Eur Biophys J DOI 10.1007/s00249-015-1077-y ORIGINAL PAPER Investigation of the free energy profiles of amantadine and rimantadine in the AM2 binding pocket Hung Van Nguyen1 · Hieu Thanh Nguyen1 · Ly Thi Le1,2 Received: 10 June 2015 / Revised: 20 August 2015 / Accepted: 30 August 2015 © European Biophysical Societies’ Association 2015 Abstract The purpose of this work was to study the mechanism of drug resistance of M2 channel proteins by analyzing the interactions between the drugs amantadine and rimantadine and M2 channel proteins (including the wild type and the three mutants V27A, S31N, and G34A) and the drug binding pathways, by use of a computational approach Our results showed that multiple drug-binding sites were present in the M2 channel, and the trajectory of the drugs through the M2 channel was determined A novel method was developed to investigate of free energy profiles of the ligand–protein complexes Our work provides a new explanation of the large amount of experimental data on drug efficacy Keywords AM2 virus · Pathway docking · Amantadine · Rimantadine · M2 pocket Introduction The influenza A M2 channel protein is a target in antiinfluenza drug design because of its importance in viral infection (Holsinger and Lamb 1991; Sugrue and Hay 1991; Takeda et al 2002) The tetrameric structure of the M2 protein forms a pH-dependent channel across the viral H Van Nguyen and H T Nguyen contributed equally to this work * Ly Thi Le ly.le@hcmiu.edu.vn Life Science Laboratory, Institute for Computational Science and Technology, Ho Chi Minh City, Vietnam School of Biotechnology of International University, Vietnam National University, Ho Chi Minh City, Vietnam membrane for control of proton conductance when the virus penetrates cells (Pinto et al 1992; Pielak and Chou 2010; Lin and Schroeder 2001) Acidification weakens electrostatic interactions between matrix proteins and ribonucleoprotein (RNP) complexes, causing the disintegration of the viral membrane and the movement of the uncoated RNP from the cytosol to the nucleus Because of its crucial involvement in influenza viral pathogenesis, a variety of M2 channel structures have been solved for structure-based drug development (Tran et al 2013) by use of different techniques, for example site-directed infrared dichroism (Kukol et al 1999), UV resonance Raman spectroscopy (Okada et al 2001), electron spin resonance, and solidstate nuclear magnetic resonance (NMR) spectroscopy (Nishimura et al 2002; Kovacs et al 2000; Tian et al 2003; Schnell and Chou 2008) The M2 channel has four identical subunits and contains 97 residues per monomer (Lamb et al 1985) in three main segments: an extracellular N-terminal segment (residues 1–23), a transmembrane (TM) segment (residues 24–46), and an intracellular C-terminal segment (residues 47–97) (Pielak and Chou 2010) The transmembrane (TM) segment is the main region responsible for proton conduction and inhibition of the channel In investigations of the proton conductance of M2 channel proteins for drug development, this segment has been studied in depth (Nishimura et al 2002; Hu et al 2007; Stouffer et al 2008; Cady and Hong 2008; Cady et al 2010; Acharya et al 2010) Residues 24–46 of the four monomers form a TM helix bundle lined by the polar residues Val27, Ser31, Gly34, His37, Trp41, Asp44, and Arg45 The tetrameric His37–Trp41 cluster is the center of acid activation and proton conductance (Tang et al 2002; Venkataraman et al 2005; Hu et al 2006) The ionizable His37 is essential for proton selectivity, and acts as a channel sensor (Wang et al 1995), whereas Trp41 is important for unidirectional conductance and acts as a proton gate (Pielak and Chou 2010; Tang et al 2002) 13 In addition to the Trp41 gate, it has been recently been proposed that Val27, together with His37, forms a secondary gate (Yi et al 2008; Nguyen and Le 2015) Several M2 inhibitors have been approved by the FDA In particular, 1-aminoadamantane hydrochloride, known as amantadine, is the first efficient drug in influenza therapeutics Amantadine indirectly frustrates virus activity by using a hydrophobic cage to prevent proton conductance by the ion-channel The other drug approved for treating influenza A, rimantadine (α-methyl-1-adamantane methylamine hydrochloride), has efficacy comparable with that of amantadine but with a greater risk of adverse side effects (Stephenson and Nicholson 2001; Jefferson et al 2004) The WHO has, however, restricted the application of amantadine and rimantadine for treatment of influenza A because of the rapidly increasing occurrence of resistant strains (Tran et al 2011; Le and Leluk 2011; Tran and Le 