Identification of a conserved drug binding pocket in TMEM16 proteins

Abstract

The TMEM16 family of calcium-activated membrane proteins includes ten mammalian paralogs (TMEM16A-K) playing distinct physiological roles with some implicated in cancer and airway diseases. Their modulators with therapeutic potential include 1PBC, a potent inhibitor with anti-tumoral properties, and the FDA-approved drug niclosamide that targets TMEM16F to inhibit syncytia formation induced by SARS-CoV-2 infection. Here, we report cryo-EM structures of TMEM16F associated with 1PBC and niclosamide, revealing that both molecules bind the same drug binding pocket. We functionally and computationally validate this binding pocket in TMEM16A as well as TMEM16F, thereby showing that drug modulation also involves residues that are not conserved between TMEM16A and TMEM16F. This study establishes a much-needed structural framework for the development of more potent and more specific drug molecules targeting TMEM16 proteins.

Figure 1. Cryo-EM analysis reveals asymmetry within the TMEM16F dimer.

(A) Cryo-EM density of the asymmetric state of the TMEM16F dimer with the monomers colored blue and light blue, respectively, and the lipid densities in grey. The gaussian filtered cryo-EM density (semitransparent) reveals distortion of the lipid nanodisc. (B) Side view of a TMEM16F monomer (blue) highlighting the trail of lipids (grey) covering the TM region. (C) Front and side view of the atomic model of the asymmetric state of TMEM16F with Ca²⁺ atoms and glycans shown in green and red, respectively. The ion conduction channel identified by HOLE is represented by spheres colored in rainbow scale based on the local width of the channel, where red <1.5 Å and blue >7.5 Å.

Figure 2. Niclosamide and 1PBC bind the same hydrophobic groove in TMEM16F.

Atomic model of the TM1-TM6 region of (A) Class1 and (B) Class 2 of the drug-free control, (C) niclosamide- and (D) 1PBC-supplemented datasets. In each case, the additional cryo-EM densities found in the area are shown. Below, zoom into the TM1-TM6 groove with the residues shown as sticks and colored by heteroatom and the additional density found within the pocket shown in semitransparent. Structures of niclosamide and 1PBC as determined by computational docking using Glide are shown in purple and green, respectively.

Figure 3. Functional validation of the drug binding site in TMEM16F.

Representative curves of live imaging of TMEM16F-dependent PS exposure (A) and (C); and Ca²⁺ influx (E) and (G). Data are represented as mean ± SEM. Scattered dot plots of time of onset of TMEM16F-dependent PS exposure [(B) and (D)] and Ca²⁺ influx [(F) and (H)]. Time of onset could not be determined for time courses with a linear rather than sigmoidal rise. The mean ± SEM is shown along with the statistical significance determined by unpaired t-test for each mutant as compared to vehicle controls (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 4. Electrophysiology-based validation of the binding site in TMEM16A.

Representative voltage clamp current traces at +70 mV with holding potential at −5 mV of wildtype mTMEM16A and alanine substitution mutants in the absence or presence of (A) 3 μM niclosamide or (B) 30 μM 1PBC recorded in 500 nM [Ca²⁺]_i or 12 mM [Ca²⁺]_i, respectively. Graphs showing the coefficient of the steady-state current measured for the wildtype control and each mutant upon addition of (C) niclosamide and (D) 1PBC divided by the maximum Intensity (I/I_max) in each case. The mean ± SEM is shown along with the statistical significance determined by unpaired t-test for each mutant as compared to wildtype controls (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 5. Comparison of the drug binding pocket in TMEM16A and TMEM16F.

(A) Schematic representation of the TMEM16F dimer (light blue and blue) embedded in a lipid bilayer (grey), where Ca²⁺ atoms are shown as green circles and the inhibitors as a purple polygon and dotted black lines represent the closed ion conduction pore. (B) Structure of the drug binding pocket in TMEM16F (blue, left) and TMEM16A (grey, right) with the side chains of the surrounding residues shown as sticks and the non-conserved residues highlighted in orange. Computationally docked structures of niclosamide and 1PBC are shown in purple and green, respectively. All atoms are colored by heteroatom.