Structural modulation study of inhibitory compounds for ribonuclease H activity of human immunodeficiency virus type 1 reverse transcriptase.

Reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) has two enzymatic functions. One of the functions is ribonuclease (RNase) H activity concerning the digestion of only RNA of RNA/DNA hybrid. The RNase H activity is an attractive target for a new class of anti-HIV drugs because no approved inhibitor is available now. In our previous studies, an agent bearing 5-nitro-furan-2-carboxylic acid ester core was found from chemical screening and dozens of the derivatives were synthesized to improve compound potency. In this work, some parts of the chemical structure were modulated to deepen our understanding of the structure-activity relationship of the analogous compounds. Several derivatives having nitro-furan-phenyl-ester skeleton were shown to be potent RNase H inhibitors. Attaching methoxy-carbonyl and methoxy groups to the phenyl ring increased the inhibitory potency. No significant cytotoxicity was observed for these active derivatives. In contrast, the derivatives having nitro-furan-benzyl-ester skeleton showed modest inhibitory activities regardless of attaching diverse kinds of functional groups to the benzyl ring. Both the modulation of the 5-nitro-furan-2-carboxylic moiety and the conversion of the ester linkage resulted in a drastic decrease in inhibitory potency. These findings are informative for designing potent inhibitors of RNase H enzymatic activity of HIV-1.

Mechanism of drug resistance of hemagglutinin of influenza virus and potent scaffolds inhibiting its function.

Highly pathogenic influenza viruses have become a global threat to humans. It is important to select an effective therapeutic option suitable for the subtypes in an epidemic or pandemic. To increase the options, the development of novel antiviral agents acting on targets different from those of the currently approved drugs is required. In this study, we performed molecular dynamics simulations on a spike protein on the viral envelop, hemagglutinin, for the wild-type and three kinds of mutants using a model system consisting of a trimeric hemagglutinin complex, viral lipid membrane, solvation waters, and ions. A natural product, stachyflin, which shows a high level of antiviral activity specific to some subtypes of influenza viruses, was examined on binding to the wild-type hemagglutinin by docking simulation. The compound potency of stachyflin is, however, easily lost due to resistant mutations. From a comparison of simulation results between the wild-type and the resistant mutants, the reason for the drug resistance of hemagglutinin was clarified. Next, 8 compounds were selected from a chemical database by in silico screening, considering the findings from the simulations. Inhibitory activities to suppress the proliferation of influenza virus were measured by cell-based antiviral assays, and two chemical scaffolds were found to be potent for an inhibitor. More than 30 derivatives bearing either of these two chemical scaffolds were synthesized, and cell culture assays were carried out to evaluate the compound potency. Several derivatives displayed a high compound potency, and 50% effective concentrations of two synthesized compounds were below 1 µM.

Structural and biochemical study on the inhibitory activity of derivatives of 5-nitro-furan-2-carboxylic acid for RNase H function of HIV-1 reverse transcriptase.

Rapid emergence of drug-resistant variants is one of the most serious problems in chemotherapy for HIV-1 infectious diseases. Inhibitors acting on a target not addressed by approved drugs are of great importance to suppress drug-resistant viruses. HIV-1 reverse transcriptase has two enzymatic functions, DNA polymerase and RNase H activities. The RNase H activity is an attractive target for a new class of antiviral drugs. On the basis of the hit chemicals found in our previous screening with 20,000 small molecular-weight compounds, we synthesized derivatives of 5-nitro-furan-2-carboxylic acid. Inhibition of RNase H enzymatic activity was measured in a biochemical assay with real-time monitoring of florescence emission from the digested RNA substrate. Several derivatives showed higher inhibitory activities that those of the hit chemicals. Modulation of the 5-nitro-furan-2-carboxylic moiety resulted in a drastic decrease in inhibitory potency. In contrast, many derivatives with modulation of other parts retained inhibitory activities to varying degrees. These findings suggest the binding mode of active derivatives, in which three oxygen atoms aligned in a straight form at the nitro-furan moiety are coordinated to two divalent metal ions located at RNase H reaction site. Hence, the nitro-furan-carboxylic moiety is one of the critical scaffolds for RNase H inhibition. Of note, the RNase H inhibitory potency of a derivative was improved by 18-fold compared with that of the original hit compound, and no significant cytotoxicity was observed for most of the derivatives showing inhibitory activity. Since there is still much room for modification of the compounds at the part opposite the nitro-furan moiety, further chemical conversion will lead to improvement of compound potency and specificity.

