Computer automated structure evaluation (CASE): a study of inhibitors of the thermolysin enzyme

The Computer Automated Structure Evaluation (CASE) program has been applied to the analysis of the inhibition of the thermolysin enzyme by derivatives of di- and poly-peptides. The inhibition constant ki, was used as a measure of the activity of the inhibitors. The program successfully identified molecular fragments relevant to the inhibitory activity of the peptides, without any assumption regarding the mechanism of inhibitory action. Utilizing these major fragments, Quantitative Structure Activity Relationship (QSAR) calculations were performed yielding a multiple linear regression equation for the prediction of inhibitory activity. A comparison of the conclusions reported in the literature regarding the structural features involved in the inhibition of thermolysin with the major fragments identified by the program is also made.     

A CASE-SAR analysis of polycyclic aromatic hydrocarbon carcinogenicity

A CASE SAR analysis was performed on a selected database of PAHs to investigate the possible use of the CASE method as an aid for preliminary assessment of carcinogenic potential of untested environmental PAHs. A data set, denoted LEARN, consisting of 78 PAHs and their experimental carcinogenicities was used to 'train' the CASE method and derive the CASE fragments. 8 activating fragments and 4 inactivating fragments were identified. These fragments predicted the activities of 94% of the LEARN set correctly. The biological significance of several of these fragments are rationalized in light of the current theories of PAH carcinogenesis. Using these fragments, the potential activities of a database of 106, mostly untested PAHs, denoted TEST, were predicted. These were compared to 'expert judgement' predictions based on mechanistic considerations in order to evaluate the extent of concordance between these two methods and their respective strengths and weaknesses. Initial poor agreement (64%) was attributed to limitations of the LEARN database involving inadequate representation of 2- and 3-ring PAH subclasses. When these subclasses were excluded from the TEST database, the concordance improved to 90%. The CASE fragments were also used to predict the activities of a database of 24 PAHs, denoted VALIDATE (not included in the LEARN set) for which carcinogenicity data were available. The total prediction accuracy of 75% (89% of the actives correctly identified), despite the structural diversity of the VALIDATE set, provided independent evidence of the utility of the present CASE results. A close examination of the CASE incorrect predictions was conducted to delineate inadequancies of these CASE results in order to provide cautionary guidance for future application of the method. Finally, the present results were compared to the results of a previous CASE analysis based on a more limited PAH data set, and were found to be of greater general utility. It is concluded that the CASE fragments derived in the current study should provide a useful tool for assisting and complementing 'expert judgement' in the preliminary screening of PAHs for carcinogenic activity.     

Structural requirements for the mutagenicity of environmental nitroarenes

The nitroarenes comprise a large group of widely distributed environmental agents some of which are extraordinarily mutagenic while others are devoid of such activity. A newly developed computer program has been used to determine which structural features of these molecules might account for this broad spectrum of activities. Using Salmonella mutagenicity data for 53 nitroarenes, 2 fragments associated with activity and 2 deactivating fragments have been identified. The coexistence of an active and a deactivating fragment on the same molecule results in a nitroarene possessing marginal or no mutagenicity. The activity of 47 of 53 nitroarenes was correctly predicted by this procedure. Most of the discrepancies involved hexa- and heptacyclic nitroarenes which were predicted to be active but reported to be inactive. The ‘CASE’ program can be used to predict the mutagenicity of the many untested nitroarenes identified in the ambient environment.     

Structure-genotoxic activity relationships of pesticides: comparison of the results from several short-term assays

The Computer-Automated Structure Evaluation (CASE) program has been applied to the analysis of the genotoxic activity of 54 pesticides (31 insecticides, 15 herbicides and 8 fungicides) in 5 different short-term test systems measuring gene mutation and DNA damage. The database contains compounds presenting diverse structures including carbamates, thiocarbamates, organophosphates, halo-aromatics and other functionalities. Some significant relationships between common structural features and the genotoxic activity displayed by these chemicals have been found. Among the most relevant fragments, automatically selected by the program, a methoxyphosphinyl and a chlorovinyl group appear as the common structural subunits responsible for the activities detected in the battery composed of the Salmonella typhimurium histidine reversion assay, the mouse lymphoma gene mutation assay and recombination in the yeast Saccharomyces cerevisiae.     

Structural requirements for the induction of the SOS repair in bacteria by nitrated polycyclic aromatic hydrocarbons and related chemicals.

