ABSTRACT
Phosphorylation-dependent protein-protein interactions play a significant role in biological signaling pathways; therefore, small molecules that are capable of influencing these interactions can be valuable research tools and have potential as pharmaceutical agents. MEMO1 (mediator of ErbB2-cell driven motility) is a phosphotyrosine-binding protein that interacts with a variety of protein partners and has been found to be upregulated in breast cancer patients. Herein, we report the first small-molecule inhibitors of MEMO1 interactions identified through a virtual screening platform and validated in a competitive fluorescence polarization assay. Initial structure-activity relationships have been investigated for these phenazine-core inhibitors and the binding sites have been postulated using molecular dynamics simulations. The most potent biochemical inhibitor is capable of disrupting the large protein interface with a KI of 2.7â µm. In addition, the most promising phenazine core compounds slow the migration of breast cancer cell lines in a scratch assay.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Phenazines/pharmacology , Small Molecule Libraries/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Drug Screening Assays, Antitumor , Female , Fluorescence Polarization , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Molecular Dynamics Simulation , Molecular Structure , Phenazines/chemical synthesis , Phenazines/chemistry , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
ErbB2 signaling pathways are linked to breast cancer formation, growth, and aggression; therefore, understanding the behavior of proteins associated with these pathways as well as regulatory factors that influence ErbB2 function is essential. MEMO1 is a redox active protein that is shown to associate with phosphorylated ErbB2 and mediate cell motility. We have developed a fluorescence polarization assay to probe the interaction between MEMO1 and an ErbB2-derived peptide containing a phosphorylated tyrosine residue. This interaction is shown to be pH-dependent and stronger with longer peptides as would be expected for protein-protein interactions. We have quantitatively mapped the binding interface of MEMO1 to the peptide using the fluorescence polarization assay and molecular dynamics simulations. We have confirmed that phosphorylation of the peptide is essential for binding and through mutagenesis have identified residues that contribute to favorable interactions. Our results highlight the importance of the protein-protein interactions of MEMO1 that complement the oxidase activity. In the future, these studies will provide a method for screening for selective modulators of MEMO1, which will allow for additional biological investigations.
