Garima Khare

University of Delhi

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Anil K. Tyagi

Mankind Pharma (India)

Rakesh Gupta

Fortis Memorial Research Institute

Vibha Gupta

University of Delhi

Prachi Nangpal

University of Delhi

Nupur Angrish

University of Delhi

Shekhar C. Mande

Savitribai Phule Pune University

Simran Kaur

University of Delhi

Dinakar M. Salunke

International Centre for Genetic Engineering and Biotechnology

Abhijit A. Sardesai

Centre for DNA Fingerprinting and Diagnostics

Swati Singh

Indian Institute of Science Education and Research Mohali

Cooperative Institutions

University of Delhi

Academy of Scientific and Innovative Research

Institute of Genomics and Integrative Biology

National Institute of Immunology

Centre for Cellular and Molecular Biology

Centre for DNA Fingerprinting and Diagnostics

Council of Scientific and Industrial Research

Indian Institute of Science Bangalore

Regional Centre for Biotechnology

Institute of Medical Sciences

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In silico virtual screening for the identification of novel inhibitors against dihydrodipicolinate reductase (DapB) of Mycobacterium tuberculosis , a key enzyme of diaminopimelate pathway

Microbiology Spectrum (2023)

Nupur Angrish Neha Lalwani Garima Khare

Non-compliance to lengthy antituberculosis (TB) treatment regimen, associated side effects, and emergence of drug-resistant strains of Mycobacterium tuberculosis (M. tb) emphasize the need to develop more effective anti-TB drugs. Here, we have evaluated the role of M. tb dihydrodipicolinate reductase (DapB), a component of the diaminopimelate pathway, which is involved in the biosynthesis of both lysine and mycobacterial cell wall. We showed that DapB is essential for the in vitro as well as intracellular growth of M. tb. We further utilized M. tb DapB, as a target for identification of inhibitors by employing in silico virtual screening, and conducted various in vitro screening assays to identify inhibitors with potential to inhibit DapB activity and in vitro and intracellular growth of M. tb with no significant cytotoxicity against various mammalian cell lines. Altogether, M. tb DapB serves as an important drug target and a hit molecule, namely, 4-(3-Phenylazoquinoxalin-2-yl) butanoic acid methyl ester has been identified as an antimycobacterial molecule in our study.

10.1128/spectrum.01359-23

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Necrosis triggered byMycobacterium tuberculosisalters macrophage triglyceride metabolism and inflammatory response in a DGAT1 – dependent manner

bioRxiv (Cold Spring Harbor Laboratory) (2017)

Neetika Jaisinghani Stanzin Dawa Kaurab Singh Ananya Nandy Dilip Menon

Abstract Tuberculosis granulomas represent a site of controlling bacterial dissemination at the cost of host tissue damage. Within the heterogenous array of TB granulomas, some contain triglyceride (TG) rich foamy macrophages, the etiology and function of which remains largely unexplained. Here we show that necrosis of tuberculosis lesions and M. tuberculosis (Mtb) infected macrophages elicits a bystander response of triglyceride storage. We elucidate a role for the RD1 region of mycobacterial genome to be a key player in this phenomenon. TG storage in necrosis associated foamy macrophages promoted the pro-inflammatory state of macrophages while silencing expression of diacylglycerol O-acyltransferase (DGAT1) suppressed expression of pro-inflammatory genes. Surprisingly, acute infection with Mtb led to lipolysis of host TG, rather than synthesis, suggesting mobilization of triglyceride stored within macrophage lipid droplets to be involved in the inflammatory response to infection. Our data is likely to open new avenues for management of the inflammatory response during infection.

10.1101/187104

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Identification of Inhibitors against Mycobacterium tuberculosis Thiamin Phosphate Synthase, an Important Target for the Development of Anti-TB Drugs

PLoS ONE (2011)

