Biochemistry: Latest Articles (ACS Publications)

Biochemistry: Latest Articles (ACS Publications)

Development of small molecule inhibitors of protein–protein interactions (PPIs) is hampered by our poor understanding of the druggability of PPI target sites. Here, we describe the combined application of alanine-scanning mutagenesis, fragment screening, and FTMap computational hot spot mapping to evaluate the energetics and druggability of the highly charged PPI interface between Kelch-like ECH-associated protein 1 (KEAP1) and nuclear factor erythroid 2 like 2 (Nrf2), an important drug target. FTMap identifies four binding energy hot spots at the active site. Only two of these are exploited by Nrf2, which alanine scanning of both proteins shows to bind primarily through E79 and E82 interacting with KEAP1 residues S363, R380, R415, R483, and S508. We identify fragment hits and obtain X-ray complex structures for three fragments via crystal soaking using a new crystal form of KEAP1. Combining these results provides a comprehensive and quantitative picture of the origins of binding energy at the interface. Our findings additionally reveal non-native interactions that might be exploited in the design of uncharged synthetic ligands to occupy the same site on KEAP1 that has evolved to bind the highly charged DEETGE binding loop of Nrf2. These include π-stacking with KEAP1 Y525 and interactions at an FTMap-identified hot spot deep in the binding site. Finally, we discuss how the complementary information provided by alanine-scanning mutagenesis, fragment screening, and computational hot spot mapping can be integrated to more comprehensively evaluate PPI druggability. Jump To The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biochem.9b00943 . Characterization data for the KEAP1 protein constructs used in this study (Figure S1), characterization data for the Nrf2 protein constructs used in this study (Figure S2), inhibition data for Nrf2 1–100 and Nrf2 34–100 (Figure S3), scheme summarizing the estimated interaction energy generated by each contact at the Nrf2–KEAP1 interface (Figure S4), intramolecular interactions involving D77 and T80 (Figure S5), new crystal form of the KEAP1 β-propeller domain suitable for ligand soaking and comparison of soaked structures of wild-type (LDEETGEFL) and T80A (LDEEAGEFL) 9-mer peptides (Figure S6), sequence alignment of Nrf2 paralogs and sequence conservation of key residues in KEAP1 (Figure S7), binding poses of the fragments reported by Astex (Figure S8), conserved water molecules located at hot spot A (Figure S9), illustration showing how hot spot A can be approached to achieve a higher binding affinity (Figure S10), crystallographic data collection and refinement statistics (Table S1), locations and strengths of FTMap CC and correlation with hot spots identified by alanine scanning (Table S2), extent of surface area that is buried by each residue in the DEETGE motif, upon binding to KEAP1, and the degree of overlap of each side chain with the FTMap CC (Table S3), and fragment hits, with chemical structures (Table S4) ( PDF ) pdf bi9b00943_si_001.pdf (8.17 MB) Terms & Conditions Electronic Supporting Information files are available without a subscription to ACS Web Editions. The American Chemical Society holds a copyright ownership interest in any copyrightable Supporting Information. Files available from the ACS website may be downloaded for personal use only. Users are not otherwise permitted to reproduce, republish, redistribute, or sell any Supporting Information from the ACS website, either in whole or in part, in either machine-readable form or any other form without permission from the American Chemical Society. For permission to reproduce, republish and redistribute this material, requesters must process their own requests via the RightsLink permission system. Information about how to use the RightsLink permission system can be found at http://pubs.acs.org/page/copyright/permissions.html . Cited By This article has not yet been cited by other publications. Partners



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