Experimental characterization of the association of ß-cyclodextrin and eight novel cyclodextrin derivatives with two guest compounds.

We investigate the binding of native ß-cyclodextrin (ß-CD) and eight novel ß-CD derivatives with two different guest compounds, using isothermal calorimetry and 2D NOESY NMR. In all cases, the stoichiometry is 1:1 and binding is exothermic. Overall, modifications at the 3' position of ß-CD, which is at the secondary face, weaken binding by several kJ/mol relative to native ß-CD, while modifications at the 6' position (primary face) maintain or somewhat reduce the binding affinity. The variations in binding enthalpy are larger than the variations in binding free energy, so entropy-enthalpy compensation is observed. Characterization of the bound conformations with NOESY NMR shows that the polar groups of the guests may be situated at either face, depending on the host molecule, and, in some cases, both orientations are populated. The present results were used in the SAMPL7 blinded prediction challenge whose results are detailed in the same special issue of JCAMD.

Differential Binding of Rimantadine Enantiomers to Influenza A M2 Proton Channel.

Rimantadine hydrochloride (α-methyl-1-adamantane-methalamine hydrochloride) is a chiral compound which exerts antiviral activity against the influenza A virus by inhibiting proton conductance of the M2 ion channel. In complex with M2, rimantadine has always been characterized as a racemic mixture. Here, we report the novel enantioselective synthesis of deuterium-labeled (R)- and (S)-rimantadine and the characterization of their protein-ligand interactions using solid-state NMR. Isotropic chemical shift changes strongly support differential binding of the enantiomers to the proton channel. Position restrained simulations satisfying distance restraints from (13)C-(2)H rotational-echo double-resonance NMR show marked differences in the hydrogen-bonding pattern of the two enantiomers at the binding site. Together these results suggest a complex set of interactions between (R)-rimantadine and the M2 proton channel, leading to a higher stability for this enantiomer of the drug in the channel pore.

Transverse relaxation dispersion of the p7 membrane channel from hepatitis C virus reveals conformational breathing.

The p7 membrane protein encoded by hepatitis C virus (HCV) assembles into a homo-hexamer that selectively conducts cations. An earlier solution NMR structure of the hexameric complex revealed a funnel-like architecture and suggests that a ring of conserved asparagines near the narrow end of the funnel are important for cation interaction. NMR based drug-binding experiments also suggest that rimantadine can allosterically inhibit ion conduction via a molecular wedge mechanism. These results suggest the presence of dilation and contraction of the funnel tip that are important for channel activity and that the action of the drug is attenuating this motion. Here, we determined the conformational dynamics and solvent accessibility of the p7 channel. The proton exchange measurements show that the cavity-lining residues are largely water accessible, consistent with the overall funnel shape of the channel. Our relaxation dispersion data show that residues Val7 and Leu8 near the asparagine ring are subject to large chemical exchange, suggesting significant intrinsic channel breathing at the tip of the funnel. Moreover, the hinge regions connecting the narrow and wide regions of the funnel show strong relaxation dispersion and these regions are the binding sites for rimantadine. Presence of rimantadine decreases the conformational dynamics near the asparagine ring and the hinge area. Our data provide direct observation of µs-ms dynamics of the p7 channel and support the molecular wedge mechanism of rimantadine inhibition of the HCV p7 channel.

Computational investigation of drug-resistant mutant of M2 proton channel (S31N) against rimantadine.

M2 proton channel is the target for treating the patients who ere suffering from influenza A infection, which facilitates the spread of virions. Amantadine and rimantadine are adamantadine-based drugs, which target M2 proton channel and inhibit the viral replication. Preferably, rimantadine drug is used more than amantadine because of its fewer side effects. However, S31N mutation in the M2 proton channel was highly resistant to the rimantadine drug. Therefore, in the present study, we focused to understand the drug-resistance mechanism of S31N mutation with the aid of molecular docking and dynamics approach. The docking analysis undoubtedly indicates that affinity for rimantadine with mutant-type M2 proton channel is significantly lesser than the native-type M2 proton channel. In addition, RMSD, RMSF, and principal component analysis suggested that the mutation shows increased flexibility. Furthermore, the intermolecular hydrogen bonds analysis showed that there is a complete loss of hydrogen bonds in the mutant complex. On the whole, we conclude that the intermolecular contact was maintained by D-44, a key residue for stable binding of rimantadine. These findings are certainly helpful for better understanding of drug-resistance mechanism and also helpful for designing new drugs for treating influenza infection against drug-resistance target.

Unusual architecture of the p7 channel from hepatitis C virus.

