The fluorescent protein iLOV as a reporter for screening of high-yield production of antimicrobial peptides in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is commonly used for the production of recombinant proteins at scale. The identification of an optimally overexpressing strain following transformation can be time and reagent consuming. Fluorescent reporters like GFP have been used to assist identification of superior producers, but their relatively big size, maturation requirements and narrow temperature range restrict their applications. Here, we introduce the use of iLOV, a flavin-based fluorescent protein, as a fluorescent marker to identify P. pastoris high-yielding strains easily and rapidly. The use of this fluorescent protein as a fusion partner is exemplified by the production of the antimicrobial peptide NI01, a difficult target to overexpress in its native form. iLOV fluorescence correlated well with protein expression level and copy number of the chromosomally integrated gene. An easy and simple medium-throughput plate-based screen directly following transformation is demonstrated for low complexity screening, while a high-throughput method using fluorescence-activated cell sorting (FACS) allowed for comprehensive library screening. Both codon optimization of the iLOV_NI01 fusion cassettes and different integration strategies into the P. pastoris genome were tested to produce and isolate a high-yielding strain. Checking the genetic stability, process reproducibility and following the purification of the active native peptide are eased by visualization of and efficient cleavage from the iLOV reporter. We show that this system can be used for expression and screening of several different antimicrobial peptides recombinantly produced in P. pastoris.

The microdynamics of spatial polarization: A model and an application to survey data from Ukraine.

Although spatial polarization of attitudes is extremely common around the world, we understand little about the mechanisms through which polarization on divisive issues rises and falls over time. We develop a theory that explains how political shocks can have different effects in different regions of a country depending upon local dynamics generated by the preexisting spatial distribution of attitudes and discussion networks. Where opinions were previously divided, attitudinal diversity is likely to persist after the shock. Meanwhile, where a clear precrisis majority exists on key issues, opinions should change in the direction of the predominant view. These dynamics result in greater local homogeneity in attitudes but at the same time exacerbate geographic polarization across regions and sometimes even within regions. We illustrate our theory by developing a modified version of the adaptive voter model, an adaptive network model of opinion dynamics, to study changes in attitudes toward the European Union (EU) in Ukraine in the context of the Euromaidan Revolution of 2013 to 2014. Using individual-level panel data from surveys fielded before and after the Euromaidan Revolution, we show that EU support increased in areas with high prior public support for EU integration but declined further where initial public attitudes were opposed to the EU, thereby increasing the spatial polarization of EU attitudes in Ukraine. Our tests suggest that the predictive power of both network and regression models increases significantly when we incorporate information about the geographic location of network participants, which highlights the importance of spatially rooted social networks.

Searching for Dual Inhibitors of the MDM2-p53 and MDMX-p53 Protein-Protein Interaction by a Scaffold-Hopping Approach.

Two libraries of substituted benzimidazoles were designed using a 'scaffold-hopping' approach based on reported MDM2-p53 inhibitors. Substituents were chosen following library enumeration and docking into an MDM2 X-ray structure. Benzimidazole libraries were prepared using an efficient solution-phase approach and screened for inhibition of the MDM2-p53 and MDMX-p53 protein-protein interactions. Key examples showed inhibitory activity against both targets.

Decreased expression of the satiety signal receptor CCKAR is responsible for increased growth and body weight during the domestication of chickens.

