Solution structures of Mycobacterium tuberculosis thioredoxin C and models of intact thioredoxin system suggest new approaches to inhibitor and drug design

Here, we report the NMR solution structures of Mycobacterium tuberculosis (M. tuberculosis) thioredoxin C in both oxidized and reduced states, with discussion of structural changes that occur in going between redox states. The NMR solution structure of the oxidized TrxC corresponds closely to that of the crystal structure, except in the C‐terminal region. It appears that crystal packing effects have caused an artifactual shift in the α4 helix in the previously reported crystal structure, compared with the solution structure. On the basis of these TrxC structures, chemical shift mapping, a previously reported crystal structure of the M. tuberculosis thioredoxin reductase (not bound to a Trx) and structures for intermediates in the E. coli thioredoxin catalytic cycle, we have modeled the complete M. tuberculosis thioredoxin system for the various steps in the catalytic cycle. These structures and models reveal pockets at the TrxR/TrxC interface in various steps in the catalytic cycle, which can be targeted in the design of uncompetitive inhibitors as potential anti‐mycobacterial agents, or as chemical genetic probes of function. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

The structure of glucose‐1‐phosphate thymidylyltransferase from Mycobacterium tuberculosis reveals the location of an essential magnesium ion in the RmlA‐type enzymes

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, continues to be a major threat to populations worldwide. Whereas the disease is treatable, the drug regimen is arduous at best with the use of four antimicrobials over a six‐month period. There is clearly a pressing need for the development of new therapeutics. One potential target for structure‐based drug design is the enzyme RmlA, a glucose‐1‐phosphate thymidylyltransferase. This enzyme catalyzes the first step in the biosynthesis of l ‐rhamnose, which is a deoxysugar critical for the integrity of the bacterium's cell wall. Here, we report the X‐ray structures of M. tuberculosis RmlA in complex with either dTTP or dTDP‐glucose to 1.6 Å and 1.85 Å resolution, respectively. In the RmlA/dTTP complex, two magnesium ions were observed binding to the nucleotide, both ligated in octahedral coordination spheres. In the RmlA/dTDP‐glucose complex, only a single magnesium ion was observed. Importantly, for RmlA‐type enzymes with known three‐dimensional structures, not one model shows the position of the magnesium ion bound to the nucleotide‐linked sugar. As such, this investigation represents the first direct observation of the manner in which a magnesium ion is coordinated to the RmlA product and thus has important ramifications for structure‐based drug design. In the past, molecular modeling procedures have been employed to derive a three‐dimensional model of the M. tuberculosis RmlA for drug design. The X‐ray structures presented herein provide a superior molecular scaffold for such endeavors in the treatment of one of the world's deadliest diseases.     

Crystal structure of the Mycobacterium tuberculosis phosphate binding protein PstS3

Mycobacterium tuberculosis evades host immune responses by colonizing macrophages. Intraphagosomal M. tuberculosis is exposed to environmental stresses such as reactive oxygen and nitrogen intermediates as well as acid shock and inorganic phosphate (Pi) depletion. Experimental evidence suggests that expression levels of mycobacterial protein PstS3 (Rv0928) are significantly increased when M. tuberculosis bacilli are exposed to Pi starvation. Hence, PstS3 may be important for survival of Mtb in conditions where there is limited supply of Pi. We report here the structure of PstS3 from M. tuberculosis at 2.3‐Å resolution. The protein presents a structure typical for ABC phosphate transfer receptors. Comparison with its cognate receptor PstS1 showed a different pattern distribution of surface charges in proximity to the Pi recognition site, suggesting complementary roles of the two proteins in Pi uptake. Proteins 2014; 82:2268–2274. © 2014 Wiley Periodicals, Inc.     

