Preclinical characterization of a non-peptidomimetic HIV protease inhibitor with improved metabolic stability.

Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents.

[Hepcidin and Erythroferrone Levels in Child-Bearing Women with Iron Deficiency Anemia].

OBJECTIVE: To detect serum hepcidin and erythroferrone levels in child-bearing women with iron deficiency anemia (IDA), and to investigate the association between them and iron status parameters. METHODS: The study consisted of 65 child-bearing women (35 with iron deficiency anemia and 30 age-matched healthy women). The levels of serum iron were detected by using automated chemistry analyzer, the contents of serum ferritin were detected by electrochemiluminescence immunoassay, and the levels of serum erythroferrone and hepcidin were detected by specific enzyme-linked immunosorbent assay (ELISA) kit. The quantitative variables between two groups were compared and analyzed by SPSS22.0 software. Spearman correlation was used to detect correlation between the parameters. RESULTS: The levels of Hb, serum iron, ferritin and transferrin saturation were significantly decreased in IDA patients as compared with in control group (P<0.001). Serum hepcidin levels in IDA patients were significant lower than those in control group (P<0.001). Serum erythroferrone levels slightly increased in IDA group (P>0.05). In IDA patients, serum hepcidin concentrations were positively correlated with hemoglobin concentration, serum iron, serum ferritin and transferrin saturation (r=0.448, r=0.496, r=0.754, r=0.491). But, serum erythroferrone concentrations showed no correlation with hemoglobin concentration, serum iron, serum ferritin, transferrin saturation and hepcidin (P>0.05). CONCLUSION: Serum hepcidin levels were significantly decreased in child-bearing women with IDA, but the serum erythroferrone levels were not obviously different between two groups, suggesting that serum erythroferrone may be not involved in the regulation of iron metabolism in child-bearing women with mild and moderate IDA.

Wogonin inhibits in vitro herpes simplex virus type 1 and 2 infection by modulating cellular NF-κB and MAPK pathways.

BACKGROUND: Wogonin, a natural flavonoid-like chemical compound, exhibits anti-inflammatory, antitumor, antiviral, neuroprotective, and anxiolytic effects by modulating a variety of cellular signaling pathways including PI3K-Akt, p53, nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK) pathways. In this study, its antiviral effect against herpes simplex virus (HSV) type 1 and 2 (HSV-1 and HSV-2) replication was investigated. RESULTS: Wogonin suppressed HSV-2-induced cytopathic effect (CPE) and reduced viral mRNA transcription, viral protein synthesis, and infectious virion particle titers in a dose-dependent manner. A time-of-drug-addition assay demonstrated that wogonin acted as a postentry viral inhibitor. Wogonin also significantly reduced HSV-induced NF-κB and MAPK pathway activation, which has previously been demonstrated to be important for viral replication. CONCLUSIONS: Our results suggest that the anti-herpes effect of wogonin may be mediated by modulation of cellular NF-κB and JNK/p38 MAPK pathways and imply that wogonin may be useful as an anti-HSV agent.

[Correlation analysis between the numbers of G-MDSCs and M-MDSCs in peripheral blood decrease and therapeutic effect in patients with colorectal cancer].

