Mtb H37Rv Research Articles

P-788. Exploring β-Lactam Interactions with DacB1: Unraveling Optimal Therapies for Combating Drug-Resistant Mycobacterium tuberculosis

Abstract Background Mtb is a global health problem. DacB1, a D, D-carboxypeptidase, involved in Mtb peptidoglycan biosynthesis, is a promising target for β-lactam antibiotics (BLs) underutilized in Mtb treatment. Dual BL therapy may inactivate multiple targets in the peptidoglycan synthesis pathway (“target redundancy”). Modeling studies with meropenem (MEM), showed that the active site of DacB1 encompasses three penicillin-binding protein motifs: S121XXK124, S176XN178, and K282TG284 (PDB 4PPR). We explored the structure-activity relations, SAR, between DacB1 and other BLs to gain insight into optimal inhibitors for this important drug target. Tab. 1: β-lactam and β-lactamase Inhibitor MICs for Mtb H37Rv, H37Ra and representative Clinical Isolates Methods Minimum inhibitory concentrations (MICs) of BLs and β-lactamase inhibitors (BLIs) against Mtb H37Ra, H37Rv, and representative clinical isolates were tested via broth microdilution. Timed electrospray ionization mass spectrometry captured acyl-enzyme adducts of DacB1 and BLs [amoxicillin (AMX), ceftriaxone (CRO), tebipenem (TBP), imipenem (IPM), MEM] or BLIs [clavulanate (CLA), sulbactam (SUL)]. Molecular docking analyses of MEM, IPM, TBP, and Michaelis-Menten complexes, were conducted.Fig.1:The 2D representation of Michaelis-Menten complexes of DacB1 and IPM (A.), MEM (B.), and TPB (C.) with the formation of initial interactions. The carbonyl group of all the carbapenems is positioned into the oxyanion hole fromed by the catalytic Ser121:N and by Tyr285 amide. the hydroxyethyl group on the β-lactam ring present on all carbapenes produce a hydrophobic interaction with Leu218 and helps the initial positioning od the carbonyl into the oxyanion hole of DacB1. The C1 methyl group in both MEM and TBP, with additional hydrophobic interactions (B. and C.), contribute to the slower acyl-enzyme formation fro those compounds. Results MICs for IPM, MEM, and CRO ranged from 0.5 → >64, 1 → 32, and 0.25 → 16 µg/mL respectively (Tab. 1). Combining IPM or MEM with CRO further lowered MICs up to 32-fold (0.13 → 4 µg/mL), all within clinically achievable concentrations. Acyl-enzyme adducts of DacB1 (MW = 34414 Da) with carbapenems were captured; but not with AMX, CRO, or BLIs even after 120 min incubation. DacB1-IPM complexes were detected with 5 min incubation (Δ+299 Da) while at 15 min and 30 min for -MEM (Δ+383 Da) and -TBP (Δ+384 Da) complexes respectively. Conclusion IPM preferentially binds to DacB1 compared to MEM and TBP, and BLs, shedding light on crucial SAR for drug development. We postulate that the carbonyl group present in all carbapenems assists with positioning into the oxyanion hole of DacB1. However, the C1 methyl in MEM and TBP with their additional hydrophobic interactions contribute to steric hindrance and slower acyl-enzyme formation (Fig. 1). This study advances our understanding of the molecular mechanisms governing DacB1 interactions with BLs, offering insights into how carbapenems may inhibit another important BL target in Mtb peptidoglycan synthesis. Disclosures All Authors: No reported disclosures

Novel isoxazole thiophene-containing compounds active against Mycobacterium tuberculosis.

Screening of the ChemDiv molecular library in cholesterol media against Mycobacterium tuberculosis (Mtb) H37Rv strain identified a novel isoxazole thiophene hit as a putative Rv1625c/Cya activator with a promising in vitro activity and good pharmacokinetic properties. Twenty-nine analogs were synthesized to assess the structure-activity relationships (SAR) to further improve potency. The most notable analog was P15, which showed an intramacrophage EC50 = 1.96 µM and exhibited 58.0 % oral bioavailability when it was dosed orally at 20 mg/kg in a mouse pharmacokinetic (PK) study. The overall medicinal chemistry campaign revealed limited SAR that did not support further investigation into this series.