Characterization of a triazole scaffold compound as an inhibitor of Mycobacterium tuberculosis imidazoleglycerol-phosphate dehydratase

Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB), employs ten enzymes including imidazoleglycerol-phosphate dehydratase (IGPD) for de novo biosynthesis of histidine. The absence of histidine-biosynthesis in humans combined with its essentiality for Mtb makes the enzymes of this pathway major anti-TB drug targets. We explored the inhibitory potential of a small molecule β-(1,2,4-Triazole-3-yl)-DL-alanine (DLA) against Mtb IGPD. DLA exhibits an in vitro inhibitory efficacy in the lower micromolar range. Higher-resolution crystal structures of native and substrate-bound Mtb IGPD provided additional structural features of this important drug target. Crystal structure of IGPD-DLA complex at a resolution of 1.75 Å, confirmed that DLA locks down the function of the enzyme by binding in the active site pocket of the IGPD mimicking the substrate-binding mode to a high degree. In our biochemical study, DLA showed an efficient inhibition of Mtb IGPD. Furthermore, DLA also showed bactericidal activity against Mtb and Mycobacterium smegmatis and inhibited their growth in respective culture medium. Importantly, owing to the favorable ADME and physicochemical properties, it serves as an important lead molecule for further derivatizations.     

Insight into the structural requirements of aminopyrimidine derivatives for good potency against both purified enzyme and whole cells of M. tuberculosis: combination of HQSAR,CoMSIA, and MD simulation studies

The Mycobacterium tuberculosis protein kinase B (PknB) is critical for growth and survival of M. tuberculosis within the host. The series of aminopyrimidine derivatives show impressive activity against PknB (IC₅₀ < .5 μM). However, most of them show weak or no cellular activity against M. tuberculosis (MIC > 63 μM). Consequently, the key structural features related to activity against of both PknB and M. tuberculosis need to be investigated. Here, two- and three-dimensional quantitative structure–activity relationship (2D and 3D QSAR) analyses combined with molecular dynamics (MD) simulations were employed with the aim to evaluate these key structural features of aminopyrimidine derivatives. Hologram quantitative structure–activity relationship (HQSAR) and CoMSIA models constructed from IC₅₀ and MIC values of aminopyrimidine compounds could establish the structural requirements for better activity against of both PknB and M. tuberculosis. The NH linker and the R₁ substituent of the template compound are not only crucial for the biological activity against PknB but also for the biological activity against M. tuberculosis. Moreover, the results obtained from MD simulations show that these moieties are the key fragments for binding of aminopyrimidine compounds in PknB. The combination of QSAR analysis and MD simulations helps us to provide a structural concept that could guide future design of PknB inhibitors with improved potency against both the purified enzyme and whole M. tuberculosis cells.     

Atomic resolution structures of Escherichia coli and Bacillus anthracis malate synthase A: Comparison with isoform G and implications for structure‐based drug discovery

Enzymes of the glyoxylate shunt are important for the virulence of pathogenic organisms such as Mycobacterium tuberculosis and Candida albicans. Two isoforms have been identified for malate synthase, the second enzyme in the pathway. Isoform A, found in fungi and plants, comprises ～530 residues, whereas isoform G, found only in bacteria, is larger by ～200 residues. Crystal structures of malate synthase isoform G from Escherichia coli and Mycobacterium tuberculosis were previously determined at moderate resolution. Here we describe crystal structures of E. coli malate synthase A (MSA) in the apo form (1.04 Å resolution) and in complex with acetyl‐coenzyme A and a competitive inhibitor, possibly pyruvate or oxalate (1.40 Å resolution). In addition, a crystal structure for Bacillus anthracis MSA at 1.70 Å resolution is reported. The increase in size between isoforms A and G can be attributed primarily to an inserted α/β domain that may have regulatory function. Upon binding of inhibitor or substrate, several active site loops in MSA undergo large conformational changes. However, in the substrate bound form, the active sites of isoforms A and G from E. coli are nearly identical. Considering that inhibitors bind with very similar affinities to both isoforms, MSA is as an excellent platform for high‐resolution structural studies and drug discovery efforts.     

Crystal structure of decaprenylphosphoryl‐β‐ D‐ribose 2'‐epimerase from Mycobacterium smegmatis

Decaprenylphosphoryl‐β‐D ‐ribose 2'‐epimerase (DprE1) is an essential enzyme in the biosynthesis of cell wall components and a target for development of anti‐tuberculosis drugs. We determined the crystal structure of a truncated form of DprE1 from Mycobacterium smegmatis in two crystal forms to up to 2.35 Å resolution. The structure extends from residue 75 to the C‐terminus and shares homology with FAD‐dependent oxidoreductases of the vanillyl‐alcohol oxidase family including the DprE1 homologue from M. tuberculosis. The M. smegmatis DprE1 structure reported here provides further insights into the active site geometry of this tuberculosis drug target. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Crystallization and preliminary crystallographic study of human CksHs1: A cell cycle regulatory protein

