ABSTRACT
Nematode parasites of humans, livestock and crops pose a significant burden on 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 present a pipeline that exploits multiple motor outputs of the model nematode C. elegans for nematicide discovery. This pipeline yielded a compound, which we call Nementin, that selectively immobilizes diverse nematode parasites. We find that Nementin induces convulsions by agonizing neuronal dense core vesicle release, which in turn agonizes cholinergic signaling. Consequently, Nementin synergistically enhances the potency of acetylcholinesterase inhibitors that are restricted agrochemicals. Nementin therefore represents a novel candidate nematicide that may improve the selectivity of broad-acting pesticides.
One-Sentence Summary A C. elegans-based screening pipeline identifies a selective nematicide that potentiates acetylcholinesterase inhibitors.
INTRODUCTION
Parasitic nematodes are a scourge to humanity. Not only do helminths currently infect more than 1.5 billion people (WHO Soil Transmitted Helminth Fact Sheet, 2022) but nematode parasitism also destroys tens of billions of dollars (USD) worth of livestock annually (1). Alarmingly, the rate of destruction is growing due to the evolution of anthelmintic resistance (2). Plant-parasitic nematodes (PPNs) are even more destructive as they ruin over 125 billions of dollars (USD) worth of food crops annually (3). Traditional nematicides that protect crops have been justifiably banned or severely restricted due to a lack of phylum selectivity(4), but these restrictions severely impact food security (5). Compounding these issues, global food demand is expected to increase by 70% by the year 2050 (6). Hence, the development of new and selective nematicides is essential to our collective welfare.
Here, we present a novel motor-centric screening pipeline to identify novel candidate nematicides. Disrupting a parasite’s motor control has repeatedly proven effective in mitigating nematode infection (7, 8). We therefore screened our collection of worm- active (wactive) compounds (9) using two successive behavioural assays of the free- living nematode Caenorhabditis elegans. We identified four molecules that fail to elicit phenotypes in off-target systems at concentrations that kill multiple nematode species. We focused on one of these, called Nementin, that induces C. elegans hyperactivity within seconds of exposure, followed by whole-body convulsions minutes later.
Nementin and its analogs selectively immobilize and/or kill nematode parasites of plants and mammals. Chemical-genetic analyses reveal that Nementin agonizes neuronal dense core vesicle release, which in turn, agonizes synaptic vesicle release. This insight led to the finding that Nementin enhances the paralytic effects of organophosphate and carbamate acetylcholinesterase inhibitors not only in C. elegans, but in nematode parasites of mammals and plants. We conclude that Nementin is a novel nematode-selective scaffold that may also improve the selectivity of broad-acting pesticides.
RESULTS
26 Compounds Disrupt Multiple C. elegans Motor Activities
To identify small molecules that disrupt nematode neuromuscular activity, we designed a 96-well plate imaging system that measures the egg-laying rate of adult C. elegans hermaphrodites (Fig. S1). The regulation of C. elegans egg-laying relies on well- characterized cholinergic, serotonergic, GABAergic and peptidergic circuits (10), and is therefore a paragon to assess neuromuscular perturbation by small molecules. Using methodology described in the Supplementary Text, we screened our collection of 486 worm-active (wactive) compounds (9) and identified 29 stimulators and 29 inhibitors of egg-laying (Fig. 1A; Data S1; Data S2). A Tanimoto structural similarity cut-off of 0.55 reveals that the 58 egg-laying modulators segregate into 11 clusters and 26 singletons that have a distinct structural scaffold (Fig. 1A). 10 of the 11 clusters are composed of molecules with the identical egg-laying phenotype, which validates the phenotypic assignments.
We reasoned that molecules that disrupt multiple motor circuits are more likely to break the parasitic nematode lifecycle. We therefore surveyed the 58 egg-laying modulators for those that disrupt the normal sinusoidal locomotion of wild type C. elegans. 26 of the 58 egg-laying modulators (45%) induce at least one locomotory phenotype such as convulsions, coiling, and paralysis (Table 1; Fig. 1A; Movies S1-S6; Data S3). The molecules that induce slow movement and paralysis are dominated (88%) by molecules that inhibit egg-laying (Fig. 1A; Table 1). This is consistent with the idea that the egg-laying inhibitor class may be enriched with acutely lethal molecules (Fig S1). By contrast, the molecules that induce convulsions, shaking, and coiling are dominated (80%) by those that enhance egg-laying (Table 1).
Nementin-1 is Effective Against Multiple Parasitic Nematodes
To prioritize the 26 neuromodulatory molecules, we eliminated those from consideration that: i) are cytotoxic to human HEK293 cells; ii) induce developmental defects in zebrafish; iii) have lackluster lethality against the five nematodes previously assayed; iv) induce subtle locomotory phenotypes (reversal-defective), or v) were closely related to the commercial nematicide fluopyram (9) (Table 1; Data S4). This left worm-active (wact) molecules 10, 13, 15, 55, 120, 128, and 444 for further consideration (Fig. 1B; Table 1).
We further assessed the activity of our seven hits against seven nematode species from three phylogenetic clades (Fig. 1C). Four of these seven species are parasitic. We also analyzed (or reanalyzed) the molecules’ activity against the non-target models D. rerio fish, HEK293 cells, and Arabidopsis thaliana plants (Fig. 1C; Fig. S2). We prioritized wact-55 because it arguably demonstrates the best combination of broad nematode activity and selectivity. SciFinder-based literature searches failed to reveal prior annotation of nematicidal activity for wact-55’s alkyl phenylpiperidine core scaffold (11).
A detailed temporal analysis showed that within 90 seconds of exposure of 60 µM wact-55, C. elegans exhibits a hyperactive nose movement phenotype (Fig. 1D). The hyperactive phenotype dissipates at the expense of spastic convulsions over the course of 2 hours (Fig. 1D). By 24 hours, 100% of the adult animals incubated in wact-55 are dead (non-moving and/or disintegrating; three trials, n = 18 animals per trial). We renamed the wact-55 molecule ‘Nementin-1’ (nematode enhancer of neurotransmission-1).
To determine whether Nementin-1 might have a canonical mechanism-of-action (MOA), we tested whether any of the C. elegans mutant strains that resist the effects of seven popular anthelmintics also resist the lethal effects of Nementin-1. All drug- resistant strains remain sensitive to Nementin-1 (Fig. S3). Furthermore, we previously showed that C. elegans cannot be easily mutated to resist Nementin-1’s lethality; 290,000 randomly mutated genomes failed to yield wact-55-resistant mutants (9). These results suggest that: i) Nementin-1 does not share an MOA with canonical anthelmintics, ii) Nementin-1’s MOA may not be limited to a single protein target, and, iii) genetic resistance to Nementin-1 may be difficult to achieve in the field. All of these properties make Nementin-1 an attractive hit for further investigation.
Nementin Convulsions are Dependent on Dense Core Vesicle Release
We reasoned that mutations in the pathway targeted by Nementin-1 might phenocopy the motor defects induced by the molecule and provide mechanistic insight. A survey of the literature yielded eight mutants that share at least some of Nementin-1’s phenotypes, including acr-2, unc-2, unc-43, unc-58, unc-93, sup-9, sup-10, and twk-18 (12–20) (Table S1). We tested the hypothesis that Nementin-1 disrupts the pathway disrupted by each of these mutants. We did this by asking whether known suppressors of the phenocopying mutants can suppress Nementin-1’s effects (see Table S1). Of the potential suppressing mutants tested, only the unc-43 gain-of-function (GF) mutant resisted Nementin-1’s convulsions (Fig. 2A). We also found the converse to be true; unc-43 reduction-of-function (RF) alleles enhance Nementin-1 convulsions (Fig. 2A).
Remarkably, the interaction between the unc-43 GF and Nementin-1 is mutual in that Nementin-1 can rescue the paralysis of unc-43 GF (Fig. 2B). This observation argues against the idea that the unc-43 GF mutant alters Nementin-1 absorption or metabolism. These results suggest that Nementin-1 may be disrupting UNC-43 pathway activity.