2014) The sites of binding of amantadine and rimantadine on the M2 channel have been a controversial issue for more than 20 years, because of the location of drug-resistant mutations at residue 26, 27, 30, 31, 34, and 38 (Hay et al 1985; Wang et al 1993) Interestingly, the side chains of amino acids 27, 31, and 34 are predicted to face the channel interior, leading to a hypothesis that the drugs bind to the inside of the channel (Pielak and Chou 2010) Schnell and Chou (2008), however, by use of nuclear Overhauser effect (NOE) experiments, detected four equivalent binding sites of rimantadine outside the channel Rimantadine acts as a link between the two adjoining helixes and indirectly keeps the channel gate closed Although recent study has suggested that binding positions of amantadine and its derivatives inside the M2 channel are more energetically favorable, the mechanism of the binding process remains unclear (Jing et al 2008; Ohigashi et al 2009) For this reason, study of the molecular mechanism of binding of M2 inhibitors to their wild type and mutant targets will provide important insight into the mechanism of M2 drug resistance In this research, we used a novel method, called “pathway docking”, to investigate the interaction between the M2 channel and amantadine and rimantadine during their entry into the channel pore of the wild type (WT) and three drug-resistant mutants (V27A, S31 N and G34A), to gain insight into the effects of mutations Eur Biophys J Table 1 Adamantane-based inhibitors The colored branches are functional groups and their z box sizes are determined by the maximum length they can reach Inhibitor Amantadine Rimantadine 5.89 7.39 Chemical structure z (Å) from complex 3C9J and rimantadine was constructed on the basis of the amantadine structure by use of GaussView 5.0; the geometry of the drugs was then optimized by use of Gaussian 09 (Table 1) (Hada et al 2004) The V27A, S31N, and G34A mutant models were generated by use of the mutagenesis tool of Visual Molecular Dynamics (VMD) (Humphrey et al 1996) The free energies of binding between the inhibitors and the M2 channels were predicted by AutoDock Vina software (Trott and Olson 2010) Finally, the conformation having the lowest energy and smallest RMSD from docking output was chosen for subsequent analysis Free energy scanning method Materials and methods The basis of this method is movement of the grid box for docking along the M2 channel from serine 22 to histidine 37 while the box size is modified to ensure the inhibitors bind to the inside of the channel only (Fig. 1) The binding energies corresponding to each step were then assembled to produce the energy profile In particular, the center of mass of serine 22 and histidine 37 were chosen to form a symmetric axis This axis was then parallelized with the z-axis by pivoting the protein, the ligands were docked into it after the center of box had been moved 0.05 Å along the z-axis from Ser22 to His37, or approximately 25 Å At each step, the x and y dimensions of the box were determined by the difference between the respective maximum and minimum of the x and y coordinates of the protein’s atoms; their z coordinates were the interval of z box size The largest length of the ligands was used to define the z size of the box and only one z box size corresponded to each inhibitor (Table 1) Materials Analytical methods The 3D structure of the M2 channel was taken from Protein Data Bank (PDB; entry 2L0J, strain A/Udon/307/1972) It was derived from a complex embedded in DMPC liposomes (Sharma et al 2010) Amantadine was extracted The free energies predicted for the interactions between the inhibitors and the M2 channels were represented as a function of the z-coordinate of the center of the box when the docking process was complete On the basis of these 13 Eur Biophys J Fig. 1 The pathway docking method moves the grid box 0.05 Å every docking step from serine 22 to histidine 37 along a symmetric axis (paralleled with the z axis) of the M2 pore (a), the z box size is determined by the largest size of the inhibitor (b) and is kept constant throughout the molecular docking process free energy values, geometric features, as functions of z, were displayed as significant when compounds “moved” from outside to inside the pore In the docking method, the ligands were separated into two parts, i.e “root” and “branches” Specifically, the geometric center of the adamantane cage represents the “root”, and that of each functional group represents a “branch” (Fig. 2) Besides the root and branches, the center of the inhibitor was associated z Branch Center Root x o y Fig. 2 Simplified rimantadine