The cell cycle regulatory protein CksHs1 has been crystallized in a form suitable for X-ray studies. CksHsl crystals were grown in the presence of vanadate, a phos-phatase inhibitor, but were also obtained with phosphate or tungstate as a cofactor. They belong to the hexagonal space group P6₁22 with unit cell dimensions: a=b=94 Å, c=131.6 Å, and γ =120. The crystals grown in the presence of vanadate diffract X-rays to at least 2.8 Å. Molecular replacement results from the homologous human CksHs2 structure reveal that a dimer forms the crystal habit, giving the unusual V_m value of 4.4 ų/Da or a solvent content of 72%. © 1995 Wiley-Liss, Inc.     

Reconstitution of oxaloacetate metabolism in the tricarboxylic acid cycle in Synechocystis sp. PCC 6803: discovery of important factors that directly affect the conversion of oxaloacetate

The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathways in nature. Oxygenic photoautotrophic bacteria, cyanobacteria, have an unusual TCA cycle. The TCA cycle in cyanobacteria contains two unique enzymes that are not part of the TCA cycle in other organisms. In recent years, sustainable metabolite production from carbon dioxide using cyanobacteria has been looked at as a means to reduce the environmental burden of this gas. Among cyanobacteria, the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) is an optimal host for sustainable metabolite production. Recently, metabolite production using the TCA cycle in Synechocystis 6803 has been carried out. Previous studies revealed that the branch point of the oxidative and reductive TCA cycles, oxaloacetate metabolism, plays a key role in metabolite production. However, the biochemical mechanisms regulating oxaloacetate metabolism in Synechocystis 6803 are poorly understood. Concentrations of oxaloacetate in Synechocystis 6803 are extremely low, such that in vivo analysis of oxaloacetate metabolism does not seem realistic. Therefore, using purified enzymes, we reconstituted oxaloacetate metabolism in Synechocystis 6803 in vitro to reveal the regulatory mechanisms involved. Reconstitution of oxaloacetate metabolism revealed that pH, Mg²⁺ and phosphoenolpyruvate are important factors affecting the conversion of oxaloacetate in the TCA cycle. Biochemical analyses of the enzymes involved in oxaloacetate metabolism in this and previous studies revealed the biochemical mechanisms underlying the effects of these factors on oxaloacetate conversion. In addition, we clarified the function of two l- malate dehydrogenase isozymes in oxaloacetate metabolism. These findings serve as a basis for various applications of the cyanobacterial TCA cycle.     

GarA is an essential regulator of metabolism in Mycobacterium tuberculosis

Alpha‐ketoglutarate is a key metabolic intermediate at the crossroads of carbon and nitrogen metabolism, whose fate is tightly regulated. In mycobacteria the protein GarA regulates the tricarboxylic acid cycle and glutamate synthesis by direct binding and regulation of three enzymes that use α‐ketoglutarate. GarA, in turn, is thought to be regulated via phosphorylation by protein kinase G and other kinases. We have investigated the requirement for GarA for metabolic regulation during growth in vitro and in macrophages. GarA was found to be essential to Mycobacterium tuberculosis, but dispensable in non‐pathogenic Mycobacterium smegmatis. Disruption of garA caused a distinctive, nutrient‐dependent phenotype, fitting with its proposed role in regulating glutamate metabolism. The data underline the importance of the TCA cycle and the balance with glutamate synthesis in M. tuberculosis and reveal vulnerability to disruption of these pathways.     

Structural studies on the enzyme complex isopropylmalate isomerase (LeuCD) from Mycobacterium tuberculosis

