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Acetylcholinesterase (AChE), one of the major therapeutic strategies for the treatment of Alzheimer's disease (AD) is to increase the acetylcholine (ACh) level in the brain by inhibiting the biological activity of AChE. In this present work, a set of alkaloids and flavonoids against AChE enzyme were screened by computational chemistry techniques. The docking results showed that among alkaloid compounds the oxindole alkaloid namely mitragynine oxidole B (MITOB) and the indole alkaloids namely mitragynine (MIT) exhibited a good binding affinity towards AChE. These two compounds were then studied by molecular dynamics (MD) simulations. The binding free energy calculation and ligand-protein binding pattern suggested that both alkaloids could interact with AChE very well. Since MIT is the main alkaloid constituent of Mytragyna speciose leaves, this compound was isolated from M. speciose leaves and tested for anti-AChE activity. As a result, the isolated MIT had an inhibitory activity with pIC50 value of 3.57. This finding provided that the mitragynine compound has the potential to be as a therapeutic agent for further anti-AChE drug development in treatment of Alzheimer's disease.

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 vitro cytotoxicity screening results demonstrated that the HEL erythroleukemia cell line was susceptible to compounds 11 and 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 25 was found to be a dual inhibitor against JAK2 and EGFR, whereas compounds 11 and 12 selectively inhibited the JAK2 protein. According to the molecular docking analysis, compounds 11, 12 and 25 formed 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 25 formed 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.

Monoclonal antibodies (mAbs) are one of the fastest-growing areas of biopharmaceutical industry and have been widely used for a broad spectrum of diseases. Meanwhile, the immunogenicity of non-human derived antibodies can generate side effects by inducing the human immune response to produce human anti-mouse-immunoglobulin antibody (HAMA). In this work, we aim to reduce the immunogenicity of muMAb A.4.6.1 by substitute human sequences for murine sequences. Humanized antibodies are constructed by grafting, specificity determining residues (SDR), complementary determining regions (CDR), and chimeric region of muMAb A.4.6.1, onto variable domain of Trastuzumab (Herceptin). The interactions between grafted antibodies and their target, Vascular endothelial growth factor (VEGF), were theoretically investigated by molecular dynamics simulation in order to evaluate the antibodies–antigen binding behavior. The obtained protein–protein interactions and calculated binding free energy suggested that the SDR–VEGF complex presented a significantly greater binding affinity, number of contact and total number of H-bonds compared to CDR and chimeric mAbs, significantly. Moreover, the Camsol program predicted that the solubility of SDR mAb exhibits the greatest solubility. This result was supported by performing a western blot analysis of the grafted mAbs with soluble and insoluble fractions in order to evaluate their solubility, in which SDR was found to have a much lower amount of insoluble proteins. Consequently, the enhanced binding affinity and solubility of the designed SDR was achieved by the single S106D mutation using computational methods. With the aim of low immunogenicity, high solubility, and high affinity, this SDR humanized antibody was expected to have greater efficacy than murine or chimeric antibodies for future use in humans.

Graphical abstract, The 3C-like main protease of SARS-CoV-2 (3CLPro) is responsible for the cleavage of the viral polyprotein. This process is essential for the viral life cycle. Therefore, 3CLPro is a promising target to develop antiviral drugs for COVID-19 prevention and treatment. Traditional enzymatic assays for the identification of 3CLPro inhibitors rely on peptide-based colorimetric or fluorogenic substrates. However, the COVID-19 pandemic has limit or delay access to these substrates, especially for researchers in developing countries attempting to screen natural product libraries. We explored the use of the fluorescent probe 8-anilinonaphthalene-1-sulfonate (ANS) as an alternative assay for inhibitor identification. Fluorescence enhancement upon binding of ANS to 3CLPro was observed, and this interaction was competitive with a peptide substrate. The utility of ANS-based competitive binding assay to identify 3CLPro inhibitors was demonstrated with the flavonoid natural products baicalein and rutin. The molecular nature of ANS and rutin interaction with 3CLPro was explored with molecular modeling. Our results suggested that ANS could be employed in a competitive binding assay to facilitate the identification of novel SARS-CoV-2 antiviral compounds.

Recently, the H3N2 influenza outbreak has caused serious global public health concern for future control of the next influenza pandemic. Since using current anti-influenza drugs targeting neuraminidase (oseltamivir and zanamivir) and the proton M2 channel (amantadine and rimantadine) leads to drug resistance, it is essential to seek new anti-viral agents that act on additional viral targets. Hemagglutinin (HA), a glycoprotein embedded in the viral surface and playing a critical role in influenza the viral replication cycle has become an attractive target. This work investigates the molecular binding mechanism of HA H3N2 of influenza virus complexed with the fusion inhibitor, arbidol and its derivative (der-arbidol), by means of molecular dynamics simulation. The result showed that the arbidol derivative could form many and strong hydrogen bonds with the HA surrounding amino acids comprising GLU103

Human carbonic anhydrase (CA) isozyme II has been used as protein target for disorder treatment including glaucoma. Current clinically used sulfonamide-based CA inhibitors can induce side effects, and so alternatives are required. This study aimed to investigate a natural CA inhibitor from Murraya paniculata. The previously developed yeast-based assay was used to screen 14 compounds isolated from M. paniculata and identified by NMR analysis for anti-human CA isozyme II (hCAII) activity. Cytotoxicity of the compounds was also tested using the same yeast-based assay but in a different cultivation condition. Two flavonoid candidate compounds, 5, 6, 7, 8, 3’, 4’, 5’-heptamethoxyflavone (4) and 3 ,5, 7, 8, 3’, 4’, 5’-heptamethoxyflavone (9), showed potent inhibitory activity against hCAII with a minimal effective concentration of 10.8 and 21.5 μM, respectively, while they both exhibited no cytotoxic effect, even at the highest concentration tested (170 μM). The results from an in vitro esterase assay of the two candidates confirmed their hCAII inhibitory activity with IC50 values of 24.0 and 34.3 μM, respectively. To investigate the potential inhibition mechanism of compound 4, in silico molecular docking was performed using the FlexX and SwissDock software. This revealed that compound 4 coordinated with the Zn2+ ion in the hCAII active site through its methoxy oxygen at a distance of 1.60 A (FlexX) or 2.29 A (SwissDock). The interaction energy of compound 4 with hCAII was -13.36 kcal/mol. Thus, compound 4 is a potent novel flavonoid-based hCAII inhibitor and may be useful for further anti-CAII design and development.

