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(利用遗传和化学方法操纵精子特异性蛋白水解酶) 项目3的总体目标是阐明男性的分子作用机制 利用遗传学和化学方法研究生殖道特异的丝氨酸蛋白酶样酶 生物学，并使用这些数据来指导新型非激素避孕药的发现。 对于药物发现工作来说，理想的生物靶点是那些具有显著和非冗余的生物靶点 作用、高度特异的分子功能和易于分子抑制的生化特征。不是 对于有效的药物靶点的识别或确认，存在单一的权威方法，并且肯定 进化的过程并不会产生许多生理上脆弱的途径，特别是 用于避孕。因此，对生育的分子机制也有深入的了解 作为一种对广泛的干预点进行抽样的方法，是可取的。作为对目标的补充 从项目1和项目2中脱颖而出的项目3将侧重于五种丝氨酸的遗传和化学分析 仅在男性生殖道表达的类蛋白水解酶。这些机械论的研究将 允许我们将这些丝氨酸蛋白酶样酶放入生殖途径，并同时 确定每种蛋白质作为避孕靶标的效用。精子带的早期研究 透明带相互作用假设精子顶体中包含的酶是 卵丘细胞层和透明带的穿透。最近，情况变得很明显，尽管 顶体反应过程中与顶体酶释放相关的胞吐过程是必不可少的。 对于受精，它既不是通过接触透明带而诱导的，也不要求发生在靠近卵透明带的地方 卵母细胞的外衣。同样，能动的精子对于成功受精也是必不可少的。我们的整体 假设丝氨酸蛋白酶样酶是男性生育所需的一类新的蛋白质，而 这些生精所需酶的小分子抑制剂的发现将导致这一发展 独一无二的男性避孕药。我们的具体目标如下：1)使用CRISPR/Cas9模型 生殖道特异的蛋白水解酶以阐明其在生殖中的需求；2)辅助表达和 从项目1、2和3中提纯用于DNA编码化学技术(DEC- Tec)亲和选择；以及3)使用DEC-Tec发现新型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和类似物是否可接受 体内避孕概念验证研究中的药代动力学特性。