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1.
J Med Chem ; 62(7): 3254-3267, 2019 04 11.
Article in English | MEDLINE | ID: mdl-30763090

ABSTRACT

We previously described the discovery of GSK5852 (1), a non-nucleoside polymerase (NS5B) inhibitor of hepatitis C virus (HCV), in which an N-benzyl boronic acid was essential for potent antiviral activity. Unfortunately, facile benzylic oxidation resulted in a short plasma half-life (5 h) in human volunteers, and a backup program was initiated to remove metabolic liabilities associated with 1. Herein, we describe second-generation NS5B inhibitors including GSK8175 (49), a sulfonamide- N-benzoxaborole analog with low in vivo clearance across preclinical species and broad-spectrum activity against HCV replicons. An X-ray structure of NS5B protein cocrystallized with 49 revealed unique protein-inhibitor interactions mediated by an extensive network of ordered water molecules and the first evidence of boronate complex formation within the binding pocket. In clinical studies, 49 displayed a 60-63 h half-life and a robust decrease in viral RNA levels in HCV-infected patients, thereby validating our hypothesis that reducing benzylic oxidation would improve human pharmacokinetics and lower efficacious doses relative to 1.


Subject(s)
Antiviral Agents/pharmacology , Boronic Acids/pharmacology , Drug Design , Hepacivirus/drug effects , Nucleic Acid Synthesis Inhibitors/pharmacology , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Boronic Acids/chemistry , Boronic Acids/pharmacokinetics , Crystallography, X-Ray , Dogs , Half-Life , Humans , Macaca fascicularis , Mice , Molecular Structure , Nucleic Acid Synthesis Inhibitors/chemistry , Nucleic Acid Synthesis Inhibitors/pharmacokinetics , Rats
2.
ISME J ; 9(9): 2046-58, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25822482

ABSTRACT

Cyanobacteria are often the dominant phototrophs in polar freshwater communities; yet, the phages that infect them remain unknown. Here, we present a genomic and morphological characterization of cyanophage S-EIV1 that was isolated from freshwaters on Ellesmere Island (Nunavut, High Arctic Canada), and which infects the polar Synechococcus sp., strain PCCC-A2c. S-EIV1 represents a newly discovered evolutionary lineage of bacteriophages whose representatives are widespread in aquatic systems. Among the 130 predicted open reading frames (ORFs) there is no recognizable similarity to genes that encode structural proteins other than the large terminase subunit and a distant viral morphogenesis protein, indicating that the genes encoding the structural proteins of S-EIV1 are distinct from other viruses. As well, only 19 predicted coding sequences on the 79 178 bp circularly permuted genome have homology with genes encoding proteins of known function. Although S-EIV1 is divergent from other sequenced phage isolates, it shares synteny with phage genes captured on a fosmid from the deep-chlorophyll maximum in the Mediterranean Sea, as well as with an incision element in the genome of Anabaena variabilis (ATCC 29413). Sequence recruitment of metagenomic data indicates that S-EIV1-like viruses are cosmopolitan and abundant in a wide range of aquatic systems, suggesting they have an important ecological role.


Subject(s)
Bacteriophages/genetics , Biological Evolution , Genome, Viral , Synechococcus/virology , Viral Proteins/genetics , Anabaena/genetics , Arctic Regions , Canada , Chloroform , Chlorophyll/chemistry , Fresh Water/microbiology , Genomics , Mediterranean Sea , Microscopy, Electron, Transmission , Open Reading Frames , Phylogeny , Sequence Analysis, DNA , Synechococcus/genetics , Synteny
3.
Bioorg Med Chem Lett ; 24(10): 2288-94, 2014 May 15.
Article in English | MEDLINE | ID: mdl-24731273

