Corrigendum to "Interaction between goniothalamin and peroxisomal multifunctional enzyme type 2 triggering endoplasmic reticulum stress" [Heliyon 6 (10) (October 2020) e05200].

[This corrects the article DOI: 10.1016/j.heliyon.2020.e05200.].

Interaction between goniothalamin and peroxisomal multifunctional enzyme type 2 triggering endoplasmic reticulum stress.

Endoplasmic reticulum stress is one of the pathways involved in cell cytotoxicity. In this study, goniothalamin, one of styryllactone compounds found in plant Goniothalamus spp., was observed to trigger ER stress in HeLa cell line. In addition, we demonstrated that peroxisomal multifunctional enzyme type2 (MFE2) was a specific goniothalamin-binding protein using an in vitro goniothalamin-linked bead pull-down assay. Since MFE2 has been reported to be an important mediator enzyme for peroxisomal ß-oxidation of a very long chain fatty acid metabolism, therefore computational molecular docking analysis was performed to confirm the binding of goniothalamin and MFE2. The results indicated that goniothalamin structure binds to scp-2 domain, enoyl-CoA hydratase 2 domain and (3R)-hydroxyacyl-CoA dehydrogenase domain of MFE2. To further determine the effect of MFE2 on ER stress induction, MFE2 knockdown by siRNA in HeLa cell was conducted. The results implied that MFE2 triggered CHOP, a key mediator of ER stress-induced apoptosis, expression. Therefore, these data inferred that goniothalamin may interrupt the MFE2 function resulting in lipid metabolism perturbation associated with ER stress-independent activation of unfolded protein response. This is the first report to show that goniothalamin binds directly to MFE2 triggering ER stress activation probably through the lipid metabolism perturbation.

Effects of the protonation state of the catalytic residues and ligands upon binding and recognition in targeted proteins of HIV-1 and influenza viruses.

The determination of the protonation state of the functional groups of ligands, and the amino acid residues with electrically charged side chains (His, Lys, Arg, Asp and Glu) or the nucleotide bases of the nucleic acids that they interact with, is important for ligand binding and recognition, the enzyme activity and reaction mechanism, and protein folding/unfolding and stability. Herein, the effects of different protonation state assignments of the small substrate and inhibitors and the critical residues on the reverse transcriptase and protease of human immunodeficiency virus type 1 (HIV-1) and the M2 proton channel of influenza A virus are reviewed. Theoretical studies on these topics are summarized and compared with the experimental data.

Why amantadine loses its function in influenza m2 mutants: MD simulations.

Molecular dynamics simulations of the drug-resistant M2 mutants, A30T, S31N, and L26I, were carried out to investigate the inhibition of M2 activity using amantadine (AMT). The closed and open channel conformations were examined via non- and triply protonated H37. For the nonprotonated state, these mutants exhibited zero water density in the conducting region, and AMT was still bound to the channel pore. Thus, water transport is totally suppressed, similar to the wild-type channel. In contrast, the triply protonated states of the mutants exhibited a different water density and AMT position. A30T and L26I both have a greater water density compared to the wild-type M2, while for the A30T system, AMT is no longer inside the pore. Hydrogen bonding between AMT and H37 crucial for the bioactivity is entirely lost in the open conformation. The elimination of this important interaction of these mutations is responsible for the lost of AMT's function in influenza A M2. This is different for the S31N mutant in which AMT was observed to locate at the pore opening region and bond with V27 instead of S31.

Combined QM/MM mechanistic study of the acylation process in furin complexed with the H5N1 avian influenza virus hemagglutinin's cleavage site.

