Chunlong Ma

Functional studies indicate amantadine binds to the pore of the influenza A virus M2 proton-selective ion channel

Influenza A and B viruses contain proton-selective ion channels, A/M2 and BM2, respectively, and the A/M2 channel activity is inhibited by the drugs amantadine and its methyl derivative rimantadine. The structure of the pore-transmembrane domain has been determined by both x-ray crystallography [Stouffer et al. (2008) Nature 451:596–599] and by NMR methods [Schnell and Chou (2008) Nature 451:591–595]. Whereas the crystal structure indicates a single amantadine molecule in the pore of the channel, the NMR data show four rimantadine molecules bound on the outside of the helices toward the cytoplasmic side of the membrane. Drug binding includes interactions with residues 40–45 with a polar hydrogen bond between rimantadine and aspartic acid residue 44 (D44) that appears to be important. These two distinct drug-binding sites led to two incompatible drug inhibition mechanisms. We mutagenized D44 and R45 to alanine as these mutations are likely to interfere with rimantadine binding and lead to a drug insensitive channel. However, the D44A channel was found to be sensitive to amantadine when measured by electrophysiological recordings in oocytes of Xenopus laevis and in mammalian cells, and when the D44 and R45 mutations were introduced into the influenza virus genome. Furthermore, transplanting A/M2 pore residues 24–36 into BM2, yielded a pH-activated chimeric ion channel that was partially inhibited by amantadine. Thus, taken together our functional data suggest that amantadine/rimantadine binding outside of the channel pore is not the primary site associated with the pharmacological inhibition of the A/M2 ion channel.

Identification of the functional core of the influenza A virus A/M2 proton-selective ion channel

The influenza A virus M2 protein (A/M2) is a homotetrameric pH-activated proton transporter/channel that mediates acidification of the interior of endosomally encapsulated virus. This 97-residue protein has a single transmembrane (TM) helix, which associates to form homotetramers that bind the anti-influenza drug amantadine. However, the minimal fragment required for assembly and proton transport in cellular membranes has not been defined. Therefore, the conductance properties of truncation mutants expressed in Xenopus oocytes were examined. A short fragment spanning residues 21–61, M2(21-61), was inserted into the cytoplasmic membrane and had specific, amantadine-sensitive proton transport activity indistinguishable from that of full-length A/M2; an epitope-tagged version of an even shorter fragment, M2(21-51)-FLAG, had specific activity within a factor of 2 of the full-length protein. Furthermore, synthetic fragments including a peptide spanning residues 22–46 were found to transport protons into liposomes in an amantadine-sensitive manner. In addition, the functionally important His-37 residue pKa values are highly perturbed in the tetrameric form of the protein, a property conserved in the TM peptide and full-length A/M2 in both micelles and bilayers. These data demonstrate that the determinants for folding, drug binding, and proton translocation are packaged in a remarkably small peptide that can now be studied with confidence.

Exploring the Size Limit of Templates for Inhibitors of the M2 Ion Channel of Influenza A Virus

Amantadine inhibits the M2 proton channel of influenza A virus, yet its clinical use has been limited by the rapid emergence of amantadine-resistant virus strains. We have synthesized and characterized a series of polycyclic compounds designed as ring-contracted or ring-expanded analogues of amantadine. Inhibition of the wild-type (wt) M2 channel and the A/M2-S31N and A/M2-V27A mutant ion channels were measured in Xenopus oocytes using two-electrode voltage clamp (TEV) assays. Several bisnoradamantane and noradamantane derivatives inhibited the wt ion channel. The compounds bind to a primary site delineated by Val27, Ala30, and Ser31, though ring expansion restricts the positioning in the binding site. Only the smallest analogue 8 was found to inhibit the S31N mutant ion channel. The structure-activity relationship obtained by TEV assay was confirmed by plaque reduction assays with A/H3N2 influenza virus carrying wt M2 protein.

