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Background: Mycobacterium tuberculosis (M.tb), the etiological agent of Tuberculosis (TB), is the second leading cause of mortality after COVID-19, with a global death toll of 1.5 million in 2020. The escalating cases of drug-resistant TB are further worsening the current situation and making TB treatment extremely challenging. Thus, it is crucial to look for new anti-TB drugs with novel mechanisms of action and high efficacy. The DnaG of M.tb replication machinery is an essential protein for pathogen survival. Also, its imperative primase activity and lack of structural homology to human proteins, make it a possible target for drug development. Methods: In this presented study, using a computational structure-based drug repurposing approach, Food and drug administration (FDA) approved drugs were virtually screened against M.tb DnaG to identify potential inhibitors. Five drugs viz. Caspofungin, Doxorubicin, Mitoxantrone, Vapreotide, and Zanamivir showed higher molecular docking scores. Further RMSD, RMSF, Rg, SASA, H-bond, and PCA analysis of these drugs and DnaG complexes. Alamar Blue Assay further evaluated the anti-TB activity of these drugs in vitro using H37Ra and H37Rv M.tb strains. Results: The top results for DnaG binding included several FDA-approved drugs, out of which five were selected and subjected to Molecular dynamic simulation and displayed their high binding affinity, stable interaction, more compactness, and reduced atomic motion. The minimum inhibitory concentration of Doxorubicin, Mitoxantrone, and Vapreotide were detected in the range of 0.19-25 µg/ml for both H37Ra, and H37Rv, respectively. Conclusions: Our findings from the study present potential repurposed drug candidates that target DnaG and inhibit M.tb survival. Thorough investigations of these compounds may lead to the discovery of new anti-TB therapeutics.

Tuberculosis (TB) is one of the prominent cause of deaths across the world and multidrug-resistant and extensively drug-resistant TB continues to pose challenges for clinicians and public health centers. The risk of death is extremely high in individuals who have compromised immune systems, HIV infection, or diabetes. Research institutes and pharmaceutical companies have been working on repurposing existing drugs as effective therapeutic options against TB. The identification of suitable drugs with multi-target affinity profiles is a widely accepted way to combat the development of resistance. Flavin-dependent thymidylate synthase (FDTS), known as ThyX, is in the class of methyltransferases and is a possible target in the discovery of novel anti-TB drugs. In this study, we aimed to repurpose existing drugs approved by Food and Drug Administration (FDA) that could be used in the treatment of TB. An integrated screening was performed based on computational procedures: high-throughput molecular docking techniques, followed by molecular dynamics simulations of the target enzyme, ThyX. After performing in silico screening using a library of 3,967 FDA-approved drugs, the two highest-scoring drugs, Carglumic acid and Mesalazine, were selected as potential candidates that could be repurposed to treat TB.Communicated by Ramaswamy H. Sarma.

Capreomycin is a second line antibiotic used for the treatment of drug resistant Tuberculosis (TB), primary reason of death from a solo infectious organism

Tuberculosis is one the oldest known affliction of mankind caused by the pathogen Mycobacterium tuberculosis. Till date, there is no absolute single treatment available to deal with the pathogen, which has acquired a great potential to develop drug resistance rapidly. BCG is the only anti-tuberculosis vaccine available till date which displays limited global efficacy due to genetic variation and concurrent pathogen infections. Extracellular vesicles or exosomes vesicle (EVs) lie at the frontier cellular talk between pathogen and the host, and therefore play a significant role in establishing pathogenesis. In the present study, an in-silico approach has been adopted to construct a multi-epitope vaccine from selected immunogenic EVs proteins to elicit a cellular as well as a humoral immune response. Our designed vaccine has wide population coverage and can effectively compensate for the genetic variation among different populations. For maximum efficacy and minimum adverse effects possibilities the antigenic, non-allergenic and non-toxic B-cell, HTL and CTL epitopes from experimentally proven EVs proteins were selected for the vaccine construct. TLR4 agonist RpfE served as an adjuvant for the vaccine construct. The vaccine construct structure was modelled, refined and docked on TLR4 immune receptor. The designed vaccine construct displayed safe usage and exhibits a high probability to elicit the critical immune regulators, like B cells, T-cells and memory cells as displayed by the in-silico immunization assays. Therefore, it can be further corroborated using in vitro and in vivo assays to fulfil the global need for a more efficacious anti-tuberculosis vaccine.

