Natural mutations in the sensor kinase of the PhoPR two-component regulatory system modulate virulence of ancestor-like tuberculosis bacilli.

The molecular factors and genetic adaptations that contributed to the emergence of Mycobacterium tuberculosis (MTB) from an environmental Mycobacterium canettii-like ancestor, remain poorly investigated. In MTB, the PhoPR two-component regulatory system controls production and secretion of proteins and lipid virulence effectors. Here, we describe that several mutations, present in phoR of M. canettii relative to MTB, impact the expression of the PhoP regulon and the pathogenicity of the strains. First, we establish a molecular model of PhoR and show that some substitutions found in PhoR of M. canettii are likely to impact the structure and activity of this protein. Second, we show that STB-K, the most attenuated available M. canettii strain, displays lower expression of PhoP-induced genes than MTB. Third, we demonstrate that genetic swapping of the phoPR allele from STB-K with the ortholog from MTB H37Rv enhances expression of PhoP-controlled functions and the capacities of the recombinant strain to colonize human macrophages, the MTB target cells, as well as to cause disease in several mouse infection models. Fourth, we extended these observations to other M. canettii strains and confirm that PhoP-controlled functions are expressed at lower levels in most M. canettii strains than in M. tuberculosis. Our findings suggest that distinct PhoR variants have been selected during the evolution of tuberculosis bacilli, contributing to higher pathogenicity and persistence of MTB in the mammalian host.

Exploring the plasticity of the InhA substrate-binding site using new diaryl ether inhibitors.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a worldwide scourge with more than 10 million people affected yearly. Among the proteins essential for the survival of Mtb, InhA has been and is still clinically validated as a therapeutic target. A new family of direct diaryl ether inhibitors, not requiring prior activation by the catalase peroxidase enzyme KatG, has been designed with the ambition of fully occupying the InhA substrate-binding site. Thus, eleven compounds, featuring three pharmacophores within the same molecule, were synthesized. One of them, 5-(((4-(2-hydroxyphenoxy)benzyl)(octyl)amino)methyl)-2-phenoxyphenol (compound 21), showed good inhibitory activity against InhA with IC50 of 0.70 µM. The crystal structure of compound 21 in complex with InhA/NAD+ showed how the molecule fills the substrate-binding site as well as the minor portal of InhA. This study represents a further step towards the design of new inhibitors of InhA.

Microtubule nucleation by γ-tubulin complexes.

Microtubule nucleation is regulated by the γ-tubulin ring complex (γTuRC) and related γ-tubulin complexes, providing spatial and temporal control over the initiation of microtubule growth. Recent structural work has shed light on the mechanism of γTuRC-based microtubule nucleation, confirming the long-standing hypothesis that the γTuRC functions as a microtubule template. The first crystallographic analysis of a non-γ-tubulin γTuRC component (γ-tubulin complex protein 4 (GCP4)) has resulted in a new appreciation of the relationships among all γTuRC proteins, leading to a refined model of their organization and function. The structures have also suggested an unexpected mechanism for regulating γTuRC activity via conformational modulation of the complex component GCP3. New experiments on γTuRC localization extend these insights, suggesting a direct link between its attachment at specific cellular sites and its activation.

Solution structure of the type I polyketide synthase Pks13 from Mycobacterium tuberculosis.