ABSTRACT

Two novel series of spirocyclic piperidine analogs appended to a pyrazolo[1,5-a]pyridine core were designed, synthesized and evaluated for their anti-HCV activity. A series of piperidine ketals afforded dispiro 6p which showed excellent in vitro anti-HCV activities (EC50 of 1.5nM and 1.2nM against genotype 1a and 1b replicons, respectively). A series of piperidine oxazolidinones afforded 27c which showed EC50's of 10.9nM and 6.1nM against 1a and 1b replicons, respectively. Both compounds 6p and 27c bound directly to non-structural NS4B protein in vitro (IC50's=10.2 and 30.4nM, respectively) and exhibited reduced potency in replicons containing resistance mutations encoding changes in the NS4B protein.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Hepacivirus/physiology , Spiro Compounds/chemistry , Spiro Compounds/pharmacology , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects , Antiviral Agents/chemical synthesis , Drug Design , Hepacivirus/drug effects , Hepacivirus/metabolism , Humans , Molecular Targeted Therapy , Spiro Compounds/chemical synthesis
4.
J Med Chem ; 57(5): 2107-20, 2014 Mar 13.
Article in English | MEDLINE | ID: mdl-23544424

ABSTRACT

We describe the preclinical development and in vivo efficacy of a novel chemical series that inhibits hepatitis C virus replication via direct interaction with the viral nonstructural protein 4B (NS4B). Significant potency improvements were realized through isosteric modifications to our initial lead 1a. The temptation to improve antiviral activity while compromising physicochemical properties was tempered by the judicial use of ligand efficiency indices during lead optimization. In this manner, compound 1a was transformed into (+)-28a which possessed an improved antiviral profile with no increase in molecular weight and only a modest elevation in lipophilicity. Additionally, we employed a chimeric "humanized" mouse model of HCV infection to demonstrate for the first time that a small molecule with high in vitro affinity for NS4B can inhibit viral replication in vivo. This successful proof-of-concept study suggests that drugs targeting NS4B may represent a viable treatment option for curing HCV infection.


Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Virus Replication/drug effects , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Area Under Curve , Disease Models, Animal , Hepacivirus/physiology , Hepatitis C/virology , Mice , Prodrugs/chemistry , Prodrugs/pharmacokinetics , Prodrugs/pharmacology
5.
ACS Med Chem Lett ; 3(7): 565-9, 2012 Jul 12.
Article in English | MEDLINE | ID: mdl-24900511

ABSTRACT

A series of imidazo[1,2-a]pyridines which directly bind to HCV Non-Structural Protein 4B (NS4B) is described. This series demonstrates potent in vitro inhibition of HCV replication (EC50 < 10 nM), direct binding to purified NS4B protein (IC50 < 20 nM), and an HCV resistance pattern associated with NS4B (H94N/R, V105L/M, F98L) that are unique among reported HCV clinical assets, suggestive of the potential for additive or synergistic combination with other small molecule inhibitors of HCV replication.

6.
Environ Sci Technol ; 43(13): 4802-9, 2009 Jul 01.
Article in English | MEDLINE | ID: mdl-19673268

ABSTRACT

Recent and historical deposition of mercury (Hg) was examined over a broad geographic area from southwestern Northwest Territories to Labrador and from the U.S. Northeast to northern Ellesmere Island using dated sediment cores from 50 lakes (18 in midlatitudes (41-50 degrees N), 14 subarctic (51-64 degrees N) and 18 in the Arctic (65-83 degrees N)). Distinct increases of Hg overtime were observed in 76% of Arctic, 86% of subarctic and 100% of midlatitude cores. Subsurface maxima in Hg depositional fluxes (microg m(-2) y(-1)) were observed in only 28% of midlatitude lakes and 18% of arctic lakes, indicating little recent reduction of inputs. Anthropogenic Hg fluxes adjusted for sediment focusing and changes in sedimentation rates (deltaF(adj,F)) ranged from -22.9 to 61 microg m(-2) y(-1) and were negatively correlated (r = -0.57, P < 0.001) with latitude. Hg flux ratios (FRs; post-1990)/pre-1850) ranged from 0.5 to 7.7. The latitudinal trend for Hg deltaF(adj,F) values showed excellent agreement with predictions of the global mercury model, GRAHM for the geographic location of each lake (r = 0.933, P < 0.001). The results are consistent with a scenario of slow atmospheric oxidation of mercury, and slow deposition of reactive mercury emissions, declining with increasing latitude away from emission sources in the midlatitudes, and support the view that there are significant anthropogenic Hg inputs in the Arctic.


