Synthesis, molecular docking study and biological evaluation of new pyrrole scaffolds as potential antitubercular agents for dual targeting of enoyl ACP reductase and dihydrofolate reductase.

In this study, new series of N'-(2-(substitutedphenoxy)acetyl)-4-(1H-pyrrol-1-yl)benzohydrazides (3a-j) 4-(2,5-dimethyl-1H-pyrrol-1-yl)-N'-(2-(substitutedphenoxy)acetyl)benzohydrazides (5a-j) were synthesized, characterized and assessed as inhibitors of enoyl ACP reductase and DHFR. Most of the compounds exhibited dual inhibition against the enzymes enoyl ACP reductase and DHFR. Several synthesized substances also demonstrated significant antibacterial and antitubercular properties. A molecular docking analysis was conducted in order to determine the potential mechanism of action of the synthesized compounds. The results indicated that there were binding interactions seen with the active sites of dihydrofolate reductase and enoyl ACP reductase. Additionally, important structural details were identified that play a critical role in sustaining the dual inhibitory activity. These findings were useful for the development of future dual inhibitors. Therefore, this study provided strong evidence that several synthesized molecules could exert their antitubercular properties at the cellular level through multi-target inhibition. By shedding light on the mechanisms through which these compounds exert their inhibitory effects, this research opens up promising avenues for the future development of dual inhibitors with enhanced antibacterial and antitubercular properties. The study's findings underscore the importance of multi-target approaches in drug design, providing a strong foundation for the design and optimization of novel compounds that can effectively target bacterial infections at the cellular level.

The Structural and Molecular Mechanisms of Mycobacterium tuberculosis Translational Elongation Factor Proteins.

Targeting translation factor proteins holds promise for developing innovative anti-tuberculosis drugs. During protein translation, many factors cause ribosomes to stall at messenger RNA (mRNA). To maintain protein homeostasis, bacteria have evolved various ribosome rescue mechanisms, including the predominant trans-translation process, to release stalled ribosomes and remove aberrant mRNAs. The rescue systems require the participation of translation elongation factor proteins (EFs) and are essential for bacterial physiology and reproduction. However, they disappear during eukaryotic evolution, which makes the essential proteins and translation elongation factors promising antimicrobial drug targets. Here, we review the structural and molecular mechanisms of the translation elongation factors EF-Tu, EF-Ts, and EF-G, which play essential roles in the normal translation and ribosome rescue mechanisms of Mycobacterium tuberculosis (Mtb). We also briefly describe the structure-based, computer-assisted study of anti-tuberculosis drugs.

Structure-Function Analysis of the Essential Mycobacterium tuberculosis P450 Drug Target, CYP121A1.

Cytochrome P450 CYP121A1 is a well-known drug target against Mycobacterium tuberculosis, the human pathogen that causes the deadly disease tuberculosis (TB). CYP121A1 is a unique P450 enzyme because it uses classical and non-classical P450 catalytic processes and has distinct structural features among P450s. However, a detailed investigation of CYP121A1 protein structures in terms of active site cavity dynamics and key amino acids interacting with bound ligands has yet to be undertaken. To address this research knowledge gap, 53 CYP121A1 crystal structures were investigated in this study. Critical amino acids required for CYP121A1's overall activity were identified and highlighted this enzyme's rigid architecture and substrate selectivity. The CYP121A1-fluconazole crystal structure revealed a novel azole drug-P450 binding mode in which azole heme coordination was facilitated by a water molecule. Fragment-based inhibitor approaches revealed that CYP121A1 can be inhibited by molecules that block the substrate channel or by directly interacting with the P450 heme. This study serves as a reference for the precise understanding of CYP121A1 interactions with different ligands and the structure-function analysis of P450 enzymes in general. Our findings provide critical information for the synthesis of more specific CYP121A1 inhibitors and their development as novel anti-TB drugs.

Mtb-Selective 5-Aminomethyl Oxazolidinone Prodrugs: Robust Potency and Potential Liabilities.