Effect of pioglitazone on various parameters of insulin resistance including lipoprotein subclass according to particle size by a gel-permeation high-performance liquid chromatography in newly diagnosed patients with type 2 diabetes.

Pioglitazone is an insulin-sensitizing agent that has been reported to have anti-arteriosclerotic effects. The aim of this study was to obtain a better understanding of the mechanism involved in the insulin sensitizing effect of pioglitazone. A total of 50 newly diagnosed patients with type 2 diabetes were enrolled in this study and divided into two groups, 25 of who were treated with 15 mg/day pioglitazone and 25 with 500 mg/day metformin for 12 weeks. Changes in various parameters of insulin resistance including lipoprotein subclass according to particle size determined by high performance liquid chromatography, as well as glucose metabolism, were monitored to determine the relationship between lipoprotein subclass and other insulin resistance parameters. Both pioglitazone and metformin treatment were associated with significant reductions in hyperglycemia, HOMA-IR and HbA1c levels. Pioglitazone treatment, but not metformin treatment resulted in significant reductions in serum large very low-density lipoprotein (VLDL: 44.5-64.0 nm) and increases in serum adiponectin levels (both <0.001). In the pioglitazone group, the change in large VLDL levels correlated positively with changes in HbA1c (r=0.468, P=0.0174), HOMA-IR (r=0.593, P=0.0014), very small LDL (r=0.714, P<0.0001) and net electronegative charged modified-LDL (r=0.412, P=0.0399), and inversely with changes in adiponectin level (r=-0.526, P=0.0061). The results in this study suggest that the hypoglycemic effect of pioglitazone is achieved mainly through improvement of hepatic insulin resistance, and that pioglitazone may have an antiatherosclerotic effect by decreasing serum atherogenic modified-LDL and by increasing adiponectin.

Synergistic effect of HLA class II loci and cytokine gene polymorphisms on the risk of gastric cancer in Japanese patients with Helicobacter pylori infection.

It has been reported that polymorphisms of human leukocyte antigen (HLA) genes and several cytokine genes are associated with an increased risk of developing gastric cancer (GC). However, the results of studies from different geographic regions, ethnic groups and study groups are inconsistent. The aim of this study was to evaluate the influence of H. pylori infection and host genetic factors on GC susceptibility in Japanese patients with GC. We analyzed genotypes for HLA class I and II, tumor necrosis factor alpha, interleukin (IL)-1beta, IL-1 receptor, IL-4, IL-4Ralpha and IL-10 in 330 H. pylori-infected noncardia patients with GC and 190 H. pylori-infected nonulcer dyspeptic controls. Haplotype analyses indicated that the frequencies of the HLA DRB1*0405 and DQB1*0401 alleles were increased in the patients with intestinal-type GC when compared with controls (both DRB1*0405 and DQB1*0401: p = 0.015, OR = 1.57, 95% CI = 1.09-2.26), but the changes were not statistically significant after correction for multiple comparisons. None of the cytokine gene polymorphisms were associated with GC susceptibility, whether patients with GC were analyzed as a group according to the histological subtype. Of interest was the comparison of controls and patients with intestinal-type GC. The frequency of an IL-10-592AA homozygote showing concomitant carriage of the HLA DRB1*0405-DQB1*0401 haplotype was significantly higher in patients with intestinal-type GC (chi(2) = 6.369, p = 0.0116, p(c) = 0.0464, OR = 2.43, 95% CI = 1.21-4.48). Our results suggest that the HLA class II and IL-10-592A/C polymorphisms synergistically affect the susceptibility to GC development of H. pylori-infected individuals in the Japanese population.