BACKGROUND: Type I polyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of a group of diverse natural compounds with biotechnological and pharmaceutical interest called polyketides. The diversity of polyketides is impressive despite the limited set of catalytic domains used by PKSs for biosynthesis, leading to considerable interest in deciphering their structure-function relationships, which is challenging due to high intrinsic flexibility. Among nineteen polyketide synthases encoded by the genome of Mycobacterium tuberculosis, Pks13 is the condensase required for the final condensation step of two long acyl chains in the biosynthetic pathway of mycolic acids, essential components of the cell envelope of Corynebacterineae species. It has been validated as a promising druggable target and knowledge of its structure is essential to speed up drug discovery to fight against tuberculosis. RESULTS: We report here a quasi-atomic model of Pks13 obtained using small-angle X-ray scattering of the entire protein and various molecular subspecies combined with known high-resolution structures of Pks13 domains or structural homologues. As a comparison, the low-resolution structures of two other mycobacterial polyketide synthases, Mas and PpsA from Mycobacterium bovis BCG, are also presented. This study highlights a monomeric and elongated state of the enzyme with the apo- and holo-forms being identical at the resolution probed. Catalytic domains are segregated into two parts, which correspond to the condensation reaction per se and to the release of the product, a pivot for the enzyme flexibility being at the interface. The two acyl carrier protein domains are found at opposite sides of the ketosynthase domain and display distinct characteristics in terms of flexibility. CONCLUSIONS: The Pks13 model reported here provides the first structural information on the molecular mechanism of this complex enzyme and opens up new perspectives to develop inhibitors that target the interactions with its enzymatic partners or between catalytic domains within Pks13 itself.

Drug screening approach against mycobacterial fatty acyl-AMP ligase FAAL32 renews the interest of the salicylanilide pharmacophore in the fight against tuberculosis.

Tuberculosis (TB) remains a global health crisis, further exacerbated by the slow pace of new treatment options, and the emergence of extreme and total drug resistance to existing drugs. The challenge to developing new antibacterial compounds with activity against Mycobacterium tuberculosis (Mtb), the causative agent of TB, is in part due to unique features of this pathogen, especially the composition and structure of its complex cell envelope. Therefore, targeting enzymes involved in cell envelope synthesis has been of major interest for anti-TB drug discovery. FAAL32 is a fatty acyl-AMP ligase involved in the biosynthesis of the cell wall mycolic acids, and a potential target for drug discovery. To rapidly advance research in this area, we initiated a drug repurposing campaign and screened a collection of 1280 approved human or veterinary drugs (Prestwick Chemical Library) using a biochemical assay that reads out FAAL32 inhibition. These efforts led to the discovery of salicylanilide closantel, and some of its derivatives as inhibitors with potent in vitro activity against M. tuberculosis. These results suggest that salicylanilide represents a potentially promising pharmacophore for the conception of novel anti-tubercular candidates targeting FAAL32 that would open new targeting opportunities. Moreover, this work illustrates the value of drug repurposing campaigns to discover new leads in challenging drug discovery fields.

Urea derivatives carrying a thiophenylthiazole moiety: Design, synthesis, and evaluation of antitubercular and InhA inhibitory activities.

The recent emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) has complicated and significantly slowed efforts to eradicate and/or reduce the worldwide incidence of life-threatening acute and chronic cases of tuberculosis. To overcome this setback, researchers have increased the intensity of their work to identify new small-molecule compounds that are expected to remain efficacious antimicrobials against Mtb. Here, we describe our effort to apply the principles of molecular hybridization to synthesize 16 compounds carrying thiophene and thiazole rings beside the core urea functionality (TTU1-TTU16). Following extensive structural characterization, the obtained compounds were initially evaluated for their antimycobacterial activity against Mtb H37Rv. Subsequently, three derivatives standing out with their anti-Mtb activity profiles and low cytotoxicity (TTU5, TTU6, and TTU12) were tested on isoniazid-resistant clinical isolates carrying katG and inhA mutations. Additionally, due to their pharmacophore similarities to the well-known InhA inhibitors, the molecules were screened for their enoyl acyl carrier protein reductase (InhA) inhibitory potentials. Molecular docking studies were performed to support the experimental enzyme inhibition data. Finally, drug-likeness of the selected compounds was established by theoretical calculations of physicochemical descriptors.

Directed evolution of SecB chaperones toward toxin-antitoxin systems.

