Importance of Binding Site Hydration and Flexibility Revealed When Optimizing a Macrocyclic Inhibitor of the Keap1-Nrf2 Protein-Protein Interaction.

Fabio Begnini,Jens Carlsson,Andreas Luttens,Anna Friedel,Stefan Schiesser,Patrik Johansson,Beate König,Richard J Lewis,Lisa Wissler,Stefan Geschwindner,Emma Danelius,Peter Sjö,Jan Kihlberg,Stijn Lenders,Pierre Matricon

doi:10.1021/acs.jmedchem.1c01975

Abstract

Upregulation of the transcription factor Nrf2 by inhibition of the interaction with its negative regulator Keap1 constitutes an opportunity for the treatment of disease caused by oxidative stress. We report a structurally unique series of nanomolar Keap1 inhibitors obtained from a natural product-derived macrocyclic lead. Initial exploration of the structure-activity relationship of the lead, followed by structure-guided optimization, resulted in a 100-fold improvement in inhibitory potency. The macrocyclic core of the nanomolar inhibitors positions three pharmacophore units for productive interactions with key residues of Keap1, including R415, R483, and Y572. Ligand optimization resulted in the displacement of a coordinated water molecule from the Keap1 binding site and a significantly altered thermodynamic profile. In addition, minor reorganizations of R415 and R483 were accompanied by major differences in affinity between ligands. This study therefore indicates the importance of accounting both for the hydration and flexibility of the Keap1 binding site when designing high-affinity ligands.

Highlights

The human proteome has been estimated to consist of 100,000−1,000,000 binary protein-protein interactions (PPIs).Compounds modulating PPIs are considered as an exciting but often challenging opportunity for drug discovery.1 The surface area buried in PPIs may reach up to 6000 Å2 with binding affinities between the two proteins ranging from high micromolar to picomolar ranges
We recently reported a novel series of inhibitors of the Kelchlike ECH-associated protein 1 (Keap1)−nuclear factor erythroid 2-related factor 2 (Nrf2) PPI discovered by docking of a set of macrocyclic cores obtained by mining of the natural product chemical space
Compounds that provided compound 1 revealed the importance of the macrocycle, its ring size, the stereochemistry of the amino acid constituents, and the dimethyl amide for inhibition of the Keap1−Nrf2 PPI (Figure 2)

Summary

■ INTRODUCTION

The human proteome has been estimated to consist of 100,000−1,000,000 binary protein-protein interactions (PPIs). The macrocyclic ring of the four ligands adopts an almost identical conformation (Figure 11A−D) This water molecule has been displaced by the substituents at the ortho-position of the macrocyclic phenylene moiety of compounds 60, 63, and 64 (Figure 12B) Displacement of this bound water by a carboxyl group has previously been reported to provide major increases in potency for other lead series.. Synthesis of Compounds 41−58a pubs.acs.org/jmc aReagents and conditions: (a) 4 M HCl in 1,4-dioxane, rt, 1 h, R-COOH, HATU or EDC·HCl, DIPEA, DMSO, rt, 2 h.

■ SUMMARY AND CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES