Identification and characterization of repurposed small molecule inhibitors of Mycobacterium tuberculosis caseinolytic protease B (ClpB) as anti-mycobacterials.

Mycobacterium tuberculosis (Mtb) caseinolytic protease B (ClpB) is a chaperone possessing a unique ability to resolubilize the aggregated proteins in vivo. ClpB has been shown to be important for the survival of Mtb within the host. Thus, it appears to be a promising target to develop new therapeutic molecules against tuberculosis. In this study, we have screened FDA approved compounds in silico to identify inhibitors against Mtb ClpB. In our screen, several compounds interacted with ClpB. The top four compounds, namely framycetin, gentamicin, ribostamycin and tobramycin showing the highest binding energy were selected for further investigation. MD simulations and tryptophan-based quenching of ClpB-drug complexes established that the selected inhibitors stably interacted with the target protein. The inhibitor and protein complexes were found to be stabilized by hydrogen bonding, and hydrophobic interactions. Although, the compounds did not affect the ATPase activity of ClpB significantly, the protein resolubilization activity of ClpB was remarkably reduced in their presence. All four compounds potently inhibited the growth of Mtb H37Ra. The antimycobacterial activity of the compounds appears to be due the inhibition of functional ClpB oligomer formation, in turn affecting its chaperonic activity.

Targeting of essential mycobacterial replication enzyme DnaG primase revealed Mitoxantrone and Vapreotide as novel mycobacterial growth inhibitors.

Tuberculosis (TB) is the second leading cause of mortality after COVID-19, with a global death toll of 1.6 million in 2021. The escalating situation of drug-resistant forms of TB has threatened the current TB management strategies. New therapeutics with novel mechanisms of action are urgently required to address the current global TB crisis. The essential mycobacterial primase DnaG with no structural homology to homo sapiens presents itself as a good candidate for drug targeting. In the present study, Mitoxantrone and Vapreotide, two FDA-approved drugs, were identified as potential anti-mycobacterial agents. Both Mitoxantrone and Vapreotide exhibit a strong Minimum Inhibitory Concentration (MIC) of ≤25µg/ml against both the virulent (M.tb-H37Rv) and avirulent (M.tb-H37Ra) strains of M.tb. Extending the validations further revealed the inhibitory potential drugs in ex vivo conditions. Leveraging the computational high-throughput multi-level docking procedures from the pool of ~2700 FDA-approved compounds, Mitoxantrone and Vapreotide were screened out as potential inhibitors of DnaG. Extensive 200 ns long all-atoms molecular dynamic simulation of DnaGDrugs complexes revealed that both drugs bind strongly and stabilize the DnaG during simulations. Reduced solvent exposure and confined motions of the active centre of DnaG upon complexation with drugs indicated that both drugs led to the closure of the active site of DnaG. From this study's findings, we propose Mitoxantrone and Vapreotide as potential anti-mycobacterial agents, with their novel mechanism of action against mycobacterial DnaG.

Unravelling the potential of Triflusal as an anti-TB repurposed drug by targeting replication protein DciA.

The increasing prevalence of drug-resistant Tuberculosis (TB) is imposing extreme difficulties in controlling the TB infection rate globally, making treatment critically challenging. To combat the prevailing situation, it is crucial to explore new anti-TB drugs with a novel mechanism of action and high efficacy. The Mycobacterium tuberculosis (M.tb)DciA is an essential protein involved in bacterial replication and regulates its growth. DciA interacts with DNA and provides critical help in binding other replication machinery proteins to the DNA. Moreover, the lack of any structural homology of M.tb DciA with human proteins makes it an appropriate target for drug development. In this study, FDA-approved drugs were virtually screened against M.tb DciA to identify potential inhibitors. Four drugs namely Lanreotide, Risedronate, Triflusal, and Zoledronic acid showed higher molecular docking scores. Further, molecular dynamics simulations analysis of DciA-drugs complexes reported stable interaction, more compactness, and reduced atomic motion. The anti-TB activity of drugs was further evaluated under in vitro and ex vivo conditions where Triflusal was observed to have the best possible activity with the MIC of 25 µg/ml. Our findings present novel DciA inhibitors and anti-TB activity of Triflusal. Further investigations on the use of Triflusal may lead to the discovery of a new anti-TB drug.

