Anti-oxidant and Anti-inflammatory Effects of Ethanol Extract from Polygala sibirica L. var megalopha Fr. on Lipopolysaccharide-Stimulated RAW264.7 Cells.

OBJECTIVE: To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L. var megalopha Fr. (EEP) on RAW264.7 mouse macrophages. METHODS: RAW264.7 cells were pretreated with 0-200 µg/mL EEP or vehicle for 2 h prior to exposure to 1 µg/mL lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) and prostaglandin (PGE2) production were determined by Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin-1beta (IL-1ß), and IL-6 were determined using reverse transcription polymerase chain reaction (RT-PCR). Western blot assay was used to determine the protein expressions of iNOS, COX-2, phosphorylation of extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK), inhibitory subunit of nuclear factor Kappa B alpha (Iκ B-α) and p38. Immunofluorescence was used to observe the nuclear expression of nuclear factor-κ B p65 (NF-κ B p65). Additionally, the anti-oxidant potential of EEP was evaluated by reactive oxygen species (ROS) production and the activities of catalase (CAT) and superoxide dismutase (SOD). The 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), superoxide anion (O2-) radical and nitrite scavenging activity were also measured. RESULTS: The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g. With EEP treatment (100 and 150 µg/mL), there was a notable decrease in NO and PGE2 production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions (P<0.01 or P<0.05). Furthermore, with EEP treatment (150 µg/mL), there was a decrease in the mRNA expression levels of TNF-α, IL-1ß and IL-6, as well as in the phosphorylation of ERK, JNK and p38 mitogen-activated protein kinase (MAPK, P<0.01 or P<0.05), by blocking the nuclear translocation of NF-κ B p65 in LPS-stimulated cells. In addition, EEP (100 and 150 µg/mL) led to an increase in the anti-oxidant enzymes activity of SOD and CAT, with a concomitant decrease in ROS production (P<0.01 or P<0.05). EEP also indicated the DPPH, OH, O2- radical and nitrite scavenging activity. CONCLUSION: EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κ B pathway and protected against oxidative stress.

E3 ligase ATL5 positively regulates seed longevity by mediating the degradation of ABT1 in Arabidopsis.

Ubiquitination is a fundamental mechanism regulating the stability of target proteins in eukaryotes; however, the regulatory mechanism in seed longevity remains unknown. Here, we report that an uncharacterized E3 ligase, ARABIDOPSIS TÓXICOS EN LEVADURA 5 (ATL5), positively regulates seed longevity by mediating the degradation of ACTIVATOR OF BASAL TRANSCRIPTION 1 (ABT1) in Arabidopsis. Seeds in which ATL5 was disrupted showed faster accelerated aging than the wild-type, while expressing ATL5 in atl5-2 basically restored the defective phenotype. ATL5 was highly expressed in the embryos of seeds, and its expression could be induced by accelerated aging. A yeast two-hybrid screen identified ABT1 as an ATL5 interacting protein, which was further confirmed by bimolecular fluorescence complementary assay and co-immunoprecipitation analysis. In vitro and in vivo assays showed that ATL5 functions as an E3 ligase and mediates the polyubiquitination and degradation of ABT1. Disruption of ATL5 diminished the degradation of translated ABT1, and the degradation could be induced by seed ageing and occurred in a proteasome-dependent manner. Furthermore, disruption of ABT1 enhanced seed longevity. Taken together, our study reveals that ATL5 promotes the polyubiquitination and degradation of the ABT1 protein posttranslationally and positively regulates seed longevity in Arabidopsis.

Investigations of Fish Assemblages Using Two Methods in Three Terminal Reservoirs of the East Route of South-to-North Water Transfer Project, China.

The terminal reservoirs of water transfer projects directly supply water for domestic, agricultural, and industrial applications, and the water quality of these reservoirs produce crucial effects on the achievement of project targets. Typically, fish assemblages are monitored as indicators of reservoir water quality, and can also be regulated for its improvement. In the present study, we compared traditional fish landing (TFL) and environmental DNA (eDNA) metabarcoding methods for monitoring fish assemblages in three terminal reservoirs of the East Route of the South-to-North Water Transfer Project, China. Results of TFL and eDNA showed similar assemblage structures and patterns of diversity and spatial distribution with obvious differences in fish composition across three examined reservoirs. Demersal and small fish were dominant in all reservoirs. In addition, a strong association between water transfer distance and assemblages and distribution of non-native fish was found. Our findings highlight the necessity of the fish assemblage monitoring and managing for water quality and revealed the impact of water diversion distance on the structure of fish assemblages and dispersal of alien species along the water transfer project.