Combined quantum mechanical/molecular mechanical (QM/MM) techniques have been applied to investigate the detailed reaction mechanism of the first step of the acylation process by furin in which the cleavage site of the highly pathogenic avian influenza virus subtype H5N1 (HPH5) acts as its substrate. The energy profile shows a simultaneous mechanism, known as a concerted reaction, of the two subprocesses: the proton transfer from Ser368 to His194 and the nucleophilic attack on the carbonyl carbon of the scissile peptide of the HPH5 cleavage site with a formation of tetrahedral intermediate (INT). The calculated energy barrier for this reaction is 16.2 kcal.mol(-1) at QM/MM B3LYP/6-31+G*//PM3-CHARMM22 level of theory. Once the reaction proceeds, the ordering of the electrostatic stabilization by protein environment is of the enzyme-substrate < transition state < INT complexes. Asp153 was found to play the most important role in the enzymatic reaction by providing the highest degree of intermediate complex stabilization. In addition, the negatively charged carbonyl oxygen of INT is well stabilized by the oxyanion hole constructed by Asn295's carboxamide and Ser368's backbone.

Source of oseltamivir resistance in avian influenza H5N1 virus with the H274Y mutation.

Molecular dynamics simulations were carried out for the mutant oseltamivir-NA complex, to provide detailed information on the oseltamivir-resistance resulting from the H274Y mutation in neuraminidase (NA) of avian influenza H5N1 viruses. In contrast with a previous proposal, the H274Y mutation does not prevent E276 and R224 from forming the hydrophobic pocket for the oseltamivir bulky group. Instead, reduction of the hydrophobicity and size of pocket in the area around an ethyl moiety at this bulky group were found to be the source of the oseltamivir-resistance. These changes were primarily due to the dramatic rotation of the hydrophilic -COO(-) group of E276 toward the ethyl moiety. In addition, hydrogen-bonding interactions with N1 residues at the -NH(3) (+) and -NHAc groups of oseltamivir were replaced by a water molecule. The calculated binding affinity of oseltamivir to NA was significantly reduced from -14.6 kcal mol(-1) in the wild-type to -9.9 kcal mol(-1) in the mutant-type.

How amantadine and rimantadine inhibit proton transport in the M2 protein channel.

To understand how antiviral drugs inhibit the replication of influenza A virus via the M2 ion channel, molecular dynamics simulations have been applied to the six possible protonation states of the M2 ion channel in free form and its complexes with two commercial drugs in a fully hydrated lipid bilayer. Among the six different states of free M2 tetramer, water density was present in the pore of the systems with mono-protonated, di-protonated at adjacent position, tri-protonated and tetra-protonated systems. In the presence of inhibitor, water density in the channel was considerably better reduced by rimantadine than amantadine, agreed well with the experimental IC(50) values. With the preferential position and orientation of the two drugs in all states, two mechanisms of action, where the drug binds to the opening pore and the histidine gate, were clearly explained, i.e., (i) inhibitor was detected to localize slightly closer to the histidine gate and can facilitate the orientation of His37 imidazole rings to lie in the close conformation and (ii) inhibitor acts as a blocker, binding at almost above the opening pore and interacts slightly with the three pore-lining residues, Leu26, Ala30 and Ser31. Here, the inhibitors were found to bind very weakly to the channel due to their allosteric hindrance while theirs side chains were strongly solvated.

Source of high pathogenicity of an avian influenza virus H5N1: why H5 is better cleaved by furin.

The origin of the high pathogenicity of an emerging avian influenza H5N1 due to the -RRRKK- insertion at the cleavage loop of the hemagglutinin H5, was studied using the molecular dynamics technique, in comparison with those of the noninserted H5 and H3 bound to the furin (FR) active site. The cleavage loop of the highly pathogenic H5 was found to bind strongly to the FR cavity, serving as a conformation suitable for the proteolytic reaction. With this configuration, the appropriate interatomic distances were found for all three reaction centers of the enzyme-substrate complex: the arrangement of the catalytic triad, attachment of the catalytic Ser(368) to the reactive S1-Arg, and formation of the oxyanion hole. Experimentally, the--RRRKK--insertion was also found to increase in cleavage of hemagglutinin by FR. The simulated data provide a clear answer to the question of why inserted H5 is better cleaved by FR than the other subtypes, explaining the high pathogenicity of avian influenza H5N1.

Understanding of known drug-target interactions in the catalytic pocket of neuraminidase subtype N1.

