Behavioral fingerprints predict insecticide and anthelmintic mode of action.

Novel invertebrate-killing compounds are required in agriculture and medicine to overcome resistance to existing treatments. Because insecticides and anthelmintics are discovered in phenotypic screens, a crucial step in the discovery process is determining the mode of action of hits. Visible whole-organism symptoms are combined with molecular and physiological data to determine mode of action. However, manual symptomology is laborious and requires symptoms that are strong enough to see by eye. Here, we use high-throughput imaging and quantitative phenotyping to measure Caenorhabditis elegans behavioral responses to compounds and train a classifier that predicts mode of action with an accuracy of 88% for a set of ten common modes of action. We also classify compounds within each mode of action to discover substructure that is not captured in broad mode-of-action labels. High-throughput imaging and automated phenotyping could therefore accelerate mode-of-action discovery in invertebrate-targeting compound development and help to refine mode-of-action categories.

NHR-176 regulates cyp-35d1 to control hydroxylation-dependent metabolism of thiabendazole in Caenorhabditis elegans.

Knowledge of how drugs are metabolized and excreted is an essential component of understanding their fate within and among target and non-target organisms. Thiabendazole (TBZ) was the first benzimidazole (BZ) to be commercially available and remains one of the most important anthelmintic drugs for medical and veterinary use. We have characterized how Caenorhabditis elegans metabolizes and excretes TBZ. We have shown that TBZ directly binds to the nuclear hormone receptor (NHR)-176 and that this receptor is required for the induction by TBZ of the cytochrome P450 (CYP) encoded by cyp-35d1. Further, RNAi inhibition of cyp-35d1 in animals exposed to TBZ causes a reduction in the quantity of a hydroxylated TBZ metabolite and its glucose conjugate that is detected in C. elegans tissue by HPLC. This final metabolite is unique to nematodes and we also identify two P-glycoproteins (PGPs) necessary for its excretion. Finally, we have shown that inhibiting the metabolism we describe increases the susceptibility of C. elegans to TBZ in wild-type and in resistant genetic backgrounds.

UV-DIB: label-free permeability determination using droplet interface bilayers.

Simple diffusion of molecular entities through a phospholipid bilayer, is a phenomenon of great importance to the pharmaceutical and agricultural industries. Current model lipid systems to probe this typically only employ fluorescence as a readout, thus limiting the range of assessable chemical matter that can be studied. We report a new technology platform, the UV-DIB, which facilitates label free measurement of small molecule translocation rates. This is based upon the coupling of droplet interface bilayer technology with implemented fiber optics to facilitate analysis via ultraviolet spectroscopy, in custom designed PMMA wells. To improve on current DIB technology, the platform was designed to be reusable, with a high sampling rate and a limit of UV detection in the low µM regime. We demonstrate the use of our system to quantify passive diffusion in a reproducible and rapid manner where the system was validated by investigating multiple permeants of varying physicochemical properties across a range of lipid interfaces, each demonstrating differing kinetics. Our system permits the interrogation of structural dependence on the permeation rate of a given compound. We present this ability from two structural perspectives, that of the membrane, and the permeant. We observed a reduction in permeability between pure DOPC and DPhPC interfaces, concurring with literature and demonstrating our ability to study the effects of lipid composition on permeability. In relation to the effects of permeant structure, our device facilitated the rank ordering of various compounds from the xanthine class of compounds, where the structure of each permeant differed by a single group alteration. We found that DIBs were stable up to 5% DMSO, a molecule often used to aid solubilisation of pharmaceutical and agrochemical compounds. The ability of our device to rank-order compounds with such minor structural differences provides a level of precision that is rarely seen in current, industrially applied technologies.

Regulation of growth by ploidy in Caenorhabditis elegans.

Some animals, such as the larvae of Drosophila melanogaster, the larvae of the Appendicularian chordate Oikopleura, and the adults of the nematode Caenorhabditis elegans, are unusual in that they grow largely by increases in cell size. The giant cells of such species are highly polyploid, having undergone repeated rounds of endoreduplication. Since germline polyploid strains tend to have large cells, it is often assumed that endoreduplication drives cell growth, but this remains controversial. We have previously shown that adult growth in C. elegans is associated with the endoreduplication of nuclei in the epidermal syncitium, hyp 7. We show here that this relationship is causal. Manipulation of somatic ploidy both upwards and downwards increases and decreases, respectively, adult body size. We also establish a quantitative relationship between ploidy and body size. Finally, we find that TGF-beta (DBL-1) and cyclin E (CYE-1) regulate body size via endoreduplication. To our knowledge, this is the first experimental evidence establishing a cause-and-effect relationship between somatic polyploidization and body size in a metazoan.

Comparative metabolism of xenobiotic chemicals by cytochrome P450s in the nematode Caenorhabditis elegans.

