A Novel Confocal Scanning Protein-Protein Interaction Assay (PPI-CONA) Reveals Exceptional Selectivity and Specificity of CC0651, a Small Molecule Binding Enhancer of the Weak Interaction between the E2 Ubiquitin-Conjugating Enzyme CDC34A and Ubiquitin.
Joanna Koszela, Nhan T Pham, Steven Shave, Daniel St-Cyr, Derek F Ceccarelli, Steven Orlicky, Anne Marinier, Frank Sicheri, Mike Tyers, Manfred Auer
Author Information
Joanna Koszela: School of Molecular Biosciences, University of Glasgow, Glasgow G12 8QQ, U.K. ORCID
Nhan T Pham: School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland EH9 3BF, U.K. ORCID
Steven Shave: School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland EH9 3BF, U.K. ORCID
Daniel St-Cyr: X-Chem Inc., Montréal, Québec H4S 1Z9, Canada. ORCID
Derek F Ceccarelli: Centre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.
Steven Orlicky: Centre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.
Anne Marinier: Institute for Research in Immunology and Cancer, University of Montreal, Montreal, Québec H3T 1J4, Canada.
Frank Sicheri: Centre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada. ORCID
Mike Tyers: Institute for Research in Immunology and Cancer, University of Montreal, Montreal, Québec H3T 1J4, Canada.
Manfred Auer: School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland EH9 3BF, U.K. ORCID
Protein-protein interactions (PPIs) are some of the most challenging target classes in drug discovery. Highly sensitive detection techniques are required for the identification of chemical modulators of PPIs. Here, we introduce PPI confocal nanoscanning (PPI-CONA), a miniaturized, microbead based high-resolution fluorescence imaging assay. We demonstrate the capabilities of PPI-CONA by detecting low affinity ternary complex formation between the human CDC34A ubiquitin-conjugating (E2) enzyme, ubiquitin, and CC0651, a small molecule enhancer of the CDC34A-ubiquitin interaction. We further exemplify PPI-CONA with an E2 enzyme binding study on CC0651 and a CDC34A binding specificity study of a series of CC0651 analogues. Our results indicate that CC0651 is highly selective toward CDC34A. We further demonstrate how PPI-CONA can be applied to screening very low affinity interactions. PPI-CONA holds potential for high-throughput screening for modulators of PPI targets and characterization of their affinity, specificity, and selectivity.
