Fig. 4: MCB-613 and KEAP1 form a molecular bridge requiring Lys97.

a Diagram depicting initial serial truncation strategy. b Immunoblot analysis of HA-Clover KEAP1 dimerization (upper bands) in 293FT cells transfected with (from left to right) no template, HA-Clover KEAP1^WT, HA-Clover KEAP1⁵⁹⁷, HA-Clover KEAP1³¹⁴, HA-Clover KEAP1¹⁷⁸, and HA-Clover KEAP1⁵⁹ and treated with vehicle (DMSO) or MCB-613 for 1 h. N = 1 experiment. c Diagram depicting follow-up serial truncation strategy. d Immunoblot analysis of HA-Clover KEAP1 dimerization (upper bands) in 293FT cells transfected with (from left to right) no template, HA-Clover KEAP1^WT, HA-Clover KEAP1¹⁷⁰ through HA-Clover KEAP1⁶⁸, and treated with vehicle (DMSO) or MCB-613 for 1 h. Representative example shown from n = 2 biologically independent experiments. e Model of covalent interaction between MCB-613 and KEAP1. f Immunoblot analysis of HA-Clover KEAP1 dimerization (upper bands) in 293FT cells transfected with either HA-Clover KEAP1 WT or HA-Clover KEAP1 K97A and treated with DMSO or MCB-613 for 1 h. All images are taken from the same membrane. Direct comparisons between WT and K97A for a given molecular weight are taken from the same film exposure. N = 1 experiment. g Relative cell viability of WZR12 cells after 72-h exposure to DMSO or two different doses of MCB-613 following ectopic overexpression of either wild-type KEAP1 or the KEAP1 K97A mutant. Data are mean ± SEM for n = 3 biologically independent experiments. P values computed using one-way ANOVA with multiple comparisons. Exact p values listed within Source Data file. Source data are provided as a Source Data file.