Mycobacterium tuberculosis (Mtb) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is indispensable for glycolysis, it also performs several critical non-metabolic functions. In the present study, we demonstrate that CRISPRi silencing of GAPDH inhibited enzyme activity and iron acquisition via human transferrin (Tf)/lactoferrin (Lf). GAPDH silencing also enhanced reactive oxygen species (ROS) and ROS induced damage suggesting its role as a redox sensor. We then examined the impact of GAPDH inhibition in Mtb using small molecule inhibitors. Vitamin C (VC) was selected considering its potent bactericidal effects against Mtb and its inhibition of human GAPDH resulting in its efficacy against cancer cells. The GAPDH inhibitors Ethyl bromopyruvate (EBP) and Koningic acid (KA) are anti-cancer agents that target the glycolytic activity of GAPDH. In contrast, TCH346 was identified as a neuroprotective agent, wherein it targets the non-metabolic function of GAPDH induced apoptotic signalling. The effects of inhibitors, alone or in combination with VC mirrored the cellular effects of GAPDH silencing, resulting in significant anti-bacterial activity. VC induced iron mobilization which coupled with GAPDH inhibitors induced a veritable “double whammy” resulting in massive increase in ROS and downstream effects. The efficacy of these treatments was assessed in a murine model, confirming that VC augmented the potent anti-tubercular activity induced by EBP and TCH346. Overall, this study identifies the crucial function of Mtb GAPDH as a redox sensor and highlights the potential of targeting its pleiotropic cellular functions towards drug discovery. In addition, the efficacy of TCH346 provides an opportunity of drug-repurposing as a strategy for therapy.

The synthesis and characterization of novel tryptanthrin hydrazide hydrazones along with their photophysical and biological applications are described. Comprehensive photophysical studies revealed that the chloro derivative of tryptanthrin hydrazide hydrazone (3b) functions as a selective and sensitive fluorescent chemosensor for picric acid (PA) detection with a lowest detection limit of 0.012 mu M, maintaining great resistance to interference from other nitro aromatic compounds (NACs). The mechanism of quenching involves (i) inner filter effect (IFE) and (ii) intermolecular hydrogen bond interactions; both mechanisms are supported by density functional theory (DFT) calculations computed at B3LYP/6-311 + G(d,p) level of theory. Analysis of real samples and onsite test strip detection of PA underscore the capability of 3b as a reliable sensor for PA, with promising applications in environmental monitoring and security-related detection. In a separate study, anti-tubercular (antiTB) activity of the compounds were also evaluated and it was found that 3d-h exhibited very low MIC of 0.125-16 mu g mL(- 1) (against Mtb H37Rv (ATCC 27294)), out of which 3d & 3e showed highest selectivity index (SI) of >200 and 160 respectively.

The limitations of existing mouse models of lung infection with Mycobacteroides abscessus impede drug discovery and development. In contrast to current animal models that introduce NTM intravenously or by intranasal/intra-tracheal instillation or via bronchoscopy-guided insufflation, we developed a dry powder inhalation (DPI) of M. abscessus ATCC 19977 that generated paucibacillary lung infection and histopathology in immunocompetent mice. Swiss outbred mice receiving similar to 1000 (3-log) colony forming units (CFU) of M. abscessus/gram lung tissue via the DPI administered by nose-only inhalation for 90 s showed peak bacterial burden of similar to 3.35-log CFU/g in the lungs after 28 days. This was maintained at similar to 2-log/g from Day 35 through 56 in the lungs, but not in the spleen. Histopathology indicated increasing severity of inflammation, fibrosis and lung consolidation. Bacteria were rarely recovered from spleen, and histopathological examination indicated partial resolution in the spleen between Days 49-56. The DPI, prepared by freeze-drying log-phase liquid culture with cryoprotectants was formulated to possess aerosol characteristics suitable for alveolar deposition. Aerosol exposure to inoculum mimics natural airborne infection. Non-invasive aerosol infection is convenient, inexpensive, does not require special equipment or extensive training and mitigates stress to animals, but biosafety level 3 containment is recommended to mitigate risk to experimenters.

