.2, 37.1, 36.4, 35.2, 33.5,

32.3, 31.0, 30.2, 29.6, 28.9, 28.0, 27.4, 26.1, 24.3, 23.6, 21.8, 20.0, 19.2, 17.9, 15.2; LRMS (ESI) m/z

764.44 [M+H+].; HPLC purity: > 95% (tr = 7.20 min).

168

6.2 Biology experimental section

6.2.1 Turbidimetric Solubility6

Solubility of the test compounds was determined using the turbidimetric method described by

Bevan and Lloyd.7 Stock solutions of test and control compounds were prepared by dissolving an

accurately weighed amount of each in HPLC-grade DMSO to a 10 mM concentration. Pre-dilutions of

the stock solutions, also in DMSO, were then prepared in clear, v-shaped bottom 96-well

polystyrene microtitre plates as illustrated in Figure 6.1.

Compound 1

(triplicate)

Compound 2

(triplicate)

Compound 3

(triplicate)

Compound 4

(triplicate)

Conc.

(mM) 1 2 3 4 5 6 7 8 9 10 11 12

0 A

0.25 B

0.5 C

1.0 D

2.0 E

4.0 F

8.0 G

10.0 H

Figure 6.1: Layout of turbidimetric solubility assay compound pre-dilution plate

For this, each stock solution was diluted serially in triplicate by first pipetting 20 μl DMSO into wells

in row G on the plate and 50 μl DMSO to the wells in the rows above (i.e. rows F, E, D…A). 80 μl of

the 10 mM stock solutions was then pipetted to the wells in row G and mixed to give a starting

concentration of 8 mM (100 μl). Serial dilution of this solution was achieved by subsequently

transferring 50 μl to the well in the row above (Row F) from Row F through to B. The final pre-

dilution concentrations obtained ranged from 0.25 mM (Row B) to 8.0 mM (Row G). All wells in Row

A contained only DMSO, while those in Row H contained the undiluted 10 mM stock solution.

From each pre-dilution solution, secondary dilutions of the compounds in both DMSO and 0.01M pH

7.4 phosphate buffered saline (PBS) were prepared in a second 96-well plate, also in triplicate

(Figure 6.2). Wells in columns 1-6 contained the test compound dissolved in DMSO while those in

169

columns 7-12 contained the same compound and concentration dissolved in PBS. The final volume in

each well was 200 μl, prepared by pipetting 4 μl each of solution from the pre-dilution plate to the

corresponding well into both DMSO and PBS (both 196 μl). The final concentration of DMSO in the

PBS aqueous buffer preparations was therefore 2% v/v. The concentrations of test compounds in

DMSO served as controls to determine potential false turbidimetric absorbance readings arising

from the compounds in solution absorbing radiation at the analysis wavelength.

DMSO 0.01M pH 7.4 PBS

Compound 1

(triplicate)

Compound 2

(triplicate)

Compound 1

(triplicate)

Compound 2

(triplicate)

Conc. (M) 1 2 3 4 5 6 7 8 9 10 11 12

0 A

5 B

10 C

20 D

40 E

80 F

160 G

200 H

Figure 6.2: Turbidimetric solubility assay plate layout

The plates were covered and left to equilibrate for 2 hrs at ambient temperature, after which UV-VIS

absorbance readings were measured at 620 nm using a SpectraMax 340PC384 microplate reader

(Molecular Devices, Sunnydale, CA). Absorbance from the wells containing only DMSO and 2% v/v

DMSO in PBS (0 μM in plate layout, Row A) served as controls and blanks. At concentrations above

the limit of solubility, undissolved particles precipitated out of solution, occluding incident radiation,

resulting in increased apparent absorbance. Therefore, the concentration at which increased

absorbance was noted was deemed the approximate solubility of the compounds. Reserpine and

hydrocortisone were used as minimum and maximum solubility standards respectively, as

established by reports from literature.7

170

6.2.2 Bacterial strains and growth conditions

For all the experiments carried out, the strain of M. smegmatis (Msm) used was M. smegmatis

mc21558, while that of Mtb was M. tuberculosis H37Rv (MA)9, from stocks maintained by the

Molecular Mycobacteriology Research Unit (MMRU) at -80 oC. In the case of Msm, the stocks were

stored in 33% glycerol (v/v). All culturing and manipulations of Mtb were performed in a

Biosafety Level 3 laboratory, in a Class II flow cabinet at negative pressure (160 - 170 kPa).

Table 6.3: Bacterial strains used in this study

Strain Description/Genotype Reference/Source

Mycobacterium smegmatis

(Msm) mc2155

High frequency transformation

mutant of Msm ATCC 706 Snapper et al, 1990

Mycobacterium tuberculosis

(Mtb) H37Rv(MA)

Virulent reference laboratory

strain of Mtb ATCC 27294 Ioerger et al, 2010

Unless otherwise specified, strains were cultured in Middlebrook 7H9 (Difco™) broth enriched with

10% oleic acid-dextrose-catalase (OADC) (Difco™), 0.5% glycerol and 0.05% Tween® 80. The agar

used for raising colonies was Middlebrook 7H10 (Difco™) medium supplemented with 10% OADC

and 0.5% glycerol. Growth medium was sterilised by autoclaving at 121 oC for 15 minutes and, in the

case of liquid broth, additionally filtered using filtration flasks fitted with 0.22 µm filter membranes.