SecB chaperones assist protein export in bacteria. However, certain SecB family members have diverged to become specialized toward the control of toxin-antitoxin (TA) systems known to promote bacterial adaptation to stress and persistence. In such tripartite TA-chaperone (TAC) systems, the chaperone was shown to assist folding and to prevent degradation of its cognate antitoxin, thus facilitating inhibition of the toxin. Here, we used both the export chaperone SecB of Escherichia coli and the tripartite TAC system of Mycobacterium tuberculosis as a model to investigate how generic chaperones can specialize toward the control of TA systems. Through directed evolution of SecB, we have identified and characterized mutations that specifically improve the ability of SecB to control our model TA system without affecting its function in protein export. Such a remarkable plasticity of SecB chaperone function suggests that its substrate binding surface can be readily remodeled to accommodate specific clients.

Protein X-ray Crystallography and Drug Discovery.

With the advent of structural biology in the drug discovery process, medicinal chemists gained the opportunity to use detailed structural information in order to progress screening hits into leads or drug candidates. X-ray crystallography has proven to be an invaluable tool in this respect, as it is able to provide exquisitely comprehensive structural information about the interaction of a ligand with a pharmacological target. As fragment-based drug discovery emerged in the recent years, X-ray crystallography has also become a powerful screening technology, able to provide structural information on complexes involving low-molecular weight compounds, despite weak binding affinities. Given the low numbers of compounds needed in a fragment library, compared to the hundreds of thousand usually present in drug-like compound libraries, it now becomes feasible to screen a whole fragment library using X-ray crystallography, providing a wealth of structural details that will fuel the fragment to drug process. Here, we review theoretical and practical aspects as well as the pros and cons of using X-ray crystallography in the drug discovery process.

Structural Insights into the Carbohydrate Binding Ability of an α-(1→2) Branching Sucrase from Glycoside Hydrolase Family 70.

The α-(1→2) branching sucrase ΔN123-GBD-CD2 is a transglucosylase belonging to glycoside hydrolase family 70 (GH70) that catalyzes the transfer ofd-glucosyl units from sucroseto dextrans or gluco-oligosaccharides via the formation of α-(1→2) glucosidic linkages. The first structures of ΔN123-GBD-CD2 in complex withd-glucose, isomaltosyl, or isomaltotriosyl residues were solved. The glucose complex revealed three glucose-binding sites in the catalytic gorge and six additional binding sites at the surface of domains B, IV, and V. Soaking with isomaltotriose or gluco-oligosaccharides led to structures in which isomaltosyl or isomaltotriosyl residues were found in glucan binding pockets located in domain V. One aromatic residue is systematically identified at the bottom of these pockets in stacking interaction with one glucosyl moiety. The carbohydrate is also maintained by a network of hydrogen bonds and van der Waals interactions. The sequence of these binding pockets is conserved and repeatedly present in domain V of several GH70 glucansucrases known to bind α-glucans. These findings provide the first structural evidence of the molecular interaction occurring between isomalto-oligosaccharides and domain V of the GH70 enzymes.

Insight into Structure-Function Relationships and Inhibition of the Fatty Acyl-AMP Ligase (FadD32) Orthologs from Mycobacteria.

Mycolic acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is one of the targets of first-line antituberculous drugs. This pathway contains a number of potential targets, including some that have been identified only recently and have yet to be explored. One such target, FadD32, is required for activation of the long meromycolic chain and is essential for mycobacterial growth. We report here an in-depth biochemical, biophysical, and structural characterization of four FadD32 orthologs, including the very homologous enzymes fromMycobacterium tuberculosisandMycobacterium marinum Determination of the structures of two complexes with alkyl adenylate inhibitors has provided direct information, with unprecedented detail, about the active site of the enzyme and the associated hydrophobic tunnel, shedding new light on structure-function relationships and inhibition mechanisms by alkyl adenylates and diarylated coumarins. This work should pave the way for the rational design of inhibitors of FadD32, a highly promising drug target.

Ser/Thr Phosphorylation Regulates the Fatty Acyl-AMP Ligase Activity of FadD32, an Essential Enzyme in Mycolic Acid Biosynthesis.