Atosiban and Rutin exhibit anti-mycobacterial activity - An integrated computational and biophysical insight toward drug repurposing strategy against Mycobacterium tuberculosis targeting its essential enzyme HemD.

With the advancements of high throughput computational screening procedures, drug repurposing became the privileged framework for drug discovery. The structure-based drug discovery is the widely used method of drug repurposing, consisting of computational screening of compounds and testing them in-vitro. This current method of repurposing leaves room for mechanistic insights into how these screened hits interact with and influence their targets. We addressed this gap in the current study by integrating highly sensitive biophysical methods into existing computational repurposing methods. We also corroborated our computational and biophysical findings on H37Rv for the anti-mycobacterial action of selected drugs in-vitro and ex-vivo conditions. Atosiban and Rutin were screened as highly interacting hits against HemD through multi-stage docking and were cross-validated in biophysical studies. The affinity of these drugs (K ~ 106 M-1) was quantified using fluorescence quenching studies. Differential Scanning Fluorimetry (DSF) and urea-based chemical denaturation studies revealed a destabilizing effect of these drugs on target which was further validated using MD simulations. Conformational rearrangements of secondary structures were established using CD spectra and intrinsic fluorescence. Furthermore, Atosiban and Rutin inhibited M.tb growth in-vitro and ex-vivo while remaining non-toxic to mice peritoneal macrophages.

Potential Repurposed Drug Candidates for Tuberculosis Treatment: Progress and Update of Drugs Identified in Over a Decade.

The devastating impact of Tuberculosis (TB) has been a menace to mankind for decades. The World Health Organization (WHO) End TB Strategy aims to reduce TB mortality up to 95% and 90% of overall TB cases worldwide, by 2035. This incessant urge will be achieved with a breakthrough in either a new TB vaccine or novel drugs with higher efficacy. However, the development of novel drugs is a laborious process involving a timeline of almost 20-30 years with huge expenditure; on the other hand, repurposing previously approved drugs is a viable technique for overcoming current bottlenecks in the identification of new anti-TB agents. The present comprehensive review discusses the progress of almost all the repurposed drugs that have been identified to the present day (∼100) and are in the development or clinical testing phase against TB. We have also emphasized the efficacy of repurposed drugs in combination with already available frontline anti-TB medications along with the scope of future investigations. This study would provide the researchers a detailed overview of nearly all identified anti-TB repurposed drugs and may assist them in selecting the lead compounds for further in vivo/clinical research.

Combating the challenges of COVID-19 pandemic: Insights into molecular mechanisms, immune responses and therapeutics against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes lethal coronavirus disease (COVID-19). SARS-CoV-2 has been the chief source of threat to public health and safety from 2019 to the present. SARS-CoV-2 caused a sudden and significant rise in hospitalization due to respiratory issues and pneumonia. We are consistently uncovering new information about SARS-CoV-2, and yet so much is to explore to implement efficient interventions to combat the emergent variants and spread of the ongoing pandemic. Information regarding the existing COVID-19 pandemic is streamlining continuously. However, clinical symptoms of SARS-CoV-2 infections spanning from asymptomatic infection to severe death-instigating disease remain consistent with preliminary reports. In this review, we have briefly introduced highlights of the COVID-19 pandemic and features of SARS-CoV-2. We have focused on current knowledge of innate and adaptive immune responses during SARS-CoV-2 infections and persisting clinical features of recovered patients. Furthermore, we have discussed how these immune responses are not tightly regulated and imbalance can direct the latter phases of COVID-19, long-COVID symptoms, and cause detrimental immunopathogenesis. COVID-19 vaccines are also discussed in detail to describe the efforts going around the world to control and prevent the infection. Overall, we have summarized the current knowledge on the immunology of SARS-CoV-2 infection and the utilization of that knowledge in the development of a suitable COVID-19 therapeutics and vaccines.

Withaferin A Protects against Primary and Recurrent Tuberculosis by Modulating Mycobacterium-Specific Host Immune Responses.