Glycerol-alanine salts supplemented with iron and Tween®80 (GAST/Fe) was made up using the

recipe outlined below, and the medium was sterilised by filtration using filtration flasks fitted with

µm filter membranes.

6.2.2.1 Culture media

All media were made to a final volume of 1L with distilled water. Media were sterilized by

autoclaving at 121 °C for 15 min, except GAST/Fe which was sterilised by filtration.

7H9/OADC

4.7 g Middlebrook 7H9 broth powder (DifcoTM, USA), 2 ml glycerol (Merck, Germany)

100 ml OADC Middlebrook Enrichment (BD Microbiology Systems, USA) added after autoclaving.

Tween®80 was added to 0.05%.

171

GAST/Fe

0.3 g of Bacto Casitone (Difco); 4.0 g of dibasic potassium phosphate; 2.0 g of citric acid; 1.0 g of L-

alanine; 1.2 g of magnesium chloride hexahydrate; 0.6 g of potassium sulfate; 2.0 g of ammonium

chloride; 0.05 g of ferric ammonium citrate; 1.8 ml of 10M NaOH; 10 ml of glycerol; Tween®80 was

added to 0.05% and pH adjusted to 6.6.

7H10/OADC agar

19 g Middlebrook 7H10 agar powder (DifcoTM, USA), 5 ml glycerol (Merck, Germany)

100 ml Middlebrook OADC Enrichment (BD Microbiology Systems, USA) added after autoclaving.

Msm liquid cultures were grown at 37 oC in a shaking incubator while Mtb liquid cultures were

placed flat in non-shaking incubators. All culturing and manipulation of Mtb strains was performed

in a Biosafety Level 3 laboratory in Class II flow cabinets at negative pressure.

6.2.2.2 Antimycobacterial screening: The Minimum Inhibitory Concentration (MIC) of FA against

Msm and Mtb

Drug susceptibility testing by broth microdilution using the Microplate Alamar Blue Assay (MABA)

The determination of the MIC of FA was conducted by broth microdilution method, using the

Microplate Alamar Blue Assay (MABA). The broth microdilution method enables the determination

of the minimum inhibitory concentration (MIC) of compounds over a range of concentrations on a

single 96-well microtitre plate.10,11 In the MABA assay, metabolic activity of the cell correlates with

the reduction of resazurin to resorufin, and the resulting change in colour from blue to pink and

fluorescent, allowing visual detection and fluorometric or colorimetric quantification. Wells with

non-viable cells thus remain blue while those with viable cells exhibit pink colouration. For the

experiments reported, visual detection was used to assess growth and growth inhibition. Prior to

addition of alamar blue (at day 3 in the case of Msm and day 7 for Mtb), growth and inhibition was

assessed by visual inspection for pellet formation.

Briefly, a 10 ml culture of Mtb was grown to an OD600 of 0.6 – 0.7. The culture was then diluted

1:500 (in the case of Mtb) or 1:1000 (in the case of Msm) in 7H9/OADC. In a 96- well, U-bottom

microtitre plate (PGRE650180, Lasec, SA), 50 μl of 7H9/OADC was added to all wells from Rows 2-12.

The compounds to be tested were added to Row 1 at a final concentration of 8 × MIC90 (for

compounds with pre-determined or estimated MIC values) and serially diluted, 2-fold, using a

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multichannel pipette, by transferring 50 μl of the liquid in Row 1 to Row 2 and aspirating to mix. 50

μl of the liquid in Row 2 was transferred to Row 3 and aspirated. The procedure was repeated until

Row 11 was reached, where 50 μl of the liquid in Row 11 was discarded to bring the final volume in

these wells to 50 μl. In Row 12, 1 mg/ml of RIF or INH was used as the control for maximal inhibition.

Finally, 50 μl of the diluted culture was added to all the wells. The microtitre plate was stored in a

secondary container and incubated at 37 °C for 3 days (Msm) and 14 days (Mtb). On Day 3 (Msm)

and Day 14 (Mtb) alamarBlue® (BUF012B, Celtic Molecular Diagnostics) was added to each well and

plates were incubated at 37 °C for 6 hours or 24 hours, respectively (in the case of Msm, in plastic

Ziploc® bags, while in the case of Mtb, with 5% CO2) without shaking. The MIC results registered

were calculated, defined as the lowest concentration of compound that inhibited visible growth. The

lowest concentration of drug which prevented the colour change of alamar blue (from blue to pink)

was considered the MIC that inhibited more than 90% of the bacterial population (MIC90).