Subject(s)
Geologic Sediments , Mercury/toxicity , Water Pollutants/analysis , Arctic Regions , Atmosphere , Canada , Environmental Monitoring/methods , Geography , Mercury/analysis , Oxygen/analysis , Time Factors , Water Pollution/analysis , Water Supply
7.
J Evol Biol ; 20(3): 971-84, 2007 May.
Article in English | MEDLINE | ID: mdl-17465908

ABSTRACT

Sympatric fish populations observed in many north temperate lakes are among the best models to study the processes of population divergence and adaptive radiation. Despite considerable research on such systems, little is known about the associations between ecological conditions and the extent of ecotypic divergence. In this study, we examined the biotic and abiotic properties of postglacial lakes in which lake whitefish, Coregonus clupeaformis, occur as a derived dwarf ecotype in sympatry with an ancestral normal ecotype. We compared 19 limnological variables between two groups of lakes known from previous studies to harbour sympatric dwarf and normal ecotypes with high and low levels of phenotypic and genetic differentiation respectively. We found clear environmental differences between the two lake groups. Namely, oxygen was the most discriminant variable, where lakes harbouring the most divergent populations were characterized by the greatest hypolimnetic oxygen depletion. These lakes also had lower zooplankton densities and a narrower distribution of zooplantonic prey length. These results suggest that the highest differentiation between sympatric ecotypes occurs in lakes with reduced habitat and prey availability that could increase competition for resources. This in turns supports the hypothesis that parallelism in the extent of phenotypic divergence among sympatric whitefish ecotypes is associated with parallelism in adaptive landscape in terms of differences in limnological characteristics, as well as availability and structure of the zooplanktonic community.


Subject(s)
Biodiversity , Biological Evolution , Salmonidae/physiology , Animals , Fresh Water/chemistry , Genetic Speciation , Genotype , Maine , Oxygen/analysis , Phenotype , Quebec , Salmonidae/anatomy & histology , Salmonidae/genetics
8.
Bioorg Med Chem Lett ; 16(17): 4554-8, 2006 Sep 01.
Article in English | MEDLINE | ID: mdl-16784854

ABSTRACT

As a continuation of our efforts to discover novel apoptosis inducers as anticancer agents using a cell-based caspase HTS assay, 2-phenyl-oxazole-4-carboxamide derivatives were identified. The structure-activity relationships of this class of molecules were explored. Compound 1k, with EC(50) of 270 nM and GI(50) of 229 nM in human colorectal DLD-1 cells, was selected and demonstrated the ability to cleave PARP and displayed DNA laddering, the hallmarks of apoptosis. Compound 1k showed 63% tumor growth inhibition in human colorectal DLD-1 xenograft mouse model at 50 mpk, bid.


Subject(s)
Amides/chemistry , Amides/pharmacology , Apoptosis/drug effects , Oxazoles/chemistry , Oxazoles/pharmacology , Amides/chemical synthesis , Animals , Cell Line, Tumor , Female , Humans , Mice , Molecular Structure , Oxazoles/chemical synthesis , Structure-Activity Relationship , Xenograft Model Antitumor Assays
9.
Bioorg Med Chem Lett ; 16(15): 4085-9, 2006 Aug 01.
Article in English | MEDLINE | ID: mdl-16725321

ABSTRACT

The synthesis of novel [1,2,4]oxadiazoles and their structure-activity relationship (SAR) for the inhibition of tryptase and related serine proteases is presented. Elaboration of the P'-side afforded potent, selective, and orally bioavailable tryptase inhibitors.