Tuberculosis (TB) is one of the prominent cause of deaths across the world and multidrug-resistant and extensively drug-resistant TB continues to pose challenges for clinicians and public health centers. The risk of death is extremely high in individuals who have compromised immune systems, HIV infection, or diabetes. Research institutes and pharmaceutical companies have been working on repurposing existing drugs as effective therapeutic options against TB. The identification of suitable drugs with multi-target affinity profiles is a widely accepted way to combat the development of resistance. Flavin-dependent thymidylate synthase (FDTS), known as ThyX, is in the class of methyltransferases and is a possible target in the discovery of novel anti-TB drugs. In this study, we aimed to repurpose existing drugs approved by Food and Drug Administration (FDA) that could be used in the treatment of TB. An integrated screening was performed based on computational procedures: high-throughput molecular docking techniques, followed by molecular dynamics simulations of the target enzyme, ThyX. After performing

Development of effective counteragents against the novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains, requires clear insights and information for understanding the immune responses associated with it. This global pandemic has pushed the healthcare system and restricted the movement of people and succumbing of the available therapeutics utterly warrants the development of a potential vaccine to contest the deadly situation. In the present study, highly efficacious, immunodominant cytotoxic T-lymphocyte (CTL) epitopes were predicted by advanced immunoinformatics assays using the spike glycoprotein of SARS-CoV2, generating a robust and specific immune response with convincing immunological parameters (Antigenicity, TAP affinity, MHC binder) engendering an efficient viral vaccine. The molecular docking studies show strong binding of the CTL construct with MHC-1 and host membrane specific TLR2 receptors. The molecular dynamics simulation in an explicit system confirmed the stable and robust binding of CTL epitope with TLR2. Steep magnitude RMSD variation and compelling residual fluctuations existed in terminal residues and various loops of the β linker segments of TLR2-epitope (residues 105-156 and 239-254) to about 0.4 nm. The reduced Rg value (3.3 nm) and stagnant SASA analysis (275 nm/S2/N after 8 ns and 5 ns) for protein surface and its orientation in the exposed and buried regions suggests more compactness due to the strong binding interaction of the epitope. The CTL vaccine candidate establishes a high capability to elicit the critical immune regulators, like T-cells and memory cells as proven by the in silico immunization assays and can be further corroborated through in vitro and in vivo assays.

Background Predicting adverse drug reactions (ADRs) has become very important owing to the huge global health burden and failure of drugs. This indicates a need for prior prediction of probable ADRs in preclinical stages which can improve drug failures and reduce the time and cost of development thus providing efficient and safer therapeutic options for patients. Though several approaches have been put forward for in silico ADR prediction, there is still room for improvement. Methods In the present work, we have used machine learning based approach for cardiovascular (CV) ADRs prediction by integrating different features of drugs, biological (drug transporters, targets and enzymes), chemical (substructure fingerprints) and phenotypic (therapeutic indications and other identified ADRs), and their two and three level combinations. To recognize quality and important features, we used minimum redundancy maximum relevance approach while synthetic minority over-sampling technique balancing method was used to introduce a balance in the training sets. Results This is a rigorous and comprehensive study which involved the generation of a total of 504 computational models for 36 CV ADRs using two state-of-the-art machine-learning algorithms: random forest and sequential minimization optimization. All the models had an accuracy of around 90% and the biological and chemical features models were more informative as compared to the models generated using chemical features. Conclusions The results obtained demonstrated that the predictive models generated in the present study were highly accurate, and the phenotypic information of the drugs played the most important role in drug ADRs prediction. Furthermore, the results also showed that using the proposed method, different drugs properties can be combined to build computational predictive models which can effectively predict potential ADRs during early stages of drug development. Electronic supplementary material The online version of this article (10.1186/s12967-019-1918-z) contains supplementary material, which is available to authorized users.

Outbreak of Human Herpes virus-5 (HHV-5) infection in emerging countries has raised worldwide health concern owing to prevalence of congenital impairments and life threatening consequences in immunocompromised individuals. Thus, there lies an impending need to develop vaccine against HHV-5. HHV-5 enters into host cells with the help of necessary components glycoprotein B (gB) and H/L. In this study, the conformational linear B-cell and T-cell epitopes for gB of HHV-5 have been predicted using conformational approaches, for their possible collective use as vaccine candidates. We examined epitope's interactions with major histocompatibility complexes using molecular docking and also investigated their stable binding with specific toll like receptor-2 (TLR2), present on host cells during HHV-5 infection. Predicted MHC-I epitope 'LVAIAVVII' with high antigenicity and large coverage of HLA alleles was found to superimpose on MHC-II epitope (Rank 1) and was also identified to be the core sequence of putative B cell epitope 'ILVAIAVVIITYLI'. Resulting epitope was found to have consistent interaction with TLR2 during long term (100 ns) MD run. We also validated this nonamer epitope for its dissimilarity with human genome and high population coverage, suggesting it to be a potential vaccine candidate with higher coverage for both the MHC alleles of Indian population. Communicated by Ramaswamy H. Sarma.