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6.3 Assessment of the effect of transcriptional silencing of fusA1 on the susceptibility of Mtb to FA

and selected analogues using the checkerboard assay 12

An adapted form of the checkerboard assay was used to assess the effect of transcriptional silencing

of fusA1 on the susceptibility of Mtb to FA and selected analogues. This assay applied the broth

microdilution method, whereby anhydrotetracycline (ATc, compound A) and FA/ selected

analogues (compound B), were serially diluted 2-fold across and down the plate, respectively13

(Figure 6.3).

1 2 3 4 5 6 7 8 9 10 11 12

A

Min

imu

m in

hib

itio

n

Maxim

um

inh

ibitio

n

B

C

D

E

F

G

H

Figure 6.3: Layout of checkerboard assay 96-well microtitre plate

Briefly, 50 μl media was added to every well in the plate, except Row 1 and 12 (minimum and

maximum inhibition controls, respectively). Additional 50 μl media was added to row B3-B11, and

column 2A-2H, and 100 μl to well B2. From 10 mM stock solutions, dilutions of FA and the FA

analogues were made to a concentration 400x MIC and ATc to a concentration of 1 µg/ml. 2 μl of

FA/FA analogue (compound B) was added to the wells B3-B11 and serially diluted (50 μl) to H (50 μl

was discarded from H). 4μl of the same compound B was added to well B2 and serially diluted (100

μl) down column 2 (100 μl was discarded form 2H). Finally, 2 μl of ATc (compound A) was added to

every well in column 3 and serially diluted (50 μl) from column 2 to column 3 until column 11 (50 μl

Compound A (ATc)

Co

mp

ou

nd

B

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was discarded from 11). Finally, 50 μl Mtb culture was added to every well. RIF at a final

concentration of 0.2 μM (50 × MIC) was used as the maximum inhibition control, while drug-free

7H9 served as the minimum inhibition control and well A2 served as a “no drug” control. The

microtitre plates were stored in secondary containers and incubated at 37°C for 13 days, after which

10 μl of alamar blue (Celtic Molecular Diagnostics) was added to each well. After incubation for 24

hr, the MIC results registered were calculated, defined as the lowest concentration of compound

that inhibited visible growth. The lowest concentration of drug which prevented the colour change

of alamar blue (from blue to pink) was considered the MIC that inhibited more than 90% of the

bacterial population (MIC90).

6.4 Generation of spontaneous resistant mutants

6.4.1 Generation of FA-resistant mutant strains of Msm

To generate FA-resistant (FAR) strains of Msm, the MIC of FA in 7H9 medium was established as 41.6

μg/ml (80 μM). A 10 ml culture of Msm was grown in 7H9 medium at 37 oC in a shaking incubator to

an OD600 of 1.0, which corresponds to a cell density of approximately 5-9 x 108 CFU/ml. The culture

was adjusted to a concentration of 109 CFU/ml by centrifugation at 4200 rpm for 10 minutes, 20 oC

and resuspended in 1 ml of 7H9 medium.

A second attempt was made following the above protocol, with higher concentrations of FA as

follows: 50x MIC (4.05 mM) and 100x MIC (8.1 mM). After incubation for 5 days, four colonies arose

at 50x MIC, while there was no visible growth in the plates containing 100x MIC of FA. The colonies

were isolated and inoculated into 200 μl of 7H9 medium, then incubated at 37 oC with shaking at

120 rpm overnight. These were then passaged into fresh 10 ml of 7H9 and incubated at 37 oC with

shaking overnight. 1 ml aliquots of the log-phase cultures were made up to 33% glycerol and stored

at -80 oC, while the remaining cultures were used to screen the colonies for the MIC of FA.

6.4.2 Generation of FAR Mtb mutants

To generate FAR strains of Mtb, the MIC of FA in 7H9 medium was established as 2.5 μg/ml (5 μM). A

10 ml culture of Mtb was grown in 7H9 medium at 37 oC in a shaking incubator to an OD600 of 1.0,

which corresponds to a cell density of approximately 5-9 x 108 CFU/ml. The culture was adjusted to a

concentration of 109 CFU/ml by centrifugation at 4200 rpm for 10 minutes, 20 oC and resuspended in

1 ml of 7H9 medium. A 100 μl aliquot of this was spread onto 7H10 agar plates containing FA at the

following concentrations above MIC in duplicate: 5X MIC (25 μM), 10X MIC (50 μM) 15X MIC (75

μM), 20X MIC (100 μM) and 25X MIC (125 μM). Two controls were plated; 7H10 plates containing

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RIF at a concentration of 2 g/ml and standard 7H10 plates (without drug) consisting of serially

diluted culture, from 100 to 10-7, also in duplicate. After spreading the inoculum using sterile loops,

the plates were left to dry for an hour at ambient temperature before storing in plastic sleeves at 37

oC for 21 days. At the end of the incubation period, single colonies arose at several concentrations of

FA: 10X MIC, 20X MIC and 25X MIC. Single colonies were isolated and inoculated in 200 l of 7H9

medium and incubated for 5 days at 37 oC in a shaking incubator then passaged into 10 ml of fresh

medium, with subsequent incubation under similar conditions. Within 5 days, the cultures had

reached mid-log phase and 1 ml aliquots of undiluted culture were stored at -80 oC for further use.