Chrysophanol alleviates acute lung injury caused by Klebsiella pneumoniae infection by inhibiting pro-inflammatory cytokine production.

Acute lung injury (ALI) caused by acute bacterial infection remains a common life-threatening lung disease. An increased inflammatory response is the basis for the occurrence and development of ALI. Most antibiotics can only reduce the bacterial load but do not protect from lung damage because of an excessive immune response. Chrysophanol (chrysophanic acid, Chr), as a natural anthraquinone extracted from Rheum palmatum L., has various biological functions, including anti-inflammatory, anti-cancer activities, and ameliorative effects on cardiovascular diseases. Considering these properties, we investigated the effect of Chr in Klebsiella pneumoniae (KP)-induced ALI mice and its potential mechanism. Our results showed that Chr had protective effects against KP-infected mice, including increased survival rate, decreased bacterial burden, reduced recruitment of immune cells, and reduced reactive oxygen species level of lung macrophages. Chr reduced the expression of inflammatory cytokines by inhibiting the toll-like receptor 4/nuclear factor kappa-B (TLR4/NF-κB) signaling pathway and inflammasome activation and strengthening autophagy. Overactivation of the TLR4/NF-κB signaling pathway by the activator Neoseptin 3 led to Chr losing control of inflammatory cytokines in cells, resulting in increased cell death. Similarly, overactivation of the c-Jun N-terminal kinase signaling pathway using the activator anisomycin resulted in Chr losing its inhibitory effect on NOD-like receptor thermal protein domain associated protein 3 (NFRP3) inflammasome activation, and cell viability was reduced. In addition, autophagy was blocked by siBeclin1, so Chr could not reduce inflammatory factors, and cell viability was markedly inhibited. Collectively, this work unravels the molecular mechanism underpinning Chr-alleviated ALI via inhibiting pro-inflammatory cytokines. Thus, Chr is a potential therapeutic agent for KP-induced ALI.

Self-propelled predator-prey of swarming Janus micromotors.

It is a long-standing challenge to accomplish bionic microrobot that acts in a similar way of white blood cell, chasing bacteria in complex environment. Without an effective external control field, most swarming microrobots systems are usually unable to perform directional movement and redirect their motion to capture the target. Here we report the predatory-prey dynamics of self-propelled clusters of Janus micromotors. The active cluster generates an oxygen bubbles cloud around itself by decomposing H2O2, which levitated it above the substrate, enhancing its mobility in solution to wander around to devour other clusters. The fast decomposition of H2O2 also induced a tubular low-concentration zone that bridges two clusters far separated from each other, resulting in a diffusio-osmotic pressure that drives the two clusters to meet. This predatory-prey phenomena mimic white blood cells chasing bacteria and swarming flocks in nature, shedding light on emergent collective intelligence in biology.

Diagnostic and Prognostic Value of Dysregulated miR-10a-3p in Patients with Severe Pneumonia.

Purpose: Previous studies have shown that microRNA is involved in regulating a variety of human inflammatory diseases. The purpose of this study was to investigate the expression of miR-10a-3p in the blood of patients with severe pneumonia and evaluate its value in the diagnosis and prognosis of severe pneumonia. Patients and Methods: Seventy patients with severe pneumonia and 75 healthy individuals were included in this study. Venous blood of all subjects was obtained for RT-qPCR analysis to obtain the relative expression level of miR-10a-5p. The diagnostic accuracy of miR-10a-5p for severe pneumonia was assessed by ROC curve. After standardized treatment, the prognosis of patients with severe pneumonia was analyzed by a 28-day follow-up method. Kaplan-Meier curve and multivariate Cox regression analysis were used to determine the basic factors influencing the prognosis of patients. Results: Compared with healthy control, serum miR-10a-3p expression in patients with severe pneumonia was distinctly upregulated (P < 0.001). Besides, ROC analysis showed that miR-10a-3p had high diagnostic accuracy for severe pneumonia, with an AUC of 0.881, sensitivity and specificity of 75.7% and 84.0%, respectively. Kaplan-Meier curve exhibited that high miR-10a-3p expression group had a higher probability of death than those with low miR-10a-3p expression. Multivariate Cox regression analysis demonstrated that miR-10a-3p and CRP were independent risk factors affecting the prognosis of patients. Conclusion: The expression of miR-10a-3p was increased in patients with severe pneumonia, and abnormally expressed miR-10a-3p has the potential to be used as a diagnostic and prognostic marker for severe pneumonia, which provides a new biological direction for the early detection and risk assessment of severe pneumonia.

