Pharmacological and phosphoproteomic studies of HIPK4-dependent spermatogenesis

本研究利用化学和生物学的尖端技术，提高我们对精子成熟的理解，并促进安全可逆的男性避孕方法的发展。目前男性避孕的选择是避孕套和输精管结扎术，它们分别受到其有效性和侵入性的限制。开发男性激素避孕药的努力受到不良副作用的阻碍，而开发中的非激素避孕药可能受到靶效应的限制。男性缺乏有效的避孕选择是美国和全球意外怀孕流行的部分原因，这对母亲和孩子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素。最近，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 - / -雄性小鼠是不育的，但在其他方面表现出正常的发育、生理和行为，这突出了Hipk4作为安全避孕靶点的潜力。此外，HIPK4主要在晚期精子中表达，这表明如果怀孕，抑制它不会对男性种系造成不可逆的损伤或引入遗传病变。因此，假设小分子抑制HIPK4将提供安全可逆的男性避孕。此外，研究人员推测，确定hipk4调控的磷酸化蛋白质组将为精子延伸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依赖性磷酸化蛋白，并表征其在精子发生中的作用。在Aim 1中，一种基于细胞的高通量检测将用于筛选HIPK4抑制剂结构多样的化学文库。命中将使用额外的细胞读数和生化分析进行验证。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，基于质谱的方法将用于阐明生殖细胞中hipk4调控的磷蛋白组。免疫荧光显微镜、western blot和丝状肌动蛋白沉淀法将用于检测HIPK4与这些假定的底物和下游效应物之间的功能相互作用。在Aim 3中，选定的磷蛋白在精子成熟中的作用将通过改变其在精原干细胞中的表达来研究。

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.