Linezolid is a drug with proven human antitubercular activity whose use is limited to highly drug-resistant patients because of its toxicity. This toxicity is related to its mechanism of action─linezolid inhibits protein synthesis in both bacteria and eukaryotic mitochondria. A highly selective and potent series of oxazolidinones, bearing a 5-aminomethyl moiety (in place of the typical 5-acetamidomethyl moiety of linezolid), was identified. Linezolid-resistant mutants were cross-resistant to these molecules but not vice versa. Resistance to the 5-aminomethyl molecules mapped to an N-acetyl transferase (Rv0133) and these mutants remained fully linezolid susceptible. Purified Rv0133 was shown to catalyze the transformation of the 5-aminomethyl oxazolidinones to their corresponding N-acetylated metabolites, and this transformation was also observed in live cells of Mycobacterium tuberculosis. Mammalian mitochondria, which lack an appropriate N-acetyltransferase to activate these prodrugs, were not susceptible to inhibition with the 5-aminomethyl analogues. Several compounds that were more potent than linezolid were taken into C3HeB/FeJ mice and were shown to be highly efficacious, and one of these (9) was additionally taken into marmosets and found to be highly active. Penetration of these 5-aminomethyl oxazolidinone prodrugs into caseum was excellent. Unfortunately, these compounds were rapidly converted into the corresponding 5-alcohols by mammalian metabolism which retained antimycobacterial activity but resulted in substantial mitotoxicity.

Durlobactam, a Diazabicyclooctane ß-Lactamase Inhibitor, Inhibits BlaC and Peptidoglycan Transpeptidases of Mycobacterium tuberculosis.

Peptidoglycan synthesis is an underutilized drug target in Mycobacterium tuberculosis (Mtb). Diazabicyclooctanes (DBOs) are a class of broad-spectrum ß-lactamase inhibitors that also inhibit certain peptidoglycan transpeptidases that are important in mycobacterial cell wall synthesis. We evaluated the DBO durlobactam as an inhibitor of BlaC, the Mtb ß-lactamase, and multiple Mtb peptidoglycan transpeptidases (PonA1, LdtMt1, LdtMt2, LdtMt3, and LdtMt5). Timed electrospray ionization mass spectrometry (ESI-MS) captured acyl-enzyme complexes with BlaC and all transpeptidases except LdtMt5. Inhibition kinetics demonstrated durlobactam was a potent and efficient DBO inhibitor of BlaC (KI app 9.2 ± 0.9 µM, k2/K 5600 ± 560 M-1 s-1) and similar to clavulanate (KI app 3.3 ± 0.6 µM, k2/K 8400 ± 840 M-1 s-1); however, durlobactam had a lower turnover number (tn = kcat/kinact) than clavulanate (1 and 8, respectively). KI app values with durlobactam and clavulanate were similar for peptidoglycan transpeptidases, but ESI-MS captured durlobactam complexes at more time points. Molecular docking and simulation demonstrated several productive interactions of durlobactam in the active sites of BlaC, PonA1, and LdtMt2. Antibiotic susceptibility testing was conducted on 11 Mtb isolates with amoxicillin, ceftriaxone, meropenem, imipenem, clavulanate, and durlobactam. Durlobactam had a minimum inhibitory concentration (MIC) range of 0.5-16 µg/mL, similar to the ranges for meropenem (1-32 µg/mL) and imipenem (0.5-64 µg/mL). In ß-lactam + durlobactam combinations (1:1 mass/volume), MICs were lowered 4- to 64-fold for all isolates except one with meropenem-durlobactam. This work supports further exploration of novel ß-lactamase inhibitors that target BlaC and Mtb peptidoglycan transpeptidases.

Inhibitors of the Thioesterase Activity of Mycobacterium tuberculosis Pks13 Discovered Using DNA-Encoded Chemical Library Screening.

DNA-encoded chemical library (DEL) technology provides a time- and cost-efficient method to simultaneously screen billions of compounds for their affinity to a protein target of interest. Here we report its use to identify a novel chemical series of inhibitors of the thioesterase activity of polyketide synthase 13 (Pks13) from Mycobacterium tuberculosis (Mtb). We present three chemically distinct series of inhibitors along with their enzymatic and Mtb whole cell potency, the measure of on-target activity in cells, and the crystal structures of inhibitor-enzyme complexes illuminating their interactions with the active site of the enzyme. One of these inhibitors showed a favorable pharmacokinetic profile and demonstrated efficacy in an acute mouse model of tuberculosis (TB) infection. These findings and assay developments will aid in the advancement of TB drug discovery.

A robust computational quest: Discovering potential hits to improve the treatment of pyrazinamide-resistant Mycobacterium tuberculosis.