The absence of the leucine biosynthesis pathway in humans makes the enzymes of this pathway in pathogenic bacteria such as Mycobacterium tuberculosis potential candidates for developing novel antibacterial drugs. One of these enzymes is isopropylmalate isomerase (IPMI). IPMI exists as a complex of two subunits: the large (LeuC) and the small (LeuD) subunit. The functional LeuCD complex catalyzes the stereospecific conversion reaction of α‐isopropylmalate to β‐isopropylmalate. Three C‐terminally truncated variants of LeuD have been analyzed by X‐ray crystallography to resolutions of 2.0 Å (LeuD_1–156), 1.2 Å (LeuD_1–168), and 2.5 Å (LeuD_1–186), respectively. The two most flexible parts of the structure are the regions of residues 30–37, the substrate discriminating loop, and of residues 70–74, the substrate binding loop. The three determined structures were also compared with the structures of other bacterial LeuDs. This comparison suggests the presence of two LeuD subfamilies. A model for the structure of the inactive enzyme complex has been obtained from solution X‐ray scattering experiments. The crystal structure of LeuD was shown to be compatible with the solution X‐ray scattering data from the small subunit. In contrast, the solution scattering results suggest that the large subunit LeuC and the LeuCD complex have overall shapes, which are radically different from the ones observed in the crystals of the functional homolog mitochondrial aconitase. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

High-level heterologous production of propionate in engineered Escherichia coli

A propanologenic (i.e., 1-propanol-producing) bacterium Escherichia coli strain was previously derived by activating the genomic sleeping beauty mutase (Sbm) operon. The activated Sbm pathway branches out of the tricarboxylic acid (TCA) cycle at the succinyl-CoA node to form propionyl-CoA and its derived metabolites of 1-propanol and propionate. In this study, we targeted several TCA cycle genes encoding enzymes near the succinyl-CoA node for genetic manipulation to identify the individual contribution of the carbon flux into the Sbm pathway from the three TCA metabolic routes, that is, oxidative TCA cycle, reductive TCA branch, and glyoxylate shunt. For the control strain CPC-Sbm, in which propionate biosynthesis occurred under relatively anaerobic conditions, the carbon flux into the Sbm pathway was primarily derived from the reductive TCA branch, and both succinate availability and the SucCD-mediated interconversion of succinate/succinyl-CoA were critical for such carbon flux redirection. Although the oxidative TCA cycle normally had a minimal contribution to the carbon flux redirection, the glyoxylate shunt could be an alternative and effective carbon flux contributor under aerobic conditions. With mechanistic understanding of such carbon flux redirection, metabolic strategies based on blocking the oxidative TCA cycle (via ∆sdhA mutation) and deregulating the glyoxylate shunt (via ∆iclR mutation) were developed to enhance the carbon flux redirection and therefore propionate biosynthesis, achieving a high propionate titer of 30.9 g/L with an overall propionate yield of 49.7% upon fed-batch cultivation of the double mutant strain CPC-Sbm∆sdhA∆iclR under aerobic conditions. The results also suggest that the Sbm pathway could be metabolically active under both aerobic and anaerobic conditions.     

Detection of anti-tuberculosis activity in some folklore plants by radiometric BACTEC assay

Aims: The anti‐tubercular drugs are less effective because of the emergence of multi‐drug resistant (MDR) and extensively drug resistant (XDR) strains of M. tuberculosis, so plants being an alternative source of anti‐microbial compounds. The aim of this study was to investigate anti‐tuberculosis potential of the plants using Mycobacterium smegmatis as a rapid screening model for detection of anti‐mycobacterial activity and further to evaluate the active plants for anti‐tuberculosis activity against M. tuberculosis using radiometric BACTEC assay. Methods and Results: The 15 plants were screened for anti‐mycobacterial activity against M. smegmatis by the disk diffusion assay. The ethanolic extracts of Mallotus philippensis, Vitex negundo, Colebrookea oppositifolia, Rumex hastatus, Mimosa pudica, Kalanchoe integra and Flacourtia ramontchii were active against M. smegmatis in primary screening. The anti‐tuberculosis potential was identified in the leaves extracts of Mallotus philippensis by radiometric BACTEC assay. The ethanolic extract of M. philippensis showed anti‐tuberculosis activity against virulent and avirulent strains of M. tuberculosis H₃₇Rv and M. tuberculosis H₃₇Ra with minimum inhibitory concentration 0·25 and 0·125 mg ml^?1, respectively. The inhibition in growth index values of M. tuberculosis was observed in the presence of ethyl acetate fraction at a minimum concentration of 0·05 mg ml^?1. Conclusion: We found that BACTEC radiometric assay is a valuable method for detection of anti‐tuberculosis activity of the plant extracts. The results indicate that ethanolic extract and ethyl acetate fraction of M. philippensis exhibited significant anti‐mycobacterial activity against M. tuberculosis. Significance and Impact of the Study: These findings provide scientific evidence to support the traditional medicinal uses of M. philippensis and indicate a promising potential of this plant for the development of anti‐tuberculosis agent.     

