Fig. 3: Parsing differential pharmacology of equipotent MTH1 inhibitors with a V5-G48E drug biosensor.

a MTH1 inhibitors tested and their reported biochemical IC₅₀ values^31,33,34,35. b A representative blot (n = 2) of induced V5-MTH1 WT or V5-MTH1 G48E in U-2 OS cells following incubated with DMSO, 2.5 µM TH588, 1 µM AZ19, 1 µM IACS-4759, or 1 µM BAY-707 for 24 hours. Black arrow – V5 MTH1; gray arrow – endogenous, WT MTH1. c A schematic depicting multiparametric CeTEAM analysis by flow cytometry of TH588 and AZ19 pharmacology by V5 (drug binding, AF-488), pHH3 Ser10 (mitotic marker, AF-647), and Hoechst (cell cycle) readouts using V5-MTH1 G48E clone 6 cells. d V5-G48E saturation profiles of TH588 (blue) and AZ19 (orange) after 24 hours by western blot. Mean of n = 3 ± range. Comparison made by two-sided extra sum-of-squares F Test (F [DFn, DFd] = 12.67 [1, 36]). Interpolated data points representing occupancy designations are shown in red and defined adjacent to data plots. e Median V5-G48E fold change (left axis; TH588 – blue, AZ19 – orange) and percent pHH3 Ser10⁺ cells (right axis; yellow) of described occupancy designations by flow cytometry (mean of n = 3 ± SD). Reference line at V5 fold change = 1. P values shown for one-way ANOVA with multiple comparisons to the DMSO control (Dunnett’s test; F_HH3 [DFn, DFd] = 122.3 [6, 14], F_V5 [DFn, DFd] = 1.625 [6, 14]). f Proportion of cells in sub-G1 (light gray), G0/G1 (black), S (white), and G2/M (dark gray) phases by Hoechst intensity. Mean of n = 3 ± SD. Red highlight – P < 0.05. P_{Pre,TH588,SubG1} > 0.9999, P_{Pre,TH588,G0/G1} = 0.9998, P_Pre,TH588,S > 0.9999, P_{Pre,TH588,G2/M} = 0.9997, P_{Sat,TH588,SubG1} > 0.9999, P_{Sat,TH588,G0/G1} = 0.9251, P_Sat,TH588,S = 0.9978, P_{Sat,TH588,G2/M} = 0.9963, P_{Lit,TH588,SubG1} > 0.9999, P_{Lit,TH588,G0/G1} = 0.1700, P_Lit,TH588,S = 0.0934, P_{Lit,TH588,G2/M} = 0.0001, P_{Pre,AZ19,SubG1} > 0.9999, P_{Pre,AZ19,G0/G1} = 0.9992, P_Pre,AZ19,S=0.9948, P_{Pre,AZ19,G2/M} = 0.9950, P_{Sat,AZ19,SubG1} > 0.9999, P_{Sat,AZ19,G0/G1} = 0.9997, P_Sat,AZ19,S = 0.9997, P_{Sat,AZ19,G2/M} = 0.9995, P_{Lit,AZ19,SubG1>}0.9999, P_{Lit,AZ19,G0/G1=}0.7657, P_Lit,AZ19,S = 0.8923, P_{Lit,AZ19,G2/M} > 0.9999 by ordinary two-way ANOVA with multiple comparisons to the DMSO control (Dunnett’s test; F [DFn, DFd]: F_Interaction [18, 56] = 3.103, F_{Row Factor} [6, 56] = 0.01093, F_{Column Factor} [3, 56] = 552.0).