Computational determination of hERG-related cardiotoxicity of drug candidates.

BACKGROUND: Drug candidates often cause an unwanted blockage of the potassium ion channel of the human ether-a-go-go-related gene (hERG). The blockage leads to long QT syndrome (LQTS), which is a severe life-threatening cardiac side effect. Therefore, a virtual screening method to predict drug-induced hERG-related cardiotoxicity could facilitate drug discovery by filtering out toxic drug candidates. RESULT: In this study, we generated a reliable hERG-related cardiotoxicity dataset composed of 2130 compounds, which were carried out under constant conditions. Based on our dataset, we developed a computational hERG-related cardiotoxicity prediction model. The neural network model achieved an area under the receiver operating characteristic curve (AUC) of 0.764, with an accuracy of 90.1%, a Matthews correlation coefficient (MCC) of 0.368, a sensitivity of 0.321, and a specificity of 0.967, when ten-fold cross-validation was performed. The model was further evaluated using ten drug compounds tested on guinea pigs and showed an accuracy of 80.0%, an MCC of 0.655, a sensitivity of 0.600, and a specificity of 1.000, which were better than the performances of existing hERG-toxicity prediction models. CONCLUSION: The neural network model can predict hERG-related cardiotoxicity of chemical compounds with a high accuracy. Therefore, the model can be applied to virtual high-throughput screening for drug candidates that do not cause cardiotoxicity. The prediction tool is available as a web-tool at http://ssbio.cau.ac.kr/CardPred .

In silico prediction of potential chemical reactions mediated by human enzymes.

BACKGROUND: Administered drugs are often converted into an ineffective or activated form by enzymes in our body. Conventional in silico prediction approaches focused on therapeutically important enzymes such as CYP450. However, there are more than thousands of different cellular enzymes that potentially convert administered drug into other forms. RESULT: We developed an in silico model to predict which of human enzymes including metabolic enzymes as well as CYP450 family can catalyze a given chemical compound. The prediction is based on the chemical and physical similarity between known enzyme substrates and a query chemical compound. Our in silico model was developed using multiple linear regression and the model showed high performance (AUC = 0.896) despite of the large number of enzymes. When evaluated on a test dataset, it also showed significantly high performance (AUC = 0.746). Interestingly, evaluation with literature data showed that our model can be used to predict not only enzymatic reactions but also drug conversion and enzyme inhibition. CONCLUSION: Our model was able to predict enzymatic reactions of a query molecule with a high accuracy. This may foster to discover new metabolic routes and to accelerate the computational development of drug candidates by enabling the prediction of the potential conversion of administered drugs into active or inactive forms.

ANTI-NOCICEPTIVE EFFECT OF AGRIMONIA EUPATORIA EXTRACT ON A CISPLATIN-INDUCED NEUROPATHIC MODEL.

BACKGROUND: Natural products including Agrimonia eupatoria are considered an incomparable source of molecular diversity that has led to the medicines, especially for pain treatment. To investigate the antinociception of Agrimonia eupatoria, we examined its activity in a rat model of cisplatin neuropathy. MATERIALS AND METHODS: Male Sprague-Dawley rats received intraperitoneal (i.p.) cisplatin twice a week at a dose of 2 mg/kg (cumulative dose, 20 mg/kg) for 4 weeks. Before each injection, 2 ml of sterile saline solution was given subcutaneously to prevent renal damage via hyperhydration. The mice were treated with gabapetin as a positive control drug with a 100mg/kg intraperitoneal injection. A. eupatoria extract of 200mg/kg was solved in saline and then treated by oral administration. RESULTS: The mice treated with A. eupatoria showed lower withdrawal duration in the pin-prick and plantar tests, and a higher withdrawal threshold in the paw-withdrawal threshold test as compared to control animals in a cisplatin-induced neuropathic model. In the case of cold-allodynia, A. eupatoria treatment increased paw-withdrawal duration in a chemical test. A. eupatoria showed a more outstanding effect than gabapentin in all used tests for preventing cisplatin-induced nerve injury for 4 weeks. CONCLUSIONS: Our results suggest that A. eupatoria extract showed an antinociceptive effect in the pin-prick test, plantar test, and paw-withdrawal threshold test using a cisplatin-induced neuropathic rat model.

