2 studies of the mode of action in 3 4 gregory t...

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In Vitro Evaluation of CBR-2092, a Novel Rifamycin-Quinolone Hybrid Antibiotic: 1

Studies of the Mode of Action in Staphylococcus aureus 2

3

Gregory T. Robertson, Eric J. Bonventre†, Timothy B. Doyle

‡, Qun Du, Leonard 4

Duncan‡, Timothy W. Morris

#, Eric D. Roche

§, Dalai Yan

¶ and A. Simon Lynch* 5

6

Cumbre Pharmaceuticals Inc., 1502 Viceroy Drive, Dallas, Texas 75235-2304. 7

8

*Corresponding author: 9

A. Simon Lynch 10

Cumbre Pharmaceuticals Inc. 11

1502 Viceroy Drive, Dallas, TX 75235-2304 12

Phone: (214) 631-4700 ext. 7510. 13

Fax: (214) 631-4710 14

E-mail: [email protected] / [email protected] 15

16

Present addresses: Department of Pharmacology, University of Texas Southwestern 17

Medical Center, Dallas, TX 75390†, Vertex Pharmaceuticals Inc., Coralville, IA 52241

‡, 18

Bausch & Lomb Inc., Rochester, NY 14609#, Healthpoint Ltd., Fort Worth, TX 76107

§, 19

Department of Microbiology & Immunology, Indiana University School of Medicine, 20

Indianapolis, IN 46202¶. 21

Running title: Mode-of-Action of CBR-2092 in S. aureus 22

Keywords: Rifampin, quinolone, hybrid antibiotic, RNA polymerase, 23

DNA topoisomerase24

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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.01649-07 AAC Accepts, published online ahead of print on 28 April 2008

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ABSTRACT 25

Rifamycins have proven efficacy in the treatment of persistent bacterial 26

infections. However, the frequency with which bacteria develop resistance to 27

rifamycin agents restricts their clinical use to antibiotic combination regimens. In a 28

program directed toward the synthesis of rifamycins with a lower propensity to 29

elicit resistance development, a series of compounds were prepared that covalently 30

combine rifamycin and quinolone pharmacophores to form stable hybrid 31

antibacterial agents. Herein we describe mode-of-action studies in Staphylococcus 32

aureus for CBR-2092, a novel hybrid that combines the rifamycin SV and 4H-4-oxo-33

quinolizine pharmacophores. In biochemical studies, CBR-2092 exhibits rifampin-34

like potency as an inhibitor of RNA polymerase and is an equipotent inhibitor of 35

DNA gyrase and DNA topoisomerase IV with retention of activity against a 36

prevalent quinolone-resistant variant. Macromolecular biosynthesis studies confirm 37

that CBR-2092 has rifampin-like effects on RNA synthesis in rifampin-susceptible 38

strains and quinolone-like effects on DNA synthesis in rifampin-resistant strains. 39

Studies of mutant strains that exhibit reduced susceptibility to CBR-2092 further 40

substantiate RNA polymerase as the primary cellular target of CBR-2092 with DNA 41

gyrase and DNA topoisomerase IV as secondary and tertiary targets, respectively, in 42

strains exhibiting pre-existing rifampin-resistance. In contrast to quinolone 43

comparator agents, no strains with altered susceptibility to CBR-2092 were found to 44

exhibit changes consistent with altered efflux properties. The combined data 45

indicate that CBR-2092 may have potential utility in monotherapy for the treatment 46

of persistent S. aureus infections. 47

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INTRODUCTION 48

Antibiotics of the rifamycin class exhibit potent antimicrobial activity against an 49

array of gram-positive bacteria including the Mycobacteria, and have been used globally 50

for the treatment of tuberculosis (TB). Rifamycins, however, exert their antibacterial 51

activity as inhibitors of a single enzyme target - DNA-dependent RNA polymerase - and 52

a variety of single point mutations in the rpoB gene (encoding the β subunit of the 53

enzyme) give rise to strains that exhibit highly elevated MICs (7, 12). This resistance 54

development liability limits the approved use of rifamycin class agents to combination 55

regimens. 56

Rifamycins, alone or in combination, exhibit activity against susceptible bacteria 57

propagated in the biofilm state, including data from in vitro biofilm assays (3, 28, 32, 37, 58

41) and animal models of biofilm-associated infections (6, 19, 20, 37). In addition, 59

clinical studies of rifampin in combination with fluoroquinolones (24, 36, 42, 43), 60

vancomycin (21, 38), fusidic acid (38) and amoxicillin (38) have led to the adoption of 61

specific rifampin-containing regimens as standard therapies for the treatment of biofilm-62

associated infections of indwelling medical devices (5). 63

The efficacy of the rifamycins in the treatment of biofilm-associated infections and 64

other persistent or latent infections that are often recalcitrant to other antibiotics is likely 65

explained by two distinct features. First, at the mechanistic level, the transcription 66

process is thought to be essential for the establishment and maintenance of bacteria in 67

alternate survival modes including biofilms and metabolically quiescent states typical of 68

latent cell populations. Second, at the physicochemical level, rifamycin agents exhibit 69

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excellent tissue distribution (1), efficiently penetrate biofilms formed in vitro (41), and 70

exhibit good activity against a number of obligate or facultative intracellular pathogens. 71

In a program directed toward the synthesis of rifamycin derivatives with improved 72

resistance development properties, a series of compounds was prepared in which 73

rifamycin and quinolone pharmacophores are covalently combined. The design of these 74

rifamycin-quinolone hybrids was such that they act as stable, dual-pharmacophore agents 75

and therein not active as pro-drugs. This strategy should ensure matched 76

pharmacokinetics, pharmacodynamics and tissue distribution of the composite 77

pharmacophores. In CBR-2092, the rifamycin SV pharmacophore is combined with a 78

quinolone pharmacophore derived from the 4H-4-oxo-quinolizine (or ‘2-pyridone’) 79

subfamily of fluoroquinolones that exhibit balanced (equipotent) activity against both 80

DNA gyrase and DNA topoisomerase IV and retain activity against ciprofloxacin-81

resistant strains (26). In an accompanying article (30), we describe the results of 82

microbiology studies to characterize the in vitro profile of activity of CBR-2092 against 83

staphylococci and streptococci. Herein we describe the results of biochemical, cell 84

biology and genetic studies undertaken to characterize the mode-of-action of CBR-2092 85

in a primary target pathogen, Staphylococcus aureus. 86

(Portions of this work were previously presented [47th

Intersci. Conf. Antimicrob. 87

Agents and Chemother., abstr. F1-2101, 2007].) 88

89

MATERIALS AND METHODS 90

Antimicrobial Agents. CBR-2092 and ABT-719 (A-86719.0) were synthesized at 91

Cumbre Pharmaceuticals Inc. Rifampin, ethidium bromide and reserpine were purchased 92

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from Sigma-Aldrich (St. Louis, MO). Ciprofloxacin, levofloxacin, gatifloxacin and 93

nadifloxacin were purchased from LKT Laboratories (St. Paul, MN). 94

Bacterial strains. Table 1 shows relevant details of a series of derivatives of S. 95

aureus ATCC 29213 (CB190) or RN4220 (CB1244) that exhibit stable resistance to 96

agents of the rifamycin and/or quinolone classes that were isolated and characterized as 97

described below. 98

Isolation and characterization of resistant mutants. Single-step selections of 99

antibiotic resistant mutants were undertaken by standard agar-based methods. In all 100

cases, mutants were purified through drug-free passage and the initial antibiotic 101

resistance phenotype then verified to ensure that stable, true-breeding mutants had been 102

obtained. Step-wise passage for multi-step resistance selection was undertaken in glass 103

tubes with MHII medium plus or minus 0.002% (vol:vol) Polysorbate-80 (P-80) and were 104

inoculated with 106 CFU/mL at sub-MIC doses of test agents; see legends to Tables 5A 105

and 5B for details. Cultures were incubated with shaking at 37°C for 20-24 hours. 106