Capreomycin is a second line antibiotic used for the treatment of drug resistant Tuberculosis (TB), primary reason of death from a solo infectious organism, Mycobacterium tuberculosis (M.tb). Capreomycin targets the ribosome of bacteria and is known to bind at the interface where the large and small ribosomal subunits interact in M.tb using an S-Adenosyl Methionine (SAM) dependent methyltransferase, TlyA (Rv1794). Besides the methyltransferase activity, TlyA has also been found to show substantial haemolytic activity. The dual activity of TlyA highlights its crucial role in pathogenesis and virulence of M.tb. In the present study, docking and molecular dynamics (MD) simulations were carried out to explore the impact of mutations in a conserved SAM binding motif, 90GASTG94, on the affinity of TlyA enzyme for SAM. Two already reported mutations, A91E and S92L, and the remaining wild type residues, Gly90, Thr93, Gly94 mutated to alanine were taken into consideration resulting in a total of six systems, wild type + SAM, G90A + SAM, A91E + SAM, S92L + SAM, T93A + SAM and G94A + SAM that were subjected to 100 ns MD simulations. Docking scores and MD simulations analyses revealed that in contrast to wild type, mutants reduced the affinity of SAM for TlyA with most prominent effect observed in case of alanine mutants. Mutations also led to the loss of hydrogen bond and hydrophobic interactions and large-scale movement of atoms evident from the principal component analyses indicating their destabilizing impact on TlyA. The present study gives insights into influence of mutations on binding of SAM to TlyA in M.tb and promoting capreomycin resistance. Communicated by Ramaswamy H. Sarma

Development of effective counteragents against the novel coronavirus disease caused by SARS CoV-2 strains requires clear insights for understanding immune responses associated with it. The succumbing of available therapeutics utterly warrants the development of a potential vaccine to contest the deadly situation. Herein, we report Cytotoxic T-cell Lymphocytes immunomodulator by advanced immunoinformatics avenues for spike-glycoprotein of SARS CoV-2, which can generate robust immune response with convincing immunological parameters (Antigenicity, TAP affinity, MHC-binder) engendering an efficient viral vaccine. Strong binding of the CTL construct with MHC-1 and membrane-specific TLR2 was conferred through molecular docking and molecular dynamics simulation in an explicit system. Steep magnitude RMSD variation and compelling residual fluctuations existed in terminal residues and various loops of the β linker segments of TLR2-epitope (residues 105-156 and 239-254) to about 0.4nm. The reduced Rg value (3.3nm) and stagnant SASA analysis (275nm/S2/N after 8ns and 5ns) for protein surface and its orientation in exposed and buried regions suggests more compactness by strong binding of epitope. The CTL vaccine candidate establishes a high capability to elicit critical immune regulators, like T-cells and memory cells as proven by in silico immunization assays and can be further corroborated through in vitro and in vivo assays.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (M.tb), causes highest number of deaths globally for any bacterial disease necessitating novel diagnosis and treatment strategies. High-throughput sequencing methods generate a large amount of data which could be exploited in determining multi-drug resistant (MDR-TB) associated mutations. The present work is a computational framework that uses artificial intelligence (AI) based machine learning (ML) approaches for predicting resistance in the genes rpoB, inhA, katG, pncA, gyrA and gyrB for the drugs rifampicin, isoniazid, pyrazinamide and fluoroquinolones. The single nucleotide variations were represented by several sequence and structural features that indicate the influence of mutations on the target protein coded by each gene. We used ML algorithms - naïve bayes, k nearest neighbor, support vector machine, and artificial neural network, to build the prediction models. The classification models had an average accuracy of 85% across all examined genes and were evaluated on an external unseen dataset to demonstrate their application. Further, molecular docking and molecular dynamics simulations were performed for wild type and predicted resistance causing mutant protein and anti-TB drug complexes to study their impact on the conformation of proteins to confirm the observed phenotype.