The remaining cultures were used to screen against FA to establish the shift in MIC.

In addition to FA, selected FA analogues as well as a panel of standard anti-TB drugs and

experimental compounds were also tested against wild-type and GKFA isolates of Mtb to identify

any potent compounds against the GKFA isolates.

DNA was extracted from the GKFA isolate and subjected to both whole genome and targeted

sequencing. Mtb fusA (EF-G) was amplified by PCR and sequenced against wild-type Mtb H37Rv

(MA) as the reference strain.

6.4.3 DNA extraction, purification and amplification

6.4.3.1 CTAB (cetyltrimethylammonium bromide, ICN Biomedicals, Aurora, Ohio) genomic DNA

isolation from Msm and Mtb

The reagent CTAB, which lends its name to the DNA extraction procedure commonly applied in the

isolation of genomic DNA, was prepared as follows: 4.1 g NaCl was dissolved in 80 ml of Merck

Millipore-filtered distilled water (dH2O). While stirring, 10 g of N-cetyl-N, N, N-trimethyl ammonium

bromide (CTAB) was added. The solution was heated to 65 oC and the volume adjusted to 100 ml,

filter-sterilized while still warm and stored at room temperature.

6.4.3.2 DNA extraction procedure

A 10 ml culture of each of the mutant Msm or Mtb strains was grown to approximately 108 CFU/ml,

corresponding to an OD600 of 1.0. The culture was divided into two 5 ml aliquots which were

centrifuged at 3200 rpm, 4 oC for 10 minutes. The medium was discarded, and the pellet was

resuspended in 0.5 ml TE (1 mM Tris-HCl and 1 mM Na2EDTA, pH 7.4) buffer in 1.5 ml vials. 50 l of

10 mg/ml lysozyme was added and the vials incubated overnight in a shaking incubator at 37 oC.

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The contents of each vial were mixed using a vortex machine, then 70 μl of 10% Sodium dodecyl

sulfate (SDS) and 6 μl of 10 mg/ml proteinase K were added. The resulting solutions were left to

shake at 300 rpm 60 oC in a thermomixer for one hour, after which 100 μl of pre-heated 5M NaCl

was added to each vial and mixed thoroughly by manually inverting each vial. 80 μl of 10% CTAB was

added next, and the suspension mixed thoroughly by inverting by hand. The vials were then

incubated at 60 oC for 15 minutes with low-mode (300 rpm) shaking in the thermomixer, frozen for

15 minutes at -80 oC then left to thaw at ambient temperature before a second cycle of low-mode

shaking in a thermomixer at 60 oC.

6.4.3.3 DNA harvesting

The vials were left to cool at ambient temperature for 10 minutes before 700 μl of chloroform:

isoamyl alcohol (24:1 v/v)was added to each vial (to degrade residual proteins), which was then

inverted by hand 25 times to form a white suspension consisting of an aqueous and organic phase.

The vials were then centrifuged at 13200 rpm at ambient temperature and the upper (aqueous)

phase was transferred to a vial containing 700 μl of ice-cold isopropanol. Each vial was tilted upside

down 5 times to ensure homogeneity of the mixture. The vials were left to stand at 4 oC overnight

for DNA to precipitate, after which they were centrifuged for 10 minutes at ambient temperature.

The supernatant was discarded and the pellets washed with ice-cold 70% ethanol before

centrifuging at 13200 rpm and ambient temperature for 10 minutes. The supernatant (ethanol) was

discarded and the vials set to dry under vacuum in a speedvac DNA concentrator at 16100 g for 20

minutes. 55 μl of dH2O was added to each vial, mixed by aspiration using a pipette tip and incubated

at 42 oC in a heating block for 10 minutes prior to analysis by gel electrophoresis.

6.4.3.4 DNA analysis

To ensure the quality, purity and size of the genomic DNA, gel electrophoresis was carried out using

1% agarose gel in TE buffer at ambient temperature. Gels were prepared using 1x TAE (40 mM Tris-

acetic acid, 1 mM Na2EDTA pH 8.0), agarose powder (Sigma-Aldrich) and 0.5 µg/ml ethidium

bromide. To prepare samples for electrophoresis, 2 μl of DNA, 8 μl of dH2O and 2 μl of

tracking/loading dye (0.025% bromophenol blue in 30% glycerol) were mixed using a pipette. To

make up a sample for each well, were mixed by pipetting and each loaded into individual wells in the

agarose gel. Fragment sizes were assessed using lambda DNA molecular weight marker IV; Roche

Applied Science, Germany). Gels were electrophoresed in a Mini-Sub Cell GT mini gel horizontal

submarine unit (Bio-Rad) at 100 volts, until dye markers had migrated an appropriate distance

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allowing visualisation of the DNA. Visualization was performed under UV light using ta Gel Doc

(WealTeach Keta Imaging System). Once the purity and size were ascertained, the DNA samples

were stored at -20 oC awaiting whole genome sequencing, while in the case of targeted sequencing,

PCR amplification and subsequent purification using NucleoSpin gel and PCR clean-up kit was

carried out prior to sequencing.

