Jian Wan

Binding Interaction Analysis of the Active Site and Its Inhibitors for Neuraminidase (N1 Subtype) of Human Influenza Virus by the Integration of Molecular Docking, FMO Calculation and 3D-QSAR CoMFA Modeling

Recently, the worldwide spread of A/H5N1 avian influenza with high virulence has highlighted the potential threat of human influenza pandemic. Tamiflu and Relenza are currently the only two anti-influenza drugs targeting the neuraminidase (NA) enzyme of human influenza virus. Reports of the emergence of drug resistance further make the development of new potent anti-influenza inhibitors a priority. The X-ray crystallographic study of A/H5N1 avian influenza NA subtypes (Russell, R. J. Nature 2006, 443, 45-49) has demonstrated that there exist two genetically distinct groups, group-1 (N1, N4, N5 and N8) and group-2 (N2, N3, N6, N7 and N9), whose conformations are substantially different. The detailed comparison of their active sites has established, heretofore, the most accurate and solid molecular basis of structure and mechanism for the development of new anti-influenza drugs. In the present study, a three-dimensional structure of N1 subtype of human influenza type A virus (N1hA) has been generated by homology modeling using the X-ray crystallographic structure of N1 subtype of avian influenza virus (N1aA) as the template. Binding interaction analysis between the active site and its inhibitors has been performed by combining ab initio fragment molecular orbital (FMO) calculations and three-dimensional quantitative structure-activity relationship with comparative molecular field analysis (3D-QSAR CoMFA) modeling. Integrated with docking-based 3D-QSAR CoMFA modeling, molecular surface property (electrostatic and steric) mapping and FMO pair interaction analysis, a set of new receptor-ligand binding models and bioaffinity predictive models for rational design and virtual screening of more potent inhibitors of N1hA are established. In addition, the flexibility of the loop-150 of N1hA and N1aA has been examined by a series of molecular dynamics simulations.

Structure-Based Rational Screening of Novel Hit Compounds with Structural Diversity for Cytochrome P450 Sterol 14α-Demethylase from Penicillium digitatum

Cytochrome P450 sterol 14alpha-demethylases (CYP51s) are essential enzymes in sterol biosynthesis and well-known as the target of antifungal drugs. All fungal CYP51s are integral membrane proteins, making structural and biophysical characterization more challenging. The X-ray crystallographic structure of CYP51 isolated from Mycobacterium tuberculosis (MT-CYP51) is the unique reported one hitherto. In the present study, a homology modeling three-dimensional structure of CYP51 from Penicillium digitatum (PD-CYP51) was generated by CPHmodels, in which the accuracy of sequence alignment could be improved by taking into account further structural conservation information, using MT-CYP51 as the template. Interaction mechanism between the active site of PD-CYP51 and its inhibitors were further investigated by molecular dynamics simulating and molecular docking. With the effective docking process and interaction analysis information, structure-based virtual screening was performed to pick out the thirty new potential inhibiting compounds with structural diversity by using a new virtual screening strategy including Flex-Pharm/PMF/GOLD//FlexX/PMF/GOLD molecular docking procedures, and finally, seven new hit compounds out of SPECs database with potent inhibitory ability were validated by bioaffinity assays at enzyme level and on P. digitatum in vitro. The positive results indicated that all modeling strategies and screening processes presented in the current study most like to be an encouraging way in search of novel lead compounds with structural diversity for the specifically individual fungal CYP51s of both plants and human pathogens in the future.

Structure-based design and screen of novel inhibitors for class II 3-hydroxy-3-methylglutaryl coenzyme A reductase from Streptococcus pneumoniae.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a primary target in the current clinical treatment of hypercholesterolemia with specific inhibitors of statin family. Statins are excellent inhibitors of the class I (human) enzyme but relatively poor inhibitors of the class II enzyme, which are well-known as a potential target to discover drugs fighting against the invasive diseases originated from S. pneumoniae . However, no significantly effective inhibitors of class II HMGR have been reported so far. In the present study, the reasonable three-dimensional (3D) structure of class II HMGR from S. pneumoniae (SP-HMGR-II) was built by Swissmodel. On the basis of the modeling 3D structure in close flap domain form, several novel potential hit compounds out of SPECs database were picked out by using structure-based screening strategy. Especially the compounds 4, 3, and 11 exhibit highly inhibitory activities, with IC50 values of 11.5, 18.5, and 18.1 μM, respectively. Furthermore, the hit compounds were chosen as probe molecules, and their probable interactions with the corresponding individual residues have been examined by jointly using the molecular docking, site-directed mutagenesis, enzymatic assays, and fluorescence spectra, to provide an insight into a new special binding-model located between the HMG-CoA and NADPH pockets. The good agreement between theoretical and experimental results indicate that the modeling strategies and screening processes in the present study are very likely to be a promising way to search novel lead compounds with both structural diversity and high inhibitory activity against SP-HMGR-II in the future.