The rise of pyrazinamide (PZA)-resistant strains of Mycobacterium tuberculosis (MTB) poses a major challenge to conventional tuberculosis (TB) treatments. PZA, a cornerstone of TB therapy, must be activated by the mycobacterial enzyme pyrazinamidase (PZase) to convert its active form, pyrazinoic acid, which targets the ribosomal protein S1. Resistance, often associated with mutations in the RpsA protein, complicates treatment and highlights a critical gap in the understanding of structural dynamics and mechanisms of resistance, particularly in the context of the G97D mutation. This study utilizes a novel integration of computational techniques, including multiscale biomolecular and molecular dynamics simulations, physicochemical and medicinal chemistry predictions, quantum computations and virtual screening from the ZINC and Chembridge databases, to elucidate the resistance mechanism and identify lead compounds that have the potential to improve treatment outcomes for PZA-resistant MTB, namely ZINC15913786, ZINC20735155, Chem10269711, Chem10279789 and Chem10295790. These computational methods offer a cost-effective, rapid alternative to traditional drug trials by bypassing the need for organic subjects while providing highly accurate insight into the binding sites and efficacy of new drug candidates. The need for rapid and appropriate drug development emphasizes the need for robust computational analysis to justify further validation through in vitro and in vivo experiments.

Third Generation Solid Dispersion-Based Formulation of Novel Anti-Tubercular Agent Exhibited Improvement in Solubility, Dissolution and Biological Activity.

The long treatment period and development of drug resistance in tuberculosis (TB) necessitates the discovery of new anti-tubercular agents. The drug discovery program of the institute leads to the development of an anti-tubercular lead (IIIM-019), which is an analogue of nitrodihydroimidazooxazole and exhibited promising anti-tubercular action. However, IIIM-019 displays poor aqueous solubility (1.2 µg/mL), which demands suitable dosage form for its efficient oral administration. In the present study, third generation solid dispersion-based formulation was developed to increase the solubility and dissolution of IIIM-019. The solubility profile of IIIM-019 using various polymeric carriers was determined and subsequently, PVP K-30 and P-407 were selected for preparation of binary and ternary solid dispersion. The third-generation ternary solid dispersion comprising PVP K-30 and P-407 revealed a remarkable enhancement in the aqueous solubility of IIIM-019. Physicochemical characterization of the developed formulations was done by employing FTIR spectroscopy, scanning electron microscopy, X-ray diffraction analysis, differential scanning calorimetry, and dynamic light scattering analysis. The dissolution study indicated an impressive release profile with the optimized formulation. The optimized formulation was further examined for cytotoxicity, cellular uptake, and hemolytic activity. The results indicated that the formulation had no apparent cytotoxicity on Caco-2 cells and was non-hemolytic in nature. Moreover, the optimized formulation showed significantly improved anti-tubercular activity compared to the native molecule. These findings showed that the developed third generation ternary solid dispersion could be a promising option for the oral delivery of investigated anti-tubercular molecule.

Investigating the Impact of First-Line Anti-Tuberculosis Drugs Encapsulated in a Eugenol-Based Nanoemulsion on Human Serum Albumin.

BACKGROUND: Eugenol exhibits broad-spectrum antibacterial and anti-inflammatory properties. However, cytotoxicity at high concentrations limits the full utilization of eugenol-based drug complexes. Formulations of multidrug-loaded eugenol-based nanoemulsions have reduced cytotoxicity; however, it remains crucial to understand how these eugenol complexes interact with primary human carrier proteins to design and develop therapeutic alternatives. Consequently, this study primarily aims to investigate the impact on Human Serum Albumin (HSA) when it interacts with eugenol-based complexes loaded with first-line anti-tuberculosis drugs. METHODS: This study used various spectroscopic such as UV-visible spectroscopy, Fluorescence spectroscopy, Fourier-transform infrared spectroscopy and computational methods such as molecular docking and 100 ns molecular simulation to understand the impact of eugenol-based first-line anti-tuberculosis drug-loaded nanoemulsions on HSA structure. RESULTS: The binding of the HSA protein and eugenol-based complexes was studied using UV-visible spectroscopic analysis. Minor changes in the fluorophores of the protein further confirmed binding upon interaction with the complexes. The Fourier-transform infrared spectra showed no significant changes in protein structure upon interaction with eugenol-based multidrug-loaded nanoemulsions, suggesting that this complex is safe for internal administration. Unlike eugenol or first-line anti-tuberculosis alone, molecular docking revealed the strength of the binding interactions between the complexes and the protein through hydrogen bonds. The docked complexes were subjected to a 100 ns molecular dynamics simulation, which strongly supported the conclusion that the structure and stability of the protein were not compromised by the interaction. CONCLUSIONS: From the results we could comprehend that the eugenol (EUG)-drug complex showed greater stability in HSA protein structure when compared to HSA interacting with isoniazid (INH), rifampicin (RIF), pyrazinamide (PYR), or ethambutol (ETH) alone or with EUG alone. Thus, inferring the potential of EUG-based drug-loaded formulations for a safer and efficient therapeutic use.