Objective To detect the frequencies of myeloid-derived suppressor cells (MDSCs) in the peripheral blood of patients with colorectal cancer (CRC) in an attempt to investigate the relationship between the change of MDSC frequencies and clinicopathologic characteristics of CRC patients. Methods The frequencies of granulocytic MDSCs (G-MDSCs) and monocytic MDSCs (M-MDSCs) in the peripheral blood of 82 CRC patients and 30 healthy volunteers were detected by flow cytometry. ANOVA and t-test were used to explore the relationship between the frequencies of MDSCs in the peripheral blood and the TNM stages, lymph node metastasis, tumor sites, histological grade as well as common therapeutic measures of CRC patients. Correlation analysis was performed to evaluate the correlation between the frequencies of G-MDSCs and M-MDSCs. Results Compared with the 30 healthy volunteers, the frequencies of G-MDSCs and M-MDSCs in the peripheral blood of the 82 CRC patients were statistically higher. The frequencies of G-MDSCs and M-MDSCs in the peripheral blood from TNF stage III and IV CRC patients were obviously higher than those from stage I and II patients, and CRC patients with lymph node metastasis had remarkably higher frequencies of G-MDSCs and M-MDSCs than the patients without lymph node metastasis. There were no statistical differences in the frequencies of G-MDSCs and M-MDSCs in the peripheral blood between CRC patients with different tumor sites as well as histological grades. Radical resection and effective adjuvant chemotherapy significantly decreased the frequencies of G-MDSCs and M-MDSCs in the peripheral blood of CRC patients. No statistical correlation was found between the frequencies of circulating G-MDSCs and M-MDSCs in CRC patients. Conclusion G-MDSC and M-MDSC frequencies in the peripheral blood of CRC patients significantly increased, and were closely associated with TNM stages and lymph node metastasis. Radical resection and effective adjuvant chemotherapy significantly reduced the frequencies of circulating G-MDSCs sand M-MDSCs of CRC patients.

[Expression of COX-1 and COX-2 in the Platelet of Iron Deficiency Anemia Women at Childbearing Age and Its Clinical Significance].

OBJECTIVE: To detect the expression level of cyclooxygenase-1（COX-1） and cyclooxygenase-2（COX-2） in the platelet of iron deficiency anemia（IDA）women at childbearing age and to explore its correlation with the different indexes of anemia and platelets. METHODS: Forty female IDA patients at childbearing age and 35 healthy controls were enrolled in this study. The Flow cytometry was used to detect the expression of platelet COX-1 and COX-2，the platelet aggregation function as examined by turbidimetric method，and the levels of serum ferritin were analyzed by electrochemical luminescence method，the leval of serum iron was determined by ELISA，and the correlation of different indexes was analyzed. RESULTS: Compared with healthy controls，the levels of platelet COX-1 and COX-2 were significantly lower in female IDA patients at Childbearing age（P<0.05），but platelet count（Plt），mean platelet volume（MPV） and platelet aggregation rate（PAgT）were not statistically different between the 2 groups（P > 0.05）. The expression level of platelet COX-1 positively correlated with those of Hb（r =0.623，P<0.01），serum iron（r =0.321，P<0.05） and HCT（r=0.305，P<0.05）. but the platelet COX-2 expression did not corelate with these indexs. CONCLUSION: The expression of platelet COX-1 and COX-2 in female IDA patients at Childbearing age markedly decrease，and the expression level of platelet COX-1 closely relates with the severity of anemia，that possesses reference value for clinical diagnosis of female IDA patients at Childbearing age..

miR-1179 promotes cell invasion through SLIT2/ROBO1 axis in esophageal squamous cell carcinoma.

MiR-1179, a new identified miRNA highly associated with metastasis of colorectal cancer which was never reported in esophageal squamous cell carcinoma (ESCC). Here we measured the expression levels of miR-1179 and the candidate target gene in tissues from 40 patients with ESCC. Transwell, Dual-luciferase reporter assay and immunocytochemistry assay were employed to detect the function role of miR-1179 in vitro. We found that miR-1179 was up-regulated in human ESCC tumor tissues. Bioinformatics analysis indicated that SLIT2 acting as a new potential target of miR-1179 which was confirmed by luciferase reporter assay. Down-regulation of miR-1179 suppressed cell invasion in vitro with an increasing level of SLIT2 and ROBO1, besides, the up-regulation of SLIT2 decreased cell invasion through ROBO1. Taken together, these findings will shed light the role to mechanism of miR-1179 in regulating cell invasion via SLIT2/ROBO1 axis.

miR-1271 promotes non-small-cell lung cancer cell proliferation and invasion via targeting HOXA5.

