X-ray crystal structure localizes the mechanism of inhibition of an IL-36R antagonist monoclonal antibody to interaction with Ig1 and Ig2 extra cellular domains.

Cellular signaling via binding of the cytokines IL-36α, ß, and γ along with binding of the accessory protein IL-36RAcP, to their cognate receptor IL-36R is believed to play a major role in epithelial and immune cell-mediated inflammation responses. Antagonizing the signaling cascade that results from these binding events via a directed monoclonal antibody provides an opportunity to suppress such immune responses. We report here the molecular structure of a complex between an extracellular portion of human IL-36R and a Fab derived from a high affinity anti-IL-36R neutralizing monoclonal antibody at 2.3 Å resolution. This structure, the first of IL-36R, reveals similarities with other structurally characterized IL-1R family members and elucidates the molecular determinants leading to the high affinity binding of the monoclonal antibody. The structure of the complex reveals that the epitope recognized by the Fab is remote from both the putative ligand and accessory protein binding interfaces on IL-36R, suggesting that the functional activity of the antibody is noncompetitive for these binding events.

Assessment of bacterial dependence on marine primary production along a northern latitudinal gradient.

Recent observations in polar marine waters have shown that a large fraction of primary production may be lost to respiration by planktonic bacteria due to very low bacterial growth efficiencies in cold waters. Here we report that sea temperature may be a key factor (but not the only one) influencing the interaction between bacteria and primary production in North Atlantic and Arctic waters, suggesting that low primary production rates could not sustain bacterial carbon demand in the coldest Arctic waters. The use of freshly produced phytoplankton exudate by bacteria in early- and mid-summer was assessed, together with the bacterial uptake of dissolved inorganic nitrogen (DIN = nitrate and ammonium), in surface waters along a latitudinal gradient from the North Sea to the Arctic sea ice. Bacterial production was independent of the low primary production measured in the coldest waters. Under these conditions, heterotrophic bacteria can consume a large fraction of DIN and N-rich organic matter, making them strong contributors to N fluxes in these waters.

Development of Three Orthogonal Assays Suitable for the Identification and Qualification of PIKfyve Inhibitors.

FYVE-type zinc finger-containing phosphoinositide kinase (PIKfyve) catalyzes the formation of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) from phosphatidylinositol 3-phosphate (PI(3)P). PIKfyve has been implicated in multiple cellular processes, and its role in the regulation of toll-like receptor (TLR) pathways and the production of proinflammatory cytokines has sparked interest in developing small-molecule PIKfyve inhibitors as potential therapeutics to treat autoimmune and inflammatory diseases. We developed three orthogonal assays to identify and qualify small-molecule inhibitors of PIKfyve: (1) a purified component microfluidic enzyme assay that measures the conversion of fluorescently labeled PI(3)P to PI(3,5)P2 by purified recombinant full-length human 6His-PIKfyve (rPIKfyve); (2) an intracellular protein stabilization assay using the kinase domain of PIKfyve expressed in HEK293 cells; and (3) a cell-based functional assay that measures the production of interleukin (IL)-12p70 in human peripheral blood mononuclear cells stimulated with TLR agonists lipopolysaccharide and R848. We determined apparent Km values for both ATP and labeled PI(3)P in the rPIKfyve enzyme assay and evaluated the enzyme's ability to use phosphatidylinositol as a substrate. We also tested four reference compounds in the three assays and showed that together these assays provide a platform that is suitable to select promising inhibitors having appropriate functional activity and confirmed cellular target engagement to advance into preclinical models of inflammation.

Structural studies unravel the active conformation of apo RORγt nuclear receptor and a common inverse agonism of two diverse classes of RORγt inhibitors.

The nuclear receptor retinoid acid receptor-related orphan receptor γt (RORγt) is a master regulator of the Th17/IL-17 pathway that plays crucial roles in the pathogenesis of autoimmunity. RORγt has recently emerged as a highly promising target for treatment of a number of autoimmune diseases. Through high-throughput screening, we previously identified several classes of inverse agonists for RORγt. Here, we report the crystal structures for the ligand-binding domain of RORγt in both apo and ligand-bound states. We show that apo RORγt adopts an active conformation capable of recruiting coactivator peptides and present a detailed analysis of the structural determinants that stabilize helix 12 (H12) of RORγt in the active state in the absence of a ligand. The structures of ligand-bound RORγt reveal that binding of the inverse agonists disrupts critical interactions that stabilize H12. This destabilizing effect is supported by ab initio calculations and experimentally by a normalized crystallographic B-factor analysis. Of note, the H12 destabilization in the active state shifts the conformational equilibrium of RORγt toward an inactive state, which underlies the molecular mechanism of action for the inverse agonists reported here. Our findings highlight that nuclear receptor structure and function are dictated by a dynamic conformational equilibrium and that subtle changes in ligand structures can shift this equilibrium in opposite directions, leading to a functional switch from agonists to inverse agonists.