Garima Khare Ritika Kar Bahal Anil K. Tyagi

Tuberculosis (TB) continues to pose a serious challenge to human health afflicting a large number of people throughout the world. In spite of the availability of drugs for the treatment of TB, the non-compliance to 6–9 months long chemotherapeutic regimens often results in the emergence of multidrug resistant strains of Mycobacterium tuberculosis adding to the precariousness of the situation. This has necessitated the development of more effective drugs. Thiamin biosynthesis, an important metabolic pathway of M.tuberculosis, is shown to be essential for the intracellular growth of this pathogen and hence, it is believed that inhibition of this pathway would severely affect the growth of M.tuberculosis. In this study, a comparative homology model of M.tuberculosis thiamin phosphate synthase (MtTPS) was generated and employed for virtual screening of NCI diversity set II to select potential inhibitors. The best 39 compounds based on the docking results were evaluated for their potential to inhibit the MtTPS activity. Seven compounds inhibited MtTPS activity with IC50 values ranging from 20 – 100 µg/ml and two of these exhibited weak inhibition of M.tuberculosis growth with MIC99 values being 125 µg/ml and 162.5 µg/ml while one compound was identified as a very potent inhibitor of M.tuberculosis growth with an MIC99 value of 6 µg/ml. This study establishes MtTPS as a novel drug target against M.tuberculosis leading to the identification of new lead molecules for the development of antitubercular drugs. Further optimization of these lead compounds could result in more potent therapeutic molecules against Tuberculosis.

10.1371/journal.pone.0022441

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An Insight into the Importance of Ferritins in the Physiology of Mycobacterium tuberculosis: Unique Structural and Functional Properties

Springer eBooks (2020)

Garima Khare Prachi Nangpal Anil K. Tyagi

10.1007/978-981-15-5530-5_17

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Crystal Structure of Bfr A from Mycobacterium tuberculosis: Incorporation of Selenomethionine Results in Cleavage and Demetallation of Haem

PLoS ONE (2009)

Vibha Gupta Rakesh Gupta Garima Khare Dinakar M. Salunke Anil K. Tyagi

Emergence of tuberculosis as a global health threat has necessitated an urgent search for new antitubercular drugs entailing determination of 3-dimensional structures of a large number of mycobacterial proteins for structure-based drug design. The essential requirement of ferritins/bacterioferritins (proteins involved in iron storage and homeostasis) for the survival of several prokaryotic pathogens makes these proteins very attractive targets for structure determination and inhibitor design. Bacterioferritins (Bfrs) differ from ferritins in that they have additional noncovalently bound haem groups. The physiological role of haem in Bfrs is not very clear but studies indicate that the haem group is involved in mediating release of iron from Bfr by facilitating reduction of the iron core. To further enhance our understanding, we have determined the crystal structure of the selenomethionyl analog of bacterioferritin A (SeMet-BfrA) from Mycobacterium tuberculosis (Mtb). Unexpectedly, electron density observed in the crystals of SeMet-BfrA analogous to haem location in bacterioferritins, shows a demetallated and degraded product of haem. This unanticipated observation is a consequence of the altered spatial electronic environment around the axial ligands of haem (in lieu of Met52 modification to SeMet52). Furthermore, the structure of Mtb SeMet-BfrA displays a possible lost protein interaction with haem propionates due to formation of a salt bridge between Arg53-Glu57, which appears to be unique to Mtb BfrA, resulting in slight modulation of haem binding pocket in this organism. The crystal structure of Mtb SeMet-BfrA provides novel leads to physiological function of haem in Bfrs. If validated as a drug target, it may also serve as a scaffold for designing specific inhibitors. In addition, this study provides evidence against the general belief that a selenium derivative of a protein represents its true physiological native structure.

Cleavage (geology)

10.1371/journal.pone.0008028

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Dissecting the Role of Critical Residues and Substrate Preference of a Fatty Acyl-CoA Synthetase (FadD13) of Mycobacterium tuberculosis

PLoS ONE (2009)

Garima Khare Vibha Gupta Rakesh Gupta Radhika Gupta Rajiv Bhat

Newly emerging multi-drug resistant strains of Mycobacterium tuberculosis (M.tb) severely limit the treatment options for tuberculosis (TB); hence, new antitubercular drugs are urgently needed. The mymA operon is essential for the virulence and intracellular survival of M.tb and thus represents an attractive target for the development of new antitubercular drugs. This study is focused on the structure-function relationship of Fatty Acyl-CoA Synthetase (FadD13, Rv3089) belonging to the mymA operon. Eight site-directed mutants of FadD13 were designed, constructed and analyzed for the structural-functional integrity of the enzyme. The study revealed that mutation of Lys(487) resulted in approximately 95% loss of the activity thus demonstrating its crucial requirement for the enzymatic activity. Comparison of the kinetic parameters showed the residues Lys(172) and Ala(302) to be involved in the binding of ATP and Ser(404) in the binding of CoenzymeA. The influence of mutations of the residues Val(209) and Trp(377) emphasized their importance in maintaining the structural integrity of FadD13. Besides, we show a synergistic influence of fatty acid and ATP binding on the conformation and rigidity of FadD13. FadD13 represents the first Fatty Acyl-CoA Synthetase to display biphasic kinetics for fatty acids. FadD13 exhibits a distinct preference for C(26)/C(24) fatty acids, which in the light of earlier reported observations further substantiates the role of the mymA operon in remodeling the cell envelope of intracellular M.tb under acidic conditions. A three-dimensional model of FadD13 was generated; the docking of ATP to the active site verified its interaction with Lys(172), Ala(302) and Lys(487) and corresponded well with the results of the mutational studies. Our study provides a significant understanding of the FadD13 protein including the identification of residues important for its activity as well as in the maintenance of structural integrity. We believe that the findings of this study will provide valuable inputs in the development of inhibitors against the mymA operon, an important target for the development of antitubercular drugs.

