Studies of the membrane influenza A/M2 protein with aminoadamantane drugs using experimental and computational biophysics

Chapter 1 refers to the description of the basic features for influenza A virus replication, with emphasis on the function of influenza A M2. In Chapter 2, is described the structure of influenza A matrix 2 (M2) wild type (WT) proton protein channel, which is an archetypal ion channel. It is the target of the antiviral drugs amantadine and rimantadine. A number of methods were used to understand structural and functional features of this channel included neutron diffraction, electrophysiology, solution NMR spectroscopy, solid state NMR (ssNMR) spectroscopy, X-ray crystallography etc during an adventure of three decades with a lot of controversies. The experimental structure of influenza A M2(22-46) transmembrane domain (M2TM), the pore of the M2 protein channel, was solved in 2000 and X-ray structures of its complexes with amantadine, rimantadine etc were published by 2018. Till now basic characteristics of the influenza A M2 conductance domain (CD) protein M2CD or M2AH including M2TM and the amphipathic helices (46-62) have been also solved using ssNMR.The basic features of the experimental biophysical methods used in this PhD thesis, i.e. Differential Scanning Calorimetry (DSC), X-ray scattering at small and wide angles (SAXS/WAXS) and ssNMR are discussed in Chapter 3. Aminoadamantane drugs, e.g. amantadine and rimantadine, are lipophilic amines that bind to membrane embedded influenza A WT M2 protein. In Chapter 4, are investigated the comparative perturbation effects exerted by the influenza M2 WT protein inhibitors amantadine and it’s spiro[pyrrolidine-2,2'-adamantane] variant AK13 to membrane bilayers using biophysical methods and molecular dynamics (MD) simulations. This is a work performed in close collaboration with Professor’s Thomas Mavromoustakos and Professor’s Costas Demetzos groups as well as Dr Barbara Sartori and Professor Heinz Amenitsch. The experimental biophysical methods used included, DSC, X-ray scattering and ssNMR. All three experimental methods pointed out that the two analogs perturbed drastically the DMPC bilayers with AK13 to be more effective at high concentrations. At high ligand concentrations AK13 was tolerated in lipid bilayers while Amt was crystallized. This is an important consideration in possible formulations of these drugs as it designates a limitation of aminoadamantane drug incorporation. MD simulations provided details about the strong interactions of the drugs in the interface region between glycerol backbone and lipophilic segments. The two drugs form hydrogen bonding with both water and sn-2 carbonyls or phosphate oxygens. Such localization of the drugs explains their strong perturbing effect evidenced by all biophysical methodologies applied.In Chapter 5, is described our work to investigate the interactions of M2TM WT with bilayers. This is a work performed in close collaboration with Professor Thomas Mavromoustakos, Professor Costas Demetzos, groups as well as Dr Barbara Sartori and Professor Heinz Amenitsch. The M2TM peptide was synthesized by Professor Thedoros Tselios group. We focused on (a) the characterization of changes in bilayer organization from changes in micromolar concentrations of M2TM WT without or with aminoadamantane (Aamt) ligands, and from changes in Aamt ligand structure included with M2TM, (b) exploring how common biophysical methods can be applied to identify the membrane perturbations effected by the protein without or with the ligand.A variety of biophysical methods, including DSC, SAXS/WAXS, MD simulations, and one-dimension (1D) ssNMR, were used to study two micromolar concentrations of M2TM without or with a small excess of amantadine or its spiro-pyrrolidine analogue, AK13, in DMPC bilayers.DSC and SAXS showed that at a low micromolar M2TM concentration, two lipid domains are observed, which likely correspond to M2TM boundary lipids and bulk lipids. At a higher M2TM concentration, only one domain is identified, indicating that all of the lipids behave as boundary lipids. 1H and 31P ssNMR showed that M2TM in either apo or drug-bound form spans the membrane, interacting strongly with lipid acyl chain-tails and the phosphate groups of the polar head surface. The 13C ssNMR experiments allowed the inspection of excess drug molecules and the assessment of their impact on the lipid head group region.According to SAXS, WAXS, and DSC, in the absence of M2TM both aminoadamantane drugs exert a similar perturbing effect on the bilayer at low concentrations, i.e., mole fractions (relative to lipid) of x=0.05-0.08. At the same concentrations of the drug when M2TM is present, the amantadine and, to a lesser extent, AK13 cause a significant disordering of chain-stacking. This different effect between the two drugs is likely due, according to the MD simulations, to the preference of the excess of the more lipophilic AK13 to locate closer to M2TM. In contrast, amantadine perturbs the lipids through the stronger ionic interactions of its ammonium group with phosphate groups (compared with the buried ammonium group in AK13) and influences the formation of two lipid domains. The preference of AK13 to concentrate inside the lipid may contribute to its six-fold higher binding affinity (compared to amantadine) if drug binding occurs from the lipid by way of a path between the transmembrane helices.The results