Discovery and optimization of a potent and selective triazolopyridinone series of c-Met inhibitors

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of a structurally diverse series of carbon-linked quinoline triazolopyridinones, which demonstrates nanomolar inhibition of c-Met kinase activity. This novel series of inhibitors exhibits favorable pharmacokinetics as well as potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.     

A Synergetic Screening Approach with Companion Effector for Combination Therapy: Application to Retinoblastoma

For many cancers, the lack of potency and the toxicity of current drugs limits the dose achievable in patients and the efficacy of treatment. Among them, retinoblastoma is a rare cancer of the eye for which better chemotherapeutic options are needed. Combination therapy is a compelling approach to enhance the efficacy of current treatment, however clinical trials to test rationally designed combinations of approved drugs are slow and expensive, and limited by our lack of in-depth knowledge of drug specificity. Since many patients already turn to nutraceuticals in hopes of improving their condition, we hypothesized that certain approved drugs could potentially synergize with widely consumed supplements. Following this hypothesis, we devised an alternative screening strategy aimed at taking advantage of a bait compound such as a nutraceutical with potential therapeutic benefits but low potency, by screening chemical libraries for approved drugs that synergize with this companion effector. As a proof of concept, we sought to identify approved drugs with synergetic therapeutic effects toward retinoblastoma cells in combination with the antioxidant resveratrol, popular as a supplement. We systematically tested FDA-approved drugs and known bioactives seeking to identify such pairs, which led to uncovering only a few additive combinations; but to our surprise, we identified a class of anticancer drugs widely used in the clinic whose therapeutic effect is antagonized with resveratrol. Our observations could explain in part why some patients do not respond well to treatment. Our results validate this alternative approach, and we expect that our companion effector strategy could significantly impact both drug discovery and the nutraceutical industry.     

High-performance liquid chromatographic assay for the determination of sulfadoxine and N-acetyl sulfadoxine in plasma from patients infected with sensitive and resistant Plasmodium falciparum malaria

A reversed-phase high-performance liquid chromatographic method using a mobile phase of acetonitrile-methanol-trifluoroacetic acid-water (16.1:7.2:0.1:76.6, v/v/v/v) at a flow rate of 1.0 ml min(-1) on a LiChrospher RP-18 column with UV (254 nm) detection has been developed for the separation of sulfadoxine and its metabolite N-acetyl sulfadoxine in plasma. No interferences due to endogenous compounds or common antimalarial drugs were noticed. The limit of detection for sulfadoxine and N-acetyl sulfadoxine was 0.01 microg ml(-1) with a signal-to-noise ratio of 5:1 while the limit of quantification was 2.5 microg ml(-1). Intra-day mean relative standard deviations (RSD's) for sulfadoxine and N-acetyl sulfadoxine were 2.6 and 2.8%, respectively, while mean inter-day RSD's for sulfadoxine and N-acetyl sulfadoxine were 2.4 and 2.8%, respectively. Extraction recoveries averaged 90.6% for sulfadoxine and 86.9% for N-acetyl sulfadoxine. The method was applied for the assay of sulfadoxine and its metabolite N-acetyl sulfadoxine in plasma from Plasmodium falciparum malaria patients. Mean plasma sulfadoxine concentrations on day 2 (51 h) from samples collected from sensitive and resistant P. falciparum patients treated with three tablets of Fansidar were 62.8 and 60.5 microg ml(-1), respectively. Mean ratio of N-acetyl sulfadoxine to sulfadoxine was 9.1% for responders and 13.9% for non-responders which revealed that higher amounts of the metabolite N-acetyl sulfadoxine were present in non-responders. The method described should find an application in the therapeutic monitoring of malaria patients.     

Production of neutralizing antibody to rabies and vesicular stomatitis viruses by hybrid cell lines

Hybrid cell lines were obtained following fusion of P 3 × 63 Ag-8 myeloma cells with spleen cells derived from BALB/c mice immunized either with rabies virus or with vesicular stomatitis virus. Hybrid cell lines were selected which continued to secrete rabies virus or vesicular stomatitis virus neutralizing antibody specifically directed against coat glycoprotein of respective viruses.     

A network of conserved interactions regulates the allosteric signal in a glutamine amidotransferase

We have combined equilibrium and steered molecular dynamics (SMD) simulations with principal component and correlation analyses to probe the mechanism of allosteric regulation in imidazole glycerol phosphate (IGP) synthase. An evolutionary analysis of IGP synthase revealed a conserved network of interactions leading from the effector binding site to the glutaminase active site, forming conserved communication pathways between the remote active sites. SMD simulations of the undocking of the ribonucleotide effector N1-[(5'-phosphoribulosyl)-formino]-5'-aminoimidazole carboxamide ribonucleotide (PRFAR) resulted in a large scale hinge-opening motion at the interface. Principal component analysis and a correlation analysis of the equilibration protein motion indicate that the dynamics involved in the allosteric transition are mediated by coupled motion between sites that are more than 25 A apart. Furthermore, conserved residues at the substrate-binding site, within the barrel, and at the interface were found to exhibit highly correlated motion during the allosteric transition. The coupled motion between PRFAR unbinding and the directed opening of the interface is interpreted in combination with kinetic assays for the wild-type and mutant systems to develop a model of allosteric regulation in IGP synthase that is monitored and investigated with atomic resolution.     