One‐pot synthesis and antitubercular activity of imidazo[2,1‐b] thiazol‐1H‐pyrazolylphosphonates.

Herein, we present an effective one‐pot method for the synthesis of imidazo[2,1‐b]thiazole‐pyrazole phosphonates from 5‐imidazo[2,1‐b] thiazole carboxaldehydes. The reaction involves a 1,3‐dipolar cycloaddition between in situ generated α,β‐unsaturated ketones and the Bestmann–Ohira reagent (BOR), facilitated by a base, leading to the formation of pyrazole phosphonates as the sole regioisomer with excellent yields. The in vitro evaluation of the newly synthesized compounds 4(a‐x) against Mycobacterium tuberculosis H37Rv (Mtb) identified compound 4o as the most effective analogue (MIC: 6.25μg/mL; 10.40µM).

Challenging the Biginelli scaffold to surpass the first line antitubercular drugs: Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt) inhibition activity and molecular modelling studies

New Biginelli adducts were rationalised, via the introduction of selected anti-tubercular (TB) pharmacophores into the dihydropyrimidine (DHPM) ring of deoxythymidine monophosphate (dTMP), the natural substrate of Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt). Repurposing was one of the design rationale strategies for some selected mimics of the designed compounds. The anti-TB activity was screened against the Mtb H37Rv strain where 11a was superior to ethambutol (EMB), and was 9-fold more potent than pyrazinamide (PZA). Additionally, compounds 11b, 4a, 4b, 13a, 13b and 14a elicited higher anti-TB activity than PZA, showing better safety profiles than EMB against RAW 264.7 cells’ growth. The in vitro TMPKmt inhibition assay released compounds 11a, 11b and 13b as the most potent inhibitors. Docking studies presumed the binding modes and molecular dynamics (MD) simulation revealed the dynamic stability of 11a-TMPKmt complex over 100 ns. In silico prediction of the chemo-informatics properties of the most active compounds was conducted.

You Win Some, You Lose Some: Modifying the Molecular Periphery of Nitrofuran-Tagged Diazaspirooctane Reshapes Its Antibacterial Activity Profile.

The use of the concept of privileged structures significantly accelerates the search for new leads and their optimization. 6-(methylsulfonyl)-8-(4-methyl-4H-1,2,4-triazol-3-yl)-2-(5-nitro-2-furoyl)-2,6-diazaspiro[3.4]octane 1 has been identified as a lead, with MICs of 0.0124-0.0441 μg/mL against MTb multiresistant strains. Several series of structural analogues have been synthesized, including variations in the periphery and simplifications of their scaffolds. All synthesized compounds were tested against the MTb H37Rv strain and ESKAPE panel of pathogens using serial broth dilutions. However, an attempt to optimize structure of 1 did not lead to the development of more active compounds which can work against MTb, but to substances with high activity against S. aureus. Induced-fit docking and MM-GBSA calculations determined a change in the likely biotarget from deazaflavin-dependent nitroreductase to azoreductases. The privileged nature of the scaffold was demonstrated by the detection of a different type of activity.

Design, Synthesis and Biological Evaluation of Ethyl 5-(1-benzyl-1H-indol-5-yl) Isoxazole-3-Carboxylates as Antimycobacterial Agents.

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 μg/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 μg/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.

Novel Triazolo[4,3-a]pyrazines as Potential MmpL3 Inhibitors: Design, Synthesis, Antitubercular Evaluation and Molecular Docking Studies

A series of biologically active novel triazolo[4,3-a]pyrazines (5a-f) were synthesized and characterized by spectroscopic techniques. The synthesized compounds 5a-f were evaluated for their in vitro anti-tubercular (anti-TB) activity against drug-sensitive Mtb H37Rv (ATCC 27294) strain as well as two drug-resistant Mtb strains viz. Mtb H37Rv ATCC 35822 (INH-resistant) and Mtb H37Rv ATCC 35837 (ETH-resistant). The synthesized compounds 5a-f displayed good to moderate anti-TB activity against drug-sensitive, INH-resistant and ETH-resistant Mtb H37Rv strains. Among all the compounds tested, compound 5c was found to be significantly potent. It inhibited the growth of drug-sensitive, INH-resistant and ETH-resistant Mtb H37Rv strains with MIC values 0.59 ± 0.11 µM, 20.83 ± 0.67 µM and 15.37 ± 0.14 µM, respectively. Moreover, compounds 5a-f were also tested for their cytotoxic efficacy against the mammalian Vero cell line at a maximum concentration of 50 µM. Molecular docking experiments of compounds 5a-f with the mycobacterial membrane protein large 3 (MmpL3) were performed to justify the biological activity and provide insight into the possible mechanism of action and binding mode of compounds. In silico predictions were used to validate compound toxicity descriptors, drug scores and drug-likeness properties.