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 3CLpro was 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 3CLpro active 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.

Graphical abstract, Highlights • The binding affinity towards SARS-CoV-2 nsp16 of SFG is higher than that of SAH. • Asp99 is a key binding residue for SAH and SFG via charge-charge attraction. • SFG could electrostatically interact with the 2’-OH and N3 groups of adenosine moiety of RNA substrate. • The distance between 2’-OH of RNA and −NH3 + (at 6’ position) of SFG mimics the methyl transfer reaction., The recent ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to rapidly spread across the world. To date, neither a specific antiviral drug nor a clinically effective vaccine is available. Among the 15 viral non-structural proteins (nsps), nsp16 methyltransferase has been considered as a potential target due to its crucial role in RNA cap 2’-O-methylation process, preventing the virus detection by cell innate immunity mechanisms. In the present study, molecular recognition between the two natural nucleoside analogs (S-adenosyl-L-homocysteine (SAH) and sinefungin (SFG)) and the SARS-CoV-2 nsp16/nsp10/m7GpppAC5 was studied using all-atom molecular dynamics simulations and free energy calculations based on MM/GBSA and WaterSwap approaches. The binding affinity and the number of hot-spot residues, atomic contacts, and H-bond formations of the SFG/nsp16 complex were distinctly higher than those of the SAH/nsp16 system, consistent with the lower water accessibility at the enzyme active site. Notably, only SFG could electrostatically interact with the 2’-OH and N3 groups of RNA’s adenosine moiety, mimicking the methyl transfer reaction of S-adenosyl-L-methionine substrate. The atomistic binding mechanism obtained from this work paves the way for further optimizations and designs of more specific SARS-CoV-2 nsp16 inhibitors in the fight against COVID-19.

A flavonoid is a versatile core structure with various cellular, immunological, and pharmacological effects. Recently, flavones have shown anti-dengue activities by interfering with viral translation and replication. However, the molecular target is still elusive. Here we chemically modified apigenin by adding an alkyne moiety into the B-ring hydroxyl group. The alkyne serves as a chemical tag for the alkyne-azide cycloaddition reaction for subcellular visualization. The compound located at the perinuclear region at 1 and 6 h after infection. Interestingly, the compound signal started shifting to vesicle-like structures at 6 h and accumulated at 24 and 48 h after infection. Moreover, the compound treatment in dengue-infected cells showed that the compound restricted the viral protein inside the vesicles, especially at 48 h. As a result, the dengue envelope proteins spread throughout the cells. The alkyne-tagged apigenin showed a more potent efficacy at the EC50 of 2.36 ± 0.22, and 10.55 ± 3.37 µM, respectively, while the cytotoxicities were similar to the original apigenin at the CC50 of 70.34 ± 11.79, and 82.82 ± 11.68 µM, respectively. Molecular docking confirmed the apigenin binding to the previously reported target, ribosomal protein S9, at two binding sites. The network analysis, homopharma, and molecular docking revealed that the estrogen receptor 1 and viral NS1 were potential targets at the late infection stage. The interactions could attenuate dengue productivity by interfering with viral translation and suppressing the viral proteins from trafficking to the cell surface.

Clostridium difficile infection (CDI) is the most common hospital acquired diarrheal disease with its increasing incidence and mortality rate globally. DNA Gyrase B (GyrB) is a key component of DNA replication process across all bacterial genera; thus, this offers a potential target for the treatment of CDI. In the present study, several virtual screening approaches were employed to identify a novel C. difficile GyrB inhibitor. The 139 known metabolites were screened out from the 480 flavonoids in PhytoHub database. Molinspiration and PROTOX II servers were used to calculate the ADME properties and oral toxicity of the metabolites, whereas mutagenicity, tumorigenicity, irritant, and reproductive effect were predicted using DataWarrior program. The binding mode and the binding efficiency of the screened flavonoids against the GyrB were studied using FlexX docking program. From virtual screening of 139 metabolites, we found 25 flavonoids with no mutagenicity, tumorigenicity, irritant, and reproductive effect. Docking study suggested that flavonoids 1030 ((-)-epicatechin 3'-O-sulfate), 1032 ((-)-epicatechin 4'-O-sulfate), 1049 (3'-O-methyl-(-)-epicatechin 4-O-sulfate), 1051 (3'-O-methyl-(-)-epicatechin 7-O-sulfate), 1055 (4'-O-methyl-(-)-epicatechin 7-O-sulfate) and 1317 (quercetin sulfate) have significantly higher binding affinity than the known GyrB inhibitor novobiocin. The results from molecular dynamics simulation and free energy calculations based on solvated interaction energy suggested that (-)-epicatechin 3'-O-sulfate could be a potential drug candidate in the management of CDI.

The impact of autophagy on cancer treatment and its corresponding responsiveness has galvanized the scientific community to develop novel inhibitors for cancer treatment. Importantly, the discovery of inhibitors that targets the early phase of autophagy was identified as a beneficial choice. Despite the number of research in recent years, screening of the DrugBank repository (9591 molecules) for the Vacuolar protein sorting 34 (VPS34) has not been reported earlier. Therefore, the present study was designed to identify potential VPS34 antagonists using integrated pharmacophore strategies. Primarily, an energy-based pharmacophore and receptor cavity-based analysis yielded five (DHRRR) and seven featured (AADDHRR) pharmacophore hypotheses respectively, which were utilized for the database screening process. The glide score, the binding free energy, pharmacokinetics and pharmacodynamics properties were examined to narrow down the screened compounds. This analysis yielded a hit molecule, DB03916 that exhibited a better docking score, higher binding affinity and better drug-like properties in contrast to the reference compound that suffers from a toxicity property. Importantly, the result was validated using a 50 ns molecular dynamics simulation study. Overall, we conclude that the identified hit molecule DB03916 is believed to serve as a prospective antagonist against VPS34 for cancer treatment.