To provide detailed information and insight into the drug-target interaction, structure, solvation, and dynamic and thermodynamic properties, the three known-neuraminidase inhibitors-oseltamivir (OTV), zanamivir (ZNV), and peramivir (PRV)-embedded in the catalytic site of neuraminidase (NA) subtype N1 were studied using molecular dynamics simulations. In terms of ligand conformation, there were major differences in the structures of the guanidinium and the bulky groups. The atoms of the guanidinium group of PRV were observed to form many more hydrogen bonds with the surrounded residues and were much less solvated by water molecules, in comparison with the other two inhibitors. Consequently, D151 lying on the 150-loop (residues 147-152) of group-1 neuraminidase (N1, N4, N5, and N8) was considerably shifted to form direct hydrogen bonds with the --OH group of the PRV, which was located rather far from the 150-loop. For the bulky group, direct hydrogen bonds were detected only between the hydrophilic side chain of ZNV and residues R224, E276, and E277 of N1 with rather weak binding, 20-70% occupation. This is not the case for OTV and PRV, in which flexibility and steric effects due to the hydrophobic side chain lead to the rearrangement of the surrounded residues, that is, the negatively charged side chain of E276 was shifted and rotated to form hydrogen bonds with the positively charged moiety of R224. Taking into account all the ligand-enzyme interaction data, the gas phase MM interaction energy of -282.2 kcal/mol as well as the binding free energy (DeltaG(binding)) of -227.4 kcal/mol for the PRV-N1 are significantly lower than those of the other inhibitors. The ordering of DeltaG(binding) of PRV < ZNV < OTV agrees well with the ordering of experimental IC(50) value.

Insight into analysis of interactions of saquinavir with HIV-1 protease in comparison between the wild-type and G48V and G48V/L90M mutants based on QM and QM/MM calculations.

Saquinavir (SQV) was the first HIV-1 PR inhibitor licensed for clinical use and widely used for acquired immunodeficiency syndrome (AIDS) therapy. Its effectiveness, however, has been hindered by the emergence of resistant mutations. The two most important HIV-1 PR mutants are G48V and G48V/L90M. Inhibition studies of SQV on these mutants demonstrated 13.5- and 419-fold reductions of susceptibility, respectively. In this study, an analysis of energetic binding affinity between saquinavir and the HIV-1 PR wild-type and these two mutants has been performed in detail based on density functional theory and the hybrid quantum mechanical/molecular mechanical (QM/MM) calculations. We have found that the interaction of SQV with flap residue 48 of the mutants is significantly perturbed, as shown by the reduced stability of binding between SQV and residue 48 for the G48V and G48V/L90M mutants over the wild-type. This was associated with conformational changes of the inhibitor and the enzyme, leading to the loss of hydrogen bonding between the binding subsite P2 and the backbone carbonyl of residue 48. Moreover, the G48V/L90M mutations cause the repositioning of the residues close to residues 48 and 90, at important locations as a part of the flap and catalytic regions, respectively. The repositioning of these residues consequently perturbed the binding affinity of SQV in the pocket as indicated by the decreasing interaction energies. In addition to the loss of inhibitor/enzyme binding, it is interesting to observe that the mutation leads significantly to an increase of the stability of the enzyme.

Structural analysis of lead fullerene-based inhibitor bound to human immunodeficiency virus type 1 protease in solution from molecular dynamics simulations.

