High-throughput screens for eEF-2 kinase.
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.
1Texas Screening Alliance for Cancer Therapeutics, The University of Texas at Austin, TX, USA.
Ashwini K
AK
Devkota
Mangalika
M
Warthaka
Ramakrishna
R
Edupuganti
Clint D J
CD
Tavares
William H
WH
Johnson
Bulent
B
Ozpolat
Eun Jeong
EJ
Cho
Kevin N
KN
Dalby
D000970:Antineoplastic Agents / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor; D054736:Elongation Factor 2 Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D008163:Luminescent Measurements / Q000379:methods; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015203:Reproducibility of Results; D013379:Substrate Specificity
D000970:Antineoplastic Agents; D010455:Peptides; D047428:Protein Kinase Inhibitors; D054736:Elongation Factor 2 Kinase
Sox; assay development; eEF-2K inhibitors; high throughput screen; luminescence