Parasitic nematodes infect one quarter of the world's population and impact all humans through widespread infection of crops and livestock. Resistance to current anthelmintics has prompted the search for new drugs. Traditional screens that rely on parasitic worms are costly and labour intensive and target-based approaches have failed to yield novel anthelmintics. Here, we present our screen of 67,012 compounds to identify those that kill the non-parasitic nematode Caenorhabditis elegans. We then rescreen our hits in two parasitic nematode species and two vertebrate models (HEK293 cells and zebrafish), and identify 30 structurally distinct anthelmintic lead molecules. Genetic screens of 19 million C. elegans mutants reveal those nematicides for which the generation of resistance is and is not likely. We identify the target of one lead with nematode specificity and nanomolar potency as complex II of the electron transport chain. This work establishes C. elegans as an effective and cost-efficient model system for anthelmintic discovery.

Small-molecule inhibitors of protein function are powerful tools for biological analysis and can lead to the development of new drugs. However, a major bottleneck in generating useful small-molecule tools is target identification. Here we show that Caenorhabditis elegans can provide a platform for both the discovery of new bioactive compounds and target identification. We screened 14,100 small molecules for bioactivity in wild-type worms and identified 308 compounds that induce a variety of phenotypes. One compound that we named nemadipine-A induces marked defects in morphology and egg-laying. Nemadipine-A resembles a class of widely prescribed anti-hypertension drugs called the 1,4-dihydropyridines (DHPs) that antagonize the alpha1-subunit of L-type calcium channels. Through a genetic suppressor screen, we identified egl-19 as the sole candidate target of nemadipine-A, a conclusion that is supported by several additional lines of evidence. egl-19 encodes the only L-type calcium channel alpha1-subunit in the C. elegans genome. We show that nemadipine-A can also antagonize vertebrate L-type calcium channels, demonstrating that worms and vertebrates share the orthologous protein target. Conversely, FDA-approved DHPs fail to elicit robust phenotypes, making nemadipine-A a unique tool to screen for genetic interactions with this important class of drugs. Finally, we demonstrate the utility of nemadipine-A by using it to reveal redundancy among three calcium channels in the egg-laying circuit. Our study demonstrates that C. elegans enables rapid identification of new small-molecule tools and their targets.

The resistance of Caenorhabditis elegans to pharmacological perturbation limits its use as a screening tool for novel small bioactive molecules. One strategy to improve the hit rate of small-molecule screens is to preselect molecules that have an increased likelihood of reaching their target in the worm. To learn which structures evade the worm's defenses, we performed the first survey of the accumulation and metabolism of over 1,000 commercially available drug-like small molecules in the worm. We discovered that fewer than 10% of these molecules accumulate to concentrations greater than 50% of that present in the worm's environment. Using our dataset, we developed a structure-based accumulation model that identifies compounds with an increased likelihood of bioavailability and bioactivity, and we describe structural features that facilitate small-molecule accumulation in the worm. Preselecting molecules that are more likely to reach a target by first applying our model to the tens of millions of commercially available compounds will undoubtedly increase the success of future small-molecule screens with C. elegans.

Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows for excellent microscopic visualization. Their discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, the nonmotile nature of the cells allows for the retrograde flow to be analyzed in the absence of cell translocation. In this study we have begun an analysis of the retrograde flow mechanism by characterizing its kinetic and structural properties. The supramolecular organization of actin and myosin II was investigated using light and electron microscopic methods. Light microscopic immunolocalization was performed with anti-actin and anti-sea urchin egg myosin II antibodies, whereas transmission electron microscopy was performed on platinum replicas of critical point-dried and rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical actin network, which feeds into an extensive array of radial bundles in the interior. These actin bundles terminate in a perinuclear region, which contains a ring of myosin II bipolar minifilaments. Retrograde flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin cytoskeleton to separate from the cell margin and undergo a finite retrograde retraction. In contrast, inhibition of myosin II function either with the wide-spectrum protein kinase inhibitor staurosporine or the myosin light chain kinase-specific inhibitor KT5926 stopped flow in the cell center, whereas normal retrograde flow continued at the cell periphery. These differential results suggest that the mechanism of retrograde flow has two, spatially segregated components. We propose a "push-pull" mechanism in which actin polymerization drives flow at the cell periphery, whereas myosin II provides the tension on the actin cytoskeleton necessary for flow in the cell interior.

This protocol describes a procedure for screening small molecules for bioactivity and a genetic approach to target identification using the nematode Caenorhabditis elegans as a model system. Libraries of small molecules are screened in 24-well plates that contain a solid agar substrate. On top of the agar mixture, one small-molecule species is deposited into each well, along with worm food (E. coli), and two third-stage or fourth-stage larval worms using a COPAS (Complex Object Parametric Analyzer and Sorter) Biosort. Three to five days later the plates are screened for phenotype. Images of the wells are acquired and archived using a HiDI 2100 automated imaging system (Elegenics). Up to 2,400 chemicals can be screened per week. To identify the predicted protein target of a bioactive molecule, wild-type worms are mutagenized using ethylmethanesulfonate (EMS). Progeny are screened for individuals resistant to the molecules effects. The candidate mutant target that confers resistance is then identified. Target identification might take months.