The fate of Mycobacterium tuberculosis infection is governed by immune signaling pathways that can either eliminate the pathogen or result in tuberculosis (TB). Anti-TB therapy (ATT) is extensive and is efficacious only against active, drug-sensitive strains of M. tuberculosis. Due to severe side effects, ATT often causes impairment of host immunity, making it imperative to use novel immunotherapeutics for better clinical outcomes. In this study, we have explored the immunomodulatory potential of withaferin A (WA) as an immunotherapeutic against TB. Here, we demonstrate that WA can constrain intracellular drug-sensitive and -resistant strains of M. tuberculosis by augmenting host immune responses. We also established the potential of WA treatment in conjunction with isoniazid. We show that WA directs the host macrophages toward defensive M1 polarization and enhances TH1 and TH17 immune responses against M. tuberculosis infection. The reduced bacterial burden upon T cell adoptive transfer further corroborated the augmented T cell responses. Interestingly, WA stimulated the generation of T cell memory populations by instigating STAT signaling, thereby reducing the rate of TB recurrence due to reactivation and reinfection. We substantiate the prospects of WA as a potent adjunct immunomodulator that enriches protective memory cells by prompting STAT signaling and improves host defense against M. tuberculosis. IMPORTANCE Despite being extensive, conventional antituberculosis therapy (ATT) is barely proficient in providing sterile immunity to tuberculosis (TB). Failure to constrain the escalating global TB burden due to the emergence of drug-resistant bacterial strains and immune dampening effects of ATT necessitates adjunct immunotherapeutics for better clinical outcomes. We evaluated the prospects of withaferin A (WA), an active constituent of Withania somnifera, as an adjunct immunomodulator against diverse M. tuberculosis strains. WA efficiently restricts the progression of TB by stimulating antimycobacterial host responses, protective immune signaling, and activation of diverse immune cell populations. Protective effects of WA can be attributed to the enrichment of memory T cells by induction of STAT signaling, thereby enhancing resistance to reinfections and reactivation of disease. We ascertained the immunotherapeutic potential of WA in boosting host immune responses against M. tuberculosis.

Berberine governs NOTCH3/AKT signaling to enrich lung-resident memory T cells during tuberculosis.

Stimulation of naïve T cells during primary infection or vaccination drives the differentiation and expansion of effector and memory T cells that mediate immediate and long-term protection. Despite self-reliant rescue from infection, BCG vaccination, and treatment, long-term memory is rarely established against Mycobacterium tuberculosis (M.tb) resulting in recurrent tuberculosis (TB). Here, we show that berberine (BBR) enhances innate defense mechanisms against M.tb and stimulates the differentiation of Th1/Th17 specific effector memory (TEM), central memory (TCM), and tissue-resident memory (TRM) responses leading to enhanced host protection against drug-sensitive and drug-resistant TB. Through whole proteome analysis of human PBMCs derived from PPD+ healthy individuals, we identify BBR modulated NOTCH3/PTEN/AKT/FOXO1 pathway as the central mechanism of elevated TEM and TRM responses in the human CD4+ T cells. Moreover, BBR-induced glycolysis resulted in enhanced effector functions leading to superior Th1/Th17 responses in human and murine T cells. This regulation of T cell memory by BBR remarkably enhanced the BCG-induced anti-tubercular immunity and lowered the rate of TB recurrence due to relapse and re-infection. These results thus suggest tuning immunological memory as a feasible approach to augment host resistance against TB and unveil BBR as a potential adjunct immunotherapeutic and immunoprophylactic against TB.

Epigenetic code during mycobacterial infections: therapeutic implications for tuberculosis.

Epigenetics involves changing the gene function without any change in the sequence of the genes. In the case of tuberculosis (TB) infections, the bacilli, Mycobacterium tuberculosis (M.tb), uses epigenetics as a tool to protect itself from the host immune system. TB is a deadly disease-causing maximum death per year due to a single infectious agent. In the case of TB, there is an urgent need for novel host-directed therapies which can effectively target the survival and long-term persistence of the bacteria without developing drug resistance in the bacterial strains while also reducing the duration and toxicity associated with the mainstream anti-TB drugs. Recent studies have suggested that TB infection has a significant effect on the host epigenome thereby manipulating the host immune response in the favor of the pathogen. M.tb alters the activation status of key genes involved in the immune response against TB to promote its survival and subvert the antibacterial strategies of the host. These changes are reversible and can be exploited to design very efficient host-directed therapies to fight against TB. This review has been written with the purpose of discussing the role of epigenetic changes in TB pathogenesis and the therapeutic approaches involving epigenetics, which can be utilized for targeting the pathogen.