6.4.4 PCR amplification of genomic DNA

All PCR reactions were carried out in the MyCycler™ thermal cycler (Bio-Rad) with

oligonucleotide primers purchased from the Molecular and Cell Biology Department, University

of Cape Town.

6.4.5 Sequencing

Targeted sequencing of PCR products was carried out as a pay-for service by the Central Analytical

Facilities at Stellenbosch University. Whole genome sequencing was performed by Professor Tom R.

Ioerger and James C. Sacchettini at the Department of Computer Science, Texas A&M University in

Texas, USA.

6.4.6 Targeted sequencing of MsmfusA1 and fusA2

In Msm, the translation elongation factor G (EF-G) is encoded by fusA1 (MSMEG_1400), while fusA2

(MSMEG_6535), which closely resembles fusA1, encodes EFG-2. Genomic DNA extracted from both

wild-type Msm and the four isolates was amplified and sequenced against both fusA1 and fusA2

using the following primers:

MsmfusA1 5’GCGTATCTGCTTCGTCAACA 3’ (forward primer)

MsmfusA1 5’ GATGCCGATGAAGTCGTTCT 3’ (reverse primer)

MsmfusA2 5’CTTCCTGCACTCTGTCCATA 3’ (forward primer)

MsmfusA2 5’ GCGCATGGATCTGATGTTCA 3’ (reverse primer)

For both fusA1 and fusA2, ~200 ng of the DNA template was used for each of four 50 μl reactions

containing 5 μl of 2 mM dNTPs, 1.5 μl of DMSO, 5 μl of buffer 10X with MgCl2), 5 μl of 10 μM each of

forward and reverse primers, 2 μl of Fastart Taq polymerase and dH20 to make up the volume to 50

μl. The reaction conditions included heating at 95 °C for 5 minutes, followed by 30 cycles of

incubations at 95 °C for 30 seconds, 65 °C for 30 s, 72 °C for 1 minute, and final extension at 72 °C for

7 minutes. The PCR products (2.1 kb for both genes) were analysed by gel electrophoresis using dye

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markers III and IV for confirmation of appropriate size, then stored at -20 oC awaiting sequencing.

The PCR products were purified and sequenced at the Central Analytical Facilities at Stellenbosch

University as a pay-for service. Primers for sequencing were designed every 400 base pairs (bp),

starting and ending 100 bp upstream and downstream respectively (Appendix 2). The sequence data

were analysed using CLC Main Workbench version 7 to align the sequences of the strains generated,

using M. smegmatis mc2155 (wild- type) as the reference strain to identify Single Nuclear

Polymorphisms (SNPs).

6.4.7 Targeted sequencing of Mtb fusA1

MtufusA1 (Rv0684) which encodes EF-G in Mtb was amplified using the following primers:

MtufusA1 5’ CTACCACGAGGTTGACTCCT 3’ (forward primer)

MtufusA1 5’ GGTTGTATCTACTCGCCAGT 3’ (reverse primer)

~200ng of the DNA template was used for each of four 50 μl reactions containing 5 μl of 2 mM

dNTPs, 1.5 μl of DMSO, 5 μl of buffer 10x with MgCl2), 5 μl of 10 μM forward and reverse primers,

2 μl of Fastart Taq polymerase and dH2O to make up the volume to 50 μl. The reaction conditions

included heating at 95 °C for 5 minutes, followed by 30 cycles of incubations at 95 °C for 30 seconds,

61 °C for 30 s, 72 °C for 1 minute, and final extension at 72 °C for 7 minutes. The PCR products (2.1

kb) so obtained were analysed by gel electrophoresis using dye markers III and IV for confirmation of

appropriate size, then stored at -20 oC awaiting sequencing. Appendix 1 illustrates the wild-type and

FAR DNA amplicons. The PCR products were purified and sequenced at the Central Analytical

Facilities at Stellenbosch University, South Africa. Primers for sequencing were designed every 300

bp, starting and ending 100 bp upstream and downstream respectively (Appendix 2). The sequence

data was analysed using CLC Main Workbench version 7 to align the sequences of the strains

generated, using M. tuberculosis H37Rv (MA) (wild-type) as the reference strain to identify SNPs.

