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The identification of novel Mycobacterium tuberculosis DHFR inhibitors and the investigation of their binding preferences by using molecular modelling.

ABSTRACT: It is an urgent need to develop new drugs for Mycobacterium tuberculosis (Mtb), and the enzyme, dihydrofolate reductase (DHFR) is a recognised drug target. The crystal structures of methotrexate binding to mt- and h-DHFR separately indicate that the glycerol (GOL) binding site is likely to be critical for the function of mt-DHFR selective inhibitors. We have used in silico methods to screen NCI small molecule database and a group of related compounds were obtained that inhibit mt-DHFR activity and showed bactericidal effects against a test Mtb strain. The binding poses were then analysed and the influence of GOL binding site was studied by using molecular modelling. By comparing the chemical structures, 4 compounds that might be able to occupy the GOL binding site were identified. However, these compounds contain large hydrophobic side chains. As the GOL binding site is more hydrophilic, molecular modelling indicated that these compounds were failed to occupy the GOL site. The most potent inhibitor (compound 6) demonstrated limited selectivity for mt-DHFR, but did contain a novel central core (7H-pyrrolo[3,2-f]quinazoline-1,3-diamine), which may significantly expand the chemical space of novel mt-DHFR inhibitors. Collectively, these observations will inform future medicinal chemistry efforts to improve the selectivity of compounds against mt-DHFR.

SUBMITTER: Hong W

PROVIDER: S-EPMC4607890 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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The identification of novel Mycobacterium tuberculosis DHFR inhibitors and the investigation of their binding preferences by using molecular modelling.

Hong Wei W Wang Yu Y Chang Zhe Z Yang Yanhui Y Pu Jing J Sun Tao T Kaur Sargit S Sacchettini James C JC Jung Hunmin H Lin Wong Wee W Fah Yap Lee L Fong Ngeow Yun Y Paterson Ian C IC Wang Hao H

Scientific reports 20151016

It is an urgent need to develop new drugs for Mycobacterium tuberculosis (Mtb), and the enzyme, dihydrofolate reductase (DHFR) is a recognised drug target. The crystal structures of methotrexate binding to mt- and h-DHFR separately indicate that the glycerol (GOL) binding site is likely to be critical for the function of mt-DHFR selective inhibitors. We have used in silico methods to screen NCI small molecule database and a group of related compounds were obtained that inhibit mt-DHFR activity a ...[more]

PMID: 26471125

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Project description:Mycobacterium tuberculosis kills approximately 2 million people each year and presents an urgent need to identify new targets and new antitubercular drugs. Thymidylate synthase (TS) enzymes from other species offer good targets for drug development and the M. tuberculosis genome contains two putative TS enzymes, a conventional ThyA and a flavin-based ThyX. In M. tuberculosis, both TS enzymes have been implicated as essential for growth, either based on drug-resistance studies or genome-wide mutagenesis screens. To facilitate future small molecule inhibitors against these proteins, a detailed enzymatic characterization was necessary.After cloning, overexpression, and purification, the thymidylate-synthesizing ability of ThyA and ThyX gene products were directly confirmed by HPLC analysis of reaction products and substrate saturation kinetics were established. 5-Fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) was a potent inhibitor of both ThyA and ThyX, offering important clues to double-targeting strategies. In contrast, the folate-based 1843U89 was a potent inhibitor of ThyA but not ThyX suggesting that it should be possible to find ThyX-specific antifolates. A turnover-dependent kinetic assay, combined with the active-site titration approach of Ackermann and Potter, revealed that both M. tuberculosis enzymes had very low k(cat) values. One possible explanation for the low catalytic activity of M. tuberculosis ThyX is that its true biological substrates remain to be identified. Alternatively, this slow-growing pathogen, with low demands for TMP, may have evolved to down-regulate TS activities by altering the turnover rate of individual enzyme molecules, perhaps to preserve total protein quantities for other purposes. In many organisms, TS is often used as a part of larger complexes of macromolecules that control replication and DNA repair.Thus, the present enzymatic characterization of ThyA and ThyX from M. tuberculosis provides a framework for future development of cell-active inhibitors and the biological roles of these TS enzymes in M. tuberculosis.

Project description:Mycobacterial efflux pumps play a major role in the emergence of antimycobacterial drug resistance. Of particular interest is the proteinaceous multi-drug efflux pump protein Rv1258c that encodes a tetracycline/aminoglycoside resistance (TAP-2)-like efflux pump which is active in susceptible and drug resistant Mycobacterium tuberculosis. Rv1258c is implicated in drug resistance to numerous antimycobacterials including first line drugs rifampicin and isoniazid as well as fluoroquinolone and aminoglycoside antibiotic classes. To date, compounds like verapamil and piperine have been shown to inhibit Rv1258c but no direct evidence for binding or mode of action exist. Therefore in the present study we generated an accurate 3D model of Rv1258c using MODELLER and validated its structure using molecular dynamic simulation studies with GROMACS software. The 3D-structures of Rv1258c and the homologous template 1pw4 were simulated within a POPE/POPG lipid bilayer and found to behave similar. Another important finding was the identification of one local energy minima state of the apo protein, which speaks to the flexibility of the protein and will be investigated further. Extraction of one of the open channel conformations of Rv1258c and blind docking of various structurally diverse putative inhibitors and substrates, allowed for the identification of a probable binding site. Spectinamide was found to bind to a different location on the outside surface of the protein suggesting its ability to avoid the efflux channel. We further identified 246 putative compounds that showed higher binding affinity values to Rv1258c compared to piperine and verapamil. Interaction analysis of the top 20 purchasable compounds identified crucial hydrogen bond interactions with Ser26, Ser45 and Glu243 as well as a pi-pi stacking interaction with Trp32 that accounted for the strong affinity of these compounds for Rv1258c. Future studies will entail purchasing a number of compounds for in vitro activity testing against Mycobacterium tuberculosis.

Dataset Information

The identification of novel Mycobacterium tuberculosis DHFR inhibitors and the investigation of their binding preferences by using molecular modelling.

Publications

The identification of novel Mycobacterium tuberculosis DHFR inhibitors and the investigation of their binding preferences by using molecular modelling.

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