Sirtuin inhibitors reduce intracellular growth of M. tuberculosis in human macrophages via modulation of host cell immunity

Host-directed therapies aiming to strengthen the body’s immune system, represent an underexplored opportunity to improve treatment of tuberculosis (TB). We have previously shown in Mycobacterium tuberculosis (Mtb)-infection models and clinical trials that treatment with the histone deacetylase (HDAC) inhibitor, phenylbutyrate (PBA), can restore Mtb-induced impairment of antimicrobial responses and improve clinical outcomes in pulmonary TB. In this study, we evaluated the efficacy of different groups of HDAC inhibitors to reduce Mtb growth in human immune cells. A panel of 21 selected HDAC inhibitors with different specificities that are known to modulate infection or inflammation was tested using high-content live-cell imaging and analysis. Monocyte-derived macrophages or bulk peripheral blood cells (PBMCs) were infected with the green fluorescent protein (GFP)-expressing Mtb strains H37Ra or H37Rv and treated with HDAC inhibitors in the micromolar range in parallel with a combination of the first-line antibiotics, rifampicin, and isoniazid. Host cell viability in HDAC inhibitor treated cell cultures was monitored with Cytotox-red. Seven HDAC inhibitors were identified that reduced Mtb growth in macrophages > 45–75% compared to average 40% for PBA. The most effective compounds were inhibitors of the class III HDAC proteins, the sirtuins. While these compounds may exhibit their effects by improving macrophage function, one of the sirtuin inhibitors, tenovin, was also highly effective in extracellular killing of Mtb bacilli. Antimicrobial synergy testing using checkerboard assays revealed additive effects between selected sirtuin inhibitors and subinhibitory concentrations of rifampicin or isoniazid. A customized macrophage RNA array including 23 genes associated with cytokines, chemokines and inflammation, suggested that Mtb-infected macrophages are differentially modulated by the sirtuin inhibitors as compared to PBA. Altogether, these results demonstrated that sirtuin inhibitors may be further explored as promising host-directed compounds to support immune functions and reduce intracellular growth of Mtb in human cells.

Distinct gene expression patterns of mono-isoniazid resistant Mycobacterium tuberculosis uncover divergent responses to isoniazid in host-mimicked condition

Isoniazid stands as a frontline antibiotic utilized in the treatment of tuberculosis (TB), predominantly impacting the mycolic acid component within the cell wall of Mycobacterium tuberculosis (Mtb). It also affects the formation of lipoarabinomannan (LAM), an essential glycolipid in the cell envelope of Mtb. Despite the effectiveness of antibiotics for TB treatment, drug tolerance development in mycobacteria frequently stems from their adaptation to the hostile environment within the host, leading to treatment failure. Herein, we investigate mycobacterial adaptation to the isoniazid exposure in the host-mimicked conditions by focusing on the stress response genes (virS, icl1, whiB3, tgs1) and LAM-related genes (lprG, p55, lmeA, mptA, embC). Mtb H37Rv and mono-isoniazid resistant (INH-R) strains were cultivated in the host-mimicked multi-stress condition (MS) with or without isoniazid and the relative expressions of these gene candidates were measured using real-time PCR. In the INH-R strain, treatment with isoniazid in multi-stress conditions caused significant upregulation of tgs1, and LAM precursor-lipomannan (LM) synthesis and its transport genes (lprG, p55, lmeA, embC). In the case of H37Rv, all LAM-related genes and tgs1 were downregulated whereas other stress response genes were upregulated, remarkably in icl1 and whiB3. These findings highlight differences in gene expression patterns between drug-sensitive and resistant strains in multi-stress environments with drug pressure. Notably, stress response genes, particularly tgs1, may play a crucial role in regulating LAM production in the INH-R strain in response to isoniazid exposure. This study enhances our understanding of the mechanisms underlying drug resistance, offering valuable insights that could contribute to the development of new strategies for treating and eliminating TB.