Artificial Intelligence Algorithm-Based Computerized Tomography Image Features Combined with Serum Tumor Markers for Diagnosis of Pancreatic Cancer.

The objective of this study was to analyze the value of artificial intelligence algorithm-based computerized tomography (CT) image combined with serum tumor markers for diagnoses of pancreatic cancer. In the study, 68 hospitalized patients with pancreatic cancer were selected as the experimental group, and 68 hospitalized patients with chronic pancreatitis were selected as the control group, all underwent CT imaging. An image segmentation algorithm on account of two-dimensional (2D)-three-dimensional (3D) convolution neural network (CNN) was proposed. It also introduced full convolutional network (FCN) and UNet network algorithm. The diagnostic performance of CT, serum carbohydrate antigen-50 (CA-50), serum carbohydrate antigen-199 (CA-199), serum carbohydrate antigen-242 (CA-242), combined detection of tumor markers, and CT-combined tumor marker testing (CT-STUM) for pancreatic cancer were compared and analyzed. The results showed that the average Dice coefficient of 2D-3D training was 84.27%, which was higher than that of 2D and 3D CNNs. During the test, the maximum and average Dice coefficient of the 2D-3D CNN algorithm was 90.75% and 84.32%, respectively, which were higher than the other two algorithms, and the differences were statistically significant (P < 0.05). The penetration ratio of pancreatic duct in the experimental group was lower than that in the control group, the rest were higher than that in the control group, and the differences were statistically significant (P < 0.05). CA-50, CA-199, and CA-242 in the experimental group were 141.72 U/mL, 1548.24 U/mL, and 83.65 U/mL, respectively, which were higher than those in the control group, and the differences were statistically significant (P < 0.05). The sensitivity, specificity, positive predictive value, and authenticity of combined detection of serum tumor markers were higher than those of CA-50, CA-199, and CA-242, and the differences were statistically significant (P < 0.05). The results showed that the proposed algorithm 2D-3D CNN had good stability and image segmentation performance. CT-STUM had high sensitivity and specificity in diagnoses of pancreatic cancer.

Chromosome-level genome assembly of the Chinese longsnout catfish Leiocassis longirostris.

The Chinese longsnout catfish ( Leiocassis longirostris Günther) is one of the most economically important freshwater fish in China. As wild populations have declined sharply in recent years, it is also a valuable model for research on sexual dimorphism, comparative biology, and conservation. However, the current lack of high-quality chromosome-level genome information for the species hinders the advancement of comparative genomic analysis and evolutionary studies. Therefore, we constructed the first high-quality chromosome-level reference genome for L. longirostris. The total genome was 703.19 Mb, with 389 contigs and contig N50 length of 4.29 Mb. Using high-throughput chromosome conformation capture (Hi-C) data, the genome sequences (685.53 Mb) were scaffolded into 26 chromosomes ranging from 17.36 to 43.97 Mb, resulting in a chromosomal anchoring rate for the genome of 97.44%. In total, 23 708 protein-coding genes were identified in the genome. Phylogenetic analysis indicated that L. longirostris and its closest related species P. fulvidraco diverged approximately 26.6 million years ago. This high-quality reference genome of L. longirostris should pave the way for future genomic comparisons and evolutionary research.

Full-length transcript sequencing accelerates the transcriptome research of Gymnocypris namensis, an iconic fish of the Tibetan Plateau.

