Discovery and characterization of an iminocoumarin scaffold as an inhibitor of MEKK2 (MAP3K2)

• Published in: Biochemical and biophysical research communications Vol. 496; no. 1; pp. 205 - 211
• Main Authors: Ahmad, Syed, St. Hilaire, Valentine R., Dandepally, Srinivasa R., Johnson, Gary L., Williams, Alfred L., Scott, John E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 29.01.2018
• Subjects: 60 APPLIED LIFE SCIENCES; adenosine diphosphate; adenosine triphosphate; ADP; ATP; Cells, Cultured; Coumarins - administration & dosage; Coumarins - chemistry; Coumarins - metabolism; dimethyl sulfoxide; DMSO; Drug Delivery Systems - methods; Drug Discovery; Drug Evaluation, Preclinical - methods; ELISA; enzyme-linked immunosorbent assay; high throughput screening; HTS; Humans; Inhibitor; Kinase; MAP Kinase Kinase Kinase 2 - antagonists & inhibitors; MAP Kinase Kinase Kinase 2 - metabolism; MAP3K2; MAPK; mass spectrometry; MEKK2; METASTASES; mitogen-activated protein kinase; NEOPLASMS; PHOSPHOTRANSFERASES; Protein Interaction Mapping - methods; Protein Kinase Inhibitors - administration & dosage; Protein Kinase Inhibitors - chemistry; Protein Kinase Inhibitors - metabolism; SAR; standard deviation; structure-activity relationship; STRUCTURE-ACTIVITY RELATIONSHIPS; time-resolved fluorescence resonance energy transfer; TR-FRET; MAP3K2; DMSO; SAR; MS; HTS; MEKK2; MAPK; ADP; mitogen-activated protein kinase; SD; dimethyl sulfoxide; Kinase; adenosine diphosphate; high throughput screening; adenosine triphosphate; enzyme-linked immunosorbent assay; mass spectrometry; Inhibitor; time-resolved fluorescence resonance energy transfer; ATP; structure-activity relationship; TR-FRET; standard deviation; ELISA
• ISSN: 0006-291X; 1090-2104; 1090-2104
• DOI: 10.1016/j.bbrc.2018.01.027

The kinase MEKK2 (MAP3K2) activates the MEK5/ERK5 cell signaling pathway and may play an important role in tumor growth and metastasis. Thus, MEKK2 may represent a novel kinase target for cancer. In order to identify inhibitors of MEKK2, we screened a library of compounds using a high throughput MEKK2 intrinsic ATPase enzyme assay. We identified two hits with validated structures and confirmed activity in the primary assay (IC50 values = 322 nM and 7.7 μM) and two orthogonal MEKK2 biochemical assays. Compound 1, the more potent hit, was the subject of further investigation. Limited structure-activity relationship (SAR) studies were performed on this iminocoumarin hit which resulted in ≥20-fold more potent analogs (e.g. 8 and 16 nM IC50). Two analogs had improved selectivity in a 50-member kinase profiling panel compared to the hit. These studies suggested that substitutions around the phenoxy ring of this scaffold can impart improved potency and selectivity for MEKK2. Analog Compound 1s (16 nM IC50) was further verified by external testing to inhibit MEKK2 and MEKK3 with similar potencies. Compound 1s displayed activity in cell-based assays in which it inhibited ERK5 pathway activation in cells and inhibited cell migration in a scratch assay. Thus, we have identified a scaffold that has promising potential to be developed into a highly selective and potent inhibitor of MEKK2. Information from these SAR studies provides specific guidance for the future design of MEKK2 inhibitor probes. •Two novel small molecule inhibitors of MEKK2 discovered from screening.•Structures validated and activities confirmed in multiple MEKK2 biochemical assays.•Synthesis of analogs for iminocoumarin scaffold led to ≥20-fold more potent analogs.•Kinase profiling indicated that scaffold modifications could increase selectivity.•One analog inhibited ERK5 pathway activation in cells and cell migration.