Macrophage-derived extracellular vesicles from Ascaris lumbricoides antigen exposure enhance Mycobacterium tuberculosis growth control, reduce IL-1β, and contain miR-342-5p, miR-516b-5p, and miR-570-3p that regulate PI3K/AKT and MAPK signaling pathways.

Helminth coinfection with tuberculosis (TB) can alter the phenotype and function of macrophages, which are the major host cells responsible for controlling Mycobacterium tuberculosis (Mtb). However, it is not known whether helminth infection stimulates the release of host-derived extracellular vesicles (EVs) to induce or maintain their regulatory network that suppresses TB immunity. We previously showed that pre-exposure of human monocyte-derived macrophages (hMDMs) with Ascaris lumbricoides protein antigens (ASC) results in reduced Mtb infection-driven proinflammation and gained bacterial control. This effect was entirely dependent on the presence of soluble components in the conditioned medium from helminth antigen-pre-exposed macrophages. Our objective was to investigate the role of EVs released from helminth antigen-exposed hMDMs on Mtb-induced proinflammation and its effect on Mtb growth in hMDMs. Conditioned medium from 48-h pre-exposure with ASC or Schistosoma mansoni antigen (SM) was used to isolate EVs by ultracentrifugation. EVs were characterized by immunoblotting, flow cytometry, nanoparticle tracking assay, transmission electron microscopy, and a total of 377 microRNA (miRNA) from EVs screened by TaqMan array. Luciferase-expressing Mtb H37Rv was used to evaluate the impact of isolated EVs on Mtb growth control in hMDMs. EV characterization confirmed double-membraned EVs, with a mean size of 140 nm, expressing the classical exosome markers CD63, CD81, CD9, and flotillin-1. Specifically, EVs from the ASC conditioned medium increased the bacterial control in treatment-naïve hMDMs and attenuated Mtb-induced IL-1β at 5 days post-infection. Four miRNAs showed unique upregulation in response to ASC exposure in five donors. Pathway enrichment analysis showed that the MAPK and PI3K-AKT signaling pathways were regulated. Among the mRNA targets, relevant for regulating inflammatory responses and cellular stress pathways, CREB1 and MAPK13 were identified. In contrast, SM exposure showed significant regulation of the TGF-β signaling pathway with SMAD4 as a common target. Overall, our findings suggest that miRNAs in EVs released from helminth-exposed macrophages regulate important signaling pathways that influence macrophage control of Mtb and reduce inflammation. Understanding these interactions between helminth-induced EVs, miRNAs, and macrophage responses may inform novel therapeutic strategies for TB management.

Sirtuin2 blockade inhibits replication of Human Immunodeficiency Virus-1 and Mycobacterium tuberculosis in macrophages and humanized mice.

Coinfections with Mycobacterium tuberculosis (Mtb) and HIV-1 present a critical health challenge and require treatment for survival. We found that human M1 macrophages inhibit Mtb growth, while M2 macrophages, characterized by elevated Sirt2 expression, permit Mtb growth. Further, we found that HIV-1 augmented Sirt2 gene expression in MФs. Therefore, we explored the therapeutic potential of sirtuin-modulating drugs in MФs. Sirtinol, a Sirt2 inhibitor, significantly reduced HIV-1 growth in M0, M1, and M2-MФs by >1 log10 over 7 days. Conversely, individual doses of resveratrol and SRT1460, which activate Sirt1, did not affect HIV-1. However, their combination showed a strong synergistic inhibition of HIV-1. The combination of sirtinol with resveratrol was neither synergistic nor antagonistic. Sirtinol upregulated iNOS and ATG5 mRNA in HIV-1 infected MФs in a phenotype-dependent manner. In a humanized mouse model (Hu-NSG-SGM3) co-infected with Mtb H37Rv and the HIV-1 BAL strain, treatment with sirtinol alone, or in combination with combination antiretroviral therapy (cART), showed promising results; Sirtinol alone reduced Mtb growth, while its combination with cART effectively inhibited HIV-1 replication in the organs. We propose that Sirt2 blockade and Sirt1-activation represent a novel dual therapeutic strategy for treating HIV-1 and Mtb coinfections.