Tuberculosis (TB) is a leading cause of death worldwide and its impact has intensified due to the emergence of multi drug-resistant (MDR) and extensively drug-resistant (XDR) TB strains. Protein phosphorylation plays a vital role in the virulence of Mycobacterium tuberculosis (M.tb) mediated by protein kinases. Protein tyrosine phosphatase A (MptpA) undergoes phosphorylation by a unique tyrosine-specific kinase, protein tyrosine kinase A (PtkA), identified in the M.tb genome. PtkA phosphorylates PtpA on the tyrosine residues at positions 128 and 129, thereby increasing PtpA activity and promoting pathogenicity of MptpA. In the present study, we performed an extensive investigation of the conformational behavior of the intrinsically disordered domain (IDD) of PtkA using replica exchange molecular dynamics simulations. Long-term molecular dynamics (MD) simulations were performed to elucidate the role of IDD on the catalytic activity of kinase core domain (KCD) of PtkA. This was followed by identification of the probable inhibitors of PtkA using drug repurposing to block the PtpA-PtkA interaction. The inhibitory role of IDD on KCD has already been established; however, various analyses conducted in the present study showed that IDDPtkA had a greater inhibitory effect on the catalytic activity of KCDPtkA in the presence of the drugs esculin and inosine pranobex. The binding of drugs to PtkA resulted in formation of stable complexes, indicating that these two drugs are potentially useful as inhibitors of M.tb.

The global health emergency caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to alarming numbers of fatalities across the world. So far the researchers worldwide have not been able to discover a breakthrough in the form of a potent drug or an effective vaccine. Therefore, it is imperative to discover drugs to curb the ongoing menace. In silico approaches using FDA approved drugs can expedite the drug discovery process by providing leads that can be pursued. In this report, two drug targets, namely the spike protein and main protease, belonging to structural and non-structural class of proteins respectively, were utilized to carry out drug repurposing based screening. The exposed nature of the spike protein on the viral surface along with its instrumental role in host infection and the involvement of main protease in processing of polyproteins along with no human homologue make these proteins attractive drug targets. Interestingly, the screening identified a common high efficiency binding molecule named rutin. Further, molecular dynamics simulations in explicit solvent affirmed the stable and sturdy binding of rutin with these proteins. The decreased Rg value (4 nm for spike-rutin and 2.23 nm for main protease-rutin) and stagnant SASA analysis (485 nm/S2/N in spike-rutin and 152 nm/S2/N in main protease-rutin) for protein surface and its orientation in the exposed and buried regions suggests a strong binding interaction of the drug. Further, cluster analysis and secondary structure analysis of complex trajectories validated the conformational changes due to binding of rutin., Graphical Abstract Communicated by Ramaswamy H. Sarma

Streptomycin was the first antibiotic used for the treatment of tuberculosis by inhibiting translational proof reading. Point mutation in gidB gene encoding S-adenosyl methionine (SAM)-dependent 7-methylguanosine (m7G) methyltransferase required for methylation of 16S rRNA confers streptomycin resistance. As there was no structural substantiation experimentally, gidB protein model was built by threading algorithm. In this work, molecular dynamics (MD) simulations coupled with binding free energy calculations were performed to outline the mechanism underlying high-level streptomycin resistance associated with three novel missense mutants including S70R, T146M, and R187M. Results from dynamics analyses suggested that the structure distortion in the binding pocket of gidB mutants modulate SAM binding affinity. At the structural level, these conformational changes bring substantial decrease in the number of residues involved in hydrogen bonding and dramatically reduce thermodynamic stability of mutant gidB-SAM complexes. The outcome of comparative analysis of the MD simulation trajectories revealed lower conformational stability associated with higher flexibility in mutants relative to the wild-type, turns to be major factor driving the emergence of drug resistance toward antibiotic. This study will pave way toward design and development of resistant defiant gidB inhibitors as potent anti-TB agents.

Mycobacterium tuberculosis (Mtb) resistance toward anti-tuberculosis drugs is a widespread problem. Pyrazinamide (PZA) is a first line antitubercular drug that kills semi-dormant bacilli when converted into its activated form, that is, pyrazinoic acid (POA) by Pyrazinamidase (PZase) enzyme coded by pncA gene. In this study, we conducted several analyses on native and mutant structures (W68R, W68G) of PZase before and after docking with the PZA drug to explore the molecular mechanism behind PZA resistance caused due to pncA mutations. Structural changes caused by mutations were studied with respect to their effects on functionality of protein. Docking was performed to analyze the protein-drug binding and comparative analysis was done to observe how the mutations affect drug binding affinity and binding site on protein. Native PZase protein was observed to have the maximum binding affinity in terms of docking score as well as shape complementarity in comparison to the mutant forms. Molecular dynamics simulation analyses showed that mutation in the 68th residue of protein results in a structural change at its active site which further affects the biological function of protein, that is, conversion of PZA to POA. Mutations in the protein thereby led to PZA resistance in the bacterium due to the inefficient binding.

