Extended Data Fig. 1: Additional in vitro characterization of compound 3.
From: Mutant-selective AKT inhibition through lysine targeting and neo-zinc chelation
a, Deconvoluted intact-protein mass spectra of AKT2(WT) (1 µM) treated with vehicle or 1–3 (5 µM, 37 °C, 15 min) and then reduced with NaBH4 (10 mM, 5 min). b, Dissociation of preformed AKT2(WT)-ligand complexes (1 µM AKT2(WT), 5 µM ligand) was initiated by the addition of excess ARQ092 (50 µM) with continuous incubation at 37 °C. At the indicated time points, the percentage of covalently modified AKT2(WT) was determined by intact-protein MS after quenching with NaBH4 (10 mM, 5 min). Duplicate measurements for each time point were plotted, and dissociation half-times were determined using an exponential decay function. Kinetics of AKT1 dissociation are reproduced from Fig. 1d. c, The melting temperature (Tm) of AKT2(WT) (2 µM) treated with DMSO, 3 (10 µM), or ARQ092 (10 µM) was assessed by differential scanning fluorimetry (DSF, mean ± s.d., n = 3). ΔTm was calculated relative to the DMSO control (mean ± s.d.). Apparently missing errors bars indicate that the error is too small to be visualized. Data for AKT1 are reproduced from Fig. 1e. d, IC50 values from biochemical AKT kinase activity assays (10 μM ATP, radioisotope filter binding, 10-pt dose response, Reaction Biology). e, Modified site identification by tryptic LC-MS/MS. Purified recombinant AKT1(E17K) or AKT1(WT) (f), was treated with compound 3 (4 µM, 15 min) and reduced with NaBH4 (5 mM). The sample was reduced with DTT, alkylated with iodoacetamide and then digested with trypsin. The tryptic peptides were analysed by LC-MS/MS and modified sites identified using MSFragger. The modified sites with the greatest MS1 intensity were Lys17 for AKT1(E17K) (e) and Lys297 for AKT1(WT) (f). The annotated MS2 spectra with b- and y-ions for the relevant modified peptides are shown.