The CASE (computer-automated structure evaluation) methodology was used to investigate the structural basis of the SOS-inducing activity of 56 nitrated polycyclic aromatic hydrocarbons (nitroarenes, nPAH) and the unsubstituted parent PAH molecules. Based upon the presence and/or absence of structural features, CASE identified 5 activating (biophores) and 4 inactivating (biophobes) fragments responsible for the SOS-inducing activity. Based upon these fragments, CASE correctly calculated the genotoxicity of 94.6% of the molecules in the training set (sensitivity = 0.85, specificity = 1.0). Disregarding the questionable experimental results of the unexpected very weak direct-acting activity of the unsubstituted benzo[a]pyrene, dibenzo[a,h]anthracene and 7,12-dimethylbenz[a]anthracene, the concordance of the prediction was 100%, i.e., sensitivity = 1.0, specificity = 1.0. Additionally, the quantitative analysis of the SOS-inducing potency showed a good correlation between the experimental and predicted results. The present analyses indicate an identity in the structural determinants responsible for SOS induction in E. coli PQ37 (SOS chromotest) and mutagenicity in Salmonella typhimurium.     

Structural basis of the in vivo induction of micronuclei.

The structural basis of the in vivo induction of micronuclei was examined with CASE, a structure-activity relational method. The CASE program identified a number of structures associated with this activity. When used to predict the activity of chemicals not included in the learning set, these structural determinants gave a concordance in excess of 83%. The existence of a structural basis for the induction of micronuclei will permit an investigation of the mechanistic basis of this phenomenon.     

Structural basis of carcinogenicity in rodents of genotoxicants and non-genotoxicants

A set of 189 chemicals tested in the National Toxicology Program Cancer Bioassay was subjected to analysis by CASE, the Computer-Automated Structure Evaluation system. In the data set, 63% of the chemicals were carcinogens, approx. 40% of the carcinogens were non-genotoxic, i.e., they possessed neither "structural alerts" for DNA reactivity as defined by Ashby and Tennant, 1988, nor were they mutagenic for Salmonella. The data base can be characterized as a "combined rodent" compilation as chemicals were characterized as "carcinogenic" if they were carcinogenic in either rats or mice or both. CASE identified 23 fragments which accounted for the carcinogenicity, or lack thereof, of most of the chemicals. The sensitivity and specificity were unexpectedly high: 1.00 and 0.86, respectively. Based upon the identified biophores and biophobes, CASE performed exceedingly well in predicting the activity of chemicals not included among the 189 in the original set. CASE predicted correctly the carcinogenicity of non-genotoxic carcinogens thereby suggesting a structural commonality in the action of this group of carcinogens. As a matter of fact biophores restricted to non-genotoxic carcinogens were identified as were "non-electrophilic" biophores shared by genotoxic and non-genotoxic carcinogens. The findings suggest that the CASE program may help in the elucidation of the basis of the action of non-genotoxic carcinogens.     

Recombinant antibodies: towards a new generation of antivenoms?

Poisoning by scorpion venoms is a major health hazard in tropical and subtropical regions and serum therapy, which was discovered in 1894, remains the only specific treatment. No real progress has been made since this time and the therapeutic use of antivenoms which still consists in polyclonal antibody fragments from the sera of immunized animals may be associated with major drawbacks. Protein engineering now allows to design novel recombinant antibody fragments which are superior to polyclonal antivenoms in homogeneity, specific activity and possibly safety. Several single-chain antibody fragments (scFvs) which neutralize scorpion toxins have been produced and characterized over the last few years. These scFvs can also be used as building blocks to engineer more complex structures including multivalent monospecific antibody fragments (diabodies, triabodies) and bispecific molecules (tandem-scFv). Some of these molecules neutralize scorpion neurotoxins and protect mice from experimental envenoming. Thus, research projects currently underway suggest that new strategies might soon be available to treat poisonings in the absence of socio-economic considerations.     

Identification of novel neuroprotective agents using pharmacophore modeling

In addition to its endocrine function, for which it was named, thyrotropin-releasing hormone (TRH) has substantial neuroprotective actions as well as other physiological effects. We have developed a number of modified TRH analogues as well as cyclic dipeptides structurally related to a major metabolic product of TRH, which have enhanced neuroprotective activity but none of the other major physiological effects of TRH. The extensive structure-activity data developed with these compounds were used to develop a pharmacophore model. Subsequently, a web-based pharmacophore searching program was used to query several large three-dimensional databases. Of the 219 compounds identified whose structures met the pharmacophore model, 15 were chosen for study in a classical model of neuronal cell death in vitro; five of these, 2-6, showed neuroprotective activity. Thus, pharmacophore modeling developed from neuroprotective small peptides can be used to identify novel lead compounds as neuroprotective agents.     

