Project Summary Tuberculosis (TB) remains a major global health burden. In 2021, the number of deaths from TB increased to 1.6 million, once again making it the leading cause of death from infectious disease. Current anti-tubercular drugs are mainly effective against metabolically active and replicating bacteria. In addition to the need for new antibiotics that are effective against both drug sensitive and drug resistant strains, there is an urgent need for new therapeutics that can shorten therapy. We identified several series of molecules using a target-based, high-throughput, phenotypic screen for inhibitors of protein secretion. We evaluated these series for tractability and druglike properties and completed preliminary structure-activity relationship (SAR) studies. We identified two high priority series for hit-to-lead progression. Our data suggest these molecules target protein synthesis and importantly, have activity against both replicating and non-replicating bacteria. The aim of this proposal is to develop new anti-tubercular drugs that work by targeting protein secretion and to determine the mechanism by which these agents kill Mycobacterium tuberculosis. We will complete lead generation for our priority series and generate proof of concept of in vivo efficacy in the mouse model of infection. We will determine how inhibition of LepB-mediated secretion leads to rapid cell death in M. tuberculosis, and why this is more pronounced under non-replicating conditions. In addition, we will determine the in vitro and in vivo mechanisms of resistance to LepB inhibitors that will be relevant in the clinical setting. The outcomes of this project would be (i) identification of a molecule for lead optimization; (ii) identification of the mode of action and mechanism of kill of the series; and (iii) identification of in vitro and in vivo mechanism(s) of resistance. Demonstration of in vivo activity and tractability of the series would lead to a full drug development project in the future.