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AbstractC. difficileor Clostridioides difficileinfection (CDI) is currently one of the major causes of epidemics worldwide. Toxin B from Clostridioides difficiletoxin B (TcdB) infection is the main target protein inhibiting CDI recurrence. Clinical research suggested that bezlotoxumab’s (Bez) efficiency is significantly reduced in neutralizing the B2 strain compared to the B1 strain. The monoclonal antibody (mAb) functions by binding to the epitope 1 and 2 regions in the combined repetitive oligopeptide (CROP) domain. Some binding residues are distinctively different between B1 and B2 strains. In this work, we aimed to elucidate and compare insights into the interaction of toxins B1 and B2 in complex with Bez by using all-atom molecular dynamics (MD) simulations and binding free energy calculations. The predicted ΔGbindingvalues suggested that the antibody (Ab) could bind to toxin B1 significantly better than B2, supported by higher salt bridge and hydrogen bonding (H-bonding) interactions, as well as the number of contact residues between the two focused proteins. The toxin B1 residues important for binding with Bez were E1878, T1901, E1902, F1905, N1941, V1946, N2031, T2032, E2033, V2076, V2077, and E2092. The lower susceptibility of Bez towards toxin B2 was primarily due to a change of residue E2033 from glutamate to alanine (A2033) and the loss of E1878 and E1902 contributions, as determined by the intermolecular interaction changes from the dynamic residue interaction network (dRIN) analysis. The obtained data strengthen our understanding of Bez/toxin B binding.

AbstractTyrosinase, a key enzyme catalyzing a rate-limiting step of the melanin production, has been the most promising target for suppressing hyperpigmentation. Although a number of tyrosinase inhibitors have been developed, most of those lack clinical efficacy as they were identified from using mushroom tyrosinase (mTyr) as the target. Previous study revealed that the inhibitory effect of isobutylamido thiazolyl resorcinol (ThiamidolTM) on human tyrosinase (hTyr) is ∼100 times higher than that on mTyr. In the present study, we aimed to investigate the structural dynamics and susceptibility of ThiamidolTMagainst hTyr and mTyr at the atomic level using molecular docking, molecular dynamics simulation, and free energy calculation based on the molecular mechanics/Poisson-Boltzmann surface area method. The obtained results revealed that the resorcinol moiety of ThiamidolTMwas found to be embedded in the catalytic copper center, interacting with H180, H202, H211, F386, and H390 residues of hTyr as well as with F264 residue of mTyr, mostly through van der Waals interactions. However, the number of destabilizing residues was found to be more pronounced in the ThiamidolTM/mTyr complex than the ThiamidolTM/hTyr system, supported by the lower binding affinity of ThiamidolTM/mTyr complex as well as the higher water accessibility and the lower number of atomic contacts at the active site of mTyr. Altogether, the structural and energetic information from this work would be useful for further optimization of more potent human tyrosinase inhibitors based on ThiamidolTMscaffold.Communicated by Ramaswamy H. Sarma

3CLprois a viable target for developing antiviral therapies against the coronavirus. With the urgent need to find new possible inhibitors, a structure-based virtual screening approach was developed. This study recognized 75 pharmacologically bioactive compounds from our in-house library of 1052 natural product-based compounds that satisfied drug-likeness criteria and exhibited good bioavailability and membrane permeability. Among these compounds, three promising sulfonamide chalcones were identified by combined theoretical and experimental approaches, with SWC423 being the most suitable representative compound due to its competitive inhibition and low cytotoxicity in Vero E6 cells (EC50= 0.89 ± 0.32 μM; CC50= 25.54 ± 1.38 μM; SI = 28.70). The binding and stability of SWC423 in the 3CLproactive site were investigated through all-atom molecular dynamics simulation and fragment molecular orbital calculation, indicating its potential as a 3CLproinhibitor for further SARS-CoV-2 therapeutic research. These findings suggested that inhibiting 3CLprowith a sulfonamide chalcone such as SWC423 may pave the effective way for developing COVID-19 treatments.

An efficient structural refinement technique for protein-RNA complexes is proposed based on a combination of AI-based modeling and flexible docking. Specifically, an enhanced sampling method called parallel cascade selection molecular dynamics (PaCS-MD) was extended to include flexible docking to construct protein-RNA complexes from those obtained by AI-based modeling (AlphaFold2). With the present technique, the conformational sampling of flexible RNA regions is accelerated by PaCS-MD, enabling one to construct plausible models for protein-RNA complexes. For demonstration, PaCS-MD constructed several protein-RNA complexes of the RNA-binding Musashi-1 (MSI1) family of proteins, which were validated by comparing a group of crucial residues for RNA-binding with experimental complexes. Our analyses suggest that PaCS-MD improves the quality of complex modeling compared to the standard protocol based on template-based modeling (Phyre2). Furthermore, PaCS-MD could also be a beneficial technique for constructing complexes of non-native RNA-binding to proteins.A new method for refining protein-RNA complex models has been proposed that combines AI-based modeling with flexible docking. The technique, called PaCS-MD, speeds up conformational sampling of flexible RNA regions, resulting in plausible complex models. The method was validated using Musashi-1 (MSI1) family proteins and showed improved modeling compared to standard methods. PaCS-MD could also be useful for constructing complexes with non-native RNA-binding proteins.

Three new phenanthrene derivatives (1, 2, 4), one new fluorenone (3), and four known compounds (5–8) were isolated from the ethyl acetate extract of Dendrobium crumenatumSw. stems using column chromatography. The chemical structures were elucidated by analysis of spectroscopic data. The absolute configuration of 4was determined by electronic circular dichroism calculation. We also evaluated the immunomodulatory effects of compounds isolated from D. crumenatumin human peripheral blood mononuclear cells from healthy individuals and those from patients with multiple sclerosis in vitro. Dendrocrumenol B (2) and dendrocrumenol D (4) showed strong immunomodulatory effects on both CD3+T cells and CD14+monocytes. Compounds 2and 4could reduce IL-2 and TNF production in T cells and monocytes that were treated with phorbol-12-myristate-13-acetate and ionomycin (PMA/Iono). Deep immune profiling using high-dimensional single-cell mass cytometry could confirm immunomodulatory effects of 4, quantified by the reduction of activated T cell population under PMA/Iono stimulation, in comparison to the stimulated T cells without treatment.

Interest has been shown in the use of melanin nanoparticles (MNPs) for drug delivery applications. This study investigates the potential use of MNPs as an oral drug delivery system. The toxicity and cellular uptake of MNPs via intestinal barrier were evaluated using the Caco-2 cell model. Doxorubicin (DOX) was selected as a representative low bioavailability drug. The DOX-release profile of drug-loaded MNPs and molecular dynamic (MD) simulation were investigated at different pH. Our results demonstrated that MNPs synthesized from dopamine HCl are suitable as nanocarriers, presenting as monodisperse particles at mean size <300 nm. MNPs at size 89, 156, 268 nm at 3.13–50 μg/mL showed a high biocompatibiltity with Caco-2 cells, since no significant changes in %cell viability, reactive oxygen species, mitochondria membrane potential, and inflammatory cytokine were observed. The TEM results exhibited cellular internalization of MNPs into Caco-2 cells. The loading capacity (LC) and entrapment efficiency (EE) of DOX-loaded MNPs were 13.3 ± 2.7 % and 30.0 ± 3.6 %, respectively. DOX-loaded MNPs possessed a pH-dependent drug release profile. At pH 1.2, the amount of liberated DOX was 1.6- and 3.8-fold increase in comparison to pH 5 and 7.4, respectively. MD simulation clearly demonstrated that the binding affinity between polydopamines of MNPs and DOX molecules was weaker at the lower pH. These results thus support the potential use of MNPs as an oral drug delivery system particularly for drugs used in cancer treatment.

AbstractHepatitis C virus (HCV) NS3/4A serine protease is a promising drug target for the discovery of anti-HCV drugs. However, its amino acid mutations, particularly A156T, commonly lead to rapid emergence of drug resistance. Paritaprevir and glecaprevir, the newly FDA-approved HCV drugs, exhibit distinct resistance profiles against the A156T mutation of HCV NS3/4A serine protease. To illustrate their different molecular resistance mechanisms, molecular dynamics simulations and binding free energy calculations were carried out on the two compounds complexed with both wild-type (WT) and A156T variants of HCV NS3/4A protease. QM/MM-GBSA-based binding free energy calculations revealed that the binding affinities of paritaprevir and glecaprevir towards A156T NS3/4A were significantly reduced by ∼4 kcal/mol with respect to their WT complexes, which were in line with the experimental resistance folds. Moreover, the relatively weak intermolecular interactions with amino acids such as H57, R155, and T156 of NS3 protein, the steric effect and the destabilized protein binding surface, which is caused by the loss of salt bridge between R123 and D168, are the main contributions for the higher fold-loss in potency of glecaprevir due to A156T mutation. An insight into the difference of molecular mechanism of drug resistance against the A156T substitution among the two classes of serine protease inhibitors could be useful for further optimization of new generation HCV NS3/4A inhibitors with enhanced inhibitory potency.Communicated by Ramaswamy H. Sarma

The coronavirus disease pandemic is a constant reminder that global citizens are in imminent danger of exposure to emerging infectious diseases. Therefore, developing a technique for inhibitor discovery is essential for effective drug design. Herein, we proposed fragment molecular orbital (FMO)-based virtual screening to predict the molecular binding energy of potential severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease inhibitors. The integration of quantum mechanical approaches and trajectory analysis from a microsecond molecular dynamics simulation was used to identify potential inhibitors. We identified brominated baicalein as a potent inhibitor of the SARS-CoV-2 main protease and confirmed its inhibitory activity in an in vitro assay. Brominated baicalein did not demonstrate significant toxicity in either in vitro or in vivo studies. The pair interaction energy from FMO-RIMP2/PCM and inhibitory constants based on the protease enzyme assay suggested that the brominated baicalein could be further developed into novel SARS-CoV-2 protease inhibitors.

AbstractDrug-resistant Pseudomonas aeruginosaefflux pump extrudes antibiotics from cells for survival. Efflux pump inhibitor (EPI) thus becomes an interesting alternative to handle the drug-resistant bacteria. Conessine, a natural steroidal alkaloid from Holarrhena antidysenterica, previously exhibited efflux pump inhibitory potential. Our molecular docking and molecular dynamics (MD) studies provided atomistic information as well as the interaction of conessine with bacterial MexB efflux pump in phospholipid bilayer membrane to further the previous experimental report. Herein, the binding site and proposed mode of action of conessine were identified compared to known/commercial EPIs such as PAβN or designed-synthetic P9D. Our results explained conessine binding mode of action as an effective agent against the MexB efflux pump. The MD simulation also suggested that conessine was able to affect glycine loop (G-loop) flexibility, and the reduced G-loop flexibility due to conessine could hinder an antibiotics extrusion. In addition, our study suggested the conessine core structure buried in a hydrophobic region in the efflux pump similar to other known EPIs. Our finding could cope as a key for the design and development of the conessine derivative as novel EPI against P. aeruginosa.Communicated by Ramaswamy H. Sarma

