Rational design,synthesis, and verification of affinity ligands to a protein surface cleft

The structure-based design, synthesis, and screening of a glucuronic acid scaffold library of affinity ligands directed toward the catalytic cleft on porcine pancreas alpha-amylase are presented. The design was based on the simulated docking to the enzyme active site of 53 aryl glycosides from the Available Chemicals Directory (ACD) selected by in silico screening. Twenty-three compounds were selected for synthesis and screened in solution for binding toward alpha-amylase using nuclear magnetic resonance techniques. The designed molecules include a handle outside of the binding site to allow their attachment to various surfaces with minimal loss of binding activity. After initial screening in solution, one affinity ligand was selected, immobilized to Sepharose (Amersham Biosciences), and evaluated as a chromatographic probe. A column packed with ligand-coupled Sepharose specifically retained the enzyme, which could be eluted by a known inhibitor.     

Porcine pancreatic alpha-amylase shows binding activity toward N-linked oligosaccharides of glycoproteins.

Porcine pancreatic alpha-amylase was shown by interaction analyses using a resonance mirror detector and alpha-amylase-immobilized Sepharose to bind with glycoproteins possessing N-glycans but not O-linked mucin-type glycans. Direct binding of three types of N-glycans to the alpha-amylase was demonstrated by surface plasmon resonance. Binding with biotin-polymer sugar probes revealed that the alpha-amylase has affinity to alpha-mannose, alpha-N-acetylneuraminic acid, and beta-N-acetyllactosamine, which are components of N-glycans. The binding of glycoproteins or carbohydrates enhanced the enzyme activity, indicating that the recognition site for N-glycans is different from its catalytic site. The binding activity was unique to porcine pancreatic alpha-amylase and was not observed for alpha-amylase from saliva, wheat, and fungus.     

Preparation of human salivary alpha-amylase specific monoclonal antibody

A mouse hybridoma cell line which produced an anti-human salivary alpha-amylase monoclonal antibody was obtained by fusion between mouse spleen cells immunized with human salivary alpha-amylase and mouse myeloma cells, followed by screening the hybridoma cells by enzyme-linked immunosorbent assay. The hybridoma cell line (27-4-1) secreted IgG. The monoclonal antibody produced by the hybridoma showed no inhibitory effect on the activity of human salivary alpha-amylase. The specificity and reactivity of this monoclonal antibody were examined by determining the activities of human salivary and pancreatic alpha-amylases bound to the monoclonal antibody immobilized on polystyrene balls or by enzyme immunoassay with the monoclonal antibody conjugated with beta-D-galactosidase. The results revealed that the monoclonal antibody produced by the hybridoma cell line was specific for salivary alpha-amylase and absolutely unreactive to pancreatic alpha-amylase.     

Cloning, sequencing, and expression of the gene encoding extracellular alpha-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

The gene encoding the hyperthermophilic extracellular alpha-amylase from Pyrococcus furiosus was cloned by activity screening in Escherichia coli. The gene encoded a single 460-residue polypeptide chain. The polypeptide contained a 26-residue signal peptide, indicating that this Pyrococcus alpha-amylase was an extracellular enzyme. Unlike the P. furiosus intracellular alpha-amylase, this extracellular enzyme showed 45 to 56% similarity and 20 to 35% identity to other amylolytic enzymes of the alpha-amylase family and contained the four consensus regions characteristic of that enzyme family. The recombinant protein was a homodimer with a molecular weight of 100,000, as estimated by gel filtration. Both the dimer and monomer retained starch-degrading activity after extensive denaturation and migration on sodium dodecyl sulfate-polyacrylamide gels. The P. furiosus alpha-amylase was a liquefying enzyme with a specific activity of 3,900 U mg-1 at 98 degrees C. It was optimally active at 100 degrees C and pH 5.5 to 6.0 and did not require Ca2+ for activity or thermostability. With a half-life of 13 h at 98 degrees C, the P. furiosus enzyme was significantly more thermostable than the commercially available Bacillus licheniformis alpha-amylase (Taka-therm).     

Barley alpha-amylase/subtilisin inhibitor: structure, biophysics and protein engineering

Bifunctional alpha-amylase/subtilisin inhibitors have been implicated in plant defence and regulation of endogenous alpha-amylase action. The barley alpha-amylase/subtilisin inhibitor (BASI) inhibits the barley alpha-amylase 2 (AMY2) and subtilisin-type serine proteases. BASI belongs to the Kunitz-type trypsin inhibitor family of the beta-trefoil fold proteins. Diverse approaches including site-directed mutagenesis, hybrid constructions, and crystallography have been used to characterise the structures and contact residues in the AMY2/BASI complex. The three-dimensional structure of the AMY2/BASI complex is characterised by a completely hydrated Ca2+ situated at the protein interface that connects the three catalytic carboxyl groups in AMY2 with side chains in BASI via water molecules. Using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC), we have recently demonstrated Ca2+-modulated kinetics of the AMY2/BASI interaction and found that the complex formation involves minimal structural changes. The modulation of the interaction by calcium ions makes it unique among the currently known binding mechanisms of proteinaceous alpha-amylase inhibitors.     