6.5 Competition assay

The assay was adapted from a method described by Bhatter et al.14 An aliquot each of the drug

susceptible (wild-type) and FAR strains was separately inoculated into 10 ml of 7H9 medium were

inoculated separately in two culture flasks, to achieve an OD600 that corresponds to an approximate

cell density of 105 CFU/ml. These served as controls to demonstrate the individual strains were not

retarded in growth. Simultaneously, aliquots of each strain in equal amounts (approximately 0.5 x

105 CFUs/ml each) were co-inoculated into a fresh flask containing 10 ml of 7H9 medium. The

separately inoculated cultures were serially diluted on days 0, 2, 4, 6, 8 and 14 and plated in

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triplicate on agar plates (without FA). The mixed cultures were serially diluted and plated in triplicate

on 7H10 agar plates with and without FA at 10 µM on days 0, 2, 4, 6, 8 and 14. The agar plates were

incubated for 21 days before CFU enumeration.

6.5.1 Calculation of relative fitness

For each competition experiment, the mean of three CFUs was used to calculate the relative

competitive fitness. At each time point, the CFU counts on FA-containing plates indicated the

number of FAR cells in the mixed cultures. The number of susceptible cells was calculated by

subtracting the number of resistant cells from the total cell numbers revealed by CFU counts of the

drug-free agar plates. To calculate the relative competitive fitness W of the drug resistant strains,

the formula below was used:

W = ( ln(Rf ÷ Ri) ) ÷ (ln(Sf ÷ Si)

Ri and Si denote resistant and susceptible cells at day 0 respectively, while Rf and Sf denote resistant

and susceptible cells at day 14 respectively.

Total CFU = cells from mixed cultures plated on plain agar.

Resistant cells = cells from mixed cultures plated on FA-supplemented agar.

Susceptible cells: Total CFU – resistant cells

The results reported were the mean of two independent experiments.

6.6 Cytotoxicity assays

6.6.1 Cytotoxicity assay in CHO

Compounds were screened for in vitro cytotoxicity against Chinese Hamster Ovarian (CHO) cells, a

mammalian cell line, using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)

assay. The MTT assay is a colorimetric assay for the assessment of cellular growth and survival which

compares well with other available assays.15,16 The tetrazolium salt MTT was used to measure all

growth and chemosensitivity. The test samples were tested in triplicate on one occasion.

Stock solutions of the test samples were prepared by dissolving each compound in 100% DMSO to a

concentration of 20 mg/ml, which were then stored at -20 oC until use. Dilutions were prepared on

the day of the experiment. Emetine, a second-line antiprotozoal drug with low cytotoxicity, was used

as the reference drug in all experiments. The initial concentration of emetine was 100 μg/ml, which

was serially diluted in complete medium with 10-fold dilutions to give 6 concentrations, the lowest

being 0.001 μg/ml. The same dilution technique was applied to the all test samples. The highest

concentration of solvent to which the cells were exposed had no measurable effect on the cell

viability. The values of the concentration that inhibited 50% of the cell population (IC50) were

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obtained from full dose-response curves, using non-linear dose-response curve fitting analysis

generated using GraphPad Prism v.4 software.

6.6.2 Cytotoxicity in THP-1 cells

6.6.2.1 Maintenance of THP-1 cells

Cytotoxicity and intracellular drug efficacy studies were carried out using THP-1 cells (ATCC TIB-202)

from stocks maintained by the MMRU at -196 oC in liquid Nitrogen. These consisted of 1ml vials each

containing 1 x 105 cells/ml suspended in RPMI culture medium (Sigma-Aldrich) supplemented with

10% foetal bovine serum (FBS) or foetal calf serum (FCS). The FBS and FCS were procured heat-

inactivated and sterilized by filtration using 0.22 µm filters prior to addition to RPMI. 10% DMSO was

added as a cryopreservant. All experiments were carried out under sterile conditions in a biosafety

cabinet. Cells were passaged a maximum of 3 times and maintained without antibiotics and

antimycotics. Initial passage involved thawing a single vial of frozen stock of THP-1 cells thawed at

room temperature and added to 9 ml of culture medium in a vented T25 culture flask (Corning®).

The cell culture was incubated at 37 oC with 5% CO2. Every 3 days, cells were washed, counted and

split to achieve the appropriate volume of culture corresponding to the scale of the assays. To wash

the cells, cultures were centrifuged at 100 g or 150 g for 5 minutes at 20 oC then the old cell culture

medium was replaced with a fresh aliquot. To count cells, 10 µl of a 1:1 ratio of cell culture to trypan

blue® was added to each chamber in a counting slide, and counted using a TC20 ™ automated cell

counter (Bio-Rad). Only cultures with a minimum of 90% viability were used. Upon attaining a cell

count of 1 x 106 cells/ml, cultures were split into separate flasks containing an appropriate volume of

fresh medium, depending on the volume of culture required.