AbstractHuman topoisomerase II alpha (TopoIIα) is a crucial enzyme involved in maintaining genomic integrity during the process of DNA replication and mitotic division. It is a vital therapeutic target for designing novel anticancer agents in targeted cancer therapy. Sulfones, members of organosulfur compounds, have been reported to possess various biological activities such as antimicrobial, anti-inflammatory, anti-HIV, anticancer, and antimalarial properties. In the present study, a series of sulfones was selected to evaluate their inhibitory activity against TopoIIα using computational approaches. Molecular docking results revealed that several sulfone analogs bind efficiently to the ATPase domain of TopoIIα. Among them, sulfones 18a, 60a, *4 b, *8 b, *3c, and 8c exhibit higher binding affinity than the known TopoII inhibitor, salvicine. Molecular dynamics simulations and free energy calculations based on MM/PB(GB)SA method demonstrated that sulfone *8 b strongly interacts with amino acid residues in the ATP-binding pocket (E87, N91, D94, I125, I141, F142, S149, G161, and A167), driven mainly by an electrostatic attraction and a strong H-bond formation at G161 residue. Altogether, the obtained results predicted that sulfones could have a high potential to be a lead molecule for targeting TopoIIα.Communicated by Ramaswamy H. Sarma

Global public health has been a critical problem by the sudden increase of the COVID-19 outbreak. The papain-like protease (PLpro) of SARS-CoV-2 is a key promising target for antiviral drug development since it plays a pivotal role in viral replication and innate immunity. Here, we employed the all-atom molecular dynamics (MD) simulations and binding free energy calculations based on MM-PB(GB)SA and SIE methods to elucidate and compare the binding behaviors of five inhibitors derived from peptidomimetic inhibitors (VIR250 and VIR251) and naphthalene-based inhibitors (GRL-0617, compound 3, and compound Y96) against SARS-CoV-2 PLpro. The obtained results revealed that all inhibitors interacting within the PLpro active site are mostly driven by vdW interactions, and the hydrogen bond formation in residues G163 and G271 with peptidomimetics and the Q269 residue with naphthalene-based inhibitors was essential for stabilizing the protein–ligand complexes. Among the five studied inhibitors, VIR250 exhibited the most binding efficiency with SARS-CoV-2 PLpro, and thus, it was chosen for the rational drug design. Based on the computationally designed ligand–protein complexes, the replacement of aromatic rings including heteroatoms (e.g., thiazolopyridine) at the P2 and P4 sites could help to improve the inhibitor-binding efficiency. Furthermore, the hydrophobic interactions with residues at P1–P3 sites can be increased by enlarging the nonpolar moieties (e.g., ethene) at the N-terminal of VIR250. We expect that the structural data obtained will contribute to the development of new PLpro inhibitors with more inhibitory potency for COVID-19.

AbstractHepatitis B virus (HBV), a small enveloped DNA virus, attacks the human liver causing both acute and chronic diseases. Current therapeutic drugs use the nucleos(t)ide analogues (NAs) as a competitive inhibitor against HBV reverse transcriptase (HBV-RT), an essential enzyme pivotally involved in viral replication. Unfortunately, this treatment still causes the development of resistant variants of HBV against NAs. As HBV-RT is homologous to the human immunodeficiency virus reverse transcriptase (HIV-RT), it is reasonable to treat HBV-RT with anti-HIV drugs. In the present study, we aimed to investigate the structural dynamics and susceptibility of the known anti-HIV drugs (stavudine [d4T], didanosine [DDI], and zidovudine [ZDV]) against HBV-RT enzyme in comparison to the anti-HBV drug lamivudine (3TC) and deoxythymidine triphosphate (dTTP) substrate using several computational approaches. The calculations revealed that seven polar residues (K32, R41, D83, S85, D205, N236, and K239) and three hydrophobic residues (A86, A87, and F88) of HBV-RT as well as the adjacent DNA strands play an important role in the ligand binding. In addition, the H-bond pattern of d4T is similar to that of 3TC, especially at the residues A86 and A87. Such interactions promote the favorable conformation of ligand in the HBV-RT binding pocket, while the several different conformations of ligand are found in the unbound state. The predicted binding free energy results based on QM/MM-GBSA and MM/GB(PB)SA methods suggested that the susceptibility towards HBV-RT of d4T and ZDV is higher than that of 3TC and dTTP. Altogether, this work sheds light on the potentiality of d4T and ZDV as a promising drug for HBV-infected patients harboring 3TC resistance.Communicated by Ramaswamy H. Sarma

Conformational change of dibnezocyclooctadiene (DBCOD) units between chair (C) and twist-boat (TB) can greatly influence the coefficient of thermal expansion of polymers. Our experimental results showed direct information of the relationship between the structure of DBCOD units and the thermal contraction behavior of the related polymers.