Antimalarial activity of nepodin isolated from Rumex crispus.

The purpose of this study is to define the antimalarial activity of Rumex crispus. To identify an active compound that is isolated from R. crispus, bioassay-based chromatographic fractionation and purification is carried out from 70 % ethanol extract of R. crispus; then, an active compound, nepodin, is identified by spectroscopic analysis. Anitmalarial activity is measured by PfNDH2 assay, cytotoxicity, and animal test. From NADH:quinone oxidoreductase enzyme (PfNDAH2) assay, nepodin exhibited significant IC50 values that were 0.74 ± 0.07 and 0.79 ± 0.06 µg/ml against P. falciparum chloroquine-sensitive (3D7) and P. falciparum chloroquine-resistant (S20), respectively. Nepodin showed a potential selective inhibition (SI index: ratio of 50 % cytotoxic concentration to 50 % effective anti-plasmodial concentration) of 161.6 and 151.4 against P. falciparum 3D7 and P. falciparum S20. In the animal test, all groups of nepodin treatment of 10, 50, and 250 mg/kg were active with a parasitemia suppression of 97.1 ± 3.3, 99.1 ± 3.7, and 99.1 ± 2.6 %, respectively. The survival time with nepodin treatment was increased by 14.6 ± 2.5, 16.2 ± 1.5, and 19.8 ± 1.7 days at each dose, respectively. This study newly identified the plant R. crispus containing nepodin, which is a potential antimalarial compound. It exhibited the inhibitory activity of PfNDH2 and prolonged the survival time on the group of nepodin treatment; moreover, it inhibited the parasitemia in the animal test.

Mucilaginibacter herbaticus sp. nov., isolated from the rhizosphere of the medicinal plant Angelica sinensis.

A strictly aerobic, Gram-staining-negative, non-motile and rod-shaped bacterial strain, DR-9(T), was isolated from rhizosphere soil of the medicinal herb Angelica sinensis. Strain DR-9(T) grew at 20-40 °C, at pH 4.0-9.0 and in the presence of 0-1 % (w/v) NaCl. The major fatty acids were iso-C15 : 0 and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c and/or iso-C15 : 0 2-OH), MK-7 was the major isoprenoid quinone, and phosphatidylethanolamine and an unidentified aminophospholipid were the major polar lipids. A phylogenetic tree based on 16S rRNA gene sequences showed that strain DR-9(T) formed a lineage within the genus Mucilaginibacter and was closely related to Mucilaginibacter polysacchareus DRP28(T) (96.1 % sequence similarity), Mucilaginibacter myungsuensis HMD1056(T) (95.9 % sequence similarity), Mucilaginibacter ximonensis XM-003(T) (95.8 %) and Mucilaginibacter boryungensis BDR-9(T) (95.1 %). The status of strain DR-9(T) as a representative of a separate species was confirmed by DNA hybridization, with 38.6, 36.3 and 29.9 % DNA-DNA relatedness with M. polysacchareus DRP28(T), M. ximonensis XM-003(T) and M. boryungensis BDR-9(T), respectively. The genomic DNA G+C content of strain DR-9(T) was 49.8 %. These data suggest that strain DR-9(T) should be considered as a representative of a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter herbaticus sp. nov. is proposed. The type strain is DR-9(T) ( = KACC 16469(T) = NBRC 108839(T)).

Protective effects of fucoidan against γ-radiation-induced damage of blood cells.

