Influenza A M2 Spans the Membrane Bilayer, Perturbs its Organization and Differentiates the Effect of Amantadine and Spiro[pyrrolidine-2,2'-adamantane] AK13 on Lipids

The investigation and observations made for the M2TM, excess aminoadamantane ligands in DMPC were made using the simpler version of biophysical methods including SDC, SAXS and WAXS, MD simulations and ssNMR. 1H, 31P ssNMR and MD simulations, showed that M2TM in apo form or drug-bound form span the membrane interacting strongly with lipid acyl chain tails and the phosphate groups of the polar head surface. The MD simulations showed that the drugs anchor through their ammonium group with the lipid phosphate and occasionally with M2TM asparagine-44 carboxylate groups. The 13C ssNMR experiments allow the inspection of excess drug molecules and the assessment of its impact on the lipid head-group region. At low peptide concentrations of influenza A M2TM tetramer in DPMC bilayer, two lipid domains were observed that likely correspond to the M2TM boundary lipids and the bulk-like lipids. At high peptide concentrations, one domain was identified which constitute essentially all of the lipids which behave as boundary. This effect is likely due, according to the MD simulations, to the preference of AK13 to locate in closer vicinity to M2TM compared to Amt as well as the stronger ionic interactions of Amt primary ammonium group with phosphate groups, compared with the secondary buried ammonium group in AK13.

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Plastidic Δ6 fatty-acid desaturases with distinctive substrate specificity regulate the pool of c18-pufas in the ancestral picoalga ostreococcus tauri

10.1101/2020.03.10.986216 ◽

2020 ◽

Author(s):

Charlotte Degraeve-Guilbault C. ◽

Rodrigo E. Gomez ◽

Cécile. Lemoigne ◽

Nattiwong Pankansem ◽

Soizic Morin ◽

...

Keyword(s):

Substrate Specificity ◽

Acyl Chain ◽

Cytochrome B5 ◽

Head Group ◽

Fatty Acid Desaturases ◽

Physiological Alterations ◽

Lipid Head Group ◽

Species Specific ◽

Native Host ◽

Δ6 Desaturase

ABSTRACTEukaryotic Δ6-desaturases are microsomal enzymes which balance the synthesis of ω-3 and ω-6 C18-polyunsaturated-fatty-acids (PUFA) accordingly to their specificity. In several microalgae, including O. tauri, plastidic C18-PUFA are specifically regulated by environmental cues suggesting an autonomous control of Δ6-desaturation of plastidic PUFA. Sequence retrieval from O. tauri desaturases, highlighted two putative Δ6/Δ8-desaturases sequences clustering, with other microalgal homologs, apart from other characterized Δ-6 desaturases. Their overexpression in heterologous hosts, including N. benthamiana and Synechocystis, unveiled their Δ6-desaturation activity and plastid localization. O. tauri lines overexpressing these Δ6-desaturases no longer adjusted their plastidic C18-PUFA amount under phosphate starvation but didn’t show any obvious physiological alterations. Detailed lipid analyses from the various overexpressing hosts, unravelled that the substrate features involved in the Δ6-desaturase specificity importantly involved the lipid head-group and likely the non-substrate acyl-chain, in addition to the overall preference for the ω-class of the substrate acyl-chain. The most active desaturase displayed a broad range substrate specificity for plastidic lipids and a preference for ω-3 substrates, while the other was selective for ω-6 substrates, phosphatidylglycerol and 16:4-galactolipid species specific to the native host. The distribution of plastidial Δ6-desaturase products in eukaryotic hosts suggested the occurrence of C18-PUFA export from the plastid.One sentence summaryOsteococcus tauri plastidic lipid C18-PUFA remodelling involves two plastid-located cytochrome-b5 fused Δ6-desaturases with distinct preferences for both head-group and acyl-chain.

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(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

10.26434/chemrxiv.11494170.v1 ◽

2020 ◽

Author(s):

Christina Tzitzoglaki ◽

Kelly McGuire ◽

Athina Konstantinidi ◽

Panagiotis Lagarias ◽

Anja Hoffmann ◽

...