The Janus kinase (JAK) and epidermal growth factor receptor (EGFR) have been considered as potential targets for cancer therapy due to their role in regulating proliferation and survival of cancer cells. In the present study, the aromatic alkyl-amino analogs of thiazole-based chalcone were selected to experimentally and theoretically investigate their inhibitory activity against JAK2 and EGFR proteins as well as their anti-cancer effects on human cancer cell lines expressing JAK2 (TF1 and HEL) and EGFR (A549 and A431). In vitrocytotoxicity screening results demonstrated that the HEL erythroleukemia cell line was susceptible to compounds 11and 12, whereas the A431 lung cancer cell line was vulnerable to compound 25. However, TF1 and A549 cells were not sensitive to our thiazole derivatives. From kinase inhibition assay results, compound 25was found to be a dual inhibitor against JAK2 and EGFR, whereas compounds 11and 12selectively inhibited the JAK2 protein. According to the molecular docking analysis, compounds 11, 12and 25formed hydrogen bonds with the hinge region residues Lys857, Leu932 and Glu930 and hydrophobically came into contact with Leu983 at the catalytic site of JAK2, while compound 25formed a hydrogen bond with Met769 at the hinge region, Lys721 near a glycine loop, and Asp831 at the activation loop of EGFR. Altogether, these potent thiazole derivatives, following Lipinski's rule of five, could likely be developed as a promising JAK2/EGFR targeted drug(s) for cancer therapy.

Aromatic antiepileptic drugs (AEDs)-induced cutaneous adverse drug reactions (cADRs) add up to the limited use of the AEDs in the treatment and prevention of seizures. Human leukocyte antigen-B(HLA-B) alleles have been linked to AEDs-induced cADRs. We investigated the association between cADRs (including Stevens–Johnson syndrome; SJS/toxic epidermal necrolysis; TEN, drug reaction with eosinophilia and systemic symptoms; DRESS, and Maculopapular eruption; MPE) caused by AEDs (phenytoin, carbamazepine, lamotrigine, phenobarbital and oxcarbazepine) and HLA-Balleles in Thai population. Through the case-control study, 166 patients with AEDs-induced cADRs, 426 AEDs-tolerant patients (AEDs-tolerant controls), and 470 healthy subjects (Thai population) were collected. The HLAgenotypes were detected using the polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) method. We also performed a meta-analysis with these data and other populations. The carrier rate of HLA-B*15:02was significantly different between AEDs-induced cADRs group and AEDs-tolerant group (Odds ratio; OR 4.28, 95% Confidence interval; CI 2.64–6.95, p< 0.001), AEDs-induced cADRs group and Thai population (OR 2.15, 95%CI 1.41–3.29, p< 0.001). In meta-analysis showed the strong association HLA-B*15:02with AEDs-induced cADRs (OR 4.77, 95%CI 1.79–12.73, p< 0.001). Furthermore, HLA-B*15:02was associated with SJS/TEN induced by AEDs (OR 10.28, 95%CI 6.50–16.28, p< 0.001) Phenytoin (OR 4.12, 95%CI 1.77–9.59, p= 0.001) and carbamazepine (OR 137.69, 95%CI 50.97–371.98, p< 0.001). This study demonstrated that genetic association for AEDs-induced cADRs was phenotype-specific. A strong association between HLA-B*15:02and AEDs-induced SJS/TEN was demonstrated with an OR of 10.79 (95%CI 5.50–21.16, p< 0.001) when compared with AEDs-tolerant group. On the other hand, the carrier rates of HLA-B*08:01, HLA-B*13:01, and HLA-B*56:02were significantly higher in the DRESS group compared with the AEDs-tolerant group (p= 0.029, 0.007, and 0.017, respectively). The HLA-B*15:02allele may represent a risk factor for AEDs-induced cADRs.

Background: Neuraminidase (NA), a major glycoprotein found on the surface of the influenza virus, is an important target for the prophylaxis and treatment of influenza virus infections. Recently, several plant-derived polyphenols, especially caffeic acid analogs, have been reported to exert the inhibitory activity against NA. Objective: Herein, we aimed to investigate the anti-influenza NA activity of caffeic acid and its hydroxycinnamate analogues, rosmarinic acid and salvianolic acid A, in comparison to a known NA inhibitor, oseltamivir. Methods: In vitro MUNANA-based NA inhibitory assay was used to evaluate the inhibitory activity of the three interested hydroxycinnamic compounds towards the influenza NA enzyme. Subsequently, allatom molecular dynamics (MD) simulations and binding free energy calculations were employed to elucidate the structural insights into the protein-ligand complexations. Results: Rosmarinic acid showed the highest inhibitory activity against NA with the IC50 of 0.40 μM compared to caffeic acid (IC50 of 0.81 μM) and salvianolic acid A (IC50 of >1 μM). From 100-ns MD simulations, the binding affinity, hot-spot residues, and H-bond formations of rosmarinic acid/NA complex were higher than those of caffeic acid/NA model, in which their molecular complexations was driven mainly by electrostatic attractions and H-bond formations from several charged residues (R118, E119, D151, R152, E227, E277, and R371). Notably, the two hydroxyl groups on both phenyl and phenylacetic rings of rosmarinic acid play a crucial role in stabilizing NA through a strongly formed Hbond( s). Conclusion: Our findings shed light on the potentiality of rosmarinic acid as a lead compound for further development of a potential influenza NA inhibitor.

