Polyphony: superposition independent methods for ensemble-based drug discovery.

BACKGROUND: Structure-based drug design is an iterative process, following cycles of structural biology, computer-aided design, synthetic chemistry and bioassay. In favorable circumstances, this process can lead to the structures of hundreds of protein-ligand crystal structures. In addition, molecular dynamics simulations are increasingly being used to further explore the conformational landscape of these complexes. Currently, methods capable of the analysis of ensembles of crystal structures and MD trajectories are limited and usually rely upon least squares superposition of coordinates. RESULTS: Novel methodologies are described for the analysis of multiple structures of a protein. Statistical approaches that rely upon residue equivalence, but not superposition, are developed. Tasks that can be performed include the identification of hinge regions, allosteric conformational changes and transient binding sites. The approaches are tested on crystal structures of CDK2 and other CMGC protein kinases and a simulation of p38α. Known interaction - conformational change relationships are highlighted but also new ones are revealed. A transient but druggable allosteric pocket in CDK2 is predicted to occur under the CMGC insert. Furthermore, an evolutionarily-conserved conformational link from the location of this pocket, via the αEF-αF loop, to phosphorylation sites on the activation loop is discovered. CONCLUSIONS: New methodologies are described and validated for the superimposition independent conformational analysis of large collections of structures or simulation snapshots of the same protein. The methodologies are encoded in a Python package called Polyphony, which is released as open source to accompany this paper [http://wrpitt.bitbucket.org/polyphony/]

Longevity of Rodenticide Bait Pellets in a Tropical Environment Following a Rat Eradication Program

Invasive rodents (primarily Rattus spp.) are responsible for loss of biodiversity in island ecosystems worldwide. Large-scale rodenticide applications are typically used to eradicate rats and restore ecological communities. In tropical ecosystems, environmental conditions rapidly degrade baits and competition for baits by non-target animals can result in eradication failure. Our objective was to evaluate persistence of rodenticide baits during a rat eradication program on Palmyra Atoll; a remote tropical atoll with intense competition for resources by land crabs. Following aerial application, bait condition was monitored in four terrestrial environments and in the canopy foliage of coconut palms. Ten circular PVC hoops were fixed in place in each of Palmyra\u27s four primary terrestrial habitats and five rodenticide pellets were placed in each hoop. Five coconut palms were selected in three distinct regions of the atoll. One rodenticide pellet was placed on each of five palm fronds in each coconut palm. Fresh baits were placed in all monitoring locations after each broadcast bait application. Bait condition and survival was monitored for 7 days after the first bait application and 6 days after second application. Bait survival curves differed between applications at most monitoring sites, suggesting a decrease in overall rat activity as a result of rodenticide treatment. One terrestrial site showed near 100%bait survival after both applications, likely due to low localized rat and crab densities. Median days to pellet disappearance were one and two days for the first and second application, respectively. Differences in survival curves were not detected in canopy sites between bait applications. Median days to pellet disappearance in canopy sites were 2 and 4 days for the first and second application, respectively. Frequent rainfall likely contributed to rapid degradation of bait pellets in coconut palm fronds

Evaluation of the palatability and toxicity of candidate baits and toxicants for mongooses (\u3ci\u3eHerpestes auropunctatus\u3c/i\u3e)

The small Indian mongoose (Herpestes auropunctatus) is an invasive pest species responsible for damage to native avian, reptile, and amphibian species on Hawaii, Croatia, Mauritius, and several Caribbean Islands, among other regions.Mongoose control has been pursued through a variety of means, with varying success. One toxicant, diphacinone, has been shown to be effective in mongooses and is co-labeled in a rodenticide bait for mongoose control in Hawaii; however, preliminary observations indicate low performance as a mongoose toxicant due likely to poor consumption. We evaluated the efficacy and palatability of 10 commercial rodenticide baits, technical diphacinone powder, and two alternative acute toxicants against mongooses in laboratory feeding trials. We observed poor acceptance and subsequent low overall mortality, of the hard grain-based pellets or block formulations typical of most of the commercial rodenticide baits. The exception was Tomcat® bait blocks containing 0.1% bromethalin, an acute neurotoxin, which achieved up to 100% mortality. Mortality among all other commercial rodenticide formulations ranged from 10 to 50%. Three-day feedings of 0.005% technical diphacinone formulated in fresh minced chicken achieved 100% mortality. One-day feedings of para-aminopropiophenone (PAPP), a chemical that reduces the oxygen-carrying capacity of the blood, achieved 100% mortality at concentrations of 0.10 to 0.15%. Bait acceptance of two sodium nitrite formulations (similar toxic mode of action as PAPP) was relatively poor, and mortality averaged 20%. In general, commercially produced rodenticide baits were not preferred by mongooses and had lower mortality rates compared to freshly prepared meat bait formulations. More palatable baits had higher consumption and achieved higher mortality rates. The diphacinone bait registered for rat and mongoose control in Hawaii achieved 20% mortality and was less effective than some of the other commercial or candidate fresh bait products evaluated in this study