showed that DSC and SAXS are useful methods to detect changes in membrane organization caused by small changes in M2TM or aminoadamantane drug concentration and structure and that WAXS and MD simulations can suggest details of ligand topology. Water-mediated interactions play key roles in drug binding. In protein sites with sparse polar functionality, a ligand-based only approach is often viewed as insufficient to achieve high affinity and specificity. In Chapter 6, are showed that small molecules, i.e. amantadine and rimantadine, can enable potent inhibition by targeting key waters using as example the M2 WT proton channel of influenza A which is the target of the antiviral drugs amantadine and rimantadine. This is a work performed in close collaboration with Professor William DeGrado and Associate Professor Jun Wang groups. Structural studies of drug binding to the channel using X-ray crystallography have been limited due to the challenging nature of the target, with the first crystal structure solved in 2008 limited to 3.5 Å resolution. We described crystal structures of amantadine bound to M2 in the Inwardclosed conformation (2.00 Å), rimantadine bound to M2 in both the Inwardclosed (2.00 Å) and Inwardopen (2.25 Å) conformations, and a spiro-adamantyl amine inhibitor bound to M2 in the Inwardclosed conformation (2.63 Å). These X-ray crystal structures of the M2 proton channel with bound inhibitors reveal that ammonium groups bind to water-lined sites, formed by two layers of waters close to Ala30 and Gly34, respectively, observed in the X-ray structures, that are hypothesized to stabilize transient hydronium ions formed in the proton-conduction mechanism. Furthermore, the ammonium and adamantyl groups of the adamantyl-amine class of drugs are free to rotate in the channel, minimizing the entropic cost of binding. The MD simulation reproduced perfectly the X-ray structures of cautiously tuned. These drug-bound complexes provide the first high-resolution structures of drugs that interact with and disrupt networks of hydrogen-bonded waters that are widely utilized throughout nature to facilitate proton diffusion within proteins.The V27A mutation confers amantadine resistance to the influenza A M2 WT proton channel and is becoming more prevalent in circulating populations of influenza A virus. In Chapter 7, is described our collaborative work with DeGrado and Wang groups to solve M2TM V27A structure in complex with a spiro-adamantyl amine inhibitor bound to M2(22-46) V27A and also to M2(21-61) V27A in the Inwardclosed conformation using X-ray crystallography and MD simulations. The spiro-adamantyl amine binding site is nearly identical for the two crystal structures. Compared to the M2 WT with valine at position 27, we observed that the channel pore is wider at its N-terminus as a result of the V27A mutation and that this removes V27 side chain hydrophobic interactions that are important for binding of amantadine and rimantadine. The spiro-adamantyl amine inhibitor blocks proton conductance in both the WT and V27A mutant channels by shifting its binding site in the pore depending on which residue is present at position 27. Additionally, in the structure of the M2(21-61) V27A construct, the C-terminus of the channel is tightly packed relative to the M2(22-46) construct. We observed that residues Asp44, Arg45, and Phe48 face the center of the channel pore and would be well-positioned to interact with protons exiting the M2 channel after passing through the His37 gate. However, the orientation of AHs after residue 48 did not reproduce the almost vertical orientation as regards the M2TM, that found by Professor Tim Cross experimentally with ssNMR experiments. The MD simulations of the M2(22-46) V27A - spiro-adamantyl amine complex predicted with accuracy the position of the ligands and waters inside the pore in the X-ray crystal structure of the M2 V27A complex.The influenza A M2 wild type proton channel is the target of the anti-influenza drug rimantadine. Rimantadine has two enantiomers, though most investigations into drug binding and inhibition have used a racemic mixture. ssNMR experiments by Professor Tim Cross have shown significant spectral differences that were interpreted to indicate tighter binding for (R)- vs. (S)- rimantadine. However, it was unclear if this is due to the specific condition of the ssNMR experiments (i.e. close to 0 oC), correlates with a functional difference in drug binding and inhibition and we undertook to investigate this in collaboration with Professor DeGrado, Associate Professor Jun Wang and Professor Jon Essex. Thus, in Chapter 8, using X-ray crystallography, we have determined that both (R)- and (S)-rimantadine bind to the M2 pore with slight differences in the hydration of each enantiomer. However, this did not result in a difference in potency or binding kinetics, as we measured similar values for kon, koff, and Kd in electrophysiological assays and EC50 values in cellular assays. We concluded that the slight differences in hydration we observed in the X-ray structures for the (R)- and (S)-rimantadine enantiomers were not relevant to drug binding or channel inhibition. To further explore the effect of the hydration of the M2 pore on binding affinity, the water structure was evaluated by waters titration calculations Grand Canonical Monte Carlo simulations as a function of the chemical potential of the water. Initially, the two layers of ordered water