PMC

Effect of olmutinib on the efflux of Rho 123, the ATPase activity and the [¹²⁵I]-IAAP photoaffinity labeling of ABCG2. (A) Time course (0, 30, 60, 90, 120 min) of Rho 123 efflux was measured in S1 and S1-MI-80 cells, with or without 1 µmol/L olmutinib. Data are expressed as mean±standard. (B) Olmutinib competed for photolabeling of ABCG2 by [¹²⁵I]-IAAP crude membranes from ABCG2-overexpressing MCF7/FLV1000 cells were incubated with [¹²⁵I]-IAAP and a range of different concentration (0–5 µmol/L) of olmutinib. The samples were then cross-linked by UV illumination, and subjected to SDS-PAGE. A representative autoradiogram from three independent experiments was shown. The relative amount of [¹²⁵I]-IAAP incorporated is plotted against the concentration of olmutinib used in the competition. 100% incorporation refers to the absence of olmutinib. (C) Effect of olmutinib on ABCG2 ATPase activity. The vanadate-sensitive ABCG2 ATPase activity in the presence of the indicated concentrations of olmutinib was evaluated. Data are expressed as mean±SD from three independent experiments.

The concentration-survival curve of olmutinib on drug-induced ABCB1-, ABCG2-, and ABCC1-overexpressing cell lines and transfected ABCB1-, ABCG2-, and ABCC1-overexpressing cell lines. (A) Concentration-survival curves of KB-3-1 and KB-C2 cell lines incubated only with olmutinib. (B) Concentration-survival curves of NCI-H460 and NCI-H460/MX20 cell lines incubated only with olmutinib. (C) Concentration-survival curves of KB-3-1 and KB-CV60 cell lines incubated only with olmutinib. (D) Concentration-survival curves of HEK293/pcDNA3.1, HEK293/G2, HEK293/ABCB1, and HEK293/MRP1 cell lines incubated only with olmunitib. Each cell line was incubated with a designated concentration gradient of olmunitib for 72 h.

ABCG2 transporter specific ATPase activity was stimulated by olmutinib. The effect of olmutinib on the subcellular localization of BCRP in NCI-H460/MX20 cells. The graph shows that ATPase activity was plotted with SD as a function of concentration of olmutinib. The inset shows stimulation of ATP hydrolysis at lower (0–10 μM) concentration of olmutinib.

Binding mode/interactions of olmutinib (shown as sticks colored by element) into EGFR 696-1022 T790M (pdb: 5X2K): (A) 3D binding mode of olmutinib overlaid with the co-crystallized ligand, WZ4003 (yellow sticks); (B) 2D binding mode of olmutinib showing different types of binding interactions.