Hexamethylene amiloride binds the SARS‐CoV‐2 envelope protein at the protein–lipid interface

The SARS‐CoV‐2 envelope (E) protein forms a five‐helix bundle in lipid bilayers whose cation‐conducting activity is associated with the inflammatory response and respiratory distress symptoms of COVID‐19. E channel activity is inhibited by the drug 5‐(N,N‐hexamethylene) amiloride (HMA). However, the binding site of HMA in E has not been determined. Here we use solid‐state NMR to measure distances between HMA and the E transmembrane domain (ETM) in lipid bilayers. (13)C, (15)N‐labeled HMA is combined with fluorinated or (13)C‐labeled ETM. Conversely, fluorinated HMA is combined with (13)C, (15)N‐labeled ETM. These orthogonal isotopic labeling patterns allow us to conduct dipolar recoupling NMR experiments to determine the HMA binding stoichiometry to ETM as well as HMA‐protein distances. We find that HMA binds ETM with a stoichiometry of one drug per pentamer. Unexpectedly, the bound HMA is not centrally located within the channel pore, but lies on the lipid‐facing surface in the middle of the TM domain. This result suggests that HMA may inhibit the E channel activity by interfering with the gating function of an aromatic network. These distance data are obtained under much lower drug concentrations than in previous chemical shift perturbation data, which showed the largest perturbation for N‐terminal residues. This difference suggests that HMA has higher affinity for the protein–lipid interface than the channel pore. These results give insight into the inhibition mechanism of HMA for SARS‐CoV‐2 E.