Identification of a druggable pocket of the calcium-activated chloride channel TMEM16A in its open state

Figures

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  Figure 1. Open state of TMEM16A.A, Ca2+-free structure (orange) of TMEM16A versus the Ca2+-bound structure (magenta). B, cryo-EM structure of TMEM16A in calcium-bound state. C, the overlap of Ca2+-bound structure (magenta) of TMEM16A and the open state of nhTMEM16 (white). D, the root-mean-square deviation of (C). E, the reconstructed two-dimensional free energy surface (kcal/mol) shows different conformational distributions of TMEM16A. F, representative conformation of TMEM16A. G, the overlap of open state and desensitized state of TMEM16A. H, the root-mean-square deviation of (G). I, the pore radius of open state and desensitized state of TMEM16A. The pore maps generated using HOLE is shown in green (radius ≈ size of water) and blue (radius > size of water).

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  Figure 2. Identification of an inhibitor-binding pocket for the open state of TMEM16A.A, the inhibitor-binding pocket of the desensitized state of TMEM16A. B, the inhibitor-binding pocket of the open state of TMEM16A. C, affinity assessment of compounds and proteins in virtual screening. D, docking conformation of Etoposide and TMEM16A. E, representative current of TMEM16A inhibited by various concentrations of Etoposide (1, 3, 10, 30, 100, 300 μM). F, concentration response curves of Etoposide inhibition of TMEM16A currents in HEK293T cells. The plot was fitted to the Hill equation (n = 5). G, I-V curve of the TMEM16A currents inhibited with different concentrations of Etoposide (n = 5).

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  Figure 3. Etoposide inhibits TMEM16A by blocking the ion conduction pore.A and B, molecular dynamics simulation systems. TMEM16A is shown in cartoon; POPC is shown in stick (gray). Etoposide is shown as yellow sticks. C, the root-mean-square fluctuation (RMSF) of the TMEM16A protein. D, radius of gyration of backbone atoms of binding pocket for the two systems. E, the overlap of Apo structure (purple, 200 ns)/Holo structure (cyan, 200 ns) and open structure of TMEM16A (white). F, the RMSD of Apo structure (purple 200 ns)/Holo structure (cyan 200 ns) and open structure. G, the RMSD of Apo structure (purple 200 ns)/Holo structure (cyan 200 ns) and desensitized state structure. H, the overlap of Apo structure (purple, 200 ns), Holo structure (cyan, 200 ns), and desensitized state structure (magenta).

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  Figure 4. Interaction mode of Etoposide with the open state of TMEM16A.A, schematic diagram of reaction coordinates. B, free energy landscape of the Etoposide binding mode. C and D, representations of the binding mode of Etoposide and TMEM16A in their lowest binding energy conformation. E, ESP mapped molecular VDW surface of etoposide. F, electron localization function of Etoposide. G, representative mutant TMEM16A whole-cell current of HEK293T cells activated by 600 nM Ca2+ in the pipette solution and inhibited by 1 mM Etoposide. H, concentration response curves of Etoposide inhibition of TMEM16A mutant currents in HEK293T cells. The plot was fitted to the Hill equation (n = 3∼5). I, IC50 values of Etoposide inhibits TMEM16A wildtype and mutants. ESP, electrostatic surface potential; VDW, van der Waals.

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  Figure 5. Interaction mode between Etoposide and desensitized state structure.A, the docking conformation of Etoposide and TMEM16A. B, overlaps of Etoposide with that of previously discovered inhibitors. C, free energy landscape of the Etoposide binding mode. D and E, representations of the binding mode of Etoposide and TMEM16A in their lowest binding energy conformation. F, possible space occupation by Etoposide in the binding pocket. G, representative mutant TMEM16A whole-cell current of HEK293T cells activated by 600 nM Ca2+ in the pipette solution and inhibited by 30 μM Etoposide. H, inhibitory effect of 30 μM Etoposide on TMEM16A wildtype and mutant (n = 5). Data are means ± SD. One-way ANOVA followed by Tukey honestly significant difference test (∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

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  Figure 6. Etoposide inhibits prostate cancer by targeting TMEM16A.A and B, Western blot images of TMEM16A expression in HEK293T and PC-3 cells. C and D, the expression of TMEM16A was normalized to the expression level of GAPDH (n = 6). E, TMEM16A currents of PC-3 cells were activated by 600 nM Ca2+ in the pipette solution and inhibited by 10 μM of T16Ainh-A01 or 100 μM of Etoposide (n = 4∼5). F, statistical results of the TMEM16A whole-cell current in (E). G, long-term colony formation assay was performed over 2 weeks on PC-3 cells (purple), cultured with the indicated concentrations of Etoposide. H and I, the statistical histogram of (G) (n = 4). J, inhibitory effect of Etoposide on the proliferation of PC-3 (n = 6). K, statistics results of cell viability after transfection with shRNA-16A (n = 6). Data are means ± SD. One-way ANOVA followed by Tukey honestly significant difference test (ns p > 0.1, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).