UNC-43 is the C. elegans ortholog of CaMKII (calcium/calmodulin-dependent protein kinase II) and is a key negative regulator of dense core vesicle (DCV) release in C. elegans neurons (13, 21) (Fig. 2C). In animals with reduced UNC-43 function, neuronal DCV content is released in excess and is likely responsible for the mutant’s convulsive and constitutive egg-laying phenotypes (12, 21). In unc-43 GF animals, DCVs accumulate, and their limited release is likely responsible for the mutant’s severely lethargic locomotion (21).
To examine DCV behaviour in response to Nementin-1, we exploited the neuropeptide reporter (INS-22::GFP) that is packaged into DCVs in cholinergic motor neurons (21). Like unc-43 RF mutants (21), worms treated with either Nementin-1 or a second analog called Nementin-12 have reduced axonal INS-22::GFP signal (p<0.05) (Fig. 2D-2H; Fig. S4). Coincidentally, INS-22::GFP accumulates in the pseudocoelomic fluid-scavenging cells, called coelomocytes, of Nementin-treated animals (Fig. 2H; Fig. S4).
We reasoned that if Nementin disrupts motor behavior by agonizing DCV release, then disruption of UNC-31 (calcium-dependent activator protein for secretion (CAPS)), which is required for DCV release (21, 22), or UNC-64 syntaxin, which is required for all vesical fusions (23, 24), should suppress the Nementin-1-driven locomotory defects.
Indeed, we find that both the weaker temperature-sensitive unc-31(e169), the unc- 31(e928) null, and the canonical unc-64(e246) RF mutants suppress the convulsions induced by Nementin-1 (Fig. 2A). Together, these data indicate that Nementin-induced DCV release is responsible for convulsions.
Collectively, these results beg the question of whether UNC-43 is likely to be the physiologically relevant target of Nementin-1. Several observations argue against this. First, we note that the terminal phenotype of Nementin-1 is death, yet presumptive null alleles of unc-43 (ce685 and n1186) are viable (13, 21). Second, if Nementin-1 inhibits UNC-43, then Nementin-1 should not be able to enhance the convulsion phenotype of unc-43 null mutants. We find that Nementin-1 does indeed enhance presumptive unc-43 null mutants (ce685 and n1186) (Fig. 2A). Third, if UNC-43 was Nementin-1’s sole physiological target, then unc-43 GF mutations would have been identified in our genetic suppressor screens (9), but no resistant mutants arose. Finally, the unc-43 GF mutant (and unc-31 and unc-64 RF mutants) remain sensitive to the lethal effects of Nementin-1 (Fig. S5). Together, these data argue against the idea that UNC-43 is Nementin-1’s sole physiologically relevant target. Instead, the similarities between Nementin-1-induced phenotypes and the unc-43 RF mutants suggest that the compound targets a component(s) that acts with UNC-43 (Fig. 2C, purple glow).
Nementin-1 Agonizes Cholinergic Signaling
We next investigated whether cholinergic signaling is also required for Nementin-1 activity. We tested whether mutants of the UNC-17 vesicular acetylcholine transporter (VAChT), which loads synaptic vesicles (SVs) with acetylcholine, or mutants of UNC-13, which is specifically required for SV release (25, 26), could suppress Nementin-1- induced convulsions. Reduction-of-function mutants of UNC-17 and UNC-13 remained sensitive to Nementin-1 (Fig. 3A). Incidentally, we found that Nementin-1 could partially rescue the lethargic locomotion of unc-17 RF mutants (Fig. 3B). These data show that despite cholinergic signalling being dispensable for Nementin-1-induced convulsions, Nementin-1 may nevertheless agonize cholinergic signalling.
We further investigated the idea that Nementin-1 agonizes cholinergic signalling. To do so, we used the acetylcholinesterase (AChE) inhibitor aldicarb, which is a well- characterized tool used to investigate perturbations of cholinergic signaling in C. elegans (27). AChE catabolizes acetylcholine at the neuromuscular junction (NMJ) and its inhibition increases acetylcholine levels that in turn paralyzes the animal (28) (see Fig. 2C). We found that Nementin-1 sensitizes animals to the paralytic effects of aldicarb (Fig. 3C), suggesting that Nementin-1 agonizes acetylcholine release at the NMJ. The relationship between Nementin-1 and aldicarb is synergistic in nature, yielding a zero-interaction potency (ZIP) δ-score of 31.1 with a δ-score of 64.8 over the most synergistic set of concentrations (ZIP scores >10 are considered synergistic (29, 30) (Fig. 3D). Nementin-1 also enhances the paralysis induced by dichlorvos and trichlorfon, which are two other AChE inhibitors that are also commercial anthelmintics, by 12.7 and 18.7-fold, respectively (Fig. 3C). By contrast, Nementin-1 fails to enhance the effects of an AChE-inhibitor on the fruit fly D. melanogaster (Fig. 3C). These data provide additional support for the idea that Nementin-1 agonizes cholinergic signalling and does so in a nematode-selective manner.
Cholinergic Agonism is a Secondary Effect of DCV Release
Neuropeptides released from DCVs are known to interact with G-protein coupled- receptors (GPCRs) that signal through G-proteins (31). In C. elegans neurons, the Gαq protein EGL-30 stimulates DAG production via the EGL-8 and PLC-3 phospholipases (32) (see Fig. 2C). By contrast, the Gαo protein GOA-1 reduces DAG abundance by triggering its phosphorylation by diacylglycerol kinase DGK-1 (33). Neuronal DAG primes both DCV and SV release via interaction with PKC-1(PKC-ε/η) (34) and UNC-13 (35), respectively. The bioamines and neuropeptides that are released from the DCVs can have both paracrine and autocrine activity (36, 37). This circuitry raised the possibility that Nementin-1-induced DCV release may initiate a positive feedback loop that amplifies DCV release and agonizes SV release as a secondary consequence (see green and yellow pathways in Fig. 2C).
We tested this hypothesis in three ways. First, we examined the impact of mutant components of the ‘green’ circuit depicted in Fig. 2C. We found that egl-30 RF, which reduces DAG production, suppresses Nementin-1 convulsions (Fig. 3A). By contrast, goa-1 RF and dgk-1 loss-of-function (LF) mutants, which lack regulation of membrane DAG, enhance Nementin-1 convulsions. We also found that disruption of PKC-1 reduces Nementin-1 convulsions (Fig. 3A). Second, we asked whether the cell permeable DAG mimetic phorbol myristate acetate (PMA) enhances Nementin-1 convulsions and found that it did (Fig. 3A). PMA does not induce convulsions on its own, suggesting that PKC-1-agonism alone cannot severely disrupt motor activity. Finally, we asked whether Nementin-1’s agonism of cholinergic signaling is dependent on the machinery needed for DCV release. Indeed, we found that the unc-31(e169) RF mutation and the unc-43(n498) GF mutation suppress Nementin-1’s agonism of cholinergic signaling (Fig. 3C). Together, these observations are consistent with a model whereby Nementin-1 agonizes DCV release whose contents may stimulate autocrine/paracrine G-protein signaling, which in turn agonizes both more DCV release and cholinergic signaling.
Nementin Disrupts Parasitic Nematodes of Plants and Animals
We carried out a small structure-activity relationship (SAR) analysis of Nementin analogs assayed against free-living nematodes, parasitic nematodes, and non-target models (Table S2; Data S4). We purchased 18 commercially available analogs of Nementin-1 and found that Nementin-12 was the most potent inducer of acute motor phenotypes (Table S2). We expanded the SAR by synthesizing 22 analogs of Nementin-12 (see Materials and Methods). Nementins 1, 12-5, 13 and 14 demonstrated broad anthelmintic activity without inducing obvious phenotype against non-target organisms. In addition, Nementin analogs were identified with improved activity in each parasitic nematode model tested. These data suggest that the Nementin scaffold may be further refined to improve selectivity and potency.