geometric features represented by the centers of the root (red), branch (yellow), and whole compound (green), and the angles between the rotatable bond (blue) and the z axis, θ, and the x axis, φ with a relative binding position in the M2 pore The orientation of each compound was represented the angle, θ, between the rotatable bond and the z-axis, and the angle, φ, between the rotatable bond and the x-axis To determine their differences, however, the Δθ and Δφ values were a better choice for characterization of the change of state of the ligands; these were defined as: ∆θi = θi+1 − θi (1) ∆φi = φi+1 − φi (2) Results and discussion Image of the penetration of amantadine and rimantadine: the coexistence of two binding sites of different energy inside the M2 channel and their motion along the M2 pore As shown in Figs. 3 and 4, the images of the penetration of amantadine and rimantadine were characterized by profiles of the binding free energies and by Δθ and Δφ Here, the free energy values, which were indicative of strong interaction of the inhibitors binding along the pore, and the differences between the angles indicated the stable structure of M2 channels This means that the positions at the bound states were stable when the difference between the angles 13 Eur Biophys J kCal/mol A His37 -4 A ∆E Val27 -2 15 CEN Root Branch Val27 12 ∆θ Z (Å) 120 Ser31 -120 Degree 21 18 Ser31 Gly34 -6 Degree N C ∆o 120 Gly34 -3 -6 -120 -9 -6 -3 12 15 18 21 -9 24 -6 -3 kCal/mol B ∆E Gly34 -120 21 12 15 18 21 Ser31 Gly34 -3 ∆o 120 18 Val27 ∆θ 120 15 CEN Root NH2 CH3 12 Ser31 Z (Å) Degree -6 Degree 15 Val27 -4 12 21 18 -2 Z (Å) Z (Å) B -6 -9 -120 -9 -6 -3 12 15 18 21 24 -6 -3 Z (Å) Z (Å) Fig. 3 Free energy profile (top) and differences between the z angles θ (middle) and x angles φ (bottom) along the channel from the C to N-terminus (blue left–right arrow) were predicted by pathway docking for amantadine (a) and rimantadine (b) was close to To clarify the relationship between the position of the box and the positions of amantadine and rimantadine, the z coordinates of the root, the branches, and whole molecule were represented as a function of the z coordinate for the center of the box As is apparent from Figs. 3 and 4, the pathway along which amantadine enters the pore (above His37) from outside (Ser22) was described as follows The drug was first isolated by water molecules; it then interacted with residues 22–26 Next, it stayed in front of the Val27 barrier (14–17 Å) until it crossed the barrier and went deeper into the pore There would be a region between Val27 and Ser31 (8–10 Å) where the binding energy gradually declined from the maximum close to Val27 to a lower value at Ser31 When moving through 13 Fig. 4 The z position of the center of the roots, branches, and the whole amantadine (a) and rimantadine (b) molecules were represented as a function of the z position in alignment with the center of the docking box this area, the order of the root, branches, and the center of the drug were unchanged (Fig. 4a) Here, the result shows that the region between Val27 and Ser31 has stronger binding affinity for amantadine After passing Val27, there were two positions that amantadine had bound to, which ranged from −3 to 0 Å and from to 7 Å; they were separated by the Gly34 residue Between these two positions, the bound state at the Ser31 pore was more stable and the free energy (−6.1 kCal/mol) was higher than at the other (−6.0 kCal/mol) Finally, it stopped in front of the sensor His37 and Trp41 gate These findings confirmed previous results: the existence of the secondary gate Val27 permeable not only to water molecules but also to amantadine (Yi et al 2008); Eur Biophys J the region between Val27 and Ser31 could be a provisional binding position before amantadine reached the most stable or the real bound state (Sansom and Kerr 1993); and two positions, Ser31–Gly34 (SG) (Cady et al 2009) and Gly34–His37 (GH) (Gandhi et al 1999), which were potential binding sites of amantadine New information from this representation were also valuable: although the plots of energies and angles indicated that amantadine should be at pore SG, a minor difference in energies suggests that it was also binding to the GH site In other words, there could be two significant binding sites, resulting in a high possibility that amantadine could move from one binding site to the other as a result of thermal motion When amantadine penetrated the M2 channel via the pathway with the lowest energy, its orientation along the path had rotated the hydrophobic cage and the hydrophilic head (Fig. 4a) In front of residue Val27, its root—the adamantane