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

• Grant number: 21/06718-7
• Support Opportunities: Research Grants - Young Investigators Grants - Phase 2
• Start date: October 01, 2022
• End date: September 30, 2027
• Field of knowledge: Biological Sciences - Pharmacology - Biochemical and Molecular Pharmacology
• Principal Investigator: Erick José Ramo da Silva
• Grantee: Erick José Ramo da Silva
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Associated researchers: Fabíola Freitas de Paula Lopes ; Isadora Marques Paiva ; John Howl ; Marcelo Tigre Moura ; Margarida Sâncio da Cruz Fardilha ; Mariano G Buffone ; Thiago Mattar Cunha
• Associated research grant: 15/08227-0 - A study on EPPIN as a drug target for male contraception: developing a new animal model to test the efficacy of male contraceptive drugs, AP.JP
• Associated research grant(s): 23/06885-6 - Multi-user equipment approved in grant 21/06718-7: Axiovert 5 inverted microscope for bright field, phase contrast, Plas-Dic, and fluorescence, AP.EMU
• Associated scholarship(s): 25/04157-9 - Dengue Virus (DENV-2)-Induced Epididymitis in Mice: Overview of the Inflammatory Profile and Immune Cell Recruitment., BP.DR; 24/19000-5 - Exploring the role of the NLRP3 inflammasome in epididymitis: consequences for tissue damage and cell death pathways., BP.MS; 25/02400-3 - Implementation and operationalization of Multi-user equipment approved in grant 21/06718-7: Axiovert 5 inverted microscope for bright field, phase contrast, Plas-Dic, and fluorescence, BP.TT; + associated scholarships 25/02448-6 - Developing EPPIN-binding peptides with spermostatic activity for male contraception, BP.PD; 24/16122-2 - Analysis of the bioportide Modified Stop Sperm 1 (MSS1) on mouse sperm motility, BP.IC; 23/04496-2 - EPPIN as a binding site for the seminal plasma protein SVS2 on mouse spermatozoa: effects on capacitation, mechanism of action and applications in male contraception, BP.DD; 23/03529-4 - A novel CRISPR/Cas9 transgenic mouse model for in vivo studies on EPPIN, a sperm-based male contraceptive drug target., BP.PD; 23/00125-0 - Dissecting the roles of EPPIN/SVS2 binding in the modulation of mouse sperm motility., BP.IC - associated scholarships

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)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

SPADELLA, MARIA ANGELICA; SILVA, ERICK J. R.; CHIES, AGNALDO BRUNO; DE ALMEIDA, LAINE ANDREOTTI. Insights Into Antioxidant Strategies to Counteract Radiation-Induced Male Infertility. Antioxidants & Redox Signaling, v. 40, n. 13-15, p. 26-pg., 2023-12-22. (21/06718-7, 19/09488-2)

GOMES, ANTONIEL A. S.; SANTOS, NATALIA C. M.; ROSA, LEONARDO R.; BORGES, RAFAEL J.; FONTES, MARCOS R. M.; HAMIL, KATHERINE G.; O'RAND, MICHAEL G.; SILVA, ERICK J. R.. Interactions of the male contraceptive target EPPIN with semenogelin-1 and small organic ligands. SCIENTIFIC REPORTS, v. 13, n. 1, p. 11-pg., 2023-09-01. (21/06718-7, 15/08227-0, 21/04746-3)

ANDRADE, ALEXANDRE D.; ALMEIDA, PRISCILA G. C.; MARIANI, NOEMIA A. P.; SANTOS, NATALIA C. M.; CAMARGO, ISABELA A.; MARTINI, POLIANA V.; KUSHIMA, HELIO; AI, DINGDING; AVELLAR, MARIA CHRISTINA W.; MEINHARDT, ANDREAS; et al. Regional modulation of toll-like receptor signaling pathway genes in acute epididymitis in mice. ANDROLOGY, v. 12, n. 5, p. 14-pg., 2024-03-18. (21/06718-7, 21/04746-3)