Given that Nementin-1 synergistically enhances the activity of AChE inhibitors in C. elegans, we tested whether it might act similarly against parasitic nematodes. We tested Nementin-1’s interaction with the organophosphate dichlorvos against Strongyloides ratti, a nematode parasite of mammals. Indeed, we find that Nementin-1 synergistically enhances dichlorvos’ activity against S. ratti (Fig. 3E). Nementin-1 was also found to synthetically paralyze the plant-parasitic nematode Meloidogyne hapla with the carbamate AChE inhibitor oxamyl (Fig. 3F). Both dichlorvos and oxamyl are approved for field use (38, 39). The Nementins may therefore have added benefit against parasitic nematodes when used in combination with pesticides that are AChE inhibitors.
DISCUSSION
Here, we have developed a C. elegans-based pipeline to identify novel anthelmintic small molecules with phylum-selectivity. The pipeline was designed to yield broad- acting anthelmintics that acutely affect nematode behaviour. The strategy yielded four small molecules that exhibit phylum-selectivity within the limited range of organisms tested. We focused on one of these molecules, called Nementin-1, which induces C. elegans hyperactivity within seconds of exposure, followed by spastic convulsions minutes later. Several Nementin analogs affect parasitic nematodes from distinct clades at concentrations that fail to elicit obvious phenotypes from non-target organisms. Despite significant effort, we were not able to generate C. elegans mutants that resist the lethal effects of Nementin, suggesting that resistance in the wild may be difficult to achieve.
Our data support a model whereby Nementin exposure initiates DCV release, which in turn agonizes SV release that promotes cholinergic signaling. Nementin-induced convulsions depend on DCV release but not cholinergic signaling, indicating that agonized DCV release is key to Nementin’s ability to disrupt motor behavior. How Nementin agonizes DCV release remains unclear. Despite the phenotypic similarities between Nementin exposure and unc-43 RF mutants, our data suggests that Nementin is unlikely to inhibit UNC-43 in any canonical fashion. However, because of CaMKII’s complexity and its many isoforms (13, 40), we cannot rule out the possibility that Nementin interacts with CaMKII in a complex and perhaps tissue-specific manner.
Nementin has several features that make it an attractive anthelmintic for further development. First, in vitro tests demonstrate that it exhibits potentially broad-spectrum activity while maintaining phylogenetic selectivity. Such a feature is key to the development of an environmentally safe anthelmintic. Second, Nementin analogs exhibit an in vitro potency that is comparable to several commercial anthelmintics. Third, the Nementin synthesis route is relatively simple and uses inexpensive starting materials (‘Chemistry’ in Supplementary Materials) (41). Finally, Nementin has the potential to reduce the amount of non-selective pesticides that are notoriously released into the environment.
Despite the toxicity concerns over indiscriminate activity, many AChE inhibitors such as trichlorfon, dichlorvos, coumaphos, aldicarb, fosthiazate, chlorpyrifos and oxamyl remain approved for nematicidal field use as of 2021 (38) (see the FDA Approved Animal Drug Products at https://tinyurl.com/by83ks98; and the US Environmental Protection Agency at https://tinyurl.com/3n3kf34n). Because of Nementin’s nematode selectivity, combining Nementin with these AChE inhibitors has the potential to reduce the amount of pesticide applied while maintaining selectivity against the nematode parasites. Combining compounds that enhance acetylcholine release with AChE inhibitors is a novel strategy that may also prove effective in other pesticide contexts.
Funding
Research in the I.Z. lab is partially supported by the United States Department of Agriculture, Agricultural Research Service. P.J.R. is supported by CIHR project grants (313296 and 173448). P.J.R. is a Canada Research Chair (Tier 1) in Chemical Genetics.
Author Contributions
Conceptualization: PJR, SH
Methodology: SH, JJK, K-L C, AA, MK, CH, JP, JRV, MG, JS, VW, BMP, EMR, ASV,
Investigation: SH, JJK, AB, MK, CH, JP, C’DA, Y-H K, JRV, MG, JS, VW, BMP, EMR, ADS
Visualization: PJR, SH
Funding acquisition: PJR
Project administration: PJR
Supervision: PJR, JG, IS, SRC, JJD, CMY, JK, IZ, ML
Writing – original draft: PJR, SH, JJK, K-LC, AA, SRC, DK, JK
Writing – review & editing: PJR, SH, AB, JJK, SC, DK, JK, IZ, ML
Competing interests
Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. S.H., J.K., A.R.B., K-L.C., J.P., M.L. and P.J.R. have patents pending related to Nementin.
Data and materials availability
Original data for all analyses presented are included in Data S1-S4. Original images for analyses can be made available per request through the corresponding author P.J.R.
Supplementary Materials
Supplementary Text
Materials and Methods
Figs. S1 to S6
Table S1
References 42-56
Movies S1 to S6
Data S1 to S4
Supplementary Materials
Other Supplementary Materials for this manuscript include the following
Movies S1 to S6
Data S1. Egg-laying rates with and without a stimulatory cocktail. Data S2. The Wactive Library egg-laying screen data.
Data S3. The locomotory survey of the egl-modulators. Data S4. Nematode and Counter-Screen Bioassay Data.
Supplementary Text
To identify egg-laying stimulators, we screened small molecules in the background of M9 worm buffer (1). Animals incubated in M9-vehicle control wells lay 1.1 eggs per hour on average (Fig. S1C-D; Data S1). To identify egg-laying inhibitors, we screened small molecules in the background of exogenous serotonin plus nicotine that stimulates animals to lay an average of 7.5 eggs per hour (see methods; Fig. S1C-D; Data S1).
We cross-referenced the list of 58 acute egg-laying modulators to the list of 247 wactive molecules that are lethal in six-day viability assays (1). We found a 1.8-fold enrichment of lethality among the stimulators (p<0.001) (Fig. S1F), suggesting that excessive neuromuscular modulation may lead to death. A 1.4-fold enrichment of lethality among the inhibitors was also found (p<0.05), which is not unexpected given that egg-laying inhibition is certain to be a consequence of molecules that kill rapidly.
Ethics Statement
DK: Experiments on D. immitis and N. brasiliensis were performed in the laboratories of Bayer Animal Health GmbH (Monheim, Germany) in accordance with the local Animal Care and Use Committee and governmental authorities. JK: The generation of N. americanis, T. muris, S. ratti, and H. polygyrus and in vitro studies were carried out at the Swiss Tropical Institute (Basel, Switzerland), in accordance with both cantonal (license no. 2070) and Swiss national regulations on animal experimentation. JD: All zebrafish experiments were performed in compliance with any relevant ethical regulations, specifically following an institutionally reviewed and approved animal use protocol as well as the policies and guidelines of the Canadian Council on Animal Care and Animals for Research Act of Ontario.
Materials and Methods
Free-Living Nematode Strains and Culture
All nematode strains were cultured using standard methods at 20 °C unless otherwise indicated (2). The N2 (wild-type) strain of Caenorhabditis elegans, Caenorhabditis briggsae strain AF16 and Pristionchus pacificus strain PS312 were all obtained from the C. elegans Genetic Center (CGC; University of Minnesota). Rhabditophanes sp. KR3021 was obtained from Marie-Anne Félix (Institute of Biology of the Ecole Normale Supérieure (IBENS), Paris, France). Mutant C. elegans strains were also obtained from the C. elegans Genetic Center.
C. elegans Small Molecule Screens and Phenotypic Analyses
A library of 486 small-molecules (Chembridge) previously found to induce phenotypes in C. elegans (486 worm actives, aka wactives; 26108372) were tested for their ability to modulate C. elegans egg-laying. Drug dilutions were prepared from stock plates using a 96-well pinner tool (FP3S200 V&P Scientific, Inc.) transferring 0.3 mL of drug stock solution prepared in DMSO.
