Author
Fusuke Hasegawa, Shiori Inoue, Maki Onda, Department of Biological Chemistry, Graduate School of Science, Osaka Metropolitan University
Keywords
chemical chaperones, folding intermediates, protein misfolding diseases, SBDD, fluorescence microplate assays, HDX-MS
Abstract
Conformationally dynamic proteins, e.g., folding intermediates and intrinsically disordered proteins, are important key molecules to develop drugs for cancer and misfolding diseases that include Alzheimer’s and Parkinson’s diseases. Although drug discovery targeting dynamic molecules needs a paradigm shift from traditional methods that is based on stable protein structures, recent breakthroughs in computational science drive progress in this field. Here we present the development of a chemical chaperone to treat a familial misfolding disease α1-antitrypsin deficiency that results from point mutations of the protein. Using five late-folding intermediate structures predicted by MD as templates, 455 candidate compounds were selected by in silico screening from 210,116 compounds. High-throughput screening with fluorescence microplate assays and CD measurements allowed to identify a promising lead compound from 455 candidates at low cost, quickly and efficiently. For lead optimization, the mechanisms of action of the lead compound were examined by Trp-fluorescence measurements and HDX-MS that can analyze folding rates and processes by peptide fragments. The results showed that the chemical chaperone preferentially binds to a folding intermediate of the pathogenic mutant rather than that of the wild-type at earlier folding stage than the intermediates predicted by MD simulation, and assists correct folding by accelerating folding rates of the region of C-terminal half molecule that includes the pathogenic mutation and the druggable pocket. Drug discovery targeting dynamic proteins relies on predicted model structures, hence identifying the mechanisms of action of lead compound is crucial. HDX-MS and fluorescence measurements are accessible and powerful methods taking over crystallography.