Animal domestication has resulted in changes in growth and size. It has been suggested that this may have involved selection for differences in appetite. Divergent growth between chickens selected for egg laying or meat production is one such example. The neurons expressing AGRP and POMC in the basal hypothalamus are important components of appetite regulation, as are the satiety feedback pathways that carry information from the intestine, including CCK and its receptor CCKAR (CCK1 receptor). Using 16 generations of a cross between a fast and a relatively slow growing strain of chicken has identified a region on chromosome 4 downstream of the CCKAR gene, which is responsible for up to a 19% difference in body weight at 12 wk of age. Animals possessing the high-growth haplotype at the locus have lower expression of mRNA and immunoreactive CCKAR in the brain, intestine, and exocrine organs, which is correlated with increased levels of orexigenic AGRP in the hypothalamus. Animals with the high-growth haplotype are resistant to the anorectic effect of exogenously administered CCK, suggesting that their satiety set point has been altered. Comparison with traditional breeds shows that the high-growth haplotype has been present in the founders of modern meat-type strains and may have been selected early in domestication. This is the first dissection of the physiological consequences of a genetic locus for a quantitative trait that alters appetite and gives us an insight into the domestication of animals. This will allow elucidation of how differences in appetite occur in birds and also mammals.

Family-wide chemical profiling and structural analysis of PARP and tankyrase inhibitors.

Inhibitors of poly-ADP-ribose polymerase (PARP) family proteins are currently in clinical trials as cancer therapeutics, yet the specificity of many of these compounds is unknown. Here we evaluated a series of 185 small-molecule inhibitors, including research reagents and compounds being tested clinically, for the ability to bind to the catalytic domains of 13 of the 17 human PARP family members including the tankyrases, TNKS1 and TNKS2. Many of the best-known inhibitors, including TIQ-A, 6(5H)-phenanthridinone, olaparib, ABT-888 and rucaparib, bound to several PARP family members, suggesting that these molecules lack specificity and have promiscuous inhibitory activity. We also determined X-ray crystal structures for five TNKS2 ligand complexes and four PARP14 ligand complexes. In addition to showing that the majority of PARP inhibitors bind multiple targets, these results provide insight into the design of new inhibitors.

Collaboration versus outsourcing: the need to think outside the box.

As has been widely reviewed elsewhere, the pharmaceutical industry is experiencing an 'innovation deficit' as evidenced by the decline in new chemical entity output. This decline, compounded by increased costs and regulatory requirements highlights the need to significantly revise strategic options across the drug-discovery spectrum. Within such revision(s), much of the focus has been on outsourcing to reduce, or at least contain, costs, but if the underlying predominance of 'closed collaborations' is not challenged to allow better use of combined knowledge and, thus, move towards a more genuine collaborative process then a 'numbers only' approach will not bring medium-to-long-term survival. There are many problems to confront in evolving new sustainable strategies, a real need to think differently exists and should to be cultivated. This article reviews current outsourcing and collaboration strategies to provide a perspective on how great knowledge sharing could help revise the drug-discovery process.

Ask the experts: future of the pharmaceutical industry. Interview by Future Medicinal Chemistry.

The pharmaceutical industry is facing numerous, well-documented challenges - from the effects of patent expirations to high attrition rates in the drug-development pipeline. Future Medicinal Chemistry has invited a group of leaders from academia and industry to express their views on where the industry is heading and speculate as to what role medicinal chemists will play in the future.

Towards a more robust approach to selecting and prosecuting promising targets and compounds.

There are many factors that influence predictivity in drug discovery and impact on productivity within the pharmaceutical industry. This article will concentrate on just two aspects; first, the role of investigating target modulation within a (human) disease setting, from target selection through screening to animal models, and second, potential developments in the analysis and probing of the chemical space appropriate for drug discovery and, in particular, steps to improve predictivity thus moving to a more forward-looking process. The activities associated with target selection should develop significantly over the next 5-10 years leading to a more robust association of target modulation with disease modification. In addition, better understanding of the opportunities for target modulators should drive and improve the selection of ligands suitable for therapeutic applications. Within these areas it will be important to move away from a retrospective consideration of druggable targets towards a forward-looking approach based on holistic (disease context) profiling of both (progressable) targets and subsequently their ligands. Improvements in the predictive analysis and probing of the chemical space will be needed to confront both safety and efficacy end points that currently remain major reasons for failure in the clinic. It is hoped that improvements in data visualization together with chemocentric mining of the literature will facilitate better interrogation of development and clinical data, potentially modifying research project plans to better address these key issues.