Cloning and Expression of a Schwanniomyces occidentalis alpha-Amylase Gene in Saccharomyces cerevisiae

An alpha-amylase gene (AMY) was cloned from Schwanniomyces occidentalis CCRC 21164 into Saccharomyces cerevisiae AH22 by inserting Sau3AI-generated DNA fragments into the BamHI site of YEp16. The 5-kilobase insert was shown to direct the synthesis of alpha-amylase. After subclones containing various lengths of restricted fragments were screened, a 3.4-kilobase fragment of the donor strain DNA was found to be sufficient for alpha-amylase synthesis. The concentration of alpha-amylase in culture broth produced by the S. cerevisiae transformants was about 1.5 times higher than that of the gene donor strain. The secreted alpha-amylase was shown to be indistinguishable from that of Schwanniomyces occidentalis on the basis of molecular weight and enzyme properties.     

Discovery of novel inhibitors for DHODH via virtual screening and X-ray crystallographic structures

Amino-benzoic acid derivatives 1–4 were found to be inhibitors for DHODH by virtual screening, biochemical, and X-ray crystallographic studies. X-ray structures showed that 1 and 2 bind to DHODH as predicted by virtual screening, but 3 and 4 were found to be structurally different from the corresponding compounds initially identified by virtual screening.     

Mode of alpha-amylase production by the shochu koji mold Aspergillus kawachii

Aspergillus kawachii produces two kinds of alpha-amylase, one is an acid-unstable alpha-amylase and the other is an acid-stable alpha-amylase. Because the quality of the shochu depends strongly on the activities of the alpha-amylases, the culture conditions under which these alpha-amylases are produced were examined. In liquid culture, acid-unstable alpha-amylase was produced abundantly, but, acid-stable alpha-amylase was not produced. The acid-unstable alpha-amylase was produced significantly when glycerol or glucose was used as a carbon source, similarly to the use of inducers such as starch or maltose. In liquid culture, A. kawachii assimilated starch at pH 3.0, but no alpha-amylase activity was recognized in the medium. Instead, the alpha-amylase was found to be trapped in the cell wall. The trapped form was identified as acid-unstable alpha-amylase. Usually, acid-unstable alpha-amylase is unstable at pH 3.0, so its stability appeared to be due to its immobilization in the cell wall. In solid-state culture, both kinds of alpha-amylase were produced. The production of acid-stable alpha-amylase seems to be solid-state culture-specific and was affected by the moisture content in the solid medium.     

Application of a statistical design to the optimization of parameters and culture medium for alpha-amylase production by Aspergillus oryzae CBS 819.72 grown on gruel (wheat grinding by-product)

The production optimization of alpha-amylase (E.C.3.2.1.1) from Aspergillus oryzae CBS 819.72 fungus, using a by-product of wheat grinding (gruel) as sole carbon source, was performed with statistical methodology based on three experimental designs. The optimisation of temperature, agitation and inoculum size was attempted using a Box-Behnken design under the response surface methodology. The screening of nineteen nutrients for their influence on alpha-amylase production was achieved using a Plackett-Burman design. KH(2)PO(4), urea, glycerol, (NH(4))(2)SO(4), CoCl(2), casein hydrolysate, soybean meal hydrolysate, MgSO(4) were selected based on their positive influence on enzyme formation. The optimized nutrients concentration was obtained using a Taguchi experimental design and the analysis of the data predicts a theoretical increase in the alpha-amylase expression of 73.2% (from 40.1 to 151.1 U/ml). These conditions were validated experimentally and revealed an enhanced alpha-amylase yield of 72.7%.     

Secretion of alpha-amylase and multiple forms of glucoamylase by the yeast Trichosporon pullulans

Trichosporon pullulans IGC 3488 produced extracellular alpha-amylase and glucoamylase activities when grown in batches in a medium containing corn steep liquor and soluble starch or corn starch. alpha-Amylase, unlike glucoamylase activity, was secreted biphasically. For both amylases the maximum concentration was found in stationary phase cultures. The amylolytic enzymes, previously concentrated by ammonium sulfate precipitation, were separated into a glucoamylase fraction and an alpha-amylase fraction by Ultrogel AcA 54 gel filtration. Pullulanase activity was located in the glucoamylase fraction, whereas cyclodextrinase activity was restricted to the alpha-amylase fraction. Isoamylase and alpha-glucosidase were not detected. Electrophoretic analysis showed that alpha-amylase activity was due to a single protein. Glucoamylase, however, occurred in multiple forms. The four glucoamylases and the alpha-amylase were glycoproteins.     

Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings

We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small molecule inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compounds from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymatic HTS assay was developed and optimized to experimentally screen approximately 41,000 compounds from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding analysis using surface plasmon resonance (SPR). The campaign identified a reversible small molecule inhibitor exhibiting mixed-type inhibition with a K_i value of 11.1 μM. Together, these results validate our protocols as suitable approaches to screen virtual and chemical libraries, and the newly identified compound reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development.     