C. elegans Egg-laying Assay
To screen for small molecules that modulate egg laying, ∼20 young-adult wild-type (N2 Bristol) animals were pipetted in 15 mL of M9 buffer to 96-well flat-bottom polystyrene plates (2024-06 TC plate – Sarstedt) to a final volume of 50 mL containing 60 μM of test molecules in either 1) M9 buffer to identify egg-laying stimulators; or 2) a combination of 12.3 mM serotonin creatine sulfate monohydrate (H775 - Sigma-Aldrich) and 7.7 mM nicotine (N3876 - Sigma-Aldrich) in M9 buffer that induces a robust egg-laying response to identify egg-laying inhibitors (aka NS condition). Screen molecules were transferred to test wells using a 96-well pinner tool (FP3S200 V&P Scientific, Inc.) transferring 0.3 mL of drug stock solution prepared in DMSO. Plates were incubated for 1 hour at room- temperature. After 1 hour 182 mL of solution containing 50mM sodium azide (71289 – Sigma Aldrich) and 0.25% sodium dodecyl sulfate (SDS001.100 - BioShop) in M9 buffer using a multi-channel pipette followed by 182 mL of M9 buffer to raise the volume of each well such that a flat meniscus is produced. Plates were immediately imaged on the 2020 Imager and were separated by 5 minutes to allow time for preparation of subsequent plates for imaging. After imaging egg-laying data was extracted from raw captured images using the ‘Egg & Worm Counter’ ImageJ plugin described above. Stimulators were considered molecules that stimulated egg-laying ≥ 2-fold greater than two proximal controls in the benign M9 buffer conditions with significance (p < 0.05 unpaired heteroscedastic t-test) over triplicate measurement (3 test compared against 6 control wells). Egl inhibitors were considered molecules that suppressed egg-laying ≤ 0.5 the normalized egg-laying rate of proximal controls in the ‘nicotine (7.7 mM) + serotonin (12.3 mM)’ condition described above with significance (p < 0.05 unpaired heteroscedastic t-test) over triplicate measurement (3 test compared against 6 control wells). Primary egg-laying modulators that reached the above threshold in at least 1 of 2 additional tests were considered bonafide ‘Egl modulators’. As a more stringent criteria to narrow our focus on robust Egl modulators we limited our inquiry of Egl stimulators that induced Egl ≥ 2 fold that of control over 3 trials (primary screen + 2 retests) or stimulated Egl 3-fold relative to control over at least 2 of 3 trials and Egl inhibitors that suppressed Egl ≤ 2 fold control over all 3 re-tests or molecules that suppressed Egl ≤ 3 fold that of control in at least 2 of 3 trials.
Construction of the ‘2020 Imager’
High-content brightfield data were acquired on a custom brightfield 96 well-plate imager. The well-plate was mounted on a stationary platform, while the imaging setup travelled parallel to the bottom of the well-plate via a motorized stage. The plate was illuminated from above using a 10 W white LED. Underneath the plate, an Olympus 4x objective (Olympus, UPLFLN4XPH) was used with a 150 mm tube lens (Thorlabs, AC254-150-A- ML) to create an effective magnification of 3.33x. A mirror was used to maintain a low- profile imaging setup and minimize distortions induced by the displacement of the imaging optics. The magnified image was projected onto a 4K line scan camera (Dalsa, P2-23-04K40), resulting in an effective resolution of 3.00 µm/px. The camera had a single line of 4096 px which captured an area of 12.3mm by 3 µm with a bit depth of 10 bits. To capture a row of 12 wells, the image acquisition software (EPIX Inc, XCAP-Ltd) was setup to acquire scans at 3000 lines/s. This resulted in a 4K by 48K image that was acquired in about 15 seconds. To sequentially image each row in of a well-plate, an Arduino UNO was used to synchronize the image acquisition with the movement of the motorized stage. For additional details see Aaron Au’s Master’s thesis titled: ‘Optical Imaging Strategies for High-Content Studies of Development’ available through the URL https://tspace.library.utoronto.ca/bitstream/1807/91538/3/Au_Aaron_K_201811_MAS_th esis.pdf.
ImageJ Analysis of C. elegans Egg-Laying Rate
A custom ImageJ (version 1.52i) plugin was used to quantify the number of worms and eggs present in each well (versions used available on github: github.com/seanph16/WormScanner3000/upload). Prior to object counting a threshold was applied based on the mean pixel intensity in each well image. The number of worms in a well was determined by measuring the area covered by non-egg shaped objects divided by the average area of a worm. Worm-like objects were recognized by the built-in ImageJ analyze particles function identifying objects greater than 11450 px in area with a circularity of 0.029–0.80 (the approximate minimum single adult worm size and range of shape circularities adopted by a worm) divided by the median worm area. Single eggs were counted by first creating a mask of egg and egg clump shaped objects (objects that are 300-6000 px in area with circularity of 0.15-1.00), applying the built-in ImageJ ‘Watershed’ function to recognize single eggs within clumps and counting objects with a circularity of 0.5–1 with a size of 300-2500 px. The number of egg objects divided by the number of worms was used as a read-out of egg-laying behaviour.
C. elegans Locomotory Survey
Locomotor phenotype analyses were done in 24-well plates with 1 mL of MYOB substrate (27.5 g Trizma HCl, 12 g Trizma Base, 230 g bacto tryptone, 10 g NaCl, 0.4 g cholesterol (95%)) seeded with 25 µL of OP50 Escherichia coli on each well. Each compound was added to the MYOB substrate before pouring to achieve the desired final concentrations of 30 µM or 60 µM after diffusion through the media. The final concentration of dimethyl sulfoxide (DMSO) in each of the wells was 1% v/v. Young adult or late fourth-staged larval worms are transferred into each well using a platinum wire pick. A Leica MZ75 stereomicroscope was used to visualize the movement of worms on the solid substrate. The specific locomotor phenotype (i.e. ‘rubber-band’ or ‘coiler’) was noted, and a qualitative assessment of the severity was made based on the degree of locomotor incapacitation and penetrance of the phenotype. Samples deemed ‘Severe’ indicated a strong perturbation and high penetrance, ‘moderate’ indicated a strong phenotype with low penetrance or a weak phenotype that is highly penetrant, and ‘mild’ indicated a weak phenotype that has low penetrance. Paralysis was distinguished from death by the presence of pharyngeal pumping.
C. elegans Motor Phenotype Analyses
The intensity of nementin-induced convulsions is tightly correlated with the degree of paralysis (i.e. animals that exhibit paralysis invariably convulse). We therefore used the degree of paralysis as a conveniently measurable proxy of convulsion intensity. In our survey of the effects of nementin analogs across C. elegans, P. pacificus and R. diutinus, animals were scored as convulsive if they failed to back at least ½ a body length after a touch on the head with a platinum wire. A more stringent scoring method was employed to compare convulsive phenotypes exhibited by C. elegans mutants. Animals were scored as convulsive if animals failed to demonstrate a sinusoidal wave form before or after a touch on the head with a platinum wire and failed to back ½ a body length. Like the convulsion scoring method above, animals treated with acetylcholinesterase inhibitors that failed to demonstrate a sinusoidal wave form before or after a touch on the head with a platinum wire that also fail to back ½ a body length were scored as paralyzed.
Locomotory Radiation Assay
∼150 L4/young adult worms were pipetted in a 15 µL droplet onto the centre of a standard 10 cm round culture plate containing MYOB media + agar containing small- molecule with 1% DMSO. Media with drug were prepared in 50 mL falcon tube inverted 10x before pouring. Plates were dried for 90 minutes before being supplemented with a full lawn of OP50 bacteria seeded from a saturated culture of OP50 grown in LB broth. Plates were left uncovered adjacent to a flame until the (∼15 minutes). 3 hours after pipetting worms onto plates the plates were flash frozen at -80°C for 3 minutes to freeze worms in place. The fraction of worms that travelled at least 1.65 cm (diameter after measuring 1cm from the edge of the droplet) from centre of the plate were recorded.
Developmental Growth Assay
C. elegans larval development assays were conducted in 96-well flatbottom clear flatbottom plates. ∼20 L1 larvae in 10 μL of M9 buffer were pipetted into each test wells containing 40 μL of NGM media supplemented with HB101 E. coli with the desired test compound (+0.6% dimethylsulfoxide (DMSO; Sigma-Aldrich product ID: D8418) as the chemical solvent). Plates were wrapped in 3 layers of brown paper towels soaked with water. After either 3 or 6 days of incubation the number of C. elegans animals of different larval stages were recorded using a Leica MZ75 stereomicroscope.
