Covalent Inhibitors of KEAP1 with Exquisite Selectivity

Abstract

The NRF2-KEAP1 interaction is central for cytoprotection against stresses, giving it high clinical significance. Covalent modification of KEAP1 is an efficient approach, but the covalent inhibitors used in the clinic carry undesired side effects originating in their moderate selectivity. Starting with a phenotypic screen, we identified a new covalent inhibitor chemotype that was optimized to deliver a series of potent and highly selective KEAP1 binders. While the developed compounds showed both cellular and in vivo activity, upregulating antioxidant response element-dependent target genes, they showed no genotoxicity in vitro. The lead compound exhibited broad selectivity in activity-based protein profiling and showed no significant interaction with a panel of commonly studied receptors nor with a broad panel of kinases. The nature of its interaction with KEAP1 and the origin of its selectivity were revealed by X-ray crystallography.

Figure 1

Selected covalent inhibitors of the NRF2-KEAP1 interaction.

Figure 2

Validated hits selected for further optimization and sites targeted for modification.

Figure 3

General synthesis of 2,4-bis(aryl/alkyl)sulphonyl-thiazoles. Reagents and conditions: (i) Pd(dppf)Cl₂, K₃PO₄, DMA, reflux; (ii) TMPMgCl·LiCl, THF, −40 °C; (iii) CBr₄, −78 to −10 °C; (iv) R²-SNa, DMF, rt or R²-SH, K₂CO₃, MeCN, 45 °C; (v) NCS, MeCN, reflux; (vi) H₂O₂, AcOH, reflux; (vii) MeCN or MeCN/EtOH, reflux; (viii) formamide, toluene, rt; (ix) PCl₅, DCM, 10 °C; (x) R¹SO₂Na, MeCN, rt; (xi) PCl₅, 120 °C; (xii) thiourea, MeCN, reflux; and (xiii) pyridine, toluene, rt.

Figure 4

X-ray structure of the KEAP1 BTB+3 box dimer with bound inhibitors 23 and 25. Electron density 2Fo–Fc map contoured at 1.5 σ, colored orange showing covalent linkage between Cys151 and ligands. Models and electron densities shown are from chain A in both structures. KEAP1 protein and compounds are colored blue and orange, respectively.

Figure 5

Waterfall plots of the isoTOP ABPP results for 23 and the negative control 28 in MDA-MB-231 cells. Competed peptides with a ligandability cutoff R > 4.5 are labeled.