Arctic marine phytobenthos of northern Baffin Island.

Global climate change is expected to alter the polar bioregions faster than any other marine environment. This study assesses the biodiversity of seaweeds and associated eukaryotic pathogens of an established study site in northern Baffin Island (72° N), providing a baseline inventory for future work assessing impacts of the currently ongoing changes in the Arctic marine environment. A total of 33 Phaeophyceae, 24 Rhodophyceae, 2 Chlorophyceae, 12 Ulvophyceae, 1 Trebouxiophyceae, and 1 Dinophyceae are reported, based on collections of an expedition to the area in 2009, complemented by unpublished records of Robert T. Wilce and the first-ever photographic documentation of the phytobenthos of the American Arctic. Molecular barcoding of isolates raised from incubated substratum samples revealed the presence of 20 species of brown seaweeds, including gametophytes of kelp and of a previously unsequenced Desmarestia closely related to D. viridis, two species of Pylaiella, the kelp endophyte Laminariocolax aecidioides and 11 previously unsequenced species of the Ectocarpales, highlighting the necessity to include molecular techniques for fully unraveling cryptic algal diversity. This study also includes the first records of Eurychasma dicksonii, a eukaryotic pathogen affecting seaweeds, from the American Arctic. Overall, this study provides both the most accurate inventory of seaweed diversity of the northern Baffin Island region to date and can be used as an important basis to understand diversity changes with climate change.

Using Molecular Networking for Microbial Secondary Metabolite Bioprospecting.

The oceans represent an understudied resource for the isolation of bacteria with the potential to produce novel secondary metabolites. In particular, actinomyces are well known to produce chemically diverse metabolites with a wide range of biological activities. This study characterised spore-forming bacteria from both Scottish and Antarctic sediments to assess the influence of isolation location on secondary metabolite production. Due to the selective isolation method used, all 85 isolates belonged to the phyla Firmicutes and Actinobacteria, with the majority of isolates belonging to the genera Bacillus and Streptomyces. Based on morphology, thirty-eight isolates were chosen for chemical investigation. Molecular networking based on chemical profiles (HR-MS/MS) of fermentation extracts was used to compare complex metabolite extracts. The results revealed 40% and 42% of parent ions were produced by Antarctic and Scottish isolated bacteria, respectively, and only 8% of networked metabolites were shared between these locations, implying a high degree of biogeographic influence upon secondary metabolite production. The resulting molecular network contained over 3500 parent ions with a mass range of m/z 149-2558 illustrating the wealth of metabolites produced. Furthermore, seven fermentation extracts showed bioactivity against epithelial colon adenocarcinoma cells, demonstrating the potential for the discovery of novel bioactive compounds from these understudied locations.

Bacterial Diversity Associated with the Coccolithophorid Algae Emiliania huxleyi and Coccolithus pelagicus f. braarudii.

Coccolithophores are unicellular calcifying marine phytoplankton that can form large and conspicuous blooms in the oceans and make significant contributions to oceanic carbon cycling and atmospheric CO2 regulation. Despite their importance, the bacterial diversity associated with these algae has not been explored for ecological or biotechnological reasons. Bacterial membership of Emiliania huxleyi and Coccolithus pelagicus f. braarudii cultures was assessed using cultivation and cultivation-independent methods. The communities were species rich compared to other phytoplankton cultures. Community analysis identified specific taxa which cooccur in all cultures (Marinobacter and Marivita). Hydrocarbon-degrading bacteria were found in all cultures. The presence of Acidobacteria, Acidimicrobidae, Schlegelella, and Thermomonas was unprecedented but were potentially explained by calcification associated with coccolith production. One strain of Acidobacteria was cultivated and is closely related to a marine Acidobacteria isolated from a sponge. From this assessment of the bacterial diversity of coccolithophores, a number of biotechnological opportunities are evident, from bioprospecting for novel taxa such as Acidobacteria to helping understand the relationship between obligate hydrocarbonoclastic bacteria occurrence with phytoplankton and to revealing bacterial taxa that have a specific association with algae and may be suitable candidates as a means to improve the efficiency of mass algal cultivation.

