Targeting Mycobacterium Tuberculosis Enoyl-Acyl Carrier Protein Reductase Using Computational Tools for Identification of Potential Inhibitor and their Biological Activity.

Enoyl-acyl carrier protein reductase (InhA) of type II fatty acid synthase system is involved in the synthesis of mycolic acids which is a major component of the bacterial cell wall. Since they are the key enzymes playing a very significant role in the FASII pathway of the bacterium. In this study, we have developed a workflow for identification of InhA inhibitors by utilizing in silico virtual screening approaches based on various machine learning algorithms followed by pharmacophore based virtual screening. The hits screened from the models were further subjected to molecular docking. Further, based on the XP docking score best twenty compounds were subjected to molecular dynamics study. Finally, nine compounds were shortlisted on the basis of best stable ligand RMSD, c-alpha RMSD, and RMSF plot for biological evaluation studies. Experimental validation of the shortlisted compounds identified one compound JFD01724 having potent inhibitory activity and was able to inhibit the growth of mycobacterium tuberculosis. Further medicinal chemistry efforts may help to improve the inhibitory potency of the identified compound.

Synthesis of 2-methoxy-3-(thiophen-2-ylmethyl)quinoline containing amino carbinols as antitubercular agents.

We have designed and synthesized 2-methoxy-3-(thiophen-2-ylmethyl)quinoline containing amino carbinols as possible anti-tubercular agents to combat the disease. These molecules were synthesized by tethering amino ether linkage with hydroxyl group to diarylquinoline skeleton; hydroxyl and amine chains were engrafted on diaryl ring. They were evaluated against strain (H37Ra) of Mycobacterium tuberculosis and most of compounds showed in vitro antitubercular activity. Two compounds having diaryl quinoline hydroxyl amino ether scaffold and three compounds having diaryl amino alkyl carbinol core showed activities at 6.25 µg/mL. This study explores diaryl carbinol prototype as inhibitor against Mycobacterium tuberculosis.

hsa-let-7b-5p facilitates Mycobacterium tuberculosis survival in THP-1 human macrophages by Fas downregulation.

Tuberculosis continues to be one of the deadliest infectious diseases worldwide. MicroRNAs (miRNAs) are small non-coding entities that play critical role as post-transcriptional regulators and are transcriptionally deregulated upon mycobacterial infection. In this study, we found significant upregulation of hsa-let-7b-5p in Mycobacterium tuberculosis (MTB) infected THP-1 human macrophages. Concomitantly, we detected the reduced level of Fas protein, one of the targets of hsa-let-7b-5p, in MTB-infected THP-1 macrophages. Using luciferase assay, a direct interaction between hsa-let-7b-5p and the Fas 3΄-untranslated region (3΄-UTR) was established. Inhibition of hsa-let-7b-5p augmented the apoptosis of THP-1 cells enabling enhanced clearance of MTB. Our findings suggest that hsa-let-7b-5p helps intracellular survival of MTB in THP-1 cells by downregulating Fas protein level. This highlights hsa-let-7b-5p as a potential therapeutic target for tuberculosis treatment.

Characterization of Mycobacterium smegmatis sigF mutant and its regulon: overexpression of SigF antagonist (MSMEG_1803) in M. smegmatis mimics sigF mutant phenotype, loss of pigmentation, and sensitivity to oxidative stress.

In Mycobacterium smegmatis, sigF is widely expressed during different growth stages and plays role in adaptation to stationary phase and oxidative stress. Using a sigF deletion mutant of M. smegmatis mc(2) 155, we demonstrate that SigF is not essential for growth of bacterium. Deletion of sigF results in loss of carotenoid pigmentation which rendered increased susceptibility to H2 O2 induced oxidative stress in M. smegmatis. SigF modulates the cell surface architecture and lipid biosynthesis extending the repertoire of SigF function in this species. M. smegmatis SigF regulon included variety of genes expressed during exponential and stationary phases of growth and those responsible for oxidative stress, lipid biosynthesis, energy, and central intermediary metabolism. Furthermore, we report the identification of a SigF antagonist, an anti-sigma factor (RsbW), which upon overexpression in M. smegmatis wild type strain produced a phenotype similar to M. smegmatis mc(2) 155 ΔsigF strain. The SigF-anti-SigF interaction is duly validated using bacterial two-hybrid and pull down assays. In addition, anti-sigma factor antagonists, RsfA and RsfB were identified and their interactions with anti-sigma factor were experimentally validated. Identification of these proteins will help decode regulatory circuit of this alternate sigma factor.