Gymnocypris namensis, the only commercial fish in Namtso Lake of Tibet in China, is rated as nearly threatened species in the Red List of China's Vertebrates. As one of the highest-altitude schizothorax fish in China, G. namensis has strong adaptability to the plateau harsh environment. Although being an indigenous economic fish with high value in research, the biological characterization, genetic diversity, and plateau adaptability of G. namensis are still unclear. Here, we used Pacific Biosciences single molecular real time long read sequencing technology to generate full-length transcripts of G. namensis. Sequences clustering analysis and error correction with Illumina-produced short reads to obtain 319,044 polished isoforms. After removing redundant reads, 125,396 non-redundant isoforms were obtained. Among all transcripts, 103,286 were annotated to public databases. Natural selection has acted on 42 genes for G. namensis, which were enriched on the functions of mismatch repair and Glutathione metabolism. Total 89,736 open reading frames, 95,947 microsatellites, and 21,360 long non-coding RNAs were identified across all transcripts. This is the first study of transcriptome in G. namensis by using PacBio Iso-seq. The acquisition of full-length transcript isoforms might accelerate the transcriptome research of G. namensis and provide basis for further research.

Knockdown of TRIM28 inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and migration.

Atherosclerosis is a common type of cardiovascular disease (CVD), remaining one of the leading causes of global death. Tripartite motif-containing 28 (TRIM28) is a member of TRIM family that has been found to be involved in atherosclerosis. However, the role of TRIM28 in atherosclerosis remains unknown. This study aimed to investigate the effects of TRIM28 on the phenotypic switching of human aortic smooth muscle cells (HASMCs), which is considered as a fundamental event during the development of atherosclerosis. The results showed that TRIM28 was highly expressed in human atherosclerotic tissues, as well in cultured HASMCs stimulated by platelet-derived growth factor subunit B homodimer (PDGF-BB). Knockdown of TRIM28 by transfection with siRNA targeting TRIM28 (si-TRIM28) significantly suppressed the PDGF-BB-induced cell proliferation and migration of HASMCs. Besides, knockdown of TRIM28 inhibited the expressions of matrix metalloproteinase (MMP)-2 and MMP-9. The VSMC markers including α-smooth muscle actin (α-SMA), calponin and SM22α were upregulated in TRIM28 knocked down HASMCs. Furthermore, knockdown of TRIM28 blocked PDGF-BB-induced NF-κB activation in HASMCs. Collectively, knockdown of TRIM28 prevented PDGF-BB-induced phenotypic switching of HASMCs, which might be mediated by the regulation of NF-κB signaling pathway.

High fat diet-induced oxidative stress blocks hepatocyte nuclear factor 4α and leads to hepatic steatosis in mice.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease with manifestation of over-accumulation of fat in liver. Increasing evidences indicate that NAFLD may be in part caused by malfunction of very low density lipoprotein (VLDL) secretion. Hepatocyte nuclear factor 4α (HNF4α), a nuclear receptor protein, plays an important role in sustain hepatic lipid homeostasis via transcriptional regulation of genes involved in secretion of VLDL, such as apolipoprotein B (ApoB). However, the exact functional change of HNF4α in NAFLD remains to be elucidated. In the present study, we found that high fat diet (HFD) induced cytoplasmic retention of HNF4α in hepatocytes, which led to down-regulation of hepatic ApoB expression and its protein level in serum, as well as reduced secretion of VLDL. We further revealed that oxidative stress, elevated in fatty liver, was the key factor inducing the cytoplasmic retention of HNF4α in hepatocytes by activating protein kinase C (PKC)-mediated phosphorylation in HNF4α. Thus, our findings reveal a novel mechanism underlying HFD-induced fatty liver that oxidative stress impairs function of HNF4α on ApoB expression and VLDL secretion via PKC activation, eventually promoting fat accumulation in the liver. Therefore, oxidative stress/PKC/HNF4α pathway may be a novel target to treat diet-induced fatty liver.

Development and Qualification of an Opsonophagocytic Killing Assay To Assess Immunogenicity of a Bioconjugated Escherichia coli Vaccine.

