%0 Journal Article
%A Devkota, Ashwini
K.
%A D. J. Tavares, Clint
%A Warthaka, Mangalika
%A Abramczyk, Olga
%A Marshall, Kyle D.
%A Kaoud, Tamer S.
%A Gorgulu, Kivanc
%A Ozpolat, Bulent
%A Dalby, Kevin N.
%D 2012
%T Investigating the Kinetic
Mechanism of Inhibition
of Elongation Factor 2 Kinase by NH125: Evidence of a Common in Vitro
Artifact
%U https://acs.figshare.com/articles/journal_contribution/Investigating_the_Kinetic_Mechanism_of_Inhibition_of_Elongation_Factor_2_Kinase_by_NH125_Evidence_of_a_Common_in_Vitro_Artifact/2541040
%R 10.1021/bi201787p.s001
%2 https://acs.figshare.com/ndownloader/files/4184053
%K NH 125 exhibits
%K kinase assay conditions
%K NH 125
%K eEF 2 phosphorylation
%K ATP
%K kinase assay
%K 549 lung cancer
%K Elongation Factor 2 Kinase
%K TRPM
%K HEK
%K Western blot approach
%K IC 50
%K elongation factor 2 kinase
%K ERK
%X Evidence that elongation factor 2 kinase (eEF-2K) has
potential
as a target for anticancer therapy and possibly for the treatment
of depression is emerging. 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 a 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 a nonspecific colloidal aggregator in
vitro, a notion further supported by the observation that NH125 inhibits
other protein kinases, such as ERK2 and TRPM7 in a manner similar
to that of 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 a decrease in the level
of eEF2 phosphorylation was observed up to 12 h following addition
of NH125 to the media. Furthermore, contrary to the previously reported
literatures, NH125 induced the phosphorylation of eEF-2.
%I ACS Publications