Study reveals that 1PBC and niclosamide bind the same drug-binding pocket

By Neha MathurReviewed by Danielle Ellis, B.Sc.Feb 14 2022

In a recent preprint posted to Research Square* and under consideration at a Nature Portfolio Journal, researchers used cryo-electron microscopy (EM) to demonstrate that 1-hydroxy-3-(trifluoromethyl)pyrido[1,2-a]benzimidazole-4-carbonitrile (1PBC), and the drug niclosamide, both inhibitors of transmembrane proteins 16 (TMEM16) family have therapeutic potential and may be used for coronavirus disease 2019 (COVID-19) treatment.

The TMEM16 family of calcium-activated membrane proteins occurs in mammals in 10 distinct forms, ranging from TMEM16A-K. These assemble as dimers, with each subunit having ten transmembrane helices (TMs) containing an ion conduction pore enclosed and surrounded by TM3-7. However, it is unknown whether two monomers of TMEM16 work independently or cooperatively. Recent studies have indicated that drug modulators, 1PBC, and niclosamide bind the same drug-binding pocket in TMEM16A and TMEM16F. Subsequently, these have emerged as important pharmacological targets for the COVID-19 treatment.

*Important notice: Research Square publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

About the study

In the present study, researchers determined multiple cryo-EM structures of TMEM16F, representing different conformations of TMEM16F in unliganded states, in the presence of Ca²⁺ ions, and TMEM16A and TMEM16F activator, phosphatidylinositol 4,5-biphosphate (PIP2).

They also determined structures of TMEM16F bound to niclosamide and 1PBC. Using computational docking and mutagenesis analyses, they validated the binding site of these modulators as a hydrophobic groove between TM1 and TM6.

Notably, TMEM16F functions as a Ca²⁺-activated ion channel and a Ca²⁺-activated lipid scramblase. Through its lipid scrambling activity, TMEM16F allows the movement of diverse lipids between the inner and outer leaflets of the plasma membrane.

Study findings

Previous studies imposed C2 symmetry during cryo-EM data processing of TMEM16 proteins in lipid nanodiscs. In the current study, the researchers did not impose C2 symmetry to identify three distinct coexisting states with major differences in the conformation of TM6 and the number of Ca²⁺ atoms bound in each monomer of TMEM16F. 

These states revealed that straightening of TM6 correlated with binding of the second Ca²⁺ ion, whereas kinking of TM6 was associated with an outward rigid body motion of the intracellular domain that brought it closer to the nanodisc. Moreover, bending of TM6 directly correlated with distortion of the nanodisc and significant thinning of the membrane at the kinking position. Overall, these observations support the notion that kinking of TM6 at P628 causes membrane distortion.

Further, cryo-EM structures showed that drug modulators/antagonists niclosamide and 1PBC bind the same, conserved hydrophobic pocket in TMEM16A and TMEM16F. They directly contacted the extracellular-proximal end of TM6, showing that residues on this helix are critical for drug binding. As TM6 is the main gating element in both TMEM16A and TMEM16F, it gets locked by niclosamide and 1PBC in a closed configuration when they bind the upper part of TM6. 

The data also showed that the individual contribution of different hydrophobic residues to the interaction was distinct in TMEM16A and TMEM16F. Moreover, the non-conserved residues, such as T606 and K370 in TMEM16F, were also important for the inhibitory effects of 1PBC and niclosamide. Therefore, structural studies revealed that while the TMEM16A binding pocket consisted almost exclusively of hydrophobic residues, the equivalent site in TMEM16F had several charged and OH-containing side chains.

Further, the structural model revealed that 1PBC and niclosamide binding sites had maximum membrane distortion and thinning in the TMEM16F structures. Additionally, this site corresponded precisely with the entry and exit point of the lipids as they transitioned between the inner and outer leaflets of the plasma membrane. These structural revelations illustrated how 1PBC and niclosamide directly inhibited TMEM16F scramblase activity by physically obstructing the path of the lipids across the membrane, thereby significantly reducing lipid densities along this path.

Additionally, the study reconstructions showed glycans and conserved disulfide bonds in the extracellular region, the presence of a third Ca²⁺ ion coordinated by E395 on TM2, and S854 and D859 on TM10 near the dimer interface in the intracellular region of TMEM16F.

Conclusions 

The study established a structural framework of TMEM16F dimer to provide insights into the underlying mechanism behind its calcium-activated chloride channel (CaCC) that regulates several physiological properties in response to changes in intracellular Ca²⁺ concentration.

Niclosamide is a highly hydrophobic molecule that presents extremely poor solubility in aqueous solutions. The cryo-EM structures showed non-conserved hydrophilic residues within the drug-binding pocket of TMEM16F. Developing niclosamide analogs with better pharmacological properties that might exclusively target TMEM16F would help design potent and specific TMEM16 antagonists for asthma, cancer, and COVID-19 treatment.

*Important notice: Research Square publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.