Human sweet taste receptor (hSTR) recognizes a wide array of sweeteners, resulting in sweet taste perception. Maltitol and lactitol have been extensively used in place of sucrose due to their capability to prevent dental caries. Herein, several molecular modeling approaches were applied to investigate the structural and energetic properties of these two polyols/hSTR complexes. Triplicate 500 ns molecular dynamics (MD) simulations and molecular mechanics/generalized Born surface area (MM/GBSA)-based free energy calculations revealed that the TAS1R2 monomer is the preferential binding site for maltitol and lactitol rather than the TAS1R3 region. Several polar residues (D142, S144, Y215, D278, E302, R383, and especially N143) were involved in polyols binding through electrostatic attractions and H-bond formations. The molecular complexation process not only induced the stable form of ligands but also stimulated the conformational adaptation of the TAS1R2 monomer to become a close-packed structure through an induced-fit mechanism. Notably, the binding affinity of the maltitol/TAS1R2 complex (ΔGbindof −17.93 ± 1.49 kcal/mol) was significantly higher than that of the lactitol/TAS1R2 system (−8.53 ± 1.78 kcal/mol), in line with the experimental relative sweetness. These findings provide an in-depth understanding of the differences in the sweetness response between maltitol and lactitol, which could be helpful to design novel polyol derivatives with higher sweet taste perception.

Since the emergence of a novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported from Wuhan, China, neither a specific vaccine nor an antiviral drug against SARS-CoV-2 has become available. However, a combination of two HIV-1 protease inhibitors, lopinavir and ritonavir, has been found to be effective against SARS-CoV, and both drugs could bind well to the SARS-CoV 3C-like protease (SARS-CoV 3CLpro). In this work, molecular complexation between each inhibitor and SARS-CoV-2 3CLprowas studied using all-atom molecular dynamics simulations, free energy calculations, and pair interaction energy analyses based on MM/PB(GB)SA and FMO-MP2/PCM/6-31G* methods. Both anti-HIV drugs interacted well with the residues at the active site of SARS-CoV-2 3CLpro. Ritonavir showed a somewhat higher number atomic contacts, a somewhat higher binding efficiency, and a somewhat higher number of key binding residues compared to lopinavir, which correspond with the slightly lower water accessibility at the 3CLproactive site. In addition, only ritonavir could interact with the oxyanion hole residues N142 and G143 via the formation of two hydrogen bonds. The interactions in terms of electrostatics, dispersion, and charge transfer played an important role in the drug binding. The obtained results demonstrated how repurposed anti-HIV drugs could be used to combat COVID-19.

Darunavir, a frontline treatment for HIV infection, faces limitations due to emerging multidrug resistant (MDR) HIV strains, necessitating the development of analogs with improved activity. In this study, a combinatorial in silico approach was used to initially design a series of HIV-1 PI analogs with modifications at key sites, P1′ and P2′, to enhance interactions with HIV-1 PR. Fifteen analogs with promising binding scores were selected for synthesis and evaluated for the HIV-1 PR inhibition activity. The variation of P2′ substitution was found to be effective, as seen in 5aa(1.54 nM), 5ad(0.71 nM), 5ac(0.31 nM), 5ae(0.28 nM), and 5af(1.12 nM), featuring halogen, aliphatic, and alkoxy functionalities on the phenyl sulfoxide motif exhibited superior inhibition against HIV-1 PR compared to DRV, with minimal cytotoxicity observed in Vero and 293T cell lines. Moreover, computational studies demonstrated the potential of selected analogs to inhibit various HIV-1 PR mutations, including I54M and I84V. Further structural dynamics and energetic analyses confirmed the stability and binding affinity of promising analogs, particularly 5ae, which showed strong interactions with key residues in HIV-1 PR. Overall, this study underscores the importance of flexible moieties and interaction enhancement at the S2′ subsite of HIV-1 PR in developing effective DRV analogs to combat HIV and other global health issues.

The three-dimensional distribution function (DF) and the potential of mean force (PMF) of water and hydronium ions in five protonated states of the influenza A M2 channel are calculated by means of the three-dimensional reference interaction site model (3D-RISM) theory in order to clarify the proton conduction mechanism of the channel. Each protonated state, denoted as iH, where i= 0−4, has a different number of protonated histidines, from 0 to 4. The DF of water in each state exhibits closed structures of 0H, 1H, and 2H and open structures in 3H and 4H. In the closed form, the DF and PMF indicate that hydronium ions are excluded from the channel. In contrast, the ion can distribute throughout the opened channel. The barrier in PMF of 3H, ∼3−5 kJ/mol, is lower than that of 4H, 5−7 kJ/mol, indicating that 3H has higher permeability to protons. On the basis of the radial DFs of water and hydronium ions around the imidazole rings of His37, we propose a new mechanism of proton transfer through the gating region of the channel. In this process, a hydronium ion hands a proton to a non-protonated histidine through a hydrogen bond between them, and then the other protonated histidine releases a proton to a water molecule via a hydrogen bond. The process transfers a proton effectively from one water molecule to another.

AbstractTwo important glycoproteins on the influenza virus membrane, hemagglutinin (HA) and neuraminidase (NA), are relevant to virus replication. As previously reported, HA has a substrate specificity towards SIA-2,3-GAL-1,4-NAG (3SL) and SIA-2,6-GAL-1,4-NAG (6SL) glycans, while NA can cleave both types of linkages. However, the substrate binding into NA and its preference are not well understood. In this work, the glycan binding and specificity of human and avian NAs were evaluated by classical molecular dynamics (MD) simulations, whilst the conformational diversity of 3SL avian and 6SL human glycans in an unbound state was investigated by replica exchange MD simulations. The results indicated that the 3SL avian receptor fits well in the binding cavity of all NAs and does not require a conformational change for such binding compared to the flexible shape of the 6SL human receptor. From the QM/MM-GBSA binding free energy and decomposition free energy data, 6SL showed a much stronger binding towards human NAs (H1N1, H2N2 and H3N2) than to avian NAs (H5N1 and H7N9). This suggests that influenza NAs have a substrate specificity corresponding to their HA, indicating the functional balance between the two important glycoproteins. Both linkages show distinct glycan topologies when complexed with NAs, while the flexibility of torsion angles between GAL and NAG in 6SL results in the various shapes of glycan and different binding patterns. Lower conformational diversities of both glycans when bound to NA compared to the unbound state were found, and were required in order to be accommodated within the NA cavity.Communicated by Ramaswamy H. Sarma

