Extended Data Fig. 10: Characterization of COX5A as a bona fide off-target candidate of asciminib.
From: Small-molecule binding-site discovery using silyl ether-enabled chemoproteomics
A, Structural representation of COX5A-labeled sites (G(T)79, Y80 and D81) in close proximity with a defined lipid-binding pocket with phosphotidylsulfocholine (PSC) ligand (PDB: 1V54 for bovine heart cytochrome C oxidase structure). B-C, In-gel fluorescence ‘B’ and immunoblot analysis ‘C’ of HEK293T cells transiently overexpressing FLAG-tagged GFP or COX5A treated with 5b (1 μM, 10 μM, 1 h) in ‘A’ or asciminib/ dasatinib (10 μM, 1 h) in ‘B’ in a UV-dependent manner. D, AP-MS analysis of HEK293T cells transiently overexpressing FLAG-tagged GFP, wildtype (WT) and mutant COX5A, showing protein intensities (n = 3 biological replicates). Data presented are mean ± SD. E, Co-immunoprecipitation of COX5A-HA overexpressing HEK293T stable cells that were transiently transfected with COX5A-FLAG, treated with asciminib or dasatinib (10 μM, 1 h). F, Mean ratios of assembled complex IV to Complex II assessed by Blue Native Polyacrylamide Gel Electrophoresis (BNGE) for HEK293T cells treated with asciminib or dasatinib (10 μM, 6 h). Quantitative assembly blots were shown in ‘Supplementary Fig. 7F’. Data presented are mean ± SD with significance values between experimental conditions calculated using paired student’s t-test (n = 6 biological replicates; from left to right, p = 0.008397 and 0.336080) with *p < 0.05, **p < 0.01,***p < 0.001, and NS (not significant) p > 0.05. For ‘B,C,E’, data presented are representative n = 3 (B,C) and n = 2 (E) independent experiments. All MS data can be found in Supplementary Table 9.
