Accurate calculation of affinity changes to the close state of influenza A M2 transmembrane domain in response to subtle structural changes of adamantyl amines using free energy perturbation methods in different lipid bilayers.

Experimental binding free energies of 27 adamantyl amines against the influenza M2(22-46) WT tetramer, in its closed form at pH 8, were measured by ITC in DPC micelles. The measured Kd's range is ~44 while the antiviral potencies (IC50) range is ~750 with a good correlation between binding free energies computed with Kd and IC50 values (r = 0.76). We explored with MD simulations (ff19sb, CHARMM36m) the binding profile of complexes with strong, moderate and weak binders embedded in DMPC, DPPC, POPC or a viral mimetic membrane and using different experimental starting structures of M2. To predict accurately differences in binding free energy in response to subtle changes in the structure of the ligands, we performed 18 alchemical perturbative single topology FEP/MD NPT simulations (OPLS2005) using the BAR estimator (Desmond software) and 20 dual topology calculations TI/MD NVT simulations (ff19sb) using the MBAR estimator (Amber software) for adamantyl amines in complex with M2(22-46) WT in DMPC, DPPC, POPC. We observed that both methods with all lipids show a very good correlation between the experimental and calculated relative binding free energies (r = 0.77-0.87, mue = 0.36-0.92 kcal mol-1) with the highest performance achieved with TI/MBAR and lowest performance with FEP/BAR in DMPC bilayers. When antiviral potencies are used instead of the Kd values for computing the experimental binding free energies we obtained also good performance with both FEP/BAR (r = 0.83, mue = 0.75 kcal mol-1) and TI/MBAR (r = 0.69, mue = 0.77 kcal mol-1).

Molecular investigation of artificial and natural sweeteners as potential anti-inflammatory agents.

Repurposing existing drugs, as well as natural and artificial sweeteners for novel therapeutic indications could speed up the drug discovery process since numerous associated risks and costs for drug development can be surpassed. In this study, natural and artificial sweeteners have been evaluated by in silico and experimental studies for their potency to inhibit lipoxygenase enzyme, an enzyme participating in the inflammation pathway. A variety of different methods pinpointed that aspartame inhibits the lipoxygenase isoform 1 (LOX-1). In particular, "LOX-aspartame" complex, that was predicted by docking studies, was further evaluated by Molecular Dynamics (MD) simulations in order to assess the stability of the complex. The binding energy of the complex has been calculated after MD simulations using Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method. Furthermore, Quantum Mechanics/Molecular Mechanics (QM/MM) calculations have been applied for geometry optimization of the "enzyme-ligand" complex. After having fully characterized the "LOX-aspartame" complex in silico, followed in vitro biological assays confirmed that aspartame inhibits LOX-1 (IC50=50 ± 3.0 µΜ) and blocks its biological response. The atomic details of aspartame's interaction profile with LOX-1 were revealed through Saturation Transfer Difference (STD) NMR (Nuclear Magnetic Resonance). Finally, aspartame was also tested with Molecular Docking and Molecular Dynamics studies for its potent binding to a number of different LOX isoforms of many organisms, including human. The in silico methods indicated that aspartame could serve as a novel starting point for drug design against LOX enzyme. Communicated by Ramaswamy H. Sarma.

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.

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.

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.

The boundary lipid around DMPC-spanning influenza A M2 transmembrane domain channels: Its structure and potential for drug accommodation.

We have investigated the perturbation of influenza A M2TM in DMPC bilayers. We have shown that (a) DSC and SAXS detect changes in membrane organization caused by small changes (micromolar) in M2TM or aminoadamantane concentration and aminoadamantane structure, by comparison of amantadine and spiro[pyrrolidine-2,2'-adamantane] (AK13), (b) that WAXS and MD can suggest details of ligand topology. DSC and SAXS show that at a low M2TM micromolar concentration in DPMC bilayers, two lipid domains are observed, which likely correspond to M2TM boundary lipids and bulk-like lipids. At higher M2TM concentrations, one domain only is identified, which constitutes essentially all of the lipid molecules behaving as boundary lipids. 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. At the same concentrations of the drug when M2TM is present, amantadine and, to a lesser extent, AK13 cause, according to WAXS, a significant disordering of chain-stacking, which also leads to the formation of two lipid domains. This effect is likely due, according to MD simulations, to the preference of the more lipophilic AK13 to locate closer to the lateral surfaces of M2TM when compared to amantadine, which forms stronger ionic interactions with phosphate groups. The preference of AK13 to concentrate inside the lipid bilayer close to the exterior of the hydrophobic M2TM helices may contribute to its higher binding affinity compared to amantadine.

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 applied.

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.

Molecular Dynamics Simulations on the Bioactive Molecule of hIAPP22-29 (NFGAILSS) and Rational Drug Design.

This chapter includes information about the structure in equilibrium of the bioactive molecule hIAPP22-29 (NFGAILSS). The experimental structure was derived using X-ray and its 2D NOESY NMR experiments in d 6-DMSO and d-HFIP solvents. This molecule contains eight of the ten amino acids of the 20-29 region of the human islet amyloid polypeptide (hIAPP) often referred as the "amyloidogenic core." Amyloid deposits are well-known to cause as many as 20 pathological neurodegenerative disorders such as Alzheimer, Parkinson, Huntington, and Creutzfeldt-Jakob. The experimental structure was relaxed using molecular dynamics (MD) in simulation boxes consisting in DMSO and HFIP; the latter not provided by the applied software. The calculations were performed in GPUs and supercomputers, and some basic scripting is described for reference. The simulations confirmed the inter- and intramolecular forces that led to an "amyloidogenic core" observed from NOE experiments. The results showed that in DMSO and HFIP environment, Phe is not in spatial proximity with Leu or Ile, and this is consistent with an amyloidogenic core. However, in an amphipathic environment such as the model lipid bilayers, this communication is possible and may influence peptide amyloidogenic properties. The knowledge gained through this study may contribute to the rational drug design of novel peptides or organic molecules acting by modifying preventing amyloidogenic properties of the hIAPP peptide.

Unraveling the Binding, Proton Blockage, and Inhibition of Influenza M2 WT and S31N by Rimantadine Variants.

Recently, the binding kinetics of a ligand-target interaction, such as the residence time of a small molecule on its protein target, are seen as increasingly important for drug efficacy. Here, we investigate these concepts to explain binding and proton blockage of rimantadine variants bearing progressively larger alkyl groups to influenza A virus M2 wild type (WT) and M2 S31N protein proton channel. We showed that resistance of M2 S31N to rimantadine analogues compared to M2 WT resulted from their higher koff rates compared to the kon rates according to electrophysiology (EP) measurements. This is due to the fact that, in M2 S31N, the loss of the V27 pocket for the adamantyl cage resulted in low residence time inside the M2 pore. Both rimantadine enantiomers have similar channel blockage and binding kon and koff against M2 WT. To compare the potency between the rimantadine variants against M2, we applied approaches using different mimicry of M2, i.e., isothermal titration calorimetry and molecular dynamics simulation, EP, and antiviral assays. It was also shown that a small change in an amino acid at site 28 of M2 WT, which does not line the pore, seriously affects M2 WT blockage kinetics.