Structure-based drug design, synthesis and screening of MmaA1 inhibitors as novel anti-TB agents.

Tuberculosis is one of the leading causes of death across the world. The treatment regimens for tuberculosis are well established, but still the control of the disease faces many challenges such as lengthy treatment protocols, drug resistance and toxicity. In the present work, mycolic acid methyl transferase (MmaA1), a protein involved in the maturation of mycolic acids in the biochemical pathway of the Mycobacterium, was studied for novel drug discovery. The homology model of the MmaA1 protein was built and validated by using computational techniques. The MmaA1 protein has 286 amino acid residues consisting of 10 α-helices and 7 ß-sheets. The active site of the MmaA1 protein was identified using CASTp, SiteMap and PatchDock. Virtual screening studies were performed with two small molecule ligand databases: Asinex synergy and Diverse_Elite_Gold_Platinum databases having a total of 43,446 molecules and generated 1,30,814 conformers against the predicted and validated active site of the MmaA1 protein. Binding analysis showed that the residues ASP 19, PHE 22, TRP 30, TYR 32, TRP 74 and ALA 77 of MmaA1 protein have consistent interactions with the ligands. The hit ligands were further filtered by in silico ADME properties to eliminate potentially toxic molecules. Of the top 10 molecules, 3-(2-morpholinoacetamido)-N-(1,4-dihydro-4-oxoquinazolin-6-yl) benzamide was synthesised and screened for in vitro anti-TB activity against Mtb H37Rv using MABA assay. The compound and its intermediates exhibited good in vitro anti-TB activity which can be taken up for future lead optimisation studies. Structure based virtual screening study was performed using a validated homology model against small molecules from two virtual compound libraries. Synthesised the lead compound 3-(2-morpholinoacetamido)-N-(1,4-dihydro-4-oxoquinazolin-6-yl)benzamide obtained from virtual screening. In vitro activity against Mtb H37Rv has given a promising result.

Design, synthesis, antimycobacterial activity and molecular docking studies of novel 3- (N-substituted glycinamido) benzoic acid analogues as anti tubercular agents.

We have recently identified mycolic acid methyl transferase (MmaA1) enzyme inhibitors as potential antitubercular agents using in silico modelling techniques. In continuation of that study, we synthesised a series of novel 3-(N-substituted glycinamido) benzoic acid derivatives with an aim to optimise the lead molecule. The newly synthesised compounds were evaluated for their in vitro antimycobacterial activity against M. tuberculosis H37Rv. Among these, 5 compounds A3, A4, A5, A6 and A10 exhibited most potent activity with an MIC value of 1.6 µg/ml. Further molecular docking studies were carried out to investigate the binding mode of the ligands with MmaA1 protein. The docking studies revealed that the ligands made strong interactions with the catalytic site residues TRP30, TYR 32, GLY 71, TRP 74, GLY 76, ALA 77 and GLU 136 of MmaA1 protein. Druglikeness and leadlikeness properties of the compounds were also studied using computational tools. The results of in silico and in vitro studies indicate that these novel compounds are propitious leads for tuberculosis therapy.

Identification of novel selective Mtb-DHFR inhibitors as antitubercular agents through structure-based computational techniques.

Inhibition of dihydrofolate reductase from Mycobacterium tuberculosis-dihydrofolate reductase (Mtb-DHFR) has emerged as a promising approach for the treatment of tuberculosis. To identify novel Mtb-DHFR inhibitors, structure-based virtual screening (SBVS) of the Molecular Diversity Preservation International (MolMall) database was performed using Glide against the Mtb-DHFR and h-DHFR enzymes. On the basis of SBVS, receptor fit, drug-like filters, and ADMET (absorption, distribution, metabolism, excretion, and toxicity) analysis, 16 hits were selected and tested for their antitubercular activity against the H37 RV strain of M. tuberculosis. Five compounds showed promising activity with compounds 11436 and 15275 as the most potent hits with IC50 values of 0.65 and 12.51 µM, respectively, against the H37 RV strain of M. tuberculosis. The two compounds were further tested in the Mtb-DHFR and h-DHFR enzymatic assay for selectivity and were found to be three- to eight-fold selective towards Mtb-DHFR over h-DHFR with minimum inhibitory concentration values of 5.50, 73.89 µM and 42.00, 263.00 µM, respectively. In silico simulation studies also supported the stability of the protein-ligand complex formation. The present study demonstrates the successful utilization of in silico SBVS tools for the identification of novel and potential Mtb-DHFR inhibitors and compound 11436 ((2,4-dihydroxyphenyl)(3,4,5-trihydroxyphenyl)methanone) as a potential lead for the development of novel Mtb-DHFR inhibitors.

