Mycobacterium phenotypic hits are a good reservoir for new chemotypes for the treatment of tuberculosis. However, the absence
of defined molecular targets and modes of action could lead to failure in drug development. Therefore, a combination of
ligand-based and structure-based chemogenomic approaches followed by biophysical and biochemical validation have been used to
identify targets for Mycobacterium tuberculosis phenotypic hits. Our approach identified EthR and InhA as targets for several hits,
with some showing dual activity against these proteins. From the 35 predicted EthR inhibitors, eight exhibited an IC50 below 50
μM against M. tuberculosis EthR and three were confirmed to be also simultaneously active against InhA. Further hit validation was
performed using X-ray crystallography yielding eight new crystal structures of EthR inhibitors. Although the EthR inhibitors attain
their activity against M. tuberculosis by hitting yet undefined targets, these results provide new lead compounds that could be
further developed to be used to potentiate the effect of EthA activated pro-drugs, such as ethionamide, thus enhancing their
bactericidal effect.GM is grateful to the European Molecular Biology Laboratory and Marie Sklodowska-Curie Actions for funding this work. VM and MB
acknowledge Bill & Melinda Gates Foundation [subcontract by the Foundation for the National Institutes of Health (NIH)]
(OPP1024021). VM and MS acknowledge the European Community’s Seventh Framework Programme [grant number 260872]. GP
would like to acknowledge the Wellcome Trust and the European Molecular Biology Laboratory for funding. JPO was funded by the
member nation states of the European Molecular Biology Laboratory. TLB acknowledges The Wellcome Trust for funding and
support (grant number 200814/Z/16/Z)