Mycobacterium tuberculosis (M.tb), the pathogen causing tuberculosis, is a major threat to human health worldwide. Nearly 10% of M.tb genome encodes for a unique family of PE/PPE/PGRS proteins present exclusively in the genus Mycobacterium. The functions of most of these proteins are yet unexplored. The PGRS domains of these proteins have been hypothesized to consist of Ca2+ binding motifs that help these intrinsically disordered proteins to modulate the host cellular responses. Ca2+ is an important secondary messenger that is involved in the pathogenesis of tuberculosis in diverse ways. This study presents the calcium-dependent function of the PGRS domain of Rv0297 (PE_PGRS5) in M.tb virulence and pathogenesis. Tandem repeat search revealed the presence of repetitive Ca2+ binding motifs in the PGRS domain of the Rv0297 protein (Rv0297PGRS). Molecular Dynamics simulations and fluorescence spectroscopy revealed Ca2+ dependent stabilization of the Rv0297PGRS protein. Calcium stabilized Rv0297PGRS enhances the interaction of Rv0297PGRS with surface localized Toll like receptor 4 (TLR4) of macrophages. The Ca2+ stabilized binding of Rv0297PGRS with the surface receptor of macrophages enhances its downstream consequences in terms of Nitric Oxide (NO) production and cytokine release. Thus, this study points to hitherto unidentified roles of calcium-modulated PE_PGRS proteins in the virulence of M.tb. Understanding the pathogenic potential of Ca2+ dependent PE_PGRS proteins can aid in targeting these proteins for therapeutic interventions.

Fluoroquinolones are among the most important classes of highly effective antibacterial drugs, exhibiting wide range of activity to cure infectious diseases. Ofloxacin is second generation fluoroquinolone approved by FDA for the treatment of tuberculosis by selectively inhibiting DNA gyrase. However, the emergence of drug resistance owing to mutations in DNA gyrase poses intimidating challenge for the effective therapy of this drug. The double mutants GyrAA90V GyrBD500N and GyrAA90V GyrBT539N are reported to be implicated in conferring higher levels of OFX resistance. The present study was designed to unravel the molecular principles behind development of resistance by the bug against fluoroquinolones. Our results highlighted that polar interactions play critical role in the development of drug resistance and highlight the significant correlation between the free energy calculations predicted by MM-PBSA and stability of the ligand-bound complexes. Modifications at the OFX binding pocket due to amino acid substitution leads to fewer hydrogen bonds in mutants DNA gyrase-OFX complex, which determined the low susceptibility of the ligand in inhibiting the mutant protein. This study provides a structural rationale to the mutation-based resistance to ofloxacin and will pave way for development potent fluoroquinolone-based resistant-defiant drugs. J. Cell. Biochem. 118: 2950-2957, 2017. © 2017 Wiley Periodicals, Inc.

DNA gyrase is a validated target of fluoroquinolones which are key components of multidrug resistance tuberculosis (TB) treatment. Most frequent occurring mutations associated with high level of resistance to fluoroquinolone in clinical isolates of TB patients are A90V, D94G, and A90V-D94G (double mutant [DM]), present in the larger subunit of DNA Gyrase. In order to explicate the molecular mechanism of drug resistance corresponding to these mutations, molecular dynamics (MD) and mechanics approach was applied. Structure-based molecular docking of complex comprised of DNA bound with Gyrase A (large subunit) and Gyrase C (small subunit) with moxifloxacin (MFX) revealed high binding affinity to wild type with considerably high Glide XP docking score of -7.88 kcal/mol. MFX affinity decreases toward single mutants and was minimum toward the DM with a docking score of -3.82 kcal/mol. Docking studies were also performed against 8-Methyl-moxifloxacin which exhibited higher binding affinity against wild and mutants DNA gyrase when compared to MFX. Molecular Mechanics/Generalized Born Surface Area method predicted the binding free energy of the wild, A90V, D94G, and DM complexes to be -55.81, -25.87, -20.45, and -12.29 kcal/mol, respectively. These complexes were further subjected to 30 ns long MD simulations to examine significant interactions and conformational flexibilities in terms of root mean square deviation, root mean square fluctuation, and strength of hydrogen bond formed. This comparative drug interaction analysis provides systematic insights into the mechanism behind drug resistance and also paves way toward identifying potent lead compounds that could combat drug resistance of DNA gyrase due to mutations.

Thymidylate synthase A (ThyA) is the key enzyme involved in the folate pathway in Mycobacterium tuberculosis. Mutation of key residues of ThyA enzyme which are involved in interaction with substrate 2′-deoxyuridine-5′-monophosphate (dUMP), cofactor 5,10-methylenetetrahydrofolate (MTHF), and catalytic site have caused para-aminosalicylic acid (PAS) resistance in TB patients. Focusing on R127L, L143P, C146R, L172P, A182P, and V261G mutations, including wild-type, we performed long molecular dynamics (MD) simulations in explicit solvent to investigate the molecular principles underlying PAS resistance due to missense mutations. We found that these mutations lead to (i) extensive changes in the dUMP and MTHF binding sites, (ii) weak interaction of ThyA enzyme with dUMP and MTHF by inducing conformational changes in the structure, (iii) loss of the hydrogen bond and other atomic interactions and (iv) enhanced movement of protein atoms indicated by principal component analysis (PCA). In this study, MD simulations framework has provided considerable insight into mutation induced conformational changes in the ThyA enzyme of Mycobacterium.

Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (M.tb), takes one human life every 15 s globally. Disease relapse occurs due to incomplete clearance of the pathogen and reactivation of the antibiotic tolerant bacilli. M.tb, like other bacterial pathogens, creates an ecosystem of biofilm formed by several proteins including the cyclophilins. We show that the M.tb cyclophilin peptidyl-prolyl isomerase (PpiB), an essential gene, is involved in biofilm formation and tolerance to anti-mycobacterial drugs. We predicted interaction between PpiB and US FDA approved drugs (cyclosporine-A and acarbose) by in-silico docking studies and this was confirmed by surface plasmon resonance (SPR) spectroscopy. While all these drugs inhibited growth of Mycobacterium smegmatis (M.smegmatis) when cultured in vitro, acarbose and cyclosporine-A showed bacteriostatic effect while gallium nanoparticle (GaNP) exhibited bactericidal effect. Cyclosporine-A and GaNP additionally disrupted M.tb H37Rv biofilm formation. Co-culturing M.tb in their presence resulted in significant (2–4 fold) decrease in dosage of anti-tubercular drugs- isoniazid and ethambutol. Comparison of the cyclosporine-A and acarbose binding sites in PpiB homologues of other biofilm forming infectious pathogens revealed that these have largely remained unaltered across bacterial species. Targeting bacterial biofilms could be a generic strategy for intervention against bacterial pathogens. Tuberculosis, caused by Mycobacterium tuberculosis, is the leading cause of death due to a single infectious agent. New therapeutic options are needed, and repurposing clinically approved drugs to destroy biofilms is an attractive approach, as these microbial communities are often less susceptible to antibiotics. A team lead by Seyed Hasnain at the Indian Institute of Technology Delhi identified an enzyme, PpiB, from M. tuberculosis that promoted biofilm formation and showed that PpiB interacts with several drugs that are currently used to treat diabetes, immunological diseases and cancer. These drugs destabilise M. tuberculosis biofilms in culture and enhanced the potency of two current anti-tuberculosis antibiotics. Future work is needed to test these medications against tuberculosis in humans, but given PpiB is found in different bacteria, there may be broader promise of using these repurposed drugs to combat other infections.

Mycobacterium tuberculosis (M.tb), the cause of deadly disease tuberculosis, is an opportunistic pathogen that primarily infects host alveolar macrophages. M.tb has developed several mechanisms to persist in infected host cells and to disseminate the disease. Its pathogenesis mainly depends on its competence to modulate the host machinery for its own benefit. One of the cellular responses triggered by M.tb is endoplasmic reticulum stress in infected macrophages, which eventually disturbs the physiological functioning of the ER. Uncontrolled ER stress activates IRE1, PERK, and ATF6 pathway to induce apoptosis of infected cells. Although apoptosis is known to control and clear the infection in primary stages of infection, in case of M.tb-infected ER stressed macrophages, apoptosis is able to disseminate the pathogen during its advanced stages. M.tb-infected lung granulomas are the preferential site of accumulation of apoptotic macrophages, thereby increasing the risk of disease dissemination. The present chapter will describe the mechanism for ER stress response generated by known M.tb virulence factors such as ESAT-6, HBHA, 38-kDa antigen, and PE_PGRS5. Future insights to describe M.tb infection in respect of eliciting ER stress response-mediated apoptosis and host interacting partners has the potential in identifying novel targets for vaccination and drugs to combat the disease.

Mycobacterium tuberculosis (M.tb) is a pathogen of incredible international prominence owing to its persistence for long duration inside human host in both active and latent form, complex eradication methods and imposing long-term treatment procedures. The mechanisms employed by M.tb to adjust and survive inside extreme host environment and to evade the immune system of host need to be explored in greater depth in order to enable the rational design of novel treatment strategies. Methylation of biomolecules plays a significant role in almost every kingdom of life but has not been extensively addressed in the case of M.tb. The genome of M.tb codes for 121 methyltransferases (MTases) in spite of the reductive evolution of its genome. In the present chapter, we will discuss in detail about various MTases modifying DNA, RNA, protein, mycolic acid and other biomolecules of M.tb along with the host. This will also shed light on how methylation is implicated in virulence and influences the mechanism of pathogenesis of M.tb.