Expression profiling of metalloproteinases and their inhibitors in synovium and cartilage

Cartilage destruction in osteoarthritis (OA) is thought to be mediated by two main enzyme families; the matrix metalloproteinases (MMPs) are responsible for cartilage collagen breakdown, whereas enzymes from the 'a disintegrin and metalloproteinase domain with thrombospondin motifs' (ADAMTS) family mediate cartilage aggrecan loss. Tissue inhibitors of metalloproteinases (TIMPs) regulate the activity of these enzymes. Although cartilage destruction in OA might be driven by the chondrocyte, low-grade synovitis is reported in patients with all grades of this disease.     

Habitat degradation affects the summer activity of polar bears

Understanding behavioral responses of species to environmental change is critical to forecasting population-level effects. Although climate change is significantly impacting species’ distributions, few studies have examined associated changes in behavior. Polar bear (Ursus maritimus) subpopulations have varied in their near-term responses to sea ice decline. We examined behavioral responses of two adjacent subpopulations to changes in habitat availability during the annual sea ice minimum using activity data. Location and activity sensor data collected from 1989 to 2014 for 202 adult female polar bears in the Southern Beaufort Sea (SB) and Chukchi Sea (CS) subpopulations were used to compare activity in three habitat types varying in prey availability: (1) land; (2) ice over shallow, biologically productive waters; and (3) ice over deeper, less productive waters. Bears varied activity across and within habitats with the highest activity at 50–75% sea ice concentration over shallow waters. On land, SB bears exhibited variable but relatively high activity associated with the use of subsistence-harvested bowhead whale carcasses, whereas CS bears exhibited low activity consistent with minimal feeding. Both subpopulations had fewer observations in their preferred shallow-water sea ice habitats in recent years, corresponding with declines in availability of this substrate. The substantially higher use of marginal habitats by SB bears is an additional mechanism potentially explaining why this subpopulation has experienced negative effects of sea ice loss compared to the still-productive CS subpopulation. Variability in activity among, and within, habitats suggests that bears alter their behavior in response to habitat conditions, presumably in an attempt to balance prey availability with energy costs.     

Pathogenicity and Transmissibility of North American Triple Reassortant Swine Influenza A Viruses in Ferrets

North American triple reassortant swine (TRS) influenza A viruses have caused sporadic human infections since 2005, but human-to-human transmission has not been documented. These viruses have six gene segments (PB2, PB1, PA, HA, NP, and NS) closely related to those of the 2009 H1N1 pandemic viruses. Therefore, understanding of these viruses'' pathogenicity and transmissibility may help to identify determinants of virulence of the 2009 H1N1 pandemic viruses and to elucidate potential human health threats posed by the TRS viruses. Here we evaluated in a ferret model the pathogenicity and transmissibility of three groups of North American TRS viruses containing swine-like and/or human-like HA and NA gene segments. The study was designed only to detect informative and significant patterns in the transmissibility and pathogenicity of these three groups of viruses. We observed that irrespective of their HA and NA lineages, the TRS viruses were moderately pathogenic in ferrets and grew efficiently in both the upper and lower respiratory tracts. All North American TRS viruses studied were transmitted between ferrets via direct contact. However, their transmissibility by respiratory droplets was related to their HA and NA lineages: TRS viruses with human-like HA and NA were transmitted most efficiently, those with swine-like HA and NA were transmitted minimally or not transmitted, and those with swine-like HA and human-like NA (N2) showed intermediate transmissibility. We conclude that the lineages of HA and NA may play a crucial role in the respiratory droplet transmissibility of these viruses. These findings have important implications for pandemic planning and warrant confirmation.     

Aeromonas hydrophila and Aeromonas veronii Predominate among Potentially Pathogenic Ciprofloxacin- and Tetracycline-Resistant Aeromonas Isolates from Lake Erie

Members of the genus Aeromonas are ubiquitous in nature and have increasingly been implicated in numerous diseases of humans and other animal taxa. Although some species of aeromonads are human pathogens, their presence, density, and relative abundance are rarely considered in assessing water quality. The objectives of this study were to identify Aeromonas species within Lake Erie, determine their antibiotic resistance patterns, and assess their potential pathogenicity. Aeromonas strains were isolated from Lake Erie water by use of Aeromonas selective agar with and without tetracycline and ciprofloxacin. All isolates were analyzed for hemolytic ability and cytotoxicity against human epithelial cells and were identified to the species level by using 16S rRNA gene restriction fragment length polymorphisms and phylogenetic analysis based on gyrB gene sequences. A molecular virulence profile was identified for each isolate, using multiplex PCR analysis of six virulence genes. We demonstrated that Aeromonas comprised 16% of all culturable bacteria from Lake Erie. Among 119 Aeromonas isolates, six species were identified, though only two species (Aeromonas hydrophila and A. veronii) predominated among tetracycline- and ciprofloxacin-resistant isolates. Additionally, both of these species demonstrated pathogenic phenotypes in vitro. Virulence gene profiles demonstrated a high prevalence of aerolysin and serine protease genes among A. hydrophila and A. veronii isolates, a genetic profile which corresponded with pathogenic phenotypes. Together, our findings demonstrate increased antibiotic resistance among potentially pathogenic strains of aeromonads, illustrating an emerging potential health concern.