Discovery of Novel Dual Inhibitors of the Wild-Type and the Most Prevalent Drug-Resistant Mutant, S31N, of the M2 Proton Channel from Influenza A Virus

Anti-influenza drugs, amantadine and rimantadine, targeting the M2 channel from influenza A virus are no longer effective because of widespread drug resistance. S31N is the predominant and amantadine-resistant M2 mutant, present in almost all of the circulating influenza A strains as well as in the pandemic 2009 H1N1 and the highly pathogenic H5N1 flu strains. Thus, there is an urgent need to develop second-generation M2 inhibitors targeting the S31N mutant. However, the S31N mutant presents a huge challenge to drug discovery, and it has been considered undruggable for several decades. Using structural information, classical medicinal chemistry approaches, and M2-specific biological testing, we discovered benzyl-substituted amantadine derivatives with activity against both S31N and WT, among which 4-(adamantan-1-ylaminomethyl)-benzene-1,3-diol (44) is the most potent dual inhibitor. These inhibitors demonstrate that S31N is a druggable target and provide a new starting point to design novel M2 inhibitors that address the problem of drug-resistant influenza A infections.

The Interplay of Functional Tuning, Drug Resistance, and Thermodynamic Stability in the Evolution of the M2 Proton Channel from the Influenza A Virus

We explore the interplay between amino acid sequence, thermodynamic stability, and functional fitness in the M2 proton channel of influenza A virus. Electrophysiological measurements show that drug-resistant mutations have minimal effects on M2s specific activity, and suggest that resistance is achieved by altering a binding site within the pore rather than a less direct allosteric mechanism. In parallel, we measure the effects of these mutations on the free energy of assembling the homotetrameric transmembrane pore from monomeric helices in micelles and bilayers. Although there is no simple correlation between the evolutionary fitness of the mutants and their stability, all variants formed more stable tetramers in bilayers, and the least-fit mutants showed the smallest increase in stability upon moving from a micelle to a bilayer environment. We speculate that the folding landscape of a micelle is rougher than that of a bilayer, and more accommodating of conformational variations in nonoptimized mutants.

An amantadine-sensitive chimeric BM2 ion channel of influenza B virus has implications for the mechanism of drug inhibition

Influenza A virus M2 (A/M2) and the influenza B virus BM2 are both small integral membrane proteins that form proton-selective ion channels. Influenza A virus A/M2 channel is the target of the antiviral drug amantadine (and its methyl derivative rimantadine), whereas BM2 channel activity is not affected by the drug. The atomic structure of the pore–transmembrane (TM) domain peptide has been determined by x-ray crystallography [Stouffer et al. (2008) Nature 451:596–599] and of a larger M2 peptide by NMR methods [Schnell and Chou (2008) Nature 451:591–595]. The crystallographic data show electron density (at 3.5 Å resolution) in the channel pore, consistent with amantadine blocking the pore of the channel. In contrast, the NMR data show 4 rimantadine molecules bound on the outside of the helices toward the cytoplasmic side of the membrane. Drug binding includes interactions with residues 40–45 and a polar hydrogen bond between rimantadine and aspartic acid residue 44 (D44). These 2 distinct drug-binding sites led to 2 incompatible drug inhibition mechanisms. We have generated chimeric channels between amantadine-sensitive A/M2 and amantadine-insensitive BM2 designed to define the drug-binding site. Two chimeras containing 5 residues of the A/M2 ectodomain and residues 24–36 of the A/M2 TM domain show 85% amantadine/rimantadine sensitivity and specific activity comparable to that of WT BM2. These functional data suggest that the amantadine/rimantadine binding site identified on the outside of the 4 helices is not the primary site associated with the pharmacologic inhibition of the A/M2 ion channel.

Exploring organosilane amines as potent inhibitors and structural probes of influenza a virus M2 proton channel.