Expression and homology modelling of sterol 14α-demethylase of Magnaporthe grisea and its interaction with azoles

BACKGROUNDnMagnaporthe grisea (Hebert) ME Barr infection is one of the most serious diseases for cultivated rice in the world. Sterol 14alpha-demethylase (CYP51) is an important drug target for microbial pathogenic infections. To exploit specific and effective fungicides for M. grisea better, the authors have analysed the characteristics of interaction between sterol 14alpha-demethylase from M. grisea (MGCYP51) and azoles. MGCYP51 with truncation of N-terminal residues was cloned and expressed in E. coli, difference binding spectra of MGCYP51 induced by addition of four commercial azoles were determined and molecular modelling of MGCYP51 based on the crystal structure of Mycobacterium tuberculosis Lehmann & Newman and docking with the azoles were performed.nnnRESULTSnThe affinity of the azoles for MGCYP51 was positively correlated with their hydrophobicity. Amino acid residues Tyr112, Phe120, Phe220, His308 and Phe497 of MGCYP51, forming a large hydrophobic cavity, are the key residues interacting with azole fungicides. Furthermore, Phe220 and Phe497 are fungus and species specific respectively.nnnCONCLUSIONnThe results suggest that the more potent azole fungicides for MGCYP51 should possess more hydrophobic groups interacting with residues Phe220 and Phe497.

A Rational Design, Synthesis, Biological Evaluation and Structure–Activity Relationship Study of Novel Inhibitors against Cyanobacterial Fructose-1,6-bisphosphate Aldolase

In the present study, a series of novel maleimide derivatives were rationally designed and optimized, and their inhibitory activities against cyanobacteria class-II fructose-1,6-bisphosphate aldolase (Cy-FBA-II) and Synechocystis sp. PCC 6803 were further evaluated. The experimental results showed that the introduction of a bigger group (Br, Cl, CH3, or C6H3-o-F) on the pyrrole-2,5-dione ring resulted in a decrease in the Cy-FBA-II inhibitory activity of the hit compounds. Generally, most of the hit compounds with high Cy-FBA-II inhibitory activities could also exhibit high in vivo activities against Synechocystis sp. PCC 6803. Especially, compound 10 not only shows a high Cy-FBA-II activity (IC50 = 1.7 μM) but also has the highest in vivo activity against Synechocystis sp. PCC 6803 (EC50 = 0.6 ppm). Thus, compound 10 was selected as a representative molecule, and its probable interactions with the surrounding important residues in the active site of Cy-FBA-II were elucidated by the joint use of molecular docking, molecular dynamics simulations, ONIOM calculations, and enzymatic assays to provide new insight into the binding mode of the inhibitors and Cy-FBA-II. The positive results indicate that the design strategy used in the present study is very likely to be a promising way to find novel lead compounds with high inhibitory activities against Cy-FBA-II in the future. The enzymatic and algal inhibition assays suggest that Cy-FBA-II is very likely to be a promising target for the design, synthesis, and development of novel specific algicides to solve cyanobacterial harmful algal blooms.

Optimised expression and spectral analysis of the target enzyme CYP51 from Penicillium digitatum with possible new DMI fungicides

BACKGROUNDnSterol 14α-demethylase (CYP51), a key target of azole (DMI) fungicides, can be expressed in both prokaryotes and eukaryotes. Green mould of citrus, caused by Penicillium digitatum (Pers.) Sacc., is a serious post-harvest disease. To develop specific and more effective fungicides against this disease, the characteristics of the interaction between sterol 14α-demethylase from P. digitatum (PdCYP51) and possible new fungicides were analysed. The cyp51 gene of P. digitatum was cloned and expressed under different conditions in Escherichia coli (Mig.) Cast. & Chalm., and the binding spectra of PdCYP51 were explored by the addition of two commercial azoles and four new nitrogen compounds.nnnRESULTSnThe yield of soluble protein (PdCYP51) was largest when expressed in Rosetta (DE3) induced by 0.5 mM IPTG for 8 h at 30 °C. Compound B (7-methoxy-2H-benzo[b][1,4]thiazine-3-amine) showed the strongest binding activity of the four new nitrogen compounds, with a K(d) value of 0.268 µM. The K(d) values of the six compounds were significantly correlated with their EC(50) values.nnnCONCLUSIONnThe spectral analysis and bioassay results could be used to screen the new chemical entities effectively. Compound B, selected by virtual screening from a commercial chemical library, is a candidate for a new DMI fungicide. These results provide a theoretical basis and new ideas for efficient design and development of new antifungal agents.