cage and its branch—the primary amine group, was horizontal to the z-axis or at the same z position (12–18 Å) After crossing the barrier, they had turned vertically, the branch pointed to Ser31 and the root pointed to Val27 (9–12 Å) This orientation was maintained for a short time before the root turned back and pointed toward the C-terminal (7–9 Å) Next, its branch turned back and pointed toward Gly34 whereas the root pointed at Ser31 (0–7 Å) In addition, the primary amine group bound at that position until the root crossed Gly34 and pointed to His37 (−2 to 0 Å) The sequential orientation above was considered reasonable only if the root and the branch of amantadine exchanged their role as a “hook” The cage would hook around Val27 (11 Å) and the primary amine group rotated around it It then held at Ser31 (6.3 Å) and the cage rotated around it Finally, the primary amine group hooked to Gly34 (0.7 Å) and the Adamantane cage could stay above it in the most stable bound state or rotated around it to move to the second stable state This explains why experimental structures of the complex of amantadine and the M2 channel have different positions (Stouffer et al 2008; Yi et al 2008; Cady et al 2009; Gandhi et al 1999) and orientations (Stouffer et al 2008; Cady et al 2009; Gandhi et al 1999) They were simply positions in the “walking” process of the inhibitors The “walking” process of amantadine is shown in Fig. 5 These results explain why the resistant mutations span more than three helical turns, whereas amantadine has a diameter of only Å and cannot interact with the entire N-terminal half of the M2 channel (Pielak and Chou 2010) For rimantadine, the Val27, Ser31, and Gly34 residues were still used as crucial factors for analysis of the process of penetration of the inhibitor into the M2 pore (Fig. 4b) Compared with amantadine, the free energy values and binding sites were very different: Root Branch Val27 Ser31 Gly34 His37 Fig. 5 Illustration of the walking process of amantadine in the M2 pore Most of the points in the free energy plots for rimantadine were substantially lower than for amantadine, and the plots of the angles for rimantadine also fluctuated less than for amantadine (Fig. 3) This means rimantadine bound more strongly than amantadine, in good agreement with the experimental observation that rimantadine is more efficient than amantadine for treatment of influenza A infection (Stephenson and Nicholson 2001; Jefferson et al 2004) The binding sites of rimantadine were also shrunk and were more specific than for amantadine in the SG and GH regions, except for the area in front of Val27 (Fig. 3) After addition of a methyl group, the hydrophobicity of the functional group including the primary amine group, had substantially increased Rimantadine bound closer to Val27 (a hydrophobic side chain) or at the Ser22–Val27 (SV) region than amantadine The binding site at SG and GH was narrower and focused on Ser31 and Gly34 (a polar and hydrophobic side chain) for the same purpose Therefore, the difference between the energy of the bound state for SG or GH was larger for rimantadine than for amantadine In other words, rimantadine bound strongly at S31 resi- 13 Eur Biophys J due, and, as a result, the probability of transition from SG to the GH bound state was limited Effects of mutations on penetration: loss of the inhibitor trap Val27, the unstable binding place Asn31, and the third gate Ala34 blocked the channel to amantadine and rimantadine The major importance of the Val27, Ser31, and Gly34 residues and their mutants V27A, S31N, and G34A was strongly confirmed by the mechanism of resistance to amantadine (Wang et al 1995; Hay et al 1985) In this part of our work the mechanisms of drug resistance of mutants In more detail, the results showed that: WT S31N Val27 -3 -8 ∆E (kCal/mol) V27A: the barrier at residue 27 had vanished; S31N: the binding free energies in the SG region were wider and deeper; and G34A: when mutation occurred at the Gly34 residue (low barrier) to become Ala34 (steep barrier), the inhibitors needed more energy to cross the barrier, because the steep barrier of the Ala34 residue was higher than that of Val27 residue and even higher than that of the His37 residue A S31N WT 120 Ser31 Gly34 -6 ∆θ A of the M2 channel were determined from the energy profile or from the process of penetration of the inhibitors On the basis of the energy profile, we found that mutated residues led to different effects of amantadine and rimantadine on the M2 channel (Fig. 6) It was found that: V27A -3 -120 -8 -6 120 G34A o -3 -8 -6 -120 -6 -4 13 19 15 21 24 -6 Z (Å) WT S31N Gly34 ∆E (kCal/mol) 