Development of RBM46 Small Molecular Degraders for Reversible Male Contraception

Yaxia Yuan¹, Guiming Li¹, Alex Taylor¹, Daohong Zhou¹, Stanton McHardy², Corinna Ross³, Christopher Geyer⁴, Brian Hermann⁵

1. Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229

2. Department of Chemistry, University of Texas at San Antonio, San Antonio, TX 78249

3. Texas Biomedical Research Institute, San Antonio, TX 78227

4. Department of Anatomy and Cell Biology, East Carolina University, Greenville, NC 27834

5. Department of Neuroscience, Developmental and Regenerative Biology, University of Texas at San Antonio, San Antonio, TX 78249

 

Abstract Text:

Development of non-steroidal male contraceptive drugs to-date has met with poor success, often due to the difficulty of accessing targets in meiotic spermatocytes, post-meiotic spermatids, and sperm located inside the blood-testis barrier (BTB). A particularly appealing alternative approach for male contraception is to target a protein of interest (POI) expressed by differentiating germ cells outside the BTB because: 1) it would obviate consideration of BTB transit for contraceptive compounds; 2) differentiating germ cells (spermatogonia and early primary spermatocytes) are distinct from the spermatogonial stem cell (SSC) pool, and thus can be targeted without eliminating the germline; and 3) these cells earlier in spermatogenesis are considerably less numerous, increasing the likelihood of contraceptive effectiveness. We identified a highly promising contraceptive POI – the RNA binding protein RBM46. RBM46 is only expressed in germ cells, binds and regulates mRNAs encoding key spermatogenic proteins, and is essential for both spermatogonial differentiation and meiosis. Indeed, Rbm46 knockout (KO) mice are sterile and their testes still contain SSCs and undifferentiated progenitor spermatogonia. Importantly, Rbm46 KO mice have no other phenotype except infertility, indicating a high safety threshold and positioning RBM46 as a validated male contraceptive target. Although targeting RBM-containing proteins with conventional small molecule inhibitors can be a challenge due to their planar functional domains, we made the recent discovery that RBM46 exists as a dimer and predicted that monomeric RBM46 would be unstable and thus prone to rapid degradation. This finding prompted us to identify small molecules with the capacity to both bind RBM46 and disrupt dimerization. Using a high-throughput HiBiT-based assay, we identified multiple small molecules that – with DC₅₀ in the sub-micromolar range – significantly reduced RBM46 protein levels without cytotoxicity. Taken together, these results validate the feasibility of using small molecules to degrade RBM46 by disrupting dimerization as a reversible means of male contraception.