甘油醛3-磷酸脱氢酶(GAPDHS)人类精子特异性亚型高通量筛选方法的开发与实现

Current chemical genomics Pub Date : 2011-01-01 Epub Date: 2011-07-04 DOI:10.2174/1875397301105010030
Jonathan Z Sexton, Polina V Danshina, David R Lamson, Mark Hughes, Alan J House, Li-An Yeh, Deborah A O'Brien, Kevin P Williams
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引用次数: 11

摘要

局限于男性生殖系的糖酵解同工酶是开发可逆、非激素男性避孕药的潜在目标。GAPDHS是甘油醛-3-磷酸脱氢酶的精子特异性异构体,是糖酵解的必需酶,使其成为合理药物设计的一个有吸引力的靶标。为了实现这一目标,我们对384孔格式的GAPDHS高通量分光光度法进行了优化和验证。随着时间的推移,该试验稳定且耐DMSO。整个平板验证实验的Z值>0.8,表明对HTS的检测是可靠的。从Prestwick收集的测试筛选中鉴定并确认了两种化合物。该实验用于筛选多种化学文库,并鉴定出14个调节重组纯化GAPDHS活性的小分子,其IC50值在1.8至42µM之间。这些化合物可能为研究GAPDHS在精子运动中的作用提供有用的分子工具,并为开发有效的、选择性的GAPDHS抑制剂提供新的避孕药物。
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Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS).

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.

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