Mechanistic Analysis of an Extracellular Signal-Regulated Kinase 2-Interacting Compound that Inhibits Mutant BRAF-Expressing Melanoma Cells by Inducing Oxidative Stress.

Constitutively active extracellular signal-regulated kinase (ERK) 1/2 signaling promotes cancer cell proliferation and survival. We previously described a class of compounds containing a 1,1-dioxido-2,5-dihydrothiophen-3-yl 4-benzenesulfonate scaffold that targeted ERK2 substrate docking sites and selectively inhibited ERK1/2-dependent functions, including activator protein-1-mediated transcription and growth of cancer cells containing active ERK1/2 due to mutations in Ras G-proteins or BRAF, Proto-oncogene B-RAF (Rapidly Acclerated Fibrosarcoma) kinase. The current study identified chemical features required for biologic activity and global effects on gene and protein levels in A375 melanoma cells containing mutant BRAF (V600E). Saturation transfer difference-NMR and mass spectrometry analyses revealed interactions between a lead compound (SF-3-030) and ERK2, including the formation of a covalent adduct on cysteine 252 that is located near the docking site for ERK/FXF (DEF) motif for substrate recruitment. Cells treated with SF-3-030 showed rapid changes in immediate early gene levels, including DEF motif-containing ERK1/2 substrates in the Fos family. Analysis of transcriptome and proteome changes showed that the SF-3-030 effects overlapped with ATP-competitive or catalytic site inhibitors of MAPK/ERK Kinase 1/2 (MEK1/2) or ERK1/2. Like other ERK1/2 pathway inhibitors, SF-3-030 induced reactive oxygen species (ROS) and genes associated with oxidative stress, including nuclear factor erythroid 2-related factor 2 (NRF2). Whereas the addition of the ROS inhibitor N-acetyl cysteine reversed SF-3-030-induced ROS and inhibition of A375 cell proliferation, the addition of NRF2 inhibitors has little effect on cell proliferation. These studies provide mechanistic information on a novel chemical scaffold that selectively regulates ERK1/2-targeted transcription factors and inhibits the proliferation of A375 melanoma cells through a ROS-dependent mechanism. SIGNIFICANCE STATEMENT: Constitutive activation of the extracellular signal-regulated kinase (ERK1/2) pathway drives the proliferation and survival of many cancer cell types. Given the diversity of cellular functions regulated by ERK1/2, the current studies have examined the mechanism of a novel chemical scaffold that targets ERK2 near a substrate binding site and inhibits select ERK functions. Using transcriptomic and proteomic analyses, we provide a mechanistic basis for how this class of compounds inhibits melanoma cells containing mutated BRAF and active ERK1/2.

Rationally Designed Polypharmacology: α-Helix Mimetics as Dual Inhibitors of the Oncoproteins Mcl-1 and HDM2.

Protein-protein interactions (PPIs), many of which are dominated by α-helical recognition domains, play key roles in many essential cellular processes, and the dysregulation of these interactions can cause detrimental effects. For instance, aberrant PPIs involving the Bcl-2 protein family can lead to several diseases including cancer, neurodegenerative diseases, and diabetes. Interactions between Bcl-2 pro-life proteins, such as Mcl-1, and pro-death proteins, such as Bim, regulate the intrinsic pathway of apoptosis. p53, a tumor-suppressor protein, also has a pivotal role in apoptosis and is negatively regulated by its E3 ubiquitin ligase HDM2. Both Mcl-1 and HDM2 are upregulated in numerous cancers, and, interestingly, there is crosstalk between both protein pathways. Recently, synergy has been observed between Mcl-1 and HDM2 inhibitors. Towards the development of new anticancer drugs, we herein describe a polypharmacology approach for the dual inhibition of Mcl-1 and HDM2 by employing three densely functionalized isoxazoles, pyrazoles, and thiazoles as mimetics of key α-helical domains of their partner proteins.

Kröhnke pyridines: Rapid and facile access to Mcl-1 inhibitors.

The tumorigenic activity of upregulated Mcl-1 is manifested by binding the BH3 α-helical death domains of opposing Bcl-2 family members, neutralizing them and preventing apoptosis. Accordingly, the development of Mcl-1 inhibitors largely focuses on synthetic BH3 mimicry. The condensation of α-pyridinium methyl ketone salts and α,ß-unsaturated carbonyl compounds in the presence of a source of ammonia, or the Kröhnke pyridine synthesis, is a simple approach to afford highly functionalized pyridines. We adapted this chemistry to rapidly generate low-micromolar inhibitors of Mcl-1 wherein the 2,4,6-substituents were predicted to mimic the i, i + 2 and i + 7 side chains of the BH3 α-helix.