Molecular basis of inhibition and resistance of the influenza M2 ion channel by aminoadamantane drugs and discovery of novel resistance-breaking inhibitors targeting the mutant M2 proton channel

Η αμανταδίνη (Amt ) και η ριμανταδίνη (Rim) είναι αναστολείς της μεταφοράς πρωτονίων διαμέσου του ιοντικού διαύλου της πρωτεΐνης Μ2 του ιού influenza A, και αποτέλεσαν εγκεκριμένα μέσα πρόληψης και θεραπείας έναντι των ιών της γρίπης Α άγριου τύπου (WT). Ωστόσο, από το 2005, η μετάλλαξη του αμινοξέος σερίνη σε ασπαραγίνη στη θέση 31 (S31N) στην Μ2 πρωτεΐνη, δημιούργησε ένα στέλεχος ανθεκτικό στην Amt το οποίο έχει κυριαρχήσει παγκοσμίως, καταργώντας την κλινική χρησιμότητα της αμανταδίνης και πιθανότατα και άλλων προαναφερθέντων, στη διεθνή βιβλιογραφία, αναστολέων της Μ2. Επομένως, είναι απαραίτητη η ανάπτυξη νέων μορίων για την καταπολέμηση των ανθεκτικών στελεχών της γρίπης. H κύρια θέση πρόσδεσης της Amt (1) και της Rim (2) είναι η διαμεμβρανική περιοχή (transmembrane domain, TM) των αμινοξέων 22-46 του συμπλέγματος των τεσσάρων α-ελίκων της Μ2 που σχηματίζει τον δίαυλο μεταφοράς πρωτονίων. Σύμφωνα με δομές υψηλής διαχωριστικότητας που προέκυψαν από πειράματα κρυσταλλογραφίας ακτίνων-Χ και φασματοσκοπίας πυρηνικού μαγνητικού συντονισμού στερεάς κατάστασης (solid state NMR, ssNMR) και δημοσιεύτηκαν μεταξύ 2008-2011 από τους ερευνητές και καθηγητές Tim Cross, William DeGrado και Mei Hong στα πιο έγκριτα περιοδικά όπως Nature, JACS κλπ, η Amt (1) και η Rim (2) δρουν φράσσοντας τον πόρο του διαύλου M2TM. Ο κλωβός του αδαμαντανίου των ενώσεων αυτών περικλείεται από τις τέσσερεις πλευρικές αλυσίδες των V27 και της A30 του τετραμερούς M2TM προκαλώντας τον στερεοχημικό αποκλεισμό της μεταφοράς πρωτονίων και αποτρέποντας την συνέχεια του κύκλου ζωής του ιού. Τα αποτελέσματα ssNMR έδειξαν επίσης ότι η ομάδα αμμωνίου αυτών των φαρμάκων έχει προσανατολισμό προς τα τέσσερα αμινοξέα της His37 δηλαδή προς το C-τελικό άκρο.9 Αυτός ο προσανατολισμός σταθεροποιείται μέσω ενός δικτύου δεσμών υδρογόνου μεταξύ προσδέτη και (α) μορίων νερού εντός του πόρου του ιοντικού διαύλου, που βρίσκονται μεταξύ ιμιδαζολίου της H37 και προσδέτη, και, ενδεχομένως, (β) με το καρβονύλιο της A30 σύμφωνα με πειραματικά αποτελέσματα και προσομοιώσεις μοριακής δυναμικής (MD).Δεδομένου ότι η M2TM αποτελεί το απλούστερο μοντέλο πρόσδεσης της Amt (1) και Rim (2) για την M2, οι προηγούμενες πειραματικές δομές υψηλής ανάλυσης μπορούν να χρησιμοποιηθούν για το σχεδιασμό και ανάπτυξη νέων αναστολέων που να συνδέονται αποτελεσματικότερα με τον πόρο Μ2ΤΜ, μέσω προσομοιώσεων μοριακής δυναμικής (MD) 19ή ακριβέστερα με υπολογισμούς ελεύθερης ενέργειας.Amantadine (Amt (1)) and rimantadine (Rim (2)) are blockers of proton transport by influenza A virus Μ2 ion channel protein, and approved as prophylactics and therapeutics against influenza A wild type (WT) viruses. However, since 2005, the amantadine-insensitive Ser-to-Asn mutation at position 31 (S31N) in M2 protein has become globally prevalent, abrogating the clinical usefulness of amantadine3,4and possibly other previously reported M2 inhibitors. Thus, new agents are needed to combat drug-resistant strains of influenza. The primary binding site of amantadine and rimantadine is the transmembrane domain lumen (TM, amino acids 22-46) in the four-helix bundle of tetrameric M2 WT, that forms the proton transport path. According to high resolution structures from X-ray and solid state NMR (ssNMR) experiments published between 2008-2011 by Tim Cross, William DeGrado, and Mei Hong in the highest impact factor Journals i.e., Nature, JACS etc, the M2TM protein channel is blocked by amantadine and rimantadine via a pore-binding mechanism. The adamantyl cage in these molecules is tightly contacted on all sides by V27 and A30 side chains, producing a steric occlusion of proton transport and thereby preventing the continuation of the viral life cycle. The ssNMR results also demonstrated that the ammonium group of these drugs is pointing towards the four H37 residues at the C-end. 9 This orientation can be stabilized only through hydrogen bonds between the ligand and (a) with water molecules in the channel lumen between the imidazoles of H37 and the ligand and (b) possibly with A30 carbonyls in the vicinity, according to experimental and molecular dynamics (MD) simulations data. Provided that M2TM is a minimal model for M2 binding, these experimental high resolution structures can be used for the prediction of new ligands binding more effectively to the M2TM pore for example through MD simulations19 or more precisely by binding free energy calculations