Subject(s)
Enzyme Inhibitors/pharmacology , Serine Endopeptidases/drug effects , Administration, Oral , Biological Availability , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/chemistry , Models, Molecular , Structure-Activity Relationship , Tryptases
10.
J Mol Biol ; 329(1): 93-120, 2003 May 23.
Article in English | MEDLINE | ID: mdl-12742021

ABSTRACT

An extensive structural manifold of short hydrogen bond-mediated, active site-directed, serine protease inhibition motifs is revealed in a set of over 300 crystal structures involving a large suite of small molecule inhibitors (2-(2-phenol)-indoles and 2-(2-phenol)-benzimidazoles) determined over a wide range of pH (3.5-11.4). The active site hydrogen-bonding mode was found to vary markedly with pH, with the steric and electronic properties of the inhibitor, and with the type of protease (trypsin, thrombin or urokinase type plasminogen activator (uPA)). The pH dependence of the active site hydrogen-bonding motif is often intricate, constituting a distinct fingerprint of each complex. Isosteric replacements or minor substitutions within the inhibitor that modulate the pK(a) of the phenol hydroxyl involved in short hydrogen bonding, or that affect steric interactions distal to the active site, can significantly shift the pH-dependent structural profile characteristic of the parent scaffold, or produce active site-binding motifs unique to the bound analog. Ionization equilibria at the active site associated with inhibitor binding are probed in a series of the protease-inhibitor complexes through analysis of the pH dependence of the structure and environment of the active site-binding groups involved in short hydrogen bond arrays. Structures determined at high pH (>11), suggest that the pK(a) of His57 is dramatically elevated, to a value as high as approximately 11 in certain complexes. K(i) values involving uPA and trypsin determined as a function of pH for a set of inhibitors show pronounced parabolic pH dependence, the pH for optimal inhibition governed by the pK(a) of the inhibitor phenol involved in short hydrogen bonds. Comparison of structures of trypsin, thrombin and uPA, each bound by the same inhibitor, highlights important structural variations in the S1 and active sites accessible for engineering notable selectivity into remarkably small molecules with low nanomolar K(i) values.


Subject(s)
Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology , Thrombin/antagonists & inhibitors , Urokinase-Type Plasminogen Activator/antagonists & inhibitors , Animals , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Binding Sites , Cattle , Crystallography, X-Ray , Drug Design , Humans , Hydrogen Bonding , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Protein Conformation , Static Electricity , Structure-Activity Relationship , Thrombin/chemistry , Trypsin/chemistry , Trypsin Inhibitors/chemistry , Trypsin Inhibitors/pharmacology , Urokinase-Type Plasminogen Activator/chemistry
11.
Naturwissenschaften ; 87(3): 137-41, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10798200

ABSTRACT

The Ward Hunt Ice Shelf (83 degrees N, 74 degrees W) is the largest remaining section of thick (> 10 m) land-fast sea ice along the northern coastline of Ellesmere Island, Canada. Extensive meltwater lakes and streams occur on the surface of the ice and are colonized by photosynthetic microbial mat communities. This High Arctic cryo-ecosystem is similar in several of its physical, biological and geochemical features to the McMurdo Ice Shelf in Antarctica. The ice-mats in both polar regions are dominated by filamentous cyanobacteria but also contain diatoms, chlorophytes, flagellates, ciliates, nematodes, tardigrades and rotifers. The luxuriant Ward Hunt consortia also contain high concentrations (10(7)-10(8) cm-2) of viruses and heterotrophic bacteria. During periods of extensive ice cover, such as glaciations during the Proterozoic, cryotolerant mats of the type now found in these polar ice shelf ecosystems would have provided refugia for the survival, growth and evolution of a variety of organisms, including multicellular eukaryotes.