All assays were conducted in 96-well format (96-well flat-bottom microtiter plate (PGRE655180,

Lasec, SA), by seeding 100 µl of cell culture were a final count of 1 x 104 cells/ml per well. As the

outer wells are prone to evaporation, culture medium without cells was added to these wells. In

order for the cells to differentiate and adhere to the surface of the wells, the RPMI medium was

supplemented with phorbol myristate acetate (PMA) to a concentration of 200 nM, and the plates

were incubated over 24 hours. After cell differentiation, cells were washed twice with PBS, and cells

from representative wells were counted. To detach cells from the wells, 100 µl of chilled 5 mM EDTA

dissolved in PBS was added and the plates incubated for 15-20 minutes at room temperature prior

to cell counting.

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6.6.3 Macrophage cytotoxicity assay

Two-fold serial dilution of the compounds was performed in RPMI medium to a final concentration

of 200 M for each compound. Each compound was tested in triplicate by adding 100 μl of RPMI

containing compound to each well. The assay plates were incubated for 72 hours, after which 20 μl

of alamar blue was added to each well. After 24 hours, the cells were assessed for growth and

inhibition by visual inspection, defined as the lowest concentration of compound that inhibited

visible growth. The lowest concentration of drug which prevented the colour change of alamar blue

(from blue to pink) was considered the MIC that inhibited more than 90% of the bacterial population

(IC90).

6.7 Macrophage efficacy assay

6.7.1 Infection of THP-1 with Mtb

From a 1 ml freezer stock, Mtb was maintained in 7H9/OADC and sub-cultured once to attain an

OD600 of 0.5 (corresponds to approximately 1x 108 CFU/ml) on the day of infection. The culture was

washed twice by centrifugation at 4000 rpm and resuspended in RPMI medium. To achieve a

multiplicity of infection (MOI) of 10 bacilli:1 macrophage, the Mtb culture was diluted 100-fold to

achieve approximate 1 x 106 CFU/ml. 100 µl of this culture was added to each well of the assay plate

containing THP-1 cells. The plates were incubated for 3 to 4 hours at 37 oC with 5% CO2 to enable

uptake of bacteria by the macrophages.

Preparation of compounds

Prior to the experiment, a 10 mM stock solution of each test compound in DMSO was diluted using

RPMI culture medium, ensuring a final concentration of DMSO ≤ 0.5%. The concentrations tested for

each compound were 1X, 2X, and 5X MIC. Isoniazid at 2X MIC and untreated cells were used as

positive and negative controls respectively.

Treatment with compound

The cells were viewed under the microscope at 40x and 100x magnification to ensure bacterial

uptake. The cells were then washed twice with PBS before addition of 100 µl of RPMI medium

containing the test compounds. The cells were incubated at 37 oC with 5% CO2.The cell culture

containing compounds was replenished every two days. At days 0, 2, 4 and 6, cells were washed

twice with PBS and lysed using distilled water with 0.05% Tween®80. The plates were incubated at

room temperature for at least 10 minutes to ensure lysis. The lysates were serially diluted 10-fold in

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standard 7H9 growth medium and plated on 7H10.The plates were then incubated for 21 to 28 days

prior to CFU enumeration.

6.8 Evaluation of mycobacterial metabolism of FA and selected FA analogues

Three flasks each containing 50 ml of GAST/Fe were inoculated with Mtb H37Rv MA and grown to

an O.D600 of 0.5, after which the cultures were centrifuged at 4000 rpm for 10 minutes at room

temperature. The supernatant was discarded and the pellets resuspended in an equal volume of PBS

(0.05% Tween®80) pH 7.4. After a second cycle of centrifugation, the supernatant was discarded and

the pellets were resuspended in a 10-fold lower volume of PBS. The bulk culture was split into 5 ml

aliquots in tubes (Corning™ Falcon™) ensure triplicate samples for each compound to be tested. For

the heat-killed cultures, the washing procedure above was preceded by heating the GAST/Fe

cultures in a water bath at 80 oC for 1 hour.

10 mM DMSO stocks of each compound were diluted to achieve a concentration of 0.5X MIC and a

DMSO ≤ 1% in the final PBS cultures. After each compound was added in triplicate, the samples were

incubated at 37 oC in a shaking incubator. At 0, 6, 12, 24 and 48 hours, 100 µl was transferred from

each sample tube to a deep well 96-well plate (Nunc™) containing 300 µl of ice-cold acetonitrile

containing 200 nM of ME-29 (Figure 6.4), a fusidic acid derivative well-characterized by LC-MS as an

internal standard. This was carried out in order to precipitate protein and extract the compounds.

Figure 6.4: Structure of ME-29.

The plates were centrifuged at 4000 rpm for 10 minutes at 4 oC, and then stored at -20 oC awaiting

LC-MS analysis.

