A Chemical Probe Strategy for Interrogating Inhibitor Selectivity Across the MEK Kinase Family

Abstract

MEK4 is an upstream kinase in MAPK signaling pathways where it phosphorylates p38 MAPK and JNK in response to mitogenic and cellular stress queues. MEK4 is overexpressed and induces metastasis in advanced prostate cancer lesions. However, the value of MEK4 as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK4 have not been developed. Despite a high level of sequence homology in the ATP-binding site, most reported MEK inhibitors are selective for MEK1/2 and display reduced potency toward other MEKs. Here, we present the first functional and binding selectivity-profiling platform of the MEK family. We applied the platform to profile a set of known kinase inhibitors and used the results to develop an in silico approach for small molecule docking against MEK proteins. The docking studies identified molecular features of the ligands and corresponding amino acids in MEK proteins responsible for high affinity binding versus those driving selectivity. WaterLOGSY and saturation transfer difference (STD) NMR spectroscopy techniques were utilized to understand the binding modes of active compounds. Further minor synthetic manipulations provide a proof of concept by showing how information gained through this platform can be utilized to perturb selectivity across the MEK family. This inhibitor-based approach pinpoints key features governing MEK family selectivity and clarifies empirical selectivity profiles for a set of kinase inhibitors. Going forward, the platform provides a rationale for facilitating the development of MEK-selective inhibitors, particularly MEK4 selective inhibitors, and repurposing of kinase inhibitors for probing the structural selectivity of isoforms.

Figure 1

(A) MEK proteins and their signaling pathways. (B) Integrated approaches to profile selectivity of MEK isoforms.

Figure 2

MEK isoform selectivity panel screen. (A) Staurosporine control data for each MEK isoform, assay validation. (B) Heat map of collected IC₅₀ data on all MEK isoforms against selected inhibitors. Cells contain IC₅₀ values in micromolarity. (C) Structural homology of MEK isoforms as a percentage of similarity (%). MEK3 and MEK5 missing in structural comparison due to lack of crystal structures. Red, blue, and green highlight the three most structurally homologous isoforms.

Figure 3

In silico analysis of inhibitors. General standard kinase active site representations for MEK4 and MEK6/7. (A) PLX4720 (cyano, stick) docked poses in MEK4 (green, cartoon) and MEK6 (pink, cartoon). (B) AST487 (magenta, stick) docked poses MEK4 (green) and MEK7 (gray). (C) Pazopanib (purple, stick) docked binding modes in MEK4 (green, cartoon) and MEK6 (pink, cartoon).

Figure 4

NMR analysis of the interactions between MEK4 and AST-487/pazopanib. (A) WaterLOGSY NMR spectra of the AST-487 interaction with recombinant MEK4. For reference, the 1D NMR spectrum of the downfield region of AST-487 is shown. (B) WaterLOGSY NMR competition assay to demonstrate competitve binding between inhibitor and ATP. Schematic diagrams showing relative STD percentages (C) AST-487 and (D) Pazopanib-01 bound to recombinant MEK4.

Figure 5

Synthetic analogs to modulate selectivity along the p38/JNK modules. (A) Analogs of AST-487 with selective alterations of the alkyl amine group to selectively target MEK4 and MEK7. (B) Analogs of pazopanib focused on trying to target back pocket uniquely between MEK4 and MEK6. (C) Analogs of PLX-4720 focused of providing simple information on the selectivity difference seen between MEK4 and MEK6. Tables contain IC₅₀ values in micromolarity determined by optimized selectivity ADPGlo assay. Blue arrows indicate a decrease, while the red arrows indicate an increase in IC₅₀ values. A comprehensive list of analogs can be found in Supporting Information, Table S4.