Background: Tuberculosis is the second leading cause of death from an infectious disease worldwide after HIV, thus reasoning the expeditions in antituberculosis research. The rising number of cases of infection by resistant forms of M. tuberculosis has given impetus to the development of novel drugs that have different targets and mechanisms of action against the bacterium. Methods: In this study, we have used machine learning algorithms on the available high throughput screening data of inhibitors of fructose bisphosphate aldolase, an enzyme central to the glycolysis pathway in M. tuberculosis, to build predictive classification models to identify actives against Mycobacterium tuberculosis, the causative organism of tuberculosis. We used Naive Bayes, Random Forest and C4.5 J48 algorithms available from Weka were used for building predictive classification models. Additionally, a set of most relevant attributes was selected using genetic search algorithm which offered improved model performance by avoiding over fitting and generating faster and cost effective models. Results: The model built using machine learning methods in this study provided good accuracy of classification of test compounds which suggests that in silico methods can be successfully used for screening of large datasets to identify potential drug leads. The substructure fragment analysis serves to further potentiate the M. tuberculosis drug development process as it would facilitate identification of structural fragments that are responsible for biological activity against this crucial glycolysis pathway target.

The latest resurrection of drug resistance poses serious threat to the treatment and control of the disease. Mutations have been detected in panD gene in the Mycobacterium tuberculosis (Mtb) strains. Mutation of histidine to arginine at residue 21 (H21R) and isoleucine to valine at residue 29 (I49V) in the non-active site of panD gene has led to PZA resistance. This study will help in reconnoitering the mechanism of pyrazinamide (PZA) resistance caused due to double mutation identified in the panD gene of M. tuberculosis clinical isolates. It is known that panD gene encodes aspartate decarboxylase essential for β-alanine synthesis that makes it a potential therapeutic drug target for tuberculosis treatment. The knowledge about the molecular mechanism conferring drug resistance in M. tuberculosis is scarce, which is a significant challenge in designing successful therapeutic drug. In this study, structural and dynamic repercussions of H21R-I49V double mutation in panD complexed with PZA have been corroborated through docking and molecular dynamics based simulation. The double mutant (DM) shows low docking score and thus, low binding affinity for PZA as compared to the native protein. It was observed that the mutant protein exhibits more structural fluctuation at the ligand binding site in comparison to the native type. Furthermore, the flexibility and compactness analyses indicate that the double mutation influence interaction of PZA with the protein. The hydrogen-bond interaction patterns further supported our results. The covariance and PCA analysis elucidated that the double mutation affects the collective motion of residues in phase space. The results have been presented with an explanation for the induced drug resistance conferred by the H21R-I49V double mutation in panD gene and gain valuable insight to facilitate the advent of efficient therapeutics for combating resistance against PZA.

Tuberculosis is a devastating disease, taking one human life every 20 seconds globally. We hypothesize that professional pathogens such as M.tb have acquired specific features that might assist in causing infection, persistence and transmissible pathology in their host. We have identified 121 methyltransferases (MTases) in the M.tb proteome, which use a variety of substrates - DNA, RNA, protein, intermediates of mycolic acid biosynthesis and other fatty acids - that are involved in cellular maintenance within the host. A comparative analysis of the proteome of the virulent strain H37Rv and the avirulent strain H37Ra identified 3 MTases, which displayed significant variations in terms of N-terminal extension/deletion and point mutations, possibly impacting various physicochemical properties. The cross-proteomic comparison of MTases of M.tb H37Rv with 15 different Mycobacterium species revealed the acquisition of novel MTases in a MTB complex as a function of evolution. Phylogenetic analysis revealed that these newly acquired MTases showed common roots with certain extremophiles such as halophilic and acidophilic organisms. Our results establish an evolutionary relationship of M.tb with halotolerant organisms and also the role of MTases of M.tb in withstanding the host osmotic stress, thereby pointing to their likely role in pathogenesis, virulence and niche adaptation.

The failure of drugs for effective treatment against infectious diseases can be attributed to resistant forms of causative agents. The evasive nature of Mycobacterium tuberculosis is partly associated to its physical features, such as having a thick cell wall and incorporation of beneficial mutations leading to drug resistance. The pro drug Isoniazid (INH) interacts with an enzyme catalase peroxidase to get converted into its active form and upon activation stops the cell wall synthesis thus killing the Mycobacterium. The most common mutation i.e. S315T leads to high degree of drug resistance by virtue of its position in the active site. Here, we have characterized the prominent attributes of two double mutant isolates S315 T/D194G and S315T/M624V which are multi drug resistant and extremely drug resistant, respectively. Protein models were generated using the crystal structure which were then subjected to energy minimization and long term molecular dynamics simulations. Further, computational analysis showed decreasing ability of INH binding to the mutants in order of: Native > S315T/D194G > S315T/M624V. Also, a trend was observed that as the docking score and binding area decreased, there was a significant increase in the distortion of the 3D geometry of the mutants as observed by PCA analysis.

