~ l ~ Chapter 5

, : · . ·Identification of inhibitors of Mycobacterium tuberculosis

Isocitrate Lyase

Cfiapter5 MtuiC£ In!tiDitors

5.1 Introduction

An important aspect in anti-TB drug discovery is the identification of new compounds

with the potential to reduce chemotherapy durations. Several lines of evidence suggest

that pathogenic mycobacteria primarily utilize fatty acids, rather than carbohydrates, as

carbon substrates during infection (Munoz-Elias & McKinney, 2005). Two pathways are

specifically required for utilization of fatty acids: the catabolic beta-oxidation cycle that

degrades fatty acids to acetyl-CoA (C2) units and the anaplerotic glyoxylate cycle. When

glycolytic substrates are absent or sparse, microorganisms rely on the glyoxylate cycle to

replenish TCA cycle intermediates during growth on fatty acid substrates. The glyoxylate

cycle in M tuberculosis apparently comprises a single gene encoding malate synthase

and two genes encoding isocitrate lyases (McKinney, et a/., 2000). Isocitrate lyases

catalyze an essential reaction of the glyoxylate shunt, an anaplerotic pathway that

bypasses the C02-generating steps of the tricarboxylic acid cycle and enables bacteria to

synthesize carbohydrates and replenish tricarboxylic acid cycle intermediates from fatty

acid-derived acetyl-coenzyme A (Sharma, eta/., 2000).

Isocitrate lyase can be broadly classified as oxo-acid-lyases, which specifically cleaves

carbon-carbon bond and catalyzes the reaction shown in Equation 1. M tuberculosis

codes for two isocitrate lyases ic/1 (Rv0467) and ic/2 (Rv1915), enzymes involved in

fatty acid metabolism (Munoz-Elias & McKinney, 2005). Recently, ic/1 has also been

shown to play role in propionate metabolism via methylcitrate cycle by functioning as 2-

methyisocitrate lyase (Gould. eta/., 2006).

ICL

Isocitrate succinate + glyoxylate (Eq. 1)

5.1.1 Domain Organization of M. tuberculosis ICL

The crystal structures of icll is available in apoenzyme form (Pdb code: 1F61) and with

bound substrate 3-Bromopyruvate (Pdb code: 1F8M). To analyze the domain

organization of both the ICLs, the homology model of ic/2 was prepared. The details of

113

Cliapter5 :MtuiC£ Infzi6itors

the methods used for ic/2 model development are described in experimental section. The

domain organization of both the ICL is found to be similar for the active site. The ICL

catalytic signature motif KKCGH (AA 193-203 in ic/1; AA 213-217 in icl2) present in

Domain I is conserved in icll as well as in ic/2 (Fig 5.1). Comparative analysis based on

icll crystal structure and icl2 homology model shows that domain II and IV are present in

icl2 while absent in icll. Domain I and III are present in both the homologs and both the

structures are similar with rmsd value of 1.4 A.

Fig 5.1 ICL Domain Organization. Superimposition of in si/ico-modeled ic/2 monomer

(in red) onto the X-ray crystal structure of icll monomer 1 F61 (in blue). All ICL contains

four domains. Domains I and III are present in all eukaryotic as well as prokaryotic ICLs.

Domain I contains the conserved catalytic motif KKCGH. Domain II is present in fungal

and plant ICLs and in mycobacterial ic/2, but absent in mycobacterial ic/1. Domain IV

(not shown) is unique to mycobacterial ic/2 and cannot be modeled as no homologous

structure is available in PDB (Protein Data Bank).

In M tuberculosis both the genes icll and ic/2 are essential for maintenance of

persistence in mouse models (Munoz-Elias & McKinney, 2005). Some other studies have

also implicated the role of glyoxylate cycle in the virulence of bacterial and fungal

pathogens (Idnurm & Howlett, 2002; Lorenz & Fink, 2001; Solomon, et al., 2004; Wang,

114

Cha.pter5 :Mtul CL I nlii6itors

eta/., 2003). Thus, the chemical inhibitors of this pathway could have broad therapeutic

utility. One of our lab's projects revolves around the identification of novel classes of

inhibitors against ic/1 from M tuberculosis (referred as MtuiCL) using virtual screening

techniques and structure elucidation of the inhibitor-enzyme complex. Against this

backdrop, the present chapter reports the identification of novel inhibitors against M

tuberculosis isocitrate lyase (ic/1) using molecular docking approaches. A virtual library

of compounds, belonging to three different classes' viz. galactosyl propanolamines,

cyclopropylphenyl methanes and nitrobutanoates, was evaluated by molecular docking

approaches using both the open and closed conformations of the enzyme. Promising

molecules were then evaluated for ICL inhibition using a high-throughput assay

developed by Dr. R. Shrivastava 's group, Microbiology Division, CDR!, Lucknow.