10.1371/journal.pone.0008387

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Citations (29)

Unique Residues at the 3-Fold and 4-Fold Axis of Mycobacterial Ferritin Are Involved in Oligomer Switching

Biochemistry (2013)

Garima Khare Prachi Nangpal Anil K. Tyagi

To identify the crucial residues involved in the self-assembly and function of BfrB, one of the important iron storage proteins of Mycobacterium tuberculosis, we constructed various mutants by employing site-directed mutagenesis. The analysis of mutants led to the identification of "interface hot-spot residues" (R69, L129, and F159) that act as "switch points" for BfrB oligomerization, and our observations show the importance of 4-fold axis residues in assembly formation. Moreover, we demonstrate that single-point mutations Q51A, Q126A, and E135A can enhance the thermal stability of the protein without affecting its assembly. Importantly, a comparative analysis of various mutations revealed that the function of various homologous positions in different ferritins could be at variance; hence, predicting the function of a residue just based on sequence-structure comparisons may not be appropriate. Thus, we report the identification of novel residues in the assembly formation and function of BfrB and show that single-point mutations have a remarkable potential for alteration of multiple properties of ferritins. Besides, "switch residues" or "interface hot spots" identified in this study could also prove to be helpful for the rational design of interfacial inhibitors.

Oligomer

10.1021/bi301189t

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A combination of docking and cheminformatics approaches for the identification of inhibitors against 4′ phosphopantetheinyl transferase ofMycobacterium tuberculosis

RSC Advances (2018)

Akshay Rohilla Garima Khare Anil K. Tyagi

We integrated virtual screening,in vitroandex vivoapproaches to identify numerous potent inhibitory scaffolds againstM. tbPptT.

Cheminformatics

Docking (animal)

10.1039/c7ra11198c

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Citations (11)

Identification of Mycobacterium tuberculosis BioA inhibitors by using structure-based virtual screening

DOAJ (DOAJ: Directory of Open Access Journals) (2018)

Swati Singh Garima Khare Bahal RK Ghosh Pc Tyagi Ak

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The heparin-binding hemagglutinin protein of Mycobacterium tuberculosis is a nucleoid-associated protein

Journal of Biological Chemistry (2023)

Chetkar Chandra Keshavam Saba Naz Aanchal Gupta Priyadarshini Sanyal Manisha Kochar

Nucleoid-associated proteins (NAPs) regulate multiple cellular processes such as gene expression, virulence, and dormancy throughout bacterial species. NAPs help in the survival and adaptation of Mycobacterium tuberculosis (Mtb) within the host. Fourteen NAPs have been identified in Escherichia coli; however, only seven NAPs are documented in Mtb. Given its complex lifestyle, it is reasonable to assume that Mtb would encode for more NAPs. Using bioinformatics tools and biochemical experiments, we have identified the heparin-binding hemagglutinin (HbhA) protein of Mtb as a novel sequence-independent DNA-binding protein which has previously been characterized as an adhesion molecule required for extrapulmonary dissemination. Deleting the carboxy-terminal domain of HbhA resulted in a complete loss of its DNA-binding activity. Atomic force microscopy showed HbhA-mediated architectural modulations in the DNA, which may play a regulatory role in transcription and genome organization. Our results showed that HbhA colocalizes with the nucleoid region of Mtb. Transcriptomics analyses of a hbhA KO strain revealed that it regulates the expression of ∼36% of total and ∼29% of essential genes. Deletion of hbhA resulted in the upregulation of ∼73% of all differentially expressed genes, belonging to multiple pathways suggesting it to be a global repressor. The results show that HbhA is a nonessential NAP regulating gene expression globally and acting as a plausible transcriptional repressor.

Nucleoid

10.1016/j.jbc.2023.105364

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Citations (3)