Discovery of Allosteric MEK Inhibitors

Wallace, Eli M.; Blake, James F.

doi:10.1002/9780470524961.ch9

Cited by 2 publications

(1 citation statement)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We developed assays for both the active and nonactive forms of the kinase. In addition, because three of these allosteric MEK inhibitors (AZD6244, PD0325901, and PD184352 [CI-1040]) have been shown to bind in an uncompetitive manner with respect to ATP—namely, that they bind with higher affinity in the presence of ATP 9–11 —we developed methods to test the displacement of the active site probe in the presence of subsaturating concentrations of ATP. After optimization of assay conditions in the absence of nucleotide, we titrated both ATP and adenosine diphosphate (ADP) to determine IC 50 values for nonactivated, nonphosphorylated MEK1 (npMEK1) and activated, in vitro phosphorylated MEK1 (pMEK1).…”

Section: Resultsmentioning

confidence: 99%

Detection of Allosteric Kinase Inhibitors by Displacement of Active Site Probes

Lebakken¹,

Reichling²,

Ellefson³

et al. 2012

SLAS Discovery

View full text Add to dashboard Cite

Non-adenosine triphosphate (ATP) competitive, allosteric inhibitors provide a promising avenue to develop highly selective small-molecule kinase inhibitors. Although this class of compounds is growing, detection of such inhibitors can be challenging as standard kinase activity assays preferentially detect compounds that bind to active kinases in an ATP competitive manner. We have previously described a time-resolved fluorescence resonance energy transfer (TR-FRET)-based kinase binding assay using the competitive displacement of ATP competitive active site fluorescent probes ("tracers"). Although this format has gained acceptance, published data with this and related formats are almost entirely without examples of non-ATP competitive compounds. Thus, this study addresses whether this format is useful for non-ATP competitive inhibitors. To this end, 15 commercially available non-ATP competitive inhibitors were tested for their ability to displace ATP competitive probes. Despite the diversity of both compound structures and their respective targets, 14 of the 15 compounds displaced the tracers with IC(50) values comparable to literature values. We conclude that such binding assays are well suited for the study of non-ATP competitive inhibitors. In addition, we demonstrate that allosteric inhibitors of BCR-Abl and MEK bind preferentially to the nonphosphorylated (i.e., inactive) form of the kinase, indicating that binding assays may be a preferred format in some cases.

show abstract

Section: Resultsmentioning

confidence: 99%