Luminescence screening assays for the identification of sensitizers for lanthanides based on the controlled formation of ternary lanthanide complexes with DTPA-bisamide ligands.

The formation of ternary complexes of lanthanide-diethylenetriamine pentaacetic acid (DTPA)-bisamide complexes with different aromatic acids and their application in luminescent screening assays are presented. The europium complexes of DTPA-bisethylamide (BEA), DTPA-bisbutylamide (BUA), DTPA-bis(2-norbornyl) (NBA), and DTPA-bis(1-adamantyl) (ADA) have been isolated and the sensitization of emission upon 1:1 formation with aromatic acids has been studied by luminescence spectroscopy. The ternary complexes show stronger luminescence with picolinate (PCA) rather than phthalate (PTA) or benzoate (BZA), with the latter forming 1:2 complexes. Isophthalate and dipicolinate sensitizers do not show a 1:1 formation of the ternary complexes. Electrospray mass spectra show characteristic peaks that confirm the formation of the ternary complexes, and NMR spectroscopic studies demonstrated a conformational locking effect upon formation of the ternary complex. It is shown that europium complexes with bulkier amide arms provide complexes with stronger luminescence enhancement that is clearly attributable to the bulkiness of the amide arms. In a luminescence screening experiment, different substitutions on BZA and PCA were examined. Sensitizers with a long alkyl chain show greater luminescent enhancements than the nonsubstituted acids, which can be attributed to an "umbrella" effect of the alkyl chain that protects the europium luminescent center from the quenching of secondary high-energy vibrations, in particular O-H from water molecules. The same effect is presented for the quinoline derivatives as sensitizers. Quinoline derivatives with salicylate binding units were identified as the best sensitizers when combined with EuADA, which is even bulkier than EuNBA, with HQ-3COO showing a 50-fold enhancement of emission upon formation of 1:1 complexes.

How to achieve confidence in drug discovery and development: managing risk (from a reductionist to a holistic approach).

Confidence in mechanism: Creating a more holistic understanding of disease pathophysiology and an early confidence in the mechanism under investigation could help facilitate the selection of not only the most appropriate targets but also the best mechanisms for disease intervention and how to select and optimise the best compounds. Drug target and candidate selection are two of the key decision points within the drug discovery process for which all companies use certain selection criteria to make decisions on which targets to accept into their discovery pipelines and which compounds will pass into development. These steps not only help define the overall productivity of every company but they are also decisions taken without full predictive knowledge of the risks that lie ahead or how best to manage them. In particular, the process of selecting new targets does not normally involve full evaluation of the risk(s) in the mechanism under investigation (the modulation of the target), which may result in an inability to fully connect in vitro and animal model results to the disease (clinical) setting. The resulting poor progression statistics of many compounds in the clinic is at least partially the result of a lack of understanding of disease pathophysiology. Notably, the lack of efficacy is still a major reason for failure in the clinic.1 Creating a more holistic understanding of disease pathophysiology and an early confidence in the mechanism under investigation could help facilitate the selection of not only the most appropriate targets but also the best mechanisms for disease intervention and how to select and optimise the best compounds.

Modeling the pore structure of voltage-gated sodium channels in closed, open, and fast-inactivated conformation reveals details of site 1 toxin and local anesthetic binding.