&beta, Glucosidases and &beta, mannosidases hydrolyze substrates that differ only in the epimer of the nonreducing terminal sugar moiety, but most such enzymes show a strong preference for one activity or the other. Rice Os3BGlu7 and Os7BGlu26 &beta, glycosidases show a less strong preference, but Os3BGlu7 and Os7BGlu26 prefer glucosides and mannosides, respectively. Previous studies of crystal structures with glucoimidazole (GIm) and mannoimidazole (MIm) complexes and metadynamic simulations suggested that Os7BGlu26 hydrolyzes mannosides via the B2,5 transition state (TS) conformation preferred for mannosides and glucosides via their preferred 4H3/4E TS conformation. However, MIm is weakly bound by both enzymes. In the present study, we found that MIm was not bound in the active site of crystallized Os3BGlu7, but GIm was tightly bound in the &minus, 1 subsite in a 4H3/4E conformation via hydrogen bonds with the surrounding residues. One-microsecond molecular dynamics simulations showed that GIm was stably bound in the Os3BGlu7 active site and the glycone-binding site with little distortion. In contrast, MIm initialized in the B2,5 conformation rapidly relaxed to a E3/4H3 conformation and moved out into a position in the entrance of the active site, where it bound more stably despite making fewer interactions. The lack of MIm binding in the glycone site in protein crystals and simulations implies that the energy required to distort MIm to the B2,5 conformation for optimal active site residue interactions is sufficient to offset the energy of those interactions in Os3BGlu7. This balance between distortion and binding energy may also provide a rationale for glucosidase versus mannosidase specificity in plant &beta, glycosidases.

MCR (mobile colistin resistance) enzymes catalyse phosphoethanolamine (PEA) addition to bacterial lipid A, threatening the "last-resort" antibiotic colistin. Molecular dynamics and density functional theory simulations indicate that monozinc MCR supports PEA transfer to the Thr285 acceptor, positioning MCR as a mono- rather than multinuclear member of the alkaline phosphatase superfamily.

This study reports UV–Visible spectra and electronic structures of indigo (IG) in aqueous solution using a combination of experimental and theoretical methods. In the visible region, the experimental absorption spectrum of the solution showed a broad peak with the longest wavelength of maximal absorption (λmax) value at 708 nm. For the theoretical method, a trans-IG monomer and a trans-IG bound with two water molecules (IG.2W) were optimized in the ground state using the B3LYP and B3LYP-D3 calculations with the 6-31 + G(d,p) basis set and the SCRF-CPCM model for taking solvent effect into account was also applied. Sequentially, the UV–Visible spectra and λmax of the optimized trans-IG and IG.2W models in the implicit water were simulated by the time-dependent density functional theory (TD-DFT) calculations. The TD-DFT methods including BLYP, B3LYP, PBE0, CAM-B3LYP, M06-2X, ωB97XD, LC-BLYP, and LC-ωPBE functionals without and with the D3 correction and the 6-31 + G(d,p) basis set were selected. The results pointed out that BLYP and BLYP-D3 were the best methods because they could reproduce the experimental λmax value of IG in aqueous solution. The predicted λmax values of IG.2W were almost equal to 708 nm (the experimental data), indicating that IG.2W could be responsible for optical properties of IG.

Cancer stem cells (CSCs) are unique populations of cells that can self-renew and generate different cancer cell lineages. Although CSCs are believed to be a promising target for novel therapies, the specific mechanisms by which these putative therapeutics could intervene are less clear. Nitric oxide (NO) is a biological mediator frequently up-regulated in tumors and has been linked to cancer aggressiveness. Here, we search for targets of NO that could explain its activity. We find that it directly affects the stability and function of octamer-binding transcription factor 4 (Oct4), known to drive the stemness of lung cancer cells. We demonstrated that NO promotes the CSC-regulatory activity of Oct4 through a mechanism that involves complex formation between Oct4 and the scaffolding protein caveolin-1 (Cav-1). In the absence of NO, Oct4 forms a molecular complex with Cav-1, which promotes the ubiquitin-mediated proteasomal degradation of Oct4. NO promotes Akt-dependent phosphorylation of Cav-1 at tyrosine 14, disrupting the Cav-1:Oct4 complex. Site-directed mutagenesis and computational modeling studies revealed that the hydroxyl moiety at tyrosine 14 of Cav-1 is crucial for its interaction with Oct4. Both removal of the hydroxyl via mutation to phenylalanine and phosphorylation lead to an increase in binding free energy (ΔG(bind)) between Oct4 and Cav-1, destabilizing the complex. Together, these results unveiled a novel mechanism of CSC regulation through NO-mediated stabilization of Oct4, a key stem cell transcription factor, and point to new opportunities to design CSC-related therapeutics.

Several quinone-based metabolites of anticancer drugs and naturally occurring quinone-containing compounds have been characterized as potent inhibitors toward topoisomerase IIα (TopoIIα), an essential enzyme involved in maintaining genomic integrity during DNA replication and mitotic division. Mansonone G (MG), a naphthoquinone-containing compound extracted from the heartwood of Mansonia gagei , exhibits various biological activities including antitumor potential. In the present study, MG and its semi-synthetic derivatives were selected to study the preferential binding site and dynamics behavior as well as to predict the inhibitory activity against TopoIIα using molecular modeling approaches. The molecular docking results revealed that the entire series of MG preferentially target to the ATPase domain. Among all studied MGs, the ester derivative MG14 containing C-10 length exhibited the highest binding affinity against TopoIIα and greater than that of the ATP-competitive inhibitor salvicine as well as 1,4-benzoquinone. Interestingly, the MG14 binding could induce the closed form of the turn region (residues 147–151) inside ATP-binding pocket, implying that this event might be one of the crucial mechanisms underlying TopoIIα inhibition. The obtained theoretical information is useful as rational guide for further development of new anticancer agents containing naphthoquinone moiety against TopoIIα.