MicroRNAs (miRNAs) are short, non-coding RNAs (∼ 22 nt) that play important roles in the pathogenesis of human diseases by negatively regulating numerous target genes at posttranscriptional level. However, the role of microRNAs in lung cancer, particularly non-small-cell lung cancer (NSCLC), has remained elusive. In this study, two microRNAs, miR-1271 and miR-628, and their predicted target genes were identified differentially expressed in NSCLC by analyzing the miRNA and mRNA expression data from NSCLC tissues and their matching normal controls. miR-1271 and its target gene HOXA5 were selected for further investigation. CCK-8 proliferation assay showed that the cell proliferation was promoted by miR-1271 in NSCLC cells, while miR-1271 inhibitor could significantly inhibited the proliferation of NSCLC cells. Interestingly, migration and invasion assay indicated that overexpression of miR-1271 could significantly promoted the migration and invasion of NSCLC cells, whereas miR-1271 inhibitor could inhibited both cell migration and invasion of NSCLC cells. Western blot showed that miR-1271 suppressed the protein level of HOXA5, and luciferase assays confirmed that miR-1271 directly bound to the 3'untranslated region of HOXA5. This study indicated indicate that miR-1271 regulates NSCLC cell proliferation and invasion, via the down-regulation of HOXA5. Thus, miR-1271 may represent a potential therapeutic target for NSCLC intervention.

Structure-activity relationships of diamine inhibitors of cytochrome P450 (CYP) 3A as novel pharmacoenhancers, part I: core region.

Ritonavir (RTV), an HIV-1 protease inhibitor (PI), is also a potent mechanism-based inhibitor of human cytochrome P450 3A (CYP3A) and has been widely prescribed as a pharmacoenhancer. As a boosting agent for marketed PIs, it reduces pill burden, and improves compliance. Removal of the hydroxyl group from RTV reduces, but does not eliminate HIV PI activity and does not affect CYP3A inhibition. Herein we report the discovery of a novel series of CYP3A inhibitors that are devoid of antiviral activity. The synthesis and evaluation of analogs with extensive modifications of the 1,4-diamine core along with the structure activity relationships with respect to anti-HIV activity, CYP3A inhibitory activity, selectivity against other CYP enzymes and the human pregnane X receptor (PXR) will be discussed.

Preclinical characterization of GS-9669, a thumb site II inhibitor of the hepatitis C virus NS5B polymerase.

GS-9669 is a highly optimized thumb site II nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, with a binding affinity of 1.35 nM for the genotype (GT) 1b protein. It is a selective inhibitor of HCV RNA replication, with a mean 50% effective concentration (EC(50)) of ≤ 11 nM in genotype 1 and 5 replicon assays, but lacks useful activity against genotypes 2 to 4. The M423T mutation is readily generated clinically upon monotherapy with the thumb site II inhibitors filibuvir and lomibuvir, and it is notable that GS-9669 exhibited only a 3-fold loss in potency against this variant in the genotype 1b replicon. Rather than M423T, resistance predominantly tracks to residues R422K and L419M and residue I482L in GT 1b and 1a replicons, respectively. GS-9669 exhibited at least additive activity in combination with agents encompassing four other direct modes of action (NS3 protease, NS5A, NS5B via an alternative allosteric binding site, and NS5B nucleotide) as well as with alpha interferon or ribavirin in replicon assays. It exhibited high metabolic stability in in vitro human liver microsomal assays, which, in combination with its pharmacokinetic profiles in rat, dog, and two monkey species, is predictive of good human pharmacokinetics. GS-9669 is well suited for combination with other orally active, direct-acting antiviral agents in the treatment of genotype 1 chronic HCV infection. (This study has been registered at ClinicalTrials.gov under registration number NCT01431898.).

Mutations in multiple domains of Gag drive the emergence of in vitro resistance to the phosphonate-containing HIV-1 protease inhibitor GS-8374.

