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的基因。这些蛋白质在减数分裂后精子细胞的睾丸中选择性表达。小鼠TSSK 1和2的缺失导致雄性不育，在鞭毛发生期间发生精子发生停滞。TSSK 1和2底物TSKS在人和小鼠中定位于鞭毛发生期间精子细胞的中心体，并且TSKS持续存在于成熟精子的中心粒中。TSSK2磷酸化第二鞭毛底物，精子轴丝中央器蛋白SPAG16L。SPAG16L的缺乏导致与受损的精子活力相关的男性不育。这些关键发现证实了TSSK 1和2以及TSSK 1和2途径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：TSSK 1和2的小分子抑制剂的施用将通过损害鞭毛发生和精子活力而导致男性不育。在拟议的研究中，将进行两个目标，以确定TSSK 1和2的主要抑制剂的目标。 目的1确定人TSSK 1、2和TSKS的三维结构.此外，将获得TSSK 1和2与底物、肽和药物配体的共晶体的结构。目标二：通过标准的结构功能分析、有机合成和组合文库筛选，鉴定与TSSK 1和2相互作用的化合物。注意力将被引导到催化位点、调节位点和底物抑制剂。我们已经建立了一个定制的组合库的基础上提供的专有技术葛兰素史克和将筛选这个库，以确定化合物抑制TSSK 1和2。该文库是专门开发的，含有激酶抑制剂，并将通过固相和基于细胞的测定进行筛选。这项研究计划将产生有关人类精子发生过程中信号转导事件的新信息，并预计将确定具有避孕潜力的化合物，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。 公众相关性：该项目将确定位于人类睾丸中的2种激酶[TSSK 1和2]及其底物[TSKS]的晶体结构。该项目将使用蛋白质结构知识来识别与这些激酶结合的化合物。TSSK 1和2和TSKS在精子产生的最后一步的睾丸中被发现，并且已经在小鼠中显示对于适当的精子发育和功能是必需的。抑制这些激酶的化合物预期用作男性避孕药。

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.