Relationship between insulin resistance and inflammatory markers and anti-inflammatory effect of losartan in patients with type 2 diabetes and hypertension.

BACKGROUND: It is now well established that vascular inflammation and endothelial dysfunction associated with cardiovascular diseases contributes to insulin resistance. METHODS: We investigated the relationship between the homeostasis model assessment-insulin resistance index (HOMA-R) and various serum inflammatory markers and the effect of losartan on serum concentrations of these markers in patients with type 2 diabetes and hypertension. The patients were divided into 2 groups according to the value of HOMA-R with 60 patients with values=2.4 in Group A and 44 patients with values>2.5 in Group B. The variables were measured at baseline and after 6 months of treatment with losartan (50 mg/day). RESULTS: The HOMA-R concentrations were positively related to TNF-alpha (r=0.336, P<0.01) and inversely related to adiponectin (r=-0.405, P<0.01) and extracellular-superoxide dismutase (EC-SOD) (r=-0.452, P<0.01). Stepwise multiple regression analysis showed a significant relationship between HOMA-R and adiponectin (F=8.74) and EC-SOD (F=14.39). In Group B, losartan treatment significantly increased the serum concentrations of EC-SOD and adiponectin and decreased TNF-alpha and HOMA-R. CONCLUSION: Serum EC-SOD concentrations may be a sensitive biochemical marker of insulin resistance in patients with type 2 diabetes and hypertension and that losartan improves insulin sensitivity by increasing EC-SOD and adiponectin production and decreasing TNF-alpha production.

Clinical and genetic characteristics of diabetic patients with high-titer (>10,000 U/ml) of antibodies to glutamic acid decarboxylase.

We investigated the clinical aspects and genetic background of 13 diabetic patients with high-titers (>10,000 U/ml) of anti-glutamic acid decarboxylase antibody (Group A) and compared these 28 middle-aged (35-51 years, Group B) and 13 elderly (66-79 years, Group C) patients with anti-GAD(+) (<1100 U/ml) who were diagnosed initially as having type 2 diabetes. The mean age and mean age at onset of Group A were 70.8 +/- 3.9 years (range, 64-78) and 50.4 +/- 5.4 years (range, 43-61), respectively. In Group A, the prevalence of insulin-deficient patients was significantly lower (30.8%, 4 of 13) than in Group C (96.3%, 27 of 28, P < 0.001). Patients in Group A had a significantly longer interval between the clinical onset of diabetes to initiation insulin therapy (21.8 +/- 2.3 years) compared to patients in both Group B (1.8+/-1.1 years, P < 0.001) and Group C (14.8 +/- 7.1 years, P = 0.049). The frequency of DRB1*0405-DQB1*0401/DRB1*1502-DQB1*0601 or DRB*1501-DQB*0602 heterozygous genotypes in Group A (53.8%, 7 of 13) was significantly higher than in both Group B (3.6%, 1 of 28, P < 0.01) and Group C (7.7%, 1 of 13, P < 0.05). Compared with Group B, Group A had an increased frequency of the TNFA-U01 haplotype and the IL-10 -592 C allele (TNFA-U01; 53.8% versus 30.4%, P = 0.05 and IL-10 -592 C; 57.7% versus 33.9 %, P = 0.042). All sera from Group A reacted with GAD(65) protein on Western blots. We conclude that adult-onset diabetic patients with a high-titer of anti-GDAab differ from patients with latent autoimmune diabetes mellitus in adult (LADA) with respect to beta-cell function, cellular autoimmunity and genetic background. Our study also showed that high-titers of antibodies to glutamic acid decarboxylase (anti-GADab) were not predictive of later development of insulin deficiency in adult and/or elderly patients with type 2 diabetes. Furthermore, our results suggest that HLA-DRB1*1502-DQB1*0601 or DRB1*1501-DQB1*0602/DRB1*0405-DQB1*0401 heterozygous genotypes may be associated with high production of anti-GADab that recognizes the linear epitope(s) on the GAD(65) protein.