Phenotypic characterization of resistant Val36 variants of hepatitis C virus NS3-4A serine protease.

In patients chronically infected with hepatitis C virus (HCV) strains of genotype 1, rapid and dramatic antiviral activity has been observed with telaprevir (VX-950), a highly selective and potent inhibitor of the HCV NS3-4A serine protease. HCV variants with substitutions in the NS3 protease domain were observed in some patients during telaprevir dosing. In this study, purified protease domain proteins and reconstituted HCV subgenomic replicons were used for phenotypic characterization of many of these substitutions. V36A/M or T54A substitutions conferred less than eightfold resistance to telaprevir. Variants with double substitutions at Val36 plus Thr54 had approximately 20-fold resistance to telaprevir, and variants with double substitutions at Val36 plus Arg155 or Ala156 had >40-fold resistance to telaprevir. An X-ray structure of the HCV strain H protease domain containing the V36M substitution in a cocomplex with an NS4A cofactor peptide was solved at a 2.4-A resolution. Except for the side chain of Met36, the V36M variant structure is identical to that of the wild-type apoenzyme. The in vitro replication capacity of most variants was significantly lower than that of the wild-type replicon in cells, which is consistent with the impaired in vivo fitness estimated from telaprevir-dosed patients. Finally, the sensitivity of these replicon variants to alpha interferon or ribavirin remained unchanged compared to that of the wild-type.

The use of physical and virtual infrastructures for the validation of algal cryopreservation methods in international culture collections.

Two cryopreservation methods, colligative cryoprotection coupled with controlled cooling and vitrification-based, encapsulation-dehydration were validated by five members of the EU research infrastructure consortium, COBRA, and two independent external validators. The test strain Chlorella vulgaris SAG 211-11b was successfully cryopreserved using two-step cooling employing passive (Mr Frosty) and Controlled Rate Freezers (CRF) attaining the desired recovery target within 15% of the median viability level (94%). Significant differences (p < 0.05) between cooling regimes were observed where Mr Frosty was more variable (Inter-Quartile Range being 21.5%, versus 13.0% for CRF samples). Viability assessment using fluorescein diacetate gave significantly (P < 0.0001) higher survival than growth in agar with median values being 96% and 89%, respectively. On employing encapsulation-dehydration, greater variability between some validators was observed, with six labs observing recovery in 100% of the beads (84-95% of cells surviving) and one lab observing survival in 80% of the treated beads. Bead disruption followed by algal growth in agar was considered the most reliable and accurate method of assessing cell survival for encapsulation-dehydration.

Development of a protease production platform for structure-based drug design.

Structure-based drug design (SBDD) has played an integral role in the development of highly specific, potent protease inhibitors resulting in a number of drugs in clinical trials and on the market. Possessing biochemical assays and structural information on both the target protease and homologous family members helps ensure compound selectivity. We have redesigned the path from clone to protein eliminating many of the traditional bottlenecks associated with protein production to ensure a constant supply to feed many diverse protease drug discovery programs. The process was initiated with the design of a multi-system vector, capable of expression in both eukaryotic and prokaryotic hosts; this vector also facilitated high-throughput cloning, expression and purification. When combined into an expression screen, supplemented with salvage screens for detergent extraction and refolding, a route for protein production was established rapidly. Using this process-orientated approach we have successfully expressed and purified all mechanistic classes of active human and viral proteases for enzymatic assays and crystallization studies. While exploiting recent developments in high-throughput biochemistry, we still employ classical biophysical techniques such as light-scattering and analytical ultracentrifugation, to ensure the highest quality protein enters crystallization trials. We have drawn on examples from our own research programs to illustrate how these strategies have been successfully used in the production of proteases for SBDD.

Phenotypic and structural analyses of hepatitis C virus NS3 protease Arg155 variants: sensitivity to telaprevir (VX-950) and interferon alpha.

