Egg-laying and locomotory screens with C. elegans yield a nematode-selective small molecule stimulator of neurotransmitter release

Abstract

Nematode parasites of humans, livestock and crops dramatically impact human health and welfare. Alarmingly, parasitic nematodes of animals have rapidly evolved resistance to anthelmintic drugs, and traditional nematicides that protect crops are facing increasing restrictions because of poor phylogenetic selectivity. Here, we exploit multiple motor outputs of the model nematode C. elegans towards nematicide discovery. This work yielded multiple compounds that selectively kill and/or immobilize diverse nematode parasites. We focus on one compound that induces violent convulsions and paralysis that we call nementin. We find that nementin stimulates neuronal dense core vesicle release, which in turn enhances cholinergic signaling. Consequently, nementin synergistically enhances the potency of widely-used non-selective acetylcholinesterase (AChE) inhibitors, but in a nematode-selective manner. Nementin therefore has the potential to reduce the environmental impact of toxic AChE inhibitors that are used to control nematode infections and infestations.

Fig. 1. The identification of 58 small molecules that modulate the C. elegans nervous system.

a, b Two perspectives of the ‘2020 Imager’ showing one 96-well plate in the imager holder (arrow). c Representative half-well images of vehicle controls in the basal (M9-only) background (left) and the ‘stimulatory cocktail’ of 12.3 mM serotonin creatine sulfate and 7.7 mM nicotine (right) after 1 h. A blue arrow points to an adult hermaphrodite and a red arrow points to a pair of embryos. d The distribution of counts of eggs/worm/hour for the basal background (blue) and the stimulatory background (red) from the indicated well-coordinates over 22 experimental replicates (96-well plates). The mean and SEM is shown, n = 66 biologically independent samples with ~20 animals per data point. e A screen of the worm active (wactive) library yields 29 inhibitors (purple) and 29 stimulators (green) of the C. elegans egg-laying rate. Mean Egl rate and SEM is shown from n = 3 independent biological replicates, conducted in technical triplicate with ~20 animals per technical replicate. f Venn diagram showing the overlap of the wactive molecules that are lethal (at 60 µM) with those that modulate egg-laying among the 486 molecules assayed in both assays. g A structure similarity network of the 58 egg-laying modulators. Each node represents a numbered wactive molecule. Connecting lines indicate shared structural similarity. Additional information is provided in Fig. 2. Primary data for this figure can be found in Supplementary Data 1.

Fig. 2. C. elegans locomotor phenotype and phylogenetic activity profile of identified egg-laying modulators.

a The Chembridge Inc. identification (ID) numbers are indicated. b Egg-laying phenotype; Egl-S egg-laying stimulators; Egl-I egg-laying inhibitors. c The observed acute motor phenotypes are indicated. The concentration at which strong and moderate phenotypes appear are indicated in bright green. The concentration at which weaker phenotypes appear are indicated in light gray. ‘n’ indicates no phenotype was observed. d Larval lethality phenotypes; the previously reported results of liquid-based larval lethal assays (for C. elegans, C. briggsae, and P. pacificus) are shown. The lowest concentration at which 100% of the larvae die/arrest is shown. Colors highlight relatively potent activity. e HEK293 cell proliferation summary; compounds that reduce HEK293 proliferation below one standard of deviation from the mean at 30 or 60 µM as previously reported are indicated as purple ‘30’ or ‘60’, respectively (see for details). f Danio rerio (zebrafish) developmental defect summary; molecules that induce cardiac defects (card), developmental defects (dev) or death (dead) at a concentration of 10 µM as previously reported are summarized here. Throughout the figure, ‘n’ reports no lethality or phenotype observed. Primary data for this figure can be found in Supplementary Data 1.

Fig. 3. The motor-centric approach yields convulsion-inducing nementin-1.

a Structures of the seven prioritized neuromodulators. b Bioactivity of the seven prioritized molecules. Data are EC50s (details of assays are provided in the methods) except for the A. thaliana greening assay, which is the lowest concentration that yields a discernible difference from control in at least two of the three biological replicates tested (n = 3). c A time-course analysis of 60 µM nementin-1-induced locomotory phenotypes. Data are the mean of biological triplicate measurement of 18 animals per each time point over three biologically independent replicates (n = 3). The SEM is shown. Primary data for this figure can be found in Supplementary Data 2.