GS-8374 is a potent HIV protease inhibitor (PI) with a unique diethyl-phosphonate moiety. Due to a balanced contribution of enthalpic and entropic components to its interaction with the protease (PR) active site, the compound retains activity against HIV mutants with high-level multi-PI resistance. We report here the in vitro selection and characterization of HIV variants resistant to GS-8374. While highly resistant viruses with multiple mutations in PR were isolated in the presence of control PIs, an HIV variant displaying moderate (14-fold) resistance to GS-8374 was generated only after prolonged passaging for >300 days. The isolate showed low-level cross-resistance to darunavir, atazanavir, lopinavir, and saquinavir, but not other PIs, and contained a single R41K mutation in PR combined with multiple genotypic changes in the Gag matrix, capsid, nucleocapsid, and SP2 domains. Mutations also occurred in the transframe peptide and p6* domain of the Gag-Pol polyprotein. Analysis of recombinant HIV variants indicated that mutations in Gag, but not the R41K in PR, conferred reduced susceptibility to GS-8374. The Gag mutations acted in concert, since they did not affect susceptibility when introduced individually. Analysis of viral particles revealed that the mutations rendered Gag more susceptible to PR-mediated cleavage in the presence of GS-8374. In summary, the emergence of resistance to GS-8374 involved a combination of substrate mutations without typical resistance mutations in PR. These substrate changes were distributed throughout Gag and acted in an additive manner. Thus, they are classified as primary resistance mutations indicating a unique mechanism and pathway of resistance development for GS-8374.

In vitro characterization of GS-8374, a novel phosphonate-containing inhibitor of HIV-1 protease with a favorable resistance profile.

GS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to the para position of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (K(i) = 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4(+) T cells (50% effective concentration [EC(50)] = 3.4 to 11.5 nM), and macrophages (EC(50) = 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC(50)s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs, in vitro hepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boosting in vivo. In summary, results from this broad in vitro pharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients.

Up-regulate HLA class I expression following hepatitis B virus transfection in a hepatocellular carcinoma cell line BEL7405.

Chronic hepatitis B virus infection is associated with a high risk of developing into hepatocellular carcinoma, while tumor recognition is important during the immune surveillance process that prevents cancer development in humans. The mechanisms of immune evasion and the role of the early immune response in chronic infection caused by hepatitis B virus (HBV) are still unclear. In the present study, 1 copy or 1.2 copies of HBV genome was transfected into a hepatocellular carcinoma cell line BEL7405. RT-PCR, Western blot and flow cytometry analysis were used to evaluate the expression of HLA class I molecules and transporter associated with antigen processing 1 (TAP1). Finally, the cytotoxic activity of natural killer (NK) cells against HBV transfected liver cells was detected by MTT colorimetry method. Following transfection of 1 copy or 1.2 copies of HBV genome, HLA class I expression was up-regulated in BEL7405 cell line in a dose-dependent manner. Furthermore, increased the surface HLA class I expression were caused by enhanced expression of TAP1 at mRNA and protein levels in those transfected cells. Consequently, a significantly down-regulated cytotoxic activity of NK cells against HBV transfected liver cells was observed. These results may demonstrate a way by which HBV avoids recognition by NK cells that might be associated with the establishment of chronic infection and tumor formation.

IRF-1 and p65 mediate upregulation of constitutive HLA-A antigen expression by hepatocellular carcinoma cells.

Malignant transformation of hepatocytes is frequently associated with upregulation of HLA-A expression. Currently there is no information available regarding the mechanisms underlying this phenotypic change. We investigated HLA-A expression in 165 paraffin embedded tissues and 21 fresh tissues from liver cancer patients. Utilizing truncated HLA-A promoter-reporter constructs and gel-shift assay we had identified the regulatory elements and transcription factors required for HLA-A upregulation. 54% of the paraffin embedded tissues showed increased HLA-A expression in their cancerous part. 43% of the fresh liver cancer tissues had increased HLA-A complex expression with the HLA-A heavy chain gene demonstrating the highest level of upregulation (62%). Enhanced HLA-A expression in the liver cell lines QGY7701 and BEL7402 was found to be mediated by binding of interferon regulatory factor 1 (IRF-1) to interferon stimulated response element, and of nuclear transcription factor p65 binding to enhancer A element in the HLA-A promoter of these cell lines. The in vivo relevance of these findings was indicated by the association of the enhanced expression of IRF-1 and accumulation of nuclear p65 with HLA-A upregulation in 8 of the 21 liver cancer lesions investigated. Our results indicated that HLA-A upregulation in liver cancer was mediated by both increased nuclear aggregation of transcription factor p65 and upregulation of transcription factor IRF-1.