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

• 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
• 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

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.

该建议使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，并推动安全可逆的男性避孕药的发展。男性避孕的当前选择是避孕套和输精管切除术，分别受其有效性和侵入性的限制。不希望的副作用阻碍了开发雄性激素避孕药的努力，而发育中的非激素避孕药可能受到目标影响的限制。男性缺乏有效的避孕选择部分是导致美国和全球意外怀孕的普遍性，这对母亲和儿童都有负面的健康状况。为了满足这种未满足的需求，需要发现，表征和靶向新的精子发生因素。最近，同源域相互作用蛋白激酶4（HIPK4）被确定为精子发生的必要驱动力。 HIPK4 - / - 雄性小鼠是不育的，但其他裸露的正常发育，生理和行为，强调了HIPK4作为安全目标的潜力。此外，HIPK4主要在晚期精子中表达，表明其抑制作用不会不可逆转地损害男性种系或引入遗传病变，如果达到妊娠。因此，假设小分子对HIPK4的抑制作用将提供安全且可逆的男性避孕药。此外，假设表征HIPK4调节的磷蛋白组将为精子伸长的分子机制提供新的见解，并可能揭示其他避孕靶标。这项工作将识别和优化HIPK4抑制剂，并鉴定HIPK4依赖性磷蛋白，并表征其在精子发生中的作用。在AIM 1中，将使用基于细胞的高通量测定法来筛选HIPK4抑制剂结构多样的化学库。使用其他蜂窝读数和生化测定将验证命中率。 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。在AIM 2中，将使用基于质谱的方法来阐明生殖细胞中HIPK4调节的磷酸蛋白酶。免疫荧光显微镜，蛋白质印迹和丝状肌动蛋白沉淀将用于检查HIPK4与这些假定的底物和下游效应之间的功能相互作用。在AIM 3中，将通过改变其精子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。