Investigating the Kinetic Mechanism of Inhibition of Elongation Factor 2 Kinase by NH125: Evidence of a Common in Vitro Artifact

Abstract

Evidence is emerging that elongation factor 2 kinase (eEF-2K) has potential as a target for anti-cancer therapy and possibly for the treatment of depression. Here the steady-state kinetic mechanism of eEF-2K is presented using a peptide substrate and is shown to conform to an ordered sequential mechanism with ATP binding first. Substrate inhibition by the peptide was observed and revealed to be competitive with ATP, explaining the observed ordered mechanism. Several small molecules are reported to inhibit eEF-2K activity with the most notable being the histidine kinase inhibitor NH125, which has been used in a number of studies to characterize eEF-2K activity in cells. While NH125 was previously reported to inhibit eEF-2K in vitro with an IC50 of 60 nM its mechanism of action was not established. Using the same kinetic assay the ability of an authentic sample of NH125 to inhibit eEF-2K was assessed over a range of substrate and inhibitor concentrations. A typical dose-response curve for the inhibition of eEF-2K by NH125 is best fit to an IC50 of 18 ± 0.25 µM and a Hill coefficient of 3.7 ± 0.14, suggesting that NH125 is a weak inhibitor of eEF-2K under the experimental conditions of a standard in vitro kinase assay. To test the possibility that NH125 is a potent inhibitor of eEF2 phosphorylation we assessed its ability to inhibit the phosphorylation of eEF2. Under standard kinase assay conditions NH125 exhibits similar weak ability to inhibit the phosphorylation of eEF2 by eEF-2K. Notably, the activity of NH125 is severely abrogated by the addition of 0.1 % triton to the kinase assay through a process that can be reversed upon dilution. These studies suggest that NH125 is as a non-specific colloidal aggregator in vitro, a notion further supported by the observation that NH125 inhibits other protein kinases, such as ERK2 and TRPM7 in a similar manner to eEF-2K. As NH125 is reported to inhibit eEF-2K in a cellular environment its ability to inhibit eEF2 phosphorylation was assessed in MDA-MB-231 breast cancer, A549 lung cancer and HEK-293T cell lines using a Western blot approach. No sign of decrease in the level of eEF2 phosphorylation was observed up to 12 hours following addition of NH125 to the media. Furthermore, contrary to the previously reported literatures, NH125 induced the phosphorylation of eEF-2.

Targeted Silencing of Elongation Factor 2 Kinase Suppresses Growth and Sensitizes Tumors to Doxorubicin in an Orthotopic Model of Breast Cancer

“…As potent small molecule inhibitors of eEF-2K are not currently available [57], we investigated the potential of targeting eEF-2K by siRNA. The delivery of therapeutics to tumor cells and vasculature is recognized as a potential approach for the treatment of cancer [29].…”

Eukaryotic elongation factor 2 kinase as a drug target in cancer, and in cardiovascular and neurodegenerative diseases

“…Subsequently, NH125, an imidazolium derivative, was reported to be an eEF2K inhibitor [31] , but subsequent studies showed it actually increases eEF2 phosphorylation within cells, probably by acting as a non-specific protein-aggregating agent [32,33] . A thiopyran-dicarbonitrile analog was described as an eEF2K inhibitor by Devkota et al [34] .…”

The Molecular Mechanism of Eukaryotic Elongation Factor 2 Kinase Activation

“…We expressed eEF-2K in Escherichia coli and purified it to homogeneity in a form free of phosphate that is capable of being stimulated by Ca 2ϩ /CaM to display high catalytic activity (33). This allowed us to identify five major Ca 2ϩ /CaM-stimulated autophosphorylation sites in eEF-2K (Thr-348, Thr-353, Ser-445, Ser-474, and Ser-500) (31) and characterize the kinetic mechanism for the phosphorylation of a peptide substrate (34). We found that Thr-348 is the first site to be autophosphorylated and is important for eEF-2K activation (31,33).…”