The cation channel of sperm (CatSper) is a calcium ion channel specifically located on the flagellar membrane of mature sperm. Genetic knockout of any pore-forming subunit of CatSper abrogates sperm Ca2+ current and hyperactivated motility, leading to infertility in male mice, which makes it an attractive target for male contraception. To identify potential scaffolds as CatSper blockers, an HTS campaign with over 72,000 compounds was performed with our in-house library using a FLIPR Ca2+ influx assay. The primary screen used high pH/K+ to elicit Ca2+ influx, which provided 220 compounds that showed ≥ 60% inhibition at a concentration of 10 µM. Of the 220 scaffolds, 104 compounds displayed well-defined dose-response curves with IC50 values ≤55 µM. After further triage of these compounds based on fluorescence interference, unfavorable physicochemical properties, and potency, 12 compounds were prioritized for repurchase and retest. 8 compounds were reconfirmed in the FLIPR assay in which the Ca2+ influx was induced by progesterone. The removal of the non-selective dopamine agonist, apomorphine, from these 8 compounds provided 7 validated hits. The cytotoxicity of hits was tested in sperm and IMR-90 cells. Patch-clamp and FLIPR assays were performed to evaluate their selectivity over hSlo3, hERG, hCav1.2, and hNav1.5. Hit compound 7a was identified as a potent CatSper inhibitor with high selectivity for CatSper over hCav1.2 and hNav1.5, moderate selectivity over hSlo3 and hERG, and low cytotoxicity, and is therefore the most promising inhibitor identified in this study. A brief SAR study was also established for the 7 hit compounds. The discovery and characterization of these new CatSper blockers provide useful starting points for chemical probe development and drug discovery efforts.

Discovery and characterization of multiple classes of human CatSper blockers

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ACS Fall 2022

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