Unit:
Department of PharmacyLibrary of the School of Science
Author:
Stampolaki Marianna
Dissertation committee:
Αντώνιος Κολοκούρης, Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Ιωάννης Παπαναστασίου, Αναπληρωτής Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Νικόλαος Λουγιάκης, Επίκουρος Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Εμμανουήλ Μικρός, Καθηγητής - Διευθυντής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Θωμάς Μαυρομούστακος, Καθηγητής, Τμήμα Χημείας, ΕΚΠΑ
Loren Andreas, Καθηγητής - Group Leader, Department of NMR-based Structural Biology, Max Planck Institute for Multidisciplinary Sciences
Christian Griesinger, Καθηγητής - Διευθυντής, Department of NMR-based Structural Biology, Max Planck Institute for Multidisciplinary Sciences
Original Title:
Structure-based drug discovery research on the MmpL3 transporter of Mycobacterium tuberculosis and the M2 proton channel of Influenza A virus
Translated title:
Structure-based drug discovery research on the MmpL3 transporter of Mycobacterium tuberculosis and the M2 proton channel of Influenza A virus
Summary:
SQ109 is a tuberculosis drug candidate that has high potency against Mycobacterium tuberculosis and is thought to function at least in part by blocking cell wall biosynthesis by inhibiting the MmpL3 transporter. It also has activity against bacteria and protozoan parasites that lack MmpL3, where it can act as an uncoupler, targeting lipid membranes and Ca2+ homeostasis. Here, we showed using molecular dynamics (MD) simulations that the binding profile of nine SQ109 analogs was consistent with the X-ray structure of MmpL3 – SQ109 complex. We showed that rotation of SQ109 around carbon–carbon bond in the monoprotonated ethylenediamine unit favors two gauche conformations as minima in water and lipophilic solvent using density functional theory (DFT) calculations as well as inside the transporter’s binding area using MD simulations. Our results from MD simulations suggested that sizeable C-2 adamantyl adducts of SQ109 can fill a lipophilic region between Tyr257, Tyr646, Phe260 and Phe649 in MmpL3. This was confirmed quantitatively by our calculations of the relative binding free energies using the thermodynamic integration coupled with MD simulations method with a mean assigned error of 0.74 kcal mol−1 compared to the experimental values. We synthesized 18 analogs of SQ109 and tested them against M. smegmatis, M. tuberculosis, M. abscessus, Bacillus subtilis, and Escherichia coli, as well as against the protozoan parasites Trypanosoma brucei, T. cruzi, Leishmania donovani, L. mexicana, and Plasmodium falciparum. Activity against the mycobacteria was generally less than with SQ109 and was reduced by increasing the size of the alkyl adduct. Τwo analogs were ∼4−8-fold more active than SQ109 against M. abscessus, including a highly drug resistant strain harboring an A309P mutation in MmpL3. There was also better activity than found with SQ109 with other bacteria and protozoa. Of particular interest, we found that the adamantyl C-2 ethyl, butyl, phenyl, and benzyl analogs had 4−10× increased activity against P. falciparum asexual blood stages, together with low toxicity to a human HepG2 cell line, making them of interest as new antimalarial drug leads. We also used surface plasmon resonance (SPR) to investigate the binding of inhibitors to MmpL3 and differential scanning calorimetry to investigate binding to lipid membranes. There was no correlation between MmpL3 binding and M. tuberculosis or M. smegmatis cell activity, suggesting that MmpL3 is not a major target in mycobacteria. However, some of the more active species decreased lipid phase transition temperatures, indicating increased accumulation in membranes, which is expected to lead to enhanced uncoupler activity.
The tetrameric influenza A M2 proton channel is critical for viral replication. Proton conductance of the wild type (WT) protein, with serine at position 31, is inhibited by cage alkyl amines, like amantadine, rimantadine and analogs, which bind in the channel pore. Conjugates of a cage alkyl amine with a methylene-linked aryl group have been shown to block the channel of the prevalent S31N strain with a different mechanism. Here, we investigate the binding and blocking of WT and S31N M2 (Udorn strain) by an expanded set of cage alkyl containing amines. Using a liposomal proton flux assay, we verified the channel-blocking activity of rimantadine (Rmt), its methyl analog AK59, and the tetramethyltetracyclodec-1-ylmethylamine (RL208), an isomer of amantadine for the conductance domain of WT M2 (M2CD; residues 18-60). The binding behavior of the ligands was explored using magic angle spinning NMR by recording 2D and 3D 1H-15N and 1H-13C-15N spectra of M2CD reconstituted in DPhPC lipids. Ligand binding to WT M2 was detected via chemical shift perturbation (CSP). High kinetic energy barriers associated with entry into the M2CD pore were overcome by increased temperature or a pH shift protocol which proved to be more efficient. To assess the binding mode, MD simulations were performed. Cage alkyl amines Rmt and AK59 are known to block WT M2 with their ammonium group binding to water-lined sites at Gly34 or Ala30. By contrast, MD simulations show that the conjugates with an aryl group bind outward in S31N M2CD targeting Asn31. For S31N, the His37-His37 hydrogen bonding was not disrupted according to NMR, nor was the channel blocked in the liposomal proton flux assay. Our data suggest that there is a substantial structural barrier in the amphipathic helicix (AH)-bearing CD construct, which limits entry of RL208 and aryl conjugates.
Main subject category:
Science
Other subject categories:
Health Sciences
Keywords:
SQ109, MmpL3, MD simulations, alchemical relative binding free energy, MM-GBSA, binding affinity, organic synthesis, calorimetry, tuberculosis, malaria, leishmaniasis, adamantyl amine, cage alkyl amine, influenza A, S31N M2, WT M2, magic angle spinning, solid state NMR, liposomal proton flux assay
Number of references:
283