C. elegans Confocal Microscopy
C. elegans KG4247 expressing ceIs201 [unc-17p::ins-22::Venus + unc-17p::RFP + unc- 17p::ssmCherry + myo-2p::RFP] were incubated on 6 cm MYOB media + 2% agar plates containing either 60 μM Nementin-1 or 60 μM Nementin-12 with 1% DMSO or 1% DMSO alone for 4 hours at room temperature. Wells were seeded with OP50 E. coli bacteria and used the day after preparation (see locomotory survey for further details on the preparation of media + drug plates). After incubation, animals were picked onto a 5% agar pad, 10 μL 10 mM tetramisole hydrochloride (prepared from 99% (−)- tetramisole hydrochloride, Sigma-Aldrich product ID: L9756) solvated in standard M9 buffer was pipetted onto the pad and a cover glass put on top. Animals were imaged using a Leica DMI 6000 B confocal microscope with a Hamatsu C9100-31 camera with a 100x oil immersion objective. A 491 nm laser was used to excite INS-22::Venus and images were captured with 25ms of exposure. A 510 nm laser was used to excite ssmCherry and RFP and images were captured with 100 ms of exposure. Images were captured after anterior and dorsal nerve cord features were brought into focus in the red channel (the RFP remained stable for the duration of imaging). Anterior, midbody and posterior regions containing respective coelomocytes (ccPR + ccAR, ccPL + ccAL & ccDL respectively) were captured. Images were captured over a 30 μm Z-stack captured with a 0.5 μm step and all images were captured within 25 minutes of slide preparation. A maximal projection containing the ventral and dorsal nerve cords and coelomocyte was generated for each captured section in ImageJ (version 1.52i).
Tracings of captured axonal sections and coelomocytes were manually drawn and fluorescence signal measured in ImageJ. Due to the variability in coelomocyte endocytic/lysosomal vesicle content, coelomocytes were reported as the measurement of mean fluorescence signal of the GFP channel compared to the RFP channel. For axonal segments, the mean of two measurements of each region and representative background were collected to adjust for variability in manual measurement. Regions of interest for at least 15 animals were captured over several imaging sessions, at least 3 control animals were captured in each imaging session.
Parasitic Nematode Assays
Cooperia oncophora Assay: Fresh cattle feces containing eggs of an ivermectin- resistant strain of C. oncophora were kindly supplied by Dr. Doug Colwell and Dawn Gray (Lethbridge Research Station, Agriculture and Agri-Food Canada). Established methods were used to carry out the experimental cattle infections, and these methods were approved by the Lethbridge AAFC Animal Care committee and conducted under animal use license ACC1407. Cattle faeces containing C. oncophora eggs were stored anaerobically at room temperature for a maximum of 6 days before use. Eggs were isolated from faeces using a standard saturated salt flotation method immediately before the egg hatch assay. 80 µl of distilled and deionized water was added to each well of a 96-well culture plate, and then 1 µL of chemical at the appropriate concentration in DMSO was added to each well using a multichannel pipette.
Approximately 50 eggs were added per well in 20 µL of water for a final volume of 100 µL in each well; the final DMSO concentration was 1% (v/v). The eggs were incubated in the chemicals for 2 days at room temperature, after which hatching was stopped by the addition of 1 µL iodine tincture to each well. The number of hatched larvae was counted at each concentration, and eggs that failed to hatch were scored as dead. “Relative viability” values were calculated by dividing the fraction of eggs that hatched at each concentration by the fraction of eggs that hatched in the corresponding DMSO control well. Two biological replicates were performed for each dose-response experiment, and the relative viability values were averaged across the biological replicates. The average hatch rate for the DMSO control wells was greater than 93% for both biological replicates.
Dirofilaria immitis Assay: Experiments on D. immitis microfilariae were performed in the laboratories of Bayer Animal Health GmbH (Monheim, Germany). The Missouri D. immitis isolate used for all assays was originally isolated from an infected dog from Missouri (USA). From 2005 onwards, the isolate was maintained and passaged in beagle dogs at the University of Georgia (Athens, GA, USA). From 2012 onward, the isolate was also maintained at the laboratories of Bayer Animal Health GmbH in Monheim, Germany. For the experiments with microfilariae, blood was sampled from beagle dogs (Marshall BioResources, North Rose, NY, USA) with patent infections, and microfilariae were purified according to the protocol described by the FR3.
Approximately 250 freshly purified microfilariae were cultured in single wells of a 96- well microtiter plate containing supplemented RPMI 1640 medium. Microfilariae exposed to medium substituted with 1% DMSO were used as negative controls. Motility of microfilariae was evaluated after 72 hours of drug exposure using an image-based approach – DiroImager, developed by Bayer Technology Services. This device is a fully automated high-throughput platform, allowing high-resolution optical imaging of an entire 96-well microtiter plate. The DiroImager integrates a high-resolution video camera (Prosilica GT6600; Allied Vision) with a telecentric lens (S5LPJ3005; Sill Optics) that prevents perspective distortion of the recorded images, ensuring high accuracy of measured values across all samples. In brief, a series of 20 high-resolution images were recorded (one per second). In a first step, image processing filters were used that discriminate larger objects to avoid the detection of crystallized or undissolved particles. In the actual calculation, pixel-wise differences between sequential images were calculated to determine worm movement between single images of a series; test compound activity was determined as the reduction of motility in comparison to the solvent control. Based on the evaluation of a wide concentration range, concentration– response curves as well as IC50 values were calculated were applicable.
Nippostrongylus brasiliensis acetylcholine esterase secretion assay: This assay has been previously described in detail (3). AChE is secreted by many parasitic nematodes, including N. brasiliensis. Assaying a small molecule’s impact on AChE secretion is therefore a proxy for its ability to modulate the nematode’s nervous system. Methods have been previously developed to assay AChE secretion from Nippostrongylus using colourimetric determination of AChE activity in the culture medium (4). Briefly, test compounds were dissolved in DMSO at a concentration of X, Y, Z and serial dilutions were performed in DMSO resulting in stock solutions of A, B, C. Stock solutions were stored at -20 °C until they were diluted 1:200 with culture medium (20 g/l Bacto Casitone, 10 g/l yeast extract, 5 g/l glucose, 0.8 g/l KH2PO4, 0.8 g/l K2HPO4, 10 μg/ml sisomycin and 1 μg/ml clotrimazole, pH 7.2). Final drug concentrations were E, F, G µM in 1.0 % DMSO. Because secretion of AChE is gender and body weight specific two female and three male adult worms were placed in each well containing 1 ml of pre- warmed medium with drugs plus vehicle and incubated at 37 °C and 95% relative humidity for five days (5). All drug concentrations were performed in duplicate. From each well 25 μl medium were transferred into a 96 well plate. Then, 250 μl 5,5′-dithio- bis (2-nitrobenzoic acid) (0.25 μM) and 25 μl acetylthiocholine (4 mM) were added.
AChE cleaves acetylthiocholine into acetate and thiocholine. In a consecutive reaction, thiocholine reacts with 5,5′-dithio-bis(2- nitrobenzoic acid) to thionitrobenzoate.
Thionitrobenzoate is a yellow dye and its concentration can be determined by measuring the absorption at 405 nm. The A405 was measured after two and seven minutes of incubation at RT using an Expert 96 plate reader (Asys-Hitech, Salzburg,
Austria) and the software MikroWin 2000 (Mikrotek, Overrath, Germany). The difference in absorption between both time points was taken as measure of AChE activity. The arithmetic mean of 12 no drug control wells was set to 100% activity, and reduction of AChE activity in percentage relative to the negative control was calculated for each test compound concentrations. Within an assay, every drug concentration was performed in duplicate, and the software reported the mean of these duplicates.
Strongyloides ratti L3 larvae lethality: Data are the measurement of the % of Larval stage 3 (L3) worms (as indicated) that respond to 80°C hot water stimulus after 24 hours or 72 hours of incubation in wells containing the indicated compound. Data are the mean measurement of 30-40 larvae incubated in a dark box at room temperature for 24 or 72h over duplicate biological replicate conducted in triplicate.
