Development of WEE2 kinase inhibitors as novel non-hormonal female contraceptives that target meiosis

Carol B. Hanna, Deepti Mudaliar, Kristen John, C. Leigh Allen, Luxin Sun, Jon E. Hawkinson, Ernst Schönbrunn, Gunda I. Georg, Jeffrey T. Jensen

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

WEE2 oocyte meiosis inhibiting kinase is a well-conserved oocyte specific kinase with a dual regulatory role during meiosis. Active WEE2 maintains immature, germinal vesicle stage oocytes in prophase I arrest prior to the luteinizing hormone surge and facilitates exit from metaphase II arrest at fertilization. Spontaneous mutations at the WEE2 gene locus in women have been linked to total fertilization failure indicating that selective inhibitors to this kinase could function as non-hormonal contraceptives. Employing co-crystallization with WEE1 G2 checkpoint kinase inhibitors, we revealed the structural basis of action across WEE kinases and determined type I inhibitors were not selective to WEE2 over WEE1. In response, we performed in silico screening by FTMap/FTSite and Schrodinger SiteMap analysis to identify potential allosteric sites, then used an allosterically biased activity assay to conduct high-throughput screening of a 26 000 compound library containing scaffolds of known allosteric inhibitors. Resulting hits were validated and a selective inhibitor that binds full-length WEE2 was identified, designated GPHR-00336382, along with a fragment-like inhibitor that binds the kinase domain, GPHR-00355672. Additionally, we present an in vitro testing workflow to evaluate biological activity of candidate WEE2 inhibitors including; (1) enzyme-linked immunosorbent assays measuring WEE2 phosphorylation activity of cyclin dependent kinase 1 (CDK1; also known as cell division cycle 2 kinase, CDC2), (2) in vitro fertilization of bovine ova to determine inhibition of metaphase II exit, and (3) cell-proliferation assays to look for off-target effects against WEE1 in somatic (mitotic) cells.

Original languageEnglish (US)
Pages (from-to)368-377
Number of pages10
JournalBiology of reproduction
Volume103
Issue number2
DOIs
StatePublished - Aug 1 2020

Bibliographical note

Funding Information:
Bill & Melinda Gates Foundation OPP1178119, NIH P51OD011092, NICHD U54HD055744 and U01HD076542.

Publisher Copyright:
© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Keywords

  • Fertilization
  • Meiosis
  • Non-hormonal contraceptive
  • Oocyte maturation
  • WEE2 kinase

Fingerprint

Dive into the research topics of 'Development of WEE2 kinase inhibitors as novel non-hormonal female contraceptives that target meiosis'. Together they form a unique fingerprint.

Cite this