Amantadine variant - aryl conjugates that inhibit multiple M2 mutant - amantadine resistant influenza a viruses

Influenza A viruses can cause a serious future threat due to frequent mutations. Amantadine and rimantadine inhibit influenza A M2 wild-type (WT) viruses by binding and blocking M2 WT channel-mediated proton current. The resistant to the drugs amantadine and rimantadine influenza A viruses bearing the S31 N mutant in the M2 proton channel can be inhibited by amantadine - aryl conjugates, in which amantadine and an aryl group are linked through a methylene, which block M2 S31 N channel-mediated proton current. However, the M2 amantadine/rimantadine resistant viruses bearing one of the four mutations L26F, V27A, A30T, G34E in residues that line the M2 protein pore pose an additional concern for public health. Here, we designed 33 compounds based on the structure of three previously published and potent amantadine-aryl conjugates against M2 S31 N virus, by replacing amantadine with 16 amantadine variants. The compounds were tested against M2 WT and the five M2 amantadine-resistant viruses aiming at identifying inhibitors against multiple M2 mutant - amantadine resistant viruses. We identified 16 compounds that inhibited in vitro two influenza A viruses with M2 WT or L26F channels. Additionally, compounds 21 or 32 or 33, which are conjugates of the rimantadine variant with CMe2 (instead of CHMe in rimantadine) or the diamantylamine or the 4-(1-adamantyl)benzenamine with the 2-hydroxy-4-methoxyphenyl aryl group, were in vitro inhibitors against three influenza A viruses with M2 WT or L26F or S31 N, while compound 21 inhibited also in vitro the M2 G34E virus and 32 inhibited also in vitro the M2 A30T virus. For these compounds we performed a preliminary drug metabolism and pharmacokinetics study. Also, using electrophysiology, we showed that compound 21 was an efficient blocker of the M2 WT and M2 L26F channels, compound 32 blocked efficiently the M2 WT channel and compound 33 blocked the M2 WT, L26F and V27A channels. The drug metabolism and pharmacokinetics studies showed these compounds need further optimization

Μοριακή βάση αναστολής και ανθεκτικότητας του ιοντικού διαύλου Influenza M2 από τα αμινοαδαμαντανικά φάρμακα και ανακάλυψη καινοτόμων αναστολέων των μεταλλαγμένων Μ2 πρωτεϊνών των ανθεκτικών στελεχών του ιού

