Manipulation of sperm-specific proteases using genetic and chemical approaches

项目3概要（使用遗传和化学方法操纵精子特异性蛋白酶） 项目3的总体目标是阐明雄性激素作用的分子机制， 生殖道特异性丝氨酸蛋白酶样酶通过使用遗传学和化学 生物学，并使用这些数据来指导新的非激素避孕药的发现。 药物发现工作的理想生物靶标是那些具有显著和非冗余生物活性的靶标。 作用，高度特异性的分子功能，和生物化学特征服从分子抑制。没有 存在一种确定的方法来鉴定或验证有效的药物靶点，当然， 进化的过程并不是为了产生许多生理上脆弱的途径， 用于避孕。因此，对控制生育的分子机制的透彻理解 作为一种对广泛的介入点阵列进行采样的方法，这是所期望的。作为目标的补充 从项目1和2中产生，项目3将重点关注五种丝氨酸的遗传和化学分析， 蛋白酶样酶，表达仅限于男性生殖道。这些机械研究将 使我们能够将这些丝氨酸蛋白酶样酶置于生殖途径中，同时 确定这些蛋白质作为避孕靶点的效用。早期对精子的研究 透明体相互作用假设精子顶体中含有的酶是 穿透卵丘细胞层和透明层。最近，很明显，尽管 与顶体酶释放相关的胞吐事件（在顶体反应期间）是必需的 对于受精，它既不是通过与透明体接触而诱导的，也不需要在透明体附近发生。 卵母细胞的外膜。同样，能动的精子对于成功受精至关重要。我们的整体 假设丝氨酸蛋白酶样酶是男性生育力所需的一类新的蛋白质， 这些精子发生所需酶的小分子抑制剂的发现将导致 独特的男性避孕药。我们的具体目标如下：1）使用CRISPR/Cas9模型， 生殖道特异性蛋白酶，以阐明它们在生殖中的需求; 2）帮助表达， 纯化来自项目1、2和3的重组蛋白用于DNA编码的化学技术（DEC- Tec）亲和选择;和3）使用DEC-Tec来发现新丝氨酸的小分子探针和抑制剂 生育所需的蛋白酶样酶，并评估早期药物样先导化合物和类似物， 体内概念验证避孕研究中的药代动力学特性。

PROJECT 3 SUMMARY (Manipulation of sperm-specific proteases using genetic and chemical approaches) The overall goals of Project 3 are to elucidate the molecular mechanisms of action of male reproductive tract-specific serine protease-like enzymes through the use of genetics and chemical biology, and to use these data to guide discovery of novel non-hormonal contraceptive agents. Biological targets ideal for drug discovery endeavors are those with significant and non-redundant biological effects, highly specific molecular functions, and biochemical features amenable to molecular inhibition. No single definitive methodology exists for the identification or validation of efficacious drug targets, and certainly the processes of evolution are not driven to yield numerous physiologically vulnerable pathways, particularly for contraception. Therefore, a thorough understanding of the molecular mechanisms governing fertility as well as an approach that samples a wide array of intervention points is desirable. As a complement to targets emerging from Projects 1 and 2, Project 3 will focus on the genetic and chemical analyses of five serine protease-like enzymes with expression limited to the male reproductive tract. These mechanistic studies will allow us to place these serine protease-like enzymes into reproductive pathways and simultaneously determine the utility of each of these proteins as a contraceptive target. Early studies of the sperm-zona pellucida interaction assumed that enzymes contained within the sperm acrosome were required for penetration of cumulus cell layers and the zona pellucida. More recently, it has become apparent that whereas the exocytotic event associated with release of acrosomal enzymes (during the acrosome reaction) is essential for fertilization, it is neither induced by contact with the zona pellucida, nor required to occur in proximity of the outer vestments of the oocyte. Similarly, motile sperm are essential for successful fertilization. Our overall hypothesis is that serine protease-like enzymes are a novel class of proteins required for male fertility and the discovery of small-molecule inhibitors for these spermatogenic-required enzymes will lead to the development of unique male contraceptives. Our Specific Aims are as follows: 1) Use CRISPR/Cas9 models of male reproductive tract-specific proteases to clarify their requirement in reproduction; 2) Aid in the expression and purification of recombinant proteins from Projects 1, 2, and 3 for DNA-encoded chemistry technology (DEC- Tec) affinity selections; and 3) Use DEC-Tec to uncover small-molecule probes and inhibitors of novel serine protease-like enzymes required for fertility and evaluate early drug-like leads and analogues with acceptable pharmacokinetic properties in proof-of-concept contraceptive studies in vivo.