PROJECT SUMMARY A pharmacologic approach for male contraception remains a longstanding challenge in medicine. Recent research from our laboratories has shown that the small-molecule inhibitor JQ1 of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins is a potent inhibitor of BRDT and that the observed contraceptive effect of JQ1 is completely reversible. However, JQ1 is a pan-BET inhibitor and therefore selective BRDT inhibitors are needed that can act as safe, effective, and reversible non-hormonal male contraceptive therapeutic agents. We thus propose research directed at the discovery, optimization, biochemical characterization, mechanistic understanding, in vivo evaluation of contraceptive effect reversibility, and clinical translation of BRDT inhibitors. In Specific Aim 1, we propose four chemical platforms to discover BRDT-selective bromodomain inhibitors for male contraception: 1) novel non-obvious mimetics of acetyl-lysine; 2) inhibitors that specifically target a unique residue in human BRDT; 3) unique ligands that can bind selectivly to both bromodomains of BRDT; and 4) bifunctional small molecules designed to direct selective degradation of the BRDT protein. Structure-based drug design, biochemical, and biophysical studies will be used to drive selectivity improvements. For Specific Aim 2, the selective BDRT inhibitors identified from Specific Aim 1 will be further optimized for cellular potency and selectivity for BRDT through an iterative process that will also involve in vitro testing for ADMET properties. Specific Aim 3 will explore the clinical potential of the inhibitors through in vivo pharmacokinetic studies and pre-clinical evaluation for male contraception, involving definitive studies of spermatogenesis, fertility, and reversibility in male mice.