Fucoidan, a sulfated polysaccharide purified from brown algae including Fucus vesiculosus and Laminaria japonica, has a variety of biological activities, including antioxidant and antitumor activities. Here, we investigated the radioprotective effects of fucoidan on human monoblastic leukemia cell line U937. Further, animal tests were carried out using Balb/c mice in order to determine the radiation-induced changes in the counts of blood cells, including thrombocytes, erythrocytes, leukocytes and hematocrit. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wherein fucoidan (1, 10, and 100 µg/mL) was observed to improve recovery from damage caused by 8-Gy radiation in a dose dependent manner. The viability of U937 cells pre-treated with fucoidan also increased in a dose dependent manner. Furthermore, fucoidan at 100 mg/kg was found to protect against changes in the counts of blood cells as follows: on day 28 after irradiation, the thrombocyte count in the irradiated controls decreased to 45% compared with the non-irradiated controls, while that in the fucoidan-treated group was 60%. The hematocrit in the fucoidan-treated group recovered to 75% on day 28, while that in the irradiated control was 68%. The erythrocyte count in the irradiated controls consistently ranged from 64% to 67% throughout the experiment, but that in the fucoidan-treated group increased gradually, ranging from 75% to 80%. The mean number of survival days and 50-day actuarial survival rate increased dose dependently in the fucoidan-treated group. The mean number of survival days and the 50-day actuarial survival rate in this group was 16, 21, and 29 days and 12%, 20%, and 30% at fucoidan doses of 1, 10, and 100 mg/kg. The values of these parameters in the control group were 9 days and 0%, although the difference between the test and control groups was not statistically significant. Our results may prove valuable in the field of radioprotection.

Identification of Streptomyces sp. KH29, which produces an antibiotic substance processing an inhibitory activity against multidrug-resistant Acinetobacter baumannii.

The Actinomycete strain KH29 is antagonistic to the multidrug-resistant Acinetobacter baumannii. Based on the diaminopimelic acid (DAP) type, and the morphological and physiological characteristics observed through the use of scanning electron microscopy (SEM), KH29 was confirmed as belonging to the genus Streptomyces. By way of its noted 16S rDNA nucleotide sequences, KH29 was found to have a relationship with Streptomyces cinnamonensis. The production of an antibiotic from this strain was found to be most favorable when cultured with glucose, polypeptone, and yeast extract (PY) medium for 6 days at 27 degrees C. The antibiotic produced was identified, through comparisons with reported spectral data including MS and NMR as a cyclo(L-tryptophanyl-L-tryptophanyl). Cyclo(L-Trp-L-Trp), from the PY cultures of KH29, was seen to be highly effective against 41 of 49 multidrugresistant Acinetobacter baumannii. Furthermore, cyclo(LTrp- L-Trp) had antimicrobial activity against Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Saccharomyces cerevisiae, Aspergillus niger, and Candida albicans, However, it was ineffective against Streptomyces murinus.

Radioprotective effect of cyclo(L-phenylalanyl-L-prolyl) on irradiated rat lung.

In the present study, we investigated the radioprotective effect of cyclo(L-phenylalanyl-L-prolyl) on irradiated rat lungs to determine its potential as a radioprotective agent. We found that early lung damage induced by irradiation was reduced by treatment with 40 mg/kg of cyclo(Lphenylalanyl- L-prolyl) in the latent and early pneumonitis phases. Expression of TNF-alpha and TGF-beta1 at 2 and TGF-beta1 at 8 weeks post-irradiation was decreased in animals that received both radiation and cyclo(L-phenylalanyl-L-prolyl) compared with animals that received radiation alone. Evidence indicated that the proinflammatory cytokine TNF-alpha and the fibrogenic cytokine TGF-beta1 likely play a role in the radioprotective effect of cyclo(L-phenylalanyl-L-prolyl). However, besides TNF-alpha and TGF-beta1 expressions, the precise mechanism by which cyclo(L-phenylalanyl-L-prolyl) ameliorates the induced radiation damage is not clear.

Correlative effect between in vivo hollow fiber assay and xenografts assay in drug screening.

