Extended Data Fig. 8: Tepoxalin mechanistic studies.
From: Discovering the anticancer potential of non-oncology drugs by systematic viability profiling
a, ABCB1 western blot with and without overexpression of ABCB1 in the Kuramochi ovarian cancer cell line. Western blot was performed once with three independent samples. b, Cellular viability of Kuramochi wild type and ABCB1-overexpressing cells after treatment with tepoxalin for 8 days. Mean viability across 3 independently treated wells is shown, with standard deviation indicated by error bars. c, (Left) Single-agent dose-response curves for LS1034 cells treated with tepoxalin and zosuquidar for 5 days. Two replicates were averaged. (Middle) Dose response curves for tepoxalin in combination with varying doses of zosuquidar (indicated by different colors) for 5 days. Data are shown for tepoxalin doses above 470 nM (the range indicated by the vertical dashed lines above). (Right) Bliss synergy scores estimated for each dose combination, showing strong antagonism by zosuquidar at tepoxalin doses above ~5 μM. Two combinations were not tested (NT). d, Maximum synergy score is shown across several drug combinations measured in both LS1034 and REC1 cell lines. Both tariquidar and elacridar were strongly synergistic in combination with paclitaxel, while all MDR1 inhibitors tested were antagonistic in combination with tepoxalin. e, Cellular permeability study of tepoxalin and RWJ20142 in LS1034 colon cancer cell lines with and without ABCB1 knockout. Each indicated cell line or media-only control was treated with 20 μM tepoxalin or RWJ20142 for 3 hours. Tepoxalin and RWJ20142 concentrations in cell lysate or medium were determined by liquid chromatography-mass spectrometry. Mean viability across 3 independently treated wells is shown, with standard deviation indicated by error bars. Shaded dots indicate underlying data. f, Stability study of tepoxalin in three different vehicles over 72 hours. Percent of tepoxalin remaining is indicated at each timepoint. g, ABCB1 antagonism assay using calcein AM fluorescence. MDCKII cells were treated with indicated concentration of tepoxalin or RWJ20142. Mean percent ABCB1 inhibition across 3 replicates is shown. h, ABCB1 transport-based activity assay. Basolateral transport of the ABCB1 substrate loperamide was assessed using a monolayer of MDCK-ABCB1 cells in the presence of tepoxalin or RWJ20142. Mean percent ABCB1 inhibition across 2 replicates is shown.