Structural basis of the mutagenicity of phenylazoaniline dyes

The CASE (Computer-Automated Structure Evaluation) methodology was used to gain an understanding of the basis of mutagenicity of phenylazoanilines. It was found that the activity of these molecules is dependent upon an intact moiety that spans the azo linkage, i.e., the azo bond must remain intact for mutagenicity. The study also addressed the effect of sulfonation on the activity of these azo dyes. It was revealed that sulfonation at only certain sites resulted in loss of mutagenicity. Sulfonation of the structural moiety responsible for the activity of phenylazoaniline dyes did not necessarily result in complete elimination of activity as this substitution could generate new structural moieties which contribute to the activity of the molecules.     

HCV抗原表位预测

应用网络生物信息资源查找丙型肝炎病毒基因组全序列,用软件Lasergene中的EditSeq将来自中国河北株mRNA序列翻译为氨基酸序列,尔后用程序Protean进行氨基酸序列分析,对HCV各区段的B细胞抗原指数进行预测。同时又在两个网站对中国汉族人中频率较高的HLA基因型进行CD8和CD4T细胞表位预测。B细胞和T细胞抗原表位预测结果对于HCV诊断试剂和疫苗研制有重要的指导意义。     

Design of peptide-based inhibitors of human islet amyloid polypeptide fibrillogenesis

Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in the pancreas of over 90% of all cases of type-2 diabetes. We have generated a series of overlapping hexapeptides to target an amyloidogenic region of IAPP (residues 20-29) and examined their effects on fibril assembly. Peptide fragments corresponding to SNNFGA (residues 20-25) and GAILSST (residues 24-29) were strong inhibitors of the beta-sheet transition and amyloid aggregation. Circular dichroism indicated that even at 1:1 molar ratios, these peptides maintained full-length IAPP (1-37) in a largely random coil conformation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP resulted in the formation of only semi-fibrous aggregates and loss of the typical high density and morphology of IAPP fibrils. This inhibitory activity, particularly for the SNNFGA sequence, also correlated with a reduction in IAPP-induced cytotoxicity as determined by cell culture studies. In contrast, the peptide NFGAIL (residues 22-27) enhanced IAPP fibril formation. Conversion to the amyloidogenic beta-sheet was immediate and the accompanying fibrils were more dense and complex than IAPP alone. The remaining peptide fragments either had no detectable effects or were only weakly inhibitory. Specificity of peptide activity was illustrated by the fragments, SSNNFG and AILSST. These differed from the most active inhibitors by only a single amino acid residue but delayed the random-to-beta conformational change only when used at higher molar ratios. This study has identified internal IAPP peptide fragments which can regulate fibrillogenesis and may be of therapeutic use for the treatment of type-2 diabetes.     

A novel molecular marker for the polyphenol oxidase gene located on chromosome 2B in common wheat

Polyphenol oxidase (PPO) is a major cause of time-dependent darkening and discoloration in Asian noodles and other wheat-based products. One of the best ways to reduce this undesirable darkening is to breed new wheat cultivars with low PPO activity using efficient and reliable markers. Based on the sequence of a PPO gene SSPPO-B1 (GenBank accession no. AB254804) located on chromosome 2B of common wheat, 26 pairs of primers were designed to detect polymorphisms between wheat cultivars with low and high PPO activity. F-8, one of these primer pairs, amplified double fragments (band ??a?? of approximately 400?bp and band ??b?? of approximately 600?bp) in the cultivars with low PPO activity, and a single fragment (only band a) in the cultivars with high PPO activity. The differences between the fragments a and b include five indels and several single nucleotide polymorphisms, which occurred in intron II of the PPO gene. F-8 can be used as a sequence-tagged site marker to discriminate between two alleles Ppo-B1a (GQ303713) and Ppo-B1b (AB254804). The screening of 284 accessions of the core collection of Chinese wheat germplasms using the marker F-8 showed that the double fragments were present in 188 accessions, and the single fragments were present in the remaining 96 accessions. Statistical analysis revealed that the cultivars with the double fragments had significantly lower mean PPO activity than those with the single fragments. We also screened the 284 accessions using two additional markers, PPO18 for Ppo-A1 on chromosome 2A and STS01 for Ppo-D1 on chromosome 2D. Results showed that the combination of markers F-8, PPO18, and STS01 could reliably predict PPO activity. These markers can be used in wheat breeding programs for low PPO activity selection to improve the quality of wheat-based products.     