Constitutive α-amylase producing mutant and recombinant haploid strains of thermophilic fungusThermomyces lanuginosus

Morphological, developmental and antimetabolite-resistant mutants of T. lanuginosus were characterized and used for screening with the aim to develop constitutive alpha-amylase-hyperproducing strains. The protoplast fusion of two spontaneous mutants of T. lanuginosus, characterized as asporulating and resistant to 2-deoxy-D-glucose (2DG), resulted in sporulating, 2DG sensitive heterokaryotic fusants. A recombinant haploid strain F64fB developed there from produced alpha-amylase constitutively in glucose-containing medium. Constitutive alpha-amylase-hyperproducing mutant (III8) obtained after cyclic mutagenesis and screening yielded approximately 20 fold more alpha-amylase in a glycerol-containing medium than the wild strain.     

Production of α-amylase by microscopic fungi

The amylolytic activity and especially the production of alpha-amylase (EC 3.2.1.1) and alpha-glucosidase (EC 3.2.1.20) was screened in imperfect fungi, mucoral fungi and some ascomycetes. The character of the polysaccharide system, which is responsible for the utilization of alpha (1 to 4) glucan, was specified with a concomitant screening of growth on soluble starch. The amylolytic activity was found in 29 strains out of the 49 tested.     

Restriction map and alpha-amylase activity variation among Drosophila mutation accumulation lines

The specific activities of alpha-amylase were measured for two sets of mutation accumulation lines, each set having originated from a different lethal-carrying second chromosome and SM1(Cy) chromosome and having been maintained by a balanced lethal system for about 300 generations. Significant variation was found to have accumulated among lines of both sets. Because of dysgenic crosses in the early generations of mutation accumulation, insertions or deletions of transposable elements in the Amy gene region were suspected of being the cause of this variation. In order to test this possibility, the structural changes in the 14 kb region of these chromosomes that includes the structural genes for alpha-amylase were investigated by restriction map analysis. We found that most part of the activity variation is due to replacements of a chromosomal region of SM1(Cy), including the structural genes for alpha-amylase, by the corresponding regions of the lethal chromosomes. One line also contained an insertion in this region but this line has an intermediate activity value. Thus, insertions of transposable elements into the Amy gene region were not found to be responsible for the new variation observed in alpha-amylase activity. If we remove those lines with structural changes from the analysis, the genetic variance of alpha-amylase specific activity among lines becomes non-significant in both sets of chromosomes.     

Characterisation of a thermostable alpha-amylase from Bacillus brevis.

Biochemical characterization of a novel heat-stable alpha-amylase, produced by a thermophilic strain of Bacillus brevis, has been made. The pattern of the enzyme action on different substrates was studied. It was found that reducing groups were rapidly liberated from amylopectin, soluble and insoluble starch compared to amylose and glycogen. B. brevis alpha-amylase acted via endo-attack producing mainly maltopentaose during the first hour of hydrolysis. The enzyme showed high activity towards maltohexaose and maltoheptaose. The alpha-amylase from B. brevis had a neutral pI and was found to be a glycoprotein, containing 9.2% (by mass) neutral sugars. The enzyme protein possessed a unique high glycine content. Calcium or sodium ions in appropriate concentrations were required for enzyme thermostability.     

Discovering severe acute respiratory syndrome coronavirus 3CL protease inhibitors: virtual screening, surface plasmon resonance, and fluorescence resonance energy transfer assays

An integrated system has been developed for discovering potent inhibitors of severe acute respiratory syndrome coronavirus 3C-like protease (SARS-CoV 3CL(pro)) by virtual screening correlating with surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) technologies-based assays. The authors screened 81,287 small molecular compounds against SPECS database by virtual screening; 256 compounds were subsequently selected for biological evaluation. Through SPR technology-based assay, 52 from these 256 compounds were discovered to show binding to SARS-CoV 3CL(pro). The enzymatic inhibition activities of these 52 SARS-CoV 3CL(pro) binders were further applied to FRET-based assay, and IC(50) values were determined. Based on this integrated assay platform, 8 new SARS-CoV 3CL(pro) inhibitors were discovered. The fact that the obtained IC(50) values for the inhibitors are in good accordance with the discovered dissociation equilibrium constants (K(D)s) assayed by SPR implied the reliability of this platform. Our current work is hoped to supply a powerful approach in the discovery of potent SARS-CoV 3CL(pro) inhibitors, and the determined inhibitors could be used as possible lead compounds for further research.     

Discovery of loperamide as an antagonist of angiopoietin1 and angiopoietin2 by virtual screening

The angiopoietin-Tie2 binding and related signal transduction pathways are crucial for vascular angiogenesis, blood vessel integrity and maturation. In this study, we preformed a virtual screening of small molecules targeting to Tie2. The binding site was selected at the extracellular ligand binding region of Tie2, rather than its conventional endocellular ATP binding region. It was found that loperamide, a widely-used antidiarrhea drug, was among the top hits. The binding between loperamide and Tie2 was confirmed by surface plasmon resonance (SPR) assay. Loperamide competitively inhibited the binding of both angiopoietin1 and angiopoietin2. These results indicate that loperamide is an antagonist of angiopoietin1 and angiopoietin2.