Andrew Burns et al. (2006) Development & Evolution Meeting "Xenobiotic Uptake and Metabolism in C. elegans"

Peter Roy, Andrew Burns, Sean Cutler

[Development & Evolution Meeting, 2006]

Parasitic nematode worms present a serious health concern to humans and other agriculturally important animals. There are many nematicides used to treat parasitic infection, but resistance to these compounds is a growing problem. Work from our lab has shown that drug uptake is necessary for observable bioactivity in C. elegans. Resistance may therefore be due to detoxifying proteins such as drug-metabolizing enzymes and xenobiotic efflux pumps that prevent drug uptake. The majority of these detoxifiers remain largely uncharacterized in C. elegans. For example, Cytochrome p450 (Cyp) enzymes are a major class of Phase I metabolic enzymes. There are approximately 80 putative Cyp genes in worms. Some of these genes have shown homology to mammalian drug-metabolizing Cyps, but all 80 remain largely uncharacterized. To assay drug uptake and metabolism in C. elegans we have developed a high-throughput high performance liquid chromatography (HT-HPLC) protocol. In brief, N2 worms are incubated in a variety of small lipophilic molecules, washed, lysed, and their contents are separated using HPLC. Drug uptake and metabolism can be directly visualized and quantified. The metabolites can be purified and analyzed by mass spectrometry to determine the modification that has been made. We are currently in the process of assaying ~2000 pharmacologically active small molecules. So far our work has shown that only a small fraction (~20%) of these small molecules are taken up to an observable extent by N2 worms, and of these about 10% are metabolized. The same HT-HPLC analysis will be performed on HA and 12 other wild-type isolates of C. elegans. Any differences between the isolates with respect to drug uptake and metabolism will be noted, and the genes involved will be mapped genetically and characterized. These analyses will lead to a better understanding of the basic mechanisms of xenobiotic detoxification in a whole animal model system. It is likely that this novel whole animal approach to studying drug uptake and metabolism may uncover new mechanisms of drug resistance and detoxification that would otherwise be missed in cell- or tissue-based assays. Differences between isolates will provide insight into C. elegans intra-species pharmacogenetic variation, and will highlight the ways in which segregated wild-type worm populations have developed diverse mechanisms of xenobiotic detoxification.

Andrew Burns et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "A Predictive Model for Small Molecule Accumulation and Bioactivity in C. elegans"

Mike TYERS, Iain Wallace, Jan Wildenhain, Corey Nislow, Guri Giaever, Peter Roy, Sean Cutler, Andrew Burns

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

We are using C. elegans as a model system to discover new small molecule tools for biological analyses. One obstacle to finding such tools is the large number of xenobiotic defense factors encoded by the worm genome (Lindblom et al., 2006). For example, in a screen of 3,265 small molecules that are bioactive in other systems, we found only 64 (2%) that induce robust phenotypes in the worm (Kwok et al., 2006). We hypothesized that this hit rate is relatively low because most small molecules fail to accumulate in worms. To test this, we developed an HPLC-based assay to measure small molecule accumulation in worm tissue. We surveyed 1,018 compounds from the Spectrum library (Microsource Inc.) for accumulation in whole worms after 6 hours of incubation in 40uM of the small molecules. To ensure confidence in our assignments we established a detection limit of 18uM. Of the 361/1018 compounds that satisfied this criterion, only 25 (6.9%) accumulate in worms. Notably, 2 out of 25 accumulating molecules induce robust phenotype in the worm, compared to 0 out of 336 non-accumulating molecules. We also assayed 25 nematicides obtained from our other small molecule screens, and found that 17 (68%) accumulate. These results support our hypothesis that worms are generally resistant to small molecule accumulation, and show that accumulating small molecules are enriched for bioactivity in C. elegans. Next, the accumulation of an additional 77 compounds from our other screens was assayed, for a complete dataset of 463 small molecules. We used this dataset to build a predictive structure-based model of accumulation. We compiled 4,166 structural descriptors of the small molecules in our dataset, and built a model using a Bayesian Classifier machine learning method. Five-fold cross validation of the model estimates a prediction accuracy of 75.36 +/- 2.04%. We used the model to rank 9,742 compounds of a DIVERSet library (Chembridge Corp.), of which 48 induce robust phenotype in the worm. Encouragingly, 12 of the top-scoring 200 molecules induce phenotype, representing a 12.2-fold enrichment for bioactivity compared to the entire library (p10). None of the bottom-scoring 200 molecules induce phenotype. These data demonstrate that our model is effective at predicting small molecule accumulation and bioactivity in C. elegans. We hope to use this model to increase our efficiency at identifying new small molecule probes for biological analysis, and to aid the development of potential drug leads using C. elegans as a model.

