High-throughput screens for eEF-2 kinase

Ashwini K. Devkota, Mangalika Warthaka, Ramakrishna Edupuganti, Clint D.J. Tavares, William H. Johnson, Bulent Ozpolat, Eun Jeong Cho, Kevin N. Dalby

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

eEF-2 kinase is a potential therapeutic target for breast cancer, gliomas, and depression. No potent inhibitors of eEF-2K have been reported, and thus development of high-throughput assay systems may expedite the process. Two high-throughput assays are described for eEF-2K using recombinant, tag-free enzyme purified from bacteria. The first is a fluorescence-based assay that uses the phosphorylation of a Sox-based peptide substrate by eEF-2K, which results in a 5-fold increase in fluorescence emission, allowing for continuous monitoring of the kinase activity. The second is a luminescence-based assay that produces a luminescence signal, which correlates with the amount of adenosine triphosphate remaining in the kinase reaction. Both assays have been optimized and miniaturized for a 384-well plate format and validated in screens. In conclusion, we demonstrated that a traditional radiolabeled assay can be readily transferred to universal spectroscopic assays that are robust and will facilitate high-throughput screening of larger size libraries for the identification of small-molecule inhibitors and significantly contribute to the development of therapies for targeting eEF2K.

Original languageEnglish (US)
Pages (from-to)445-452
Number of pages8
JournalJournal of Biomolecular Screening
Volume19
Issue number3
DOIs
StatePublished - Mar 2014

Keywords

  • assay development
  • eEF-2K inhibitors
  • high throughput screen
  • luminescence
  • Sox

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biotechnology
  • Biochemistry
  • Molecular Medicine
  • Pharmacology
  • Drug Discovery

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