Thereafter, the cell inoculum was prepared from the highest consecutive drug 107

concentration which had supported growth to an absorbance equivalent to or greater than 108

≈108 CFU per mL. Daily passages were performed until compound solubility issues 109

limited further elaboration of individual step-selection studies, or it was apparent that a 110

terminal resistance endpoint had been attained in specific selections. Genotypic analysis 111

of strains exhibiting stable resistance phenotypes was undertaken by standard methods 112

employing PCR amplification and DNA sequencing of target loci. Assessment of the 113

contribution of mutations in efflux pathways towards altered antibiotic-resistance 114

phenotypes was undertaken by determining altered sensitivity to the unrelated efflux 115

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substrate ethidium bromide and/or whether quinolone susceptibility was impacted by 116

reserpine. 117

Biochemical studies. Recombinant forms of S. aureus σA RNA polymerase 118

holoenzyme and a rifampin-resistant RpoB (H481Y) variant were prepared as previously 119

described (27). Inhibition of transcription of S. aureus σA RNA polymerase holoenzymes 120

was measured in single-round G-less cassette assays that employed the pGL2B-T7A1-G-121

less template (27). Quantitative gel electrophoresis of a single 272 nucleotide G-less 122

RNA product species was used to determine the minimal concentration necessary to 123

inhibit 50% (IC50) of the holoenzyme-specific RNA product formed in the absence of test 124

agents. Derivatives of the pET28b vector (Novagen Inc.) were constructed to express 125

recombinant forms of the S. aureus GyrA or GyrA(S84L) and ParC or ParC(S80F) 126

subunits bearing amino-terminal oligo-histidine6 and T7-Tag epitopes and carboxyl-127

terminal FLAG epitope tags. The previously described vectors pTrcHisB-SA-GyrB and 128

pTrcHisA-SA-GrlB (18) were used for expression of recombinant variants of the S. 129

aureus GyrB and ParE (GrlB) proteins, respectively, bearing amino-terminal oligo-130

histidine6 affinity tags. In all cases, the recombinant subunits were purified from E. coli 131

Rosetta cells (Novagen Inc.) by immobilized metal affinity chromatography (IMAC). 132

Peak fractions eluted from the IMAC resin via imidazole gradient were identified by 133

SDS-PAGE, pooled and further purified by combination of size-exclusion 134

chromatography, affinity chromatography and/or dialysis. DNA topoisomerase 135

holoenzymes were then reconstituted through combination of the purified subunits at 136

ratios optimized for specific activity to yield either wild-type or quinolone-resistant forms 137

of DNA gyrase and DNA topoisomerase IV. For the studies described herein, DNA 138

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gyrase preparations exhibited specific activity in the range of 200 [GyrA (S84L):GyrB] to 139

250 [GyrA:GyrB] units per µg, wherein 1 unit is equivalent to the minimal enzyme 140

necessary to effect the supercoiling of 150 ng of relaxed pBR322 DNA in 1 hr at 37ºC. 141

Wild-type [ParC:ParE] and mutant [ParC (S80F):ParE] preparations of DNA 142

topoisomerase IV enzymes exhibited specific activity of 25-30 units per µg (wherein 1 143

unit is equivalent to the minimal enzyme necessary to effect the relaxation of 150 ng of 144

supercoiled pUC19 DNA in 1 hr at 37ºC). Assays to measure the inhibition of the in 145

vitro activity of Type II topoisomerases employed relaxed (DNA gyrase) or negatively 146

supercoiled (DNA topoisomerase IV) forms of covalently closed DNAs (ccDNAs) as 147

substrates. Assays of DNA gyrase were undertaken in 50mM Tris-HCl (pH 7.5), 50mM 148

potassium glutamate, 5mM MgCl2, 5mM DTT, 1mg/mL acetylated BSA, 1.5mM ATP, 149

15 µg/mL relaxed pBR322 DNA and 100 µg/mL E. coli tRNA for one hour at 37ºC. 150

Assays of DNA topoisomerase IV were undertaken in 50mM Tris-HCl (pH 7.5), 50mM 151

potassium glutamate, 5mM MgCl2, 5mM DTT, 1mg/mL acetylated BSA, 1.5mM ATP, 152

15 µg/mL supercoiled pUC19 DNA and 100 µg/mL tRNA for one hour at 37ºC. In all 153

cases, reactions were terminated by the addition of sodium dodecyl sulfate (SDS) and 154

proteinase K. Quantitative gel electrophoresis of the linear DNA products species was 155

employed to determine the minimal concentration of inhibitor necessary to induce 50% 156

cleavage over background of a ccDNA substrate (CC50) with mean values calculated 157

from ≥ 2 independent assays. 158

Macromolecular biosynthesis assays. Effects of test agents on macromolecular 159

biosynthesis in S. aureus were determined using assays wherein the incorporation of 160

specific radiolabeled precursors was measured in a time-course fashion. The radiolabels 161

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employed were [Methyl-3H]-Thymidine (Perkin Elmer # NET-027) for DNA synthesis, 162

[5,6-3H]-Uridine (Perkin Elmer # NET-367) for RNA synthesis, L-[3,4,5-3H]-Leucine 163

(Perkin Elmer # NET-460) for protein syntheis and [2,3-3H]-D-Alanine (American 164

Radiolabeled Chemicals # ART-179) for cell wall synthesis. At each time point, the ratio 165

of radiolabel incorporated in the presence of test agent relative to that of the DMSO-166

treated control culture was used to determine the percent incorporation. Radiolabel 167

incorporated at time zero (T0) was set to 100%. 168

Antimicrobial susceptibility testing. Determination of MICs was done in 169

accordance with Clinical Laboratory Standards Institute (CLSI) methodology by either 170

the broth microdilution or agar dilution methods using cation adjusted Mueller Hinton 171

(MHII) as a base medium (10). Unless otherwise indicated, microdilution broth assays 172

employed MHII medium supplemented with 0.002% (vol:vol) P-80. 173

174

RESULTS 175

Structure of CBR-2092 and related agents. In a program directed toward the 176

synthesis and evaluation of rifamycin-based hybrid antibiotics, a series of compounds 177

was prepared in which rifamycin and quinolone pharmacophore were covalently joined. 178

In total, approximately 300 rifamycin-quinolone hybrids were synthesized in an effort 179

that entailed combination of different rifamycin backbone scaffolds with quinolones 180

representative of various sub-series, including experimental 4th

generation quinolone 181

pharmacophores. Structure-Activity-Relationships (SARs) derived from data generated 182

in a range of assays described herein revealed that the potency of the quinolone entity 183

was a critical parameter in terms of the retention of antimicrobial activity of rifamycin-184

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quinolone hybrid agents against strains exhibiting rifampin-resistance. Of the 4th

185

generation quinolone pharmacophores tested, members of the experimental 4H-4-186

oxoquinolizine subfamily (25) proved one of the most promising. In CBR-2092 (Fig. 1), 187

the rifamycin SV pharmacophore is combined via a chiral linking group with a quinolone 188

entity from the 4H-4-oxoquinolizine subfamily (25); for comparison, also shown are the 189

structures of rifampin, ciprofloxacin and a representative 4H-4-oxoquinolizine (ABT-190

719) that were employed herein as comparator agents. ABT-719 was studied as a 191

comparator as the most extensively characterized member of the 4H-4-oxoquinolizine 192

series and exhibits equipotent (balanced) biochemical activity against DNA gyrase and 193

