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.

Male contraceptive options are limited, contributing to the prevalence of unplanned pregnancy and negative health outcomes for mother and child. Through this project, I will develop inhibitors of homeodomain-interacting protein kinase 4 (HIPK4), an enzyme that is required for spermiogenesis and a potential target for male contraception. I will also elucidate the HIPK4-regulated phosphoproteome to advance our understanding of sperm maturation and potentially identify new male contraceptive targets.