Keyword(s):

Rate Constants ◽

Influenza A ◽

Md Simulations ◽

Proton Channel ◽

Head Group ◽

Polar Head Group ◽

Polar Head ◽

The Impact ◽

Low K ◽

Proton Currents

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.

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(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

10.26434/chemrxiv.11494170 ◽

2020 ◽

Author(s):

Christina Tzitzoglaki ◽

Kelly McGuire ◽

Athina Konstantinidi ◽

Panagiotis Lagarias ◽

Anja Hoffmann ◽

...

Keyword(s):

Rate Constants ◽

Influenza A ◽

Md Simulations ◽

Proton Channel ◽

Head Group ◽

Polar Head Group ◽

Polar Head ◽

The Impact ◽

Low K ◽

Proton Currents

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.

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Effects of the Nonimmobilizer Hexafluroethane on the Model Membrane Dimyristoylphosphatidylcholine

Anesthesiology ◽

10.1097/00000542-200210000-00016 ◽

2002 ◽

Vol 97 (4) ◽

pp. 848-855 ◽

Cited By ~ 15

Author(s):

Laure Koubi ◽

Mounir Tarek ◽

Sanjoy Bandyopadhyay ◽

Michael L. Klein ◽

Daphna Scharf

Keyword(s):

Physical Properties ◽

Lipid Bilayer ◽

Md Simulations ◽

Model Membrane ◽

Head Group ◽

Clinical Effects ◽

Equilibrium Configurations ◽

Area Per Lipid ◽

Lipid Head Group ◽

Chain Conformations

Background Nonimmobilizers are agents that lack anesthetic properties, although their chemical structure is very similar to known anesthetics. The primary action site of both agents, whether at the membrane or target protein level, is still a matter of debate. However, increasing evidence points to the distinct modifications of the membrane physical properties that such agents induce. Such modification may play a role in the mechanism of anesthesia, and may therefore be related to the differences in their clinical behavior. Methods Molecular dynamics (MD) computer simulations have been used to investigate the distribution of a nonimmobilizer, hexafluroethane (HFE, C(2)F(6)), in a lipid membrane. The biologically relevant liquid-crystal phase of a hydrated dimyristoyl phosphatidyl choline (DMPC) bilayer was used as a membrane model. Two MD simulations corresponding to HFE mole fractions of 6% and 25% have been performed at room temperature and constant ambient pressure, for a duration of 2 nanoseconds each. Results The equilibrium configurations of HFE in the bilayer show that the nonimmobilizer molecules are evenly distributed along the lipid hydrocarbon chains with a slight preference for the bilayer center. This partitioning induces an expansion of the bilayer thickness and a lateral contraction of the membrane (decrease of the area per lipid). The presence of HFE has essentially no effect on the lipid acyl chain conformations in agreement with nuclear magnetic resonance (NMR) measurements of the chain order parameters. The partitioning of the nonimmobilizer does not influence the orientation of the lipid head-group dipole moment. Conclusions The modifications induced by the presence of the nonimmobilizer HFE on a model membrane are distinct from those previously found for halothane (CF(3)CHBrCl), its anesthetic analogue, and appear to result from different distributions in the lipid bilayer. The results of the MD simulations show that (1) the changes in the average area per lipid and in the membrane thickness are opposite for the two agents and (2) HFE induces no change in the lipid head-group orientation, in contrast to halothane. These different effects (1) on the physical properties of the lipid bilayer and (2) on the electrostatic properties of the membrane-water interface may be linked to different clinical effects, and thus might contribute to the mechanism of general anesthesia.

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Computational and experimental evidence to the permeability of withanolides across the normal cell membrane

10.1101/802645 ◽

2019 ◽

Author(s):

R. Wadhwa ◽

N. S. Yadav ◽

S. P Katiyar ◽

T. Yaguchi ◽

C. Lee ◽

...

