Targeting sperm-specific proteins during meiosis and sperm morphogenesis

项目1总结（减数分裂和精子形态发生期间靶向精子特异性蛋白） 项目1的总体目标是使用CRISPR/Cas9来了解精子的形成- 特异性顶体和鉴定靶向生精的药物样探针和临床前候选物， 用于体内避孕作用的特定可药用蛋白质。人口数量达到了惊人的水平 数字，导致我们的气候和生态系统的变化，并可能导致全球粮食，水和 为我们的子孙后代提供其他资源。为了遏制人口的疯狂增长及其可怕的 因此，我们需要专门针对男性种系的避孕药。针对性的避孕药 睾丸特异性蛋白质应该可以消除不必要的副作用。作为避孕目标，我们将重点关注两个 减数分裂特异性蛋白和四种精子必需的顶体相关跨膜蛋白。这六 基于在小鼠中的原理验证研究选择进化上保守的蛋白质， 它们是男性生育能力所必需的，我们成功地将小分子药物用于可逆性生殖， 避孕在体内，以及这些蛋白质作为男性避孕的药物靶标的潜力。七十 FDA批准的药物中有10%靶向分泌或跨膜蛋白。此外，顶体如何 蛋白质在顶体形成过程中的相互作用尚不清楚。为了确定形成和功能的相互作用组， 顶体，我们将与Ikawa博士（项目2）合作，使用CRISPR/Cas9将标签序列插入顶体， 这四个基因为了识别小分子探针和临床前候选物，我们将与DNA- 编码化学技术（DEC-Tec）核心。我们的总体假设是CRISPR/Cas9和DEC-Tec 将帮助我们定义精子发生过程中的蛋白质相互作用组，快速识别多种小分子 针对这些基本的生精蛋白，并创造了一种口服和植入的 男性和女性的避孕药。项目1的具体目标是：1）使用CRISPR/Cas9功能性地 了解精子顶体的形成及四种精子必需跨膜蛋白的特异性关系; 2)采用DEC-Tec鉴定小分子探针和临床前候选药物，以靶向生精- 特异性"可用药"蛋白;和3）评价小分子化合物的潜在体内避孕作用 抑制剂的P01和项目1的成功依赖于与项目2和3以及DEC-Tec的互动 核心Ikawa博士和他的同事创建了几个关键模型，并将为Aim 1生成标记的等位基因。 博士Sonnenburg和他的Project 3团队将帮助我们的Aim 2 DEC-Tec生产重组蛋白 卡位Young、Huang、Sonnenburg、Simmons、Yu、Lee和Li博士是使用DEC-Tec的专家， 化学、X射线晶体学和药物代谢研究，以发现先导化合物。项目1将 对所有三个项目进行体内概念验证研究（目标3）。我们的多学科团队将 解决这一紧迫问题，并为男性和女性创造新型和可逆的非激素避孕药。

PROJECT 1 SUMMARY (Targeting sperm-specific proteins during meiosis and sperm morphogenesis) The overall goals of Project 1 are to use CRISPR/Cas9 to understand the formation of the sperm- specific acrosome and identify drug-like probes and preclinical candidates to target spermatogenic- specific druggable proteins for a contraceptive effect in vivo. The human population is reaching alarming numbers, resulting in changes in our climates and ecosystems and likely global shortages of food, water, and other resources for our children and grandchildren. To curb rampant population growth and its dire consequences, we require contraceptives that specifically target the male germline. Contraceptives that target testis-specific proteins should eliminate unwanted side effects. As contraceptive targets, we will focus on two meiosis-specific proteins and four sperm-essential, acrosome-associated transmembrane proteins. These six evolutionarily-conserved proteins were chosen based on proof-of-principle studies in the mouse showing that they are specifically required for fertility in males, our success at delivering small molecules for reversible contraception in vivo, and the potential of these proteins as druggable targets for male contraception. Seventy percent of FDA-approved drugs target either secreted or transmembrane proteins. Further, how the acrosomal proteins interact during acrosome formation is unclear. To define the interactome for formation and function of the acrosome, we will collaborate with Dr. Ikawa (Project 2) to use CRISPR/Cas9 to insert tag sequences into these four genes. To identify small-molecule probes and preclinical candidates, we will partner with the DNA- Encoded Chemistry Technology (DEC-Tec) Core. Our overall hypothesis is that CRISPR/Cas9 and DEC-Tec will help us to define the protein interactome during spermatogenesis, rapidly identify multiple small-molecules that are directed at these essential spermatogenic proteins, and create an assortment of oral and implantable contraceptives for men and women. The Specific Aims of Project 1 are: 1) Use CRISPR/Cas9 to functionally understand acrosome formation and the specific relationship of four sperm-essential transmembrane proteins; 2) Employ DEC-Tec to identify small-molecule probes and preclinical candidates to target spermatogenic- specific “druggable” proteins; and 3) Evaluate potential in vivo contraceptive effects of small-molecule inhibitors. The success of this P01 and Project 1 relies on interactions with Projects 2 and 3 and the DEC-Tec Core. Dr. Ikawa and his colleagues created several key models and will generate the tagged alleles for Aim 1. Dr. Sonnenburg and his Project 3 team will help to produce recombinant proteins for our Aim 2 DEC-Tec screens. Drs. Young, Huang, Sonnenburg, Simmons, Yu, Lee, and Li are experts at using DEC-Tec, medicinal chemistry, X-ray crystallography, and drug metabolism studies for uncovering lead compounds. Project 1 will perform the in vivo proof-of-concept studies for all three projects (Aim 3). Thus, our multidisciplinary team will tackle this urgent problem and create novel and reversible non-hormonal contraceptives for men and women.