The hepatitis C virus (HCV) has developed a small membrane protein, p7, which remarkably can self-assemble into a large channel complex that selectively conducts cations. We wanted to examine the structural solution that the viroporin adopts in order to achieve selective cation conduction, because p7 has no homology with any of the known prokaryotic or eukaryotic channel proteins. The activity of p7 can be inhibited by amantadine and rimantadine, which are potent blockers of the influenza M2 channel and licensed drugs against influenza infections. The adamantane derivatives have been used in HCV clinical trials, but large variation in drug efficacy among the various HCV genotypes has been difficult to explain without detailed molecular structures. Here we determine the structures of this HCV viroporin as well as its drug-binding site using the latest nuclear magnetic resonance (NMR) technologies. The structure exhibits an unusual mode of hexameric assembly, where the individual p7 monomers, i, not only interact with their immediate neighbours, but also reach farther to associate with the i+2 and i+3 monomers, forming a sophisticated, funnel-like architecture. The structure also points to a mechanism of cation selection: an asparagine/histidine ring that constricts the narrow end of the funnel serves as a broad cation selectivity filter, whereas an arginine/lysine ring that defines the wide end of the funnel may selectively allow cation diffusion into the channel. Our functional investigation using whole-cell channel recording shows that these residues are critical for channel activity. NMR measurements of the channel-drug complex revealed six equivalent hydrophobic pockets between the peripheral and pore-forming helices to which amantadine or rimantadine binds, and compound binding specifically to this position may allosterically inhibit cation conduction by preventing the channel from opening. Our data provide a molecular explanation for p7-mediated cation conductance and its inhibition by adamantane derivatives.

Dynamic nuclear polarization study of inhibitor binding to the M2(18-60) proton transporter from influenza A.

We demonstrate the use of dynamic nuclear polarization (DNP) to elucidate ligand binding to a membrane protein using dipolar recoupling magic angle spinning (MAS) NMR. In particular, we detect drug binding in the proton transporter M2(18-60) from influenza A using recoupling experiments at room temperature and with cryogenic DNP. The results indicate that the pore binding site of rimantadine is correlated with previously reported widespread chemical shift changes, suggesting functional binding in the pore. Futhermore, the (15)N-labeled ammonium of rimantadine was observed near A30 (13)Cß and G34 (13)Cα, suggesting a possible hydrogen bond to A30 carbonyl. Cryogenic DNP was required to observe the weaker external binding site(s) in a ZF-TEDOR spectrum. This approach is generally applicable, particularly for weakly bound ligands, in which case the application of MAS NMR dipolar recoupling requires the low temperatures to quench dynamic exchange processes. For the fully protonated samples investigated, we observed DNP signal enhancements of ~10 at 400 MHz using only 4-6 mM of the polarizing agent TOTAPOL. At 600 MHz and with DNP, we measured a distance between the drug and the protein to a precision of 0.2 Å.

The involvement of a Na⁺- and Cl⁻-dependent transporter in the brain uptake of amantadine and rimantadine.

Despite their structural similarity, the two anti-influenza adamantane compounds amantadine (AMA) and rimantadine (RIM) exhibit strikingly different rates of blood-brain barrier (BBB) transport. However, the molecular mechanisms facilitating the higher rate of in situ BBB transport of RIM, relative to AMA, remain unclear. The aim of this study, therefore, was to determine whether differences in the extent of brain uptake between these two adamantanes also occurred in vivo, and elucidate the potential carrier protein facilitating their BBB transport using immortalized human brain endothelial cells (hCMEC/D3). Following oral administration to Swiss Outbred mice, RIM exhibited 2.4-3.0-fold higher brain-to-plasma exposure compared to AMA, which was not attributable to differences in the degree of plasma protein binding. At concentrations representative of those obtained in vivo, the hCMEC/D3 cell uptake of RIM was 4.5-15.7-fold higher than that of AMA, with Michaelis-Menten constants 6.3 and 238.4 µM, respectively. The hCMEC/D3 cellular uptake of both AMA and RIM was inhibited by various cationic transporter inhibitors (cimetidine, choline, quinine, and tetraethylammonium) and was dependent on extracellular pH, membrane depolarization and Na⁺ and Cl⁻ ions. Such findings indicated the involvement of the neutral and cationic amino acid transporter B°,⁺ (ATB°,⁺) in the uptake of AMA and RIM, which was demonstrated to be expressed (at the protein level) in the hCMEC/D3 cells. Indeed, AMA and RIM appeared to interact with this transporter, as shown by a 53-70% reduction in the hCMEC/D3 uptake of the specific ATB°,⁺ substrate ³H-glycine in their presence. These studies suggest the involvement of ATB°,⁺ in the disposition of these cationic drugs across the BBB, a transporter with the potential to be exploited for targeted drug delivery to the brain.