Unravelling performance of honeycomb structures as drug delivery systems for the isoniazid drug using DFT-D3 correction dispersion and molecular dynamic simulations.

In this study, the potential of aluminum nitride (h-AlN), boron nitride (h-BN) and silicon carbide (h-SiC) nanosheets as the drug delivery systems (DDS) of isoniazid (INH) was scrutinized through density functional theory (DFT) and molecular dynamic (MD) simulations. We performed DFT periodic calculations on the geometry and electronic features of nanosheets adsorbed with INH by the DFT functional (DZP/GGA-PBE) employed in the SIESTA code. In the energetically favorable model, an oxygen atom of the C-O group of the INH molecule interacts with a Si atom of the h-SiC at 2.077 Å with an interaction energy of -1.361 eV. Charge transfer (CT) calculation by employing the Mulliken, Hirshfeld and Voronoi approaches reveals that the monolayers and drug molecules act as donors and acceptors, respectively. The density of states (DOS) calculations indicate that the HOMO-LUMO energy gap (HLG) of the h-SiC nanosheet declines significantly from 2.543 to 1.492 eV upon the adsorption of the INH molecule, which causes an electrical conductivity increase and then produces an electrical signal. The signal is linked to the existence of INH, demonstrating that h-SiC may be an appropriate sensor for INH sensing. The decrease in HLG for the interaction of INH and h-SiC is the uppermost (up to 41%) representing the uppermost sensitivity, whereas the sensitivity trend is σ(h-SiC) > σ(h-AlN) > σ(h-BN). Quantum theory of atoms in molecules (QTAIM) investigations is employed to scrutinize the nature of the INH/nanosheet interactions. The QTAIM analysis reveals that the interaction of the INH molecule and h-SiC has a partially covalent nature, while INH/h-AlN model electrostatic interaction occurs in the system and noncovalent and electrostatic interaction for the INH/h-BN model. Finally, the state-of-the-art DFT-MD simulations utilized in this study can mimic ambient conditions. The results obtained from the MD simulation show that it takes more time to bond the INH drug and h-SiC, and the INH/h-SiC system becomes stable. The results of the current research demonstrate the potential of h-SiC as a suitable sensor and drug delivery platform for INH drugs to remedy tuberculosis.

Lipids Extracted from Mycobacterial Membrane and Enveloped PLGA Nanoparticles for Encapsulating Antibacterial Drugs Elicit Synergistic Antimicrobial Response against Mycobacteria.

Tuberculosis (TB) is a chronic disease caused byMycobacterium tuberculosis (Mtb), which shows a long treatment cycle often leads to drug resistance, making treatment more difficult. Immunogens present in the pathogen's cell membrane can stimulate endogenous immune responses. Therefore, an effective lipid-based vaccine or drug delivery vehicle formulated from the pathogen's cell membrane can improve treatment outcomes. Herein, we extracted and characterized lipids fromMycobacterium smegmatis, and the extracts contained lipids belonging to numerous lipid classes and compounds typically found associated with mycobacteria. The extracted lipids were used to formulate biomimetic lipid reconstituted nanoparticles (LrNs) and LrNs-coated poly(lactic-co-glycolic acid) nanoparticles (PLGA-LrNs). Physiochemical characterization and results of morphology suggested that PLGA-LrNs exhibited enhanced stability compared with LrNs. And both of these two types of nanoparticles inhibited the growth of M. smegmatis. After loading different drugs, PLGA-LrNs containing berberine or coptisine strongly and synergistically prevented the growth of M. smegmatis. Altogether, the bacterial membrane lipids we extracted with antibacterial activity can be used as nanocarrier coating for synergistic antibacterial treatment of M. smegmatis─an alternative model of Mtb, which is expected as a novel therapeutic system for TB treatment.

