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1.  Aminoadamantanes with Persistent in Vitro Efficacy against H1N1 (2009) Influenza A 
Journal of medicinal chemistry  2014;57(11):4629-4639.
A series of 2-adamantanamines with alkyl adducts of various lengths were examined for efficacy against strains of influenza A including those having an S31N mutation in M2 proton channel that confer resistance to amantadine and rimantadine. The addition of as little as one CH2 group to the methyl adduct of the amantadine/rimantadine analogue, 2-methyl-2-amino-adamantane, led to activity in vitro against two M2 S31N viruses A/Calif/07/2009 (H1N1) and A/PR/8/34 (H1N1) but not to a third A/WS/33 (H1N1). Solid state NMR of the transmembrane domain (TMD) with a site mutation corresponding to S31N shows evidence of drug binding. But electrophysiology using the full length S31N M2 protein in HEK cells showed no blockade. A wild type strain, A/Hong Kong/1/68 (H3N2) developed resistance to representative drugs within one passage with mutations in M2 TMD, but A/Calif/07/2009 S31N was slow (>8 passages) to develop resistance in vitro, and the resistant virus had no mutations in M2 TMD. The results indicate that 2-alkyl-2-aminoadamantane derivatives with sufficient adducts can persistently block p2009 influenza A in vitro through an alternative mechanism. The observations of an HA1 mutation, N160D, near the sialic acid binding site in both 6-resistant A/ Calif/07/2009(H1N1) and the broadly resistant A/WS/33(H1N1) and of an HA1 mutation, I325S, in the 6-resistant virus at a cell-culture stable site suggest that the drugs tested here may block infection by direct binding near these critical sites for virus entry to the host cell.
PMCID: PMC4127532  PMID: 24793875
2.  Drug sensitivity, drug-resistant mutations, and structures of three conductance domains of viral porins 
Biochimica et biophysica acta  2010;1808(2):538-546.
Recent controversies associated with the structure of the M2 protein from influenza A virus and the binding site of drug molecules amantadine and rimantadine motivated the comparison here of the drug binding to three viral porins including the M2 proteins from influenza A and B as well as the viral protein ‘u’ from HIV-1. While the M2 protein from influenza B does not normally bind amantadine, chimeras with the M2 protein from influenza A show blockage by amantadine. Similarly, Vpu does not normally bind rimantadine, but the single site mutation A18H converts a non-specific channel to a selective proton channel that is sensitive to rimantadine. The comparison of structures and amino acid sequences shows that the membrane protein sample environment can have a significant influence on the structural result. While a bilayer surface bound amphipathic helix has been characterized for AM2, such a helix may be possible for BM2 although it has evaded structural characterization in detergent micelles. A similar amphipathic helix seems less likely for Vpu. Even though the A18H Vpu mutant forms rimantadine sensitive proton channels, the binding of drug and its influence on the protein structure appears to be very different from that for the M2 proteins. Indeed, drug binding and drug resistance in these viral porins appears to result from a complex set of factors.
PMCID: PMC3709975  PMID: 20655872
M2 protein; Influenza A virus; Influenza B virus; Viral protein u; HIV; PISEMA; Solid-state NMR; Membrane protein
3.  M2 Protein from Influenza A: From multiple structures to biophysical and functional insights 
Current Opinion in Virology  2012;2(2):128-133.
The M2 protein from influenza A is a proton channel as a tetramer, with a single transmembrane helix from each monomer lining the pore. Val27 and Trp41 form gates at either end of the pore and His37 mediates the shuttling of protons across a central barrier between the N- and C-terminal aqueous pore regions. Numerous structures of this transmembrane domain and of a longer construct that includes an amphipathic helix are now in the Protein Data Bank. Many structural differences are apparent from samples obtained in a variety of membrane mimetic environments. High-resolution structural results in lipid bilayers have provided novel insights into the functional mechanism of the unique HxxxW cluster in the M2 proton channel.
PMCID: PMC3322387  PMID: 22482709
4.  Insight into the Mechanism of the Influenza A Proton Channel from a Structure in a Lipid Bilayer 
Science (New York, N.y.)  2010;330(6003):509-512.
The M2 protein from the influenza A virus, an acid-activated proton-selective channel, has been the subject of numerous conductance, structural, and computational studies. However, little is known at the atomic level about the heart of the functional mechanism for this tetrameric protein, a His37-Trp41 cluster. We report the structure of the M2 conductance domain (residues 22 to 62) in a lipid bilayer, which displays the defining features of the native protein that have not been attainable from structures solubilized by detergents. We propose that the tetrameric His37-Trp41 cluster guides protons through the channel by forming and breaking hydrogen bonds between adjacent pairs of histidines and through specific interactions of the histidines with the tryptophan gate. This mechanism explains the main observations on M2 proton conductance.
PMCID: PMC3384994  PMID: 20966252
5.  Fluorescence anisotropy of diphenylhexatriene and its cationic Trimethylamino derivative in liquid dipalmitoylphosphatidylcholine liposomes: opposing responses to isoflurane 
BMC Biophysics  2012;5:5.
The mechanism of action of volatile general anesthetics has not yet been resolved. In order to identify the effects of isoflurane on the membrane, we measured the steady-state anisotropy of two fluorescent probes that reside at different depths. Incorporation of anesthetic was confirmed by shifting of the main phase transition temperature.
In liquid crystalline dipalmitoylphosphatidylcholine liposomes, isoflurane (7-25 mM in the bath) increases trimethylammonium-diphenylhexatriene fluorescence anisotropy by ~0.02 units and decreases diphenylhexatriene anisotropy by the same amount.
