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Examining Ligand-based Stabilization of Proteins in Cells with MEK1 Kinase Inhibitors

Auld, Douglas, Hill, William and Orme, Jonathon (2014) Examining Ligand-based Stabilization of Proteins in Cells with MEK1 Kinase Inhibitors. Assay Drug Development Technologies, 13 (5). pp. 266-276. ISSN 1540-658X1557-8127


Here we describe the evaluation of a cell-based ligand binding assay using DiscoveRx’s enzyme-fragment complementation technology performed in two independent laboratories. The assay is based on the ability of certain ligands to bind to a protein leading to a ligand•protein complex that has a different stability than the free protein. The assay employed a pro-labeled-tagged MEK1 kinase stably expressed in A549 cells and this was used to evaluate focused sets of compounds containing known MEK1inhibitors. An assay using a pro-labeled tagged G9A expressed in A549 cells was used as a counterscreen. In one study it was found that the majority of MEK1 inhibitors were either found as inactive (43%) or showed an inhibitory response (16%) in the cell-based MEK1 assay however seven compounds showed a specific activation response consistent with stabilization of MEK1 in cells. Examination of these stabilizing compounds showed that three of these were analogs of hypothemycin, a known covalent allosteric MEK1 inhibitor, while the remaining compounds covered one structural class. Both laboratories were able to confirm activity in the cell-based MEK1 assay for certain known MEK1 inhibitors and that this activity was highly selective over the G9a counterscreen assay. This study supports that the MEK1 cellular protein stability assay is sensitive to certain MEK1 inhibitors, often noncompetitive inhibitors with respect to ATP. The cellular stability assay format could be useful to rapidly filter kinase inhibitor hit lists for allosteric kinase inhibitors and confirm target engagement in cells.

Item Type: Article
Date Deposited: 26 Apr 2016 23:45
Last Modified: 04 Jul 2016 23:45