5.2 Experimental Section

The flowchart showing the systematic methodology used for identification of inhibitors

against MtuiCL is shown in Figure 5.2. The details of the compound synthesis and

enzyme assays are not discussed here as these are not part of the thesis. The compound

synthesis has been carried out by Dr. R. P. Tripathi 's group, MPC Division, CDR!,

Lucknow while the enzyme assays were carried out by Dr. R. Shrivastava 's Group,

Microbiology Division, CDR!, Lucknow.

5.2.1 Homology modeling of ic/2

The homology model of ic/2 was generated using X-ray structure of Aspergillus nidulans

Isocitrate lyase (PDB: 1DQY) and Mycobacterium tuberculosis ic/1 (PDB: 1F61) as

templates and Modeller8v2 (Marti-Renom, et a/., 2000) as modeling tool. Domain I and

III were modeled using M tuberculosis ic/1 while Domain II of ic/2 (AA 269-365) was

modeled after the A. nidulans ICL domain II. Domain N cannot be modeled as had no

homology to any of the known sequences in PDB (Berman, et a/., 2000). The structure

quality of modeled icl2 was verified using Procheck (Laskowski, et a/., 1996) and Whatif

server (Vriend, 1990). The stereochemical quality was satisfying all the parameters with

87.9% amino acid residues present in the core, 9.0% in allowed, 2.5% in generously

allowed and 0.6% in disallowed regions. The score expressing how well the backbone

115

Cliapter5 Mtul CL I nliiDitors

conformations of all residues (Ramachandran Z-score: -0.215), corresponding to the

known allowed areas in the Ramachandran plot was within expected ranges for well­

refmed structures. Since the active site in icll is made of residues from two adjacent

subunits, the active site of icl2 was also modeled by generating its dimer using Homology

module of Insightll (M/s. Accelrys Inc.). Prior to virtual screening experiments, all the

polar hydrogen atoms were added to the model.

5.2.2 Virtual Screening Protocol

The docking simulations were performed by using Autodock v3 (Morris, et a/., 1998) and

GOLD v2.2 (Verdonk. eta/., 2003). A combination of Simulated Annealing, Lamarkian

Genetic Algorithm and Monte Carlo algorithms were used for ligand conformational

searching.

Target Preparation:

Target preparation for AutoDock: The available structures of isocitrate lyase from M.

tuberculosis were retrieved from Protein Data Bank (http://www.rcsb.org/pdb) (Pdb

code: 1F8M, 1F81, 1F61) where dimer 1F61 is in its open conformation while tetramer

1F8m and 1F81 are its closed conformation in complex with 3-bromopyruvate and 3-

nitropropionate respectively.

Structures with PDB codes 1 F61 and 1 F8M were selected as the docking template. Prior

to docking studies, crystallographic waters and heteroatoms were removed. Polar

hydrogens were added. Kollman charges were assigned to all atoms. A 60x60x60 3D and

80x80x80 3D affinity grid centered on the active site (CYS191) with 0.375 A spacing

were calculated for 1F61 and 1F8m respectively; for each of the following atom types C,

A( aromatic C ),N, 0, S, H, F, Cl, Brande by using Autogrid3.

Target preparation for Gold: All the heteroatoms were removed and all the hydrogens

were added to the docking templates using Insightll (M/s. Accelrys Inc.).

Autodock Docking Parameters:

Ligands were drawn and optimized using Builder module of lnsightll. The ligands

translation, rotation and internal torsions were assigned. For each run, following docking

parameters were used - Trials of 100 dockings, population size of 150, random starting

116

Cftapter5 !MtuiCL Inliibitors

position and conformation translation step ranges of 1.5 A, rotation step ranges 35,

elitism of 1, mutation rate of0.02, cross-over rate of0.8, local search rate of0.06 and 10

million energy evaluations. The jobs were distributed to the SGI ORIGIN 350 cluster.