Unique C-terminal extension and interactome of Mycobacterium tuberculosis GlmU impacts it's in vivo function and the survival of pathogen.

N-acetyl glucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme involved in the biosynthesis of Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a critical precursor for the synthesis of peptidoglycan and other cell wall components. The absence of a homolog in eukaryotes makes GlmU an attractive target for therapeutic intervention. Mycobacterium tuberculosis GlmU (GlmUMt) has features, such as a C-terminal extension, that are not present in GlmUorthologs from other bacteria. Here, we set out to determine the uniqueness of GlmUMt by performing in vivo complementation experiments using RvΔglmU mutant. We find that any deletion of the carboxy-terminal extension region of GlmUMt abolishes its ability to complement the function of GlmUMt. Results show orthologs of GlmU, including its closest ortholog, from Mycobacterium smegmatis, cannot complement the function of GlmUMt. Furthermore, the co-expression of GlmUMt domain deletion mutants with either acetyl or uridyltransferase activities failed to rescue the function. However, co-expression of GlmUMt point mutants with either acetyl or uridyltransferase activities successfully restored the biological function of GlmUMt, likely due to the formation of heterotrimers. Based on the interactome experiments, we speculate that GlmUMt participates in unique interactions essential for its in vivo function.

A randomized controlled trial comparing the radiographic evaluation of crestal bone resorption in single implant versus two implant-retained overdentures.

AIM: The aim of this study was to evaluate and compare radiographically the amount of crestal bone resorption during healing and loading period in single implant versus two implant-retained mandibular overdentures in totally edentulous patients. MATERIALS AND METHODS: A total of 20 edentulous patients (12 male and 8 female) with age range of 58.6 years were included in this clinical trial which was completed in four phases (clinical and radiographic diagnosis, surgical phase, implant loading phase, and bone level measurement phase). The eligible patients were randomly allocated in two equivalent groups of 10 participants each per group. The allocation was in 1:1 ratio via randomized chit method. Group I included the case group, that is, single implant, and Group II included the control group, that is, two implants located in mandible. A total of 30 implants were placed in Group I and 20 implants in Group II. Digital intraoral peri-apical radiographs (RVG 5100) were used for measuring the bone level immediately after implant surgery, 1 month, 3 months, 4 months, and 6 months. RESULT: This study showed that there was a mean crestal bone loss of 0.7 mm between the tip of the implant and alveolar crest at the end of 6 months after implant placement in single implant Group I while 0.67 mm in case of Group II two-implant-retained mandibular overdentures. The percentage of crestal bone loss after 6 months follow-up was 6.45% in Group I which was statistically insignificant compared with Group II where 6.25% of bone loss was recorded. CONCLUSION: Single implant-retained mandibular overdentures could be used as another alternative treatment option for completely edentulous elderly patients with severely resorbed ridges and financially and systemically compromised conditions.

The bifunctional protein GlmU is a key factor in biofilm formation induced by alkylating stress in Mycobacterium smegmatis.

Living organisms have developed specific defence mechanisms to counteract hostile environmental conditions. Alkylation stress response mechanisms also occur in Mycobacterium tuberculosis (MTB) the pathogen responsible for tuberculosis. The effect of alkylating agents on the cellular growth of MTB was investigated using methyl methanesulfonate (MMS) as methyl donor demonstrating that limited doses of alkylating agents might affect MTB cell viability. A global investigation of Mycobacterium smegmatis response to alkylating stress was then pursued by differential proteomics to identify the most affected cellular pathways. Quantitative analysis of proteomic profiles demonstrated that most of the proteins upregulated in the presence of alkylating agents are involved in biofilm formation and/or cell wall biosynthesis. Tailored experiments confirmed that under stress conditions M. smegmatis elicits physical defence mechanisms by increasing biofilm formation. Among the upregulated proteins, we identified the GlmU bifunctional enzyme as a possible factor involved in biofilm production. Experiments with both conditional deletion and overexpressing glmU mutants demonstrated that down regulation of GlmU decreased M. smegmatis capabilities to produce biofilm whereas overexpression of the enzyme increased biofilm formation. These results were supported by inhibition of GlmU acetyltransferase activity with two different inhibitors, suggesting the involvement of this enzyme in the M. smegmatis defence mechanisms.