Whole Genome Sequencing (WGS) is emerging as a very powerful tool for the management, outbreak analyses, surveillance and determining drug resistance of human infectious pathogens including Mycobacterium tuberculosis and MRSA. WGS can also discriminate relapse TB from re-infection and the resolution provided by WGS has no comparison to conventional technologies. With current cost coming down to

Mycobacterium tuberculosis katG gene is responsible for production of an enzyme catalase peroxidase that peroxidises and activates the prodrug Isoniazid (INH), a first-line antitubercular agent. INH interacts with catalase peroxidase enzyme within its heme pocket and gets converted to an active form. Mutations occurring in katG gene are often linked to reduced conversion rates for INH. This study is focussed on one such mutation occurring at residue 279, where glycine often mutates to aspartic acid (G279D). In the present study, several structural analyses were performed to study the effect of this mutation on functionality of KatG protein. On comparison, mutant protein exhibited a lower docking score, smaller binding cavity and reduced affinity towards INH. Molecular dynamics analysis revealed the mutant to be more rigid and less compact than the native protein. Essential dynamics analysis determined correlated motions of residues within the protein structure. G279D mutant was found to have many residues that showed related motions and an undesirable effect on the functionality of protein.

Evolution of drug-resistant Mycobacterium strains threatens the TB treatment and control programs globally. Rifampicin (RIF) is an important first line antitubercular drug. Resistance to Rifampicin is caused mainly by mutations in its target RNA polymerase beta subunit protein (RpoB). RpoB contains a Rifampicin resistance determining region (RRDR) and has several potent sites for mutations. In this study, we have investigated mutations of a single site (H451) to eight different amino acids, involved in RIF resistance. Long-term molecular dynamics simulations were performed on wild type (WT) and mutant protein structures and various structural analysis were carried out to elucidate the dynamic behavior of WT and mutant forms. Essential dynamics uncovered the difference in conformational flexibility and collective modes of motions between WT and mutants. MMPBSA calculations and interaction pattern analysis revealed the binding site relocation in some mutants. This study presents an exhaustive analysis of RIF binding to the WT and mutant RpoB and clearly highlights structural mechanism for differences in stable binding of Rifampicin with WT than the mutant targets. J. Cell. Biochem. 118: 4594-4606, 2017. © 2017 Wiley Periodicals, Inc.

Tumour suppressor p53 is known to play a central role in prevention of tumour development, DNA repair, senescence and apoptosis which is in normal cells maintained by negative feedback regulator MDM2 (Murine Double Minute 2). In case of dysfunctioning of this regulatory loop, tumour development starts thus resulting in cancerous condition. Inhibition of p53-MDM2 binding would result in activation of the tumour suppressor. In this study, a novel robust fragment-based QSAR model has been developed for piperidinone derived compounds experimentally known to inhibit p53-MDM2 interaction. The QSAR model developed showed satisfactory statistical parameters for the experimentally reported dataset (r2 = 0.9415, q2 = 0.8958, pred_r2 = 0.8894 and F-test = 112.7314), thus judging the robustness of the model. Low standard error values (r2_se = 0.3003, q2_se = 0.4009 and pred_r2_se = 0.3315) confirmed the accuracy of the developed model. The regression equation obtained constituted three descriptors (R2-DeltaEpsilonA, R1-RotatableBondCount and R2-SssOCount), two of which had positive contribution while third showed negative correlation. Based on the developed QSAR model, a combinatorial library was generated and activities of the compounds were predicted. These compounds were docked with MDM2 and two top scoring compounds with binding affinities of −10.13 and −9.80 kcal/mol were selected. The binding modes of actions of these complexes were analyzed using molecular dynamics simulations. Analysis of the developed fragment-based QSAR model revealed that addition of unsaturated electronegative groups at R2 site and groups with more rotatable bonds at R1 improved the inhibitory activity of these potent lead compounds. The detailed analysis carried out in this study provides a considerable basis for the design and development of novel piperidinone-based lead molecules against cancer and also provides mechanistic insights into their mode of actions.

Fluoroquinolones are among the most important classes of highly effective antibacterial drugs, exhibiting wide range of activity to cure infectious diseases. Ofloxacin is second generation fluoroquinolone approved by FDA for the treatment of tuberculosis by selectively inhibiting DNA gyrase. However, the emergence of drug resistance owing to mutations in DNA gyrase poses intimidating challenge for the effective therapy of this drug. The double mutants GyrA