Trichuris muris L1 larvae experiments: T. muris eggs were collected from the feces of the infected mice (as described above) using a flotation method with saturated NaCl solution in Milli-Q water. T. muris eggs were stored in Milli-Q water in the dark for 3 months at 23-25°C, until the eggs were embryonated. T. muris L1 were obtained using a hatching procedure with E. coli(6). 30-40 larvae were placed in each well of a 96-well plate containing 175 µl culture medium and 25 µl of the test drug stock solutions. Larvae were kept at 37°C, 5% CO2 for 24 hours. To evaluate the drug effect first the total number of L1 per well was determined. Then, 50-80 µl of hot water (≈80°C) was added to each well and the larvae that responded to this stimulus were counted. The proportion of larval death was determined. Larval survival counts were averaged over duplicate biological replicate conducted in triplicate normalized to controls.
T. muris adult experiments: Mice (C57BL/6NRj) were infected with 200 embryonated
T. muris eggs. Seven weeks post-infection T. muris adult worms were collected from the intestines. Three worms were placed in each well of a 24-well plate containing 1980 µl culture medium and 20 µl of the test drugs (10 µM of a 1mM stock solution). After 72 hours of incubation at 37°C, 5% CO2 the condition of the worms was microscopically evaluated using a viability scale from 3 (normal activity) to 0 (dead). Viability scores were averaged across replicates and normalized to the control wells. The experiment was conducted in duplicate.
Heligmosomoides polygyrus L3 viability: H. polygyrus infection three-week-old female NMRI mice were obtained from Charles River (Sulzfeld, Germany). Rodents were kept under environmentally-controlled conditions (temperature: 25°C, humidity: 70%, light/dark cycle 12 h /12 h) and had free access to water (municipal tap water supply) and rodent food and were allowed to acclimatize for one week. NMRI mice were infected with 88 H. polygyrus L3. Two weeks post-infection, mice were dissected cultivating the eggs on an agar plate for 8-10 days in the dark at 24°C. For the assays, 30-40 larvae were placed in each well of a 96-well plate containing 175 µl culture medium and 25 µl of the test drug stock solutions. H. polygyrus adults and stage 3 larvae (L3) were incubated in RPMI 1640 (Gibco, Waltham MA, USA) medium supplemented with 5% amphotericin B (250 µg/ml, Sigma-Aldrich, Buchs, Switzerland) and 1% penicillin 10,000 U/ml, and streptomycin 10 mg/ml solution (Sigma-Aldrich, Buchs, Switzerland). Culture plates were kept in a dark box at room temperature for up to 72 hours. To evaluate the drug effect first the total number of L3 per well was determined. Then, 50-80 µl of hot water (≈80°C) was added to each well and the larvae that responded to this stimulus were counted. The proportion of larval death was determined. Larval survival counts were averaged over duplicate biological replicate conducted in triplicate normalized to controls.
Necator americanus L3 viability: N. americanus larvae (L3) were obtained by filtering the feces of infected hamsters and cultivating the eggs on an agar plate for 8-10 days in the dark at 24°C. Necator americanus L3 were incubated in Hanks’ balanced salt solution (HBSS; Gibco, Waltham MA, USA) supplemented with 10% amphotericin B and 1% penicillin (10,000 U/ml) and streptomycin (10 mg/ml) solution. For the assays, 30-40 larvae were placed in each well of a 96-well plate containing 175 µl culture medium and 25 µl of the test drug stock solutions. Treated Larvae were kept in a dark box at room temperature for up to 72 hours. To evaluate the drug effect first the total number L3 per well was determined. Then, 50-80 µl of hot water (≈80°C) was added to each well and the larvae that responded to this stimulus were counted. The proportion of larval death was determined. Larval survival counts were averaged over duplicate biological replicate conducted in triplicate normalized to controls.
Meloidogyne incognita assays: M. incognita infective second stage juvenile (J2) in vitro viability assays were performed in 96-well polystyrene plates. Each well contained approximately 25 J2s and compounds were added at a final concentration of 45 µM (0.5% DMSO v/v) in a total volume of 100 µL of sterile distilled water. Plates were sealed with parafilm and incubated for 72 hours at 25 °C. At the end point the fraction of viable nematodes in each drug condition and DMSO solvent controls was calculated by dividing the number of mobile nematodes by the total number of nematodes in the well. The experiment was conducted twice, with three technical replicates per treatment in each trial. M. incognita egg hatching assays were performed in sterile distilled water in 96-well plates similarly to the J2 viability assays described. Embryos were incubated in 45 µM (0.5% DMSO v/v) compound for 7 days at 25 °C. At the end point the number of hatched embryos was quantified in each condition and DMSO solvent controls. The fraction of hatched juveniles that were mobile was also quantified (‘hatchling mobility’). The experiment was conducted twice, once with 50 embryos plated per well and once with 100 embryos plated per well, with three technical replicates per treatment in each trial. M. incognita 50-day soil reproduction assays were conducted in 90 grams of soil (1:1 sand:loam mix) per compartment in 6-pack planting containers. The soil was drenched with 18 mL of deionized water containing dissolved chemical or DMSO solvent alone. Approximately 1500 J2s were inoculated into the soil in 2 mL of water, for a total volume of 20 mL. The J2s were incubated in the soil and chemical for 24 hours after which a 2-3 week old tomato seedling was transplanted into the soil. Tomatoes were grown for 8 weeks in a greenhouse under long-day conditions (16 hour photoperiod) with 26/18 °C day/night temperatures. At the end point of the assay the tomato roots were harvested and eggs were extracted by rinsing in 0.6% sodium hypochlorite solution with agitation at 300 rpm for 3 minutes. Roots were rinsed with water over nested sieves and eggs present in each root system were collected and quantified. Roots were dried in a 65 °C oven and the number of eggs per milligram of dried root material was calculated. The experiment was conducted twice, with two technical replicates per treatment in each trial.
Meloidogyne hapla motor assay: M. hapla motor assays were conducted using J2 infective larvae isolated from ornamental tomato plant roots. J2s were isolated by isolating egg masses from the root network of infected plants and hatching in deionized water at room temperature for ∼1 week. 10 μL of deionized water containing ∼15 J2s (no fewer than 10 J2s) were pipetted into 96-well polystyrene plates containing the drug condition of interest with 0.6% DMSO. Addition of J2s to wells were staggered by 35 seconds for the purpose of maintaining a stringent endpoint. Drug dilutions were prepared from stock plates using a 96-well pinner tool (FP3S200 V&P Scientific, Inc.) transferring 0.3 mL of drug stock solution prepared in DMSO. Animals were incubated at room temperature with shaking for 4 hours (100 RPM; helps concentrate J2s in the middle of wells). At the 4 hour endpoint, 30 second videos of each well were captured using a Leica FLEXACAM C1 USB camera mounted to a Leica MZ75 stereomicroscope using Leica LAS EZ image capture software (V3.4.0). Videos were sped up 5x and the number of head turns was used as a read-out of J2 motility. Motility relative to control over three or four biological replicates was calculated & used as the data read-out for the SynergyFinder 2.0 server (https://synergyfinder.fimm.fi/).
Small-Molecule Tanimoto Coefficient Pairwise Similarity
Pairwise similarity scores were calculated as the Tanimoto coefficient of shared FP2 fingerprints using OpenBabel (http://openbabel.org). A Further description of Tanimoto pairwise similarity is provided in Burns et al. 2015 (26). Network visualization for Fig. 1b was performed using Cytoscape (version 3.7.2).
Zebrafish Chemical Treatments and Phenotypic Analyses
All phenotypic analysis was performed on a stereomicroscope. At 1 dpf, 5 embryos were placed in 1 mL filter-sterilized egg water with chemicals in sterile 24-well plates (Falcon). At 3dpf, larvae were anaesthetized with ∼0.6 mM tricaine methanesulfonate (tricaine), mounted in 3% methylcellulose on glass slides and bright-field images were taken with a 4x objective using a light microscope (Olympus BX43). The morphology of embryos relative to vehicle controls was assessed including their size, presence of edema, heart rate (normal, slow, or nearly absent), and presence of necrosis.
