Sunday, June 1, 2025
6/1/2025
Reversible male contraception by inhibition of serine/threonine kinase 33 - PROJECT SUMMARY The overarching objective of this new R01 grant proposal is to perform preclinical drug development studies to create potent, selective, metabolically stable, and peripherally restricted serine/threonine kinase 33 (STK33) inhibitors that can provide a foundation as first-in-class non-hormonal contraceptive drugs for men. Kinases are key enzymatic targets for drug inhibition in the pharmaceutical industry. However, only ~70 of the 538 human kinases have FDA-approved inhibitor drugs or drug candidates. Therefore, a vast number of therapeutic opportunities exist by targeting kinases. A subset of ~160 kinases are poorly understood or studied and are designated as “dark kinases”. One of these dark kinases is STK33, which is testis-enriched and expressed in spermatocytes through transformation of spermatids. Stk33 knockout males are sterile secondary to teratozoospermia and sperm immotility, and men in a single family were discovered to have a frameshift mutation in the STK33 gene, leading to infertility that phenocopied the knockout mice. Because mice and men with these STK33 mutations are grossly normal, we hypothesize that STK33 is a pursuable target with low safety risk for contraceptive inhibition in men. To discover STK33 probe or tool compounds for in vivo chemical perturbation of STK33 function, we used DNA-Encoded Chemistry Technology (DEC-Tec) and screened 3.9 billion unique DNA-molecules with human STK33 to identify high affinity binders. We created several potent and selective kinase inhibitors, including CDD-2807, which showed picomolar biochemical inhibition of STK33 (Kd = 20 pM) and nanomolar cellular inhibition of STK33 (IC50 = 9 nM) versus other kinases. In a contraceptive fertility trial in mice, CDD-2807 induced reversible contraceptive effects in male mice without causing any observable toxic effects or greatly altering testis size. Thus, our studies prove that STK33 is a genetically and chemically validated contraceptive target and that CDD-2807 is a favorable lead chemical probe suitable for in vivo studies of STK33 function. The Specific Aims are: 1) Use DEC-Tec screens, medicinal chemistry, and X-ray crystallography to create more potent, selective, and metabolically stable STK33 inhibitors; 2) Assess the metabolic and pharmacokinetic properties, tissue distribution, and safety of STK33 inhibitors in mice and non-human primates; and 3) Evaluate the contraceptive effects of optimized STK33 lead inhibitors in mice and non-human primates. Based on our preliminary studies and our outstanding interdisciplinary team, which works at the intersection of mouse and non-human primate reproductive biology, chemical biology, medicinal chemistry, structural biology, drug metabolism, and pharmaceutical discovery, these R01 studies are highly feasible, will allow us to create optimized lead inhibitors of STK33 for an Investigational New Drug application, and will address an unmet medical need for male contraceptive therapeutics.
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