Molecular dynamics (MD) simulations of the HIV-1 protease (HIVP) complexed with lead fullerene-based inhibitor (diphenyl C60 alcohol) in the three protonated states, unprotonated (Un-), monoprotonated (Mono-), and diprotonated (Di-) states at Asp25 and Asp25' were performed. As the X-ray structure of the investigated complex is not available, it was built up starting with the X-ray crystallographic structure of the HIVP complexed with non-peptide inhibitor (PDB code: 1AID) and that of the diphenyl C60 alcohol optimized using the integrated ONIOM molecular orbital calculations. The inhibitor was, then, introduced into the enzyme pocket using a molecular docking method. Change of the HIVP binding cavity for all three states were evaluated in terms of distance between the two catalytic residues, Asp25 and Asp25' as well as those between the catalytic residues and the flap regions. The torsional angles formed by the O-C-C-O of the two carboxyl groups of the catalytic dyad show the non-planar configuration with the most frequency at about -45 degrees for the Un-, 35 degrees and -95 degrees for the Mono- and 60 degrees for the Di-systems. At equilibrium, different orientations of the fullerene-based inhibitor in the three protonation states were observed. For the Di-state, the OH group of the inhibitor stably forms hydrogen bonds with the two aspartic residues. It turns to the flap region to form hydrogen bonding to the backbone N of Ile50' for the Un-state. In contrast, the OH group turns to locate between the catalytic and the flap region for the Mono-states. Beside the molecular orientation, the rotation of the OH group of the inhibitor in the Un-state was also detected. In terms of solvation, the carboxylate oxygens of the aspartic residues in the Un- and Mono-states were solvated by one to three water molecules while the OH group in these two states was coordinated by one water molecule. This is in contrast to the Di-state in which no water molecule is available in the radius of 5-6A around the oxygen atoms of the carboxylate groups of enzyme and of the OH group of the inhibitor. The simulated results lead to the conclusion that the active site of the HIVP complexed with the diphenyl C60 alcohol is the diprotonation states on Asp25 and Asp25'.

On the lower susceptibility of oseltamivir to influenza neuraminidase subtype N1 than those in N2 and N9.

Aiming to understand, at the molecular level, why oseltamivir (OTV) cannot be used for inhibition of human influenza neuraminidase subtype N1 as effectively as for subtypes N2 and N9, molecular dynamics simulations were carried out for the three complexes, OTV-N1, OTV-N2, and OTV-N9. The three-dimensional OTV-N2 and OTV-N9 initial structures were represented by the x-ray structures, whereas that of OTV-N1, whose x-ray structure is not yet solved, was built up using the aligned sequence of H5N1 isolated from humans in Thailand with the x-ray structure of the N2-substrate as the template. In comparison to the OTV-N2 and OTV-N9 complexes, dramatic changes were observed in the OTV conformation in the OTV-N1 complex in which two of its bulky side chains, N-acethyl (-NHAc) and 1-ethylproxy group (-OCHEt2), were rotated to adjust the size to fit into the N1 catalytic site. This change leads directly to the rearrangements of the OTV's environment, which are i), distances to its neighbors, W-178 and E-227, are shorter whereas those to residues R-224, E-276, and E-292 are longer; ii), hydrogen bonds to the two nearest neighbors, R-224 and E-276, are still conserved in distance and number as well as percentage occupation; iii), the calculated ligand/enzyme binding free energies of -7.20, -13.44, and -13.29 kcal/mol agree with their inhibitory activities in terms of the experimental IC50 of 36.1-53.2 nM, 1.9-2.7 nM, and 9.5-17.7 nM for the OTV-N1, OTV-N2, and OTV-N9 complexes, respectively; and iv), hydrogen-bond breaking and creation between the OTV and neighborhood residues are accordingly in agreement with the ligand solvation/desolvation taking place in the catalytic site.

Molecular dynamic and free energy studies of primary resistance mutations in HIV-1 protease-ritonavir complexes.

To understand the basis of drug resistance of the HIV-1 protease, molecular dynamic (MD) and free energy calculations of the wild-type and three primary resistance mutants, V82F, I84V, and V82F/I84V, of HIV-1 protease complexed with ritonavir were carried out. Analysis of the MD trajectories revealed overall structures of the protein and the hydrogen bonding of the catalytic residues to ritonavir were similar in all four complexes. Substantial differences were also found near the catalytic binding domain, of which the double mutant complex has the greatest impact on conformational changes of the protein and the inhibitor. The tip of the HIV-1 protease flap of the double mutant has the greater degree of opening with respect to that of the others. Additionally, the phenyl ring of Phe82 moves away from the binding pocket S1', and the conformational change of ritonavir subsite P1' consequently affects the cavity size of the protein and the conformational energy of the inhibitor. Calculations of binding free energy using the solvent continuum model were able to reproduce the same trend of the experimental inhibition constant. The results show that the resistance mutants require hydrophobic residues to maintain the interactions in the binding pocket. Changes of the cavity volume correlate well with free energy penalties due to the mutation and are responsible for the loss of drug susceptibility.