PURPOSE: This study was carried out to assess the usage of an in vivo hollow fiber assay to screen drugs with highly predictive accuracy. MATERIALS AND METHODS: The assay systems used were the hollow fiber and xenografts assays. The hollow fiber assay was carried out with the following steps; preparation of fibers, preparation of cells, loading and implanting fibers, treatment with drugs, removal of fibers and assaying for the cell viability by the MTT assay. For the xenografts assay, cell suspensions were subcutaneously transplanted into the mice. Therapy was started when the tumor volume reached 100 approximately 200 mm(3). The tumor volumes were calculated using the formula V=[length+(width)(2)]/2, and used for evaluating the efficacy of the drugs. The drug treatment doses used were adriamycin 2.1 mg/kg, mitomycin-C 0.25 mg/kg, 5-fluorouracil 24.5 mg/kg and paclitaxel 2.5 mg/kg, and administrated intravenously five times daily. RESULTS: The correlation between the xenografts and hollow fiber assays was evaluated in 20 tumor cell lines and 4 anti-cancer agents. In the 20 tumor cell lines, the overall predictive accuracy of the hollow fiber assay for sensitivity was 83%, with a predictive accuracy for resistance of 92%. CONCLUSION: The hollow fiber assay was assessed as effective in drug efficacy evaluation, and found to be compatible with that of the xenografts assay.

Cyclic dipeptides exhibit synergistic, broad spectrum antimicrobial effects and have anti-mutagenic properties.

Cyclic dipeptides are known to have antiviral, antibiotic and antitumour properties. The aim of this study was to determine the combined effects of cyclo(L-leucyl-L-prolyl) and cyclo(L-phenylalanyl-L-prolyl) on the growth of vancomycin-resistant enterococci (VRE) and pathogenic yeasts, as well as determining their anti-mutagenic effects. This drug combination was especially effective against five VRE strains: Enterococcus faecium (K-99-38), E. faecalis (K-99-17), E. faecalis (K-99-258), E. faecium (K-01-312) and E. faecalis (K-01-511) with MIC values of 0.25-1 mg/l. It was also effective against Escherichia coli, Staphylococcus aureus, Micrococcus luteus, Candida albicans and Cryptococcus neoformans with MIC values of 0.25-0.5 mg/l. In addition, the cyclic dipeptides exerted anti-mutagenic activity against Salmonella typhimurium TA98 and TA100 strains in a Salmonella mutation assay. The number of mutant colonies of S. typhimurium strains TA98 and TA100 induced by exposure to AF-2 (0.2 microg/plate) decreased in a concentration-dependent manner in the presence of the two cyclic dipeptides (correlation 0.72 and 0.78, respectively). Here, for the first time, we report synergistic effects of the cyclic dipeptides [cyclo(L-leu-L-pro) and cyclo(L-phe-L-pro)] in inhibiting the growth of pathogenic microorganisms, as well as their anti-mutagenic effects in Salmonella strains.

Cloning, expression, and characterization of 5-aminolevulinic acid synthase from Rhodopseudomonas palustris KUGB306.

The hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned from the genomic DNA of photosynthetic bacterium Rhodopseudomonas palustris KUGB306. The deduced protein (ALAS) of this gene contained 409 amino acids. The hemA gene was subcloned into an expression vector pGEX-KG and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli BL21. The recombinant ALAS was purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose 4B resin and cleavage of the purified fusion protein by thrombin protease. The optimum pH and temperature of the recombinant ALAS was found to be at pH 7.5-8.0 and 35-40 degrees C, respectively. The Km value of the enzyme was 2.01 mM for glycine and 49.55 microM for succinyl-CoA. The enzyme activity was strongly inhibited by Pb2+, Fe2+, Co2+, Cu2+, and Zn2+ at 1 mM, but slightly affected by Mg2+ and K+. The recombinant ALAS required pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor led to complete loss of the activity. Ultraviolet-visible spectroscopy with the ALAS suggested the presence of an aldimine linkage between the enzyme and PLP.

Differential distribution of GABA(B1) and GABA(B2) receptor mRNAs in the rat brain.