Keyword(s):

Cell Membrane ◽

Chemical Properties ◽

Md Simulations ◽

Computational Method ◽

Withaferin A ◽

Head Group ◽

Polar Head Group ◽

Drug Molecules ◽

Natural Drugs ◽

The Impact

AbstractPoor bioavailability due to the inability to cross the cell membrane is one of the major reasons for the failure of a drug in the clinical trials. We have used molecular dynamics simulations to predict the membrane permeability of natural drugs - withanolides (withaferin-A and withanone) that have similar structures but remarkably differ in their cytotoxicity. We found that withaferin-A, but not withanone, could proficiently transverse through the model membrane. The free energy profiles obtained were in accordance with the physico-chemical properties of the investigated drug molecules. It was observed that the polar head group of the bilayer exhibits high resistance for the passage of withanone as compared to withaferin-A, while the interior of the membrane behaves similarly for both withanolides. The solvation analysis revealed that the high solvation of terminal O5 oxygen of withaferin-A was the major driving force. The impact of the favorable interaction of terminal oxygen (O5) of withaferin-A with the phosphate of the membrane led to its smooth passage across the bilayer. The computational predictions were validated by raising and recruiting unique antibodies that react to withaferin-A and withanone. Further, the time-lapsed analyses of control and treated human normal and cancer cells, demonstrated proficient permeation of withaferin-A, but not withanone, through normal cells. These data strongly validated our computational method for predicting permeability and hence bioavailability of candidate compounds in the drug development process.Statement of significanceWhat determines the bioavailability of a drug? Does the ability to cross cell membrane determine this? A combined simulation/experimental study of the permeability of two natural drugs - withanolides (Wi-A and Wi-N) across the cell membrane was conducted. In the computational portion of the study, steered MD simulations were performed to investigate the propensity of the two molecules to permeate across the cell. It is found that Wi-A proceeds relatively simply across the cell compared to Wi-N. This trend was found to be consistent with experiment. This work is an important step towards understanding the molecular basis of permeability of natural drug molecules.

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Spotting Differences in Molecular Dynamic Simulations of Influenza A M2 Protein-Ligand Complexes by Varying M2 construct, Lipid Bilayer and Force Field

10.26434/chemrxiv.11365943.v1 ◽

2019 ◽

Author(s):

Dimitrios Kolokouris ◽

Iris Kalenderoglou ◽

Panagiotis Lagarias ◽

Antonios Kolocouris

Keyword(s):

Molecular Dynamic ◽

Lipid Bilayer ◽

Influenza A ◽

Md Simulations ◽

Membrane Curvature ◽

Buffer Size ◽

M2 Protein ◽

Dynamic Simulations ◽

Amphipathic Helices ◽

Drug Protein Binding

We studied by molecular dynamic (MD) simulations systems including the inwardclosed state of influenza A M2 protein in complex with aminoadamantane drugs in membrane bilayers. We varied the M2 construct and performed MD simulations in M2TM or M2TM with amphipathic helices (M2AH). We also varied the lipid bilayer by changing either the lipid, DMPC or POPC, POPE or POPC/cholesterol (chol), or the lipids buffer size, 10x10 Å2 or 20x20 Å2. We aimed to suggest optimal system conditions for the computational description of this ion channel and related systems. Measures performed include quantities that are available experimentally and include: (a) the position of ligand, waters and chlorine anion inside the M2 pore, (b) the passage of waters from the outward Val27 gate of M2 S31N in complex with an aminoadamantane-aryl head blocker, (c) M2 orientation, (d) the AHs conformation and structure which is affected from interactions with lipids and chol and is important for membrane curvature and virus budding. In several cases we tested OPLS2005, which is routinely applied to describe drug-protein binding, and CHARMM36 which describes reliably protein conformation. We found that for the description of the ligands position inside the M2 pore, a 10x10 Å2 lipids buffer in DMPC is needed when M2TM is used but 20x20 Å2 lipids buffer of the softer POPC; when M2AH is used all 10x10 Å2 lipid buffers with any of the tested lipids can be used. For the passage of waters at least M2AH with a 10x10 Å2 lipid buffer is needed. The folding conformation of AHs which is defined from hydrogen bonding interactions with the bilayer and the complex with chol is described well with a 10x10 Å2 lipids buffer and CHARMM36.