Structural and dynamical properties of different protonated states of mutant HIV-1 protease complexed with the saquinavir inhibitor studied by molecular dynamics simulations.

To understand the basis of drug resistance, particularly of the HIV-1 PR, three molecular dynamics (MD) simulations of HIV-1 PR mutant species, G48V, complexed with saquinavir (SQV) in explicit aqueous solution with three protonation states, diprotonation on Asp25 and Asp25' (Di-pro) and monoprotonation on each Asp residue (Mono-25 and Mono-25'). For all three states, H-bonds between saquinavir and HIV-1 PR were formed only in the two regions, flap and active site. It was found that conformation of P2 subsite of SQV in the Mono-25 state differs substantially from the other two states. The rotation about 177 degrees from the optimal structure of the wild type was observed, the hydrogen bond between P2 and the flap residue (Val48) was broken and indirect hydrogen bonds with the three residues (Asp29, Gly27, and Asp30) were found instead. In terms of complexation energies, interaction energy of -37.3 kcal/mol for the Mono-25 state is significantly lower than those of -30.7 and -10.7kcal/mol for the Mono-25' and Di-pro states, respectively. It was found also that protonation at the Asp25 leads to a better arrangement in the catalytic dyad, i.e., the Asp25-Asp25' interaction energy of -8.8 kcal/mol of the Mono-25 is significantly lower than that of -2.6kcal/mol for the Mono-25' state. The above data suggest us to conclude that interaction in the catalytic area should be used as criteria to enhance capability in drug designing and drug screening instead of using the total inhibitor/enzyme interaction.

Structure, dynamics and solvation of HIV-1 protease/saquinavir complex in aqueous solution and their contributions to drug resistance: molecular dynamic simulations.

As it is known that the understanding of the basic properties of the enzyme/inhibitor complex leads directly to enhancing the capability in drug designing and drug discovery. Molecular dynamics simulations have been performed to examine detailed information on the structure and dynamical properties of the HIV-1 PR complexed with saquinavir in the three protonated states, monoprotonates at Asp25 (Mono-25) and Asp25'(Mono-25') and diprotonate (Di-Pro) at both Asp25 and Asp25'. The obtained results support clinical data which reveal that Ile84 and Gly48 are two of the most frequent residues where mutation toward a protease inhibitor takes place. In contrast to the Ile84 mutation due to high displacement of Ile84 in the presence of saquinavir, source of the Gly48 mutation was observed to be due to the limited space in the HIV-1 PR pocket. The Gly48 was, on one side, found to form strong hydrogen bonds with saquinavir, while on the other side this residue was repelled by the hydrophobic Phe53 residue. In terms of inhibitor/enzyme binding, interactions between saquinavir and a catalytic triad of the HIV-1 PR were calculated using the ab initio method. The results show an order of the binding energy of Mono25

Insights into saquinavir resistance in the G48V HIV-1 protease: quantum calculations and molecular dynamic simulations.

The spread of acquired immune deficiency syndrome has increasingly become a great concern owing largely to the failure of chemotherapies. The G48V is considered the key signature residue mutation of HIV-1 protease developing with saquinavir therapy. Molecular dynamics simulations of the wild-type and the G48V HIV-1 protease complexed with saquinavir were carried out to explore structure and interactions of the drug resistance. The molecular dynamics results combined with the quantum-based and molecular mechanics Poisson-Boltzmann surface area calculations indicated a monoprotonation took place on D25, one of the triad active site residues. The inhibitor binding of the triad residues and its interaction energy in the mutant were similar to those in the wild-type. The overall structure of both complexes is almost identical. However, the steric conflict of the substituted valine results in the conformational change of the P2 subsite and the disruption of hydrogen bonding between the -NH of the P2 subsite and the backbone -CO of the mutated residue. The magnitude of interaction energy changes was comparable to the experimental K(i) data. The designing for a new drug should consider a reduction of steric repulsion on P2 to enhance the activity toward this mutant strain.