We investigated the metabolic capabilities of C. elegans using compounds whose metabolism has been well characterised in mammalian systems. We find that similar metabolites are produced in C. elegans as in mammals but that C. elegans is deficient in CYP1-like metabolism, as has been seen in other studies. We show that CYP-34A9, CYP-34A10 and CYP-36A1 are the principal enzymes responsible for the metabolism of tolbutamide in C. elegans. These are related to the mammalian enzymes that metabolise this compound but are not the closest homologs suggesting that sequence comparison alone will not predict functional conservation among cytochrome P450s. In mammals, metabolite production from amytryptiline and dextromethorphan is dependent on specific cytochrome P450s. However, in C. elegans we did not find evidence of similar specificity: the same metabolites were produced but in small amounts by numerous cytochrome P450s. We conclude that, while some aspects of cytochrome P450 mediated metabolism in C. elegans are similar to mammals, there are differences in the production of some metabolites and in the underlying genetics of metabolism.

Measuring bilayer surface energy and curvature in asymmetric droplet interface bilayers.

For the past decade, droplet interface bilayers (DIBs) have had an increased prevalence in biomolecular and biophysical literature. However, much of the underlying physics of these platforms is poorly characterized. To further our understanding of these structures, lipid membrane tension on DIB membranes is measured by analysing the equilibrium shape of asymmetric DIBs. To this end, the morphology of DIBs is explored for the first time using confocal laser scanning fluorescence microscopy. The experimental results confirm that, in accordance with theory, the bilayer interface of a volume-asymmetric DIB is curved towards the smaller droplet and a lipid-asymmetric DIB is curved towards the droplet with the higher monolayer surface tension. Moreover, the DIB shape can be exploited to measure complex bilayer surface energies. In this study, the bilayer surface energy of DIBs composed of lipid mixtures of phosphatidylgylcerol (PG) and phosphatidylcholine are shown to increase linearly with PG concentrations up to 25%. The assumption that DIB bilayer area can be geometrically approximated as a spherical cap base is also tested, and it is discovered that the bilayer curvature is negligible for most practical symmetric or asymmetric DIB systems with respect to bilayer area.

Analysis and imaging of biocidal agrochemicals using ToF-SIMS.

ToF-SIMS has been increasingly widely used in recent years to look at biological matrices, in particular for biomedical research, although there is still a lot of development needed to maximise the value of this technique in the life sciences. The main issue for biological matrices is the complexity of the mass spectra and therefore the difficulty to specifically and precisely detect analytes in the biological sample. Here we evaluated the use of ToF-SIMS in the agrochemical field, which remains a largely unexplored area for this technique. We profiled a large number of biocidal active ingredients (herbicides, fungicides, and insecticides); we then selected fludioxonil, a halogenated fungicide, as a model compound for more detailed study, including the effect of co-occurring biomolecules on detection limits. There was a wide range of sensitivity of the ToF-SIMS for the different active ingredient compounds, but fludioxonil was readily detected in real-world samples (wheat seeds coated with a commercial formulation). Fludioxonil did not penetrate the seed to any great depth, but was largely restricted to a layer coating the seed surface. ToF-SIMS has clear potential as a tool for not only detecting biocides in biological samples, but also mapping their distribution.

Rheological Droplet Interface Bilayers (rheo-DIBs): Probing the Unstirred Water Layer Effect on Membrane Permeability via Spinning Disk Induced Shear Stress.

A new rheological droplet interface bilayer (rheo-DIB) device is presented as a tool to apply shear stress on biological lipid membranes. Despite their exciting potential for affecting high-throughput membrane translocation studies, permeability assays conducted using DIBs have neglected the effect of the unstirred water layer (UWL). However as demonstrated in this study, neglecting this phenomenon can cause significant underestimates in membrane permeability measurements which in turn limits their ability to predict key processes such as drug translocation rates across lipid membranes. With the use of the rheo-DIB chip, the effective bilayer permeability can be modulated by applying shear stress to the droplet interfaces, inducing flow parallel to the DIB membranes. By analysing the relation between the effective membrane permeability and the applied stress, both the intrinsic membrane permeability and UWL thickness can be determined for the first time using this model membrane approach, thereby unlocking the potential of DIBs for undertaking diffusion assays. The results are also validated with numerical simulations.

Multiplexed droplet Interface bilayer formation.

We present a simple method for the multiplexed formation of droplet interface bilayers (DIBs) using a mechanically operated linear acrylic chamber array. To demonstrate the functionality of the chip design, a lipid membrane permeability assay is performed. We show that multiple, symmetric DIBs can be created and separated using this robust low-cost approach.

The nematode Caenorhabditis elegans as a tool to predict chemical activity on mammalian development and identify mechanisms influencing toxicological outcome.

