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.

该提案使用化学和生物学的尖端技术来提高我们对精子成熟的理解，并促进安全和可逆的男性避孕的发展。目前男性避孕的选择是避孕套和输精管结扎术，这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受到了不希望的副作用的阻碍，而开发中的非激素避孕药可能会受到靶向效应的限制。男性缺乏有效的避孕选择是造成美国和全球意外怀孕流行的部分原因，这对母亲和儿童的健康都有负面影响。为了解决这一未满足的需求，需要发现、表征和靶向新的精子发生因子。最近，同源结构域相互作用蛋白激酶4（HIPK 4）被确定为精子发生的重要驱动因素。Hipk 4-/-雄性小鼠是不育的，但表现出正常的发育，生理和行为，突出了HIPK 4作为避孕的安全靶点的潜力。此外，HIPK 4主要在晚期精子中表达，这表明其抑制不会不可逆地损害雄性生殖细胞或在怀孕时引入遗传损伤。因此，假设HIPK 4的小分子抑制将提供安全且可逆的男性避孕。此外，据推测，表征HIPK 4调节磷酸化蛋白质组将提供新的见解精子细胞伸长的分子机制，并可能揭示其他避孕目标。 这项工作将鉴定和优化HIPK 4抑制剂，鉴定HIPK 4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HIPK 4抑制剂的结构多样的化学文库。将使用额外的细胞读数和生化测定来验证命中。HIPK 4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。在目标2中，基于质谱的方法将用于阐明生殖细胞中HIPK 4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HIPK 4和这些推定的底物和下游效应器之间的功能相互作用。在目标3中，将通过改变精原干细胞中磷蛋白的表达来研究所选磷蛋白在精子成熟中的作用。