molecules between the bound drug and the channel's gating His37 residues mask the drug’s chirality. As the chemical potential becomes more unfavorable and the waters from the two layers were removed from the M2 pore, the drug translocated down to the lower water layer, towards the His37 at the C-terminus of M2TM, and the interaction becomes more sensitive to chirality. These studies suggested the feasibility of displacing the upper water layer (toward the N-end close to Ala30) and specifically recognizing the lower water layers by novel chiral drugs.Chapter 1 refers to the description of the basic features for influenza A virus replication, with emphasis on the function of influenza A M2. In Chapter 2, is described the structure of influenza A matrix 2 (M2) wild type (WT) proton protein channel, which is an archetypal ion channel. It is the target of the antiviral drugs amantadine and rimantadine. A number of methods were used to understand structural and functional features of this channel included neutron diffraction, electrophysiology, solution NMR spectroscopy, solid state NMR (ssNMR) spectroscopy, X-ray crystallography etc during an adventure of three decades with a lot of controversies. The experimental structure of influenza A M2(22-46) transmembrane domain (M2TM), the pore of the M2 protein channel, was solved in 2000 and X-ray structures of its complexes with amantadine, rimantadine etc were published by 2018. Till now basic characteristics of the influenza A M2 conductance domain (CD) protein M2CD or M2AH including M2TM and the amphipathic helices (46-62) have been also solved using ssNMR.The basic features of the experimental biophysical methods used in this PhD thesis, i.e. Differential Scanning Calorimetry (DSC), X-ray scattering at small and wide angles (SAXS/WAXS) and ssNMR are discussed in Chapter 3. Aminoadamantane drugs, e.g. amantadine and rimantadine, are lipophilic amines that bind to membrane embedded influenza A WT M2 protein. In Chapter 4, are investigated the comparative perturbation effects exerted by the influenza M2 WT protein inhibitors amantadine and it’s spiro[pyrrolidine-2,2'-adamantane] variant AK13 to membrane bilayers using biophysical methods and molecular dynamics (MD) simulations. This is a work performed in close collaboration with Professor’s Thomas Mavromoustakos and Professor’s Costas Demetzos groups as well as Dr Barbara Sartori and Professor Heinz Amenitsch. The experimental biophysical methods used included, DSC, X-ray scattering and ssNMR. All three experimental methods pointed out that the two analogs perturbed drastically the DMPC bilayers with AK13 to be more effective at high concentrations. At high ligand concentrations AK13 was tolerated in lipid bilayers while Amt was crystallized. This is an important consideration in possible formulations of these drugs as it designates a limitation of aminoadamantane drug incorporation. MD simulations provided details about the strong interactions of the drugs in the interface region between glycerol backbone and lipophilic segments. The two drugs form hydrogen bonding with both water and sn-2 carbonyls or phosphate oxygens. Such localization of the drugs explains their strong perturbing effect evidenced by all biophysical methodologies applied.In Chapter 5, is described our work to investigate the interactions of M2TM WT with bilayers. This is a work performed in close collaboration with Professor Thomas Mavromoustakos, Professor Costas Demetzos, groups as well as Dr Barbara Sartori and Professor Heinz Amenitsch. The M2TM peptide was synthesized by Professor Thedoros Tselios group. We focused on (a) the characterization of changes in bilayer organization from changes in micromolar concentrations of M2TM WT without or with aminoadamantane (Aamt) ligands, and from changes in Aamt ligand structure included with M2TM, (b) exploring how common biophysical methods can be applied to identify the membrane perturbations effected by the protein without or with the ligand.A variety of biophysical methods, including DSC, SAXS/WAXS, MD simulations, and one-dimension (1D) ssNMR, were used to study two micromolar concentrations of M2TM without or with a small excess of amantadine or its spiro-pyrrolidine analogue, AK13, in DMPC bilayers.DSC and SAXS showed that at a low micromolar M2TM concentration, two lipid domains are observed, which likely correspond to M2TM boundary lipids and bulk lipids. At a higher M2TM concentration, only one domain is identified, indicating that all of the lipids behave as boundary lipids. 1H and 31P ssNMR showed that M2TM in either apo or drug-bound form spans the membrane, interacting strongly with lipid acyl chain-tails and the phosphate groups of the polar head surface. The 13C ssNMR experiments allowed the inspection of excess drug molecules and the assessment of their impact on the lipid head group region.According to SAXS, WAXS, and DSC, in the absence of M2TM both aminoadamantane drugs exert a similar perturbing effect on the bilayer at low concentrations, i.e., mole fractions (relative to lipid) of x=0.05-0.08. At the same concentrations of the drug when M2TM is present, the amantadine and, to a lesser extent, AK13 cause a significant disordering of chain-stacking. This different effect between the two drugs is likely due, according to the MD simulations, to the preference of the excess of the more lipophilic AK13 to locate closer to M2TM. In contrast, amantadine perturbs the