Synthesis and screening of clerodane diterpene analogues from 16 hydroxycleroda 3,13(14)-Z-diene 15,16-olide for potential anti-mycobacterial activity.

In recent years, clerodane diterpenes, a class of bioactive compounds, have come into the spotlight due to their amazing bioactivities. Three novel clerodane diterpene analogues were obtained by synthesising 16-hydroxycleroda-3,13(14)-Z-diene-15,16-olide (Lactone) with primary amines. Anti-tubercular activity was determined using Microplate Alamar Blue Assay. Among all the synthesised compounds from methanolic extract of seeds, results clearly showed that compounds 3 and 5 have significant anti TB activity with an MIC of 1.56 µg/ml against the Mycobacterium tuberculosis MTB H37Rv bacilli strain than the gold standard drugs pyrazinamide (3.13 µg/ml), ciprofloxacin (3.13 µg/ml), streptomycin (6.25 µg/ml) and rifampicin (6.25 µg/ml). Compound 5 exhibited significant antibacterial activity with zone of inhibition of 10.8 mm with Gram + ve and 7.95 mm with Gram -ve bacteria at a conc of 50 µg/ml respectively. In the current investigation, three novel heterocycles (compounds 3-5) of the diterpenoid were prepared, in high yield, using one-pot, efficient approach.

Synthesis, molecular docking, and biological evaluation of novel 1,2,4-triazole-isatin derivatives as potential Mycobacterium tuberculosis shikimate kinase inhibitors.

The issue of emerging resistance to antitubercular drugs has created a formidable barrier in the effective prevention and cure of tuberculosis globally. In an effort to search for new antimycobacterial agents, possibly comprising new pharmacophore, novel triazole-isatin derivatives were designed as Mycobacterium tuberculosis shikimate kinase inhibitors and synthesized by microwave-assisted method. The synthesized molecules were evaluated for their antimycobacterial activity by MABA assay against M. tuberculosis H37Rv. The molecule 5h demonstrated MIC of 0.8 µg/ml and good safety profile with higher selectivity index with HEK293 cell line. The antimycobacterial activity was further substantiated with molecular docking studies. The triazole-isatin derivatives showed significant binding interactions with amino acid residues in the active site of the enzyme. These studies revealed that molecule 5h could act as a potential lead molecule for further studies to find new target-directed molecules.

Pharmacophore modeling of pretomanid (PA-824) derivatives for antitubercular potency against replicating and non-replicating Mycobacterium tuberculosis.

Pretomanid (PA-824) is the recently (2019) approved drug for the treatment of extensively drug-resistant (XDR) TB and the multidrug-resistant (MDR) TB by US FDA. The experimental data of antitubercular activity of 543 pretomanid derivatives (total 6 datasets) against replicating (active) and non-replicating (dormant) forms of Mycobacterium tuberculosis (strain H37Rv) are available in the literature. Such vast experimental data of pretomanid derivatives against both of these endpoints, and recent approval of pretomanid molecule as a drug encouraged us to utilize this existing experimental information for the development of the 3D-pharmacophore models. The developed model (Hypo-1, MABA) showed the three physicochemical features namely, the oxygen atom of nitro group (HBA_1), fused pyran ring of imidazopyran heterocycle (HYAl_2) and the 4-fluorophenyl moiety (HYAr_3) are crucial for the antitubercular activity against replicating M. tb. Subsequently, the pharmacophore model (Hypo-1, LORA) developed against the non-replicating form of M. tb also showed the contribution of three physicochemical features namely, the 4-tri-fluoromethyl group (HYAl_2) and both the phenyl groups (HYAr_3, HYAr_4) of biaryl moiety in increasing the antitubercular activity. Both the pharmacophoric classifier models showed the classification accuracies of 82.98 and 74.42% for the training set compounds, and 63.91 and 61.60% for the test set compounds respectively, for labelling the compounds into higher and lower active classes. Both the models were also found to be retaining the higher active compounds in top 1.00% of the total number of compounds (decoys and actives), after performing the decoy set screening. Communicated by Ramaswamy H. Sarma.

Synthesis of carbohydrazides and carboxamides as anti-tubercular agents.