12 15 18 21 12 15 18 21 B 120 Val27 Ser31 -6 Z (Å) B -4 -3 V27A -120 -4 -6 120 G34A o -4 -120 -6 -6 -4 13 19 15 21 24 Z (Å) Fig. 6 Energy profiles for amantadine (a) and rimantadine (b) in the WT and three M2 channel variants: S31N, V27A, and G34A 13 -6 -3 Z (Å) Fig. 7 Angle profiles of amantadine (a) and rimantadine (b) in the WT and S31N mutant Eur Biophys J The unequal energies at the two sides of the secondary gate, Val27 (Fig. 3), created a gate trap for the inhibitors It was, therefore, easier for amantadine or rimantadine to penetrate the pore than to move in the opposite direction The loss of the secondary gate was caused by V27A involved vanishing of the trap, and the inhibitors could not stably bind inside the V27A mutant pore Furthermore, the absence of the gate Val27 increased water flux in the pore (Yi et al 2008) and indirectly reduced inhibition by the Adamantane cage The deeper energy around the 31st mutant position proved that both amantadine and rimantadine were sufficiently bound But, as is apparent from Fig. 7, the large width led to unstable binding of amantadine and rimantadine (0–10 Å) Minor differences between the profiles for the mutant S31N and wild type were revealed in our virtual experiments The mutant G34A contained in a third gate, which prevented deeper penetration of the inhibitors Each mutation has different effect on the inhibitor, but the mechanism of drug-resistance of the M2 channel was not apparent from our pathway docking results One explanation could be that the virus replaced the Val27 gate, which could longer prevent passage of the inhibitors getting through, by a new higher-energy gate at Gly34 and mutated residues in front of the third gate (S31N is typical mutant), which have strong affinity for amantadine and rimantadine Conclusions To depict penetration of the M2 channel by amantadine and rimantadine, the inhibitors were docked 500 times into the wild type and its mutants (V27A, S31N, and G34A) by use of pathway docking At every docking step, the grid box was moved 0.05 Å, in the direction from the N-terminus to the C-terminus, and the box size was adjusted to ensure the inhibitors were inside the channel The energy and angle profiles led to several significant findings There was not only one binding site for amantadine and rimantadine in the M2 channel but two positions, at Ser31 (SG region) and Gly34 (GH region) The inhibitors bound at each position with different energies or probabilities, and the binding energy in the SG region was higher than that in the other The binding energy difference was not large (0.1 kCal/mol) for amantadine, which led to ease of transition between the two positions However, it was different for rimanta- dine with the added methyl group The binding affinity along the channel for rimantadine was higher and more concentrated than for amantadine, at Ser31 and Gly34; this is explained by the lower hydrophilicity of the functional groups The hydrophobic cage and the primary amine group of amantadine and rimantadine may act as hooks by use of which the ligands step inside the channel at Val27, Ser31, and Gly34 This explains the many empirical binding positions and mutations spanning the M2 channel The mechanism of drug resistance of M2 is probably a result of three features of the mutants First, residues which interact strongly with the primary amine group of the inhibitors (Ser31 in our work) are replaced by residues with lower affinities Second, water flux is increased by loss of the secondary gate Val27 in the V27A mutation Finally, a replacement gate G34A is created to prevent deeper penetration of the inhibitors and to replace Val27 in control of water flux inside the channel These processes cause unstable binding of inhibitors, thereby reducing inhibition by the hydrophobic cage of the drugs and preserving the normal activity of the M2 channel Acknowledgments The work was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106.01-2012.66 Computing resources and support provided by the Institute for Computational Science and Technology, Ho Chi Minh City, are gratefully acknowledged We would like to thank Professors Thanh Truong and Mai Suan Ly for valuable advice 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rimantadine. .. inside the M2 channel and their motion along the M2 pore As shown in Figs. 3 and 4, the images of the penetration of amantadine and rimantadine were characterized by profiles of the binding free. .. docking for amantadine (a) and rimantadine (b) was close to To clarify the relationship between the position of the box and the positions of amantadine and rimantadine, the z coordinates of the