Structural and ligand binding analyses of the periplasmic sensor domain of RsbU in Chlamydia trachomatis support a role in TCA cycle regulation

Chlamydia trachomatis is an obligate intracellular bacteria that undergo dynamic morphologic and physiologic conversions upon gaining an access to a eukaryotic cell. These conversions likely require the detection of key environmental conditions and regulation of metabolic activity. Chlamydia encodes homologs to proteins in the Rsb phosphoregulatory partner-switching pathway, best described in Bacillus subtilis. ORF CT588 has a strong sequence similarity to RsbU cytoplasmic phosphatase domain but also contains a unique periplasmic sensor domain that is expected to control the phosphatase activity. A 1.7 Å crystal structure of the periplasmic domain of the RsbU protein from C. trachomatis (PDB 6MAB) displays close structural similarity to DctB from Vibrio and Sinorhizobium. DctB has been shown, both structurally and functionally, to specifically bind to the tricarboxylic acid (TCA) cycle intermediate succinate. Surface plasmon resonance and differential scanning fluorimetry of TCA intermediates and potential metabolites from a virtual screen of RsbU revealed that alpha-ketoglutarate, malate and oxaloacetate bound to the RsbU periplasmic domain. Substitutions in the putative binding site resulted in reduced binding capabilities. An RsbU null mutant showed severe growth defects which could be restored through genetic complementation. Chemical inhibition of ATP synthesis by oxidative phosphorylation phenocopied the growth defect observed in the RsbU null strain. Altogether, these data support a model with the Rsb system responding differentially to TCA cycle intermediates to regulate metabolism and key differentiation processes.     

Metabolic fluxes for nutritional flexibility of Mycobacterium tuberculosis

The co‐catabolism of multiple host‐derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady‐state chemostat system. We demonstrate that Mtb efficiently co‐metabolises either cholesterol or glycerol, in combination with two‐carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt.     

Multiplex PCR assay for simultaneous detection and differentiation of Mycobacterium tuberculosis, Mycobacterium avium complexes and other Mycobacterial species directly from clinical specimens

Aims: Polymerase chain reaction (PCR) is the most rapid and sensitive method for diagnosing mycobacterial infections and identifying the aetiological Mycobacterial species in order to administer the appropriate therapy and for better patient management. Methods and Results: Two hundred and thirty‐five samples from 145 clinically suspected cases of tuberculosis were processed for the detection of Mycobacterial infections by ZN (Ziehl Neelsen) smear examination, L‐J & BACTEC^TM MGIT‐960 culture and multiplex PCR tests. The multiplex PCR comprised of genus‐specific primers targeting hsp65 gene, Mycobacterium tuberculosis complex‐specific primer targeting cfp10 (Rv3875, esxB) region and Mycobacterium avium complex‐specific primer pairs targeting 16S–23S Internal Transcribed Spacer sequences. The multiplex PCR developed had an analytical sensitivity of 10 fg (3–4 cells) of mycobacterial DNA. The multiplex PCR test showed the highest (77·24%) detection rate, while ZN smear examination had the lowest (20%) detection rate, which was bettered by L‐J culture (34·4%) and BACTEC^TM MGIT‐960 culture (50·34%) methods. The mean isolation time for M. tuberculosis was 19·03 days in L‐J culture and 8·7 days in BACTEC^TM MGIT‐960 culture. Using the multiplex PCR, we could establish M. tuberculosis + M. avium co‐infection in 1·3% HIV‐negative and 2·9% HIV‐positive patients. The multiplex PCR was also highly useful in diagnosing mycobacteraemia in 38·09% HIV‐positive and 15·38% HIV‐negative cases. Conclusions: The developed in‐house multiplex PCR could identify and differentiate the M. tuberculosis and M. avium complexes from other Mycobacterial species directly from clinical specimens. Significance and Impact of the Study: The triplex PCR developed by us could be used to detect and differentiate M. tuberculosis, M. avium and other mycobacteria in a single reaction tube.     