lipids through the stronger ionic interactions of its ammonium group with phosphate groups (compared with the buried ammonium group in AK13) and influences the formation of two lipid domains. The preference of AK13 to concentrate inside the lipid may contribute to its six-fold higher binding affinity (compared to amantadine) if drug binding occurs from the lipid by way of a path between the transmembrane helices.The results showed that DSC and SAXS are useful methods to detect changes in membrane organization caused by small changes in M2TM or aminoadamantane drug concentration and structure and that WAXS and MD simulations can suggest details of ligand topology. Water-mediated interactions play key roles in drug binding. In protein sites with sparse polar functionality, a ligand-based only approach is often viewed as insufficient to achieve high affinity and specificity. In Chapter 6, are showed that small molecules, i.e. amantadine and rimantadine, can enable potent inhibition by targeting key waters using as example the M2 WT proton channel of influenza A which is the target of the antiviral drugs amantadine and rimantadine. This is a work performed in close collaboration with Professor William DeGrado and Associate Professor Jun Wang groups. Structural studies of drug binding to the channel using X-ray crystallography have been limited due to the challenging nature of the target, with the first crystal structure solved in 2008 limited to 3.5 Å resolution. We described crystal structures of amantadine bound to M2 in the Inwardclosed conformation (2.00 Å), rimantadine bound to M2 in both the Inwardclosed (2.00 Å) and Inwardopen (2.25 Å) conformations, and a spiro-adamantyl amine inhibitor bound to M2 in the Inwardclosed conformation (2.63 Å). These X-ray crystal structures of the M2 proton channel with bound inhibitors reveal that ammonium groups bind to water-lined sites, formed by two layers of waters close to Ala30 and Gly34, respectively, observed in the X-ray structures, that are hypothesized to stabilize transient hydronium ions formed in the proton-conduction mechanism. Furthermore, the ammonium and adamantyl groups of the adamantyl-amine class of drugs are free to rotate in the channel, minimizing the entropic cost of binding. The MD simulation reproduced perfectly the X-ray structures of cautiously tuned. These drug-bound complexes provide the first high-resolution structures of drugs that interact with and disrupt networks of hydrogen-bonded waters that are widely utilized throughout nature to facilitate proton diffusion within proteins.The V27A mutation confers amantadine resistance to the influenza A M2 WT proton channel and is becoming more prevalent in circulating populations of influenza A virus. In Chapter 7, is described our collaborative work with DeGrado and Wang groups to solve M2TM V27A structure in complex with a spiro-adamantyl amine inhibitor bound to M2(22-46) V27A and also to M2(21-61) V27A in the Inwardclosed conformation using X-ray crystallography and MD simulations. The spiro-adamantyl amine binding site is nearly identical for the two crystal structures. Compared to the M2 WT with valine at position 27, we observed that the channel pore is wider at its N-terminus as a result of the V27A mutation and that this removes V27 side chain hydrophobic interactions that are important for binding of amantadine and rimantadine. The spiro-adamantyl amine inhibitor blocks proton conductance in both the WT and V27A mutant channels by shifting its binding site in the pore depending on which residue is present at position 27. Additionally, in the structure of the M2(21-61) V27A construct, the C-terminus of the channel is tightly packed relative to the M2(22-46) construct. We observed that residues Asp44, Arg45, and Phe48 face the center of the channel pore and would be well-positioned to interact with protons exiting the M2 channel after passing through the His37 gate. However, the orientation of AHs after residue 48 did not reproduce the almost vertical orientation as regards the M2TM, that found by Professor Tim Cross experimentally with ssNMR experiments. The MD simulations of the M2(22-46) V27A - spiro-adamantyl amine complex predicted with accuracy the position of the ligands and waters inside the pore in the X-ray crystal structure of the M2 V27A complex.The influenza A M2 wild type proton channel is the target of the anti-influenza drug rimantadine. Rimantadine has two enantiomers, though most investigations into drug binding and inhibition have used a racemic mixture. ssNMR experiments by Professor Tim Cross have shown significant spectral differences that were interpreted to indicate tighter binding for (R)- vs. (S)- rimantadine. However, it was unclear if this is due to the specific condition of the ssNMR experiments (i.e. close to 0 oC), correlates with a functional difference in drug binding and inhibition and we undertook to investigate this in collaboration with Professor DeGrado, Associate Professor Jun Wang and Professor Jon Essex. Thus, in Chapter 8, using X-ray crystallography, we have determined that both (R)- and (S)-rimantadine bind to the M2 pore with slight differences in the hydration of each enantiomer. However, this did not result in a difference in potency or binding kinetics, as we measured similar values for kon, koff, and Kd in electrophysiological assays and EC50 values in cellular assays. We concluded that the slight differences in hyd