Identification and characterization of ARS-like sequences as putative origin(s) of replication in human malaria parasite Plasmodium falciparum.

DNA replication is a fundamental process in genome maintenance, and initiates from several genomic sites (origins) in eukaryotes. In Saccharomyces cerevisiae, conserved sequences known as autonomously replicating sequences (ARSs) provide a landing pad for the origin recognition complex (ORC), leading to replication initiation. Although origins from higher eukaryotes share some common sequence features, the definitive genomic organization of these sites remains elusive. The human malaria parasite Plasmodium falciparum undergoes multiple rounds of DNA replication; therefore, control of initiation events is crucial to ensure proper replication. However, the sites of DNA replication initiation and the mechanism by which replication is initiated are poorly understood. Here, we have identified and characterized putative origins in P. falciparum by bioinformatics analyses and experimental approaches. An autocorrelation measure method was initially used to search for regions with marked fluctuation (dips) in the chromosome, which we hypothesized might contain potential origins. Indeed, S. cerevisiae ARS consensus sequences were found in dip regions. Several of these P. falciparum sequences were validated with chromatin immunoprecipitation-quantitative PCR, nascent strand abundance and a plasmid stability assay. Subsequently, the same sequences were used in yeast to confirm their potential as origins in vivo. Our results identify the presence of functional ARSs in P. falciparum and provide meaningful insights into replication origins in these deadly parasites. These data could be useful in designing transgenic vectors with improved stability for transfection in P. falciparum.

Regulation of DNA replication proteins in parasitic protozoans: possible role of CDK-like kinases.

Regulatory roles of CDKs in fundamental processes including cell cycle progression and transcription are well conserved in metazoans. This family of proteins has undergone significant evolutionary divergence and specialization. Several CDK-like kinases have been identified and characterized in parasitic protozoans. However, clear functional role and physiological relevance of these proteins in protozoans still remain elusive. In continuation with the recent finding that CDK-like protein PfPK5 regulates important DNA replication protein like origin recognition complex subunit 1 in Plasmodium falciparum, here we have discussed the emerging significance of CDK1/2 homologs in DNA replication of parasitic protozoans. In fact, involvement of these proteins in crucial cellular processes projects them as potential drug targets. The possibilities that CDKs offer as potential therapeutic targets in controlling parasite progression have also been explored.

Regulation of Plasmodium falciparum†Origin Recognition Complex subunit 1 (PfORC1) function through phosphorylation mediated by CDK-like kinase PK5.

Plasmodium falciparum Origin Recognition Complex subunit 1 (PfORC1) has been implicated in DNA replication and var gene regulation. While the C-terminus is involved in DNA replication, the specific role of N-terminus has been suggested in var gene regulation in a Sir2-dependent manner. PfORC1 is localized at the nuclear periphery, where the clustering of chromosomal ends at the early stage of parasite development may be crucial for the regulation of subtelomeric var gene expression. Upon disassembly of telomeric clusters at later stages of parasite development, ORC1 is distributed in the nucleus and parasite cytoplasm where it may be required for its other cellular functions including DNA replication. The level of ORC1 decreases dramatically at the late schizont stage. The mechanisms that mediate regulation of PfORC1 function are largely unknown. Here we show, by the use of recombinant proteins and of transgenic parasites expressing wild type or mutant forms of ORC1, that phosphorylation of the PfORC1-N terminal domain by the cyclin-dependent kinase (CDK) PfPK5 abolishes DNA-binding activity and leads to changes in subcellular localization and proteasome-mediated degradation of the protein in schizonts. These results reveal that PfORC1 phosphorylation by a CDK is central to the regulation of important biological functions like DNA replication and var gene silencing.