We describe the use of organosilanes as inhibitors and structural probes of a membrane protein, the M2 proton channel from influenza A virus. Organosilane amine inhibitors were found to be generally as potent as their carbon analogues in targeting WT A/M2 and more potent against the drug-resistant A/M2-V27A mutant. In addition, intermolecular NOESY spectra with dimethyl-substituted organosilane amine inhibitors clearly located the drug binding site at the N-terminal lumen of the A/M2 channel close to V27.

Identification of the Pore-lining Residues of the BM2 Ion Channel Protein of Influenza B Virus

The influenza B virus BM2 proton-selective ion channel is essential for virus uncoating, a process that occurs in the acidic environment of the endosome. The BM2 channel causes acidification of the interior of the virus particle, which results in dissociation of the viral membrane protein from the ribonucleo-protein core. The BM2 protein is similar to the A/M2 protein ion channel of influenza A virus (A/M2) in that it contains an HXXXW motif. Unlike the A/M2 protein, the BM2 protein is not inhibited by the antiviral drug amantadine. We used mutagenesis to ascertain the pore-lining residues of the BM2 ion channel. The specific activity (relative to wild type), reversal voltage, and susceptibility to modification by (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide of cysteine mutant proteins were measured in oocytes. It was found that mutation of transmembrane domain residues Ser9, Ser12, Phe13, Ser16, His19, and Trp23 to cysteine were most disruptive for ion channel function. These cysteine mutants were also most susceptible to (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide modification. Furthermore, considerable amounts of dimer were formed in the absence of oxidative reagents when cysteine was introduced at positions Ser9, Ser12, Ser16, or Trp23. Based on these experimental data, a BM2 transmembrane domain model is proposed. The presence of polar residues in the pore is a probable explanation for the amantadine insensitivity of the BM2 protein and suggests that related but more polar compounds might serve as useful inhibitors of the protein.

Easily accessible polycyclic amines that inhibit the wild-type and amantadine-resistant mutants of the M2 channel of influenza A virus

Amantadine inhibits the M2 proton channel of influenza A virus, yet most of the currently circulating strains of the virus carry mutations in the M2 protein that render the virus amantadine-resistant. While most of the research on novel amantadine analogues has revolved around the synthesis of novel adamantane derivatives, we have recently found that other polycyclic scaffolds effectively block the M2 proton channel, including amantadine-resistant mutant channels. In this work, we have synthesized and characterized a series of pyrrolidine derivatives designed as analogues of amantadine. Inhibition of the wild-type M2 channel and the A/M2-S31N, A/M2-V27A, and A/M2-L26F mutant forms of the channel were measured in Xenopus oocytes using two-electrode voltage clamp assays. Most of the novel compounds inhibited the wild-type ion channel in the low micromolar range. Of note, two of the compounds inhibited the amantadine-resistant A/M2-V27A and A/M2-L26F mutant ion channels with submicromolar and low micromolar IC50, respectively. None of the compounds was found to inhibit the S31N mutant ion channel.

3-Azatetracyclo[5.2.1.15,8.01,5]undecane Derivatives: From Wild-Type Inhibitors of the M2 Ion Channel of Influenza A Virus to Derivatives with Potent Activity against the V27A Mutant

We have synthesized and characterized a series of compounds containing the 3-azatetracyclo[5.2.1.1(5,8).0(1,5)]undecane scaffold designed as analogues of amantadine, an inhibitor of the M2 proton channel of influenza A virus. Inhibition of the wild-type (WT) M2 channel and the amantadine-resistant A/M2-S31N and A/M2-V27A mutant ion channels were measured in Xenopus oocytes using two-electrode voltage clamp (TEV) assays. Most of the novel compounds inhibited the WT ion channel in the low micromolar range. Of note, several compounds inhibited the A/M2 V27A mutant ion channel, one of them with submicromolar IC50. None of the compounds was found to inhibit the S31N mutant ion channel. The antiviral activity of three novel dual WT and A/M2-V27A channels inhibitors was confirmed by influenza virus yield assays.