Injectable isoniazid-loaded bone cement based on hydrazone bonds achieving long-term release and decent mechanical properties.

A robust and easily manufactured high-strength and long-term release hydrazone-based isoniazid acrylic (HIA) bone cement is reported. The mechanical strength of HIA bone cement is similar to that of normal polymethyl methacrylate (PMMA) bone cement, far surpassing that of traditional isoniazid-containing antibiotic-loaded bone cement (INH bone cement). Isoniazid is connected to the bone cement through bioorthogonal hydrazone chemistry, and it possesses release properties superior to those of INH bone cement, allowing for the sustained release of isoniazid for up to 12 weeks. In vivo and in vitro studies also indicate that HIA cement exhibits better biocompatibility than INH bone cement. The results of this study not only signify progress in the realm of antimicrobial bone cement for addressing bone tuberculosis but also enhance our capacity to create and comprehend high-performing antimicrobial bone cement.

Novel 4-nitroimidazole analogues: synthesis, in vitro biological evaluation, in silico studies, and molecular dynamics simulation.

A new series of 4-nitroimidazole bearing aryl piperazines 7-16, tetrazole 17 and 1,3,4-thiadiazole 18 derivatives was synthesized. All derivatives were screened for their anticancer activity against eight diverse human cancer cell lines (Capan-1, HCT-116, LN229, NCI-H460, DND-41, HL-60, K562, and Z138). Compound 17 proved the most potent compound of the series inhibiting proliferation of most of the selected human cancer cell lines with IC50 values in the low micromolar range. In addition, compound 11 exhibited IC50 values ranging 8.60-64.0 µM against a selection of cancer cell lines. These findings suggest that derivative 17 can potentially be a new lead compound for further development of novel antiproliferative agents. Additionally, 17-18 were assessed for their antibacterial and antituberculosis activity. Derivatives 17 and 18 were the most potent compounds of this series against both Staphylococcus aureus strain Wichita and a methicillin resistant strain of S. aureus (MRSA), as well as against Mycobacterium tuberculosis strain mc26230. The antiviral activity of 7-18 was also evaluated against diverse viruses, but no activity was detected. The docking study of compound 17 with putative protein targets in acute myeloid leukemia had been studied. Furthermore, the molecular dynamics simulation of 17 and 18 had been investigated.

Design, Synthesis and In vitro Antitubercular Effect of New Chalcone Derivatives Coupled with 1,2,3-Triazoles: A Computational Docking Techniques.

A very interesting foundation for this study is the creation of new methods for modifying compounds with a 1,2,3-triazole and chalcone scaffolds, as these compounds are significant in organic synthesis, particularly in the synthesis of bioactive organic compounds. To contribute to the development of an efficient method for the conversion of antimicrobial and antituberculosis heterocyclics, a novel series of cyclohepta pyridinone fused 1,2,3-triazolyl chalcones were designed and synthesized. All the newly prepared scaffolds were characterized by FT-IR, NMR (1H & 13C) and mass spectrometry. Among the tested compounds, hybrids 8b, 8d, and 8f exhibited exceptional antibacterial susceptibilities with zone of inhibition 27.84±0.04, 32.27±0.02, and 38.26±0.01 mm against the tested E. faecalis bacteria, whereas 8d had better antitubercular potency against M. tuberculosis H37Rv strain with MIC value 5.25 µg/mL, compared to Streptomycin [MIC=5.01 µg/mL]. All the synthesized compounds were initially assessed in silico against the targeted protein i. e., DprE1 that indicated compound 8d, 8f and 8h along with several other 1,2,3-triazole compounds as possible inhibitors. Based on docking results, 8d showed that the amino acids His74(A), Lys76(A), Cys332(A), Asp331(A), Val307(A), Tyr357(A), Met226(A), Gln276(A), Gly75(A), Peo58(A), Leu259(A), and Lys309(A) exhibited highly stable binding to DprE1 receptor of Mycobacterium tuberculosis (PDB: 4G3 U). Moreover, these scaffolds physicochemical characteristics, filtration molecular properties, assessment of toxicity, and bioactivity scores were assessed in relation to ADME (absorption, distribution, metabolism, and excretion).

A Comprehensive Examination of Heterocyclic Scaffold Chemistry for Antitubercular Activity.