DNA topoisomerase IV enzymes, potent antimicrobial activity against both Gram-194

positive and Gram-negative pathogens including ciprofloxacin-resistant isolates of S. 195

aureus, and is efficacious in rodent infection models (26). 196

Biochemical studies of CBR-2092 and comparator agents. As shown in Table 2, 197

CBR-2092 retains rifampin-like potency against wild-type S. aureus RNAP with an IC50 198

of 0.034µM – approximately two-fold less active than rifampin (0.015µM). In contrast, 199

both rifampin and CBR-2092 exhibit no detectable activity (IC50’s of >25 µM) against a 200

mutant form of the RNAP enzyme bearing a high-level rifampin-resistant RpoB (H481Y) 201

subunit. These combined data suggest that appendage of the 4H-4-oxoquinolizine 202

pharmacophore via the 3’-position of the rifamycin SV scaffold does not significantly 203

impact the ability of CBR-2092 to interact with RNA polymerase in a manner similar to 204

that of rifampin. 205

As also shown in Table 2, biochemical studies indicate that CBR-2092 exhibits 206

equipotent (balanced) activity against wild-type S. aureus DNA topoisomerase IV and 207

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DNA gyrase enzymes with 50% cleavage concentrations (CC50’s) of 1.7 and 1.5 µM, 208

respectively, that are in the same potency range as ciprofloxacin and gatifloxacin and 209

correspond to a CC50 ratio for DNA gyrase/DNA topoisomerase IV of 0.9. The apparent 210

target balance of CBR-2092 for wild-type topoisomerase IV and DNA gyrase is 211

improved over that of ciprofloxacin (CC50 ratio of 11.5) and gatifloxacin (CC50 ratio of 212

3.2) and is similar to that of the ABT-719 compound for which we observed a CC50 ratio 213

of 1.5 and for which a value of 2.1 has previously been reported (33). However, in 214

contrast to all of the other fluoroquinolone comparators tested, CBR-2092 maintains its 215

activity against a prevalent fluoroquinolone-resistant variant of DNA topoisomerase IV 216

with CC50 values of 2.7 and 1.7 µM determined for ParC (S80F) and wild-type variants, 217

respectively. These data yield a CC50 ratio for CBR-2092 for DNA topo IV ParC 218

(S80F)/DNA topo IV (ParCWT

) of 1.6 that is improved over ciprofloxacin (77.5), 219

gatifloxacin (59.1) and ABT-719 (18.3). Retention of the activity of CBR-2092 against 220

the ParC (S80F) variant of DNA topoisomerase IV was considered a key attribute of the 221

quinolone-driven activity of the molecule as this mutation most commonly underlies 222

target mediated resistance in ciprofloxacin-resistant isolates of S. aureus. 223

Activity of CBR-2092 against S. aureus strains exhibiting rifampin and/or 224

quinolone resistance. Table 3 shows that activity of CBR-2092 and comparator agents 225

against an otherwise-isogenic set of derivatives of S. aureus CB190 (ATCC 29213) that 226

bear all possible combinations of a high-level rifampin-resistance mutation rpoB 227

(H481Y) and the prevalent quinolone-resistance mutations parC (S80F) and gyrA 228

(S84L). For CBR-2092, MIC endpoints are shown here and elsewhere from assays 229

conducted in both the agar dilution and broth microdilution formats, with the latter 230

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conducted using MHII medium supplemented with 0.002% (vol:vol) Polysorbate-80 (P-231

80). As observed with a number of other semi-synthetic antibiotics, CBR-2092 exhibits 232

apparent high avidity for plastic and glass materials. In the broth microdilution format, 233

P-80 minimizes surface loss of CBR-2092 to the plastic surface of the assay plates 234

(Robertson, Du & Lynch, unpublished) and improves the concordance of MIC endpoints 235

with those determined by the agar dilution method (wherein it is assumed that such 236

compound loss is minimized by slower diffusion through the agar matrix). 237

As anticipated from past studies, the wild-type S. aureus strain CB190 (ATCC 29213) 238

strain and its rifamycin-resistant derivative CB370 [rpoB (H481Y)] were observed to 239

exhibit marked difference in their susceptibility to rifampin with the MIC against CB370 240

(>250 µg/mL) >31,250 times higher than determined for CB190 (0.008 µg/mL). In 241

contrast, the difference in MICs observed for these two strains with CBR-2092 is 242

markedly smaller, with the microbroth MIC against CB370 (0.12 µg/mL) only some 8 243

times higher than determined for CB190 (ATCC 29213) (0.015 µg/mL). Similarly, as 244

anticipated from past studies, the wild-type S. aureus strain CB190 (ATCC 29213) and 245

its fluoroquinolone-resistant derivative CB814 [gyrA (S84L), parC (S80F)] were 246

observed to exhibit marked differences in their susceptibility to fluoroquinolone agents. 247

For instance, the ciprofloxacin MIC against CB814 (16 µg/mL) is observed to be ~67 248

times higher than determined for CB190 (ATCC 29213) (0.25 µg/mL). In contrast, no 249

difference in potency is observed with CBR-2092 against these two strains with 250

microbroth MICs of 0.015 µg/mL observed for both CB814 and CB190. These 251

combined data indicate that the primary antimicrobial activity of CBR-2092 is conferred 252

by its rifamycin pharmacophore. 253

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These data are also informative about the nature of the fluoroquinolone activity of the 254

tested agents and, in particular, in delineating the relative contributions of cellular 255

inhibition of each of the Type II DNA topoisomerase targets. Ciprofloxacin shows the 256

anticipated pattern of activity against this genetically-defined strain panel with 8-fold 257

differences in MICs observed between otherwise isogenic strains bearing either the gyrA 258

(S84L) or parC (S80F) resistance determinants. Analysis of the MICs determined for 259

CBR-2092 against the four rifamycin-resistant strains in the panel suggest that CBR-2092 260

is a relatively well balanced inhibitor of both Type II DNA topoisomerase targets in S. 261

aureus with a possible slight preference for DNA gyrase. The mechanistic basis 262

underlying the antimicrobial activity that CBR-2092 exhibits against the CB815 [rpoB 263

(H481Y), gyrA (S84L), parC (S80F)] strain cannot be directly determined from these 264

data. However, as CBR-2092 exhibits near equivalent activity in vitro as an inhibitor of 265

both wild-type and fluoroquinolone-resistant forms of S. aureus DNA topoisomerase IV 266

(Table 2), it seems reasonable to assume that the activity of CBR-2092 against strain 267

CB815 is mediated via residual effects on DNA topoisomerase IV. 268

Effects of CBR-2092 and comparator agents on macromolecular biosynthesis. 269

The effects of rifampin, ciprofloxacin and CBR-2092 on the four macromolecular 270

biosynthesis pathways studied in CB190 (ATCC 29213) are shown in Figure 2. As 271

anticipated, rifampin has a primary effect on de novo RNA synthesis with a delayed, 272

secondary effect on protein synthesis and tertiary and less significant overall effects on 273

cell wall and DNA synthesis. Similarly, consistent with past studies, ciprofloxacin has a 274

primary effect on de novo DNA synthesis with minimal effects on the other pathways 275

studied. In contrast, CBR-2092 exhibits intermediary effects that are somewhat distinct 276

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from either rifampin or ciprofloxacin. Like rifampin, CBR-2092 has a primary effect on 277

de novo RNA synthesis with delayed, secondary effects on protein and cell wall 278

synthesis. However, CBR-2092 also appears to have a secondary effect on DNA 279

synthesis that is more pronounced than the effect observed with rifampin and likely 280

reflects the secondary mode of action conferred by the quinolone pharmacophore. 281