1,2-Αnnulated Adamantane Heterocyclic Derivatives as Anti-Influenza Α Virus Agents

In this report we review our results on the development of 1,2-annulated adamantane heterocyclic derivatives and we discuss the structure-activity relationships obtained from their biological evaluation against influenza A virus. We have designed and synthesized numerous potent 1,2-annulated adamantane analogues of amantadine and rimantadine against influenza A targeting M2 protein the last 20 years. For their synthesis we utilized the key intermediates 2-(2-oxoadamantan-1-yl)acetic acid and 3-(2-oxoadamantan-1-yl)propanoic acid, which were obtained by a simple, fast and efficient synthetic protocol. The latter involved the treatment of protoadamantanone with different electrophiles and a carbon-skeleton rearrangement. These ketoesters offered a new pathway to the synthesis of 1,2-disubstituted adamantanes, which constitute starting materials for many molecules with pharmacological potential, such as the 1,2-annulated adamantane heterocyclic derivatives. To obtain additional insight for their binding to M2 protein three structurally similar 1,2-annulated adamantane piperidines, differing in nitrogen position, were studied using molecular dynamics (MD) simulations in palmitoyl-oleoyl-phosphatidyl-choline (POPC) hydrated bilayers. This work is licensed under a Creative Commons Attribution 4.0 International License