The continued prevalence of drug-resistant Mycobacterium tuberculosis (Mtb) strains, particularly against first-line antitubercular (anti-TB) drugs, presents an impending public health threat that necessitates the exploration and development of New Chemical Entities (NCEs). In search of new anti-TB leads, a library of ethyl 5-(1-benzyl-1H-indol-5-yl) isoxazole-3-carboxylates were generated through a strategy of scaffold hopping from the proven isoxazole-3-carboxylate-based anti-TB pharmacophore. We evaluated their antibacterial potential against a panel of pathogenic bacteria and Mtb H37Rv strains. The majority of the compounds exhibited notable in vitro efficacy against the H37Rv strains (MIC 0.25 to 16 mu g/mL) and were not cytotoxic with a Selectivity Index (SI) >10. Compound 5e (3,4-dichlorobenzyl substituent) was found to be optimally active in the lot (MIC 0.25 mu g/mL) and SI >200. It also displayed equipotent activity against drug-resistant Mtb (DR-Mtb) strains. In addition, it demonstrated concentration-dependent bactericidal activity in a time-kill kinetic assay similar to first-line anti-TB drugs besides exhibiting synergistic activity with Streptomycin. Moreover, it complies with the drug-likeness characteristic, making it a promising candidate for further exploration as a probable anti-TB lead.

Mycobacterium tuberculosis (Mtb) remains a major global health threat, intensified by multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. We synthesized a series of N-benzoyl-arylthiourea derivatives (IITKDA1-20) as hybrids of isoniazid/pyrazinamide and ethionamide to explore their antimycobacterial potential. Our evaluation of synthesized library members for antimycobacterial activity has identified IITKDA10 (N-benzoyl-arylthiourea possessing p-(N-Boc)-thionamide) as the maximally effective inhibitor of Mtb (1 μg/mL MIC). Further, the physicochemical properties indicated a trend of high topological polar surface area (tPSA) and partition coefficient (ClogP) in the range of 3-4 was optimal for the compounds to be active against Mtb. Molecular docking of IITKDA10 into the InhA (enoyl-[acyl-carrier-protein] reductase) active site revealed strong binding (-9.63 kcal/mol), stabilized by hydrogen bonds and π-alkyl interactions. Further, crystal packing analysis indicated that hydrogen bonding networks guided supramolecular architecture, and structural planarity (e.g., IITKDA4, IITKDA8) correlated with higher activity. In contrast, twisted or L-shaped conformations (IITKDA2, IITKDA5) showed reduced potency. This study presents a structurally and functionally diverse set of N-benzoyl-arylthioureas with promising anti-TB activity, supported by structure-activity relationships, docking, and crystallographic insights.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health concern, exacerbated by the rise of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. Pyridine-4-carbohydrazide (isoniazid), a frontline anti-TB drug, is limited by high dosage requirements and associated toxicity. To overcome these limitations, a novel series of isoniazid-based derivatives were synthesized and evaluated for antitubercular activity. Several compounds exhibited potent inhibition against Mtb H37Rv, with minimum inhibitory concentrations (MICs) ranging from 0.125 to 2 mu g/mL. Among them, compounds 5j, 5k, 5o, and 5p emerged as lead candidates, showing strong activity against MDR clinical isolates. Cytotoxicity evaluation using Vero cells revealed excellent safety profiles, with selectivity indices (SI) ranging from 400 to 800. Time-kill kinetics confirmed the bactericidal nature of compound 5p. In silico molecular docking and 3D-QSAR (3D quantitative structure-activity relationship) studies were conducted to identify key structural features responsible for activity and to validate binding interactions with M. tuberculosis target enzymes. The computational studies correlated well with experimental data and supported favorable drug-like properties. Overall, the synthesized derivatives demonstrate strong potential as next-generation anti-TB agents.

