A High-Throughput Small Molecule Screen for C. elegans Linker Cell Death Inhibitors

Abstract

Programmed cell death is a ubiquitous process in metazoan development. Apoptosis, one cell death form, has been studied extensively. However, mutations inactivating key mammalian apoptosis regulators do not block most developmental cell culling, suggesting that other cell death pathways are likely important. Recent work in the nematode Caenorhabditis elegans identified a non-apoptotic cell death form mediating the demise of the male-specific linker cell. This cell death process (LCD, linker cell-type death) is morphologically conserved, and its molecular effectors also mediate axon degeneration in mammals and Drosophila. To develop reagents to manipulate LCD, we established a simple high-throughput screening protocol for interrogating the effects of small molecules on C. elegans linker cell death in vivo. From 23,797 compounds assayed, 11 reproducibly block linker cell death onset. Of these, five induce animal lethality, and six promote a reversible developmental delay. These results provide proof-of principle validation of our screening protocol, demonstrate that developmental progression is required for linker cell death, and suggest that larger scale screens may identify LCD-specific small-molecule regulators that target the LCD execution machinery.

The origins and evolution of sleep

“…Similar approaches have been used in fruit flies, where a sleep screen testing 1280 small molecules identified numerous novel regulators of sleep including the vesicular monoamine transporter VMAT (Nall and Sehgal, 2013). Although these approaches have yet to be applied to study sleep in C. elegans, the use of small-molecule screens to study other processes and availability of high-throughput behavioral monitoring systems suggest that this approach is feasible (Schwendeman and Shaham, 2016). These approaches highlight the potential for drug discovery using small-molecule-based screens and sleep itself providing a behavioral read-out of drug efficacy.…”

Revealing the anticancer potential of candidate drugs in vivo using Caenorhabditis elegans mutant strains

“…The transparent body and short life cycle of C. elegans enable faster generation of an observable response, simplicity in detecting outcomes (e.g. fluorescence imaging), and ease in generating large amounts of worms [ 6 – 8 ; [63] , [64] – 65 ]. As observed in the phenotypic assays, results can be produced within 168 h. (7 days) wherein worm cultivation can be done in 48 hours and the subsequent observation period for 120 h.. With these known features, the nematode can be used as a model that allows large scale screening of drugs, which may reduce the number of drugs for further testing in more complicated in vivo models.…”

De novo identification of toxicants that cause irreparable damage to parasitic nematode intestinal cells

“…Thus, Leflunomide appears to have neurotoxic effects that have not previously been reported for this drug in nematodes. Toxic effects of Leflunomide on C. elegans were reported [51], but observations were made only after 12 hours, obviating detection of more immediate effects. Leflunomide is an approved drug for treatment of rheumatoid arthritis and its principle mode of action is inhibition of DHODH and mitochondria-based synthesis of pyrimidines, one of two pathways that typically supply pyrimidines to cells.…”

“…Of possible relevance here, necrotic processes can be induced in intestinal cells of C. elegans by multiple different stimuli, and apparent protease mediators of this pathology can vary according to the stimulus applied [58]. As found here, toxicity of two of the inhibitors, Leflunomide [51] and Staurosporine [59], was shown for C. elegans , thus identifying C. elegans as a possible resource to dissect mechanisms of intestinal pathology induced by these inhibitors in both species.…”