PROJECT 1 SUMMARY (Targeting sperm-specific proteins during meiosis and sperm morphogenesis) The overall goals of Project 1 are to use CRISPR/Cas9 to understand the formation of the sperm- specific acrosome and identify drug-like probes and preclinical candidates to target spermatogenic- specific druggable proteins for a contraceptive effect in vivo. The human population is reaching alarming numbers, resulting in changes in our climates and ecosystems and likely global shortages of food, water, and other resources for our children and grandchildren. To curb rampant population growth and its dire consequences, we require contraceptives that specifically target the male germline. Contraceptives that target testis-specific proteins should eliminate unwanted side effects. As contraceptive targets, we will focus on two meiosis-specific proteins and four sperm-essential, acrosome-associated transmembrane proteins. These six evolutionarily-conserved proteins were chosen based on proof-of-principle studies in the mouse showing that they are specifically required for fertility in males, our success at delivering small molecules for reversible contraception in vivo, and the potential of these proteins as druggable targets for male contraception. Seventy percent of FDA-approved drugs target either secreted or transmembrane proteins. Further, how the acrosomal proteins interact during acrosome formation is unclear. To define the interactome for formation and function of the acrosome, we will collaborate with Dr. Ikawa (Project 2) to use CRISPR/Cas9 to insert tag sequences into these four genes. To identify small-molecule probes and preclinical candidates, we will partner with the DNA- Encoded Chemistry Technology (DEC-Tec) Core. Our overall hypothesis is that CRISPR/Cas9 and DEC-Tec will help us to define the protein interactome during spermatogenesis, rapidly identify multiple small-molecules that are directed at these essential spermatogenic proteins, and create an assortment of oral and implantable contraceptives for men and women. The Specific Aims of Project 1 are: 1) Use CRISPR/Cas9 to functionally understand acrosome formation and the specific relationship of four sperm-essential transmembrane proteins; 2) Employ DEC-Tec to identify small-molecule probes and preclinical candidates to target spermatogenic- specific “druggable” proteins; and 3) Evaluate potential in vivo contraceptive effects of small-molecule inhibitors. The success of this P01 and Project 1 relies on interactions with Projects 2 and 3 and the DEC-Tec Core. Dr. Ikawa and his colleagues created several key models and will generate the tagged alleles for Aim 1. Dr. Sonnenburg and his Project 3 team will help to produce recombinant proteins for our Aim 2 DEC-Tec screens. Drs. Young, Huang, Sonnenburg, Simmons, Yu, Lee, and Li are experts at using DEC-Tec, medicinal chemistry, X-ray crystallography, and drug metabolism studies for uncovering lead compounds. Project 1 will perform the in vi...