The unabated increase in antimicrobial resistance has underlined the importance of identifying novel drug combinations which eliminate infections more potently and likely reduce the emergence of resistance. In this context, we have identified Vancomycin and many β-lactams as being potently active against drug-resistant Mycobacterium tuberculosis and non-tuberculous mycobacteria including M. abscessus, emerging as pathogens of concern owing to their inherent drug resistance profile. In this study, we have identified combinations of Vancomycin, a glycopeptide and β-lactams, especially Ceftriaxone, Ceftazidime and Meropenem, in the presence or absence of Sulbactam, a β-lactamase inhibitor, as possessing potent antimicrobial activity against several drug-resistant mycobacterial strains. The combination of Vancomycin and β-lactams exhibited potent bactericidal activity and reduced the bacterial load better than either drug alone. The molecular basis of synergy was mediated by increase in permeability of mycobacterial cell as demonstrated by ethidium bromide assay. In the murine model of mycobacterial infection, synergistic combination of Vancomycin and β-lactams outperformed clinically utilized drugs including Isoniazid, Rifampicin and Ethambutol against M. tuberculosis and Amikacin, Clarithromycin against M. abscessus. The combinations caused a significant reduction in bacterial load in various organs in M. tuberculosis and M. abscessus infected mice. Thus, the synergistic combination of Vancomycin and β-lactams could potentially be utilized for treatment of recalcitrant mycobacterial infection especially those caused due to drug-resistant pathogens. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:Development of new effective drugs against multidrug resistant Mycobacterium tuberculosis is the need of the hour to combat tuberculosis (TB) disease. MATERIALS AND METHODS:Pyridine-4-carbohydrazide and substituted pyrazole aldehydes were used to synthesize target molecules (6a-r) which were evaluated against H37Rv and drug-resistant TB strains. Time kill kinetics assay was performed to check bactericidal/bacteriostatic effect, molecular docking, dynamics simulation over 100 ns was performed against enoyl acyl carrier protein reductase (InhA) along with QSAR, ADMET profile prediction. RESULTS:All compounds displayed excellent MICs in the range of 0.125-16 µg/mL. The most potent compound, 6q, with an MIC of 0.125 µg/mL showed bactericidal effect and was effective on ethambutol and streptomycin resistant Mtb strains with an MIC of 0.03 µg/mL and rifampicin resistant Mtb strain with an MIC of 0.25 µg/mL. CONCLUSION:The pyrazole clubbed with pyridine-4-carbohydrazide is a potential scaffold for further exploration as anti-TB agent.

The escalating threat of drug-resistant Mycobacterium tuberculosis (Mtb) necessitates the discovery of novel chemotherapeutic agents. In this study, a series of dihydroindazole-based derivatives were designed, synthesized, and evaluated for their antimycobacterial potential. Among the synthesized compounds, 8u exhibited the most potent in vitro activity against Mtb H37Rv with a minimum inhibitory concentration (MIC) of 2 µg/mL, while 8i and 8q showed moderate activity (MIC = 8 µg/mL). Several analogs demonstrated MICs in the range of 16-32 µg/mL. 8u also displayed enhanced activity against single-drug-resistant Mtb strains, outperforming ethambutol and rifampicin. Structure-activity relationship analysis indicated that both the hydrazide linker and heteroaryl substitutions significantly influenced antimycobacterial activity. 8u was non-cytotoxic to Vero cells (CC₅₀ > 100 µg/mL), yielding a selectivity index (SI) > 50. Time-kill kinetics confirmed its bactericidal nature. Mechanistic investigations using molecular docking and 100-ns molecular dynamics simulations identified InhA as the probable molecular target. In silico ADMET predictions (QikProp and ProTox-3.0) supported favorable pharmacokinetic and toxicity profiles. Collectively, these findings highlight 8u as a promising lead for the development of next-generation anti-TB agents.

Acinetobacter baumannii, a Gram-negative WHO critical priority pathogen, is an opportunistic bacterial pathogen associated with increasing hospital- and community-acquired infections. The emergence of a multidrug-resistant pathogen, especially the carbapenem-resistant A. baumannii (CRAB), has left us with extremely limited treatment options and, consequently, very high morbidity and mortality rates. As per WHO, the transmissibility of A. baumannii is considered between moderate to high. This review provides a unique comprehensive insight into pathogen's global epidemiology, pathogenesis, host-pathogen interaction, tools to study the pathogen, associated diseases, available treatment options, and how the pathogen is becoming resistant to almost all the treatment options available, thus presenting a holistic picture.

Considering the escalation of drug-resistant tuberculosis, there is an emerging need to develop new anti-tubercular agents with novel targets. In this context, a series of novel pyrrole coumarin conjugates were synthesized and evaluated against a mycobacterial pathogen panel consisting of Mycobacterium tuberculosis, M. abscessus, M. fortuitum, and M. chelonae. Most of the compounds exhibited selective anti-tubercular activity against M. tuberculosis with MICs ranging from 2 to 64 mu g/mL. The active compounds were nontoxic toward Vero cells, demonstrating a favorable selectivity index. The active compounds were also tested against drug-resistant M. tuberculosis (DR-MTB) strains and found to be active against all resistant strains, with MIC values ranging from 2 to 4 mu g/mL. Molecular docking studies were performed to gain insight into their mechanism of action, elucidating the potential binding mode and interactions at the enzyme's active site. Further, molecular dynamics simulation studies were carried out to validate the results obtained from the molecular docking study.

