Interaction Energetics and Druggability of the Protein–Protein Interaction between Kelch-like ECH-Associated Protein 1 (KEAP1) and Nuclear Factor Erythroid 2 Like 2 (Nrf2)

Abstract

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 FTMapidentified hot spot deep in the binding site. Finally, we discuss how the complementary information provided by alaninescanning mutagenesis, fragment screening, and computational hot spot mapping can be integrated to more comprehensively evaluate PPI druggability.

Deconstructing Noncovalent Kelch-like ECH-Associated Protein 1 (Keap1) Inhibitors into Fragments to Reconstruct New Potent Compounds

“…20 In comparison, P1 and P2 have been suggested to be the most important subpockets for binding of Nrf2, 26 while another study found three fragments binding in P1 and P4 after soaking 11 fragment hits. 76 The latter study also provided a computational analysis suggesting additional hot spots outside the canonical P1-5 subpockets. butylpyrrolidine-1-carbonyl)cyclohexyl)phenyl)-5-cyclopropyl-1H-pyrazole-4-carboxylic acid (compound 7), 31 which we have previously synthesized and tested, 33 but whose binding mode to Keap1 Kelch has not been revealed before.…”

Fucoxanthin Prevents 6‐OHDA‐Induced Neurotoxicity by Targeting Keap1

“…Two novel effective antioxidative tripeptides GWY and QWY [47] have been designed based on 3D-QSAR models, which can improve the stability of Keap1 by interacting with the key residues Arg 415 , Arg 483 , Arg 380 , and Ser 555 in the active sites. Meanwhile, alanine scanning of both Nrf2 and Keap1 proteins shows that Nrf2 interacts with Keap1 residues Ser 363 , Tyr 380 , Tyr 415 , Tyr 483 , and Ser 508 primarily through Glu 79 and Glu 82 , and Nrf2 also binds to Keap1 Tyr 525 by pi-stacking [48]. In the present study, we investigated the possible binding mechanism of FUC-Keap1 by molecular docking method.…”

Astaxanthine attenuates cisplatin ototoxicity in vitro and protects against cisplatin-induced hearing loss in vivo

“…Thus, our results suggest that AST might exert a competitive inhibition of NRF2 binding to the Kelch domain of KEAP1 via binding to residue Arg415 for hydrogen connection. This mechanism is similar to that dimethyl fumarate competes with NRF2 to bind to the Kelch domain of KEAP1 48 .…”