PROJECT SUMMARY/ABSTRACT While hormonal contraceptive methods are highly effective, they are not the method of choice for many women. The National Health Statistic Report indicates that on-demand over-the-counter (OTC) contraceptives (i.e., spermicides and condoms) are the products of choice for many women, especially teenagers. Unfortunately, OTC methods have high failure rates. As a result, teenage pregnancies contribute to more than 70% of unintended pregnancy rates in the United States. Therefore, there is a critical need for new and improved on- demand methods for women. In this proposal, we will explore a new female contraceptive method that will target two processes: 1) blocking post-ejaculated semen and 2) inhibiting sperm motility in the female reproductive tract. After ejaculation, semen changes from a gel-like to a watery consistency via the prostate-derived serine protease called kallikrein 3 (KLK3). Therefore, the central hypothesis for this application is that pharmacologic blockade of semen liquefaction provides a contraceptive effect by blocking sperm from reaching the site of fertilization. Our previous work showed that a serine protease inhibitor effectively blocked those two processes and prevented pregnancy in female mice. Based on these findings, it is hypothesized that small molecule KLK3 inhibitors with targeted activity can be developed for use as novel, non-hormonal, fully reversible contraceptives for women. In this application, a team of investigators will test the action of small molecule KLK3 inhibitor(s) in human semen and sperm function, pregnancy prevention in female rhesus macaques, and will identify novel small molecules inhibitors of KLK3 in addition to the prototype inhibitor. The hypothesis will be tested in three Specific Aims: 1) to characterize the contraceptive potential of specific KLK3 inhibitors in human semen, 2) To evaluate the cellular toxicity of specific KLK3 inhibitor and “hit” compound(s) in human cervical/vaginal cells and 3D vaginal cultures, 3) to characterize the contraceptive efficacy and pharmacodynamics/kinetics of specific KLK3 inhibitor in female rhesus macaques, and 4) To identify novel drug- like KLK3-specific inhibitors from DNA-encoded chemical libraries. Upon completion of this study, we will be able to demonstrate whether serine protease inhibitor can be developed as the first contraceptive product (i.e., film/sponge) that prevents semen liquefaction as well as inhibits the sperm function.