The rise of drug-resistant tuberculosis (TB) has created an urgent need to discover and develop new anti-mycobacterial agents. Herein, we report the synthesis and evaluation of a library of 1,3-diaryl substituted pyrazole-based imidazo[1,2-a]pyridine carboxamides as promising anti-TB agents. In preliminary screening, 10 out of 26 compounds displayed potent in vitro inhibition against Mtb H37Rv with a MIC value of 0.03 μg/mL, which is 17-fold more potent than the first-line TB drug streptomycin, 33-fold more potent than ethambutol, and equipotent with isoniazid and rifampicin. Encouragingly, most of these compounds exhibited a selectivity index (SI) >3333.3 and CC₅₀ values >100 μg/mL against Vero cells, indicating they are over 3000 times more toxic to M. tuberculosis than to mammalian cells and demonstrate absence of cytotoxicity at concentrations effective against TB (MIC = 0.03 μg/mL). Among them, 12a, 14a, and 14d demonstrated remarkable activity against drug-resistant strains of Mtb with an MIC of 0.03 μM. Time-kill kinetic studies revealed that 12a, 14a, and 14d exhibited bacteriostatic properties. Furthermore, 12a, 14a, and 14d demonstrated synergistic effects with the FDA-approved anti-TB drugs rifampicin (ƩFIC 0.093), ethambutol (ƩFIC 0.061), and moxifloxacin (ƩFIC 0.154-0.281), exhibiting bactericidal time-kill properties in combination with these drugs. Additionally, 12a, 14a, and 14d exhibited acceptable metabolic stability (CLint 11.49-14.62 μL/min/mg microsomal protein), indicating effective drug levels and bioavailability. Also, 12a, 14a, and 14d showed stable interactions with QcrB in docking studies. These findings highlight 12a, 14a, and 14d as potential candidates for in vivo evaluation and further development as novel anti-tubercular drugs.

A series of pyrrole-thiazolidin-4-one conjugates were synthesized and evaluated for their anti-mycobacterial and anti-bacterial activities. Two compounds, 10 a and 10 k, were the most effective conjugates and produced identical MICs (0.5 mu g/mL) against M. tuberculosis H37Rv with a high selectivity index. Upon evaluation against the ESKAP bacteria panel, compound 10 g emerged most effective against S. aureus (MIC=8.0 mu g/mL) while compound 10 o produced activity against A. baumannii (MIC=4.0 mu g/mL). A molecular docking study revealed that the most active compound 10 a has similar binding interactions as those of BM212 and rimonabant, with a comparable docking score against M. tuberculosis mycolic acid transporter MmpL3. This work describes the synthesis, anti-tuberculosis, and antibacterial activities of pyrrole-thiazolidin-4-one conjugates. The best analogue exhibits an MIC of 0.5 mu g/mL against the M. tuberculosis H37Rv strain and also shows promising antibacterial activities. The docking study indicates that the M. tuberculosis mycolic acid transporter MmpL3 is a possible molecular target. image

The emergence of antibiotic resistance to S. aureus and M. tuberculosis, particularly MRSA, VRSA, and drug-resistant tuberculosis, poses a serious threat to human health. Towards discovering new antibacterial agents, we designed and synthesized a series of new naphthalimide-thiourea derivatives and evaluated them against a panel of bacterial strains consisting of E. coli, S. aureus, K. pneumoniae, P. aeruginosa, A. baumannii and various mycobacterial pathogens. Compounds 4a, 4l, 4m, 4n, 4q, 9f, 9l, 13a, 13d, 13e, 17a, 17b, 17c, 17d, and 17e demonstrated potent antibacterial activity against S. aureus with MIC 0.03-8 mu g mL-1. In addition, these compounds have also exhibited potent inhibition against MDR strains of S. aureus, including VRSA with MICs 0.06-4 mu g mL(-1). Compounds 4h, 4j, 4l, 4m, 4q, 4r, 9a, 9b, 9c, 9d, 9e, 9g, 9h, 9j, 13f and 17e also exhibited good antimycobacterial activity against M. tuberculosis with MIC 2-64 mu g mL(-1). The cytotoxicity assay using Vero cells revealed that all the compounds were non-toxic and exhibited a favorable selectivity index (SI >40). Time kill kinetics data indicated that compounds exhibited concentration-dependent killing. Furthermore, in silico studies were performed to decipher the possible mechanism of action. Comprehensively, these results highlight the potential of naphthalimide-thiourea derivatives as promising antibacterial agents.