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Mechanisms of Drug Solubilization by Polar Lipids in Biorelevant Media

10.26434/chemrxiv.12649145.v1 ◽

2020 ◽

Author(s):

Vladimir Katev ◽

Zahari Vinarov ◽

Slavka S. Tcholakova

Keyword(s):

Chain Length ◽

Acyl Chain ◽

Polar Lipids ◽

Superior Performance ◽

Head Group ◽

Formulation Development ◽

Biorelevant Media ◽

Drug Solubilization ◽

Colloidal Aggregates ◽

Class 1

Despite the widespread use of lipid excipients in both academic research and oral formulation development, rational selection guidelines are still missing. In the current study, we aimed to establish a link between the molecular structure of commonly used polar lipids and drug solubilization in biorelevant media. We studied the effect of 26 polar lipids of the fatty acid, phospholipid or monoglyceride type on the solubilization of fenofibrate in a two-stage in vitro GI tract model. The main trends were checked also with progesterone and danazol. Based on their fenofibrate solubilization efficiency, the polar lipids can be grouped in 3 main classes. Class 1 substances (n = 5) provide biggest enhancement of drug solubilization (>10-fold) and are composed only by unsaturated compounds. Class 2 materials (n = 10) have an intermediate effect (3-10 fold increase) and are composed primarily (80 %) of saturated compounds. Class 3 materials (n = 11) have very low or no effect on drug solubilization and are entirely composed of saturated compounds. The observed behaviour of the polar lipids was rationalized by using two classical physicochemical parameters: the acyl chain phase transition temperature (Tm) and the critical micellar concentration (CMC). Hence, the superior performance of class 1 polar lipids was explained by the double bonds in their acyl chains, which: (1) significantly decrease Tm, allowing these C18 lipids to form colloidal aggregates and (2) prevent tight packing of the molecules in the aggregates, resulting in bigger volume available for drug solubilization. Long-chain (C18) saturated polar lipids had no significant effect on drug solubilization because their Tm was much higher than the temperature of the experiment (T = 37 C) and, therefore, their association in colloidal aggregates was limited. On the other end of the spectrum, the short chain octanoic acid manifested a high CMC (50 mM), which had to be exceeded in order to enhance drug solubilization. When these two parameters were satisfied (C > CMC, Tm < Texp), the increase of the polar lipid chain length increased the drug solubilization capacity (similarly to classical surfactants), due to the decreased CMC and bigger volume available for solubilization. The hydrophilic head group also has a dramatic impact on the drug solubilization enhancement, with polar lipids performance decreasing in the order: choline phospholipids > monoglycerides > fatty acids. As both the acyl chain length and the head group type are structural features of the polar lipids, and not of the solubilized drugs, the impact of Tm and CMC on solubilization by polar lipids should hold true for a wide variety of hydrophobic molecules. The obtained mechanistic insights can guide rational drug formulation development and thus support modern drug discovery pipelines.

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Mechanisms of Drug Solubilization by Polar Lipids in Biorelevant Media

10.26434/chemrxiv.12649145 ◽

2020 ◽

Author(s):

Vladimir Katev ◽

Zahari Vinarov ◽

Slavka S. Tcholakova

Keyword(s):