Secretory diarrhea is one of the most common causes of death world-wide especially in children under 5 years old. Isoliquiritigenin (ISLQ), a plant-derived chalcone, has previously been shown to exert anti-secretory action in vitroand in vivoby inhibiting CFTR Cl−channels. However, its CFTR inhibition potency is considerably low (IC50 > 10 μM) with unknown mechanism of action. This study aimed to identify novel chalcone derivatives with improved potency and explore their mechanism of action. Screening of 27 chalcone derivatives identified CHAL-025 as the most potent chalcone analog that reversibly inhibited CFTR-mediated Cl−secretion in T84 cells with an IC50of ∼1.5 μM. As analyzed by electrophysiological and biochemical analyses, the mechanism of CFTR inhibition by CHAL-025 is through AMP-activated protein kinase (AMPK), a negative regulator of CFTR activity. Furthermore, Western blot analyses and molecular dynamics (MD) results suggest that CHAL-025 activates AMPK by binding at the allosteric site of an upstream kinase calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ). Interestingly, CHAL-025 inhibited both cholera toxin (CT) and bile acid-induced Cl−secretion in T84 cells and prevented CT-induced intestinal fluid secretion in mice. Therefore, CHAL-025 represents a promising anti-diarrheal agent that inhibits CFTR Cl−channel activity via CaMKKβ-AMPK pathways.

Zika virus (ZIKV) is mainly transmitted to humans by Aedesspecies mosquitoes and is associated with serious pathological disorders including microcephaly in newborns and Guillain–Barré syndrome in adults. Currently, there is no vaccine or anti-ZIKV drug available for preventing or controlling ZIKV infection. An attractive drug target for ZIKV treatment is a two-compartment (NS2B/NS3) serine protease that processes viral polyprotein during infection. Here, conventional molecular dynamics simulations of the ZIKV protease in complex with peptide substrate (TGKRS) sequence at the C-terminus of NS2B show that the substrate is in the active conformation for the cleavage reaction by ZIKV protease. Hybrid quantum mechanics/molecular mechanics (QM/MM) umbrella sampling simulations (PM6/ff14SB) of acylation results reveal that proton transfer from S135 to H51 and nucleophilic attack on the substrate by S135 are concerted. The rate-limiting step involves the formation of a tetrahedral intermediate. In addition, the single-point energy QM/MM calculations, precisely at the level of coupled cluster theory (LCCSD(T)/(aug)-cc-pVTZ), were performed to correct the potential energy profiles for the first step of the acylation process. The average computed activation barrier at this level of theory is 16.3 kcal mol–1. Therefore, the computational approaches presented here are helpful for further designing of NS2B/NS3 inhibitors based on transition-state analogues.

Thioesterase (TE) domain of fatty acid synthase (FAS) is an attractive therapeutic target for design and development of anticancer drugs. In this present work, we search for the potential FAS inhibitors of TE domain from the ZINC database based on similarity search using three natural compounds as templates, including flavonoids, terpenoids, and phenylpropanoids. Molecular docking was used to predict the interaction energy of each screened ligand compared to the reference compound, which is methyl γ-linolenylfluorophosphonate (MGLFP). Based on this computational technique, rosmarinic acid and its eight analogs were observed as a new series of potential FAS inhibitors, which showed a stronger binding affinity than MGLFP. Afterward, nine docked complexes were studied by molecular dynamics simulations for investigating protein–ligand interactions and binding free energies using MM-PB(GB)SA, MM-3DRISM-KH, and QM/MM-GBSA methods. The binding free energy calculation indicated that the ZINC85948835 (R34) displayed the strongest binding efficiency against the TE domain of FAS. There are eight residues (S2308, I2250, E2251, Y2347, Y2351, F2370, L2427, and E2431) mainly contributed for the R34 binding. Moreover, R34 could directly form hydrogen bonds with S2308, which is one of the catalytic triad of TE domain. Therefore, our finding suggested that R34 could be a potential candidate as a novel FAS-TE inhibitor for further drug design.

Hepatic C virus (HCV) is a global health problem, resulting in liver cirrhosis and inflammation that can develop to hepatocellular carcinoma and fatality. The NS5B polymerase of HCV plays an important role in viral RNA replication process, making it an attractive therapeutic target for design and development of anti-HCV drugs. To search new potent compounds against the HCV NS5B polymerase, the molecular docking and the steered molecular dynamics (SMD) simulation techniques were performed. The potential potent inhibitors of the NS5B polymerase were screened out from the ZINC database using structural similarity search and molecular docking technique. Five top-hit compounds (the ZINC compounds 49888724, 49054741, 49777239, 49793673, and 49780355) were then studied by the SMD simulations based on the hypothesis that a high rupture force relates to a high binding efficiency. The results demonstrated that the ZINC compound 49888724had a greater maximum rupture force, reflecting a good binding strength and inhibitory potency than known inhibitors and the rest four ZINC compounds. Therefore, our finding indicated that the ZINC compound 49888724is a potential candidate to be a novel NS5B inhibitor for further design. Besides, the van der Waals interaction could be considered as the main contribution for stabilizing the NS5B-ligand complex.

