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A Novel Class of Common Docking Domain Inhibitors That Prevent ERK2 Activation and Substrate Phosphorylation
Rachel M Sammons, Nicole A Perry, Yangmei Li1,2
1Torrey Pines Institute for Molecular Studies , Port St. Lucie , Florida 34987 , United States.
Abstract:
Extracellular signal-regulated kinases (ERK1/2) are mitogen-activated protein kinases (MAPKs) that play a pro-tumorigenic role in numerous cancers. ERK1/2 possess two protein-docking sites that are distinct from the active site: the D-recruitment site (DRS) and the F-recruitment site. These docking sites facilitate substrate recognition, intracellular localization, signaling specificity, and protein complex assembly. Targeting these sites on ERK in a therapeutic context may overcome many problems associated with traditional ATP-competitive inhibitors. Here, we identified a new class of inhibitors that target the ERK DRS by screening a synthetic combinatorial library of more than 30 million compounds. The screen detects the competitive displacement of a fluorescent peptide from the DRS of ERK2. The top molecular scaffold from the screen was optimized for structure-activity relationship by positional scanning of different functional groups. This resulted in 10 compounds with similar binding affinities and a shared core structure consisting of a tertiary amine hub with three functionalized cyclic guanidino branches. Compound 2507-1 inhibited ERK2 from phosphorylating a DRS-targeting substrate and prevented the phosphorylation of ERK2 by a constitutively active MEK1 (MAPK/ERK kinase 1) mutant. Interaction between an analogue, 2507-8, and the ERK2 DRS was confirmed by nuclear magnetic resonance and X-ray crystallography. 2507-8 forms critical interactions at the common docking domain residue Asp319 via an arginine-like moiety that is shared by all 10 hits, suggesting a common binding mode. The structural and biochemical insights reported here provide the basis for developing new ERK inhibitors that are not ATP-competitive but instead function by disrupting critical protein-protein interactions.
Insights
Researchers discovered new inhibitors targeting the ERK D-recruitment site (DRS) in cancer. These non-ATP-competitive drugs disrupt protein interactions, offering a novel therapeutic approach for various cancers by targeting ERK1/2 signaling pathways.
Area of Science:
- Oncology
- Biochemistry
- Medicinal Chemistry
Background:
- Extracellular signal-regulated kinases (ERK1/2) are key mitogen-activated protein kinases (MAPKs) implicated in cancer progression.
- ERK1/2 signaling is regulated by specific protein-docking sites, including the D-recruitment site (DRS), distinct from the active ATP-binding site.
- Targeting these docking sites offers a potential alternative to conventional ATP-competitive inhibitors, which face challenges in cancer therapy.
Purpose of the Study:
- To identify and characterize novel inhibitors targeting the ERK DRS.
- To develop non-ATP-competitive inhibitors that disrupt ERK1/2 pro-tumorigenic functions.
- To explore new therapeutic strategies for cancers driven by aberrant ERK signaling.
Main Methods:
- Screening of a large synthetic combinatorial library (>30 million compounds) to identify DRS-displacing molecules.
- Structure-activity relationship optimization of lead compounds, focusing on a tertiary amine hub with cyclic guanidino branches.
- Biochemical assays to assess inhibitor efficacy in blocking ERK2 phosphorylation and interaction studies (NMR, X-ray crystallography) to confirm binding mode.
Main Results:
- Identification of a new class of 10 compounds targeting the ERK DRS, sharing a common molecular scaffold.
- Compound 2507-1 demonstrated inhibition of DRS-substrate phosphorylation and MEK1-mediated ERK2 phosphorylation.
- Structural analysis of analogue 2507-8 confirmed binding to the ERK2 DRS via specific interactions, revealing a common binding mode for the identified inhibitors.
Conclusions:
- A novel class of non-ATP-competitive ERK inhibitors targeting the DRS has been discovered.
- These inhibitors function by disrupting critical protein-protein interactions mediated by the DRS.
- The findings provide a foundation for developing new cancer therapeutics that modulate ERK signaling through docking site inhibition.
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