Computational study of drug binding to the membrane-bound tetrameric M2 peptide bundle from influenza A virus.

The M2 protein of influenza A virus performs the crucial function of transporting protons to the interior of virions enclosed in the endosome. Adamantane drugs, amantadine (AMN) and rimantidine (RMN), block the proton conduction in some strains, and have been used for the treatment and prophylaxis of influenza A infections. The structures of the transmembrane (TM) region of M2 that have been solved in micelles using NMR (residues 23-60) (Schnell and Chou, 2008) and by X-ray crystallography (residues 22-46) (Stouffer et al., 2008) suggest different drug binding sites: external and internal for RMN and AMN, respectively. We have used molecular dynamics (MD) simulations to investigate the nature of the binding site and binding mode of adamantane drugs on the membrane-bound tetrameric M2-TM peptide bundles using as initial conformations the low-pH AMN-bound crystal structure, a high-pH model derived from the drug-free crystal structure, and the high-pH NMR structure. The MD simulations indicate that under both low- and high-pH conditions, AMN is stable inside the tetrameric bundle, spanning the region between residues Val27 to Gly34. At low pH the polar group of AMN is oriented toward the His37 gate, while under high-pH conditions its orientation exhibits large fluctuations. The present MD simulations also suggest that AMN and RMN molecules do not show strong affinity to the external binding sites.

Insights from studying the mutation-induced allostery in the M2 proton channel by molecular dynamics.

As an essential component of the viral envelope, M2 proton channel plays a central role in the virus replications and has been a key target for drug design against the influenza A viruses. The adamantadine-based drugs, such as amantadine and rimantadine, were developed for blocking the channel so as to suppress the replication of viruses. However, patients, especially those infected by the H1N1 influenza A viruses, are increasingly suffering from the drug-resistance problem. According to the findings revealed recently by the high-resolution NMR studies, the drug-resistance problem is due to the structural allostery caused by some mutations, such as L26F, V27A and S31N, in the four-helix bundle of the channel. In this study, we are to address this problem from a dynamic point of view by conducting molecular dynamics (MD) simulations on both the open and the closed states of the wild-type (WT) and S31N mutant M2 channels in the presence of rimantadine. It was observed from the MD simulated structures that the mutant channel could still keep open even if binding with rimantadine, but the WT channel could not. This was because the mutation would destabilize the helix bundle and trigger it from a compact packing state to a loose one. It is anticipated that the findings may provide useful insights for in-depth understanding the action mechanism of the M2 channel and developing more-effective drugs against influenza A viruses.

Energetic analysis of the two controversial drug binding sites of the M2 proton channel in influenza A virus.

Understanding the mechanism of the M2 proton channel of influenza A is crucially important to both basic research and drug discovery. Recently, the structure was determined independently by high-resolution NMR and X-ray crystallography. However, the two studies lead to completely different drug-binding mechanisms: the X-ray structure shows the drug blocking the pore from inside; whereas the NMR structure shows the drug inhibiting the channel from outside by an allosteric mechanism. Which one of the two is correct? To address this problem, we conducted an in-depth computational analysis. The conclusions drawn from various aspects, such as energetics, the channel-gating dynamic process, the pK(a) shift and its impact on the channel, and the consistency with the previous functional studies, among others, are all in favour to the allosteric mechanism revealed by the NMR structure. The findings reported here may stimulate and encourage new strategies for developing effective drugs against influenza A, particularly in dealing with the drug-resistant problems.

Structure and mechanism of the M2 proton channel of influenza A virus.

The integral membrane protein M2 of influenza virus forms pH-gated proton channels in the viral lipid envelope. The low pH of an endosome activates the M2 channel before haemagglutinin-mediated fusion. Conductance of protons acidifies the viral interior and thereby facilitates dissociation of the matrix protein from the viral nucleoproteins--a required process for unpacking of the viral genome. In addition to its role in release of viral nucleoproteins, M2 in the trans-Golgi network (TGN) membrane prevents premature conformational rearrangement of newly synthesized haemagglutinin during transport to the cell surface by equilibrating the pH of the TGN with that of the host cell cytoplasm. Inhibiting the proton conductance of M2 using the anti-viral drug amantadine or rimantadine inhibits viral replication. Here we present the structure of the tetrameric M2 channel in complex with rimantadine, determined by NMR. In the closed state, four tightly packed transmembrane helices define a narrow channel, in which a 'tryptophan gate' is locked by intermolecular interactions with aspartic acid. A carboxy-terminal, amphipathic helix oriented nearly perpendicular to the transmembrane helix forms an inward-facing base. Lowering the pH destabilizes the transmembrane helical packing and unlocks the gate, admitting water to conduct protons, whereas the C-terminal base remains intact, preventing dissociation of the tetramer. Rimantadine binds at four equivalent sites near the gate on the lipid-facing side of the channel and stabilizes the closed conformation of the pore. Drug-resistance mutations are predicted to counter the effect of drug binding by either increasing the hydrophilicity of the pore or weakening helix-helix packing, thus facilitating channel opening.