Subject(s)
Ecosystem , Ice , Water Microbiology , Water/parasitology , Animals , Arctic Regions , Canada , Ciliophora/isolation & purification , Cyanobacteria/isolation & purification , Eukaryota/isolation & purification , Nematoda/isolation & purification , Rotifera/isolation & purification , Viruses/isolation & purification
12.
Nature ; 404(6777): 484-7, 2000 Mar 30.
Article in English | MEDLINE | ID: mdl-10761913

ABSTRACT

The effect of stratospheric ozone depletion on increases in ambient levels of solar ultraviolet (UV) radiation in high-latitude regions' has raised concerns about the response of northern ecosystems to environmental change. The concentration of coloured dissolved organic material, which is derived from terrestrial vegetation and acts as a screen for ultraviolet radiation, is low in high-latitude lakes. The underwater light environment in these lakes is therefore likely to be sensitive to small variations in the supply of this material, in addition to the effects of ozone depletion. Here we use fossil diatom assemblages in combination with bio-optical models to estimate the magnitude of past variations in the underwater light regime of a lake at the boreal tree line. We find large shifts in underwater UV-B, UV-A and photosynthetically available radiation associated with changes in the input of coloured dissolved organic material into subarctic lakes during the Holocene. The inferred changes in biological exposure to UV radiation were at least two orders of magnitude greater than those associated with moderate (30%) ozone depletion. Our findings indicate that freshwater ecosystems at present located across vegetation gradients will experience significant shifts in underwater spectral irradiance through the effects of climate change on catchment vegetation and the export of coloured dissolved organic material.


Subject(s)
Climate , Diatoms/radiation effects , Ecosystem , Ozone , Ultraviolet Rays , Arctic Regions , Environment , Fossils , Fresh Water , Geologic Sediments , Greenhouse Effect , Photosynthesis
14.
J Org Chem ; 64(6): 2140-2144, 1999 Mar 19.
Article in English | MEDLINE | ID: mdl-11674316
15.
Appl Environ Microbiol ; 42(4): 565-73, 1981 Oct.
Article in English | MEDLINE | ID: mdl-16345852

ABSTRACT

Both nitrate and nitrous oxide accumulate in the hypolimnion of the oligotrophic Lake Taupo, New Zealand, throughout stratification. The two forms of oxidized nitrogen increase in concentration with increasing depth toward the sediments, where the dissolved concentrations of reduced nitrogen are two orders of magnitude higher than concentrations in the overlying water. Nitrification rates were measured by dark [C]CO(2) assays with and without the inhibitor nitrapyrin. The fastest rates were recorded for planktonic nitrifiers in the epilimnion and benthic species in the surficial 2.5 mm of the sediments. Nitrifying bacteria were least active in the deep hypolimnion. Deepwater accumulation of NO(3) in Lake Taupo must therefore be a product of benthic rather than planktonic nitrification.

16.
Can J Microbiol ; 25(12): 1401-7, 1979 Dec.
Article in English | MEDLINE | ID: mdl-534961

ABSTRACT

A diverse range of freshwater plankton communities were tested for their ability to take up [14C]methylammonium. Uptake occurred at low substrate levels by high-affinity, energy-requiring, transport systems which were competitively inhibited by ammonium but not by L-amino acids or nicotinamide. A simple competitive inhibition model was used to examine the effects of increasing ammonium levels on uptake in a eutrophic lake. Apparent K1 values for the labelled substrate markedly increased with increasing ammonium. The transport systems had an approximately five-fold greater affinity for ammonium than for methylammonium. The Vmax for methylammonium uptake was relatively insensitive to large changes in ambient ammonium levels. This kinetic parameter may be a useful comparative measure of ammonium transport capacity in natural waters, particularly where low ambient ammonium concentrations preclude the use of 15N.


Subject(s)
Plankton/metabolism , Quaternary Ammonium Compounds/metabolism , Water Microbiology , Amino Acids/pharmacology , Ammonium Chloride/pharmacology , Biological Transport, Active/drug effects , Darkness , Kinetics , Light
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