Mycolic acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is a well known source of antituberculous drug targets. Among the promising new targets in the pathway, FadD32 is an essential enzyme required for the activation of the long meromycolic chain of mycolic acids and is essential for mycobacterial growth. Following the in-depth biochemical, biophysical, and structural characterization of FadD32, we investigated its putative regulation via post-translational modifications. Comparison of the fatty acyl-AMP ligase activity between phosphorylated and dephosphorylated FadD32 isoforms showed that the native protein is phosphorylated by serine/threonine protein kinases and that this phosphorylation induced a significant loss of activity. Mass spectrometry analysis of the native protein confirmed the post-translational modifications and identified Thr-552 as the phosphosite. Phosphoablative and phosphomimetic FadD32 mutant proteins confirmed both the position and the importance of the modification and its correlation with the negative regulation of FadD32 activity. Investigation of the mycolic acid condensation reaction catalyzed by Pks13, involving FadD32 as a partner, showed that FadD32 phosphorylation also impacts the condensation activity. Altogether, our results bring to light FadD32 phosphorylation by serine/threonine protein kinases and its correlation with the enzyme-negative regulation, thus shedding a new horizon on the mycolic acid biosynthesis modulation and possible inhibition strategies for this promising drug target.

New insights on the mechanism of quinoline-based DNA Methyltransferase inhibitors.

Among the epigenetic marks, DNA methylation is one of the most studied. It is highly deregulated in numerous diseases, including cancer. Indeed, it has been shown that hypermethylation of tumor suppressor genes promoters is a common feature of cancer cells. Because DNA methylation is reversible, the DNA methyltransferases (DNMTs), responsible for this epigenetic mark, are considered promising therapeutic targets. Several molecules have been identified as DNMT inhibitors and, among the non-nucleoside inhibitors, 4-aminoquinoline-based inhibitors, such as SGI-1027 and its analogs, showed potent inhibitory activity. Here we characterized the in vitro mechanism of action of SGI-1027 and two analogs. Enzymatic competition studies with the DNA substrate and the methyl donor cofactor, S-adenosyl-l-methionine (AdoMet), displayed AdoMet non-competitive and DNA competitive behavior. In addition, deviations from the Michaelis-Menten model in DNA competition experiments suggested an interaction with DNA. Thus their ability to interact with DNA was established; although SGI-1027 was a weak DNA ligand, analog 5, the most potent inhibitor, strongly interacted with DNA. Finally, as 5 interacted with DNMT only when the DNA duplex was present, we hypothesize that this class of chemical compounds inhibit DNMTs by interacting with the DNA substrate.

Phosphorylation of KasB regulates virulence and acid-fastness in Mycobacterium tuberculosis.

Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4-6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase-dependent KasB phosphorylation in regulating the later stages of mycolic acid elongation, with important consequences in terms of acid-fast staining and pathogenicity.

Crystal structure of the enoyl-ACP reductase of Mycobacterium tuberculosis (InhA) in the apo-form and in complex with the active metabolite of isoniazid pre-formed by a biomimetic approach.

InhA is an enoyl-ACP reductase of Mycobacterium tuberculosis implicated in the biosynthesis of mycolic acids, essential constituents of the mycobacterial cell wall. To date, this enzyme is considered as a promising target for the discovery of novel antitubercular drugs. In this work, we describe the first crystal structure of the apo form of the wild-type InhA at 1.80Å resolution as well as the crystal structure of InhA in complex with the synthetic metabolite of the antitubercular drug isoniazid refined to 1.40Å. This metabolite, synthesized in the absence of InhA, is able to displace and replace the cofactor NADH in the enzyme active site. This work provides a unique opportunity to enlighten the structural adaptation of apo-InhA to the binding of the NADH cofactor or of the isoniazid adduct. In addition, a differential scanning fluorimetry study of InhA, in the apo-form as well as in the presence of NAD(+), NADH and INH-NADH was performed showing that binding of the INH-NADH adduct had a strong stabilizing effect.

First structural insights into α-L-arabinofuranosidases from the two GH62 glycoside hydrolase subfamilies.