Herein, we describe the design and synthesis of a series of C-5-substituted diazenyl derivatives of uracil, exhibiting selective and potent antileishmanial but not antibacterial or antifungal activity. The formation of the substituted derivatives was confirmed by using FTIR, 1H, 13C NMR, and HRMS analysis. Among all of the sets of tested compounds, only three [4a, 6b, and 8b] showed the highest activity against Leishmania donovani (LD) promastigote and amastigote models of LD infections. Further, the cytotoxicity assays performed using three different cell lines, Vero cells, J774 cells, and THP1 cells, along with erythrocyte hemolysis assay showed the highest biocompatibility for the 4a, making it a lead compound for further biological assays. The LD cell death associated with 4a was not linked with ergosterol depletion, a common mechanism of action of antileishmanial drugs like amphotericin B (AmB). However, the LD cell death in the presence of 4a was reversed significantly through supplementation of uridine monophosphate (UMP), indicating the specific role of uridine biosynthesis pathway as the target of 4a. Furthermore, the in silico studies predicted orotidine monophosphate decarboxylase enzyme (OMPDCase) from LD as the plausible target for 4a. The proteomics analysis showed stronger downregulation of the aforementioned OMPDCase and also for a few other enzymes that are involved in the UMP biosynthesis pathway. This indicates that OMPDCase and other enzymes that regulate the UMP biosynthesis may be the target of 4a. Overall, the C-5-substituted diazenyl derivatives of uracil are presented here as novel and potent antileishmanial agents that can be used for treating visceral leishmaniasis (VL) wherein at present drug resistance and side effects of existing drugs demand a look for safer alternatives.

In this work, a novel series of naphthalimide hydrazide derivatives were designed, synthesized and evaluated against a bacterial pathogen panel. Most of the compounds were found to exhibit potent antibacterial activity against carbapenem-resistant A. baumannii BAA 1605, with MIC ranging from 0.5 to 16 μg mL-1. Compounds 5b, 5c, 5d and 5e showed the most potent antibacterial activity, with an MIC range of 0.5-1 μg mL-1. These compounds were also found to be non-toxic to Vero cells with a high selectivity index. Further, they were active against 24 clinical isolates of MDR-AB with potent antibacterial activity. In addition, synergistic studies revealed that compound 5d exhibited synergism with FDA-approved drugs, as further validated through time-kill kinetic studies. These results highlight the potential of the synthesized compounds as promising leads for the development of novel and selective agents against carbapenem-resistant A. baumannii.

The rapid rise in antimicrobial resistance (AMR) has become a major threat to human health. Consequently, the constant search for new antibacterial agents that work against resistant organisms has become a global initiative. To discover new antibacterial agents with improved efficacy and activity against drug-resistant organisms, we designed and synthesized a series of new naphthalimide-coumarin hybrids and evaluated them for their antimicrobial activity against a panel of bacterial and mycobacterial strains. In the preliminary evaluation, compounds 7a, 7c, 7d, 7e, 7f, 7 h, 7i, 7o, 7q, 20a and 20b exhibited potent inhibitory activity against S. aureus ATCC 29213 with MICs in the range of 0.5–32 µg/mL. Compounds 7c, 7d, 7f, 7 h, 7i, 7o, 20a, and 20b also exhibited good inhibitory activity against S. epidermidis with MIC 1–16 µg/mL. All these compounds were found to be non-toxic to Vero cells (CC50 = >50) and exhibited a favorable selectivity index (SI = >12.5). Additionally, these compounds 7c, 7d, 7e, 7f, 7 h, 7i, 7o, 20a and 20b have shown potent inhibition against various multidrug-resistant strains of S. aureus, including VRSA with MICs in the range of 0.5–16 µg/mL. Interestingly, compound 17b showed potent antimycobacterial activity against M. tuberculosis H37Rv with MIC 1 µg/mL and a favorable selectivity index. It also exhibited potent inhibitory activity against DR-Mtb with MIC 1 µg/mL. Topoisomerase enzyme inhibition assay was carried out to validate the mechanism of action. Furthermore, in silico studies were performed to investigate the binding mode and interactions at the active site of the target enzyme. These results highlight the potential of naphthalimide-coumarin hybrids as novel antimicrobial agents.