Peter J Roy et al. (2005) International Worm Meeting "A Small Molecule Screen in C. elegans Yields a Potent Vertebrate L-Type Calcium Channel Inhibitor, Nemadipine A"

Elise F Stanley, Peter McCourt, Allen Chen, Regina Fraser, Trevor Kwok, Peter J Roy, Nicole Ricker, Sean Cutler

[International Worm Meeting, 2005]

We screened 10,000 small membrane permeable molecules from a diverse chemical library for the induction of defects in C. elegans. One compound that we call nemadipine A induced morphological, egg-laying, and slow growth defects in wild type worms (IC50<5.0uM). A subsequent screen of 15 related structures revealed a second molecule, nemadipine B, that induces the same phenotypes as nemadipine A. The structure of the nemadipines is similar to a class of molecules called 1,4-dihydropyridines (DHPs) that inhibit the 45;1 subunit of L-type calcium channels and are used to treat hypertension and ischemic heart disease in humans. Through candidate analysis we discovered that egl-19 loss-of-function mutants are hypersensitive to the nemadipines, while egl-19 gain-of-function mutants suppress nemadipine-induced defects. EGL-19 is the only C. elegans homologue of the 45;1 subunit of mammalian L-type calcium channels. These and other observations strongly suggest that EGL-19 is the primary target of the nemadipines. We tested four other DHP drugs, including felodipine, nicardipine, nifedipine, and nimodipine, together with representatives of three other classes of L-type calcium channel inhibitors, namely verapamil, diltiazem and bepredil. Unlike the nemadipines, none of these drugs elicited phenotype in wild type worms in our whole worm assays. Since many of these drugs can inhibit calcium channels in dissected worms, this result suggested that the nemadipines have unique properties that allow efficient entry into whole worms or are more effective than existing DHPs or both. We therefore compared the abilities of nemadipine A and nifedipine to inhibit vertebrate L-type channels. Our preliminary analysis shows that nemadipine A can specifically inhibit vertebrate L-type channels in the femtimolar range, and is at least 1000 fold more potent than nifedipine. Finally, we used nemadipine A to show that UNC-2, an 45;1 subunit homologue of N/P/Q calcium channels, functions redundantly with EGL-19 to control egg-laying. We are currently using the nemadipines for the genetic analysis of DHP action and the analysis of genetic variance in response to this class of widely-used drugs (see abstract by Kwok et al; this meeting). This study shows that C. elegans can be used to identify new small molecule tools to better understand biology and can reveal potential drug-leads.

Prasad BC et al. (1997) International C. elegans Meeting "THE C. ELEGANS unc-3 GENE ENCODES A HOMOLOGUE OF THE O/E FAMILY OF MAMMALIAN TRANSCRIPTION FACTORS"

Prasad BC, Reed RR, Seydoux GC

[International C. elegans Meeting, 1997]

The expression of specialized signal transduction components in mammalian olfactory neurons is thought to be regulated by the O/E (Olf-1/EBF) family of transcription factors. The O/E proteins are expressed in cells of the olfactory neuronal lineage throughout development, and are also expressed transiently in neurons in the developing nervous system during embryogenesis. We have identified a C. elegans homologue of the mammalian O/E proteins, which displays greater than 80% similarity over 350 amino acids. The CeO/E cDNA maps to cosmid F42D1, previously shown to encode unc-3(1). We demonstrate that CeO/E is the product of the unc-3 gene, mutations in which cause defects in the axonal outgrowth of motor neurons as well as defects in dauer formation, a process requiring chemosensory input. Like its mammalian homologues, CeO/E is expressed in certain chemosensory neurons (ASI amphid neurons) throughout development, and is also expressed transiently in developing motor neurons when these cells undergo axonal outgrowth. Currently, we are defining the DNA sequence binding specificity of the CeO/E protein. These observations suggest that the O/E family of transcription factors play a central and evolutionarily conserved role in the expression of proteins essential for axonal pathfinding and neuronal differentiation. (1) Sean Eddy, WBG 12(5):86 (February 1, 1993)