A novel series of furan/thiophene carbohydrazides and carboxamides were synthesized and evaluated for anti-TB and cytotoxic activities. All the synthesized compounds were characterized using 1H and 13C NMR and mass spectral techniques. Among the 23 compounds tested for anti-tubercular activity, seven compounds (3e, 3g, 3h, 9b, 9c, 9e and 9h) showed minimum inhibitory concentration value less than 1 µg/mL against Mycobacterium tuberculosis H37Rv and they were found to be non-toxic. Molecular docking and dynamics simulation studies of these compounds with an enzyme enoyl ACP reductase revealed the probable mechanism of action, which is similar to isoniazid. Further, all these tested compounds exhibited good absorption, distribution, metabolism and excretion and drug-likeness in-silico and thus may be considered as potential leads for further drug development.

In vitro antimycobacterial and cytotoxic data on medicinal plants used to treat tuberculosis.

This article contains data on in vitro antimycobacterial activity and cytotoxicity of hydroethanolic crude extracts from five selected medicinal plant species traditionally used to treat tuberculosis in Ghanaian ethnomedicine, see "Medicinal plants used to treat TB in Ghana" [1]. The interpretation and discussion of these data and further extensive insights into drug discovery against tuberculosis from natural products of plant biodiversity can be found in "Antimycobacterial and cytotoxic activity of selected medicinal plant extracts" [2].

In vitro antimycobacterial activity and toxicity of eight medicinal plants against pathogenic and nonpathogenic mycobacterial strains.

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a serious public health challenge towards which new hits are urgently needed. Medicinal plants remains a major source of new ligands against global infectious illnesses. In our laboratories, we are currently investigating locally used ethnobotanicals for novel compounds against zoonotic tuberculosis. The microplate alamar blue assay (MABA) was used to study the anti-TB activity while the CellTiter 96® AQueous Assay, which is composed of solutions of a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine methosulfate) PMS, was used for cytotoxic studies. Correlation coefficients (R2) were used to compare the relationship between antimycobacterial activity of the eight crude extracts against nonpathogenic strains and the pathogenic Mycobacterium bovis. Minimum inhibitory concentration (MICs) values indicated that all the eight tested medicinal plant species had activity against all the three tested mycobacterial strains. Minimum inhibitory concentration value as low as 19.5µg/mL was observed against non-pathogenic strains M. bovis. Activity of the crude extracts against M. aurum was the best predictor of natural product activity against the pathogenic Mycobacterium bovis strain, with a correlation coefficient value (R2) of 0.1371. Results obtained from the current study validate, in part, the traditional utilization of the tested medicinal plants against tuberculosis. The unripe fruits from Solanum torvum are a potential source of safe and efficacious anti-TB crude drugs as well as a source for natural compounds that act as new anti-infection agents, and thus deserve further investigation towards development of a new class of molecules with activity against sensitive and drug resistant strains of M. bovis.

Synthesis, antibacterial and antitubercular activities of benzimidazole bearing substituted 2-pyridone motifs.

A series of benzimidazole bearing 2-pyridones 5a-r were synthesized and evaluated for their in vitro antibacterial and antitubercular activity. Further, all compounds were examined for their cytotoxic study on VERO cell line and characterized by well-known spectral techniques. It was observed that the compounds 5h, 5i, 5k, 5q and 5r were found to possess significant broad spectrum antibacterial activity (12.5-100 µg/mL of MIC), while compounds 5g-5i, 5k and 5l proved to be the most potent antitubercular activity in range of 2.76-20.4 µM of MIC at low level of cytotoxicity, indicating good selectivity. From SAR studies, lipophilic profile of compounds was remarkably vital for antibacterial activity, while MIC values of antitubercular activity could not be directly correlated with lipophilicity.

Design, synthesis and 3D-QSAR studies of new diphenylamine containing 1,2,4-triazoles as potential antitubercular agents.

A new series of new diphenylamine containing 1,2,4-triazoles were synthesized from 4-arylideneamino-5-[2-(2,6-dichlorophenylamino) benzyl]-2H-1,2,4-triazole-3(4H)-thiones 3a-f. The synthesized compounds were screened for in-vitro antimycobacterial and antibacterial activities. The synthesized compounds 4a, 4e and 4d have shown potential activity against Mycobacterium tuberculosis H37Rv strain with MIC of 0.2, 1.6 and 3.125 µM respectively. To investigate the SAR of diphenylamine containing 1,2,4-triazole derivatives in more details, CoMFA (q(2)-0.432, r(2)-0.902) and CoMSIA (q(2)-0.511, r(2)-0.953) models on M. tuberculosis H37Rv were established. The generated 3D-QSAR models are externally validated and have shown significant statistical results, and these models can be used for further rational design of novel diphenylamine containing 1,2,4-triazoles as potent antitubercular agents.