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

Abstract

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.

Στο Κεφάλαιο 1 περιγράφονται τα βασικά χαρακτηριστικά του μηχανισμού αντιγραφής του ιού της γρίπης Α, με έμφαση στη λειτουργία της πρωτεΐνης Μ2 του ιού της γρίπης Α. Στο Κεφάλαιο 2 περιγράφεται η δομή του πρωτεϊνικού διαύλου πρωτονίων Μ2 (M2 WT) του ιού της γρίπης Α, ο οποίος αποτελεί αρχέτυπο ιοντικό κανάλι. Ο δίαυλος αυτός αποτελεί στόχο των αντι-ιικών φαρμάκων αμανταδίνη (amantadine) και ριμανταδίνη (rimantadine). Διάφορες μέθοδοι χρησιμοποιήθηκαν για την κατανόηση των δομικών και λειτουργικών χαρακτηριστικών αυτού του διαύλου, συμπεριλαμβανομένων της περίθλασης νετρονίων, της ηλεκτροφυσιολογίας, της φασματοσκοπίας NMR διαλύματος, της φασματοσκοπίας στερεάς κατάστασης NMR (ssNMR) και της κρυσταλλογραφίας ακτίνων Χ, σε μία ερευνητική πορεία τριών δεκαετιών γεμάτη αντιπαραθέσεις. Η πειραματική δομή του διαμεμβρανικού τομέα Μ2(22-46) (M2TM), ο οποίος αποτελεί τον πόρο του διαύλου Μ2, επιλύθηκε το 2000, ενώ οι κρυσταλλικές δομές των συμπλόκων του με την αμανταδίνη, τη ριμανταδίνη και άλλα μόρια δημοσιεύθηκαν έως το 2018. Μέχρι σήμερα, έχουν επίσης επιλυθεί βασικά χαρακτηριστικά της πρωτεΐνης του αγώγιμου τομέα του M2 (M2CD ή M2AH), που περιλαμβάνει το M2TM και τις αμφιπαθείς έλικες (46-62), χρησιμοποιώντας φασματοσκοπία ssNMR. Στο Κεφάλαιο 3 παρουσιάζονται οι βασικές αρχές των πειραματικών βιοφυσικών μεθόδων που χρησιμοποιήθηκαν σε αυτή τη διδακτορική διατριβή, δηλαδή η διαφορική θερμιδομετρία σάρωσης (DSC), η σκέδαση ακτίνων Χ σε μικρές και μεγάλες γωνίες (SAXS/WAXS) και η φασματοσκοπία ssNMR. Στο Κεφάλαιο 4 διερευνώνται οι συγκριτικές επιδράσεις που ασκούν στην κυτταρική μεμβράνη οι αναστολείς του πρωτεϊνικού διαύλου M2 WT, αμανταδίνη και η παράγωγός της AK13 (spiro[pyrrolidine-2,2'-adamantane]), χρησιμοποιώντας βιοφυσικές μεθόδους και προσομοιώσεις μοριακής δυναμικής (MD). Η έρευνα πραγματοποιήθηκε σε συνεργασία με τις ερευνητικές ομάδες των Καθηγητών Thomas Mavromoustakos και Costas Demetzos, καθώς και της Dr. Barbara Sartori και του Καθηγητή Heinz Amenitsch. Οι πειραματικές μέθοδοι που χρησιμοποιήθηκαν ήταν DSC, σκέδαση ακτίνων Χ και φασματοσκοπία ssNMR. Και οι τρεις μέθοδοι έδειξαν ότι τα δύο ανάλογα μόρια προκαλούν έντονες διαταραχές στις διπλοστιβάδες DMPC, με την AK13 να είναι πιο αποτελεσματική σε υψηλές συγκεντρώσεις. Οι προσομοιώσεις MD αποκάλυψαν λεπτομέρειες για τις αλληλεπιδράσεις των φαρμάκων με την κυτταρική μεμβράνη, καθώς και τον σχηματισμό δεσμών υδρογόνου με το νερό και τις φωσφορικές ομάδες των λιπιδίων. Στο Κεφάλαιο 5 περιγράφεται η μελέτη των αλληλεπιδράσεων του M2TM WT με τις λιπιδικές διπλοστιβάδες. Η έρευνα πραγματοποιήθηκε σε συνεργασία με τις ερευνητικές ομάδες των Καθηγητών Thomas Mavromoustakos και Costas Demetzos, καθώς και της Dr. Barbara Sartori και του Καθηγητή Heinz Amenitsch. Το πεπτίδιο M2TM συντέθηκε από την ερευνητική ομάδα του Καθηγητή Thedoros Tselios. Χρησιμοποιήθηκαν διάφορες βιοφυσικές μέθοδοι (DSC, SAXS/WAXS, MD προσομοιώσεις, φασματοσκοπία ssNMR), οι οποίες έδειξαν ότι το M2TM αλληλεπιδρά έντονα με τις λιπιδικές διπλοστιβάδες. Στο Κεφάλαιο 6 παρουσιάζεται η μελέτη της σύνδεσης των μικρών μορίων αμανταδίνης και ριμανταδίνης με το κανάλι Μ2 WT του ιού της γρίπης Α, το οποίο αποτελεί στόχο των αντι-ιικών φαρμάκων. Η έρευνα πραγματοποιήθηκε σε συνεργασία με τις ερευνητικές ομάδες των Καθηγητών William DeGrado και Jun Wang. Χρησιμοποιήθηκαν κρυσταλλογραφικές μέθοδοι, που επέτρεψαν την επίλυση κρυσταλλικών δομών των συμπλόκων των φαρμάκων με το M2. Στο Κεφάλαιο 7 περιγράφεται η μελέτη της μετάλλαξης V27A στο κανάλι M2, η οποία προσδίδει ανθεκτικότητα στην αμανταδίνη. Η έρευνα πραγματοποιήθηκε σε συνεργασία με τις ερευνητικές ομάδες των Καθηγητών DeGrado και Wang. Χρησιμοποιήθηκαν κρυσταλλογραφία ακτίνων Χ και προσομοιώσεις MD, που αποκάλυψαν τις δομικές αλλαγές του διαύλου λόγω της μετάλλαξης V27A, καθώς και την αλλαγή στη θέση σύνδεσης των φαρμάκων. Στο Κεφάλαιο 8 παρουσιάζεται η μελέτη της σύνδεσης των δύο εναντιομερών της ριμανταδίνης [(R)- και (S)-rimantadine] στο κανάλι Μ2. Η έρευνα πραγματοποιήθηκε σε συνεργασία με τις ερευνητικές ομάδες των Καθηγητών DeGrado, Jun Wang και Jon Essex. Χρησιμοποιήθηκαν κρυσταλλογραφικές μέθοδοι, ηλεκτροφυσιολογικές μετρήσεις και προσομοιώσεις μοριακής δυναμικής, που αποκάλυψαν ότι τα δύο εναντιομερή δεσμεύονται με παρόμοιο τρόπο στον δίαυλο Μ2, χωρίς να εμφανίζουν σημαντικές διαφορές στην αποτελεσματικότητά τους.