Density functional theory (DFT) calculations have been carried out to investigate the ethylene insertion pathway using a Ziegler-Natta catalyst in the absence and presence of electron donor (ED) systems on the (1 1 0) MgCl 2 surface. The coadsorptions of four different EDs i.e. Si(OEt) m Cl n ( m + n = 4) were investigated. The presence of Si(OEt) 4 on the (1 1 0) MgCl 2 surface with the preferential bidentate mode was found to have the strongest adsorption energy (Δ E ads ). The potential energy surface (PES) map indicated that the reaction mechanism of the ethylene insertion into the Ti C bond on the (1 1 0) Mg 13 Cl 26 .TiCl 3 -CH 2 CH 3 surface is pseudo-concerted. As the differences in the intrinsic activation energies (Δ E a ) obtained from all systems are so small, this energy cannot be used to fully explain the significant changes in the rates of the ethylene insertion reaction observed when an ED is employed. Here, the apparent activation energy (ΔΔ E aa ) was calculated using the PBE functional and the 6-31G(d, p) basis set for C, H, O, Mg and Cl, and the LANL2DZ basis set with an ECP function for the Ti atom. All EDs presented in this work in the ethylene insertion reaction can significantly reduce the apparent energy barrier when compared to an absence of any ED system. The obtained ΔΔ E aa for the four ED complexes were found to decrease in the following order: SiOEtCl 3 > Si(OEt) 2 Cl 2 > Si(OEt) 3 Cl > Si(OEt) 4 . The obtained data lead to the conclusion that Si(OEt) 4 is the most suitable ED to increase the productivity of PE in the presence of alkoxysilane.

The human T1R2-T1R3 sweet taste receptor (STR) plays an important role in recognizing various low-molecular-weight sweet-tasting sugars and proteins, resulting in the release of intracellular heterotrimeric G protein that in turn leads to the sweet taste perception. Xylitol and sorbitol, which are naturally occurring sugar alcohols (polyols) found in many fruits and vegetables, exhibit the potential caries-reducing effect and are widely used for diabetic patients as low-calorie sweeteners. In the present study, computational tools were applied to investigate the structural details of binary complexes formed between these two polyols and the T1R2-T1R3 heterodimeric STR. Principal component analysis revealed that the Venus flytrap domain (VFD) of T1R2 monomer was adapted by the induced-fit mechanism to accommodate the focused polyols, in which α-helical residues 233-268 moved significantly closer to stabilize ligands. This finding likely suggested that these structural transformations might be the important mechanisms underlying polyols-STR recognitions. The calculated free energies also supported the VFD of T1R2 monomer as the preferential binding site for such polyols, rather than T1R3 region, in accord with the lower number of accessible water molecules in the T1R2 pocket. The E302 amino acid residue in T1R2 was found to be the important recognition residue for polyols binding through a strongly formed hydrogen bond. Additionally, the binding affinity of xylitol toward the T1R2 monomer was significantly higher than that of sorbitol, making it a sweeter tasting molecule.

Mansonone G (MG), a plant-derived compound isolated from the heartwood of Mansonia gagei, possesses a potent antitumor effect on several kinds of malignancy. However, its poor solubility limits the use for practical applications. Beta-cyclodextrin (&beta, CD), a cyclic oligosaccharide composed of seven (1&rarr, 4)-linked &alpha, D-glucopyranose units, is capable of encapsulating a variety of poorly soluble compounds into its hydrophobic interior. In this work, we aimed to enhance the water solubility and the anticancer activity of MG by complexation with &beta, CD and its derivatives (2,6-di-O-methyl-&beta, CD (DM&beta, CD) and hydroxypropyl-&beta, CD). The 90-ns molecular dynamics simulations and MM/GBSA-based binding free energy results suggested that DM&beta, CD was the most preferential host molecule for MG inclusion complexation. The inclusion complex formation between MG and &beta, CD(s) was confirmed by DSC and SEM techniques. Notably, the MG/&beta, CDs inclusion complexes exerted significantly higher cytotoxic effect (2&ndash, 7 fold) on A549 lung cancer cells than the uncomplexed MG.

Human cytosolic serine hydroxymethyltransferase (hcSHMT) is a promising target for anticancer chemotherapy and contains a flexible “flap motif” whose function is yet unknown. Here, using size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering (SAXS), molecular dynamics (MD) simulations, and ligand-binding and enzyme-kinetic analyses, we studied the functional roles of the flap motif by comparing WT hcSHMT with a flap-deleted variant (hcSHMT/Δflap). We found that deletion of the flap results in a mixture of apo-dimers and holo-tetramers, whereas the WT was mostly in the tetrameric form. MD simulations indicated that the flap stabilizes structural compactness and thereby enhances oligomerization. The hcSHMT/Δflap variant exhibited different catalytic properties in (6S)-tetrahydrofolate (THF)-dependent reactions compared with the WT but had similar activity in THF-independent aldol cleavage of β-hydroxyamino acid. hcSHMT/Δflap was less sensitive to THF inhibition than the WT (K(i) of 0.65 and 0.27 mm THF at pH 7.5, respectively), and the THF dissociation constant of the WT was also 3-fold lower than that of hcSHMT/Δflap, indicating that the flap is important for THF binding. hcSHMT/Δflap did not display the burst kinetics observed in the WT. These results indicate that, upon removal of the flap, product release is no longer the rate-limiting step, implying that the flap is important for controlling product release. The findings reported here improve our understanding of the functional roles of the flap motif in hcSHMT and provide fundamental insight into how a flexible loop can be involved in controlling the enzymatic reactions of hcSHMT and other enzymes.

