Identification and validation of the mode of action of the chalcone anti-mycobacterial compounds

Funding Information: G.S.B is supported by The Medical Research Council ( MR/S000542/1 and MR/R001154/1 ). Funding Information: J.A.G.C. is supported by the Academy of Medical Sciences /the British Heart Foundation /the Government Department of Business, Energy and Industrial Strategy / Global Challenges Research Fund /the Wellcome Trust Springboard Award [ SBF003\1088 ]. Publisher Copyright: © 2020Objectives: The search for new TB drugs has become one of the great challenges for modern medicinal chemistry. An improvement in the outcomes of TB chemotherapy can be achieved by the development of new, shorter, cheap, safe and effective anti-TB regimens. Methods: Chalcones (1a-1o) were synthesized and evaluated for their antimycobacterial activity against Mycobacterium bovis BCG using growth inhibition assays. Compound 1a was selected as a ‘hit’ compound. The mode of action of compound 1a, was identified by mycolic acid methyl esters (MAMEs) and fatty acid methyl esters (FAMEs) analysis using thin layer chromatography. Dose dependent experiments were conducted by over-expressing components of FAS-II in M. bovis BCG to confirm the target. Ligand binding using intrinsic tryptophan assay and molecular docking were used to further validate the target. Results: MAMEs and FAMEs analysis showed dose-dependent reduction of MAMEs with the overall abundance of FAMEs suggesting that compound 1a targets mycolic acid biosynthesis. Direct binding of 1a to InhA was observed using an intrinsic tryptophan fluorescence binding assay, and a 2-fold IC50 shift was observed with an InhA overexpressing strain confirming InhA as the cellular target. Conclusion: The chalcone 1a exhibits potent antimycobacterial activity, displays a good safety profile and is a direct inhibitor of InhA, a key component in mycolic acid synthesis, validating this series for further anti-TB drug developmentPeer reviewe