Agradecimentos: We thank all members of CQMED-UNICAMP for their help and support. This work was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) (2013/50724-5 and 2014/50897-0), Embrapii (Empresa Brasileira de Pesquisa e Inovação Industrial), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) (465651/2014-3) and The Structural Genomics Consortium, a registered charity (number 1097737). R.C.F was the recipient of a CAPES (Coordenac¸ a~ o de Aperfeiçoamento de Pessoal de Nível Superior) M.Sc. fellowship (130075/2020-5); S.N.S.V. was the recipient of a FAPESP post-doctoral fellowship (2018/09475-5); C.M.C.C-P fellowship was in part funded by Promega Corporation; C.V.R was the recipient of a CAPES post-doctoral fellowship (88887.146077/2017-00). J.R.S and M.A.B were funded by Cystic Fibrosis Canada. We thank the staff of the Proteomics section of the Life Sciences Core Facility (LaCTAD), part of the University of Campinas (UNICAMP). We thank Matthew Robers and Samuel Hoare for critical review of the manuscript Ver menos

Abstract: New antibiotics are urgently needed to counter the emergence of antimicrobial resistant pathogenic bacteria. A major challenge in antibiotics drug discovery is to turn potent biochemical inhibitors of essential bacterial components into effective antimicrobials. This difficulty is underpinned by a lack of methods to investigate the physicochemical properties needed for candidate antibiotics to permeate the bacterial cell envelope and avoid clearance by the action of bacterial efflux pumps. To address these issues, here we used a target engagement assay to measure the equilibrium and kinetics binding parameters of antibiotics targeting dihydrofolate reductase (DHFR) in live bacteria. We also used this assay to identify novel DHFR ligands having antimicrobial activity from a library of compounds. We validated this approach using the Gram-negative bacteria Escherichia coli and the emerging human pathogen Mycobacterium abscessus. We expect the use of target engagement assays in bacteria to expedite the discovery and progression of novel, cell-permeable antibiotics with on-target activity Ver menos