Targeted cancer therapy has become a high potential cancer treatment. Epidermal growth factor receptor (EGFR), which plays an important role in cell signaling, enhanced cell survival and proliferation, has been suggested as molecular target for the development of novel cancer therapeutics. In this study, a series of chalcone derivatives was screened by in vitro cytotoxicity against the wild type (A431 and A549) and mutant EGFR (H1975 and H1650) cancer cell lines, and, subsequently, tested for EGFR-tyrosine kinase (TK) inhibition. From the experimental screening, all chalcones seemed to be more active against the A431 than the A549 cell line, with chalcones 1c, 2a, 3e, 4e, and 4t showing a more than 50% inhibitory activity against the EGFR-TK activity and a high cytotoxicity with IC50 values of &lt, 10 &micro, M against A431 cells. Moreover, these five chalcones showed more potent on H1975 (T790M/L858R mutation) than H1650 (exon 19 deletion E746-A750) cell lines. Only three chalcones (1c, 2a and 3e) had an inhibitory activity against EGFR-TK with a relative inhibition percentage that was close to the approved drug, erlotinib. Molecular dynamics studies on their complexes with EGFR-TK domain in aqueous solution affirmed that they were well-occupied within the ATP binding site and strongly interacted with seven hydrophobic residues, including the important hinge region residue M793. From the above information, as well as ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties, all three chalcones could serve as lead compounds for the development of EGFR-TK inhibitors.

Epidermal growth factor receptor (EGFR), overexpressed in many types of cancer, has been proved as a high potential target for targeted cancer therapy due to its role in regulating proliferation and survival of cancer cells. In the present study, a series of designed vinyl sulfone derivatives was screened against EGFR tyrosine kinase (EGFR-TK) using in silico and in vitro studies. The molecular docking results suggested that, among 78 vinyl sulfones, there were eight compounds that could interact well with the EGFR-TK at the ATP-binding site. Afterwards, these screened compounds were tested for the inhibitory activity towards EGFR-TK using ADP-Glo™ kinase assay, and we found that only VF16 compound exhibited promising inhibitory activity against EGFR-TK with the IC50 value of 7.85 ± 0.88 nM. In addition, VF16 showed a high cytotoxicity with IC50 values of 33.52 ± 2.57, 54.63 ± 0.09, and 30.38 ± 1.37 µM against the A431, A549, and H1975 cancer cell lines, respectively. From 500-ns MD simulation, the structural stability of VF16 in complex with EGFR-TK was quite stable, suggesting that this compound could be a novel small molecule inhibitor targeting EGFR-TK.

Hand foot and mouth disease (HFMD) epidemic has occurred in many countries. Coxsackievirus A16 (CV-A16) and Enterovirus A71 (EV-A71) are the main causes of HFMD. Up to now, there are no anti-HFMD drugs available. Rupintrivir, a broad-spectrum inhibitor, is a drug candidate for HFMD treatment, while other HFMD inhibitors designed from several studies have a relatively low efficiency. Therefore, in this work we aim to study the binding mechanisms of rupintrivir and a peptidic α,β-unsaturated ethyl ester (SG85) against both CV-A16 and EV-A71 3C proteases (3Cpro) using all-atoms molecular dynamics simulation. The obtained results indicate that SG85 shows a stronger binding affinity than rupintrivir against CV-A16. Both inhibitors exhibit a comparable affinity against EV-A71 3Cpro. The molecular information of the binding of the two inhibitors to the proteases will be elucidated. Thus, it is implied that these two compounds may be used as leads for further anti-HFMD drug design and development.

Defensins form an antimicrobial peptides (AMP) family, and have been widely studied in various plants because of their considerable inhibitory functions. However, their roles in rice (Oryza sativa L.) have not been characterized, even though rice is one of the most important staple crops that is susceptible to damaging infections. Additionally, a previous study identified 598 rice genes encoding cysteine-rich peptides, suggesting there are several uncharacterized AMPs in rice. We performed in silico gene expression and coexpression network analyses of all genes encoding defensin and defensin-like peptides, and determined that OsDEF7 and OsDEF8 are coexpressed with pathogen-responsive genes. Recombinant OsDEF7 and OsDEF8 could form homodimers. They inhibited the growth of the bacteria Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, and Erwinia carotovora subsp. atroseptica with minimum inhibitory concentration (MIC) ranging from 0.6 to 63μg/mL. However, these OsDEFs are weakly active against the phytopathogenic fungi Helminthosporium oryzae and Fusarium oxysporum f.sp. cubense. This study describes a useful method for identifying potential plant AMPs with biological activities.

Steroid 5α-reductase type II is a membrane-associated enzyme in an oxidoreductase family. This enzyme, which is found in male sexual organs, plays the important biological actions toward steroid metabolism. Overexpression of 5α-reductase type II has affected the balance between testosterone and dihydrotestosterone, which implicates the androgenic disorders, including prostate cancer, hirsutism, and androgenic alopecia. Lack of single-crystal X-ray structures of 5α-reductase has hindered mechanistic understanding and delayed the discovery and development of an effective inhibitor. Herein, we illustrated a comparative structure of 5α-reductase type II that derived from the homology modeling, employing a membrane-bound protein, isoprenylcysteine carboxyl methyltransferase as a homologous template. A catalytic pocket and the transmembrane site were identified. The resulting in silico structure was validated via Ramachandran plot and confirmed by docking studies of 30 known 5α-reductase type I and type II inhibitors, including finasteride and dutasteride. The comparative docking study of the derived in silico 5α-reductase type II and 5β-reductase, a reported surrogate enzyme, was conducted. Our homology model approximately fitted to the membrane-associated character and showed the reasonable docking results, which depicted the well-defined comparative three-dimensional structure applicable for steroid reductase drug design.

