Abstract
The ErbB2 receptor tyrosine kinase is overexpressed in approximately 15–20% of breast tumors and associated with aggressive disease and poor clinical outcome. p130Cas represents a nodal scaffold protein regulating cell survival, migration and proliferation in normal and pathological contexts. p130Cas overexpression in ErbB2 human breast cancer correlates with poor prognosis and metastasis formation. Recent data indicate that p130Cas association to ErbB2 protects ErbB2 from degradation, thus enhancing tumorigenesis. Therefore, inhibiting p130Cas/ErbB2 interaction might represent a new therapeutic strategy to target breast cancer. Here we demonstrate by performing Molecular Modeling, Molecular Dynamics, dot blot, ELISA and fluorescence quenching experiments, that p130Cas binds directly to ErbB2. Then, by structure-based virtual screening, we identified two potential inhibitors of p130Cas/ErbB2 interaction. Their experimental validation was performed in vitro and in ErbB2-positive breast cancer cellular models. The results highlight that both compounds interfere with p130Cas/ErbB2 binding and significantly affect cell proliferation and sensitivity to Trastuzumab. Overall, this study identifies p130Cas/ErbB2 complex as a potential breast cancer target revealing new therapeutic perspectives for protein-protein interaction (PPI).
Highlights
The ErbB2 receptor tyrosine kinase is overexpressed in approximately 15–20% of breast tumors and associated with aggressive disease and poor clinical outcome. p130Cas represents a nodal scaffold protein regulating cell survival, migration and proliferation in normal and pathological contexts. p130Cas overexpression in ErbB2 human breast cancer correlates with poor prognosis and metastasis formation
Despite the enormous interest in targeting Protein-Protein Interactions (PPIs), the discovery of drugs capable to interfere with these interactions has been proven to be very challenging
The transient nature of these interactions, moderate affinity, and promiscuity of recognition are among the many factors that have contributed to difficulty in discovering effective modulators[26,27,28]
Summary
Modeling the interaction of p130Cas and ErbB2. Literature analysis suggests that p130Cas scaffold might independently associate with ErbB2 in a direct way. The same mutants were produced as MBP-tagged recombinant proteins to perform an in vitro ELISA direct binding assay (Fig. 3C) Both experiments show that mutagenesis of key residues in PPII_ErbB2 sequence alters its binding to SH3 domain of p130Cas. Interestingly, the substitution of Pro[5] and Pro[8] to Ala strongly affects the amount of binding, while the substitution of Arg to Asp does not impair the binding suggesting that the reinforced hydrogen bond plays a minor role in the interaction. The same strategy was used to validate the PPII-binding pockets in the SH3 domain of p130Cas. Three SH3-mutants carrying mutations in the three main sites of the interaction were generated and subjected to an in vitro ELISA direct binding assay to evaluate their ability to bind ErbB2 recombinant proteins (Fig. 3D). 2 is predicted to be poorly permeable (using pkCSM predictor the threshold to distinguish highly from poorly permeable compounds is 0.90), whereas 1 is expected to be a substrate of P-glycoprotein which could adversely affect the drug candidate effectiveness
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