Gamma-aminobutyric acid receptors (GABA(B)Rs) are G-protein coupled receptors mediating the slow-onset and prolonged synaptic actions of GABA in the central nervous system (CNS). There are two subtypes, GABA(B1)R and GABA(B2)R, that are though to form heterodimers, with the GABA(B1)R subunit essential for ligand binding and the GABA(B2)R subunit for cell surface localization. We have analyzed the distribution of GABA(B1)R and GABA(B2)R transcripts by in situ hybridization. The two transcripts were generally expressed in parallel, and the highest levels were detected in the piriform cortex, hippocampus, medial habenula, and olfactory bulb. There was moderate expression in several thalamic nuclei and the cortex area. In contrast to the distribution of GABA(B2)R mRNA, GABA(B1)R gave weak signals throughout the thalamus, piriform cortex, and field CA2 of the hippocampus, but strong signals in the septum, superior colliculus, retrosplenial cortex area, and field CA1 of the hippocampus. Thus GABA(B2)R and GABA(B1)R mRNA expression overlapped considerably, consistent with the suggestion that the two subtypes assemble to form a heterodimer. There are nevertheless differences in their expression levels that suggest at the existence of unidentified receptor subtypes.

Inhibition of complement activation by recombinant Sh-CRIT-ed1 analogues.

Sh-CRIT-ed1 is a potent anti-complement peptide that inhibits the classical complement-activation pathway by interfering with the formation of the C3-convertase complex, C4b2a. C2 is an essential serum glycoprotein that provides the catalytic subunit of the C3 and C5 convertases of the classical pathways of complement activation. Because only in its C4-bound state is C2a capable of cleaving its physiological protein substrates C3 and C5, the interaction of Sh-CRIT-ed1 with C2 plays a decisive role of inhibition in the classical complement-activation process. However, the role of individual Sh-CRIT-ed1 amino acid residues in C2 binding is not fully understood. We constructed nine recombinant Sh-CRIT-ed1 (rSh1) analogues, substituted at conserved residues, and evaluated their anti-complement and C2-binding activities. Results from glutathione S-transferase (GST) pull-down and haemolytic assays suggested that residues 10K, 17E, 19K and 26Y are critical for the interaction of rSh1 with C2. We then constructed an improved anti-complement peptide by duplicating Sh-CRIT-ed1 C-terminal motifs (17H-26Y). This linear homodimer (rH17d) was more potent than rSh1 with respect to binding to C2 and anti-complement activity (the 50% inhibitory concentration value was approximately equal 1.2 micro m versus approximately equal 6.02 micro m for rSh1). Furthermore, rH17d showed higher anti-complement activity in vivo, providing additional evidence that this duplication is a more effective inhibitor of complement activation than rSh1. Taken together, these results identify four key residues in rSh1 and strongly suggest that rH17d is a potent inhibitor of complement activation that may have therapeutic applications.

Caffeine-induced neuronal death in neonatal rat brain and cortical cell cultures.

We examined the potential neurotoxicity of caffeine and. Intraperitoneal administration of caffeine (50 mg/kg, 3 times a day) produced neuronal death in various brain areas of neonatal rats 24 h later. Caffeine at doses > 300 microM was also neurotoxic in murine cortical cell cultures. Caffeine-induced neuronal death was accompanied by cell body shrinkage and attenuated by anti-apoptotic drugs including cycloheximide, high potassium, and growth factors. Two necrotic pathways, excitotoxicity and oxidative stress, did not mediate caffeine neurotoxicity. The pro-apoptotic protease caspase-3 was activated to mediate neuronal death following exposure to caffeine. The present findings suggest that caffeine may cause caspase-3-dependent neuronal cell apoptosis in neonatal rat as well as.

A hot pepper cDNA encoding ascorbate peroxidase is induced during the incompatible interaction with virus and bacteria.