Low visibility on expressways caused by heavy fog and haze is a main reason for traffic accidents. Real-time estimation of atmospheric visibility is an effective way to reduce traffic accident rates. With the development of computer technology, estimating atmospheric visibility via computer vision becomes a research focus. However, the estimation accuracy should be enhanced since fog and haze are complex and time-varying. In this paper, a total bounded variation (TBV) approach to estimate low visibility (less than 300 m) is introduced. Surveillance images of fog and haze are processed as blurred images (pseudo-blurred images), while the surveillance images at selected road points on sunny days are handled as clear images, when considering fog and haze as noise superimposed on the clear images. By combining image spectrum and TBV, the features of foggy and hazy images can be extracted. The extraction results are compared with features of images on sunny days. Firstly, the low visibility surveillance images can be filtered out according to spectrum features of foggy and hazy images. For foggy and hazy images with visibility less than 300 m, the high-frequency coefficient ratio of Fourier (discrete cosine) transform is less than 20%, while the low-frequency coefficient ratio is between 100% and 120%. Secondly, the relationship between TBV and real visibility is established based on machine learning and piecewise stationary time series analysis. The established piecewise function can be used for visibility estimation. Finally, the visibility estimation approach proposed is validated based on real surveillance video data. The validation results are compared with the results of image contrast model. Besides, the big video data are collected from the Tongqi expressway, Jiangsu, China. A total of 1,782,000 frames were used and the relative errors of the approach proposed are less than 10%.

The binding affinity of oseltamivir to the influenza B neuraminidase and to its variants with three single substitutions, E119G, R152K, and D198N, is investigated by the MM/3D-RISM method. The binding affinity or the binding free energy of ligand to receptor was found to be determined by a subtle balance of two major contributions that largely cancel out each other: the ligand-receptor interactions and the dehydration free energy. The theoretical results of the binding affinity of the drug to the mutants reproduced the observed trend in the resistivity, measured by IC50 ; the high-level resistance of E119G and R152K, and the low-level resistance of D198N. For E119G and R152K, reduction of the direct drug-target interaction, especially at the mutated residue, is the main source of high-level oseltamivir resistance. This phenomenon, however, is not found in the D198N strain, which is located in the framework of the active-site.

DNA topoisomerase I (Top1) catalyzes changes in DNA topology by cleaving and rejoining one strand of the double stranded (ds)DNA. Eukaryotic Top1s are the cellular target of the plant-derived anticancer indole alkaloid camptothecin (CPT), which reversibly stabilizes the Top1-dsDNA complex. However, CPT-producing plants, including Camptotheca acuminata , Ophiorrhiza pumila and Ophiorrhiza liukiuensis , are highly resistant to CPT because they possess point-mutated Top1. Here, the adaptive convergent evolution is reported between CPT production ability and mutations in their Top1, as a universal resistance mechanism found in all tested CPT-producing plants. This includes Nothapodytes nimmoniana , one of the major sources of CPT. To obtain a structural insight of the resistance mechanism, molecular dynamics simulations of CPT– resistant and –sensitive plant Top1s complexed with dsDNA and topotecan (a CPT derivative) were performed, these being compared to that for the CPT-sensitive human Top1. As a result, two mutations, Val617Gly and Asp710Gly, were identified in O. pumila Top1 and C. acuminata Top1, respectively. The substitutions at these two positions, surprisingly, are the same as those found in a CPT derivative-resistant human colon adenocarcinoma cell line. The results also demonstrated an increased linker flexibility of the CPT–resistant Top1, providing an additional explanation for the resistance mechanism found in CPT-producing plants. These mutations could reflect the long evolutionary adaptation of CPT-producing plant Top1s to confer a higher degree of resistance.

Rhodomyrtone, a natural antimicrobial compound, displays potent activity against many Gram-positive pathogenic bacteria, comparable to last-defence antibiotics including vancomycin and daptomycin. Our previous studies pointed towards effects of rhodomyrtone on the bacterial membrane and cell wall. In addition, a recent molecular docking study suggested that the compound could competitively bind to the main bacterial cell division protein FtsZ. In this study, we applied a computational approach (in silico),in vitro, andin vivoexperiments to investigate molecular interactions of rhodomyrtone with FtsZ. Using molecular simulation, FtsZ conformational changes were observed in both (S)- and (R)-rhodomyrtone binding states, compared with the three natural states of FtsZ (ligand-free, GDP-, and GTP-binding states). Calculations of free binding energy showed a higher affinity of FtsZ to (S)-rhodomyrtone (−35.92 ± 0.36 kcal mol−1) than the GDP substrate (−23.47 ± 0.25 kcal mol−1) while less affinity was observed in the case of (R)-rhodomyrtone (−18.11 ± 0.11 kcal mol−1).In vitroexperiments further revealed that rhodomyrtone reduced FtsZ polymerization by 36% and inhibited GTPase activity by up to 45%. However, the compound had no effect on FtsZ localization inBacillus subtilisat inhibitory concentrations and cells also did not elongate after treatment. Higher concentrations of rhodomyrtone did affect localization of FtsZ and also affected localization of its membrane anchor proteins FtsA and SepF, showing that the compound did not specifically inhibit FtsZ but rather impaired multiple divisome proteins. Furthermore, a number of cells adopted a bean-like shape suggesting that rhodomyrtone possibly possesses further targets involved in cell envelope synthesis and/or maintenance.

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.

Hepatitis C virus (HCV) NS3/4A protease is an attractive target for the development of antiviral therapy. However, the evolution of antiviral drug resistance is a major problem for treatment of HCV infected patients. Understanding of drug-resistance mechanisms at molecular level is therefore very important for the guidance of further design of antiviral drugs with high efficiency and specificity. Paritaprevir is a potent inhibitor against HCV NS3/4A protease genotype 1a. Unfortunately, this compound is highly susceptible to the substitution at D168 in the protease. In this work, molecular dynamics simulations of paritaprevir complexed with wild-type (WT) and two mutated strains (D168 N and D168Y) were carried out. Due to such mutations, the inhibitor-protein hydrogen bonding between them was weakened and the salt-bridge network among residues R123, R155 and D168 responsible for inhibitor binding was disrupted. Moreover, the per-residue free energy decomposition suggested that the main contributions from key residues such as Q80, V132, K136, G137 and R155 were lost in the D168 N/Y mutations. These lead to a lower binding affinity of paritaprevir for D168 N/Y variants of the HCV NS3/4A protease, consistent with the experimental data. This detailed information could be useful for further design of high potency anti-HCV NS3/4A inhibitors.