Cancer chemopreventive effects of polyunsaturated fatty acids.

The inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), in Raji cells as a primary screening test for anti-tumor promoters, for 22 fatty acids (as free and esterified forms), including 10 di- and polyunsaturated acids, and the inhibitory effects on activation of (+/-)-(E)-methtyl-2-[(E)-hydroxy-imino]-5-nitro-6-methoxy-3-hexemide (NOR 1), a nitric oxide (NO) donor, as a primary screening test for anti-tumor initiators, for 17 fatty acids (as methyl ester forms), were evaluated. Among the fatty acids tested, three n-3 polyunsaturated acids, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), exhibited potent inhibitory effects both on EBV-EA and NOR 1 activation. Furthermore, DHA methyl ester exhibited remarkable anti-tumor-promoting activity on an in vivo two-stage carcinogenesis test of mouse tumor using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter.

Enzyme-linked immunosorbent assay for circulating human resistin: resistin concentrations in normal subjects and patients with type 2 diabetes.

BACKGROUND: Resistin is a recently identified adipocyte-secreted hormone in rodents, and has been proposed to serve as a link between obesity and insulin resistance. The aim of this study was to develop a sensitive enzyme-linked immunosorbent assay (ELISA) for human resistin and evaluate serum resistin concentrations in normal subjects and patients with type 2 diabetes. METHODS: Using ELISA developed by two polyclonal antibodies, resistin concentrations were measured in 90 patients with type 2 diabetes and compared to 74 healthy control subjects. RESULTS: This ELISA has high specificity and sensitivity over the concentration of range 0.5-100 ng/ml with good percentage recovery (97.1 +/- 4.7%) and reproducibility (within-day assay, CV = 4.8-8.6%; between-day assay, CV = 5.6-9.7%). The mean concentration of resistin in sera from type 2 diabetic patients was significantly higher than that in normal subjects (mean +/- S.E.: 20.8 +/- 0.7 vs. 14.9 +/- 0.5 ng/ml, p < 0.001). A moderate positive correlation was observed between serum resistin levels and body mass indices in both normal subjects (r = 0.412, p < 0.0003) and patients with type 2 diabetes (r = 0.395, p < 0.0001). CONCLUSIONS: Our ELISA will be useful to confirm the physiological and pathophysiological role of resistin in humans.

Increased concentration of pentosidine, an advanced glycation end product, and interleukin-6 in the vitreous of patients with proliferative diabetic retinopathy.

To investigate the relationship between advanced glycation end products (AGEs) and interleukin-6 (IL-6) in the development of diabetic retinopathy, we determined the concentrations of pentosidine, a well-characterized AGE, and IL-6 in the vitreous of 62 patients with proliferative diabetic retinopathy (PDR) and 50 non-diabetic control subjects. We also investigated the effect of AGEs on the production of IL-6 by human retinal Müller cells. The levels of pentosidine and IL-6 in the vitreous of patients with PDR were significantly higher compared with controls. In patients with PDR with vitreous hemorrhage (VH), the mean vitreous concentration of IL-6 was significantly higher than that in PDR patients without VH. There was a strong positive correlation between the vitreous levels of pentosidine and IL-6. Levels of IL-6 were strikingly higher in the vitreous compared with the serum and there was no correlation between IL-6 concentrations in the two fluids. Treatment of Müller cells with AGEs for 48 h resulted in a dose-dependent increase of IL-6 in the culture medium. These results suggest that increased formation of AGEs in the vitreous may be involved in the development of diabetic retinopathy by inducing the production of IL-6 from retinal Müller cells.