Troglitazone reduces intracellular Mycobacterium tuberculosis survival via macrophage autophagy through LKB1-AMPKα signaling.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb), results in significant morbidity and mortality worldwide. Host-directed therapy (HDT), including conventional drugs, is a promising anti-TB strategy that shows synergistic antibacterial effects when combined with anti-TB drugs. Here, the mycobactericidal effect of three anti-diabetic drugs was examined. Of these, only Troglitazone (Trog) enhanced the antimycobacterial effect in vitro and in vivo. This was due to Trog-mediated autophagy activation. Moreover, a knock-down experiment revealed that Trog activated autophagy and exhibited antimycobacterial activity through the LKB1-AMPK signaling pathway. Molecular docking and co-immunoprecipitation experiments demonstrated that Trog promoted LKB1 phosphorylation and activation by targeting STRADA. Finally, we found that Trog inhibited the intracellular survival of clinical isoniazid (INH)-resistant Mtb, and the combination of Trog and INH showed additive antibacterial effects against Mtb H37Rv. Taken together, anti-diabetic Trog may be repurposed as an HDT candidate and combined with first-line anti-TB drugs.

A new N-acylhydrazone oxadiazole derivative with activity against mycobacteria.

Aim: To evaluate the anti-Mycobacterium tuberculosis (Mtb) potential of the hybrid oxadiazol-4-methoxynaphthalene (6n) derived from N-acylhydrazone (4k).Materials & methods: The study determined the minimal inhibitory concentration of (6n) against Mtb H37Rv and Mtb clinical isolates, potential combination of (6n) with anti-tuberculosis drugs and carried out time kill curve assay of Mtb H37Rv. Additional contribution for the analysis of (6n) was explored by in silico pharmacokinetics, and in vitro and in vivo cytotoxicity determinations.Results: The newly synthesized molecule (6n) demonstrated anti-Mtb activity, low cytotoxicity and selectivity for Mtb.Conclusion: The derivative (6n) emerges as a potential anti-TB drug candidate.

Synthesis of 6-dialkylaminopyrimidine carboxamide analogues and their anti-tubercular properties

Tuberculosis (TB) continues to be a threat to global health stability. Pyrimidine carboxamides have demonstrated potent anti-tubercular properties against clinical Mycobacterium tuberculosis, the causative agent of TB. Herein, we report a follow-up study on the synthesis of pyrimidine carboxamide molecular analogues and their anti-TB evaluation. In total, a library consisting of 37 new compounds is reported. Seven compounds (7b, 7d, 7m, 7p, 7q, 7aa, and 7ah) demonstrated excellent in vitro activities with MIC90 values below 1.00 µM. Apart from compound 7ah, compounds with improved aqueous solubility properties had lower anti-TB potency. Preliminary mode of action studies using bioluminescence assays indicate that the active compounds do not affect the integrity of mycobacterial DNA or the cell wall. The active compounds were also found to be bactericidal against replicating H37Rv Mtb strain.

Screening of novel narrow-spectrum benzofuroxan derivatives for the treatment of multidrug-resistant tuberculosis through in silico, in vitro, and in vivo approaches.

Tuberculosis remains a serious global health threat, exacerbated by the rise of resistant strains. This study investigates the potential of two benzofuroxan (Bfx) derivatives, 5n and 5b, as targeted treatments for MDR-TB using in silico, in vitro, and in vivo methodologies. In vitro analyses showed that Bfx compounds have significant activity against Mtb H37Rv, with Bfx 5n standing out with a MIC90 of 0.09 ± 0.04 μM. Additionally, their efficacy against MDR and pre-XDR strains was superior compared to commercial drugs. These Bfx compounds have a narrow spectrum for mycobacteria, which helps avoid dysbiosis of the gut microbiota, and they also exhibit high selectivity and low toxicity. Synergism studies indicate that Bfx derivatives could be combined with rifampicin to enhance treatment efficacy and reduce its duration. Scanning electron microscopy revealed severe damage to the morphology of Mtb following treatment with Bfx 5n, showing significant distortions in the bacillary structures. Whole-genome sequencing of the 5n-resistant isolate suggests resistance mechanisms mediated by the Rv1855c gene, supported by in silico studies. In vivo studies showed that the 5n compound reduced the pulmonary load by 3.0 log10 CFU/mL, demonstrating superiority over rifampicin, which achieved a reduction of 1.23 log10 CFU/mL. In conclusion, Bfx derivatives, especially 5n, effectively address resistant infections caused by Mtb, suggesting they could be a solid foundation for future therapeutic developments against MDR-TB.