Telaprevir (VX-950) is a highly selective, potent inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease. It has demonstrated strong antiviral activity in patients chronically infected with genotype 1 HCV when dosed alone or in combination with peginterferon alfa-2a. Substitutions of Arg(155) of the HCV NS3 protease domain have been previously detected in HCV isolates from some patients during telaprevir dosing. In this study, Arg(155) was replaced with various residues in genotype 1a protease domain proteins and in genotype 1b HCV subgenomic replicons. Characterization of both the purified enzymes and reconstituted replicon cells demonstrated that substitutions of Arg(155) with these residues conferred low level resistance to telaprevir (<25-fold). An x-ray structure of genotype 1a HCV protease domain with the R155K mutation, in a complex with an NS4A co-factor peptide, was determined at a resolution of 2.5A. The crystal structure of the R155K protease is essentially identical to that of the wild-type apoenzyme (Protein Data Bank code 1A1R) except for the side chain of mutated residue 155. Telaprevir was docked into the x-ray structure of the R155K protease, and modeling analysis suggests that the P2 group of telaprevir loses several hydrophobic contacts with the Lys(155) side chain. It was demonstrated that replicon cells containing substitutions at NS3 protease residue 155 remain fully sensitive to interferon alpha or ribavirin. Finally, these variant replicons were shown to have reduced replication capacity compared with the wild-type HCV replicon in cells.

Nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator, an ABC transporter, catalyze adenylate kinase activity but not ATP hydrolysis.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter family. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain. Previous biochemical reports suggest NBD1 is a site of stable nucleotide interaction with low ATPase activity, whereas NBD2 is the site of active ATP hydrolysis. It has also been reported that NBD2 additionally possessed adenylate kinase (AK) activity. Knowledge about the intrinsic biochemical activities of the NBDs is essential to understanding the Cl(-) ion gating mechanism. We find that purified mouse NBD1, human NBD1, and human NBD2 function as adenylate kinases but not as ATPases. AK activity is strictly dependent on the addition of the adenosine monophosphate (AMP) substrate. No liberation of [(33)P]phosphate is observed from the gamma-(33)P-labeled ATP substrate in the presence or absence of AMP. AK activity is intrinsic to both human NBDs, as the Walker A box lysine mutations abolish this activity. At low protein concentration, the NBDs display an initial slower nonlinear phase in AK activity, suggesting that the activity results from homodimerization. Interestingly, the G551D gating mutation has an exaggerated nonlinear phase compared with the wild type and may indicate this mutation affects the ability of NBD1 to dimerize. hNBD1 and hNBD2 mixing experiments resulted in an 8-57-fold synergistic enhancement in AK activity suggesting heterodimer formation, which supports a common theme in ABC transporter models. A CFTR gating mechanism model based on adenylate kinase activity is proposed.

In vitro studies of cross-resistance mutations against two hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061.

VX-950 is a potent, small molecule, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease and has recently been shown to possess antiviral activity in a phase I trial in patients chronically infected with genotype 1 HCV. In a previous study, we described in vitro resistance mutations against either VX-950 or another HCV NS3.4A protease inhibitor, BILN 2061. Single amino acid substitutions that conferred drug resistance (distinct for either inhibitor) were identified in the HCV NS3 serine protease domain. The dominant VX-950-resistant mutant (A156S) remains sensitive to BILN 2061. The major BILN 2061-resistant mutants (D168V and D168A) are fully susceptible to VX-950. Modeling analysis suggested that there are different mechanisms of resistance for these mutations induced by VX-950 or BILN 2061. In this study, we identified mutants that are cross-resistant to both HCV protease inhibitors. The cross-resistance conferred by substitution of Ala(156) with either Val or Thr was confirmed by characterization of the purified enzymes and reconstituted replicon cells containing the single amino acid substitution A156V or A156T. Both cross-resistance mutations (A156V and A156T) displayed significantly diminished fitness (or replication capacity) in a transient replicon cell system.

In vitro resistance studies of hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061: structural analysis indicates different resistance mechanisms.

We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3.4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3.4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3.4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. The major BILN 2061-resistant mutations at Asp(168) are fully susceptible to VX-950, and the dominant resistant mutation against VX-950 at Ala(156) remains sensitive to BILN 2061. Modeling analysis suggests that there are different mechanisms of resistance to VX-950 and BILN 2061.