Amantadine (Amt (1)) and rimantadine (Rim (2)) are blockers of proton transport by influenza A virus Μ2 ion channel protein, and approved as prophylactics and therapeutics against influenza A wild type (WT) viruses. However, since 2005, the amantadine-insensitive Ser-to-Asn mutation at position 31 (S31N) in M2 protein has become globally prevalent, abrogating the clinical usefulness of amantadine3,4and possibly other previously reported M2 inhibitors. Thus, new agents are needed to combat drug-resistant strains of influenza.The primary binding site of amantadine and rimantadine is the transmembrane domain lumen (TM, amino acids 22-46) in the four-helix bundle of tetrameric M2 WT, that forms the proton transport path. According to high resolution structures from X-ray and solid state NMR (ssNMR) experiments published between 2008-2011 by Tim Cross, William DeGrado, and Mei Hong in the highest impact factor Journals i.e., Nature, JACS etc, the M2TM protein channel is blocked by amantadine and rimantadine via a pore-binding mechanism. The adamantyl cage in these molecules is tightly contacted on all sides by V27 and A30 side chains, producing a steric occlusion of proton transport and thereby preventing the continuation of the viral life cycle. The ssNMR results also demonstrated that the ammonium group of these drugs is pointing towards the four H37 residues at the C-end. 9 This orientation can be stabilized only through hydrogen bonds between the ligand and (a) with water molecules in the channel lumen between the imidazoles of H37 and the ligand and (b) possibly with A30 carbonyls in the vicinity, according to experimental and molecular dynamics (MD) simulations data. Provided that M2TM is a minimal model for M2 binding, these experimental high resolution structures can be used for the prediction of new ligands binding more effectively to the M2TM pore for example through MD simulations19 or more precisely by binding free energy calculations.Η αμανταδίνη (Amt ) και η ριμανταδίνη (Rim) είναι αναστολείς της μεταφοράς πρωτονίων διαμέσου του ιοντικού διαύλου της πρωτεΐνης Μ2 του ιού influenza A, και αποτέλεσαν εγκεκριμένα μέσα πρόληψης και θεραπείας έναντι των ιών της γρίπης Α άγριου τύπου (WT). Ωστόσο, από το 2005, η μετάλλαξη του αμινοξέος σερίνη σε ασπαραγίνη στη θέση 31 (S31N) στην Μ2 πρωτεΐνη, δημιούργησε ένα στέλεχος ανθεκτικό στην Amt το οποίο έχει κυριαρχήσει παγκοσμίως, καταργώντας την κλινική χρησιμότητα της αμανταδίνης και πιθανότατα και άλλων προαναφερθέντων, στη διεθνή βιβλιογραφία, αναστολέων της Μ2. Επομένως, είναι απαραίτητη η ανάπτυξη νέων μορίων για την καταπολέμηση των ανθεκτικών στελεχών της γρίπης. H κύρια θέση πρόσδεσης της Amt (1) και της Rim (2) είναι η διαμεμβρανική περιοχή (transmembrane domain, TM) των αμινοξέων 22-46 του συμπλέγματος των τεσσάρων α-ελίκων της Μ2 που σχηματίζει τον δίαυλο μεταφοράς πρωτονίων. Σύμφωνα με δομές υψηλής διαχωριστικότητας που προέκυψαν από πειράματα κρυσταλλογραφίας ακτίνων-Χ και φασματοσκοπίας πυρηνικού μαγνητικού συντονισμού στερεάς κατάστασης (solid state NMR, ssNMR) και δημοσιεύτηκαν μεταξύ 2008-2011 από τους ερευνητές και καθηγητές Tim Cross, William DeGrado και Mei Hong στα πιο έγκριτα περιοδικά όπως Nature, JACS κλπ, η Amt (1) και η Rim (2) δρουν φράσσοντας τον πόρο του διαύλου M2TM. Ο κλωβός του αδαμαντανίου των ενώσεων αυτών περικλείεται από τις τέσσερεις πλευρικές αλυσίδες των V27 και της A30 του τετραμερούς M2TM προκαλώντας τον στερεοχημικό αποκλεισμό της μεταφοράς πρωτονίων και αποτρέποντας την συνέχεια του κύκλου ζωής του ιού. Τα αποτελέσματα ssNMR έδειξαν επίσης ότι η ομάδα αμμωνίου αυτών των φαρμάκων έχει προσανατολισμό προς τα τέσσερα αμινοξέα της His37 δηλαδή προς το C-τελικό άκρο.9 Αυτός ο προσανατολισμός σταθεροποιείται μέσω ενός δικτύου δεσμών υδρογόνου μεταξύ προσδέτη και (α) μορίων νερού εντός του πόρου του ιοντικού διαύλου, που βρίσκονται μεταξύ ιμιδαζολίου της H37 και προσδέτη, και, ενδεχομένως, (β) με το καρβονύλιο της A30 σύμφωνα με πειραματικά αποτελέσματα και προσομοιώσεις μοριακής δυναμικής (MD).Δεδομένου ότι η M2TM αποτελεί το απλούστερο μοντέλο πρόσδεσης της Amt (1) και Rim (2) για την M2, οι προηγούμενες πειραματικές δομές υψηλής ανάλυσης μπορούν να χρησιμοποιηθούν για το σχεδιασμό και ανάπτυξη νέων αναστολέων που να συνδέονται αποτελεσματικότερα με τον πόρο Μ2ΤΜ, μέσω προσομοιώσεων μοριακής δυναμικής (MD) 19ή ακριβέστερα με υπολογισμούς ελεύθερης ενέργειας

Amantadine Variant - Aryl Conjugates that Inhibit Multiple Amantadine Resistant M2 Mutant Influenza A Viruses

One challenge facing anti-influenza drug development is the heterogeneity of the circulating influenza A viruses, which comprise several strains with variable susceptibility to antiviral drugs. Viruses bearing the S31N mutant of the M2, such as the pandemic 2009 H1N1 and seasonal H3N2, as well as other mutants (L26F, V27A, A30T, G34E) are resistant to amantadine class of drugs. Here, we synthesized and tested many of the second generation amantadine - aryl conjugates, against the WT M2 and all the M2 amantadine resistant strains, i.e. L26F, V27A, S31N, A30T, G34E generated from WSN/33 (S31N) virus. We identified many compounds that are dual in vitro M2 WT and L26F virus inhibitors. Furthermore, few of them (21, 32, 33), having a rimantadine or diamantadine or 4-(1-adamantyl)aniline instead of amantadine in the conjugate, were in vitro inhibitors against M2 WT, L26F and S31N while one of them inhibited also the A30T virus. The electrophysiology (EP) experiments showed that these compounds blocked significantly M2 WT, L26F or even M2 V27A channels but not the M2 S31N. The observation that adamantane variants and derivatives inhibit multiple M2 mutant virus replication in cell culture, without blocking M2 channel-mediated proton current in EP is not uncommon, underlying a mechanism of antiviral activity that has not been identified

(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

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