Assessing spatial variation and overall density of aerially broadcast toxic bait during a rat eradication on Palmyra Atoll

Baits containing brodifacoum rodenticide were aerially applied to eradicate invasive black rats from Palmyra Atoll, an important biodiversity center. Bait application must be sufficient to be effective, while minimizing environmental hazards by not exceeding designated label rates, prompting our bait density assessments for two aerial drops. With few physical or human resources on this remote, uninhabited atoll, assessments were particularly challenging, requiring observations within 30 min of aerial application to avoid bait loss to rats, crabs, or elements. We estimated bait density using quadrat sampling within 13 terrestrial sampling areas. We also sampled 10 tidal flat areas to assess inadvertent bait scatter into marine aquatic environments. Of particular value for challenging sampling circumstances, our quadrats had to be lightweight and durable, which we addressed by using widely available PVC hoops (“Hula Hoops”), the size of which was ideal for sampling purposes. At 77.5 and 78.7 kg/ha, overall bait densities were very near to the target densities of 80 and 75 kg/ha, respectively. However, considerable variability in bait densities existed among sampled areas, 8.6–178.2 and 31.4–129.5 kg/ha for the respective drops. Environmental, human, and equipment factors likely accounted for this variability. Tidal flat sampling revealed variable bait scatter into aquatic environments, from 0–46.3 kg/ha across the two drops. No differences were found in average bait densities among 1-, 4-, and 7-m distances from high tide lines. Our methods might broadly assist bait density (and other) surveys under challenging circumstances

Assessing spatial variation and overall density of aerially broadcast toxic bait during a rat eradication on Palmyra Atoll

Baits containing brodifacoum rodenticide were aerially applied to eradicate invasive black rats from Palmyra Atoll, an important biodiversity center. Bait application must be sufficient to be effective, while minimizing environmental hazards by not exceeding designated label rates, prompting our bait density assessments for two aerial drops. With few physical or human resources on this remote, uninhabited atoll, assessments were particularly challenging, requiring observations within 30 min of aerial application to avoid bait loss to rats, crabs, or elements. We estimated bait density using quadrat sampling within 13 terrestrial sampling areas. We also sampled 10 tidal flat areas to assess inadvertent bait scatter into marine aquatic environments. Of particular value for challenging sampling circumstances, our quadrats had to be lightweight and durable, which we addressed by using widely available PVC hoops (“Hula Hoops”), the size of which was ideal for sampling purposes. At 77.5 and 78.7 kg/ha, overall bait densities were very near to the target densities of 80 and 75 kg/ha, respectively. However, considerable variability in bait densities existed among sampled areas, 8.6–178.2 and 31.4–129.5 kg/ha for the respective drops. Environmental, human, and equipment factors likely accounted for this variability. Tidal flat sampling revealed variable bait scatter into aquatic environments, from 0–46.3 kg/ha across the two drops. No differences were found in average bait densities among 1-, 4-, and 7-m distances from high tide lines. Our methods might broadly assist bait density (and other) surveys under challenging circumstances

Brodifacoum residues in fish three years after an island-wide rat eradication attempt in the tropical Pacific