Pharmacophore-Based Virtual Screening and Experimental Validation of Novel Inhibitors against Cyanobacterial Fructose-1,6-/Sedoheptulose-1,7-bisphosphatase

Cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphoshatase (cy-FBP/SBPase) is a potential enzymatic target for screening of novel inhibitors that can combat harmful algal blooms. In the present study, we targeted the substrate binding pocket of cy-FBP/SBPase. A series of novel hit compounds from the SPECs database were selected by using a pharmacophore-based virtual screening strategy. Most of the compounds tested exhibited moderate inhibitory activities (IC50 = 20.7-176.9 μM) against cy-FBP/SBPase. Compound 2 and its analogues 10 and 11 exhibited strong inhibitory activities, with IC50 values of 20.7, 13.4, and 19.0 μM against cy-FBP/SBPase in vitro and EC50 values of 12.3, 10.9, and 2.9 ppm against cyanobacteria Synechocystis PCC6803 in vivo, respectively. The compound 10 was selected in order to perform a refined docking study to investigate the rational binding mode of inhibitors with cy-FBP/SBPase. Furthermore, possible interactions of the residues with inhibitors were examined by site-directed mutagenesis, enzymatic assays, and fluorescence spectral analyses. The results provide insight into the binding mode between the inhibitors and the substrate binding pocket. The observed theoretical and experimental results are in concert, indicating that the modeling strategies and screening methods employed are appropriate to search for novel lead compounds having both structural diversity and high inhibitory activity against cy-FBP/SBPase.

Bioinformatics Analysis of Methyl Parathion Hydrolase MPH and the Structure Prediction with Homology Modeling

Methyl parathion hydrolase mph gene was cloned from a novel bacterium Pseudomonas stutzeri HS-D36. The phylogenetic tree was constructed and the structure was predicted by bioinformatics. Results showed MPH protein was 99.0% similar to MPD protein (from pseudomonas sp. WBC-3) and it belonged to metallo-beta-lactamases family. There is an alpha/beta barrel structure in MPH structure. Two independent subunits comprise a homodimer, each subunit is composed of an active metal center (Zn2+ and Cd2+). Homology modeling of MPH was constructed based on the crystal structure of MPD (PDBID.1P9E). Results showed that three mutation amino acid residues in MPH protein were different from MPD, but the function of MPH protein did not change compared to MPD, this indicated the three residues was not important to the activity of MPH enzyme.

Structure-Based Rational Design of Novel Inhibitors Against Fructose-1,6-Bisphosphate Aldolase from Candida albicans

Class II fructose-1,6-bisphosphate aldolases (FBA-II) are attractive new targets for the discovery of drugs to combat invasive fungal infection, because they are absent in animals and higher plants. Although several FBA-II inhibitors have been reported, none of these inhibitors exhibit antifungal effect so far. In this study, several novel inhibitors of FBA-II from C. albicans (Ca-FBA-II) with potent antifungal effects were rationally designed by jointly using a specific protocols of molecular docking-based virtual screening, accurate binding-conformation evaluation strategy, synthesis and enzymatic assays. The enzymatic assays reveal that the compounds 3c, 3e-g, 3j and 3k exhibit high inhibitory activity against Ca-FBA-II (IC50 < 10 μM), and the most potential inhibitor is 3g, with IC50 value of 2.7 μM. Importantly, the compounds 3f, 3g, and 3l possess not only high inhibitions against Ca-FBA-II, but also moderate antifungal activities against C. glabrata (MIC80 = 4-64 μg/mL). The compounds 3g, 3l, and 3k in combination with fluconazole (8 μg/mL) displayed significantly synergistic antifungal activities (MIC80 < 0.0625 μg/mL) against resistant Candida strains, which are resistant to azoles drugs. The probable binding modes between 3g and the active site of Ca-FBA-II have been proposed by using the DOX (docking, ONIOM, and XO) strategy. To our knowledge, no FBA-II inhibitors with antifungal activities against wild type and resistant strains from Candida were reported previously. The positive results suggest that the strategy adopted in this study are a promising method for the discovery of novel drugs against azole-resistant fungal pathogens in the future.

Expression, Purification, Characteristics and Homology Modeling of the HMGS from Streptococcus pneumoniae

OBJECTIVEnTo understand the molecular basis for a potential reaction mechanism and develop novel antibiotics with homology modeling for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (HMGS).nnnMETHODSnThe genetic engineering technology and the composer module of SYBYL7.0 program were used, while the HMGS three-dimensional structure was analyzed by homology modeling.nnnRESULTSnThe mvaS gene was cloned from Streptococcus pneumoniae and overexpressed in Escherichia coli from a pET28 vector. The expressed enzyme (about 46 kDa) was purified by affinity chromatography with a specific activity of 3.24 micromol/min/mg. Optimal conditions were pH 9.75 and 10 mmol/L MgCl2 at 37 degrees C. The V(max) and K(m) were 4.69 micromol/min/mg and 213 micromol/L respectively. The 3D model of S. pneumoniae HMGS was established based on structure template of HMGS of Enterococcus faecalis.nnnCONCLUSIONnThe structure of HMGS will facilitate the structure-based design of alternative drugs to cholesterol-lowering therapies or to novel antibiotics to the Gram-positive cocci, whereas the recombinant HMGS will prove useful for drug development against a different enzyme in the mevalonate pathway.