Double recombinant Mycobacterium bovis BCG strain for screening of primary and rationale-based antimycobacterial compounds.

Conventional antimycobacterial screening involves CFU analysis, which poses a great challenge due to slow growth of mycobacteria. Recombinant strains carrying reporter genes under the influence of constitutive promoters allow rapid and wide screening of compounds but without revealing their modes of action. Reporter strains using pathway-specific promoters provide a better alternative but allow a limited screening of compounds interfering with only a particular metabolic pathway. This reduces these strains to merely a second-line screening system, as they fail to identify even the more potent compounds if they are not inhibiting the pathway of interest. In this study, we have generated a double recombinant Mycobacterium bovis BCG strain carrying firefly and Renilla luciferase genes as two reporters under the control of a constitutive and an inducible mycobacterial promoter. The presence of dual reporters allows simultaneous expression and analysis of two reporter enzymes within a single system. The expression profile of the firefly luciferase gene, rendered by a constitutive mycobacterial promoter, coincides with the decline in bacterial growth in response to a wide range of antimycobacterial drugs, while the enhanced expression of Renilla luciferase mirrors the selective induction of the reporter gene expression as a result of pathway-specific inhibition. Thus, the double recombinant strain allows the screening of both primary and rationally synthesized antimycobacterial compounds in a single assay. The inhibiting response of drugs was monitored with a dual-luciferase reporter assay which can be easily adapted in high-throughput mode.

Identification and characterization of Rv0494: a fatty acid-responsive protein of the GntR/FadR family from Mycobacterium tuberculosis.

Escherichia coli FadR, a member of the GntR family of transcription factors, plays dual roles in fatty acid metabolism. FadR-DNA binding is inhibited by fatty acyl-CoAs, and thus FadR acts as a sensor of the fatty acid level in bacteria. We have identified FadR-binding sites in the upstream regions of genes showing altered expression after the disruption of fatty acid biosynthesis in Mycobacterium tuberculosis. A FadR homologue in M. tuberculosis, Rv0494, was identified, which binds to its operator in the upstream region of the kas operon. We have shown that FadRMt (Rv0494) directly binds to long-chain fatty acyl-CoA and that binding quenches the intrinsic fluorescence of the purified protein. FadR-DNA binding can be impaired by long-chain fatty acyl-CoA compounds. Overexpression of Rv0494 in Mycobacterium bovis BCG reduced the basal level expression of kas operon genes, thereby suggesting the repressor nature of this protein in fatty acid synthase II regulation. This is the first report, to the best of our knowledge, of a GntR/FadR family protein acting as a fatty acid-responsive transcriptional regulator in M. tuberculosis, suggesting a possible role for this protein in mycolic acid biosynthesis.

Synthesis and bio-evaluation of alkylaminoaryl phenyl cyclopropyl methanones as antitubercular and antimalarial agents.

A series of 4-alkylaminoaryl phenyl cyclopropyl methanones (6a-6u and 8a-8c) were synthesized from 4-fluorochalcones (3a and 3b) by cyclopropanation of double bond followed by nucleophilic substitution of F with different amines. The compounds were screened for their antitubercular and antimalarial activities against Mycobacterium tuberculosis H37Rv and Plasmodium falciparum 3D7 strains in vitro respectively. Several compounds (6a, 6d-6h, 6p, 6q and 8a-8c) exhibited good in vitro antitubercular activities with MIC values 3.12-12.5µg/mL and preferentially inhibited the growth of P. falciparum in vitro (4a, 4c, 6a-6d, 6f, 6s, 8a and 8c) with IC50 as low as 0.080 and 0.035µg/mL and SI values 4975 and 6948, respectively. Molecular docking studies and in vitro evaluation against FAS-II enzymes using reporter gene assays were carried out to elucidate the mode of action of these molecules. Two compounds 4a and 6g showed significant inhibition at 25µM concentration of the compound.

