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[TSSK1和2]是最近被证实为男性避孕目标的密切相关基因。这些蛋白质在减数分裂后的精子细胞中选择性地表达在睾丸中。在小鼠中，tssk 1和2的缺失会导致男性不育，精子发生停滞发生在鞭毛发生期间。在鞭毛发生过程中，TSK1和TSK2底物定位于人和小鼠的精子细胞中心体，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，精子轴丝中央装置蛋白SPAG16L。SPAG16L基因缺失可导致精子活力受损的男性不育，这些关键发现证实了TSSK1和TSK1及TSK1和TSK2通路在精子发生中起着不可或缺的作用，为本研究的中心假说提供了初步证据：给予TSK1和TSK2的小分子抑制剂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TSSK1和TSK2的先导抑制物。目标1将确定人TSK1和TSKS的三维结构。此外，还将获得TSSK1和TSK2与底物、多肽和药物配体的共晶结构。在目标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TSSK2相互作用的化合物。将重点关注催化部位、调节部位和底物抑制剂。我们基于葛兰素史克提供的专利技术建立了一个定制的组合库，并将对该库进行筛选，以确定抑制TSSK1和2的化合物。该库是专门开发的，以包含激酶抑制剂，并将通过固相和基于细胞的分析进行筛选。这项研究计划将提供有关人类精子发生过程中信号转导事件的新信息，并有望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。 与公众相关：该项目将确定位于人类睾丸中的两种激酶酶[TSK 1和2]及其底物[TSK]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酶结合的化合物。在精子产生的最后一步中，在睾丸中发现了tssk 1和tsk，在小鼠身上被证明是精子正常发育所必需的，抑制这些激酶的功能化合物有望作为男性避孕药。

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.