Supplementary Figure 1: Small-molecule covalent inhibition of MCL-1ΔNΔC.
(a) Fluorescence polarization dilutional binding assay of FITC−BID BH3 and MCL-1ΔNΔC in the presence and absence of N-(4-hydroxy-1-naphthalenyl)-benzenesulfonamide (CAS# 36942-42-4), a small molecule screening hit that exhibited irreversible inhibition of the BH3-binding activity of MCL-1ΔNΔC. (b) Chemical structures of MAIM1 analogs that emerged from our competitive MCL-1 SAHB A −MCL-1ΔNΔC screen. Competitive potency corresponded to compound reactivity, based on the nature of the leaving group (R1) and electron withdrawing capacity (R2). Data are mean ± s.e.m. (n = 3 technical replicates). (c) Chemical synthesis of MAIM1. (d-e) Trypsin digestion and LC-MS/MS of unmodified (d) and MAIM1-conjugated (e) MCL-1ΔNΔC. Full scan spectra (left) demonstrate the peptide containing unmodified C286 (mass: 2329.18 Da) and the MAIM1 conjugate (mass: 2483.19 Da), reflecting the added mass of cleaved MAIM1 adduct (i.e. minus tosyl). MS/MS spectra (right) further confirm MAIM1 conjugation at position C286. Whereas the masses of ions y3, y4, y5, y6, and y12 are shared between the peptides, there is a discrete mass shift for ion y15, which contains C286. For clarity, only the y ions are labeled.