To determine whether a C. elegans bioassay could predict mammalian developmental activity, we selected diverse compounds known and known not to elicit such activity and measured their effect on C. elegans egg viability. 89% of compounds that reduced C. elegans egg viability also had mammalian developmental activity. Conversely only 25% of compounds found not to reduce egg viability in C. elegans were also inactive in mammals. We conclude that the C. elegans egg viability assay is an accurate positive predictor, but an inaccurate negative predictor, of mammalian developmental activity. We then evaluated C. elegans as a tool to identify mechanisms affecting toxicological outcomes among related compounds. The difference in developmental activity of structurally related fungicides in C. elegans correlated with their rate of metabolism. Knockdown of the cytochrome P450s cyp-35A3 and cyp-35A4 increased the toxicity to C. elegans of the least developmentally active compounds to the level of the most developmentally active. This indicated that these P450s were involved in the greater rate of metabolism of the less toxic of these compounds. We conclude that C. elegans based approaches can predict mammalian developmental activity and can yield plausible hypotheses for factors affecting the biological potency of compounds in mammals.

Increased or decreased levels of Caenorhabditis elegans lon-3, a gene encoding a collagen, cause reciprocal changes in body length.

Body length in C. elegans is regulated by a member of the TGFbeta family, DBL-1. Loss-of-function mutations in dbl-1, or in genes encoding components of the signaling pathway it activates, cause worms to be shorter than wild type and slightly thinner (Sma). Overexpression of dbl-1 confers the Lon phenotype characterized by an increase in body length. We show here that loss-of-function mutations in dbl-1 and lon-1, respectively, cause a decrease or increase in the ploidy of nuclei in the hypodermal syncytial cell, hyp7. To learn more about the regulation of body length in C. elegans we carried out a genetic screen for new mutations causing a Lon phenotype. We report here the cloning and characterization of lon-3. lon-3 is shown to encode a putative cuticle collagen that is expressed in hypodermal cells. We show that, whereas putative null mutations in lon-3 (or reduction of lon-3 activity by RNAi) causes a Lon phenotype, increasing lon-3 gene copy number causes a marked reduction in body length. Morphometric analyses indicate that the lon-3 loss-of-function phenotype resembles that caused by overexpression of dbl-1. Furthermore, phenotypes caused by defects in dbl-1 or lon-3 expression are in both cases suppressed by a null mutation in sqt-1, a second cuticle collagen gene. However, whereas loss of dbl-1 activity causes a reduction in hypodermal endoreduplication, the reduction in body length associated with overexpression of lon-3 occurs in the absence of defects in hypodermal ploidy.

Adaptive and specialised transcriptional responses to xenobiotic stress in Caenorhabditis elegans are regulated by nuclear hormone receptors.

Characterisation of the pathways by which xenobiotics are metabolised and excreted in both target and non-target organisms is crucial for the rational design of effective and specific novel bioactive molecules. Consequently, we have investigated the induced responses of the model nematode Caenorhabditis elegans to a variety of xenobiotics which represent a range of putative modes of action. The majority of genes that were specifically induced in preliminary microarray analyses encoded enzymes from Phase I and II metabolism, including cytochrome P450s, short chain dehydrogenases, UDP-glucuronosyl transferases and glutathione transferases. Changes in gene expression were confirmed by quantitative PCR and GFP induction in reporter strains driven by promoters for transcription of twelve induced enzymes was investigated. The particular complement of metabolic genes induced was found to be highly contingent on the xenobiotic applied. The known regulators of responses to applied chemicals ahr-1, hif-1, mdt-15 and nhr-8 were not required for any of these inducible responses and skn-1 regulated GFP expression from only two of the promoters. Reporter strains were used in conjunction with systematic RNAi screens to identify transcription factors which drive expression of these genes under xenobiotic exposure. These transcription factors appeared to regulate specific xenobiotic responses and have no reported phenotypes under standard conditions. Focussing on nhr-176 we demonstrate the role of this transcription factor in mediating the resistance to thiabendazole.

Spiroindolines identify the vesicular acetylcholine transporter as a novel target for insecticide action.

The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. Here we describe a novel class of insecticides (Spiroindolines) encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. We confidently assign the vesicular acetylcholine transporter as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway and we anticipate that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family.

A Caenorhabditis elegans TGF-beta, DBL-1, controls the expression of LON-1, a PR-related protein, that regulates polyploidization and body length.

Using cDNA-based array analysis combined with double-stranded RNA interference (dsRNAi), we have identified yk298h6 as a target gene of Caenorhabditis elegans TGF-beta signaling. Worms overexpressing dbl-1, a TGF-beta ligand, are 16% longer than wild type. Array analysis shows yk298h6 to be one of several genes suppressed in such worms. Disruption of yk298h6 function by dsRNAi also resulted in long worms, suggesting that it is a negative regulator of body length. yk298h6 was then mapped to, and shown to be identical to, lon-1, a known gene that affects body length. lon-1 encodes a 312 amino acid protein with a motif sequence that is conserved from plants to humans. Expression studies confirm that LON-1 is repressed by DBL-1, suggesting that LON-1 is a novel downstream component of the C.elegans TGF-beta growth regulation pathway. Consistent with this, LON-1 is expressed mainly in the larval and adult hypodermis and has dose-dependent effects on body length associated with changes in hypodermal ploidy, but not hypodermal cell proliferation.