Final docked conformations were clustered by tolerance of 1.5A RMSD. The top 10% of

compounds with best simulated binding energies within standard deviation of 2 Kcal/mol

were selected for synthesis and subsequent inhibition assays.

Gold Docking Parameters:

Gold v2.2 (Genetic Optimization of Ligand Docking) also samples the ligand

conformational space using a genetie algorithm (GA). However, in contrast to AutoDock,

it uses the atomic description of the protein (or a truncated binding site) and allows for

the hydroxyl (Ser, Thr, Tyr) and ammonium (Lys) hydrogen atoms to relax upon ligand

docking. All the heteroatoms were removed and all the hydrogens were added to the

receptor file. Ligand input files were prepared and optimized by Builder module of

lnsightll (M/s. Acclerys Inc.) and saved in mol2 format. Ligands were loaded in Gold

GUI using 'Adding all files in the directory' option.

Gold trials were performed using the' default parameters (1 X speed-up, 6/12 internal

potential, no flipping of amide bonds, etc.). All docking trials for GOLD v2.2 were

performed with active site radius of 10.0 A for both the molecules. Binding site was

defined using cavity. atoms file. This file contains the list of all solvent accessible atoms

making-up the protein active site. All the parameters used by GOLD (e.g. hydrogen bond

energies, atom radii and polarisabilities, torsion potentials, hydrogen bond directionalities

etc.) are specified in gold.params file. Configuration file gold.conf reads parameter

settings from a previously saved confi~uration file and loads the parameter values into

the front end of GOLD window. GoldScore were calculated and reranking was performed

for all the docked ligand conformations.

5.2.3 Enzyme Assays

Enzyme in vitro inhibition assays:

The ICL assay measured the synthesis of glyoxylate directly by its reaction with

phenylhydrazine and the formation of glyoxylate phenylhydrazone was monitored as

I17

Cftapter5 !Mtul C.L I nliibitors

increase in absorption at 324 run (Drxon & Kornberg, 1959). The reagents and steps

included 50 mM potassium phosphate buffer, pH 7.0, 6 mM MgCh, 4 mM phenyl

hydrazine, 12 mM L-cysteine, 3 mM threo-D(s)-isocitrate and purified enzyme 2J!l in 200

J.ll reaction volume. The reaction was monitored for 10 min as increase in absorption at

324 run. The known ICL inhibitor, 3-nitropropionate (Sigma) was used at 1 OOJ!M as the

positive control.

BACTEC assays:

Stock solutions of the test compounds pr.epared in DMSO at 1 mglml were sterilized by

passage through 0.22 J.lm filters and were added (50 J.ll) to 4 ml radiometric 7H12 broth;

(Becton Dickinson Instrument System US) to achieve final concentrations. Controls

received 50 J.ll DMSO. Isoniazid and rifampcin (Sigma Chemical Co., St. Louis, MO)

were included as positive drug controls. The vials were inoculated with 104 to 105

CFU/ml of M. tuberculosis H37Rv. An additional control was inoculated with 1:100

dilution of the inoculum to represent 1% of the bacterial population (1ff to 103 CFU/ml).

The vials were incubated at 3-t>C and GI (Growth index) readings were recorded daily

until GI in 1: 100 control had reached 30. The concentration of the drug producing fmal

GI reading lower than those in 1: 100 control was considered to have inhibited more than

90% of the bacteria and was defined as the MIC (Panda et al. 2004).

118

Cliapter5

Open Conformation

(1F61)

Virtual Screening Models

Open Conformation

(1F8M)

!Mtul C£ I nlii6itors

Virtual Screening with AutoDock & Gold

I Biological Testing

/Yovel ~ffolds

BACTEC

Optimization

Fig 5.2 Flowchart showing the systematic workflow used for identification of inhibitors against MtuiCLs.

119

Cliapter5 9vftul C£ I nhiDitors

5.3 Results

It has been established that inhibitors of tll:l.e enzymes isocitrate lyase (icll), responsible

for fatty acid metabolism in mycobacterium may lead to new class of anti TB drugs to

kill the persistent bacteria (Sharma, et al. 2000). Bromopyruvic acid, itaconate, and 3-

nitropropionates are known to inhibit this enzyme at one or the other concentration.

However, none of these molecules show 1my antitubercular activity, a plausible reason

being that these molecules do not meet the c::riterion for drug like molecules.