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 49888724 had 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 49888724 is 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.

The dissociation capability of the hemagglutinin precursor (HA0) to HA1 and HA2 is related to the degree of pathogenicity of influenza virus. The H5 strains that contain polybasic residues inserted at the cleavage site by the RXR/KR motif are recognized by the subtilisin-like proteases and are associated with a high pathogenicity in humans. The available predicted amino acid sequences of the H5 subtype isolated from avian and human hosts were collected and aligned. As a result, most of the insertion types in viruses isolated from humans were also found in avian H5 isolates, but not in vice versa. With avian-to-human transmission, the unique avian H5 insertion types with the RXR/KR recognition motif were predicted as potential new candidates for the next highly pathogenic avian influenza H5 in humans. Additionally, at most the two basic residues extended from the recognition motif were found to be optimum fit with the furin-like enzyme. © 2012 Wiley Periodicals, Inc.

Evidences of oseltamivir resistant influenza patients raised the need of novel neuraminidase inhibitors. In this study, five oseltamivir analogs PMC-31–PMC-36, synthesised according to the outcomes of a rational design analysis aimed to investigate the effects of substitution at the 5-amino and 4-amido groups of oseltamivir on its antiviral activity, were screened for their inhibition against neuraminidase N1 and N3. The enzymes used as models were from the avian influenza A H7N1 and H7N3 viruses. The neuraminidase inhibition assay was carried out by using recombinant species obtained from a baculovirus expression system and the fluorogenic substrate MUNANA. The assay was validated by using oseltamivir carboxylate as a reference inhibitor. Among the tested compounds, PMC-36 showed the highest inhibition on N1 with an IC50 of 14.6 ± 3.0 nM (oseltamivir 25 ± 4 nM), while PMC-35 showed a significant inhibitory effect on N3 with an IC50 of 0.1 ± 0.03 nM (oseltamivir 0.2 ± 0.02 nM). The analysis of the inhibitory properties of this panel of compounds allowed a preliminary assessment of a structure–activity relationship for the modification of the 4-amido and 5-amino groups of oseltamivir carboxylate. The substitution of the acetamido group in the oseltamivir structure with a 2-butenylamido moiety reduced the observed activity, while the introduction of a propenylamido group was well tolerated. Substitution of the free 5-amino group of oseltamivir carboxylate with an azide, decreased the activity against both N1 and N3. When these structural changes were both introduced, a dramatic reduction of activity was observed for both N1 and N3. The alkylation of the free 5-amino group in oseltamivir carboxylate introducing an isopropyl group seemed to increase the inhibitory effect for both N1 and N3 neuraminidases, displaying a more pronounced effect against N1.

The furin (FR) complex with each of four different sequences of hemagglutinin from the highly pathogenic H5N1 strains (HPH5), which were identified during the 2004–2010 influenza outbreaks in Thailand, were evaluated by molecular dynamics simulations, so as to compare the specificity and recognition of the enzyme–substrate binding. Relative to the conventional HPH5 inserted (H5Sq1, RERRRKKR), the S5-R or S6-R arginine residue is replaced by the smaller lysine in the H5Sq2 (RERKRKKR) and H5Sq3 (REKRRKKR) strains, respectively, whereas the S3-K lysine residue is deleted in H5Sq4 (RERRR_KR). The molecular dynamics results of the intermolecular interactions, in terms of hydrogen bonds and per-residue decomposition energy, between the substrate and furin revealed that the deletion of the positively charged amino acid at the S3 position in H5Sq4 leads to a notably weaker binding and specificity with the furin active site compared with that of FR–H5Sq1. A slight change in the substrate binding was found in the FR–H5Sq2 and FR–H5Sq3 complexes as a result of the replacement of the arginine with the shorter side-chained lysine (same positive charge). Altogether, the predicted binding free energy of the enzyme–substrate complexes was found to be in the following order: FR–H5Sq1

The current pandemic of the viral 2009 H1N1 influenza and its sustained human–human transmission has raised global concern for human health. The binding of the viral glycoprotein hemagglutinin (HA) and the human α-2,6-linked sialopentasaccharide (SIA-2,6-GAL) host cell receptor is a critical step in the viral replication cycle. Here, the complex structure of the 2009 H1N1 HA bound to the SIA-2,6-GAL sialopentasaccharide receptor was constructed by using homology modeling and molecular dynamic simulations. The receptor was found to fit very well within the HA binding pocket and formed hydrogen bonds with the residues of the 130-loop, 190-helix, and 220-loop. Most receptor binding residues play a significant role in stabilizing the protein–receptor complex with major contributions being provided by V135, T136, A137, K222, and Q226. The results are similar to the human SIA-2,6-GAL sialopentasaccharide receptor binding to H1 HA subtype, but are slightly different from those of H3, H5, and H9 HAs.

To reveal the source of oseltamivir-resistance in influenza (A/H5N1) mutants, the drug-target interactions at each functional group were investigated using MD/LIE simulations. Oseltamivir in the H274Y mutation primarily loses the electrostatic and the vdW interaction energies at the -NH(3)(+) and -OCHEt(2) moieties corresponding to the weakened hydrogen-bonds and changed distances to N1 residues. Differentially, the N294S mutation showed small changes of binding energies and intermolecular interactions. Interestingly, the presence of different conformations of E276 positioned between the -OCHEt(2) group and the mutated residue is likely to play an important role in oseltamivir-resistant identification. In the H274Y mutant, it moves towards the -OCHEt(2) group leading to a reduction in hydrophobicity and pocket size, whilst in the N294S mutant it acts as the hydrogen network center bridging with R224 and the mutated residue S294. The molecular details have answered a question of how the H274Y and N294S mutations confer the high- and medium-level of oseltamivir-resistance to H5N1.