The tricarboxylic acid cycle in L₃ Teladorsagia circumcincta: metabolism of acetyl CoA to succinyl CoA

Nematodes, like other species, derive much of the energy for cellular processes from mitochondrial pathways including the TCA cycle. Previously, we have shown L₃Teladorsagia circumcincta consume oxygen and so may utilise a full TCA cycle for aerobic energy metabolism. We have assessed the relative activity levels and substrate affinities of citrate synthase, aconitase, isocitrate dehydrogenase (both NAD⁺ and NADP⁺ specific) and α-ketoglutarate dehydrogenase in homogenates of L₃T. circumcincta. All of these enzymes were present in homogenates. Compared with citrate synthase, low levels of enzyme activity and low catalytic efficiency was observed for NAD⁺ isocitrate dehydrogenase and especially α-ketoglutarate dehydrogenase. Therefore, it is likely that the activity of these to enzymes regulate overall metabolite flow through the TCA cycle, especially when [NAD⁺] limits enzyme activity. Of the enzymes tested, only citrate synthase had substrate affinities which were markedly different from values obtained from mammalian species. Overall, the results are consistent with the suggestion that a full TCA cycle exists within L₃T. circumcincta. While there may subtle variations in enzyme properties, particularly for citrate synthase, the control points for the TCA cycle in L₃T. circumcincta are probably similar to those in the tissues of their host species.     

Enhancement of pyruvate production byTorulopsis glabrata through supplement of oxaloacetate as carbon source

The capability of utilizing a TCA cycle intermediates as the sole carbon source by the multi-vitamin auxotrophic yeastTorulopsis glabrata CCTCC M202019 was demonstrated with plate count method. It is indicated thatT. glabrata could grew on a medium with one of the TCA cycle intermediates as the sole carbon source, but more colonies were observed when glucose, acetate and one of the TCA cycle intermediates coexisted in the medium. Among the intermediates of the TCA cycle examined in this study, cell growth was improved by supplementing oxaloacetate. Further investigation showed that the presence of acetate was necessary when oxaloacetate was supplemented. By supplementing with 10 g/L of oxaloacetate in pyruvate batch fermentation, dry cell weight increased from 11.8 g/L to 13.6 g/L, and pyruvate productivity was enhanced from 0.96 gL⁻¹h⁻¹ to 1.19 gL⁻¹h⁻¹ after cultivation of 56 h. The yield of pyruvate to glucose was also improved from 0.63 g/g to 0.66 g/g. These results indicate that under vitamins limitation, the productivity and yield of pyruvate could be enhancedvia an increase of cell growth by the supplementation of oxaloacetate.     

Structure-based screening and molecular dynamics simulations offer novel natural compounds as potential inhibitors of Mycobacterium tuberculosis isocitrate lyase

Mycobacterium tuberculosis is the etiological agent of tuberculosis in humans and is responsible for more than two million deaths annually. M. tuberculosis isocitrate lyase (MtbICL) catalyzes the first step in the glyoxylate cycle, plays a pivotal role in the persistence of M. tuberculosis, which acts as a potential target for an anti-tubercular drug. To identify the potential anti-tuberculosis compound, we conducted a structure-based virtual screening of natural compounds from the ZINC database (n = 1,67,748) against the MtbICL structure. The ligands were docked against MtbICL in three sequential docking modes that resulted in 340 ligands having better docking score. These compounds were evaluated for Lipinski and ADMET prediction, and 27 compounds were found to fit well with re-docking studies. After refinement by molecular docking and drug-likeness analyses, three potential inhibitors (ZINC1306071, ZINC2111081, and ZINC2134917) were identified. These three ligands and the reference compounds were further subjected to molecular dynamics simulation and binding energy analyses to compare the dynamic structure of protein after ligand binding and the stability of the MtbICL and bound complexes. The binding free energy analyses were calculated to validate and capture the intermolecular interactions. The results suggested that the three compounds had a negative binding energy with ?96.462, ?143.549, and ?122.526 kJ mol^?1 for compounds with IDs ZINC1306071, ZINC2111081, and ZINC2134917, respectively. These lead compounds displayed substantial pharmacological and structural properties to be drug candidates. We concluded that ZINC2111081 has a great potential to inhibit MtbICL and would add to the drug discovery process against tuberculosis.