5.3.1 Virtual library generation and mole~cular docking with Gold and AutoDock

With the background knowledge that inhibitors of this enzyme are more likely to emerge

from the intermediate products formed in TCA cycle and glyoxalate shunt, parallely that

glycosyl amino acid and aromatic amines do possess significant antitubercular activity in

vitro (Tripathi, et al., 2002; Grover, et al., 2004; Dwivedi, et al., 2005; Srivastava, et al.

2006), it has been concluded from the studies that hydrophobicity plays very important

role in displaying antitubercular action. Some hydrophobic classes of compounds like

nitrobutanoates, galactosylpropanolamines and cyclopropyl derivatives were taken up for

our initial modeling and docking studies. The results indicated that the all the three

classes of compounds fitted well into the "binding site of MtuiCL. A virtual library of

about 200 compounds based on derivatives of all the three classes of compounds was

generated. All the parent scaffolds and some of the selected primary substitutions are

displayed in Figure 5.3. The parent scaffolds in virtual library design for cyclopropyl

derivatives were 4-Cyclopropyl-methyl phenol (Fig 5.3 A) and 4-(Cyclopropyl-hydroxy­

methyl)-phenol (Fig 5.3 B); while the design principles were based on the aryloxyphenyl

cyclopropyl methanones series which were reported earlier as anti-mycobacterial agents

(Dwivedi, et al., 2005). The other parent s.caffolds were nitro butyric acid which was

designed based on the fact that the nitropropinates were known to inhibit icll (Sharma et

al. 2000) and Galactosyl propanolamines which were designed on the basis of

glycosylated beta amino acid series, another known anti- mycobacterial agents {Tripathi,

et al., 2002). The primary substitutions includes ethyl benzene, ethyl furan, Methyl­

tetrahydro-furo-dioxole, propenyl benzene, 4-Chlorobenza.ldehyde and 3-

pyridylcarboxaldehyde, etc. while secondary substitutions were on the primary

12:0

ChapterS !Mtul CL I nliiiiitors

substitutions and includes methanol, methyl, hydroxyl, amines, dimethyl dioxolanes etc.

side chains.

RO RO

A OH

B

R=

0

CY' Cyclopropyl derivatives

H

Ar----_/N"''''"· OH

R=

Ar=4 Chlorobenzaldehyde = 3 pyridylcarboxaldehyde Nitrobutanoates

Galactosyl propanolamines

Fig 5.3 The scheme of virtual library design for the selected compounds reported as M tuberculosis ic/1 inhibitors.

121

ChapterS !Mtu!CL Inhibitors

These compounds were docked and scored in the first instance with Autodock using both

the open and closed conformation of the structures and the acceptable docking scores are

shown in Figure S.4. The in silico screening studies were used to prioritize compounds

and compounds with docking energy lower than -7 kcallmol were subsequently selected

for synthesis and enzyme assays. The compounds selected for synthesis from the virtual

library were augmented with a view to increase the structural diversity of the library. The

synthesized compounds were assayed initially for in vitro protein activity inhibition. The

in vitro inhibition results for the selected compounds are shown in Figure S.S and the

respective docking scores and structures are shown in Table SA & SB. Successful

compounds were then evaluated for inhibition of growth of M tuberculosis in BACTEC

assays (data not shown).

5.3.2 Synergistic Effect in ICL Inhibition

An examination of the docking energies of compounds against the two conformations of

the enzyme revealed that apparently some compounds exhibited a preference for a

particular conformation of MtbiCL. The docking results suggested that pairs of certain

compounds, each with better predicted affinity for 'closed' and 'open' ICL structural

conformations, should be more effective as inhibitors than individual compounds by

themselves. A closer analysis of the docking poses suggested that this might be due to the

differences in the conformation of the active site loop which leads to better stabilization

of a docking pose with a particular conformation (Fig S.6). The ligands were selected for

further re-docking procedure with Gold instead of Autodock to further validate the

docking results. The Gold Scores were also biased towards one or the other

conformations of the enzyme {Table SA). We accordingly evaluated inhibition of

successful compounds alone and also in pairs to test for any synergistic effect. The in

vitro phenyhydrazone-based assay suggests that at least some compounds in pairs are

demonstrating a synergistic effect for enzyme inhibition and results are shown in Figure

S.S. This was further confirmed by the in vivo BACTEC assays based on M tuberculosis

growth under conditions of carbon deprivation. This novel approach offers to be effective

in the design of more potent inhibitors of theM tuberculosis isocitrate lyase (ic/1).

