Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS)

Abstract

Glycolytic isozymes that are restricted to the male germline are potential targets for the development of reversible, non-hormonal male contraceptives. GAPDHS, the sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase, is an essential enzyme for glycolysis making it an attractive target for rational drug design. Toward this goal, we have optimized and validated a high-throughput spectrophotometric assay for GAPDHS in 384-well format. The assay was stable over time and tolerant to DMSO. Whole plate validation experiments yielded Z’ values >0.8 indicating a robust assay for HTS. Two compounds were identified and confirmed from a test screen of the Prestwick collection. This assay was used to screen a diverse chemical library and identified fourteen small molecules that modulated the activity of recombinant purified GAPDHS with confirmed IC50 values ranging from 1.8 to 42 µM. These compounds may provide useful scaffolds as molecular tools to probe the role of GAPDHS in sperm motility and long term to develop potent and selective GAPDHS inhibitors leading to novel contraceptive agents.

Inhibition of delta-secretase improves cognitive functions in mouse models of Alzheimer’s disease

“…3c). Since compound 11 has been reported to inhibit GAPDH activity at high-concentrations33, we tested the specificity of compound 11 by performing a GAPDH activity assay. Although compound 11 evidently antagonized δ-secretase activity at 2 μM concentration, it failed to inhibit GAPDH, although it displayed some inhibitory effect at 50 μM concentration.…”

Characterization of EPPIN's Semenogelin I Binding Site: A Contraceptive Drug Target1

“…The development of a nonhormonal male contraceptive will provide more contraceptive choices for men and women and contribute to improving human family health. Although progress has been slow for a variety of reasons, a number of promising strategies have appeared in recent years, including therapeutic ultrasound [1], gamendazol [2,3], retinoic acid antagonist BMS-189453 [4], and the sperm proteins GAPDHS [5], EPPIN [6,7], and CATSPER [8]. Epididymal protease inhibitor (EPPIN; official symbol SPINLW1) made its debut as a target for male contraception in 2004 with the demonstration that blocking EPPIN's function in nonhuman primates with antibodies led to reversible infertility [7].…”

Male contraception

“…3-Phosphate dehydrogenase-S (GAPDS) is an enzyme essential for the sperm-specific glycolysis: disruption renders male mice infertile. [62] Several small molecule inhibitors have been identified [63]. Like CatSper, this approach also has the potential for longer and shorter acting contraception.…”