Epidermal growth factor (EGF) was used as the targeting ligand to enhance the specificity of a cancer drug delivery system (DDS) via its specific interaction with the EGF receptor (EGFR) that is overexpressed on the surface of some cancer cells. To investigate the intermolecular interaction and binding affinity between the EGF-conjugated DDS and the EGFR, 50 ns molecular dynamics simulations were performed on the complex of tethered EGFR and EGF linked to single-wall carbon nanotube (SWCNT) through a biopolymer chitosan wrapping the tube outer surface (EGFR·EGF-CS-SWCNT-Drug complex), and compared to the EGFR·EGF complex and free EGFR. The binding pattern of the EGF-CS-SWCNT-Drug complex to the EGFR was broadly comparable to that for EGF, but the binding affinity of the EGF-CS-SWCNT-Drug complex was predicted to be somewhat better than that for EGF alone. Additionally, the chitosan chain could prevent undesired interactions of SWCNT at the binding pocket region. Therefore, EGF connected to SWCNT via a chitosan linker is a seemingly good formulation for developing a smart DDS served as part of an alternative cancer therapy.

Thymidylate synthase (TS) is a promising cancer target, due to its crucial function in thymine synthesis. It performs the reductive methylation of 2′-deoxyuridine-5′-phosphate (dUMP) to thymidine-5′-phosphate (dTMP), using N-5,10-methylene-5,6,7,8-tetrahydrofolate (mTHF) as a cofactor. After the formation of the dUMP/mTHF/TS noncovalent complex, and subsequent conformational activation, this complex has been proposed to react via nucleophilic attack (Michael addition) by Cys146, followed by methylene-bridge formation to generate the ternary covalent intermediate. Herein, QM/MM (B3LYP-D/6-31+G(d)-CHARMM27) methods are used to model the formation of the ternary covalent intermediate. A two-dimensional potential energy surface reveals that the methylene-bridged intermediate is formed via a concerted mechanism, as indicated by a single transition state on the minimum energy pathway and the absence of a stable enolate intermediate. A range of different QM methods (B3LYP, MP2 and SCS-MP2, and different basis sets) are tested for the calculation of the activation energy barrier for the formation of the methylene-bridged intermediate. We test convergence of the QM/MM results with respect to size of the QM region. Inclusion of Arg166, which interacts with the nucleophilic thiolate, in the QM region is important for reliable results; the MM model apparently does not reproduce energies for distortion of the guanidinium side chain correctly. The spin component scaled-Møller–Plessett perturbation theory (SCS-MP2) approach was shown to be in best agreement (within 1.1 kcal/mol) while the results obtained with MP2 and B3LYP also yielded acceptable values (deviating by less than 3 kcal/mol) compared with the barrier derived from experiment. Our results indicate that using a dispersion-corrected DFT method, or a QM method with an accurate treatment of electron correlation, increases the agreement between the calculated and experimental activation energy barriers, compared with the semiempirical AM1 method. These calculations provide important insight into the reaction mechanism of TS and may be useful in the design of new TS inhibitors.

CeO2nanorods impregnated with 2.5 atom % of NiO (NiO/CeO2nanorods) were successfully synthesized and examined as catalysts for the NH3-selective catalytic reduction (NH3-SCR) of nitric oxide (NO). The catalytic activity of NiO/CeO2nanorods resulted in up to ∼90% NO conversion at 250 °C, which is superior to that of pure CeO2nanorods or NiO nanoparticles. Subsequently, extensive studies of the NiO/CeO2-catalyzed reduction of NO were conducted using X-ray photoelectron spectroscopy, hydrogen temperature-programmed reduction, temperature-programmed desorption, and density functional theory periodic calculations. Compared to that of the pure CeO2nanorods, the results demonstrated that the NiO/CeO2nanorods resulted in (i) a higher concentration of Ce3+chemical species, (ii) a larger amount of active Oα, (iii) lower temperature reducibility, (iv) a lower amount of energy required for oxygen vacancy distortion, and (v) a significant adsorption of and strong interaction between NO and NH3molecules. Our findings therefore elucidated considerable details of the structural properties of the NiO/CeO2nanorods that were decisive for achieving a highly efficient conversion of NO by the NH3-SCR process at low temperatures.

The outbreak of avian influenza A (H5N1) virus has raised a global concern for both the animal as well as human health. Besides vaccination, that may not achieve full protection in certain groups of patients, inhibiting neuraminidase or the transmembrane protein M2 represents the main measure of controlling the disease. Due to alarming emergence of influenza virus strains resistant to the currently available drugs, development of new neuraminidase N1 inhibitors is of utmost importance. The present paper provides an overview of the recent advances in the design of new antiviral drugs against avian influenza. It also reports findings in binding free energy calculations for nine neuraminidase N1 inhibitors (oseltamivir, zanamivir, and peramivir -carboxylate, -phosphonate, and -sulfonate) using the Linear Interaction Energy method. Molecular dynamics simulations of these inhibitors were performed in a free and two bound states – the so called open and closed conformations of neuraminidase N1. Obtained results successfully reproduce the experimental binding affinities of the already known neuraminidase N1 inhibitors, i.e. peramivir being a stronger binder than zanamivir that is in turn stronger binder than oseltamivir, or phosphonate inhibitors being stronger binders than their carboxylate analogues. In addition, the newly proposed sulfonate inhibitors are predicted to be the strongest binders – a fact to be confirmed by their chemical synthesis and a subsequent test of their biological activity. Finally, contributions of individual inhibitor moieties to the overall binding affinity are explicitly evaluated to assist further drug development towards inhibition of the H5N1 avian influenza A virus.