The metabolism of [14C]rimantadine hydrochloride in rats and dogs.

Metabolism and route of excretion of [14C]rimantadine hydrochloride was studied in male rats after single po (60 mg/kg) and iv doses (15 mg/kg) and in male dogs (5 or 10 mg/kg po and 5 mg/kg iv). Total 14C excretion in urine (po and iv) in both species reached 81-87% of the dose in 96 hr. Rimantadine was excreted in rats free (1.0% po, 1.7% iv) and conjugated (0.8% of the dose, po and iv, both in 24 hr) and in dogs, free (2.6% po, 3.0% iv) and conjugated (6.4% po, 7.7% iv, both in 48 hr). In both species, rimantadine metabolism is essentially independent of the route of administration. In rats and dogs, m-hydroxyrimantadine (mostly unconjugated) was the major metabolite, 22% (po) and 24% (iv), and 27% (po) and 21% (iv), respectively. Rats, but not dogs, excreted trans-p-hydroxyrimantadine (23.5% and 25.2%, po and iv, free plus conjugated). An oxidative pathway in dogs produced the m- and p-hydroxylated analogs with a hydroxyl in place of the amino group (3.7% and 5.7% of the dose, both conjugated). A p-hydroxylated compound with a nitro group in place of the amino group may have originated from an N-hydroxy metabolite by spontaneous oxidation during isolation. Comparison of total 14C excretion, in rats (81%, po; 82%, iv) and dogs (81%, po; 84%, iv) after po and iv administration after 96 hr indicates good absorption of rimantadine.

Determination of rimantadine and its hydroxylated metabolites in human plasma and urine.

A gas chromatographic-mass spectrometric procedure has been developed for the quantitation of the antiviral agent rimantadine and its meta- and para-hydroxylated metabolites in human plasma and urine. The assay utilizes an extractive pentafluorobenzoylation at alkaline pH with cyclohexane saturated with triethanolamine-chloroform (2:1) containing pentafluorobenzoyl chloride, selective ion monitoring, methane negative ion chemical ionization mass spectrometry and stable isotope dilution. The method has been used to measure plasma concentrations of rimantadine, m-hydroxyrimantadine and the two epimers of p-hydroxyrimantadine between 5-250, 5-100 and 2.5-50 ng/ml, respectively. Similarly, the urine concentrations of these analytes measured were between 25-1250, 25-500 and 12.5-250 ng/ml, respectively.

Pharmacokinetics and metabolism of rimantadine hydrochloride in mice and dogs.

We studied the pharmacokinetics and metabolism of rimantadine hydrochloride (rimantadine) following single-dose oral and intravenous administration in mice and dogs. Absorption of the compound in mice was rapid. Maximum concentrations in plasma occurred at less than 0.5 h after oral administration, and the elimination half-life was 1.5 h. Peak concentrations in plasma following oral administration were markedly disproportional to the dose (274 ng/ml at 10 mg/kg, but 2,013 ng/ml at 40 mg/kg). The bioavailability after an oral dose of 40 mg/kg was 58.6%. Clearance was 4.3 liters/h per kg, and the volume of distribution was 7.6 liters/kg at 40 mg/kg. In contrast to the results observed in mice, absorption of the compound in dogs was slow. Maximum concentrations in plasma occurred at 1.7 h after oral administration, and the elimination half-life was 3.3 h. A further difference was that peak concentrations in plasma were approximately proportional to the dose. Following administration of a single oral dose of 5, 10, or 20 mg/kg, maximum concentrations in plasma were 275,800, and 1,950 ng/ml, respectively. The bioavailability after an oral dose of 5 mg/kg was 99.4%. The clearance was 3.7 liters/h per kg, and the volume of distribution was 13.8 liters/kg at 5 mg/kg. Mass balance studies in mice, using [methyl-14C]rimantadine, indicated that 98.7% of the administered dose could be recovered in 96 h. Less than 5% of the dose was recovered as the parent drug in dog urine within 48 h. Finally, gas chromatography-mass spectrometry studies, done with mouse plasma, identified the presence of two rimantadine metabolites. These appeared to be ring-substituted isomers of hydroxy-rimantadine.