All chemicals were prepared in DMSO and added to filter-sterilized egg water at 0.1% of the final volume. Equal volumes of vehicle solvent were used in all conditions for a single assay. Note that methylene blue was not added to the egg water in any chemical assays. Culture plates were sealed with parafilm, wrapped in aluminum foil, and incubated at 28.5°C until the assay date.
A photoactivation assay was used to elicit movement and assess locomotion of zebrafish larvae as previously described (7). At 1 dpf, embryos in their chorions were aliquoted into 150 μL system water in 96-well plates (Falcon). Next, 50 μL of 4X chemical was added to each well to bring the volume to 200μL and 1X final concentration (either 3.75-60 μM). Plates were incubated until 3dpf, at which time any embryos still in their chorions were manually dechorionated in their wells. To assay locomotion, 10 μL of 210 μM optovin analog 6b8 (ChemDiv ID#2149-0111 or ChemBridge ID#5707191) was added to each well for a final concentration of 10 μM, incubated for 5 min, and movement tracked on the ZebraBox platform (ViewPoint) using a 30s lights on/off for 3m30s.
Arabidopsis thaliana Greening Assay
Greening experiments were performed with Arabidopsis thaliana seeds of wild type Col- 0; seeds were surface sterilized in bleach and plated onto 0.5X MS, 0.5% sucrose agar medium supplemented with compounds of interest at 5, 15 and 45µM concentrations (0.2% DMSO (v/v/)). After 4d of stratification at 4°C, plates were transferred to a growth chamber (16h / 8h, 150 µE/m2) and greening recorded after 4 days. Pictures were recorded by camera (SONY a7s) with FE1.8/55 lens (FE 55 mm F1.8 ZA; SEL55F18Z). Experiments were performed in triplicate for each treatment.
Drosophila melanogaster Dose-Response Assay
Fly food in agar substrate was prepared by mixing 100 mL of unsulfured molasses, 100 mL of cornmeal, 41.2 g of Baker’s yeast, and 14.8 g of agar into 1400 mL of distilled de- ionized water and boiling for 30 minutes. The media was allowed to cool to 56°C, at which point 5 mL was added by syringe to plastic cylindrical fly vials. 10 µL of chemical, or DMSO alone, was added to the media in each vial. The chemicals were mixed into the media by mechanical mixing using a pipette. The final DMSO concentration was 0.2% (v/v). The media was allowed to solidify at room temperature (∼22°C) overnight. The following day (Day 0), eight pairs of male and female w1118 flies were added to each vial so that there were 16 flies in total per vial. The vials were stored at room temperature for 7 days, at which point the number of mobile flies was counted. Fly mobility was scored as any observable movement after the vial had been vigorously jostled. “Relative mobility” was calculated by dividing the number of mobile flies in the treatment vials by the average number of mobile flies in two DMSO control vials. On Day 8 the 16 parental flies were removed from the vials and the progeny larvae were allowed to continue to grow and hatch into adult flies. To assess larval viability, hatched flies were counted and discarded on Days 10, 12, 14, 16, 18, and 20. The counts were summed. “Relative viability” was calculated by dividing the number of hatched flies in the treatment vials by the average number of hatched flies in the two DMSO control vials. The final “relative mobility” and “relative viability” values are an average across three experimental replicates.
HEK293 Proliferation Assay
HEK293 cells were seeded into 96 well plates, at 5000 cells per well, in 100 μL total volumes of DMEM/10%FBS/1%PS media and grown overnight at 37°C in the presence of 5% CO2. Compounds (0.5 μL volumes from appropriate source plates) were then added to cells, and growth was continued for an additional 48 hours. Following growth, 10 μL of CellTiter-Blue Viability reagent (Promega) was added to each well, and plates were incubated for an additional 4 hours at 37°C in the presence of 5% CO2. Fluorescence measurements (560 nm excitation/590 nm emission) were then performed using a CLARIOstar Plate Reader (BMG Labtech) to quantify reagent reduction and estimate cell viability.
Spicule Protraction Assay
L4 males grown overnight on Nematode Growth Media (NGM) 2% agar plates + OP50
Next day adult males plated in liquid NGM liquid culture with 1% DMSO or 60 μM Nementin-1 with 1% DMSO in polystyrene 96-well plates. Adults were observed under a Leica MZ75 stereomicroscope at indicated time points. Reported ’% Protracted’ includes partial spicule protraction.
Statistical Analyses & Synergy Modelling
Unpaired one or two-sided t-tests or one-sided ANOVA with Dunnett’s adjustment for multiple comparisons were conducted between control and treatment groups with where appropriate. Two-sided Chi-square tests with Bonferroni correction were conducted for comparison of proportional convulsion data to respective controls. Extra sum-of-squares F tests were conducted comparing EC50 curves generated for dose-response data.
Statistical analyses were conducted using GraphPad Prism (version 9). Zero-Interaction Potency (ZIP) synergy scores and heatmaps were generated using the SynergyFinder2.0 server using the default parameter set.
Chemistry
General Considerations
Unless otherwise stated, all reactions were set up under inert atmosphere (argon) utilizing glassware (or 2 dram vials) that were flame-dried and cooled under argon purging. Unless otherwise stated, flash column chromatography was performed on Silicycle® Siliaflash® P60, 40-63 μm silica gel. Starting materials and catalysts were purchased from commercial suppliers (Sigma Aldrich, Strem, Alfa Aesar, TCI or Combi- Blocks) and used without further purification unless otherwise stated. All solvents were distilled, purified, and dried according to standard procedures. Reactions were monitored using thin-layer chromatography (TLC) on EMD Silica Gel 60 F254 plates.
Visualization of the developed plates was performed under UV light (254 nm) or by immersion in Ceric Ammonium Molybdate (CAM) or Potassium Permanganate (KMnO4) stains.
NMR characterization data was collected at 296 K on a Varian Mercury 300, Varian Mercury 400, Bruker Avance III 400, Agilent DD2 500 (with cold probe), or an Agilent DD2 600 operating at 300, 400, 500, or 600 MHz for 1H NMR, and 75, 100, 125, or 150 MHz for 13C NMR. (Funded by the Canadian Foundation for Innovation, project number 19119, and the Ontario MRI). 1H NMR spectra were internally referenced to the solvent residual signal (CDCl3 = 7.26 ppm) unless otherwise stated. 13C NMR spectra were internally referenced to the residual solvent signal (CDCl3 = 77.16 ppm) unless otherwise stated. 19F NMR spectra were externally referenced to CFCl3. Data for 1H NMR are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), coupling constant (Hz), integration.
Melting point (mp) ranges were determined on a Fisher-Johns® Melting Point Apparatus and are reported uncorrected.
Infrared (IR) spectra were acquired using a Shimadzu FTIR-8400S FT-IR spectrometer as thin films (CHCl3 or CH2Cl2) or neat on NaCl plates. Data is presented in wavenumbers (νmax, cm−1).
High Resolution Mass Spectra (HRMS) were obtained on a micromass 70S-250 spectrometer (EI) or an AB SCIEX QSTAR® Mass Spectrometer (ESI) or a JEOL® AccuTOF medel JMS-T1000LC mass spectrometer equipped with an IONICS® Direct Analysis in real Time (DART) ion source at Advanced Instrumentation For Molecular Structure (AIMS) in the Department of Chemistry at the University of Toronto. Where ESI+ was employed, the values correspond to the ionic species of interest and the given ionic formula includes the charging agent (H+ or Na+); both the measured and calculated values are corrected for the mass of the electron and are reported as m/z. The DART- MS accurate mass report is generated using the
Elemental Composition Estimation feature as implemented in the JEOL Mass Centre software package. Where DART was employed, the measured values correspond to the neutral species of interest and the given molecular formula includes the charging agent (H+ or NH4+); the measured and calculated values are not corrected for the mass of the electron and are reported as neutral masses.