α-L-arabinofuranosidases are glycoside hydrolases that specifically hydrolyze non-reducing residues from arabinose-containing polysaccharides. In the case of arabinoxylans, which are the main components of hemicellulose, they are part of microbial xylanolytic systems and are necessary for complete breakdown of arabinoxylans. Glycoside hydrolase family 62 (GH62) is currently a small family of α-L-arabinofuranosidases that contains only bacterial and fungal members. Little is known about the GH62 mechanism of action, because only a few members have been biochemically characterized and no three-dimensional structure is available. Here, we present the first crystal structures of two fungal GH62 α-L-arabinofuranosidases from the basidiomycete Ustilago maydis (UmAbf62A) and ascomycete Podospora anserina (PaAbf62A). Both enzymes are able to efficiently remove the α-L-arabinosyl substituents from arabinoxylan. The overall three-dimensional structure of UmAbf62A and PaAbf62A reveals a five-bladed ß-propeller fold that confirms their predicted classification into clan GH-F together with GH43 α-L-arabinofuranosidases. Crystallographic structures of the complexes with arabinose and cellotriose reveal the important role of subsites +1 and +2 for sugar binding. Intriguingly, we observed that PaAbf62A was inhibited by cello-oligosaccharides and displayed binding affinity to cellulose although no activity was observed on a range of cellulosic substrates. Bioinformatic analyses showed that UmAbf62A and PaAbf62A belong to two distinct subfamilies within the GH62 family. The results presented here provide a framework to better investigate the structure-function relationships within the GH62 family.

Structural bases for N-glycan processing by mannoside phosphorylase.

The first crystal structure of Uhgb_MP, a ß-1,4-mannopyranosyl-chitobiose phosphorylase belonging to the GH130 family which is involved in N-glycan degradation by human gut bacteria, was solved at 1.85 Å resolution in the apo form and in complex with mannose and N-acetylglucosamine. SAXS and crystal structure analysis revealed a hexameric structure, a specific feature of GH130 enzymes among other glycoside phosphorylases. Mapping of the -1 and +1 subsites in the presence of phosphate confirmed the conserved Asp104 as the general acid/base catalytic residue, which is in agreement with a single-step reaction mechanism involving Man O3 assistance for proton transfer. Analysis of this structure, the first to be solved for a member of the GH130_2 subfamily, revealed Met67, Phe203 and the Gly121-Pro125 loop as the main determinants of the specificity of Uhgb_MP and its homologues towards the N-glycan core oligosaccharides and mannan, and the molecular bases of the key role played by GH130 enzymes in the catabolism of dietary fibre and host glycans.

4'-Phosphopantetheinyl transferase PptT, a new drug target required for Mycobacterium tuberculosis growth and persistence in vivo.

The cell envelope of Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, contains lipids with unusual structures. These lipids play a key role in both virulence and resistance to the various hostile environments encountered by the bacteria during infection. They are synthesized by complex enzymatic systems, including type-I polyketide synthases and type-I and -II fatty acid synthases, which require a post-translational modification to become active. This modification consists of the covalent attachment of the 4'-phosphopantetheine moiety of Coenzyme A catalyzed by phosphopantetheinyl transferases (PPTases). PptT, one of the two PPTases produced by mycobacteria, is involved in post-translational modification of various type-I polyketide synthases required for the formation of both mycolic acids and lipid virulence factors in mycobacteria. Here we identify PptT as a new target for anti-tuberculosis drugs; we address all the critical issues of target validation to demonstrate that PptT can be used to search for new drugs. We confirm that PptT is essential for the growth of M. bovis BCG in vitro and show that it is required for persistence of M. bovis BCG in both infected macrophages and immunodeficient mice. We generated a conditional expression mutant of M. tuberculosis, in which the expression of the pptT gene is tightly regulated by tetracycline derivatives. We used this construct to demonstrate that PptT is required for the replication and survival of the tubercle bacillus during the acute and chronic phases of infection in mice. Finally, we developed a robust and miniaturized assay based on scintillation proximity assay technology to search for inhibitors of PPTases, and especially of PptT, by high-throughput screening. Our various findings indicate that PptT meets the key criteria for being a therapeutic target for the treatment of mycobacterial infections.

Structural reorganization of the antigen-binding groove of human CD1b for presentation of mycobacterial sulfoglycolipids.