183

Prior to LC-MS analysis, the 96-well plates were centrifuged at 5000 g and the supernatants

transferred to HPLC vials. LC-MS/MS analysis was performed using an ABsciex 4000 QTrap Mass

spectrometer coupled to an Agilent 1200 HPLC. Chromatography was performed on Kinetex C6

Phenyl column (4.6 x 50 mm, 5 µM particles) using 5 mM ammonium acetate in water as the

aqueous mobile phase and 5mM ammonium acetate in acetonitrile as the organic mobile phase. The

MS runs were performed in electrospray positive mode, monitoring transitions from the derivatives

of interest, as well as fusidic acid, 3-ketofusidic acid and fusidic acid lactone, so as to allow for the

detection of any metabolites formed during incubation. Analyst 1.5 was used for instrument control

and data acquisition, and data processing was performed with Microsoft Excel 2013. The ratio of the

analyte to internal standard with time was then plotted to give a measure of the stability of the

compound over time.

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Appendix 1: genomic DNA amplified from the FAR Mtb mutant strain GKFA

186

Appendix 2: Primers used for targeted sequencing of PCR amplicons

Primers for sequencing PCR product of MsmfusA1

Primer ID Sequence 5’3’ Length Forward/reverse

MsmfusA1_F1 GGTGTGAAGAACCGCAAGCA 20 forward

MsmfusA1_F2 CAGCGTCGTGAGAAGACCAT 20 forward

MsmfusA1_F3 CAGAAGGACGTGCTTACCGA 20 forward

MsmfusA1_F4 GCGTATCTGCTTCGTCAACA 20 forward

MsmfusA1_F5 GCACTGCTGGAGAAGTACTT 20 forward

MsmfusA1_F6 GGCAAGCTGTTCCAGATGCA 20 forward

MsmfusA1_R1 GGCACCGGTGATCATGTTCT 20 reverse

MsmfusA1_R2 GATGTGTTCAGTTGTGCGGT 20 reverse

MsmfusA1_R3 CAGGTCACCGATCACTTCA 19 reverse

MsmfusA1_R4 GATGAGCACCTTCGCGAACT 20 reverse

MsmfusA1_R5 CTTGTTGGCGTGCATCTGGA 20 reverse

MsmfusA1_R6 CCTTGT TCTTGAACGCGCT 19 reverse

MsmfusA1_R7 CGATGAAGTCGTTCTCGGCA 20 reverse

Primers for sequencing PCR product of MsmfusA2

Primer ID Sequence 5’3’ Length Forward/reverse

MsmfusA2_F1 GACCTGAACAAGGTCCGCA 19 Forward

MsmfusA2_F2 GCAGCGCGTTCAAGAACAA 19 Forward

MsmfusA2_F3 GGTCTGAAGGACACCACCA 19 Forward

MsmfusA2_F4 GAGTTCAAGGTCGAGGCCA 19 Forward

MsmfusA2_F5 CTGGTGAACCTGAAGGTGA 19 Forward

MsmfusA2_F6 GCGAACTACTCCATGGTGTT 20 Forward

MsmfusA2_R1 GATGATCTCCTTCGACACGT 20 Reverse

MsmfusA2_R2 CCTTCTTCAACGCCTGTGAA 20 Reverse

MsmfusA2_R3 GACCTTGTTCTCGAACTCGT 20 Reverse

MsmfusA2_R4 GCTTCTCCTGGTCACTCTT 19 Reverse

MsmfusA2_R5 GGAACCGGATTCCACGACA 19 Reverse

MsmfusA2_R6 CGAAGTACTTCTCCAGCAGT 20 Reverse

MsmfusA2_R7 GATCTGGTTGTTGTTCCAGA 20 Reverse

187

Primers for sequencing PCR product of MtbfusA1

Primer ID Sequence 5’3’ Length Forward/reverse

MtbfusA1_F1 CTCATGTCCTGAAGGGACTT 20 Forward

MtbfusA1_F2 GAGAGTTGCAAAGGTGTGGT 20 Forward

MtbfusA1_F3 CTCGAACTTCTCGACATCAT 20 Forward

MtbfusA1_F4 CATCGAACAAAACCCGGAGA 20 Forward

MtbfusA1_F5 GGTTTCGAGTTCGTCGACAA 20 Forward

MtbfusA1_F6 CGACGAAGATCAACCTGCT 19 Forward

MtbfusA1_R1 GCGCATGGATCTGATGTTC 19 Reverse

MtbfusA1_R2 GACATCGAACGCAGATCGAT 20 Reverse

MtbfusA1_R3 GTGACACCCTTCTCCATCT 19 Reverse

MtbfusA1_R4 GCTGATGATGTCGAGAAGTT 20 Reverse

MtbfusA1_R5 CACCACACCTTTGCAACTCT 20 Reverse

MtbfusA1_R6 GTTCACCTTGATCCCGTTGT 20 Reverse

MtbfusA1_R7 CGATGTGACCTACAGCACAA 20 Reverse

188

Appendix 3: Checkerboard assay plate of FA tested against the Mtb fusA knockdown strain

189

Appendix 4: COSY NMR spectrum of AW23