Pharmacological and phosphoproteomic studies of HIPK4-dependent spermatogenesis

HIPK4 依赖性精子发生的药理学和磷酸蛋白质组学研究

基本信息

批准号：
10538548
负责人：
Steven Swick
金额：
$ 2.21万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-01 至 2023-02-03
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10538548
关键词：
Acne Acrosome Reaction Actins Address Advanced Development Agonist Behavior Binding Biochemical Biological Biological Assay Biology Cells Cellular Assay Chemistry Child Competence Computer Analysis Contraceptive Agents Contraceptive methods Crystallography Cytoskeleton Data Set Defect Development Docking Drug Targeting Enzymes Erinaceidae Event Exhibits F-Actin Fertility Fertilization in Vitro Genetic Germ Cells Goals Health Healthcare Homology Modeling Immunofluorescence Microscopy In Vitro Infertility Knock-out Laboratories Lead Lesion Link Male Contraceptive Agents Mass Spectrum Analysis Molecular Moods Mothers Mus Oocytes Outcome Pathway interactions Pharmaceutical Chemistry Phosphoproteins Phosphotransferases Physiology Pregnancy Prevalence Protein Kinase Interaction Proteins Role Sedimentation process Somatic Cell Sperm Maturation Spermatids Spermatogenesis Spermiogenesis Stem cell transplant Structure Structure-Activity Relationship Techniques Testing Testis Transgenic Animals Transplantation United States Vasectomy Vasovasostomy Western Blotting Work X-Ray Crystallography antagonist condoms contraceptive target counterscreen design high throughput screening homeodomain hormonal contraception improved in vivo inhibitor insight male male fertility maternal outcome men mouse model overexpression pharmacologic phosphoproteomics scaffold side effect small molecule small molecule inhibitor small molecule libraries sperm cell stem cells thrombotic transcription factor unintended pregnancy

项目摘要

This proposal uses cutting-edge techniques in chemistry and biology to improve our understanding of sperm maturation and advance the development of safe and reversible male contraception. Current options for male contraception are condoms and vasectomy, which are limited by their efficacy and invasiveness, respectively. Efforts to develop male hormonal contraceptives have been hindered by undesired side effects, and non-hormonal contraceptive agents in development may be constrained by on target effects. The lack of effective contraceptive options for men is in part responsible for the prevalence of unintended pregnancy in the United States and globally, which has negative health outcomes for both mother and child. In order to address this unmet need, new spermatogenetic factors need to be discovered, characterized and targeted. Recently, homeodomain-interacting protein kinase 4 (HIPK4) was identified as an essential driver for spermiogenesis. Hipk4–/– male mice are infertile but otherwise exhibit normal development, physiology, and behavior, highlighting the potential of HIPK4 to serve as a safe target for contraception. In addition, HIPK4 is expressed primarily in late-stage sperm, indicating that its inhibition would not irreversibly damage the male germline or introduce genetic lesions if pregnancy is achieved. Therefore, it is hypothesized that small-molecule inhibition of HIPK4 will provide safe and reversible male contraception. Furthermore, it is hypothesized that characterizing the HIPK4-regulated phosphoproteome will provide new insights into the molecular mechanisms of spermatid elongation and may reveal additional contraceptive targets. This work will identify and optimize HIPK4 inhibitors and identify HIPK4-dependent phosphoproteins and characterize their roles in spermiogenesis. In Aim 1, a cell-based, high-throughput assay will be used to screen a structurally diverse chemical library for HIPK4 inhibitors. Hits will be validated using additional cellular readouts and biochemical assays. The crystal structure of HIPK4 will be concurrently pursued to facilitate computationally assisted inhibitor design and optimization. In Aim 2, a mass spectrometry-based approach will be used to elucidate the HIPK4-regulated phosphoproteome in germ cells. Immunofluorescence microscopy, western blot, and filamentous actin sedimentation will be used to examine functional interactions between HIPK4 and these putative substrates and downstream effectors. In Aim 3, the roles of selected phosphoproteins in sperm maturation will be investigated by altering their expression in spermatogonial stem cells.

该提案利用化学和生物学领域的尖端技术来提高我们对精子成熟的理解，并促进安全和可逆男性避孕的发展。目前男性避孕的选择是避孕套和输精管结扎术，它们分别受到其功效和侵入性的限制。开发男性激素避孕药的努力因不良副作用而受到阻碍，而开发中的非激素避孕药可能会因缺乏有效的避孕选择而受到限制。在美国和全球范围内，男性对意外怀孕的流行负有部分责任，这对母亲和孩子的健康都有负面影响。为了解决这一未得到满足的需求，最近需要发现、描述和瞄准新的精子发生因素。，同源结构域相互作用蛋白激酶 4 (HIPK4) 被确定为精子发生的重要驱动因素，Hipk4–/– 雄性小鼠不育，但在其他方面表现出正常的发育、生理和行为，这凸显了 HIPK4 发挥精子发生作用的潜力。此外，HIPK4主要在晚期精子中表达，这表明如果实现妊娠，其抑制不会不可逆地损害男性种系或引入遗传损伤。 HIPK4 将提供安全且可逆的男性避孕，此外，研究 HIPK4 调节的磷酸化蛋白质组将为精子细胞伸长的分子机制提供新的见解，并可能揭示其他避孕靶点。这项工作将鉴定和优化 HIPK4 抑制剂，鉴定 HIPK4 依赖性磷蛋白，并表征它们在精子发生中的作用。在目标 1 中，将使用基于细胞的高通量测定来筛选结构多样化的 HIPK4 抑制剂化学库。 HIPK4 的晶体结构将同时进行，以促进计算辅助抑制剂的设计和优化。基于光谱分析的方法将用于阐明生殖细胞中 HIPK4 调节的磷酸化蛋白质组，免疫荧光显微镜、蛋白质印迹和丝状肌动蛋白沉降将用于检查 HIPK4 与这些假定底物和下游效应子之间的功能相互作用。将通过改变精原干细胞中的表达来研究选定的磷蛋白在精子成熟中的作用。