The mechanisms permitting nonpolymorphic CD1 molecules to present lipid antigens that differ considerably in polar head and aliphatic tails remain elusive. It is also unclear why hydrophobic motifs in the aliphatic tails of some antigens, which presumably embed inside CD1 pockets, contribute to determinants for T-cell recognition. The 1.9-Å crystal structure of an active complex of CD1b and a mycobacterial diacylsulfoglycolipid presented here provides some clues. Upon antigen binding, endogenous spacers of CD1b, which consist of a mixture of diradylglycerols, moved considerably within the lipid-binding groove. Spacer displacement was accompanied by F' pocket closure and an extensive rearrangement of residues exposed to T-cell receptors. Such structural reorganization resulted in reduction of the A' pocket capacity and led to incomplete embedding of the methyl-ramified portion of the phthioceranoyl chain of the antigen, explaining why such hydrophobic motifs are critical for T-cell receptor recognition. Mutagenesis experiments supported the functional importance of the observed structural alterations for T-cell stimulation. Overall, our data delineate a complex molecular mechanism combining spacer repositioning and ligand-induced conformational changes that, together with pocket intricacy, endows CD1b with the required molecular plasticity to present a broad range of structurally diverse antigens.

Crystal structure of human CD1e reveals a groove suited for lipid-exchange processes.

CD1e is the only human CD1 protein existing in soluble form in the late endosomes of dendritic cells, where it facilitates the processing of glycolipid antigens that are ultimately recognized by CD1b-restricted T cells. The precise function of CD1e remains undefined, thus impeding efforts to predict the participation of this protein in the presentation of other antigens. To gain insight into its function, we determined the crystal structure of recombinant CD1e expressed in human cells at 2.90-Å resolution. The structure revealed a groove less intricate than in other CD1 proteins, with a significantly wider portal characterized by a 2 Å-larger spacing between the α1 and α2 helices. No electron density corresponding to endogenous ligands was detected within the groove, despite the presence of ligands unequivocally established by native mass spectrometry in recombinant CD1e. Our structural data indicate that the water-exposed CD1e groove could ensure the establishment of loose contacts with lipids. In agreement with this possibility, lipid association and dissociation processes were found to be considerably faster with CD1e than with CD1b. Moreover, CD1e was found to mediate in vitro the transfer of lipids to CD1b and the displacement of lipids from stable CD1b-antigen complexes. Altogether, these data support that CD1e could have evolved to mediate lipid-exchange/editing processes with CD1b and point to a pathway whereby the repertoire of lipid antigens presented by human dendritic cells might be expanded.

In silico design, synthesis and antitubercular activity of novel 2-acylhydrazono-5-arylmethylene-4-thiazolidinones as enoyl-acyl carrier protein reductase inhibitors.

Mycobacteria regulate the synthesis of mycolic acid through the fatty acid synthase system type 1 (FAS I) and the fatty acid synthase system type-2 (FAS-II). Because mammalian cells exclusively utilize the FAS-I enzyme system for fatty acid production, targeting the FAS-II enzyme system could serve as a specific approach for developing selective antimycobacterial drugs. Enoyl-acyl carrier protein reductase enzyme (MtInhA), part of the FAS-II enzyme system, contains the NADH cofactor in its active site and reduces the intermediate. Molecular docking studies were performed on an in-house database (∼2200 compounds). For this study, five different crystal structures of MtInhA (PDB Code: 4TZK, 4BQP, 4D0S, 4BGE, 4BII) were used due to rotamer difference, mutation and the presence of cofactors. Molecular dynamics simulations (250 ns) were performed for the novel 2-acylhydrazono-5-arylmethylene-4-thiazolidinones derivatives selected by molecular docking studies. Twenty-three compounds selected by in silico methods were synthesized. Antitubercular activity and MtInhA enzyme inhibition studies were performed for compounds whose structures were elucidated by IR,1H-NMR,13C-NMR, HSQC, HMBC, MS and elemental analysis.Communicated by Ramaswamy H. Sarma.