Fig. 2 also shows data from equivalent studies undertaken in the rifampin-resistant 282

strain CB370 [rpoB (H481Y)]. As expected, rifampin has no significant effects at the 283

concentration tested (equivalent to 4× MIC for CB190 (ATCC 29213)) on any of the four 284

macromolecular biosynthesis pathways studied while ciprofloxacin continues to exhibit a 285

primary effect on de novo DNA synthesis. In contrast, CBR-2092 now appears to have a 286

primary effect on de novo DNA synthesis with minimal effects on the other pathways 287

studied. Also shown in Fig. 2 are the effects of the same compounds on macromolecular 288

biosynthesis in the quinolone-resistant strain CB814 [gyrA (S84L), parC (S80F)]. As 289

expected, ciprofloxacin has no apparent effects on de novo DNA synthesis or other 290

pathways while rifampin exhibits an activity profile that is essentially similar to that 291

observed with the CB190 (ATCC 29213) strain. CBR-2092 also exhibits effects on the 292

four macromolecular pathways in CB814 that are similar to those observed in the CB190 293

(ATCC 29213) strain with a primary effect on RNA synthesis and a somewhat 294

diminished effect on DNA synthesis that more closely resembles that of rifampin. 295

Finally, at the concentrations tested here, neither rifampin, nor CBR-2092 nor 296

ciprofloxacin had discernible effects on the macromolecular pathways studied when 297

tested in the CB815 [rpoB (H481Y), gyrA (S84L), parC (S80F)] strain (data not shown). 298

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Single-step resistance studies with CBR-2092. To further characterize the mode of 299

action of CBR-2092 in S. aureus, we undertook a series of single-step genetic selections 300

wherein spontaneous mutants that exhibit reduced susceptibility to CBR-2092 were 301

isolated and chacterized. As summarized in Table 4A, a series of agar based selections 302

undertaken with the rifampin-sensitive CB190 (ATCC 29213) strain and CBR-2092 in 303

the concentration range 0.08 to 0.12 µg/mL (5- to 8-fold MIC) gave rise to 28 304

independently isolated “first step” mutants that exhibit elevated MICs for CBR-2092 and 305

rifampin in the ranges 0.12 to 0.5 µg/mL and 0.25 to > 64 µg/mL, respectively, but 306

exhibited no apparent change in their susceptibility to ciprofloxacin. The recovery of 307

such first step resistance mutations at the indicated concentrations, but not at higher drug 308

concentrations, is wholly consistent with the anticipated low spontaneous resistance 309

potential of CBR-2092 at drug concentrations above the mutant prevention concentration 310

where the secondary antimicrobial activity of CBR-2092 exerts its effects; see 311

accompanying article (30). Genotypic characterization of these strains revealed a series 312

of mutations corresponding to amino acid substitution or insertion mutations that span 313

residues Gln468 to His481 in the RpoB subunit of RNA polymerase and therein overlap 314

with Cluster I of the previously characterized rifampin-resistance determining region 315

(RRDR) of S. aureus and other pathogens. These genetic data are again wholly 316

consistent with the notion that the primary antimicrobial activity of CBR-2092 in wild-317

type S. aureus is mediated by its rifamycin pharmacophore. 318

As shown in Table 4B, a series of agar-based selections undertaken with the rifampin-319

resistant strains CB370 [rpoB (H481Y)] or CB1522 [rpoB (H481Y)] and CBR-2092 in 320

the concentration range 0.1 to 0.25 µg/mL (1- to 2-fold MIC) gave rise to a series of 9 321

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independently isolated mutants that exhibit elevated MICs for CBR-2092 in the range 0.5 322

to 2 µg/mL. Genotypic characterization of these strains revealed a series of mutations 323

corresponding to amino acid substitutions in the gyrA gene that with one exception [gyrA 324

(S84L)] lie distal to the canonical quinolone-resistance determining region (QRDR) of 325

the S. aureus gyrA gene. Further characterization of these strains revealed minimal (1- to 326

2-fold) shifts in MICs for a variety of fluoroquinolone agents (data not shown). In 327

contrast, selections undertaken with CB370 [rpoB (H481Y)] and ABT-719 (0.05 µg/mL) 328

or gatifloxacin (0.2 µg/mL) gave rise to a series of independently isolated mutants that 329

exhibited 4 to 8 fold shifts in ciprofloxacin MICs and were found to all possess 330

previously described QRDR mutations in either the parC or parE loci, encoding the 331

subunits of DNA topoisomerase IV (data not shown). These combined data are 332

consistent with the notion that, in contrast to gatifloxacin and ABT-719, the preferred 333

DNA topoisomerase target of CBR-2092 in S. aureus is DNA gyrase. 334

As is also shown in Table 4B, a series of selections undertaken with strains CB809 335

[rpoB (H481Y), parC (S80F)], CB812 [rpoB (H481Y), gyrA (S84L)] and CB815 [rpoB 336

(H481Y), gyrA (S84L), parC (S80F)] and CBR-2092 in the concentration range 0.2 to 337

0.8 µg/mL (1- to 8-fold MIC) gave rise to a series of independently isolated mutants that 338

exhibited elevated MICs for CBR-2092 in the range 1 to >16 µg/mL. Genotypic 339

characterization of these strains revealed a series of mutations corresponding to amino 340

acid substitutions in parE, parC and gyrA including both previously described QRDR 341

mutations and a further series of novel mutations. Importantly, when strains that possess 342

both the rpoB (H481Y) and gyrA (S84L) mutations were employed, CBR-2092 selections 343

resulted exclusively in mutations in the ParC or ParE subunits of DNA topoisomerase IV 344

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including mutations outside of the previously described QRDR for DNA topoisomerase 345

IV in S. aureus. These data further substantiate DNA topoisomerase IV as the secondary 346

DNA topoisomerase target of CBR-2092 in S. aureus. 347

Finally, to determine the potential contribution of efflux class mutations in CBR-348

2092-selected mutants, MICs for the efflux substrate ciprofloxacin were determined in 349

the presence and absence of the efflux pump inhibitor reserpine. In no case was a 350

significant (>2-fold) shift in ciprofloxacin MIC observed in the presence of reserpine 351

(data not shown). Likewise, no significant shifts in MIC values were apparent for any 352

CBR-2092-selected mutants when tested against minocycline, a structurally unrelated 353

antibiotic that is subject to additional efflux pathways in S. aureus (data not shown). 354

These data suggest that efflux mechanisms do not contribute in mutational mechanisms 355

associated with first- or second-step resistance to CBR-2092 in S. aureus. 356

Multi-step passage selections undertaken with CBR-2092 and comparator agents 357

with CB190 (ATCC 29213). To delineate the genetic mechanism(s) by which a wild-358

type, drug-naïve strain of S. aureus [CB190 (ATCC 29213)] may develop higher level 359

resistance to CBR-2092, we also undertook studies involving the step-wise enrichment of 360

mutants with reduced susceptibility through serial passage in MHII broth medium 361

starting from a sub-MIC concentration range. Parallel studies were also undertaken with 362

rifampin, ciprofloxacin, and ABT-719. Table 5A shows the characterization of 363

intermediary and/or terminal isolates from each study. 364

Consistent with past literature precedents, step selections undertaken with the 365

comparator agents over the course of 2-15 days resulted in the isolation of strains that 366

exhibit resistance to benchmark agents of the parental classes that is elevated > 31,250-367

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fold (rifampin, 2 days), > 500-fold (ciprofloxacin, 15 days) and > 250-fold (ABT-719, 15 368

days; data not shown) relative to those of the parental CB190 (ATCC 29213) strain. 369

Further, in the selections with fluoroquinolone agents, it is apparent that efflux 370

mechanisms contributed to the development of stable resistance to the test agents as 371

ethidium bromide MICs were elevated 16-fold (see Table 5A). 372

Step selections undertaken with CBR-2092 over the course of 26 days of serial, step-373

wise passage enrichment resulted in the isolation of a strain (CB1884) that exhibits 374

resistance elevated ~4,000-fold or 500-fold over the parent strain [CB190 (ATCC 375

