Translational studies on the sperm-binding protein EPPIN as a male contraceptive drug target

Abstract

The alarming rate of unintended pregnancies (almost 50%) contributes to the unsustainable world population growth and can have serious health and socioeconomic consequences for women and their families. This scenario underscores the need for novel contraceptives, especially male options, since they are limited to condoms and vasectomy. Druggable proteins with crucial roles in regulating sperm function are compelling pharmacological targets for non-hormonal male contraceptives. The sperm-binding protein EPPIN fits into this male contraception profile. Under normal physiological conditions, EPPIN interaction with the seminal plasma protein SEMG1 on the human sperm surface leads to transient inhibition of sperm function after ejaculation. The ability of EPPIN ligands targeting the SEMG1-binding site to inhibit sperm motility triggered the development of fully innovative spermostatic drugs. We recently showed that EPPIN binds SVS2 (mouse SEMG1 ortholog) in mouse spermatozoa. These facts indicate that EPPIN is evolutionarily conserved in terms of protein-binding partners and roles in sperm function between humans and mice. Indeed, both SEMG1 and SVS2 are seminal plasma inhibitory factors of sperm motility and capacitation-associated events (e.g., hyperactivation and acrosome reaction) and essential for sperm survival in the uterus. These findings pave the way for using mouse models to unravel EPPIN roles on male fertility in vivo. Here, we aim to unfold the mechanisms underlying EPPIN modulation of sperm function, the relevance of its protein-protein interactions in such events using mice as translational models, and to search for novel peptide-based EPPIN ligands displaying spermostatic activity. We will perform protein-protein interaction assays to determine EPPIN/SVS2 binding sequences crucial for their interaction. To test the hypothesis that EPPIN/SVS2 binding is a key event for the regulation of sperm function, we will employ state-of-the-art functional, molecular, and live single-cell imaging methodologies to evaluate whether recombinant SVS2 truncations containing EPPIN-binding sequences inhibit functional parameters (motility, acrosome reaction, and fertilization rates) and signaling pathways (cAMP production, intracellular Ca2+ levels, intracellular pH, and protein phosphorylation profile) governing sperm fertility potential. We will also determine the functional requirement for Eppin in male fertility in vivo generating transgenic Eppin knock-out mice using the CRISPR/Cas9 technology. We will build on this knowledge to rationally design novel peptide-based EPPIN ligands using state-of-the-art microwave-assisted solid-phase peptide synthesis and test their effects on human and mouse sperm motility. This proposal creates a network of scientists from Brazil, Argentina, Portugal, and the United Kingdom in a multidisciplinary team to generate novel insights on EPPIN as a sperm drug target for male contraception, thus providing solid grounds for innovation in Reproductive Pharmacology. (AU)