Discovery of benzothiazole guanidines as novel inhibitors of thrombin and trypsin IV

In a project to find novel neutral P1 fragments for the synthesis of thrombin inhibitors with improved pharmacokinetic properties, fragments containing a benzothiazole guanidine scaffold were identified as weak thrombin inhibitors. WaterLOGSY (Water-Ligand Observed via Gradient SpectroscopY) NMR was used to detect fragments binding to thrombin and these fragments were followed up by Biacore A100 affinity measurements and enzyme assays. A crystal structure of the most potent compound with thrombin was obtained and revealed an unexpected binding mode as well as the key interactions of the fragment with the protein. Based on these results, the structure-based design and synthesis of a small series of optimized novel substituted benzothiazole guanidines with comparatively low pK(a) values was accomplished. Testing of these compounds against human trypsin I and human trypsin IV revealed unexpected inhibitory activity and selectivity of some of the compounds, making them attractive starting points for selective trypsin inhibitors.     

Effect of proteolytic digestion on the structure and function of human properdin.

Human properdin (P) was found to be sensitive to the action of trypsin, chymotrypsin, pepsin, and Streptomycetes caesipitosus protease. Incubation of P with these enzymes resulted in loss of its functional activity and the production of antigenically deficient components compared to untreated P. Upon incubation with trypin, P was initially cleaved into a minor fragment and a major fragment. Further degradation ot the fragments occurred with prolongation of inculation time. The minor fragment was highly susceptible to further proteolysis compared to the major fragment which contained the carbohydrate moiety of the molecule. SDS-polyacrylamide gel electrophoretic analysis of trypsin-digested P suggested that the subunit polypeptide chains were initially cleaved at similar points to produce the major and minor fragments. The sedimentation velocity of the major fragment was higher than that of the intact molecule. The implications of these observations of the configuration of P are discussed.     

Gene markers for grain polyphenol oxidase activity in common wheat

Polyphenol oxidase (PPO) in grain is regarded as a major factor resulting in time-dependent darkening of wheat end products, particularly for Asian noodles and steamed bread. Breeding wheat cultivars with low PPO activity using efficient and reliable markers is one of the best ways to reduce the undesirable darkening. In the present study, we developed a gene-specific marker (PPO05) for low PPO activity from the sequence AY515506. This marker detected double PCR fragments (<750 and >750 bp) in the cultivars with low PPO activity and a single PCR fragment (<750 bp) in the cultivars with high PPO activity. Screening of this marker on 235 Chinese wheat micro-core collections showed that the double fragments were present in 113 genotypes and the single fragments in the remaining 122 genotypes. Statistic analysis revealed that the cultivars with the double fragments had significantly lower mean PPO activity than those with single fragments. Through sequence analysis and blast search in NCBI, we found that the cultivars with the double fragments contained the PPO-2Ab allele, while the cultivars with the single fragments contained the PPO-2Aa allele. The PPO-2Ab and PPO-2Da alleles were associated with the low grain PPO activity and the PPO-2Aa and PPO-2Db alleles associated with the high PPO activity. The genotypes carrying both PPO-2Ab and PPO-2Da showed the lowest PPO activity, while the genotypes carrying both PPO-2Aa and PPO-2Db showed the highest PPO activity. Comparison of PPO05 and STS01 with the STS markers PPO18 and PPO29 showed that the larger and small fragments of PPO05 were equivalent to the 876- and 685-bp fragments of PPO18, respectively, and that STS01 was the complementary marker of PPO29. Thus, the STS markers PPO05 and STS01 along with PPO18 and PPO29 are the efficient and reliable markers for the evaluation of PPO activity and can be used in wheat breeding programs to improve the quality of noodles and other end products.     

Synthesis of E. faecium wall teichoic acid fragments

The first synthesis of different Enterococcus faecium wall teichoic acid (WTA) fragments is presented. The structure of these major cell wall components was elucidated recently and it was shown that these glycerolphosphate (GroP) based polymers are built up from -6-(GalNAc-α(1–3)-GalNAc-β(1–2)-GroP)- repeating units. We assembled WTA fragments up to three repeating units in length, in two series that differ in the stereochemistry of the glycerolphosphate moiety. The key GalNAc-GalNAc-GroP synthons, required for the synthesis, were generated from galactosazide building blocks that were employed in highly stereoselective glycosylation reactions to furnish both the α- and β-configured linkages. By comparing the NMR spectra of the synthesized fragments with the isolated material it appears that the hereto undefined stereochemistry of the glycerol phosphate moiety is sn-glycerol-3-phosphate. The generated fragments will be valuable tools to study their immunological activity at the molecular level.