Structural Targeting Testis Specific Kinases (TSSK) 1 & 2 for Male Contraception

描述（由申请人提供）：睾丸特异性丝氨酸苏氨酸激酶1和2 [TSSK 1和2]是最近被证实为男性避孕靶点的密切相关基因。这些蛋白在减数分裂后精子的睾丸中选择性表达。小鼠中TSSK 1和2的缺失导致雄性不育，在鞭毛发生过程中发生精子发生阻滞。TSSK 1和2底物，即TSKS，在人类和小鼠的鞭毛形成过程中定位于精子的中心体，TSKS持续存在于成熟精子的中心粒中。TSSK2磷酸化第二个鞭毛底物，精子轴突中枢装置蛋白SPAG16L。这些关键发现证实了tssk1和2以及tssk1和2通路在精子发生中不可或缺的作用，为本研究的中心假设提供了主要证据：使用tssk1和2的小分子抑制剂会通过损害鞭毛发生和精子运动而导致男性不育。在拟议的研究中，将进行两个目标，以确定tssk1和2的先导抑制剂。目的1将确定人类tsk1和tsk2的三维结构。此外，还将获得tssk1和tssk2与底物、肽和药物配体的共晶结构。在Aim 2中，将通过标准结构-功能分析、有机合成和组合文库筛选来鉴定与tssk1和2相互作用的化合物。重点将放在催化位点、调控位点和底物抑制剂上。我们基于葛兰素史克公司提供的专有技术建立了一个定制的组合文库，并将筛选该文库以识别抑制TSSK 1和2的化合物。该文库是专门开发包含激酶抑制剂，并将通过固相和细胞为基础的测定筛选。该研究项目将提供有关人类精子发生过程中信号转导事件的新信息，并预计将识别具有避孕潜力的化合物，这些化合物通过抑制精子鞭毛的形成和功能，选择性地作用于精子发生的减数分裂后阶段。

DESCRIPTION (provided by applicant): 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. PUBLIC RELEVANCE: 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.