The testis specific serine threonine kinases 1 &2 [TSSK 1 &2] are closely related genes that have recently been validated as male contraceptive targets. These proteins are expressed selectively in the testis in post-meiotic spermatids. Deletion of TSSK 1 &2 in mice causes male infertility, with arrest of spermiogenesis occurring during flagellogenesis. The TSSK 1 &2 substrate, TSKS, localizes in both humans and mice to the centrosomes of spermatids during flagellogenesis and TSKS persists in the centrioles of mature spermatozoa. TSSK2 phosphorylates a second flagellar substrate, the sperm axonemal central apparatus protein SPAG16L. Deficiency of SPAG16L causes male infertility associated with impaired sperm motility, These key findings substantiate an indispensable role for TSSK 1 &2 and the TSSK 1 &2 pathway in spermatogenesis, providing the primary evidence for this study's central hypothesis: that administration of a small molecule inhibitor of TSSK 1 &2 will result in male infertility by acting to impair flagellogenesis and sperm motility. In the proposed research two aims will be undertaken toward the goal of identifying a lead inhibitor of TSSK 1 &2.
Aim 1 will determine the three dimensional structures of human TSSK 1 &2 and TSKS. In addition, structures of co-crystals of TSSK 1 &2 with substrate, peptide, and drug ligands will be obtained.
In Aim 2, compounds interacting with TSSK 1 &2 will be identified through standard structure-function analysis, organic syntheses and combinatorial library screening. Attention will be directed to catalytic site, regulatory site and substrate inhibitors. We have built a custom combinatorial library based upon proprietary technology provided by GlaxoSmithKline and will screen this library to identify compounds that inhibit TSSK 1 &2. This library was specifically developed to contain kinase inhibitors and will be screened by both solid phase and cell based assays. This research program will yield new information concerning events of signal transduction during spermiogenesis in humans and is predicted to identify compounds with contraceptive potential that selectively act on post-meiotic stages of spermatogenesis by inhibiting sperm flagellar formation and function.
This project will determine the crystal structures of 2 kinase enzymes [TSSK 1 &2] and their substrate [TSKS] that are located in the human testis. The project will use the protein structural knowledge to identify compounds that bind to these kinase enzymes. TSSK 1 &2 and TSKS are found in the testis in the last step of sperm production and have been shown in mice to be essential for proper sperm development and function Compounds that inhibit these kinases are anticipated to act as male contraceptive.
|Smith, Ryan P; Lowe, Greg J; Kavoussi, Parviz K et al. (2012) Confocal fluorescence microscopy in a murine model of microdissection testicular sperm extraction to improve sperm retrieval. J Urol 187:1918-23|