PROJECT SUMMARY Members of the testis-specific serine/threonine kinase (TSSK) family are almost exclusively expressed in germ cells post-meiotically and remain present in mature sperm, suggesting a critical role in TSSKs male reproduction. The predicted importance of TSSKs has been confirmed using knock-out (KO) mice models. Male, but not female, TSSK6 (aka SSTK) KO and TSSK1/TSSK2 double KO mice are sterile without exhibiting somatic abnormalities. In addition, TSSK4 KO mice are sub-fertile. In the case of the double TSSK1/TSSK2 KO, the extent by which the sterility phenotype is due to TSSK1, to TSSK2, or to both is not known. Based on these findings, our central hypothesis is that a selective TSSK6 inhibitor would reversibly block fertility and therefore constitute an ideal candidate for a non-hormonal male contraceptive agent. Considering this hypothesis, the objective in this application is to identify selective TSSK6 inhibitors and to evaluate their therapeutic efficacy and reversibility using mice models. Towards this objective, we will pursue three Specific Aims. In Specific Aim 1, we will use two different high throughput screening (HTS) strategies. First, we will search for highly potent ATP- competitive inhibitors using a HTS-compatible TSSK6 kinase assay. Using this approach, we expect to find potent inhibitors (with IC50 in the low nM range). These inhibitors will be very relevant for co-crystallization with TSSK6 (see Aim 2). Second, as selectivity for the target kinase over other kinases present in the kinome is generally quite challenging for ATP-site inhibitors, we also propose to identify allosteric TSSK6 inhibitors by screening allosteric kinase, focused and diverse compound library collections for TSSK6 binders. In Specific Aim 2, we will elucidate the crystal structure of TSSK6 alone and in the presence of ATP-site kinase or in the presence of allosteric inhibitors. These TSSK6 structures will be used to achieve a better understanding of TSSK6 inhibition and will be used for structure-based design of selective TSSK6 inhibitors. Finally, in Specific Aim 3, we will test the premise that TSSK6 selective inhibitors will act as contraceptives in male mice without producing behavioral changes or affecting somatic functions. These results enable the identification of a novel development candidate and support the long-term goal of clinical translation and marketing of a non-hormonal agent based on the reversible inhibition of the highly testis-specific TSSK6 protein. Toward this general goal, we will collaborate with Drs. Georg, Hawkinson, Schönbrunn and Wolgemuth. Each of them has expertise in different areas directly related to this proposal. Those include medicinal chemistry, HTS assays, protein expression and crystallography and in vivo analysis of contraceptive targets.