Invasive rats are known to threaten natural resources and human health and safety. Island-wide rat eradication attempts have been increasing in number and scale during the past several decades, as has the frequency of eradication success. The most common method to remove all rats from an island is to broadcast anticoagulant rodenticide bait into every rat’s home range on the island. Broadcast of toxicants can put humans and other nontarget species in marine and terrestrial environments at risk of exposure. The persistence of anticoagulant residues is somewhat unknown, particularly in marine environments. Three years after ~ 18,000 kg of 25 mg/kg brodifacoum bait was broadcast across Wake Atoll to eliminate rats, we collected whole-body fish samples from six near-shore sites and one intermittently land- locked pond to test for brodifacoum residues. Of the 69 samples tested using high- performance liquid chromatography with fluorescence detection, 20 were suspected of brodifacoum contamination and therefore subject to more selective liquid chromatography-mass spectrometry analysis. Of those 20 fish, brodifacoum was detected in two individuals of blacktail snapper (Lutjanus fulvus), although at levels too low to be accurately quantified. Both fish containing detectable brodifacoum residues were caught within an intermittently land-locked pond in an area of the island that received heavy brodifacoum baiting, and not truly in the “marine environment”. Brodifacoum was not detected in any of the samples collected within the lagoon of the atoll or within near-shore waters outside the lagoon. These results demonstrate that under some circumstances very low levels of brodifacoum can occur in a low proportion of fish tissues for as long as three years after the application of the rodenticide to an environment. Such information is valuable in assessing the relative environmental risks associated with rodenticide use in rodent eradications for protection of threatened species and restoration of island ecosystems. The overall result is one of declining incidence and magnitude of residue concentrations over time and within limited environmental circumstances

Cell morphology drives spatial patterning in microbial communities

The clearest phenotypic characteristic of microbial cells is their shape, but we do not understand how cell shape affects the dense communities, known as biofilms, where many microbes live. Here, we use individual-based modeling to systematically vary cell shape and study its impact in simulated communities. We compete cells with different cell morphologies under a range of conditions and ask how shape affects the patterning and evolutionary fitness of cells within a community. Our models predict that cell shape will strongly influence the fate of a cell lineage: we describe a mechanism through which coccal (round) cells rise to the upper surface of a community, leading to a strong spatial structuring that can be critical for fitness. We test our predictions experimentally using strains of Escherichia coli that grow at a similar rate but differ in cell shape due to single amino acid changes in the actin homolog MreB. As predicted by our model, cell types strongly sort by shape, with round cells at the top of the colony and rod cells dominating the basal surface and edges. Our work suggests that cell morphology has a strong impact within microbial communities and may offer new ways to engineer the structure of synthetic communities

Short-chain lipid peroxidation products form covalent adducts with pyruvate kinase and inhibit its activity in vitro and in breast cancer cells

Pyruvate kinase catalyses the last step in glycolysis and has been suggested to contribute to the regulation of aerobic glycolysis in cancer cells. It can be inhibited by oxidation of cysteine residues in vitro and in vivo, which is relevant to the more pro-oxidant state in cancer and proliferating tissues. These conditions also favour lipid peroxidation and the formation of electrophilic fragmentation products, including short-chain aldehydes that can covalently modify proteins. However, as yet few studies have investigated their interactions with pyruvate kinase, so we investigated the effects of three different aldehydes, acrolein, malondialdehyde and 4-hydroxy-2(E)-hexenal (HHE), on the structure and activity of the enzyme. Analysis by LC-MS/MS showed unique modification profiles for each aldehyde, but Cys152, Cys423 and Cys474 were the residues most susceptible to electrophilic modification. Analysis of enzymatic activity under these conditions showed that acrolein was the strongest inhibitor, and at incubation times longer than 2 h, pathophysiological concentrations induced significant effects. Treatment of MCF-7 cells with the aldehydes caused similar losses of pyruvate kinase activity to those observed in vitro, and at lower concentrations than those required to cause cell death, with time and dose-dependent effects; acrolein adducts on Cys152 and Cys358 were detected. Cys358 and Cys474 are located at or near the allosteric or active sites, and formation of adducts on these residues probably contributes to loss of activity at low treatment concentrations. This study provides the first detailed analysis of the structure-activity relationship of C3 and C6 aldehydes with pyruvate kinase, and suggests that reactive short-chain aldehydes generated in diseases with an oxidative aetiology or from environmental exposure such as smoking could be involved in the metabolic alterations observed in cancer cells, through alteration of pyruvate kinase activity