Capsicum annuum L. is infected by a number of viruses, including the tobacco mosaic virus (TMV). To study the defense-related genes that are induced by TMV in hot peppers, the pepper plant, which is susceptible to P1.2 but resistant to the P0 pathotype of TMV, was inoculated with TMV-P0. Differential screening isolated the genes that were specifically up- or down-regulated during the hypersensitive response (HR). The CaAPX1 cDNA clone that putatively encodes a polypeptide of cytosolic ascorbate peroxidase was selected as an up-regulated gene. It was isolated for further study. The full-length cDNA for CaAPX1, which is 972 bp long, contained the open-reading frame of 250-amino acid residues. A genomic Southern blot analysis showed that there were only limited copies of the CaAPX1 gene in the hot pepper genome. In hot pepper cv. Bugang, which is resistant to TMV-P0 and susceptible to TMV-P1.2, the CaAPX1 gene transcript was accumulated by TMV-P0, but not by TMV-P1.2 inoculation. CaAPX1 transcripts began to accumulate 24 h post-inoculation of TMV-P0, and increased gradually until 96 h. To investigate whether each transcript is induced by other stimuli, the plants were treated with various chemicals and wounding. A striking induction of the CaAPX1 transcript was observed at 2 h. It subsided 12 h after salicylic acid (SA), ethephon, and methyl jasmonate (MeJA) treatments. The response of the gene upon other pathogen infection was also examined by a bacterial pathogen (Xanthomonas campestris pv. vesicatoria race 3) inoculation. The CaAPX1 gene was induced in a hot pepper (C. annuum cv. ECW 20R) that was resistant to this bacterial pathogen, but not in a susceptible hot pepper (C. annuum cv. ECW). These results suggest the possible role(s) for the CaAPX1 gene in plant defense against viral and bacterial pathogen.

Refolding of the catalytic and hinge domains of human MT1-mMP expressed in Escherichia coli and its characterization.

The catalytic and hinge domain (Tyr112-Ile318) of the human membrane type-1 matrix metalloproteinase (MT1-MMP; MMP-14), containing hexa-histidines at the C-terminus (chMT1-MMP), was overexpressed in Escherichia coli. The expressed polypeptide was almost exclusively found in the inclusion body, and then purified by a single Ni2+-NTA agarose column chromatography after solubilization with 6 M urea. During refolding, the 26.9 kDa chMT1-MMP was processed to a 24.3 kDa intermediate form and then to a 22.2 kDa mature form. By Western blot analysis and mass spectrometry combined with N-terminal sequencing, the intermediate form was identified as a mixture of the Tyr112-Thr299 with a translation-initiating methionine and Ile114-Thr299, and that the mature form corresponds to Ile114-Pro290. These results demonstrate that the mature form was generated by successive autoproteolysis of the N- and C-terminal sites between Thr299-Thr300, Ala113-Ile114, and Pro290-Thr291 during refolding. Catalytic activity of the mature chMT1-MMP was demonstrated by a peptide cleavage assay. In addition, it has gelatinolytic activity and is able to activate proMMP-2 to the mature MMP-2. These results indicate that the refolded chMT1-MMP retains characteristics of MT1-MMP.

Isolation and characterization of Streptomyces sp KH-614 producing anti-VRE (vancomycin-resistant enterococci) antibiotics.

The actinomycete strain KH-614 was antagonistic to vancomycin-resistant enterococci (VRE). Based on the diaminopimelic acid (DAP) type, morphological and physiological characteristics examined by scanning electron microscopy (SEM), KH-614 was confirmed as belonging to the genus Streptomyces. Based on the 16S rDNA nucleotide sequences, Streptomyces sp. KH-614 was found to have a relationship with Streptomyces lydicus. The production of antibiotic from this strain was most favorable when cultured in glucose, polypeptone, yeast extract (PY) medium for 6 days at 27 degrees C. The antibiotic was identified as a cyclo(L-leucyl-L-prolyl) by comparing it with the reported spectral data including MS and NMR. Cyclo(leu-pro) was found to be active against twelve VRE strains, including E. faecium (vanA, vanB), and E. faecalis (vanA, vanB), that had been isolated over a period three years (1998-2000). Cyclo(leu-pro) was especially effective against VRE strains such as E. faecalis (K-99-34), E. faecalis (K-00-184), E. faecalis (K-00-221), and the MIC values were 12.5 microg/ml. Moreover, cyclo(leu-pro) was effective against three leukemic cell lines at concentrations below 100 microg/ml. At 100 mg/ml cyclo(leu-pro), K562, HL60, and U937 leukemic cell lines showed growth inhibition of 95, 91, and 93%, respectively. In a normal cell line, MDBK, cyclo(leu-pro) exerted 24% growth inhibition at a concentration of 100 microg/ml, and showed no inhibitory activity at concentrations below 10 microg/ml. These results indicate that cyclo(leu-pro) is a potential anti-leukemic and anti-VRE agent.