Manipulation of sperm-specific proteases using genetic and chemical approaches

使用遗传和化学方法操作精子特异性蛋白酶

• 批准号: 10164829
• 负责人: Thomas Garcia
• 金额: $ 31.12万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10164829
• 关键词: Acrosome; Acrosome Reaction; Affinity; Binding; Biochemical; Biological; Biology; CRISPR/Cas technology; Cells; Chemicals; Chemistry; Collaborations; Collection; Complement; Contraceptive Agents; Contraceptive methods; DNA; Data; Defect; Development; Drug Kinetics; Drug Targeting; Ensure; Enzymes; Epididymis; Event; Evolution; Female; Fertility; Fertilization; Genes; Genetic; Genetic Models; Genomic DNA; Germ Cells; Goals; Herpes zoster disease; Impairment; In Vitro; Infertility; Intervention; Knock-out; Knockout Mice; Laboratories; Lead; Libraries; Male Contraceptive Agents; Methodology; Modeling; Molecular; Molecular Mechanisms of Action; Mus; Oocytes; Pathway interactions; Penetration; Peptide Hydrolases; Pharmaceutical Preparations; Phenotype; Physiological; Process; Property; Proteins; Recombinant Proteins; Reproduction; Sampling; Serine Protease; Sperm Motility; Sperm Penetration; Surface; Technology; Testing; Testis; Therapeutic Intervention; Transgenic Mice; Validation; Zona Pellucida; acrosin; analog; contraceptive target; design; drug discovery; egg; functional genomics; guided inquiry; in vitro Model; in vivo; inhibitor/antagonist; male; male fertility; medical specialties; men; migration; mouse model; mutant; novel; paralogous gene; pre-clinical; reproductive; reproductive tract; scaffold; screening; small molecule; small molecule inhibitor; small molecule therapeutics; sperm cell; trypsin-like serine protease

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.

项目3摘要（使用遗传和化学方法操纵精子特异性蛋白酶） 项目3的总体目标是阐明男性的分子机制 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，并使用这些数据指导发现新型非荷尔蒙避孕药的发现。 生物学靶标是药物发现努力的理想选择，是那些具有重要且非冗余生物学的生物学目标 作用，高度特异性的分子功能和生化特征，可应对分子抑制作用。不 存在识别或验证有效药物靶标的单一确定方法，当然 进化过程没有被驱动以产生许多生理脆弱的途径，尤其是 用于避孕。因此，对育肥的分子机制也有透彻的理解 作为采样各种干预点的方法，是必需的。作为目标的补充 项目3从项目1和2中出现，将重点关注五个丝氨酸的遗传和化学分析 蛋白酶样酶的表达限于雄性生殖道。这些机械研究将 允许我们同时将这些丝氨酸蛋白酶样酶放入生殖途径中 确定每种蛋白质作为避孕靶标的效用。精子 - 佐纳的早期研究 Pellucida相互作用假设需要精子中包含的酶 积云细胞层和Zona Pellucida的渗透。最近，显然是 与释放的杂色事件有关（在Acrosos反应期间）是必不可少的 为了受精，既不是与Zona pellucida接触，也不需要在接近 卵母细胞的外部服装。同样，运动精子对于成功的施肥至关重要。我们的整体 假设是丝氨酸蛋白酶样酶是雄性生育力所需的一类新型蛋白质 为这些精子生成的酶发现小分子抑制剂将导致发展 独特的男性避孕药。我们的具体目的如下：1）使用男性CRISPR/CAS9模型 生殖道特异性蛋白酶以阐明其在繁殖方面的需求； 2）辅助表达和 用于DNA编码化学技术的项目1、2和3的重组蛋白的纯化（DEC- TEC）选择性选择； 3）使用DEC-TEC发现新丝氨酸的小分子探针和抑制剂 生育需要的蛋白酶状酶，并评估可接受的早期药物样铅和类似物 概念验证避孕研究中的药代动力学特性。