Differential expression of sigH paralogs during growth and under different stress conditions in Mycobacterium smegmatis.

SigH regulates a transcriptional network that responds to heat and oxidative stress in mycobacteria. Seven sigH paralogs are reported to exist in the Mycobacterium smegmatis genome. A comprehensive real-time reverse transcriptase PCR analysis during different stages of growth and upon exposure to various stress conditions and antimycobacterial compounds showed differential expression of sigH paralogs during stationary phase and severalfold increases in the levels of transcription of sigH1, sigH4, sigH5, sigH6, and sigH7 under specific stress conditions.

Conservation of sigma F in mycobacteria and its expression in Mycobacterium smegmatis.

Alternate sigma factor SigF controls the expression of virulence-associated genes and is believed to contribute to the pathology of tuberculosis. It was reported to be absent in fast-growing nontuberculous mycobacteria until its orthologs were reported recently in a database. In this study, we demonstrate the presence of sigF gene in few commonly studied nonpathogenic mycobacterial species. Further, we studied the sigF expression in Mycobacterium smegmatis and observed that unlike its late-stage expression in M. tuberculosis and M. bovis, found in earlier studies, sigF is expressed throughout the growth in M. smegmatis, by and large, at the same level, but its expression varies upon exposure to different stress conditions. The presence of sigF orthologs in nontuberculous mycobacteria and its continued expression throughout the growth suggests that apart from regulating the expression of virulence factor genes in pathogenic mycobacteria, SigF is likely to have more roles in the mycobacterial physiology.

A mouse gene encoding a novel member of the WD family of proteins is highly conserved and predominantly expressed in the testis (Wdr13).

Wdr13, a novel member of the WD family of proteins and the mouse homolog of WDR13 is localized to the locus XA1.1 and is predominantly expressed in the testis. The expression begins at the early stages of gonadal development and is maintained throughout the adult life with a predominant expression in the germ cells of adult testis. RNA in situ hybridization on the testis and brain sections indicated a cytoplasmic expression of the transcript. The alternatively spliced transcripts of the gene are generated by different methods and showed a differential pattern of expression, suggesting functional diversity. The expression of the gene in the unfertilized egg and in the neural stem cells indicated the functional significance of the gene from the early stages of development. The nuclear localization of the mouse WDR13 protein suggested a regulatory function. Evolutionary analysis of the gene indicated an extensive functional conservation across diverse species. Comparison of the genomic organization of the different homologs revealed a varied organization in the invertebrate homolog and the retention of the functionally significant introns in the same.

A highly conserved human gene encoding a novel member of WD-repeat family of proteins (WDR13).

We have identified and characterized a novel member of the WD-repeat motif gene family, WDR13, which contains 9 exons and 8 introns. The gene has been mapped to the genomic locus Xp11.23 by fluorescent in situ hybridization and in silico mapping. Sequence analysis has revealed a continuous open reading frame (ORF) encoding for 485 amino acids with six WD motifs. The expression of this gene has been detected in all the tissues analyzed with significantly varied expression levels among the tissues studied. Analysis of EST clones from various tissues, showing significant homology to WDR13, has identified two spliced variants. The transcription start point has been mapped. Promoter analysis has identified high activity in the 5' UTR, which interestingly showed a testis-specific activity in the transgenic animals studied. The subcellular localization of the WDR13 protein in the nucleus suggests that it may also have a regulatory role in nuclear function along with protein-protein interaction like other members of the WD family of proteins.