The recent outbreak of the novel strain of influenza A (H1N1) virus has raised a global concern of the future risk of a pandemic. To understand at the molecular level how this new H1N1 virus can be inhibited by the current anti-influenza drugs and which of these drugs it is likely to already be resistant to, homology modeling and MD simulations have been applied on the H1N1 neuraminidase complexed with oseltamivir, and the M2-channel with adamantanes bound. The H1N1 virus was predicted to be susceptible to oseltamivir, with all important interactions with the binding residues being well conserved. In contrast, adamantanes are not predicted to be able to inhibit the M2 function and have completely lost their binding with the M2 residues. This is mainly due to the fact that the M2 transmembrane of the new H1N1 strain contains the S31N mutation which is known to confer resistance to adamantanes.

Two pyridoacridine alkaloids, including a known petrosamine and a new 2-bromoamphimedine were isolated from a Thai marine sponge Petrosia n. sp. The alkaloids were characterized on the basis of 1D and 2D NMR, MS, and IR spectroscopy. Only petrosamine showed strong acetylcholinesterase inhibitory activity approximately six times higher than that of the reference galanthamine. A computational docking study of petrosamine with the enzyme from the electric eel Torpedo californica (TcAChE) showed the major contribution to the petrosamine-TcAChE interaction to be arising from the quaternary ammonium group of petrosamine.

The pathogenic dengue virus (DV) is a growing global threat, particularly in South East Asia, for which there is no specific treatment available. The virus possesses a two-component (NS2B/NS3) serine protease that cleaves the viral precursor proteins. Here, we performed molecular dynamics simulations of the NS2B/NS3 protease complexes with six peptide substrates (capsid, intNS3, 2A/2B, 4B/5, 3/4A and 2B/3 containing the proteolytic site between P(1) and P(1)' subsites) of DV type 2 to compare the specificity of the protein-substrate binding recognition. Although all substrates were in the active conformation for cleavage reaction by NS2B/NS3 protease, their binding strength was somewhat different. The simulated results of intermolecular hydrogen bonds and decomposition energies suggested that among the ten substrate residues (P(5)-P(5)') the P(1) and P(2) subsites play a major role in the binding with the focused protease. The arginine residue at these two subsites was found to be specific preferential binding at the active site with a stabilization energy of-10 kcal mol(-1). Besides, the P(3), P(1)', P(2)' and P(4)' subsites showed a less contribution in binding interaction (-2 kcal mol(-1)). The catalytic water was detected nearby the carbonyl oxygen of the P(1) reacting center of the capsid, intNS3, 2A/2B and 4B/5 peptides. These results led to the order of absolute binding free energy (ΔGbind) between these substrates and the NS2B/NS3 protease ranked as capsidintNS32A/2B4B/53/4A2B/3 in a relative correspondence with previous experimentally derived values.

Hepatitis C virus (HCV) causes an infectious disease that manifests itself as liver inflammation, cirrhosis, and can lead to the development of liver cancer. Its NS3/4A serine protease is a potent target for drug design and development since it is responsible for cleavage of the scissile peptide bonds in the polyprotein important for the HCV life cycle. Herein, the ligand-target interactions and the binding free energy of the four current NS3/4A inhibitors (boceprevir, telaprevir, danoprevir, and BI201335) were investigated by all-atom molecular dynamics simulations with three different initial atomic velocities. The per-residue free energy decomposition suggests that the key residues involved in inhibitor binding were residues 41-43, 57, 81, 136-139, 155-159, and 168 in the NS3 domain. The van der Waals interactions yielded the main driving force for inhibitor binding at the protease active site for the cleavage reaction. In addition, the highest number of hydrogen bonds was formed at the reactive P1 site of the four studied inhibitors. Although the hydrogen bond patterns of these inhibitors were different, their P3 site was most likely to be recognized by the A157 backbone. Both molecular mechanic (MM)/Poisson-Boltzmann surface area and MM/generalized Born surface area approaches predicted the relative binding affinities of the four inhibitors in a somewhat similar trend to their experimentally derived biological activities.

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 cyclin dependent kinases (CDKs), each with their respective regulatory partner cyclin that are involved in the regulation of the cell cycle, apoptosis, and transcription, are potentially interesting targets for cancer therapy. The CDK6 complex with cyclin D (CDK6/cycD) drives cellular proliferation by phosphorylation of specific key target proteins. To understand the flavonoids that inhibit the CDK6/cycD functions, molecular dynamics simulations (MDSs) were performed on three inhibitors, fisetin (FST), apigenin (AGN), and chrysin (CHS), complexed with CDK6/cycD, including the two different binding orientations of CHS: FST-like (CHS_A) and deschloro-flavopiridol-like (CHS_B). For all three inhibitors, including both CHS orientations, the conserved interaction between the 4-keto group of the flavonoid and the backbone V101 nitrogen of CDK6 was strongly detected. The 3'- and 4'-OH groups on the flavonoid phenyl ring and the 3-OH group on the benzopyranone ring of inhibitor were found to significantly increase the binding and inhibitory efficiency. Besides the electrostatic interactions, especially through hydrogen bond formation, the van der Waals (vdW) interactions with the I19, V27, F98, H100, and L152 residues of CDK6 are also important factors in the binding efficiency of flavonoids against the CDK6/cycD complex. On the basis of the docking calculation and MM-PBSA method, the order of the predicted inhibitory affinities of these three inhibitors toward the CDK6/cycD was FSTAGNCHS, which is in good agreement with the experimental data. In addition, CHS preferentially binds to the active CDK6 in a different orientation to FST and AGN but similar to its related analog, deschloro-flavopiridol. The obtained results are useful as the basic information for the further design of potent anticancer drugs specifically targeting the CDK6 enzyme.