In this work molecular modeling was applied to generate homology models of the pore region of the Na(v)1.2 and Na(v)1.8 isoforms of human voltage-gated sodium channels. The models represent the channels in the resting, open, and fast-inactivated states. The transmembrane portions of the channels were based on the equivalent domains of the closed and open conformation potassium channels KcsA and MthK, respectively. The critical selectivity loops were modeled using a structural template identified by a novel 3D-search technique and subsequently merged with the transmembrane portions. The resulting draft models were used to study the differences of tetrodotoxin binding to the tetrodotoxin-sensitive Na(v)1.2 (EC50: 0.012 microM) and -insensitive Na(v)1.8 (EC50: 60 microM) isoforms, respectively. Furthermore, we investigated binding of the local anesthetic tetracaine to Na(v)1.8 (EC50: 12.5 microM) in resting, conducting, and fast-inactivated state. In accordance with experimental mutagenesis studies, computational docking of tetrodotoxin and tetracaine provided (1) a description of site 1 toxin and local anesthetic binding sites in voltage-gated sodium channels. (2) A rationale for site 1 toxin-sensitivity versus -insensitivity in atomic detail involving interactions of the Na(v)1.2 residues F385-I and W943-II. (3) A working hypothesis of interactions between Na(v)1.8 in different conformational states and the local anesthetic tetracaine.

Genetic, ophthalmic, morphometric and histopathological analysis of the Retinopathy Globe Enlarged (rge) chicken.

PURPOSE: To identify the locus responsible for rge (retinopathy globe enlarged) in chickens and further characterise the rge phenotype. METHODS: A colony of chickens carrying the rge mutation was rederived from a single heterozygous animal of the original line. The eyes of blind, heterozygous and normal birds were subjected to ophthalmic, morphometric and histopathological examination to confirm and extend published observations. DNA samples were obtained and subjected to a whole genome linkage search. RESULTS: From 138 classified backcross progeny, 56 birds were blind and 82 sighted. Heterozygous birds were indistinguishable from wild type, but homozygotes had sluggish or unresponsive pupils, posterior sub-capsular lens opacities and an atrophic pecten. The fundus appeared normal with no significant pigmentary disturbance, but axial length and eye weight were increased. Pathology revealed focal retinal lesions. Linkage analysis placed the rge locus in a small region of chicken chromosome 1. CONCLUSIONS: rge is a severe recessive retinal dystrophy in chickens, with associated globe enlargement. Linkage mapping has highlighted chicken chromosome 1 in a region most probably homologous to human chromosomes 7q31-35, 21q21 or 22q12-21. Candidate disease loci include RP10 (IMPDH1) and uncharacterised Ushers (USH1E) and glaucoma (GLC1F) loci.

Analysis of the rdd locus in chicken: a model for human retinitis pigmentosa.

PURPOSE: To identify the locus responsible for the blind mutation rdd (retinal dysplasia and degeneration) in chickens and to further characterise the rdd phenotype. METHODS: The eyes of blind and sighted birds were subjected to ophthalmic, morphometric and histopathological examination to confirm and extend published observations. Electroretinography was used to determine age of onset. Birds were crossed to create pedigrees suitable for genetic mapping. DNA samples were obtained and subjected to a linkage search. RESULTS: Measurement of IOP, axial length, corneal diameter, and eye weight revealed no gross morphological changes in the rdd eye. However, on ophthalmic examination, rdd homozygotes have a sluggish pupillary response, atrophic pecten, and widespread pigmentary disturbance that becomes more pronounced with age. Older birds also have posterior subcapsular cataracts. At three weeks of age, homozygotes have a flat ERG indicating severe loss of visual function. Pathological examination shows thinning of the RPE, ONL, photoreceptors and INL, and attenuation of the ganglion cell layer. From 77 classified backcross progeny, 39 birds were blind and 38 sighted. The rdd mutation was shown to be sex-linked and not autosomal as previously described. Linkage analysis mapped the rdd locus to a small region of the chicken Z chromosome with homologies to human chromosomes 5q and 9p. CONCLUSIONS: Ophthalmic, histopathologic, and electrophysiological observations suggest rdd is similar to human recessive retinitis pigmentosa. Linkage mapping places rdd in a region homologous to human chromosomes 9p and 5q. Candidate disease genes or loci include PDE6A, WGN1, and USH2C. This is the first use of genetic mapping in a chicken model of human disease.