122

-N ',..)

0

-1

·2 - ·3 .J 0-4 ~ -5 ::J.o < -7 u~ ~ .--9

'-'·10 <I

r::

I U"'S\1 I 1~'61

. - -

-14~------------------------------------------------------------------------~ 1 3 S 7 9 11 13 15 17 19 21 23 25 2? 2931 D )5 37 39 41 43 45 47 .tg 51 Sl!S 57 59&1 63 6S 67 6911 73 ?5 n 79 8183 8S 87 89 91 93

___ ...,. COMPOUNl> ll>ENTIFIER

Figure 5.4 Docking energy graph. The plot between the binding energy calculated by AutoDock and compound identifiers. The two curves represent binding energy plots for the two different conformation of the enzyme and the compounds below the dashed lines were chosen for the inhibition assays. The compounds reported here are

indicated by an asterisk(*) viz. first* is 534, second* is 685, third* is 710 and fourth* is 689.

Cliapter5 9.1 tul CL I nliibitors

70

60 ~ •% INHIBITION H f-4 50 H IQ H

I 40 H

-.!. 30

20

10

0

!0<-, (c) ""(;;) "'\

~~ <-,

Fig 5.5 The plot showing in vitro inhibition (in percentage) of the compounds. The % inhibition on y-axis is plotted against the compound codes on x-axis. Some compounds are showing better in vitro enzyme inhibition in pairs. NP is nitropropionate.

Table SA Docking Energy table. Compounds with docking energy above nitropropionate (NPN) were selected for in-vitro assays. 1F61 is the open while 1F8M is the closed conformation of the ic/1. The docking score calculated by Autodock as well as Gold are shown in the table.

Compound Docking Scores S.No.

Autodock Docking Energy (kcallmol) GoldScore codes

I 1F61 1F8M ic/2 1F61 1F8M

1 NPN I -5.04 -8.02 -8.83 19.23 27.99

2 710 I -10.5 -11.07 -12.33 45.42 36.49

3 685 -10.8 -10.67 -13 .25 46.82 48.61 ~- - ·--

'

4 534 ' -8.87 -11.4 -11.28 40.62 -' -- ---- ~

5 689 -13.3 -12.12 -15.22 54.48 49.71

6 168 -8.42 -9.66 -11.78 35.32 26.95 ~ ~ - -- , ___ -

124

Cliapter5 9vf tul CL I nhi6itors

Table SB. Structures of the compounds shown in Table 5A. The first three compounds belongs to the cyclopropyl class, forth is combination of galctosylpropanolarnines and cyclopropyl while fifth molecule belongs to nitrobutanoates class of compounds.

S.No.

1

2

CompoundiD

710

4-( 4-Cyclopropylmethyl­phenoxymethyl )-1 ,2-dimethoxy­

benzene

534

Cyclopropyl-[ 4-(furan-2-ylmethoxy)-phenyl]-methanone

689

Hexadecanoic acid cyclopropyl-3 {4-[5-(2,2-dimethyl-

4

5

[ 1,3 ]dioxolan-4-yl)-2,2-dimethyl­tetrahydro-furo[2,3-d][1 ,3 ]dioxol-

6-yloxy]-phenyl}-methyl ester

685 1-[3-( 4-Cyclopropylmethyl­

phenoxy)-5,5-dimethyl­hexahydro-cyclopenta[b] furan-2-

yl ]-ethane-1 ,2-diol

168

3-Nitromethyl-5-phenyl-pent-4-enoic acid ethyl ester

Structures

___ ,,X ··'

# ........ ~

0

\. •+01111110

;:;;, ~

COOEt

125

Cliapter5 :M.tu!C£ Inlii6itors

FIG 5.6 Top panel shows a cartoon representation of the ICL structure, with the active site area at the center of the box. The Left panels represents ICL in open conformation (PDB code: 1F61) while right panels shows closed conformation ofthe same enzyme (PDB code: 1F8M). Predicted docking conformations of the inhibitors 685 (2nd Panel), 710 (3rd Panel) and 534 (4th Panel) within the ICL active site are shown.

