Screening Of New Anti - Tuberculosis Pilot And Molecular Pharmacology

Tuberculosis, which is caused predominantly by Mycobacterium tuberculosis, is a widespread infectious disease leading to 1.5 million deaths each year. In addition, antibiotic resistance is a growing problem in TB infections, with-MDR and-XDR TB infections being reported worldwide. Unfortunately, only two new clinically relevant drugs, bedaquiline and delamanid, have been introduced to the market in the past 50 years. Therefore, there is an urgent need to develop new anti-TB drugs with low toxicity and high efficacy.Aminoacyl tRNA synthetases (AaRS) catalyze the attachment of amino acids to their cognate tRNAs and are essential for protein synthesis. The aminoacylation of tRNA is carried out in two steps. In the first step, ATP and the amino acid react to form an aminoacyl-AMP complex, releasing pyrophosphate. In the second step, the aminoacyl-tRNAs are assembled by esterification at the acceptor arm of the cognate tRNA. When AaRS are inhibited, it leads to protein synthesis inhibition and consequently impairs many important physiological processes, eventually resulting in the inhibition of bacterial growth. Tyrosyl-tRNA synthetase (TyrRS) is one type of AaRS that was reported to be a target of a series of anti-Gram-positive bacterial compounds. There is no such report of anti-TB compounds targeting M. tuberculosis TyrRS. We therefore consider TyrRS to be a promising new anti-TB target. It is an ideal target because it is indispensable for the growth of M. tuberculosis and it shares low homology to human TyrRS.In this study, we identified the compound IMB-T130 (5-chloro-N-[4-(pyridin-2-yl)-1,3-thiazol-2-yl] thiophene-2-carboxamide) through the screening of a library of 100,000 compounds. Compound IMB-T130 inhibited the activity of TyrRS in vitro, and showed similar antibacterial potency as first-line anti-TB drugs, and even better inhibitory effects when tested against drug-resistant strains. The interaction between IMB-T130 and TyrRS was confirmed by a surface plasmon resonance (SPR) assay, then analyzed further by molecular docking experiments. Along with its low cytotoxicity against mammalian cells, our findings indicate that IMB-T130 is a promising new anti-TB compound.However, it seemed that the concentration of IMB-T130 required to inhibit the growth of M. tuberculosis was lower than that required to inhibit the aminoacylation activity of MtTyrRS in vitro. A possible reason is that IMB-T130 may be a multitarget compound. So another target protein of the compound was found by aid of computer. After comparing a series of molecular docking models, we focused on the 3-dehydroquinate synthase (DHQS) which catalyzes the second step of shikimic acid pathway. DHQS is an indispensable part for the metabolism of the amino acid and is also essential for the growth of the bacterial. We expressed and purified the DHQS protein in vitro. DHQS activity was measured by detecting the free phosphates produced by the reaction. After that, compound IMB-T130 was proven to inhibit the activity of DHQS in vitro. The interaction between IMB-T130 and DHQS was also confirmed by SPR assay.In conclusion, we identified an anti-TB compound IMB-T130, which could inhibit the activity of TyrRS and DHQS. This compound showed selective inhibition against M. tuberculosis and displayed low toxicity against mammalian cells and rats.