Chain Length ◽

Acyl Chain ◽

Polar Lipids ◽

Superior Performance ◽

Head Group ◽

Formulation Development ◽

Biorelevant Media ◽

Drug Solubilization ◽

Colloidal Aggregates ◽

Class 1

Despite the widespread use of lipid excipients in both academic research and oral formulation development, rational selection guidelines are still missing. In the current study, we aimed to establish a link between the molecular structure of commonly used polar lipids and drug solubilization in biorelevant media. We studied the effect of 26 polar lipids of the fatty acid, phospholipid or monoglyceride type on the solubilization of fenofibrate in a two-stage in vitro GI tract model. The main trends were checked also with progesterone and danazol. Based on their fenofibrate solubilization efficiency, the polar lipids can be grouped in 3 main classes. Class 1 substances (n = 5) provide biggest enhancement of drug solubilization (>10-fold) and are composed only by unsaturated compounds. Class 2 materials (n = 10) have an intermediate effect (3-10 fold increase) and are composed primarily (80 %) of saturated compounds. Class 3 materials (n = 11) have very low or no effect on drug solubilization and are entirely composed of saturated compounds. The observed behaviour of the polar lipids was rationalized by using two classical physicochemical parameters: the acyl chain phase transition temperature (Tm) and the critical micellar concentration (CMC). Hence, the superior performance of class 1 polar lipids was explained by the double bonds in their acyl chains, which: (1) significantly decrease Tm, allowing these C18 lipids to form colloidal aggregates and (2) prevent tight packing of the molecules in the aggregates, resulting in bigger volume available for drug solubilization. Long-chain (C18) saturated polar lipids had no significant effect on drug solubilization because their Tm was much higher than the temperature of the experiment (T = 37 C) and, therefore, their association in colloidal aggregates was limited. On the other end of the spectrum, the short chain octanoic acid manifested a high CMC (50 mM), which had to be exceeded in order to enhance drug solubilization. When these two parameters were satisfied (C > CMC, Tm < Texp), the increase of the polar lipid chain length increased the drug solubilization capacity (similarly to classical surfactants), due to the decreased CMC and bigger volume available for solubilization. The hydrophilic head group also has a dramatic impact on the drug solubilization enhancement, with polar lipids performance decreasing in the order: choline phospholipids > monoglycerides > fatty acids. As both the acyl chain length and the head group type are structural features of the polar lipids, and not of the solubilized drugs, the impact of Tm and CMC on solubilization by polar lipids should hold true for a wide variety of hydrophobic molecules. The obtained mechanistic insights can guide rational drug formulation development and thus support modern drug discovery pipelines.

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A comprehensive in silico study towards understanding the inhibitory mechanism of Lactoperoxidase by Dapsone and Propofol

Current Computer - Aided Drug Design ◽

10.2174/1573409916666200628104134 ◽

2020 ◽

Vol 16 ◽

Author(s):

Rameez Jabeer Khan ◽

Rajat Kumar Jha ◽

Gizachew Muluneh Amera ◽

Jayaraman Muthukumaran ◽

Rashmi Prabha Singh ◽

...

Keyword(s):

Molecular Docking ◽

Md Simulation ◽

Binding Free Energy ◽

Protoporphyrin Ix ◽

Md Simulations ◽

Prosthetic Group ◽

Drug Molecules ◽

Group A ◽

Complex Forms ◽

Covalently Linked

Introduction: Lactoperoxidase (LPO) is a member of mammalian heme peroxidase family and is an enzyme of innate immune system. It possesses a covalently linked heme prosthetic group (a derivative of protoporphyrin IX) in its active site. LPO catalyzes the oxidation of halides and pseudohalides in the presence of hydrogen peroxide (H2O2) and shows a broad range of antimicrobial activity. Methods: In this study, we have used two pharmaceutically important drug molecules, namely dapsone and propofol, which are earlier reported as potent inhibitors of LPO. Whereas the stereochemistry and mode of binding of dapsone and propofol to LPO is still not known because of the lack of the crystal structure of LPO with these two drugs. In order to fill this gap, we utilized molecular docking and molecular dynamics (MD) simulation studies of LPO in native and complex forms with dapsone and propofol. Results: From the docking results, the estimated binding free energy (ΔG) of -9.25 kcal/mol (Ki = 0.16 μM) and -7.05 kcal/mol (Ki = 6.79 μM) was observed for dapsone, and propofol, respectively. The standard error of Auto Dock program is 2.5 kcal/mol; therefore, molecular docking results alone were inconclusive. Conclusion: To further validate the docking results, we performed MD simulation on unbound, and two drugs bounded LPO structures. Interestingly, MD simulations results explained that the structural stability of LPO-Propofol complex was higher than LPO-Dapsone complex. The results obtained from this study establish the mode of binding and interaction pattern of the dapsone and propofol to LPO as inhibitors.

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