Inhibitors of the M2 Proton Channel Engage and Disrupt Transmembrane Networks of Hydrogen-Bonded Waters.

Water-mediated interactions play key roles in drug binding. In protein sites with sparse polar functionality, a small-molecule approach is often viewed as insufficient to achieve high affinity and specificity. Here we show that small molecules can enable potent inhibition by targeting key waters. The M2 proton channel of influenza A is the target of the antiviral drugs amantadine and rimantadine. Structural studies of drug binding to the channel using X-ray crystallography have been limited because of the challenging nature of the target, with the one previously solved crystal structure limited to 3.5 Å resolution. Here we describe crystal structures of amantadine bound to M2 in the Inwardclosed conformation (2.00 Å), rimantadine bound to M2 in both the Inwardclosed (2.00 Å) and Inwardopen (2.25 Å) conformations, and a spiro-adamantyl amine inhibitor bound to M2 in the Inwardclosed conformation (2.63 Å). These X-ray crystal structures of the M2 proton channel with bound inhibitors reveal that ammonium groups bind to water-lined sites that are hypothesized to stabilize transient hydronium ions formed in the proton-conduction mechanism. Furthermore, the ammonium and adamantyl groups of the adamantyl-amine class of drugs are free to rotate in the channel, minimizing the entropic cost of binding. These drug-bound complexes provide the first high-resolution structures of drugs that interact with and disrupt networks of hydrogen-bonded waters that are widely utilized throughout nature to facilitate proton diffusion within proteins

Inhibitors of the M2 Proton Channel Engage and Disrupt Transmembrane Networks of Hydrogen-Bonded Waters.

Water-mediated interactions play key roles in drug binding. In protein sites with sparse polar functionality, a small-molecule approach is often viewed as insufficient to achieve high affinity and specificity. Here we show that small molecules can enable potent inhibition by targeting key waters. The M2 proton channel of influenza A is the target of the antiviral drugs amantadine and rimantadine. Structural studies of drug binding to the channel using X-ray crystallography have been limited because of the challenging nature of the target, with the one previously solved crystal structure limited to 3.5 Å resolution. Here we describe crystal structures of amantadine bound to M2 in the Inwardclosed conformation (2.00 Å), rimantadine bound to M2 in both the Inwardclosed (2.00 Å) and Inwardopen (2.25 Å) conformations, and a spiro-adamantyl amine inhibitor bound to M2 in the Inwardclosed conformation (2.63 Å). These X-ray crystal structures of the M2 proton channel with bound inhibitors reveal that ammonium groups bind to water-lined sites that are hypothesized to stabilize transient hydronium ions formed in the proton-conduction mechanism. Furthermore, the ammonium and adamantyl groups of the adamantyl-amine class of drugs are free to rotate in the channel, minimizing the entropic cost of binding. These drug-bound complexes provide the first high-resolution structures of drugs that interact with and disrupt networks of hydrogen-bonded waters that are widely utilized throughout nature to facilitate proton diffusion within proteins

X‑ray Crystal Structures of the Influenza M2 Proton Channel Drug-Resistant V27A Mutant Bound to a Spiro-Adamantyl Amine Inhibitor Reveal the Mechanism of Adamantane Resistance

The V27A mutation confers adamantane resistance on the influenza A matrix 2 (M2) proton channel and is becoming more prevalent in circulating populations of influenza A virus. We have used X-ray crystallography to determine structures of a spiro-adamantyl amine inhibitor bound to M2(22-46) V27A and also to M2(21-61) V27A in the Inwardclosed conformation. The spiro-adamantyl amine binding site is nearly identical for the two crystal structures. Compared to the M2 "wild type" (WT) with valine at position 27, we observe that the channel pore is wider at its N-terminus as a result of the V27A mutation and that this removes V27 side chain hydrophobic interactions that are important for binding of amantadine and rimantadine. The spiro-adamantyl amine inhibitor blocks proton conductance in the WT and V27A mutant channels by shifting its binding site in the pore depending on which residue is present at position 27. Additionally, in the structure of the M2(21-61) V27A construct, the C-terminus of the channel is tightly packed relative to that of the M2(22-46) construct. We observe that residues Asp44, Arg45, and Phe48 face the center of the channel pore and would be well-positioned to interact with protons exiting the M2 channel after passing through the His37 gate. A 300 ns molecular dynamics simulation of the M2(22-46) V27A-spiro-adamantyl amine complex predicts with accuracy the position of the ligands and waters inside the pore in the X-ray crystal structure of the M2(22-46) V27A complex

Rimantadine binds to and inhibits the influenza A M2 proton channel without enantiomeric specificity