Hepatitis C virus (HCV) infections are a serious viral health problem globally, causing liver cirrhosis and inflammation that can develop to hepatocellular carcinoma and death. Since the HCV NS3/4A protease complex cleaves the scissile peptide bond in the viral encoded polypeptide to release the non-structural proteins during the viral replication process, this protease is then an important target for drug design. The computer-aided drug design and screening targeted at NS3/4A protease of HCV were reviewed. In addition, using steered molecular dynamics simulations, potent inhibitors of the NS3/4A complex were searched for by screening the ZINC database based upon the hypothesis that a high rupture force indicates a high binding efficiency. Nine top-hit compounds (59500093, 59784724, 13527817, 26660256, 29482733, 25977181, 28005928, 13527826 and 13527826) were found that had the same or a greater maximum rupture force (and so assumed binding strength and inhibitory potency) than the four current drugs and so are potential candidates as anti- HCV chemotherapeutic agents. In addition, van der Waals interactions were found to be the main contribution in stabilizing the ligand- NS3/4A complex.

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.

The morphology effect of ZrO2–CeO2on the performance of MnOx/ZrO2–CeO2catalyst for the selective catalytic reduction of NO with ammonia was investigated. The catalytic tests showed that the MnOx/ZrO2–CeO2nanorods achieved significantly higher NO conversions than the nanocubes and nanopolyhedra. The catalytic tests also showed that the MnOx/ZrO2–CeO2nanorods achieved a significantly higher rate constant with respect to NO conversion than that of the nanocubes and nanopolyhedra. On the nanorods, the apparent activation energy is 25 kJ mol–1, which was much lower than the values of nanocubes and nanopolyhedra (42 and 43 kJ mol–1). The high resolution transmission electron microscopy showed that the nanorods predominately exposed {110} and {100} planes. It was demonstrated that the ZrO2–CeO2nanorods had a strong interaction with MnOxspecies, which resulted in great superiority for the selective catalytic reduction of NO. The excellent catalytic activity of the MnOx/ZrO2–CeO2nanorods should be attributed to the Mn4+species, adsorbed surface oxygen and oxygen vacancies which are associated with their exposed {110} and {100} planes.

Manganese oxides (MnOx) supported on Ce0.9Zr0.1O2(MnOx/Ce0.9Zr0.1O2) nanorods were synthesized and tested for low-temperature selective catalytic reduction of NO with ammonia. The catalysts were characterized by transmission electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and hydrogen temperature-programmed reduction. The structure and morphology results show that the MnOxwas highly dispersed on the surface of Ce0.9Zr0.1O2nanorods. Various species, such as Mn2+, Mn3+, and Mn4+, were exposed due to a strong interaction between manganese and cerium oxides. Thus, the MnOx/Ce0.9Zr0.1O2nanorods exhibited a better catalytic performance (90% NO conversion at 150 °C) compared with that of the as-prepared Ce0.9Zr0.1O2nanorods. Density functional theory (DFT) calculations clearly demonstrated that the MnOxon the surface of supporting nanorods or Mn@CeO2(110) could easily form an oxygen vacancy distortion. Furthermore, the Mn@CeO2(110) model in the DFT analysis showed a prominent effect on the NO and NH3adsorption which resulted in a stronger nitrite intermediate (NOO*) formation and more attractive interaction with the NH3gas compared with those observed with the CeO2(110) model. Therefore, a thorough understanding of the structure and catalytic performance of MnOx/Ce0.9Zr0.1O2nanorods was successfully achieved by a combination of experimental and theoretical studies.

While the seasonal influenza viruses spreading around the world cause the annual epidemics, the recent outbreaks of influenza A virus subtype H5N1 and pandemic H1N1 have raised global human health concerns. In this review, the applicabilities of computational techniques focused on three important targets in the viral life cycle: hemagglutinin, neuraminidase and M2 proton channel are summarized. Protein mechanism of action, substrate binding specificity and drug resistance, ligand-target interactions of substrate/ inhibitor binding to these three proteins either wild-type or mutant strains are discussed and compared. Advances on the novel antiinfluenza agents designed specifically to combat the avian H5N1 and pandemic H1N1 viruses are introduced. A better understanding of molecular inhibition and source of drug resistance as well as a set of newly designed compounds is greatly useful as a rotational guide for synthetic and medicinal chemists to develop a new generation of anti-influenza drugs.

Using computer-assisted combinatorial chemistry techniques, we have designed a virtual library of antiinfluenza agents, analogs of inhibitor A-315675, containing a novel pyrrolidine core, which effectively inhibits both wild type and common oseltamivir-resistant mutant forms of the neuraminidase (NA) subtype N1 of avian influenza virus H5N1. A target-specific Potential of Mean Force (PMF) scoring function parameterized on a training set of 13 known pyrrolidine-based inhibitors of NA and validated on 3 others was used to predict the N1 inhibition constants for the focused library of A-315675 analogs. Nine virtual hits (best pyrrolidine inhibitors designed in the present study) are predicted to exhibit inhibition constants in the low picomolar range, up to 200 fold lower than the parent inhibitor A- 315675 while displaying favorable predicted ADME-related properties. Proposed small highly-focused combinatorial subsets composed of R-groups most frequently occurring in the 200 most active analogs can be useful as a guide for synthetic and medicinal chemists who are developing a new generation of drugs against the avian influenza virus H5N1 by focusing their attention on this small portion of the chemical space.

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 Ser368to 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.

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