PMID- 33109619 OWN - NLM STAT- MEDLINE DCOM- 20210208 LR - 20211204 IS - 1521-0103 (Electronic) IS - 0022-3565 (Print) IS - 0022-3565 (Linking) VI - 376 IP - 1 DP - 2021 Jan TI - Mechanistic Analysis of an Extracellular Signal-Regulated Kinase 2-Interacting Compound that Inhibits Mutant BRAF-Expressing Melanoma Cells by Inducing Oxidative Stress. PG - 84-97 LID - 10.1124/jpet.120.000266 [doi] AB - 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. CI - Copyright (c) 2020 by The Author(s). FAU - Martinez, Ramon 3rd AU - Martinez R 3rd AD - Department of Pharmaceutical Sciences, University of Maryland, Baltimore- School of Pharmacy, Baltimore, Maryland. FAU - Huang, Weiliang AU - Huang W AD - Department of Pharmaceutical Sciences, University of Maryland, Baltimore- School of Pharmacy, Baltimore, Maryland. FAU - Samadani, Ramin AU - Samadani R AD - Department of Pharmaceutical Sciences, University of Maryland, Baltimore- School of Pharmacy, Baltimore, Maryland. FAU - Mackowiak, Bryan AU - Mackowiak B AD - Department of Pharmaceutical Sciences, University of Maryland, Baltimore- School of Pharmacy, Baltimore, Maryland. FAU - Centola, Garrick AU - Centola G AD - Department of Pharmaceutical Sciences, University of Maryland, Baltimore- School of Pharmacy, Baltimore, Maryland. FAU - Chen, Lijia AU - Chen L AD - Department of Pharmaceutical Sciences, University of Maryland, Baltimore- School of Pharmacy, Baltimore, Maryland. FAU - Conlon, Ivie L AU - Conlon IL AD - Department of Pharmaceutical Sciences, University of Maryland, Baltimore- School of Pharmacy, Baltimore, Maryland. FAU - Hom, Kellie AU - Hom K AUID- ORCID: 0000-0001-7951-1396 AD - Department of Pharmaceutical Sciences, University of Maryland, Baltimore- School of Pharmacy, Baltimore, Maryland. FAU - Kane, Maureen A AU - Kane MA AD - Department of Pharmaceutical Sciences, University of Maryland, Baltimore- School of Pharmacy, Baltimore, Maryland. FAU - Fletcher, Steven AU - Fletcher S AD - Department of Pharmaceutical Sciences, University of Maryland, Baltimore- School of Pharmacy, Baltimore, Maryland. FAU - Shapiro, Paul AU - Shapiro P AD - Department of Pharmaceutical Sciences, University of Maryland, Baltimore- School of Pharmacy, Baltimore, Maryland pshapiro@rx.umaryland.edu. LA - eng GR - T32 GM066706/GM/NIGMS NIH HHS/United States GR - R25 GM055036/GM/NIGMS NIH HHS/United States GR - R01 CA120215/CA/NCI NIH HHS/United States GR - F31 GM100693/GM/NIGMS NIH HHS/United States GR - P41 GM111135/GM/NIGMS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20201027 PL - United States TA - J Pharmacol Exp Ther JT - The Journal of pharmacology and experimental therapeutics JID - 0376362 RN - 0 (Antineoplastic Agents) RN - 0 (MAS1 protein, human) RN - 0 (Proto-Oncogene Mas) RN - EC 2.7.11.1 (BRAF protein, human) RN - EC 2.7.11.1 (Proto-Oncogene Proteins B-raf) RN - EC 2.7.11.24 (Mitogen-Activated Protein Kinase 1) SB - IM MH - Antineoplastic Agents/*chemistry/pharmacology MH - Catalytic Domain MH - Cell Proliferation/drug effects MH - HeLa Cells MH - Humans MH - Jurkat Cells MH - MAP Kinase Signaling System/*drug effects MH - Melanoma/*metabolism MH - Mitogen-Activated Protein Kinase 1/chemistry/*metabolism MH - *Oxidative Stress MH - Protein Binding MH - Proto-Oncogene Mas MH - Proto-Oncogene Proteins B-raf/genetics PMC - PMC7788356 EDAT- 2020/10/29 06:00 MHDA- 2021/02/09 06:00 PMCR- 2021/01/01 CRDT- 2020/10/28 05:31 PHST- 2020/08/03 00:00 [received] PHST- 2020/10/06 00:00 [accepted] PHST- 2020/10/29 06:00 [pubmed] PHST- 2021/02/09 06:00 [medline] PHST- 2020/10/28 05:31 [entrez] PHST- 2021/01/01 00:00 [pmc-release] AID - jpet.120.000266 [pii] AID - JPET_AR2020000266 [pii] AID - 10.1124/jpet.120.000266 [doi] PST - ppublish SO - J Pharmacol Exp Ther. 2021 Jan;376(1):84-97. doi: 10.1124/jpet.120.000266. Epub 2020 Oct 27.