Abstract
We used a progressive elimination strategy to identify oocyte-specific WEE2 kinase inhibitors for potential non-hormonal contraceptives that target meiosis. Beginning with an in-house library of over 300,000 compounds, virtual high throughput screening identified 57 WEE2 inhibitors with preferential predicted binding over the somatic variant WEE1. Seven compounds were further evaluated in vitro by enzyme-linked immunosorbent assay to measure biochemical inhibition on WEE1 and WEE2 phosphorylation of CDK1. To assess specificity, we evaluated WEE2-mediated inhibition of meiosis using in vitro oocyte fertilization, and WEE1-mediated inhibition of mitosis using a somatic cell proliferation assay. Our results from these assays identified three candidates for further development: 6-(2,6-dichlorophenyl)-2-((4-(2-(diethylamino)ethoxy) phenyl)amino)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (2), 6-(2,6-dichlorophenyl)-8-methyl-2-((4-morpholinophenyl) amino)pyrido[2,3-d]pyrimidin-7(8H)-one (12), and 3-((6-(2,6-dichlorophenyl)-8-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)benzoic acid (16).
Highlights
In somatic cells, WEE1, a serine/threonine protein kinase, is a critical negative regulator of the eukaryotic cell cycle that functions as a checkpoint to ensure adequate cell growth has been achieved before entry into mitosis.[1]
Within the germinal vesicle (GV), WEE2 regulates CDK1 similar in action to WEE1 and prevents complexing with cyclin B, inactivating M-phase promoting factor (MPF)
A homology model of WEE2 was generated based on a solved crystal structure of WEE1 with inhibitor PD352396 (PDB:3BI6) using Schrodinger’s molecular modeling suite Maestro, which allows for determination of ligand fit in the binding pocket and assigns a quantitative docking score to each ligand binding pose so that output can be ranked according to best predicted binding
Summary
WEE1, a serine/threonine protein kinase, is a critical negative regulator of the eukaryotic cell cycle that functions as a checkpoint to ensure adequate cell growth has been achieved before entry into mitosis.[1]. WEE2 plays a dual regulatory role in oocyte meiosis by preventing premature resumption prior to ovulation and permitting metaphase II exit at fertilization.[3] Mutations in the WEE2 gene are associated with female infertility and have been identified in women who experience recurrent fertilization failure.[4] During prophase I of meiosis, premature oocytes in growing ovarian follicles are arrested in the germinal vesicle (GV) nuclear configuration until recruited for ovulation. This arrest is maintained by elevated levels of cAMP which activate protein kinase A (PKA). When MPF is inactive, it blocks chromosome reduction to a haploid configuration (Figure 1A).[5]
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