Benzyl/phenyl‐1,2,3‐Triazole Tethered 3‐Acetyl Coumarins as Potential Drug‐Resistant Antitubercular Agents: Synthesis, Biology, and in Silico Investigations as Mtb DNA Gyrase Inhibitors

AbstractOwing to the emergence of multi‐drug resistant tuberculosis, there is a need for the exploration of new antitubercular agents. In this context, new coumarin‐based 1,2,3‐triazole hybrids were developed and evaluated for their antimicrobial activity against ESKAPE pathogens and the Mtb H37Rv strain. Among them, compounds 9 c and 12 showed MICs of 1 and 2 μg/mL, respectively, against the Mtb strain. The lead compounds exhibited a good selectivity index against Vero cells and were equally effective against ETB‐resistant and RIF‐resistant Mtb strains. Time‐kill kinetic studies revealed the bacteriostatic properties of the lead compounds, while combination studies using FDA‐approved antibiotics showed no drug interactions. Based on the structural similarity, it was envisaged that they might inhibit the DNA gyrase, which was further proved by the DNA supercoiling inhibition assay. Additionally, in silico docking studies, binding energy calculations, and ADME/T studies for the synthesized conjugates showed favourable pharmacokinetic and physicochemical characteristics. Hence, these molecules could further pave the way for discovering new potent antitubercular agents to combat AMR.

Microwave-Assisted Synthesis and Characterization of Novel 1,3,4-Oxadiazole Derivatives and Evaluation of In Vitro Antimycobacterial Activity.

Objective The study's goal was to come up with and make a new group of 1,3,4-oxadiazole derivatives (3a-3e) and test how well they could kill Mycobacterium tuberculosis (Mtb) H37Rv strain. Additionally, molecular docking and pharmacokinetic properties were analyzed using computational software to identify potential inhibitors, followed by in vitro antimycobacterial assays. Methods A group of 1,3,4-oxadiazoles was prepared by reacting acyl hydrazides with alanine, an N-protected α-amino acid, and a small amount of POCl3. This was carried out under microwave treatment. The structural characterization of the newly synthesized compounds was performed using infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry. The in vitro antimycobacterial activity of the 1,3,4-oxadiazole derivatives (3a-3e) was assessed using the microplate Alamar Blue assay against the Mtb H37Rv strain. The synthesized compounds were subjected to molecular docking investigations in order to gain insights into their interaction mechanisms with the mycobacterial enzyme InhA (enoyl-acyl carrier protein reductase). Computational analysis of pharmacokinetic properties was performed to predict the oral bioavailability and drug-likeness of the compounds. Results All synthesized compounds inhibited the growth of Mtbat concentrations of 50 and 100 μg/mL. At a concentration of 50 μg/mL, compounds 3c and 3d exhibited the most prominent antimycobacterial action. Molecular docking results revealed that compound 3d exhibited the highest binding energy interaction with the InhA enzyme (-9.1 kcal/mol). Pharmacokinetic predictions indicated that all compounds possess favorable drug-like properties suitable for oral administration. Conclusion This study successfully synthesized a novel series of oxadiazole derivatives (3a-3e) using a microwave-assisted method with high yields. The synthesized compounds demonstrated significant antimycobacterial activity, particularly compounds 3c and 3d. Molecular docking and pharmacokinetic analyses further confirmed the potential of these compounds as promising leads for the development of anti-tubercular agents.

Design, synthesis and antimycobacterial activity of novel benzothiazinones with improved water solubility

Nitrobenzothiazinones (BTZs) represent a novel type of antitubercular agents targeting DprE1. Two clinical candidates BTZ043 and PBTZ169, as well as many other BTZs showed potent anti-TB activity, but they are all highly lipophilic and their poor aqueous solubility is still a serious issue need to be addressed. Here, we designed and synthesized a series of new BTZ derivatives, wherein a hydrophilic COOH or NH2 group is directly attached to the oxime moiety of TZY-5-84 discovered in our lab, through various linkers. Two compounds 1a and 3 were first reported to possess excellent activity against MTB H37Rv and MDR-MTB strains (MIC: <0.029–0.095 μM), low toxicity and acceptable oral PK profiles, as well as significantly improved water solubility (1200 and > 2000 μg/mL, respectively), suggesting they may serve as promising hydrophilic BTZs for further antitubercular drug discovery.