Tuberculosis is a communicable disease which affects humans particularly the lungs and is transmitted mainly through air. Despite two decades of intensive research aimed at understanding and combating tuberculosis, persistent biological uncertainties continue to hinder progress. Nowadays, heterocyclic compounds have proven themselves in effective treatment of tuberculosis because of their wide range of biological and pharmacological activities. Antituberculosis or antimycobacterial agents encompass a broad array of compounds utilized singly or in conjunction to combat Mycobacterium infections, spanning from tuberculosis to leprosy. Here, we summarize the synthesis of various heterocyclic compounds which includes the greener synthetic route as well as use of nano compounds as catalyst along with their anti TB activities.

Fucoidan coated liposomes loaded with novel antituberculosis agent: preparation, evaluation, and cytotoxicity study.

In this article, we described a novel antituberculosis imidazotetrazine derivative designed in fucoidan-coated liposomes to reduce its cytotoxicity and investigate its mucoadhesive properties. Firstly, fucoidan extracted from Ascophyllum nodosum was used for additional stabilization of liposomal suspensions and to give it mucoadhesive properties. PEG-600 and/or Tween-80 were used to increase the shelf life of liposomal suspension. The ratio of the fucoidan: lipids 1:2 was found to be the optimum that produces stable fucoidan-coated liposomes. The particle size of the optimum formulation was 336.3 ± 5.4, the PDI was 0.33, and the zeta potential was -39.6. This size and the practical spherical shape of the particles were confirmed by atomic force microscopy. In addition, the in vitro release profiles from uncoated and fucoidan-coated liposomes revealed significant and faster release compared to free antituberculosis agent. Using the MTT assay test, the fucoidan-coated liposomes exhibited fourteen times lower cytotoxicity (IC50 7.14 ± 0.91 µg/ml) than the free drug (IC50 0.49 ± 0.06). Moreover, the mucoadhesive capabilities of these liposomal formulations were also confirmed using snail mucin, which highlighting their potential use as an effective delivery system for antituberculosis therapy, with notable improvements in dissolution rate and reduced cytotoxicity.

New polycyclic tetramate macrolactams with antimycobacterial activity produced by marine-derived Streptomyces sp. KKMA-0239.

During our screening for anti-mycobacterial agents against Mycobacterium avium complex (MAC), two new polycyclic tetramate macrolactams (PTMs), named hydroxycapsimycin (1) and brokamycin (2), were isolated along with the known PTM, ikarugamycin (3), from the culture broth of marine-derived Streptomyces sp. KKMA-0239. The relative structures of 1 and 2 were elucidated by spectroscopic data analyses, including 1D and 2D NMR. Furthermore, the absolute configuration of 1 was confirmed by a single-crystal X-ray diffraction analysis. Compounds 2 and 3 exhibited moderate antimycobacterial activities against MAC, including clinically isolated drug-resistant M. avium.

Molecular docking, molecular dynamics simulations and binding free energy studies of interactions between Mycobacterium tuberculosis Pks13, PknG and bioactive constituents of extremophilic bacteria.

Mycobacterial pathogens present a significant challenge to disease control efforts globally due to their inherent resistance to multiple antibiotics. The rise of drug-resistant strains of Mycobacterium tuberculosis has prompted an urgent need for innovative therapeutic solutions. One promising way to discover new tuberculosis drugs is by utilizing natural products from the vast biochemical space. Multidisciplinary methods can used to harness the bioactivity of these natural products. This study aimed to evaluate the antimycobacterial efficacy of functional crude extracts from bacteria isolated from gold mine tailings in South Africa. Bacterial strains were identified using 16S rRNA sequencing. The crude extracts obtained from the bacteria were tested against Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis mc2155, and Mycobacterium aurum A+. Untargeted HPLC-qTOF and molecular networking were used to identify the functional constituents present in extracts that exhibited inhibitory activity. A virtual screening workflow (VSW) was used to filter compounds that were strong binders to Mycobacterium tuberculosis Pks13 and PknG. The ligands returned from the VSW were subjected to optimization using density functional theory (DFT) at M06-2X/6-311++ (d,p) level of theory and basis set implemented in Gaussian16 Rev.C01. The optimized ligands were re-docked against Mycobacterium tuberculosis Pks13 and PknG. Molecular dynamics simulation and molecular mechanics generalized born surface area were used to evaluate the stability of the protein-ligand complexes formed by the identified hits. The hit that showed promising binding characteristics was virtually modified through multiple synthetic routes using reaction-driven enumeration. Three bacterial isolates showed significant activity against the two strains of Mycobacterium, while only two, Bacillus subtilis and Bacillus licheniformis, exhibited activity against both Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis mc2155, and Mycobacterium aurum A+. The tentatively identified compounds from the bacterial crude extracts belonged to various classes of natural compounds associated with antimicrobial activity. Two compounds, cyclo-(L-Pro-4-OH-L-Leu) and vazabitide A, showed strong binding against PknG and Pks13, with pre-MD MM-GBSA values of - 42.8 kcal/mol and - 47.6 kcal/mol, respectively. The DFT-optimized compounds exhibited the same docking scores as the ligands optimized using the OPSL-4 force field. After modifying vazabitide A, its affinity to the Pks13 binding site increased to - 85.8 kcal/mol, as revealed by the post-MD MM-GBSA analysis. This study highlights the potential of bacteria isolates from gold mine tailings as a source of new scaffolds for designing and optimizing anti-Mycobacterium agents. These agents synthesized in-silico can be further tested in-vitro to evaluate their efficacy.