Fig. 4. Nementin stimulates dense core vesicle release.

a Convulsions induced by nementin-1 (Nem-1) (60 µM) in the indicated genetic background after 80 min (n = 3 biologically independent samples scoring 18 animals per trial), with the exception of 60 μM wild-type nementin (n = 7 scoring 18 animals per sample). b The mean percentage of animals of the indicated genotype (n = 3 biologically independent trials with ~150 animals per trial) that locomote to zone 2 over 180 min after being placed in the center of a plate (see schematic on right). For A and B, black and purple asterisks represent p < 0.05 and p < 0.001, Chi-square test of independence with Bonferroni correction relative to control (open circle); the standard error of the mean (SEM) is shown. c A schematic of neuronal signaling pathways relevant to nementin activity. Purple glow indicates area of possible nementin action; the green glow indicates how DCV neurotransmitter cargo may activate Gα_q and Gα_o signaling pathways; the yellow glow indicates how the secondary messenger diacylglycerol (DAG) can also promote synaptic vesicle release. d Schematic of adult (strain KG4247) expressing INS-22::GFP (green dots; packaged into DCVs) and secreted mCherry (fuchsia) in cholinergic motor neurons (cell bodies are oval). mCherry is constitutively secreted and taken up by the coelomocytes (three pairs of pink objects in the animal). Box indicates the area shown in E and F. Anterior is the left and dorsal is up. e, f Images of the midbody region of control and 60 μM nementin-1-treated KG4247 animals after 4 h. dc, dorsal cord; vc, ventral cord; co, coelomocytes; cb, cell bodies. g, h Quantification of (g) the midbody dorsal cord fluorescence (relative to background tissues) and (h) mid-body coelomocytes (ratio of measured GFP/RFP). AU, arbitrary units., 1-way ANOVA with Dunnett correction for multiple comparisons; **p < 0.01; ***p < 0.001; SEM is shown. Other regions of the animal are quantified in Supplementary Fig. 3. Primary data for this figure can be found in Supplementary Data 3.

Fig. 5. Nementin enhances AChE-inhibitor activity via DCV release.

a Convulsions induced after 80 min of exposure to nementin-1 (Nem-1) (60 μM) or vehicle control in the background of the indicated mutant gene, or in the background of 24 h of preincubation with the DAG memetic PMA. Showing the mean and SEM of n = 3 biologically independent trials (n = 10 for 60 μM wild-type) measuring 18 animals per trial. b The ability of wild type (WT) or unc-17 mutant animals to productively locomote to zone 2 over 180 min after being placed in the center of a plate (see schematic in Fig. 3b) in the indicated nementin concentration. Showing the mean of n = 4 biologically independent trials with ~150 animals per trial. For both A and B, black asterisk p < 0.01, red asterisk p < 0.001, Bonferroni corrected Chi-square test of independence relative to control (open circle); SEM is shown. c Acetylcholinesterase inhibitor (AChEi) EC50s for different species and genetic backgrounds. Oxamyl is the only AChEi tested that exhibited activity against flies. EC50s are generated from a concentration series tested over n = 3 biologically independent trials scoring 18 animals per condition. d, f, and h Double dose-response analyses of nementin-1 (y-axis) vs the indicated AChEi (x-axis) in the indicated nematode species. The scale for the percentage of the population paralyzed applies to d–f. Each concentration was tested over n = 3 biologically independent trials for elegans with 18 animals within each trial, n = 6 trials for ratti with ≥30 animals within each trial, n ≥ 3 trials for hapla with ≥15 animals within each trial. e, g, and i The corresponding zero-interaction potency (ZIP) synergy score heatmaps for the double dose-response analyses presented in d–f. Max ZIP-score is defined as the maximal ZIP-score obtained from a combination of three concentrations of each compound in the double dose response. Shading highlights the topology of the plot and differs between g–i. Primary data for this figure can be found in Supplementary Data 5.

Fig. 6. Nementin structure-activity relationship analysis.

a The analog structures are represented by the Markush structure at the top and includes atom and substituent identifiers. b Data for the free-living nematodes are EC50s (µM) and color-coded based on the potency scale at the bottom of this section. EC50s are generated from tests of a concentration series over n = 3 biologically independent trials. c, d Nematode parasites of mammals or plants were tested with a limited number of compound concentrations. In each cell in these sections, the lowest concentration (µM) tested that exhibited a phenotype is shown, and the percentage of animals that exhibit the phenotype is indicated by the color scale at the bottom of this section. e Data for models of non-targeted systems is reported either as an EC50 (µM) (for the HEK293 cells) or as the lowest concentration tested (µM) that exhibited a discernible phenotype compared to wild-type at the indicated concentration; the percentage of animals that exhibit the phenotype is indicated by the color scale at the bottom of this section. np no phenotype (<20% effect), nt not tested. See the Methods for a description of each assay. Primary data for this figure can be found in Supplementary Data 6.