Current Chemical Genomics and Translational Medicine

2011, 5 : 30-41
Published online 2011 July 4. DOI: 10.2174/1875397301105010030
Publisher ID: CCGTM-5-30

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

Jonathan Z Sexton,1,2 , Polina V Danshina,3 , David R Lamson,1 , Mark Hughes1 , Alan J House1 , Li-An Yeh1,2 , Deborah A O’Brien, *,3 and Kevin P Williams, *,1,2
1 Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA
2 Department of Pharmaceutical Sciences, North Carolina Central University, Durham, NC 27707, USA, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA
3 Department of Cell and Developmental Biology, Laboratories for Reproductive Biology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA

* Address correspondence to these authors at the Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, Room 1021, North Carolina Central University, Durham, North Carolina 27707, USA; Tel: 919-530-7726; Fax: 919-530-6600; E-mail: kpwilliams@nccu.edu

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.

Keywords:

Glycolysis, GAPDHS, high throughput screening, sperm, contraceptive.