Mutational analysis of the rpoB gene of Mycobacterium Tuberculosis Strains

Tuberculosis is an endemic. The menace of tuberculosis has affected one third of human population. Pakistan ranks 6th among high burden TB countries and 4th among the countries with highest rate of MDR-TB. Mycobacterium tuberculosis, the etiological agent of tuberculosis, has an incubation time of 8 weeks. Its fast and rapid diagnosis is required for its effective control. Molecular methods have been developed for the rapid diagnosis of TB yet the importance of conventional methods cannot be denied. LJ Culturing is considered gold standard. In the present study, a total of 90 sputum samples were taken from different cities of Punjab, Pakistan. After decontamination, the samples were subjected to LJ culturing and fluorescent microscopy. GeneXpert MTB/RIF Assay of the samples was also performed. The sensitivity of GeneXpert in comparison to culturing and microscopy was calculated. PCR of 20 rifampicin resistant samples was also performed using the primers for rpoB gene and the purified amplicons were sequenced. The sequences were analysed through various bioinformatics tools. Phylogenetic tree was constructed in comparison with wild type H37Rv MTB strain and mutations leading to rifampicin resistance were determined. The results exhibited greater sensitivity of GeneXpert MTB/RIF Assay over gold standard LJ culturing as well as fluorescent microscopy. The 49 suspected samples were found to be MTB positive with GeneXpert, 23 with microscopy and 20 with LJ culturing. Out of the 49 MTB positive samples, 23 were rifampicin resistant. Mutational analysis showed mutations in the 445th codon of RRDR of rpoB gene which inhibits the activity of RNA polymerase beta subunit. GeneXpert was found to be more sensitive than both LJ Culturing and smear microscopy. Mutational analysis showed mutations most frequently at codon 531. Mutations were also reported in other codons such as 533, 526 and 516 but these mutations were less frequent.

Design, synthesis and docking studies of new molecular hybrids bearing benzimidazole and thiazolidine-2,4-dione as potential antitubercular agents

Finding novel therapeutic medications to combat tuberculosis (TB) is crucial, as evidenced by the disease's growth as a worldwide health concern in recent decades and the rise of drug-resistant forms of Mycobacterium tuberculosis (Mtb). In this article, we describe the synthesis and design of a novel class of thiazolidine-2,4-dione derivatives (5a-i) based on 1H-benzo [d]imidazoles as antitubercular drugs. Spectroscopic techniques and elemental analysis were used to characterize each of the newly synthesized molecules. The antitubercular activity of all the newly synthesized title compounds was assessed against drug-sensitive Mtb H37Rv, multidrug-resistant (MDR-TB), and extensively drug-resistant (XDR-TB) tuberculosis. Compounds 5e and 5 h had the most antitubercular activity among all the newly synthesized hybrids against drug-sensitive, MDR-TB, and XDR-TB strains, with MIC values ranging from 0.21 to 47.84 μM. When it comes to drug-sensitive, drug-resistant, and XDR Mtb strains, compound 5 h with a trifluoromethyl group is 1.71, 10.86, and 3.50 times more potent, whereas compound 5e with a nitro group is 1.12, 8.50, and 2.61 times more active. Remarkably, the compounds' in vitro cytotoxicity test demonstrated good selectivity indices, highlighting their safety on the normal lung fibroblast (WI-38) cell line experiment. To further understand the interactions between potent hybrids and the target enzyme, molecular docking investigations were conducted against the decaprenyl-phosphoryl-ribose 2′-epimerase (DprE1) enzyme. The target protein exhibited preferentially positive interactions with the potent compounds 5e, 5f, 5 h, and 5i. Relationships between structure-activity as well as drug-likeness were used to connect the freshly synthesized compounds' physical and biological properties. When considered collectively, these results suggest that compounds 5e and 5 h might be promising candidates for the development of drug-sensitive and drug-resistant TB therapies in the future.