Identification of Novel Antimicrobial Compounds Targeting Mycobacterium tuberculosis S-Adenosyl-L-Homocysteine Hydrolase Using Dual Hierarchical In Silico Structure-Based Drug Screening.

The emergence of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis (M. tuberculosis) has become a major medical problem. S-adenosyl-L-homocysteine hydrolase (MtSAHH) was selected as the target protein for the identification of novel anti-TB drugs. Dual hierarchical in silico Structure-Based Drug Screening was performed using a 3D compound structure library (with over 150 thousand synthetic chemicals) to identify compounds that bind to MtSAHH's active site. In vitro experiments were conducted to verify whether the nine compounds selected as new drug candidates exhibited growth-inhibitory effects against mycobacteria. Eight of the nine compounds that were predicted by dual hierarchical screening showed growth-inhibitory effects against Mycobacterium smegmatis (M. smegmatis), a model organism for M. tuberculosis. Compound 7 showed the strongest antibacterial activity, with an IC50 value of 30.2 µM. Compound 7 did not inhibit the growth of Gram-negative bacteria or exert toxic effects on human cells. Molecular dynamics simulations of 40 ns using the MtSAHH-Compound 7 complex structure suggested that Compound 7 interacts stably with the MtSAHH active site. These in silico and in vitro results suggested that Compound 7 is a promising lead compound for the development of new anti-TB drugs.

Triple combination dry powder formulation of pretomanid, moxifloxacin, and pyrazinamide for treatment of multidrug-resistant tuberculosis.

Both latent and multidrug-resistant tuberculosis (TB) have been causing significant concern worldwide. A novel drug, pretomanid (PA-824), has shown a potent bactericidal effect against both active and latent forms of Mycobacterium tuberculosis (MTb) and a synergistic effect when combined with pyrazinamide and moxifloxacin. This study aimed to develop triple combination spray dried inhalable formulations composed of antitubercular drugs, pretomanid, moxifloxacin, and pyrazinamide (1:2:8 w/w/w), alone (PaMP) and in combination with an aerosolization enhancer, L-leucine (20 % w/w, PaMPL). The formulation PaMPL consisted of hollow, spherical, dimpled particles (<5 µm) and showed good aerosolization behaviour with a fine particle fraction of 70 %. Solid-state characterization of formulations with and without L-leucine confirmed the amorphous nature of moxifloxacin and pretomanid and the crystalline nature of pyrazinamide with polymorphic transformation after the spray drying process. Further, the X-ray photoelectron spectroscopic analysis revealed the predominant surface composition of L-leucine on PaMPL dry powder particles. The dose-response cytotoxicity results showed pyrazinamide and moxifloxacin were non-toxic in both A549 and Calu-3 cell lines up to 150 µg/mL. However, the cell viability gradually decreased to 50 % when the pretomanid concentration increased to 150 µg/mL. The in vitro efficacy studies demonstrated that the triple combination formulation had more prominent antibacterial activity with a minimum inhibitory concentration (MIC) of 1 µg/mL against the MTb H37Rv strain as compared to individual drugs. In conclusion, the triple combination of pretomanid, moxifloxacin, and pyrazinamide as an inhalable dry powder formulation will potentially improve treatment efficacy with fewer systemic side effects in patients suffering from latent and multidrug-resistant TB.