Pharmacokinetics of a single dose of rimantadine in young adults and children.

Single doses of rimantadine were given to children and young adults to evaluate the safety and pharmacokinetics of this antiviral compound. The half-life of rimantadine in young adults was 27.7 +/- 4.9 h for tablets and 27.8 +/- 8.0 h for syrup preparations. A total of 10 children, 5 to 8 years old, received a syrup preparation of rimantadine. The half-life of rimantadine in children was 24.8 +/- 9.4 h. A single dose of rimantadine was well tolerated in young adults and children.

Rimantadine pharmacokinetics after single and multiple doses.

Twenty-four healthy adult male volunteers were randomly assigned to one of two rimantadine regimens. The 12 volunteers assigned to regimen 1 orally received a single 100-mg rimantadine tablet followed 5 days later by 100 mg of rimantadine twice a day for 10 days. Volunteers assigned to regimen 2 ingested a single 100-mg rimantadine tablet followed 5 days later by 100 mg once a day for 10 days. The results of the study suggest that the pharmacokinetics of rimantadine are linear and accumulation of the drug during repetitive multiple doses is predictable.

Comparative single-dose pharmacokinetics of amantadine hydrochloride and rimantadine hydrochloride in young and elderly adults.

The single-dose pharmacokinetics of amantadine hydrochloride and rimantadine hydrochloride were compared in a randomized, two-period, crossover study involving six young (less than or equal to 35 years) and six elderly (less than or equal to 60 years) adults. Subjects ingested single 200-mg oral doses after an overnight fast, and serial plasma (0 to 96 h), nasal mucus (0 to 8 h), and urine (0 to 24 h) samples were collected for assay of drug concentration by electron capture gas chromatography. For both groups combined, rimantadine differed significantly from amantadine in peak plasma concentration (mean +/- standard deviation, 0.25 +/- 0.06 versus 0.65 +/- 0.22 micrograms/ml), plasma elimination half-life (36.5 +/- 15 versus 16.7 +/- 7.7 h), and percentage of administered dose excreted unchanged in urine (0.6 +/- 0.8 versus 45.7 +/- 15.7%). No significant age-related differences were noted for rimantadine. Urinary excretion (0 to 24 h) of rimantadine and its hydroxylated metabolites averaged 19% of the administered dose. The maximum nasal mucus drug concentration was similar for both drugs (0.42 +/- 0.25 versus 0.45 +/- 0.32 micrograms/g), and the ratio of maximum nasal mucus to plasma concentration was over twofold higher after rimantadine than after amantadine. These findings may in part explain the clinical effectiveness of rimantadine in influenza A virus infections at dosages that have lower toxicity than those of amantadine.

Clinical applications of antiviral agents for chemophrophylaxis and therapy of respiratory viral infections.

Table III summarizes clinical applications of antiviral agents in respiratory viral infections. (table: see text) For influenza A virus infections, both oral amantadine and rimantadine are effective when used for seasonal prophylaxis and for prophylaxis in institutional populations. Both of these drugs, as well as aerosolized ribavirin, have antiviral and therapeutic effects in uncomplicated influenza. It remains to be determined whether any of these modalities or possibly their combined use [44] will be useful in treating severe influenza hospitalized patients or whether they can prevent the development of complications in high risk patients. Unfortunately, there is no parenteral formulation of amantadine or rimantadine for use in critically ill patients. Aerosolized ribavirin has also been shown to have modest therapeutic effects in influenza B virus infection. However, a major need exists for an antiviral which is active against influenza B virus and which can be used on an outpatient basis. Controlled clinical trials have shown that aerosolized ribavirin therapy improves arterial oxygenation and modifies the severity of respiratory syncytial virus bronchiolitis and pneumonia [3,5]. Its role in treating life-threatening disease or in modifying the long-term sequelae of RSV infections are unknown at the present time. Again, a specific antiviral agent is needed for outpatient use in preventing or treating RSV infections. Finally, after over a decade of work since the original observation that intranasal interferon could prevent experimental rhinovirus infection [11], recent studies have established that intranasal rIFN-a2 is effective in the postexposure prophylaxis of rhinovirus colds in families [42]. This strategy needs to be studied with regard to the prevention of infection and its complications in high risk patients and it remains to be determined whether intranasal interferon will have therapeutic activity in established colds.