29213)] as determined by broth microdilution or agar dilution MIC assays, respectively. 376

Table 5A includes a summary of the phenotypic and genotypic characterization of stable, 377

true-breeding mutants derived from days 2, 5, 10, 15 and 26 of the CBR-2092 step-378

selection study. 379

On day 2 of the CBR-2092 step-selection study, a mutant derivative (CB1880) was 380

isolated that had highly elevated resistance to rifampin and genotypic analysis revealed 381

an rpoB (R484H) mutation in the canonical RRDR of rpoB. A secondary mutational 382

event was apparent on day 5 as MICs for CBR-2092 are further elevated 4- to 8-fold and 383

genotypic analysis of a purified day 5 isolate (CB1881) revealed a deletion mutation in 384

the gyrA gene [gyrA (∆L520)] corresponding to the leucine residue at position 520 of 385

GyrA. Interestingly, this residue lies outside of the known QRDR of gyrA and is not 386

associated with a significant change in the MIC for ciprofloxacin (Table 5A). 387

A tertiary mutational event was apparent on Day 10 of the CBR-2092 step-selection 388

study with a further 2-fold shift in the MICs determined for CBR-2092. Genotypic 389

analysis of a purified day 10 isolate (CB1882) revealed a duplication mutation in the 390

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parC gene [parC (R236dupl)] corresponding to a tandem duplication of the arginine 391

residue at position 236 of the ParC subunit of DNA topoisomerase IV. Similarly, this 392

residue also lies outside of the canonical QRDR of parC and is not associated with a 393

significant change in the MIC for ciprofloxacin. A further mutational event was apparent 394

on day 15 with CBR-2092 MICs elevated a further 2- to 8-fold. Genotypic analysis of a 395

purified day 15 isolate (CB1883) revealed a substitution mutation in the gyrA gene [gyrA 396

(S84L)] corresponding to a serine to leucine substitution at residue 84 of GyrA subunit 397

and corresponds to a prevalent QRDR mutation in fluoroquinolone-resistant S. aureus 398

isolates. A final mutational event was apparent on day 26 as the CBR-2092 MICs are 399

elevated a further 4- to 8-fold and genotypic analysis of a purified day 26 isolate 400

(CB1884) revealed a substitution mutation in the parC gene [parC (H103Y)] 401

corresponding to a histidine to tyrosine substitution at residue 103 of the ParC subunit of 402

DNA gyrase. While apparently atypical in fluoroquinolone-resistant clinical isolates, this 403

mutation has previously been reported in laboratory studies of resistance development to 404

non-fluorinated quinolones (31) and lies within the canonical QRDR of parC in S. 405

aureus. Hence, as expected, an 8-fold shift in the ciprofloxacin MIC is observed between 406

the day-15 (CB1883) and day-26 (CB1884) isolates. 407

During the course of the CBR-2092 step-selection study it was apparent that the 408

mutant derivatives that were isolated and characterized exhibited slower growth in vitro 409

than the parental strain. Table 5A also shows the doubling times for each characterized 410

isolate when growing in MHII broth medium at 37ºC with good aeration. Interestingly, 411

the doubling time of the terminal day-26 isolate (CB1884), which bears five mutations 412

[rpoB (R484H), gyrA (∆L520), gyrA (S84L) parC (R236dupl) & parC (H103Y)], is 65 413

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minutes and therein significantly longer than that of the parent CB190 (ATCC 29213) 414

strain (38 minutes). The significance of these findings with regard to the in vivo fitness 415

or pathogenicity of strains exhibiting decreased susceptibility to CBR-2092 remains to be 416

determined. 417

Finally, in contrast to the fluoroquinolone comparator agents employed, it does not 418

appear that the mutational activation of efflux systems is a contributory factor in the 419

stepwise development of CBR-2092 resistance. In the case of ciprofloxacin and ABT-420

719, the terminal day-15 isolates exhibit MICs for ethidium bromide of 64 µg/mL that are 421

16-fold elevated over that of the parent strain (CB190 (ATCC 29213), 4 µg/mL). In 422

contrast, no decrease in susceptibility to ethidium bromide is apparent for CBR-2092 423

selectants through the day-26 terminal endpoint. 424

Multi-step passage selections undertaken with strains with pre-existing 425

rifamycin and/or quinolone resistance mutations. In a follow-up study, the day-26 426

terminal isolate from the CBR-2092 step-selection study (CB1884) was used as the 427

starting strain for a step-selection undertaken with ABT-719 in MHII medium. As shown 428

in Table 5B, after 20 days of step-wise passage in increasing concentrations of ABT-719, 429

a strain (CB1887) was isolated that has broth microdilution MICs for CBR-2092 and 430

ciprofloxacin of > 250 and 64 µg/mL, respectively. Genotypic analysis of strain 431

CB1887 confirmed that each of the five mutations previously characterized in strain 432

CB1884 had been stably maintained and also revealed two further substitution mutations 433

in parC [parC (E84G) and (S80Y)] corresponding to glutamate to glycine and serine to 434

tyrosine substitutions at residues 84 and 80 of the ParC subunit of DNA topoisomerase 435

IV, respectively, and a further substitution mutation in gyrA [gyrA (E88K)] 436

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corresponding to a glutamate to lysine substitution at residue 88 of the GyrA subunit of 437

DNA gyrase. These combined data are consistent with the notion that the mode of action 438

of CBR-2092 in the concentration ranges employed herein - up to 250 and 8 µg/mL in the 439

broth and agar conditions, respectively - can be accounted for by combined effects on the 440

cellular function of RNA polymerase, DNA gyrase and DNA topoisomerase IV. 441

A final series of step-selections were undertaken with CBR-2092 in studies 442

employing starting strains that bear pre-existing rifampin- and/or quinolone-resistance 443

mutations. In these studies, MHII medium was supplemented with 0.002% (vol:vol) P-444

80 to enable selections involving higher concentrations of CBR-2092. Table 5B also 445

shows a summary of the phenotypic and genotypic characterization of stable, true-446

breeding mutants derived from the terminal isolates resulting from each of these step-447

selections. In toto, the combined data from this last set of genetic studies further 448

substantiate conclusions from previously described genetic selections including the 449

notion that CBR-2092 acting as a quinolone (in rifamycin-resistant strains) elicits 450

resistance mutations in DNA gyrase and DNA topoisomerases IV that are atypical of 451

traditional quinolones. In addition, the selection of secondary mutations in both GyrA 452

and ParC subunits in each of two independent selections undertaken with a rifampin-453

resistant strain (CB815) bearing pre-existing canonical fluoroquinolone-resistance alleles 454

[gyrA (S84L) and parC (S80F)] supports the notion that CBR-2092 exhibits residual 455

activity against the prevalent fluoroquinolone-resistant variants and is consistent with the 456

afore mentioned biochemical data. Finally, it is noted again that the terminal isolates 457

resulting from all CBR-2092 step-selections exhibit no change with regard to their 458

susceptibility to ethidium bromide suggesting that the mutational activation of efflux 459

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systems is not a contributory factor in genotypic adaptations associated with decreased 460

CBR-2092 susceptibility in vitro. 461

462

DISCUSSION 463

Examination of rifampin-RNAP co-crystal structures suggest that the tight rifampin 464

binding interaction is mediated by key hydrogen bonds formed by hydroxyl groups at C-465

1, C-8, C-21 and C-23 as well as the carbonyl oxygen of the C-25 acetoxy group (Figure 466