The influenza A M2 wild-type (WT) proton channel is the target of the anti-influenza drug rimantadine. Rimantadine has two enantiomers, though most investigations into drug binding and inhibition have used a racemic mixture. Solid-state NMR experiments using the full length-M2 WT have shown significant spectral differences that were interpreted to indicate tighter binding for (R)- vs (S)-rimantadine. However, it was unclear if this correlates with a functional difference in drug binding and inhibition. Using X-ray crystallography, we have determined that both (R)- and (S)-rimantadine bind to the M2 WT pore with slight differences in the hydration of each enantiomer. However, this does not result in a difference in potency or binding kinetics, as shown by similar values for kon, koff, and Kd in electrophysiological assays and for EC50 values in cellular assays. We concluded that the slight differences in hydration for the (R)- and (S)-rimantadine enantiomers are not relevant to drug binding or channel inhibition. To further explore the effect of the hydration of the M2 pore on binding affinity, the water structure was evaluated by grand canonical ensemble molecular dynamics simulations as a function of the chemical potential of the water. Initially, the two layers of ordered water molecules between the bound drug and the channel’s gating His37 residues mask the drug’s chirality. As the chemical potential becomes more unfavorable, the drug translocates down to the lower water layer, and the interaction becomes more sensitive to chirality. These studies suggest the feasibility of displacing the upper water layer and specifically recognizing the lower water layers in novel drugs

Comparative Perturbation Effects Exerted by the Influenza A M2 WT Protein Inhibitors Amantadine and the Spiro[pyrrolidine-2,2′-adamantane] Variant AK13 to Membrane Bilayers Studied Using Biophysical Experiments and Molecular Dynamics Simulations

Aminoadamantane drugs are lipophilic amines that block the membrane-embedded influenza A M2 WT (wild type) ion channel protein. The comparative effects of amantadine ( Amt) and its synthetic spiro[pyrrolidine-2,2'-adamantane] (AK13) analogue in dimyristoylphosphatidylcholine (DMPC) bilayers were studied using a combination of experimental biophysical methods, differential scanning calorimetry (DSC), X-ray diffraction, solid-state NMR (ssNMR) spectroscopy, and molecular dynamics (MD) simulations. All three experimental methods pointed out that the two analogues perturbed drastically the DMPC bilayers with AK13 to be more effective at high concentrations. AK13 was tolerated in lipid bilayers at very high concentrations, while Amt was crystallized. This is an important consideration in the formulations of drugs as it designates a limitation of Amt incorporation. MD simulations verify provided details about the strong interactions of the drugs in the interface region between phosphoglycerol backbone and lipophilic segments. The two drugs form hydrogen bonding with both water and sn-2 carbonyls in their amine form or water and phosphate oxygens in their ammonium form. Such localization of the drugs explains the DMPC bilayers reorientation and their strong perturbing effect evidenced by all biophysical methodologies applie

(2-Hydroxy-4-methoxy)benzyl Aminoadamantane Conjugates as Probes to Investigate Specificity Determinants in Blocking Influenza M2 S31N and M2 WT Channels with Binding Kinetics and Simulations

In an attempt to synthesize potent blockers of the influenza A M2 S31N proton channel with modifications of amantadine, we used MD simulations and MM-PBSA calculations to project binding modes of compounds 2-5, which are analogues of 1, a dual blocker. Blocking both the S31N mutant and the wild type (WT) M2, 1 is composed of amantadine linked to an aryl head group, (4-methoxy-2-hydroxy)-benzyl. Compound 6, used as control, has an 3-(thiophenyl)isoxazolyl aryl head group, and selectively blocks M2 S31N (but not WT) in an aryl head group “out” (i.e. N-ward) binding orientation. We then tested 1-6 as anti-virals in cell culture and for M2 binding efficacy with electrophysiology (EP). The new molecules 2-5 have a linker between the adamantane and amino group which can be as small as a CMe2 in rimantadine derivative 2, or longer like phenyl in 3. Alternatively, we explored the impact of expanding the diameter of adamantane with diamantyl or triamantyl in 4 and 5, respectively. Antiviral effects against A/WSN/33 and its M2 WT revertant (M2 N31S) were seen for all six compounds except for 5 vs. the native (S31N) virus and (as predicted from previous studies) 6 vs. the WT revertant. Compounds 1-5, projected to bind in a polar head group “in” (C-ward) orientation, strongly block proton currents through M2 WT expressed in voltage-clamped oocytes with fast association rate constants (kon), and slow dissociation rate constants (koff). Surprisingly, 2-5, projected to bind in a polar head group out orientation, do not effectively block M2 S31N-mediated proton currents in EP. The results from MD and MM-PBSA calculations suggested that compounds 2-5 can be fully effective at blocking the M2 channel when present. The low degree of blocking in M2 S31N is due to their kinetics of binding observed in EP, i.e. two orders of magnitude reduction in kon compared to 6, and a fast off rate constant similar to that of 6, which is consistent with steered-MDsimulations. The low kon values can be interpreted from MD simulations, which suggest distortions to V27 cluster of the M2 S31N caused by the longer (even by one methylene) hydrophobic segment from adamantane to aryl head group, appropriate to fit from G34 to V27. The deformations in the N-terminus may be sufficiently energetic for 2-5 (compared to 6) to cause the observed low kon. </p

