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Information × Registration Number 0223U004829, 0120U104795 , R & D reports Title Site-directed mutagenesis as a novel strategy to investigate and overcome Mycobacterium tuberculosis antibiotic resistance popup.stage_title Head Yarmoliuk Serhii M., Доктор хімічних наук Registration Date 05-12-2023 Organization Institute of Molecular Biology and Genetics of NAS of Ukraine popup.description2 For the first time we have obtained two mutated forms of M. tuberculosis MetRS – Q24A and L293A. It was found that these mutations caused almost complete loss of the effectiveness of the MetRS inhibitors which were developed by us during Project realization. It means that these amino acid residues in aminoacyl-adenylate binding site are important for the interactions with inhibitors. In order to find novel M. tuberculosis MetRS inhibitors we have performed biochemical screening of 93 compounds from different chemical classes in aminoacylation assay and found four compounds which inhibit synthetase activity by more than 50% at a concentration of 100 μM. We performed in silico site-directed mutagenesis of M. tuberculosis LeuRS synthetic site in order to identify the most critical amino acid residues for the interaction with substrate. We used umbrella sampling (US) algorithm for the calculation of the binding free energy (ΔG) of leucyl-adenylate analogue with LeuRS wild type and mutated forms. According to molecular dynamics calculation results we have selected two amino acid residues for experimental site-directed mutagenesis – His89 and Ile697. Two mutated forms of M. tuberculosis LeuRS – H89A and I697A, were obtained. It was revealed that these amino acid residues are important for the interaction with several known inhibitors. We have carried out biochemical screening of 50 compounds from different chemical classes toward M. tuberculosis LeuRS, among which 25 novel inhibitors were found that decrease synthetase activity by more than 50% at a concentration of 100 μM. We have developed 4 inhibitors targeting simultaneously M. tuberculosis MetRS and LeuRS. Among them three compounds belong to the derivatives of benzo[b]oxepine-4-carboxylic acid(5-benzyl-thiazol-2-yl)-amide and one – to the derivative of 1-oxo-3-phenyl-isochroman-6-carboxylic acid benzothiazol-2-ylamide. Product Description popup.authors popup.nrat_date 2023-12-05 Close