The global burden of disease caused by extraintestinal pathogenic Escherichia coli (ExPEC) is increasing as the prevalence of multidrug-resistant strains rises. A multivalent ExPEC O-antigen bioconjugate vaccine could have a substantial impact in preventing bacteremia and urinary tract infections. Development of an ExPEC vaccine requires a readout to assess the functionality of antibodies. We developed an opsonophagocytic killing assay (OPA) for four ExPEC serotypes (serotypes O1A, O2, O6A, and O25B) based on methods established for pneumococcal conjugate vaccines. The performance of the assay was assessed with human serum by computing the precision, linearity, trueness, total error, working range, and specificity. Serotypes O1A and O6A met the acceptance criteria for precision (coefficient of variation for repeatability and intermediate precision, ≤50%), linearity (90% confidence interval of the slope of each strain, 0.80, 1.25), trueness (relative bias range, -30% to 30%), and total error (total error range, -65% to 183%) at five serum concentrations and serotypes O2 and O25B met the acceptance criteria at four concentrations (the lowest concentration for serotypes O2 and O25B did not meet the system suitability test of maximum killing of ≥85% of E. coli cells). All serotypes met the acceptance criteria for specificity (opsonization index value reductions of ≤20% for heterologous serum preadsorption and ≥70% for homologous serum preadsorption). The assay working range was defined on the basis of the lowest and highest concentrations at which the assay jointly fulfilled the target acceptance criteria for linearity, precision, and accuracy. An OPA suitable for multiple E. coli serotypes has been developed, qualified, and used to assess the immunogenicity of a 4-valent E. coli bioconjugate vaccine (ExPEC4V) administered to humans.

Meta-analysis of serum non-high-density lipoprotein cholesterol and risk of coronary heart disease in the general population.

BACKGROUND: Non-high-density lipoprotein cholesterol (non-HDL-C) has been proposed an independent risk factor for coronary heart disease (CHD). However, the magnitude of this association in the general population varied considerably. OBJECTIVE: To investigate the association of baseline non-HDL-C level with CHD risk in the general population by conducting a meta-analysis. METHODS: A comprehensive literature search was performed in the Pubmed and Embase until January 2017. Prospective observational studies that investigated the association between baseline non-HDL-C level and CHD risk in the general population were included. Pooled hazard ratio (HR) and corresponding 95% confidence interval (CI) were calculated for the highest vs. the lowest non-HDL-C category. RESULTS: Seven articles with 9 independent prospective studies involving 448,732 individuals were included. Meta-analysis showed that individuals with the highest non-HDL-C level at baseline were associated with greater risk of CHD (RR 1.79; 95% CI 1.68-1.91) than those in the lowest non-HDL-C level category. Subgroup analysis suggested that the risk of CHD seemed more pronounced among men (HR 1.98; 95% CI 1.70-2.30) than among women (HR 1.63; 95% CI 1.35-1.96). CONCLUSIONS: Higher baseline non-HDL-C level is associated with increased risk of CHD in the general population. This risk seems more pronounced in men than in women. However, gender difference in non-HDL-C level associated with CHD risk should be verified by more well-designed prospective studies.

Guidable Thermophoretic Janus Micromotors Containing Gold Nanocolorifiers for Infrared Laser Assisted Tissue Welding.

Current wound sealing systems such as nanoparticle-based gluing of tissues allow almost immediate wound sealing. The assistance of a laser beam allows the wound sealing with higher controllability due to the collagen fiber melting which is defined by loss of tertiary protein structure and restoration upon cooling. Usually one employs dyes to paint onto the wound, if water absorption bands are absent. In case of strong bleeding or internal wounds such applications are not feasible due to low welding depth in case of water absorption bands, dyes washing off, or the dyes becoming diluted within the wound. One possible solution of these drawbacks is to use autonomously movable particles composing of biocompatible gold and magnetite nanoparticles and biocompatible polyelectrolyte complexes. In this paper a proof of principle study is presented on the utilization of thermophoretic Janus particles and capsules employed as dyes for infrared laser-assisted tissue welding. This approach proves to be efficient in sealing the wound on the mouse in vivo. The temperature measurement of single particle level proves successful photothermal heating, while the mechanical characterizations of welded liver, skin, and meat confirm mechanical restoration of the welded biological samples.

Leucocyte Membrane-Coated Janus Microcapsules for Enhanced Photothermal Cancer Treatment.

Polyelectrolyte multilayer (PEM) capsules are promising candidates for many kinds of cancer detection and treatment but are usually intended to deliver cargo to specific sites or to destroy cancer cells based on photothermal effects from the outside. In this publication we prove that it is possible to kill cancer cells from the inside based on phagocytosed PEM capsules. In addition we show how to open the cells and bring the PEM capsules to the surface of cancer cells based on photothermal effects and rapid evaporation of water. Diffusion-based temperature determinations of the photothermal effect up to the evaporation temperature of water are presented.

