The central goal of this project is to identify and characterize small molecule inhibitors of Ubiquitin specific peptidase 51 (USP51), a deubiquitinating (DUB) enzyme exclusive to sperm, as a means to develop a reversible, non-hormonal male contraceptive. The ubiquitin system is fundamental to ensure protein turnover within mammalian cells. Ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, and ubiquitin protein ligases catalyze the attachment of ubiquitin molecules to proteins, a process that targets them to the proteasome for degradation. DUBs catalyze the removal of ubiquitin to facilitate their recycling. We have identified USP51 to be specifically expressed in late elongating spermatids and epididymal sperm. USP51 depletion in mice using Vivo-Morpholinos results in male infertility due to a complete block of sperm motility. Our overall hypothesis is tat USP51 is a viable contraceptive target and that small molecules existing in nature or modified in the laboratory will effectively and reversibly inhibit USP51 function in epididymal sperm. We will pursue the following Specific Aims to address this hypothesis: 1) Validate the targeting of USP51 as an effective strategy for contraceptive development. 2) Screen small molecule libraries in silico and in vitro to identify hit compounds. 3) Perform hit-to-lead medicinal chemistry optimization and assess the exposure of sperm, testes and animals to candidates for efficacy and reversibility. These studies will advance our basic understanding of spermatogenesis and sperm function, as well as provide the potential for developing new contraceptive agents that reversibly inhibit a critical sperm enzyme.