Influenza A M2 Spans the Membrane Bilayer, Perturbs its Organization and Differentiates the Effect of Amantadine and Spiro[pyrrolidine-2,2\u27-adamantane] AK13 on Lipids

The investigation and observations made for the M2TM, excess aminoadamantane ligands in DMPC were made using the simpler version of biophysical methods including SDC, SAXS and WAXS, MD simulations and ssNMR. 1H, 31P ssNMR and MD simulations, showed that M2TM in apo form or drug-bound form span the membrane interacting strongly with lipid acyl chain tails and the phosphate groups of the polar head surface. The MD simulations showed that the drugs anchor through their ammonium group with the lipid phosphate and occasionally with M2TM asparagine-44 carboxylate groups. The 13C ssNMR experiments allow the inspection of excess drug molecules and the assessment of its impact on the lipid head-group region. At low peptide concentrations of influenza A M2TM tetramer in DPMC bilayer, two lipid domains were observed that likely correspond to the M2TM boundary lipids and the bulk-like lipids. At high peptide concentrations, one domain was identified which constitute essentially all of the lipids which behave as boundary. This effect is likely due, according to the MD simulations, to the preference of AK13 to locate in closer vicinity to M2TM compared to Amt as well as the stronger ionic interactions of Amt primary ammonium group with phosphate groups, compared with the secondary buried ammonium group in AK13.<br /

Chemical Probes for Blocking of Influenza A M2 Wild-type and S31N Channels

We report on using the synthetic aminoadamantane-CH2-aryl derivatives 1-6 as sensitive probes for blocking M2 S31N and influenza A virus (IAV) M2 wild-type (WT) channels as well as virus replication in cell culture. The binding kinetics measured using electrophysiology (EP) for M2 S31N channel are very dependent on the length between the adamantane moiety and the first ring of the aryl headgroup realized in 2 and 3 and the girth and length of the adamantane adduct realized in 4 and 5. Study of 1-6 shows that, according to molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations, all bind in the M2 S31N channel with the adamantyl group positioned between V27 and G34 and the aryl group projecting out of the channel with the phenyl (or isoxazole in 6) embedded in the V27 cluster. In this outward binding configuration, an elongation of the ligand by only one methylene in rimantadine 2 or using diamantane or triamantane instead of adamantane in 4 and 5, respectively, causes incomplete entry and facilitates exit, abolishing effective block compared to the amantadine derivatives 1 and 6. In the active M2 S31N blockers 1 and 6, the phenyl and isoxazolyl head groups achieve a deeper binding position and high kon/low koff and high kon/high koff rate constants, compared to inactive 2-5, which have much lower kon and higher koff. Compounds 1-5 block the M2 WT channel by binding in the longer area from V27-H37, in the inward orientation, with high kon and low koff rate constants. Infection of cell cultures by influenza virus containing M2 WT or M2 S31N is inhibited by 1-5 or 1-4 and 6, respectively. While 1 and 6 block infection through the M2 block mechanism in the S31N variant, 2-4 may block M2 S31N virus replication in cell culture through the lysosomotropic effect, just as chloroquine is thought to inhibit SARS-CoV-2 infection