Fig. 5

Effects of mutagenesis of the outer and central sites on activity of inhibitors.

A View of C2.2 (cyan) bound in the outer site. Distances between atoms are shown as yellow dashed lines. B Same view as (A) for C2.36 (gold) bound in the outer site of DrOTOP1_C2.36. H574 from DrOTOP1_apo is shown as a transparent stick. C View of C2.2 (cyan) bound in the central site of DrOTOP1_C2.2. D Inhibition by 10 µM C2.2 in cells expressing mutations as indicated (n = 5–9 cells per construct). E Representative traces showing dose-dependent inhibition of G130W by C2.2. F Dose-response data for inhibition by C2.2 for mutants G130W, E384A, and M573W, fitted to the Hill equation (n = 8, 7, and 5 cells, respectively); WT data is from Fig. 2F. IC₅₀s and Hill coefficients: E384A: 15.2 µM, h = 1.18; M573W: 30.7 µM, h = 1.24; G130W: 130.4 µM, h = 1.15. G Representative traces showing the lack of inhibition in L134W (blue) by 10 µM C2.2 and vehicle control (0.01% DMSO, gray), compared with the effect of WT (black). H Inhibition by C2.2 or vehicle from experiments as in G measured from recovery of current (WT; n = 10 cells, L134W; n = 5 cells). I, J Representative traces showing dose-dependent inhibition by C2.2 in G130W/A388W and G130W/E384A mutants. K Dose-response data for inhibition by C2.2 for mutants G130W/A388W and G130W/E384A, fit to the Hill equation (n = 6 and 8 cells); G130W, E384A, and WT data from (F) and single mutant data from Fig. 2F. IC₅₀s and Hill slopes: G130W/A388W: 397.4 µM, h = 1.10; G130W/E384A saturates at ~ 50%. L Inhibition by 100 µM C11 in cells expressing central, outer, or double mutants (n = 5–11 cells). M Representative traces showing inhibition by 100 µM C2.36 of WT (black) and G130W (blue), compared with the effect of vehicle control (0.1% DMSO). in gray. N Inhibition by C2.36 (WT and G130W) or vehicle (G130W) (WT; n = 8 cells, G130W + C2.36; n = 5 cells, G130W + Veh; n = 4 cells). Significance determined by one-way ANOVAs with Dunnett’s correction (mutants vs. WT for D, L; double mutants vs. G130W for D), unpaired two-tailed t tests with Welch correction (H, N) and paired two-tailed t test (H; C2.2 vs. vehicle on L134W). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for all statistical tests. All data presented as mean ± SEM. Experiments performed at V_m = −80 mV. Source data are provided as a Source Data file.