The anisotropy data suggest that isoflurane decreases non-axial dye mobility in the headgroup region, while increasing it in the tail region. We propose that these results reflect changes in the lateral pressure profile of the membrane.
PMCID: PMC3359235  PMID: 22444827
Biochimica et biophysica acta  2010;1808(2):516-521.
Amantadine-sensitive proton uptake by liposomes is currently the preferred method of demonstrating M2 functionality after reconstitution, to validate structural determination with techniques such as solid-state NMR. With strong driving forces (two decades each of both [K+] gradient-induced membrane potential and [H+] gradient), M2(22-62) showed a transport rate of 78 H+/ tetramer-s (pHo 6.0, pHi 8.0, nominal Vm = -114 mV), higher than previously measured for similar, shorter, and full-length constructs. Amantadine sensitivity of the conductance domain at pH 6.8 was also comparable to other published reports. Proton flux rate was optimal at protein densities of 0.05-1.0% (peptide wt% in lipid). Rundown of total proton uptake after addition of valinomycin and CCCP, as detected by delayed addition of valinomycin, indicated M2-induced K+ flux of 0.1 K+/tetramer-s, and also demonstrated that the K+ permeability, relative to H+, was 2.8×10-6. Transport rate, amantadine and cyclooctylamine sensitivity, acid activation, and H+ selectivity were all consistent with full functionality of the reconstituted conductance domain. Decreased external pH increased proton uptake with an apparent pKa of 6.
PMCID: PMC3005097  PMID: 20969830
M2 protein; influenza A virus; liposome assay; acid activation; specific transport activity; membrane protein
7.  Free-Energy Profiles for Ions in the Influenza M2-TMD Channel 
Proteins  2009;76(4):794-807.
M2 transmembrane domain channel (M2-TMD) permeation properties are studied using molecular dynamics simulations of M2-TMD (1NYJ) embedded in a lipid bilayer (DMPC) with 1 mol/kg NaCl or KCl saline solution. This study allows examination of spontaneous cation and anion entry into the selectivity filter. Three titration states of the M2-TMD tetramer are modeled for which the four His37 residues, forming the selectivity filter, are net uncharged, +2 charged, or +3 charged. M2-TMD structural properties from our simulations are compared with the properties of other models extracted from NMR and X-ray studies. During 10 ns simulations, chloride ions rarely occupy the positively-charged selectivity filter, whereas from umbrella sampling simulations, Cl− has a lower free-energy barrier in the selectivity-filter region than either Na+ or NH4+, and NH4+ has a lower free-energy barrier than Na+. For Na+ and Cl−, the free-energy barriers are less than 5 kcal/mol, suggesting that the 1NYJ conformation would probably not be exquisitely proton selective. We also point out a rotameric configuration of Trp41 that could fully occlude the channel.
PMCID: PMC2760258  PMID: 19296508
Interhelical distances; Side chain rotamers; Potential of mean force; Influenza A virus; Membrane channel; Salt
8.  Computational Studies of Gramicidin Permeation: An Entryway Sulfonate Enhances Cation Occupancy at Entry Sites 
Biochimica et biophysica acta  2009;1788(6):1404-1412.
The impact on the cation-transport free-energy profile of replacing the C-terminal ethanolamine in the gramicidin A channel with a taurine residue is studied using molecular dynamics simulations of gramicidin A (1JNO) embedded in a lipid bilayer (DMPC) with 1 mol/kg NaCl saline solution. The potential of mean force for ion transport is obtained by umbrella sampling. The presence of a negatively charged sulfonate group at the entrance of the gramicidin channel affects the depth and the location of the binding sites, producing a strong attraction for the cations in the bulk. The potential of mean force by the sulfonate acting directly through electrostatics and van der Waals interactions on the test ion is highly modulated by indirect effects (i.e., sulfonate effects on other components of the system that, in turn, affect the ion free-energy profile in the channel). Because the “entry” sites are located symmetrically at both entry and exit of the channel, the deeper free-energy wells should inhibit exit. Given that the channel has increased conductance experimentally, the simulation results suggest that the channel conductance is normally entry limited.
PMCID: PMC2701196  PMID: 19361485
Ion channel; Potential of mean force; Sulfonate parametrization; Water layering; Molecular dynamics simulations
9.  The Gramicidin Channel Ion Permeation Free-Energy Profile: Direct and Indirect Effects of CHARMM Force Field Improvements 
A revised CHARMM force field for tryptophan residues is studied as well as the effect of the new peptide torsion-energy cross-correlation correction algorithm (CMAP) using molecular dynamics simulations of gramicidin A (1JNO) embedded in a lipid bilayer (DMPC) with 1 mol/kg NaCl or 1 mol/kg KCl saline solution. The new tryptophan force field produces, in the decomposition, a Na+ PMFTrp that is consonant with the prediction from the experimental results, analyzed with rate theory by Durrant et al. (2006), in stark contrast to the prediction of the original CHARMM, version 22, tryptophan side-chain force field. However, the effect is lost in the complete PMF due to indirect effects mediated by other components of the system (peptide, lipid, ions, and water). CMAP reduces the excessive translocation barrier. Decomposition demonstrates that this effect is due to effects on the K+ PMFH2O rather than on K+ PMFgA. Both represent examples of indirect effects (i.e., consequences of the force field mediated by “innocent bystander” molecules). The results have been confirmed to be robust using an alternative umbrella potential method.
PMCID: PMC2806686  PMID: 20084184
Independent transport coordinate; Relative transport coordinate; Potential of mean force; Highly occupied site; Torsion angles; NPAT; CMAP

Results 1-9 (9)