1), while the C3-appended piperazine functionality of rifampin is spatially oriented away 467

from the RNAP binding surface and appears to be solvent accessible (4, 9). As all of the 468

chemical features of the rifampin pharmacophore identified as critical elements in RNAP 469

interaction are preserved in CBR-2092, and the quinolone moiety is appended to the C3 470

position via a secondary hydrazone functionality identical to that of rifampin, it is 471

perhaps not surprising that CBR-2092 exhibits in vitro potency as an RNAP inhibitor that 472

is nearly equivalent to that of rifampin. Preliminary evidence for distinct features of the 473

interaction of CBR-2092 with RNA polymerase is suggested by the retention of activity 474

of CBR-2092 against derivatives of a high-level quinolone resistant strain [CB1623, gyrA 475

(S84L), parC (S80F), parE (D434V), norAUP

] bearing intermediary level rifampin-476

resistance alleles including rpoB (H481N), rpoB (S464P) or rpoB (I527F) (Du, Duncan, 477

Robertson & Lynch, unpublished observations). 478

The activity of CBR-2092 against rifampin-resistant strains, combined with the range 479

of gyrA, parE and parC mutations resulting from genetic selections undertaken with 480

CBR-2092 in strains with pre-existing high-level rifampin resistance, suggest that CBR-481

2092 exhibits secondary activity against S. aureus via its quinolone pharmacophore and 482

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that DNA gyrase is the preferred topoisomerase target in vivo. In this regard, it is 483

interesting to note that other hybrid quinolone antibiotics, including quinolone dimers 484

(14, 22, 23, 40), sulfonamide-quinolones (2, 29) and oxazolidinone-quinolone hybrids 485

(13, 15, 16), similarly appear to preferentially target DNA gyrase in gram-positive 486

bacteria. These data could be interpreted as indicating that the quinolone-binding pocket 487

of DNA gyrase in ternary complexes may be more accommodating of compounds that in-488

effect bear large, bulky substitutions extending from position 7 of the classical 4-489

quinolone core nucleus. 490

DNA gyrase and DNA topoisomerase IV mutations characterized in strains resulting 491

from genetic selections employing CBR-2092 include an array of mutational changes that 492

have not previously been reported in studies of traditional quinolone agents and as such 493

lie outside of the classical quinolone-resistance determining regions. The selection of 494

such mutants could reflect distinct aspects of the binding interaction between CBR-2092 495

and the DNA topoisomerase target proteins. Alternately, these atypical mutations may 496

simply attenuate the activity of the enzyme such as to alter the sensitivity of the mutant 497

toposiomerase to inhibition by CBR-2092. In this regard, it is of interest to note that past 498

studies of laboratory isolated quinolone-resistant mutants of S. aureus have similarly 499

revealed mutations outside of the classical QRDRs and have been found to either reduce 500

the expression of DNA topoisomerase IV (17) or yield enzymes with apparent reduced 501

catalytic function (X. Zhang and D. Hooper, unpublished observations cited in (17)). 502

Finally, the mutational activation of efflux systems has been shown to be a common 503

mechanism that contributes to the development of fluoroquinolone resistance in a variety 504

of human pathogens. Studies of S. aureus suggest that up to ~50% of fluoroquinolone-505

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resistant strains of clinical origin exhibit an enhanced quinolone efflux phenotype (11, 34, 506

35, 39) and that mutations affecting the expression of two specific efflux pumps - NorA, 507

a member of the major facilitator superfamily (MFS) class and MepA, a multidrug and 508

toxic extrusion (MATE) family member - are most commonly found. However, in 509

contrast to most fluoroquinolones, including the comparator agents studied herein, the 510

mutational activation of efflux systems does not appear to be a contributory factor in the 511

development of resistance to CBR-2092. Further, in an accompanying article (30), we 512

show through the use of a panel of genetically-defined mutant strains of S. aureus that the 513

increased expression of either norA or mepA has no discernable impact on the in vitro 514

activity of CBR-2092. It is of interest to note that quinolone dimer agents linked via the 515

C7 position also appear to exhibit a lower propensity for quinolone efflux in S. aureus 516

(14, 22, 23). In addition, the improved antimicrobial activity of other quinolone hybrid 517

agents versus gram-positive cocci when compared to parent agents (or cocktails thereof) 518

may also be explained by circumvention of intrinsic or mutationally-activated efflux 519

systems (8, 13, 15, 16). One possible explanation for this apparent commonality among 520

quinolone hybrid antibiotics (including CBR-2092) is that the MFS and MATE class 521

pumps involved in quinolone efflux in gram-positive cocci exhibit relatively narrow 522

substrate specificities when compared to the resistance-nodulation-division (RND) efflux 523

systems principally involved in quinolone efflux in gram-negative pathogens. In light of 524

the increasing prevalence of efflux-mediated resistance traits in gram-positive cocci, this 525

feature may confer a selective advantage of CBR-2092 over rifamycin-fluoroquinolone 526

cocktail combinations with regard to its activity against fluoroquinolone-resistant clinical 527

isolates. 528

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529

530

ACKNOWLEDGEMENTS 531

The authors wish to thank David Hooper for provision of plasmids used for the 532

inducible over-expression of S. aureus GyrB and ParE subunits in E. coli, Ian Chopra for 533

providing strain RN4220 and Douglas Beeman and Katrina Chapo for experimental 534

contributions in the early stages of the work. The authors also acknowledge past 535

contributors to the rifamycin-quinolone program at Cumbre Pharmaceuticals including 536

Donghui Bao, Keith Combrink, Jing Li, Zhenkun Ma and Paul Renick and the 537

contributions of current colleagues Charles Ding, Steve Madden and William Weiss. 538

539

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38. Stein, A., M. Drancourt, and D. Raoult. 2000. Management of Infected 686

Orthopedic Implants. Infections Associated with Indwelling Devices (3rd edition) 687

Edited by Waldvogel and Bisno (ASM Press). 688

39. Tanaka, M., T. Wang, Y. Onodera, Y. Uchida, and K. Sato. 2000. Mechanism 689

of quinolone resistance in Staphylococcus aureus. J. Infect. Chemother. 6:131-9. 690

40. Zhao, X., B. Quinn, R. Kerns, and K. Drlica. 2006. Bactericidal activity and 691

target preference of a piperazinyl-cross-linked ciprofloxacin dimer with 692

Staphylococcus aureus and Escherichia coli. J. Antimicrob. Chemother. 58:1283-693

1286. 694

41. Zheng, Z., and P. S. Stewart. 2002. Penetration of rifampin through 695

Staphylococcus epidermidis biofilms. Antimicrob Agents Chemother 46:900-3. 696

42. Zimmerli, W., A. Trampuz, and P. E. Ochsner. 2004. Prosthetic-joint 697

infections. N. Engl. J. Med. 351:1645-54. 698

43. Zimmerli, W., A. F. Widmer, M. Blatter, R. Frei, and P. E. Ochsner. 1998. 699

Role of rifampin for treatment of orthopedic implant-related staphylococcal 700

infections: a randomized controlled trial. Foreign-Body Infection (FBI) Study 701

Group. JAMA. 279:1537-41. 702

703

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704

TABLE 1. Bacterial strains used in this study. 705

Strain Relevant Genotype Relevant Characteristics Source or

Reference

CB190 Wild-type ATCC 29213 Parent strain for isogenic strain panel ATCC

CB370 rpoB (H481Y) Spontaneous rifampin-resistant variant of CB190 This work

CB808 parC (S80F) Spontaneous ciprofloxacin-resistant variant of CB190 This work

CB809 rpoB (H481Y), parC (S80F) Spontaneous ciprofloxacin-resistant variant of CB370 This work

CB811 gyrA (S84L) Spontaneous nadifloxacin-resistant variant of CB190 This work

CB812 rpoB (H481Y), gyrA (S84L) Spontaneous nadifloxacin-resistant variant of CB370 This work

CB814 parC (S80F), gyrA (S84L) Spontaneous higher level ciprofloxacin-resistant variant of CB808 This work

CB815 rpoB (H481Y), parC (S80F), gyrA (S84L) Spontaneous higher level ciprofloxacin-resistant variant of CB809 This work