126

Cfiapter5 9rftul CL I nfzi6itors

5.4 Discussion

The structure-based drug design strategy has been successfully used to identify three

novel classes of inhibitors viz. nitrobutanoates, cyclopropyls and galactosyl

propanaolamines active against MtuiCL. The docking results suggested that selected

compounds used in 'pairs' could be more effective for inhibition rather than individual

compounds. Interestingly, the in vitro inhibition assays support the apparent 'synergistic'

effect of pairs of molecules being better inhibitors than individual compounds. This was

further confirmed through the in vivo BACTEC assays. We are not aware of any other

group having identified such 'pairs of compounds' showing better inhibition compared to

single ICL inhibitors. The synergistic approach developed here offers a new paradigm in

the design of more potent inhibitors of the M tuberculosis isocitrate lyase. The results are

currently being refmed for the identification of more potent inhibitors of the enzyme.

Earlier, it was demonstrated that both the genes ic/1 and ic/2 are necessary for the

maintenance of persistence (Munoz-Elias and McKinney, 2005). Molecular modeling

and biochemical characterization suggested that both icl genes are similar in terms of

binding site architectures. It is therefore to be expected that inhibitors of the ic/1 gene

product should also be effective against its homolog ic/2. Indeed nitropropionate was

found to inhibit the activity of both the icl gene. products (Munoz-Elias and McKinney,

2005). The present evidence suggests that the present results should broadly hold true for

the ic/2 gene product too. Nevertheless, the group is purifying the MtuiCL2 protein and

the present results against MtuiCL1 will be verified against the homolog also.

5.5 References

Berman, H. M., eta/. (2000) The Protein Data Bank. Nucleic Acids Res., 28, 235-242.

Dwivedi, N., et a/. (2005) An efficient synthesis of aryloxyphenyl cyclopropyl methanones: a new class of anti-mycobacterial agents. Bioorg Med Chem Lett, 15, 4526-30.

Dixon, G. H. & Kornberg, H. L. (1959). Assay methods for key enzymes of the glyoxylate cycle. Biochem J, 72, 3.

Gould, T.A., et a/. (2006) Dual role of isocitrate lyase 1 in the glyoxylate and methylcitrate cycles in Mycobacterium tuberculosis. Mol Microbiol, 61, 940-947.

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Cfiapter5 !MtuiCL Inhibitors

Grover, R.K.; et a/. (2004) Novel phenyl cyclopropyl methanones useful as Antitubercular agents Ind. Patent 0636DEL2004

Idnurm, A. & Howlett, B.J. (2002) Isocitrate lyase is essential for pathogenicity of the fungus Leptosphaeria maculans to canola (Brassica napus). Eukaryot Cell, 1, 719-724.

Laskowski, R.A., eta/. (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR, 8, 477-486.

Lorenz, M.C. and Fink, G.R. (2001) The glyoxylate cycle is required for fungal virulence. Nature, 412, 83-86.

Marti-Renom, M.A., et al. (2000) Comparative protein structure modeling of genes and genomes. Annu Rev Biophys Biomol Struct, 29, 291-325.

Morris, G.M., eta/. (1998) Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem, 19, 1639-1662.

McKinney, J.D., eta/. (2000) Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature, 406, 735-738.

Munoz-Elias, E.J. & McKinney, J.D. (2005) Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat Med, 11, 638-644.

Panda, G., et a/. (2004) Diaryloxy methano phenanthrenes : a new class of antituberculosis agents. Bioorg Med Chem, 12, 5269-5276.

Sharma, V., et al. (2000) Structure of isocitrate lyase, a persistence factor of Mycobacterium tuberculosis. Nat Struct Bioi, 1, 663-668.

Solomon, P.S., et a/. (2004) Pathogenicity of Stagonospora nodorum requires malate synthase. Mol Microbiol, 53, 1065-1073.

Srivastava, R; et a/. (2006) Process for the preparation of novel 4-Nitrobtanoate Inhibitors of Isocitrate Lyase from Mycobacterium tuberculosis. Ind. Patent 067DEL2006.

Tripathi, R.P., eta/. (2002) Synthesis of glycosylated beta-amino acids as new class of antitubercular agents. Eur J Med Chem, 37, 773-781.

Verdonk, M.L., eta/. (2003) Improved protein-ligand docking using GOLD. Proteins, 52,609-623.

Vriend, G. (1990) WHAT IF: a molecular modeling and drug design program. J Mol Graph, 8, 52-56.

Wang, Z.Y., eta/. (2003) The glyoxylate cycle is required for temporal regulation of virulence by the plant pathogenic fungus Magnaporthe grisea. Mol Microbiol, 47, 1601-1612.

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