AEG-1/MTDH-activated autophagy enhances human malignant glioma susceptibility to TGF-ß1-triggered epithelial-mesenchymal transition.

Autophagy is a tightly regulated process activated in response to metabolic stress and other microenvironmental changes. Astrocyte elevated gene 1 (AEG-1) reportedly induces protective autophagy. Our results indicate that AEG-1 also enhances the susceptibility of malignant glioma cells to TGF-ß1-triggered epithelial-mesenchymal transition (EMT) through induction of autophagy. TGF-ß1 induced autophagy and activated AEG-1 via Smad2/3 phosphorylation in malignant glioma cells. Also increased was oncogene cyclin D1 and EMT markers, which promoted tumor progression. Inhibition of autophagy using siRNA-BECN1 and siRNA-AEG-1 suppressed EMT. In tumor samples from patients with malignant glioma, immunohistochemical assays showed that expression levels of TGF-ß1, AEG-1, and markers of autophagy and EMT, all gradually increase with glioblastoma progression. In vivo siRNA-AEG-1 administration to rats implanted with C6 glioma cells inhibited tumor growth and increased the incidence of apoptosis among tumor cells. These findings shed light on the mechanisms underlying the invasiveness and progression of malignant gliomas.

How Leucocyte Cell Membrane Modified Janus Microcapsules are Phagocytosed by Cancer Cells.

Modern drug delivery systems rely on either antibody-based single-surface recognition or on surface-hydrophobicity-based approaches. For a tumor showing various surface mutations, both approaches fail. This publication hereby presents Janus capsules based on polyelectrolyte multilayer microcapsules exhibiting human leucocyte (THP-1 cell line) cell membranes for discriminating HUVEC cells from three different cancer cell lines. Despite destroying the cellular integrity of leucocyte cells, the modified Janus capsules are able to adhere to cancer cells. Leucocyte cell-membrane-coated Janus capsules are phagocytosed with the cellular membrane part pointing to the cells.

Assessment of the combination of doripenem plus a fluoroquinolone against non-susceptible Acinetobacter baumannii isolates from nosocomial pneumonia patients.

Carbapenem- and fluoroquinolone-non-susceptible Acinetobacter baumannii were obtained from four nosocomial pneumonia patients who were clinically cured following combination therapy with doripenem/levofloxacin or ciprofloxacin. In vitro synergy of doripenem/levofloxacin or ciprofloxacin was evaluated using time-kill analysis. In vivo synergy was tested using a mouse lethal infection model. In time-kill studies, doripenem and levofloxacin were both bactericidal when tested at Cmax; at ½Cmax, the combination showed synergy up to 8 hours. Ciprofloxacin, alone or combined with doripenem, was not bactericidal. For mouse septicemia, doripenem (100 mg/kg) was ≥90% effective in preventing death in all four isolates. Levofloxacin (200 mg/kg) was 73% effective, and ciprofloxacin (35 mg/kg) was ineffective in preventing death. At lower drug concentrations, increased efficacy was observed for doripenem/levofloxacin, but not for doripenem/ciprofloxacin. Overall, the results suggest that a doripenem/levofloxacin combination may have clinical utility in treating some non-susceptible A. baumannii infections.

Discovery of pyrido[2,3-d]pyrimidine-based inhibitors of HCV NS5A.

Efforts to improve the genotype 1a potency and pharmacokinetics of earlier naphthyridine-based HCV NS5A inhibitors resulted in the discovery of a novel series of pyrido[2,3-d]pyrimidine compounds, which displayed potent inhibition of HCV genotypes 1a and 1b in the replicon assay. SAR in this system revealed that the introduction of amides bearing an additional 'E' ring provided compounds with improved potency and pharmacokinetics. Introduction of a chiral center on the amide portion resulted in the observation of a stereochemical dependence for replicon potency and provided a site for the attachment of functional groups useful for improving the solubility of the series. Compound 21 was selected for administration in an HCV-infected chimpanzee. Observation of a robust viral load decline provided positive proof of concept for inhibition of HCV replication in vivo for the compound series.