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.

Forbidden Coherence Transfer of 19F Nuclei to Quantitatively Measure the Dynamics of a CF3-Containing Ligand in Receptor-Bound States

“…In particular, NOEs were observed for the pyrimidine group and the p38α methyl protons deeper in the ATP-binding cleft (orange spheres in Figure 2 d). This NOE pattern is consistent with the binding mode of the AST-487–mitogen-activated protein kinase kinase 4 (MEK4) complex, which was previously analyzed by computational docking and saturation transfer difference (STD) NMR experiments, where the pyrimidine group of AST-487 resides deep in the binding cleft [ 32 ]. In contrast, we detected no intermolecular NOEs for the trifluoromethyl-phenyl ring and 4-ethyl-piperazin groups.…”

Identification of Novel Src Inhibitors: Pharmacophore-Based Virtual Screening, Molecular Docking and Molecular Dynamics Simulations

“…The hits obtained from the pharmacophore-based virtual screening were subjected to molecular docking studies; the top 10 molecules with the highest docking scores were selected for the study of binding modes ( Table 1 ). Among these 10 molecules, ZINC23247639 and ZINC10479320 have been reported as broad-spectrum kinase inhibitors that interacted with the highly conserved ATP-binding sites of many human protein kinases [ 22 , 23 , 24 ]; thus, we filtered them out in this investigation. As we know, hydrogen bonds established between receptor and ligand play a major role in the functionality and stability of the complex.…”

Synthesis and Biological Evaluation of 3‐Arylindazoles as Selective MEK4 Inhibitors

“…Recognizing that MEK4 represents a novel and validated therapeutic target we sought to identify and characterize selective MEK4 inhibitors. Previously, we developed a platform for mapping the pharmacological relatedness of all seven MEK kinase family members to understand compound selectively . Herein we discuss leveraging that foundational platform to screen compounds and identify a potent and selective hit molecule.…”

“…Finally, to investigate if 6 a exhibits specificity for MEK4 relative to the other six MEK isoforms (which have 38–61 % homology to MEK4), an ADP‐Glo MEK assay panel was used to determine the potency of this inhibitor against all seven MEK enzymes. The assays were run at a saturating concentration of full‐length protein substrates (Erk2, Erk5, p38α, or Jnk1β), and an ATP concentration below the K M of ATP for each enzyme.…”

“…Previously,w ed eveloped ap latform for mapping the pharmacological relatedness of all seven MEK kinase family members to understand compound selectively. [11] Herein we discuss leveraging that foundational platform to screen compounds andi dentify ap otent and selective hit molecule. Optimization and biological evaluation gave further insight into potentialu se of this series of compoundsa ss elective MEK4 inhibitors.…”