CB1244 Wild-type RN4220 Parent strain: rsbU, agr, restriction minus, methylation

plus laboratory strain I. Chopra

CB1522 rpoB (H481Y) Spontaneous rifampin-resistant variant of CB1244 This work

706

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TABLE 2. Effects of CBR-2092 and comparator agents on the in vitro activity of target enzymes from S. aureus. 707

σA

RNA Polymerase

(IC50 in µM) Type II DNA Topoisomerases (CC50 in µM, or ratios of CC50 values)

DNA Topoisomerase IV DNA Gyrase CC50 Ratios Test Agent

Rif-S

(RpoBWT

)

Rif-R

(RpoBH481Y

) ParCWT

ParCS80F

GyrAWT

GyrAS84L

GyrA

WT

/ ParCWT

ParCS80F

/ ParCWT

CBR-2092 0.034 > 25 1.7 2.7 1.5 >150 0.9 1.6

Rifampin 0.015 > 25 nr nr nr nr nr nr

Ciprofloxacin nr nr 0.4 31 4.6 > 150 11.5 77.5

Gatifloxacin nr nr 0.22 13 0.7 18 3.2 59.1

ABT-719 nr nr 0.06 1.1 0.09 0.9 1.5 18.3

708

Abbreviations: nr, not relevant. 709 ACCEPTED on July 15, 2018 by guest

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TABLE 3. Antibacterial activity of CBR-2092 against isogenic Staphylococcus aureus strains bearing combinations of rifamycin- or 710

quinolone resistance mutations. 711

MIC (µg/mL) for Strain, Genotype

CB190, CB811, CB808, CB814, CB370, CB812, CB809, CB815,

Test Agent

(condition)a

WT gyrAS84L

parCS80F

gyrA

S84L

parCS80F

rpoB

H481Y

rpoBH481Y

gyrAS84L

rpoBH481Y

parCS80F

rpoBH481Y

gyrAS84L

parCS80F

CBR-2092 (Agar) 0.008 0.008 0.008 0.008 0.06 0.12 0.06 0.25

CBR-2092 0.015 0.015 0.015 0.015 0.12 0.50 0.25 2

Rifampin 0.008 0.008 0.008 0.008 > 250 > 250 > 250 > 250

Ciprofloxacin 0.25 0.25 2 16 0.25 0.25 2 16

Levofloxacin 0.25 0.25 1 8 0.25 0.25 1 8

Gatifloxacin 0.06 0.12 0.25 8 0.06 0.12 0.25 8

ABT-719 0.015 0.03 0.03 0.50 0.015 0.03 0.03 0.25

712

aMinimum inhibitory concentrations (MICs) in µg/mL were determined by the broth microdilution method in the presence of 0.002 % 713

(v:v) polysorbate-80 unless otherwise indicated. 714


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TABLE 4A. Characterization of CBR-2092 single-step mutants derived from a rifampin-sensitive strain. 715

Microbroth MIC (µg/mL) Mutation Identified

in rpoB

Number of

Independent

Isolations Rifampin CBR-2092 Ciprofloxacin

WT Parent nr 0.008 0.015 0.25

H481Y 12 >64 0.12 0.25

Q468L 1 >64 0.25 0.25

Q468K 1 >64 0.50 0.25

H481D 1 >64 0.25 0.25

<H472> (insertion) 1 32 0.12 0.25

D471Y 2 16 0.25 0.50

<E473> (insertion) 1 8 0.25 0.25

H481N 1 2 0.25 0.25

N474K 2 2 0.25 0.25

A477V 2 1 0.12 0.25

D471G 4 0.25 0.25 0.25

716


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TABLE 4B. Characterization of CBR-2092 single-step mutants derived from rifampin-resistant strains. 717

Strain

Parent

Strain Parent Strain Genotype

Selection

Conc.

(µg/mL)

Ending Strain Genotype

CBR-2092

Microbroth

MIC (µg/mL)

CB1522 CB1244 rpoBH481Y

na na 0.25


0.25 rpoBH481Y

gyrAA26V

1


0.25 rpoBH481Y

gyrAG773V

2


0.1 rpoBH481Y

gyrAL795S

0.5


0.1 rpoBH481Y

gyrAG532V

0.5


0.1 rpoBH481Y

gyrAD705N

0.5


0.1 rpoBH481Y

gyrAG572D

1


0.1 rpoBH481Y

gyrAG584V

0.5


0.2 rpoBH481Y

gyrAS784F

0.5


0.2 rpoBH481Y

gyrAS84L

0.5


parCS80F

0.18 rpoBH481Y

parCS80F

gyrAS84L

1


gyrAS84L

0.18 rpoBH481Y

parEE474K

gyrAS84L

16


parCS80F

gyrAS84L

0.8 rpoBH481Y

parCS80F

parEL517F

gyrAS84L

16


parCS80F

gyrAS84L

0.8 rpoBH481Y

parCS80F, Q27H

gyrAS84L

8


parCS80F

gyrAS84L

0.8 rpoBH481Y

parCS80F

parEE474Q

gyrAS84L

> 16


parCS80F

gyrAS84L

0.53 rpoBH481Y

parCS80F

parEV458G

gyrAS84L

16


parCS80F

gyrAS84L

0.53 rpoBH481Y

parCS80F

parER455H

gyrAS84L

16

718

Abbreviations: na, not applicable. 719


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TABLE 5A. Characterization of mutants from step-selections undertaken with wild-type strains. 720

MIC in µg/mL for:

CBR-2092 Strain

Selection

Agenta

Dayb Mutations Identified

Broth Agar Rifampin Cipro EtBr

Doubling

Time

(mins)c

CB190 CBR-2092 na Wild-Type 0.015 0.008 0.008 0.25 4 38

CB1880 CBR-2092 2 rpoBR484H

0.12 0.03 > 250 0.25 4 43

CB1881 CBR-2092 5 rpoBR484H

gyrA∆L520

0.5 0.25 > 250 0.5 2 41

CB1882 CBR-2092 10 rpoBR484H

gyrA∆L520

parCR236dupl.

1 0.5 > 250 1 2 45

CB1883 CBR-2092 15 rpoBR484H

gyrA∆L520, S84L

parCR236dupl.

8 1 > 250 1 2 47

CB1884 CBR-2092 26 rpoBR484H

gyrA∆L520, S84L

parCR236dupl., H103Y

64 4 > 250 8 2 65

CB1871 Rifampin 2 rpoBH481Y

0.12 nd > 250 0.25 8 nd

CB1875 Ciprofloxacin 15 gyrAS84L

parCR298K, <LNVIKEE>461

0.008 nd 0.016 > 128 64 nd

CB1891 ABT-719 15 gyrAS84L

parCE84K

parEP587S, F638V

0.03 nd 0.008 > 64 64 nd

721

aStep-selections were undertaken in MHII broth medium without P-80 supplementation. 722

bThe day on which the indicated mutant strain was purified. 723

cThe time necessary for the OD600 of a logarithmic culture propagated at 37ºC in MHII broth medium to double. 724

Abbreviations: na, not applicable; Cipro, ciprofloxacin; EtBr, Ethidium Bromide.725


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TABLE 5B. Characterization of mutants from step-selections undertaken with strains exhibiting resistance to rifamycin and/or 726

quinolone agents. 727

MIC in µg/mL for:

CBR-2092 Strain Selectiona Relevant genotype

Broth Agar Rifampin Cipro

b EtBr

c

CB1884 Parent rpoBR484H

gyrA∆L520, S84L

parC<R236>, H103Y

64 4 > 250 8 2

CB1887 CB1884 plus 20-days

selection with ABT-719 rpoB

R484H gyrA

∆L520, S84L, E88K parC

<R236>, H103Y, S80Y, E84G > 250 > 8 > 250 64 nd

CB370 Parent rpoBH481Y

0.12 0.03 > 250 0.25 4

CB1947 CB370 plus 20 days

selection with CBR-2092 rpoB

H481Y gyrA

S84L, ∆E697 parC

G167V 4 1 > 250 2 4

CB814 Parent rpoBWT

gyrAS84L

parCS80F

0.015 0.06 0.008 16 4



S486L gyrA

S84L, E88Q, G532S parC

S80F, E84L 31 4 > 250 64 4

CB815 Parent rpoBH481Y

gyrAS84L

parCS80F

1 0.25 > 125 16 4



H481Y gyrA

S84L, V598I parC

S80F, R570H 16 2 > 125 64 4



H481Y gyrA

S84L, ∆K809 parC

S80F, A523D 16 2 > 125 64 4

728


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aStep-selections were undertaken in MHII broth medium with (CBR-2092) or without (ABT-719) supplementation with 0.002% (v/v) 729

polysorbate-80. 730

bCipro, ciprofloxacin. 731

cEtBr, ethidium bromide.732


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FIGURE 1. 733

N

N

F

O

H2N

ABT-719

7

8

O

O

OH

OHOH

NH

O

O

OHOH

O

O

O

N

N

N N

N

F

O

O

O

OH

OHOH

NH

O

O

OHOH

O

O

O

N

N

N

CBR-2092

Rifampin

NN

HN

F

O O

OH

Ciprofloxacin

6

7

O

OH

O

OH

3

3

18

2123

25

734

735

FIG. 1. Chemical structures of CBR-2092 and related molecules. 736

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FIGURE 2. 737

CB190 (Wild-type) 738

739

740

741

742

743

CB370 (Rifamycin-resistant, rpoBH481Y

) 744

745

746

747

748

749

CB814 (Quinolone-resistant, gyrAS84L

plus parCS80F

) 750

751

752

753

754

755

FIG. 2. Effects of CBR-2092 and comparator agents on de novo macromolecular 756

biosynthesis in S. aureus. The radiolabels employed were [Methyl-3H]-Thymidine 757

(closed circles) for DNA synthesis, [5,6-3H]-Uridine (open circles) for RNA synthesis, 758

L-[3,4,5-3H]-Leucine (closed triangles, dashed line) for protein syntheis and [2,3-3H]-D-759

0 50 100 150 200

0

50

100

150

Rifampin(0.032 µg/ml)

Time (min)

0 50 100 150 200

0

50

100

150

CBR-2092(0.5 µg/ml)

Time (min)

0 50 100 150 200

0

50

100

150

DNA

Cell Wall

Protein

RNA

Ciprofloxacin(2 µg/ml)

Time (min)

Lab

el I

nco

rpo

rati

on

%

(CP

Md

rug/C

PM

DM

SO)

0 50 100 150 200

0

50

100

150


Time (min)

0 50 100 150 200

0

50

100

150

CBR-2092(0.5 µg/ml)

Time (min)

0 50 100 150 200

0

50

100

150

DNA

Cell Wall

Protein

RNA


Time (min)

Lab

el I

nco

rpo

rati

on

%

(CP

Md

rug/C

PM

DM

SO)

Lab

el I

nco

rpo

rati

on

%

(CP

Md

rug/C

PM

DM

SO)

0 50 100 150 200

0

50

100

150


Time (min)

0 50 100 150 200

0

50

100

150

CBR-2092(0.5 µg/ml)

Time (min)

0 50 100 150 200

0

50

100

150

DNA

Cell Wall

Protein

RNA


Time (min)

Lab

el I

nco

rpo

rati

on

%

(CP

Md

rug/C

PM

DM

SO)

0 50 100 150 200

0

50

100

150


Time (min)

0 50 100 150 200

0

50

100

150

CBR-2092(0.5 µg/ml)

Time (min)

0 50 100 150 200

0

50

100

150

DNA

Cell Wall

Protein

RNA


Time (min)

0 50 100 150 200

0

50

100

150


Time (min)

0 50 100 150 200

0

50

100

150

CBR-2092(0.5 µg/ml)

Time (min)

0 50 100 150 200

0

50

100

150

DNA

Cell Wall

Protein

RNA


Time (min)

Lab

el I

nco

rpo

rati

on

%

(CP

Mdru

g/C

PM

DM

SO)

0 50 100 150 200

0

50

100

150


Time (min)

0 50 100 150 200

0

50

100

150

CBR-2092(0.5 µg/ml)

Time (min)

0 50 100 150 200

0

50

100

150

DNA

Cell Wall

Protein

RNA


Time (min)

Lab

el I

nco

rpo

rati

on

%

(CP

Mdru

g/C

PM

DM

SO)ACCEPTED


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Alanine (open triangles, dashed line) for cell wall synthesis. At each time point, the ratio 760

of radiolabel incorporated in the presence of test agent relative to that of the DMSO-761

treated control culture was used to determine the percent incorporation. Radiolabel 762

incorporated at time zero (T0) was set to 100%. 763

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N

N

F

O

H2N

ABT-719

7

8

O

O

OH

OHOH

NH

O

O

OHOH

O

O

O

N

N

N N

N

F

O

O

O

OH

OHOH

NH

O

O

OHOH

O

O

O

N

N

N

CBR-2092

Rifampin

NN

HN

F

O O

OH

Ciprofloxacin

6

7

O

OH

O

OH

3

3

18

2123

25

AAC01649-07 v2: FIGURE 1


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CB190 (Wild-type)

CB370 (Rifamycin-resistant, rpoBH481Y)

CB814 (Quinolone-resistant, gyrAS84L plus parCS80F)

0 50 100 150 200

0

50

100

150

Rifampin(0.032 µµµµg/mL)

Time (min)

0 50 100 150 200

0

50

100

150

CBR-2092(0.5 µµµµg/mL)

Time (min)

0 50 100 150 200

0

50

100

150DNA

Cell Wall

Protein

RNA

Ciprofloxacin(2 µµµµg/mL)

Time (min)

Lab

el

Inc

orp

ora

tio

n %

(C

PM

dru

g/C

PM

DM

SO)

0 50 100 150 200

0

50

100

150


Time (min)

0 50 100 150 200

0

50

100

150


Time (min)

0 50 100 150 200

0

50

100

150DNA

Cell Wall

Protein

RNA


Time (min)

Lab

el

Inc

orp

ora

tio

n %

(C

PM

dru

g/C

PM

DM

SO)

La

bel In

co

rpo

rati

on

%

(CP

Md

rug/C

PM

DM

SO)

0 50 100 150 200

0

50

100

150


Time (min)

0 50 100 150 200

0

50

100

150


Time (min)

0 50 100 150 200

0

50

100

150DNA

Cell Wall

Protein

RNA


Time (min)

La

bel In

co

rpo

rati

on

%

(CP

Md

rug/C

PM

DM

SO)

0 50 100 150 200

0

50

100

150


Time (min)

0 50 100 150 200

0

50

100

150


Time (min)

0 50 100 150 200

0

50

100

150DNA

Cell Wall

Protein

RNA


Time (min)

0 50 100 150 200

0

50

100

150


Time (min)

0 50 100 150 200

0

50

100

150


Time (min)

0 50 100 150 200

0

50

100

150DNA

Cell Wall

Protein

RNA


Time (min)

Lab

el In

co

rpo

rati

on

%

(CP

Md

rug/C

PM

DM

SO)

0 50 100 150 200

0

50

100

150


Time (min)

0 50 100 150 200

0

50

100

150


Time (min)

0 50 100 150 200

0

50

100

150DNA

Cell Wall

Protein

RNA


Time (min)

Lab

el In

co

rpo

rati

on

%

(CP

Md

rug/C

PM

DM

SO)

AAC01649-07 v2: FIGURE 2


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