General Procedures for the Synthesis
General Procedure A – Wittig Reaction
The phosphonium salt (1.2 equiv.) was first dissolved in THF (0.3 M). KOtBu (1.2 equiv.) was then added and the mixture was stirred at room temperature for 30 minutes. The pyridinecarboxaldehyde (1 equiv.) was then added in three portions and the reaction was refluxed at 70 °C for 3 hours. The mixture was then allowed to cool to room temperature and was filtered through a Celite pad eluting with pentanes. The filtrate was concentrated in vacuo and the product (mixture of isomers) was purified by flash column chromatography, eluting with a mixture of EtOAc:pentanes.
General Procedure B – Suzuki Reaction (8)
Substituted aryl boronic acid (1.5 equiv.) and Pd(OAc)2 (1 mol%) were added to a mixture of the substituted bromopyridine (1 equiv.) in water (0.5 M). iPr2NH (2 equiv.) was then added and the reaction was refluxed at 100 °C for 16 hours. The mixture was allowed to cool to room temperature and brine was added. The aqueous phase was extracted with ethyl acetate. The combined organics was washed with brine, dried with MgSO4, and concentrated in vacuo. The product was purified by flash column chromatography, eluting with a mixture of EtOAc:pentanes.
General Procedure C – Hydrogenation
The vinylbiarene was dissolved in EtOAc (0.15 M). Pd/C (3% Pd, total 10 mol% Pd used) was added. Three cycles of evacuation and backfill with argon, followed by H2 from a balloon was carried out. The reaction was stirred at room temperature under a H2 atmosphere (balloon) for 16 hours. The contents of the flask were filtered over a Celite pad eluting with EtOAc and concentrated in vacuo. The product was purified by flash column chromatography, eluting with a mixture of EtOAc:pentanes.
2-(4-((difluoromethyl)thio)phenyl)-5-propylpyridine [nementin-12-5(C), YHK01- 077F1]
xx was synthesized according to General Procedure A, B, and C using 6-bromonicotinaldehyde, ethyltriphenylphosphonium bromide, and (4- ((difluoromethyl)thio)phenyl)boronic acid with an overall yield of 21%.
4-(difluoromethoxy)-4’-propyl-1,1’-biphenyl [nementin-12-6(D), KLC10-089F1]
xx was synthesized according to General Procedure A, B, and C using 4-bromobenzaldehyde, ethyltriphenylphosphonium bromide, and (4- (difluoromethoxy)phenyl)boronic acid with an overall yield of xx%.
5-(4-(difluoromethoxy)phenyl)-2-propylpyridine [nementin-12-7, YHK01-104F1]
xx was synthesized according to General Procedure A, B, and C using 5-bromopicolinaldehyde, ethyltriphenylphosphonium bromide, and (4- (difluoromethoxy)phenyl)boronic acid with an overall yield of 16%.
5-(difluoromethoxy)-2-(4-propylphenyl)pyridine [nementin-12-8, YHK01-102F1]
2-bromo-5-(difluoromethoxy)pyridine (Sx): The procedure was adapted from Geng et al. (9). 6-bromopyridin-3-ol (348 mg, 2 mmol, 1 equiv.) was dissolved in THF (8 mL, 0.25 M) and cooled to 0 °C. NaH (800 mg, 20 mmol, 60%, 10 equiv.) was added and the mixture was stirred at 0 °C for 30 minutes. H2O (1.80 mL, 100 mmol, 50 equiv.) was added dropwise and the mixture was stirred at 0 °C for 10 minutes. Diethyl (bromodifluoromethyl)phosphonate (0.71 mL, 4 mmol, 2 equiv.) was added and the reaction was allowed to stir from 0 °C to room temperature for 30 minutes. H2O was added and the aqueous phase was extracted with EtOAc. The combined organics was washed with brine, dried with MgSO4, and concentrated in vacuo. The crude mixture was purified by flash column chromatography, eluting with 5% (v/v) EtOAc:pentanes to give xx (161.1 mg, 0.72 mmol, 36%).
5-(difluoromethoxy)-2-(4-propylphenyl)pyridine (xx): Suzuki reaction was carried out to couple Sx (161.1 mg, 0.72 mmol, 1 equiv.) and (4-propylphenyl)boronic acid (177.1 mg, 1.08 mmol, 1.5 equiv.) according to General Procedure B to give Sx (140.0 mg, 0.53 mmol, 74%).
2-(difluoromethoxy)-5-(4-propylphenyl)pyridine [nementin-12-9, YHK01-095F1]
5-bromo-2-(difluoromethoxy)pyridine (Sx): The procedure was adapted from Ando et al. (10). 5-bromopyridin-2-ol (870 mg, 5 mmol, 1 equiv.) was dissolved in MeCN (25 mL,
0.2 M). 2,2-difluoro-2-(fluorosulfonyl)acetic acid (0.67 mL, 6 mmol, 1.2 equiv.) was added, followed by Na2SO4 (7.1 mg, 0.5 mmol, 0.1 equiv.). The reaction was quenched with saturated NaHCO3(aq) solution and extracted with EtOAC. The combined organics was washed with brine, dried with MgSO4, and concentrated in vacuo. The crude mixture was purified by flash column chromatography, eluting with a 5 to 10% (v/v) EtOAc:pentanes gradient to give Sx (521.9 mg, 2.3 mmol, 47%).
2-(difluoromethoxy)-5-(4-propylphenyl)pyridine (xx): Suzuki reaction was carried out to couple Sx (112.0 mg, 0.5 mmol, 1 equiv.) and (4-propylphenyl)boronic acid (123.0 mg, 0.75 mmol, 1.5 equiv.) according to General Procedure B to give xx (114.0 mg, 0.433 mmol, 87%).
2-(4-(difluoromethoxy)phenyl)-5-ethylpyridine [nementin-12-10(E), YHK01-040F1]
xx was synthesized according to General Procedure A, B, and C using 6-bromonicotinaldehyde, methyltriphenylphosphonium bromide, and (4- (difluoromethoxy)phenyl)boronic acid with an overall yield of 4%.
2-(4-(difluoromethoxy)phenyl)-5-methylpyridine [nementin-12-11, YHK01-122Rec]
xx was synthesized according to General Procedure B using 2-bromo-5-methylpyridine and (4-(difluoromethoxy)phenyl)boronic acid with a yield of 14%.
2-(4-(difluoromethoxy)phenyl)pyridine [nementin-12-12, YHK01-007C2F1]
xx was synthesized according to General Procedure B using 2- bromopyridine and (4-(difluoromethoxy)phenyl)boronic acid with a yield of 31%.
2-(4-(difluoromethoxy)phenyl)-5-(prop-1-yn-1-yl)pyridine [nementin-12-14, YHK01- 082, 085, 091, 094F1]
2-bromo-5-((triisopropylsilyl)ethynyl)pyridine (Sx): 2-bromo-5-iodopyridine (283.4 mg, 1 mmol, 1 equiv.), PdCl2(PPh3)2 (35.1 mg, 0.05 mmol, 5 mol%), and CuI (9.5 mg,
0.05 mmol, 5 mol%) were weighed into a flame-dried round bottom flask. The contents were purged under nitrogen for 5 minutes. THF (6 mL, 0.2 M) was then added to the flask. Et3N (1 mL, 1 M) and ethynyltriisopropylsilane (0.27 mL, 1.2 mmol, 1.2 equiv.) were added subsequently. The reaction was stirred at 50 °C for 3 hours. The mixture was cooled to room temperature and then filtered through celite, eluting with EtOAc. The filtrate was concentrated in vacuo and the crude mixture was purified by flash column chromatography, eluting with 10% (v/v) EtOAc:pentanes to give Sx (235.6 mg, 0.70 mmol, 70%)
2-(4-(difluoromethoxy)phenyl)-5-((triisopropylsilyl)ethynyl)pyridine (Sx): Suzuki reaction was carried out to couple Sx (235.6 mg, 0.70 mmol, 1 equiv.) and (4- (difluoromethoxy)phenyl)boronic acid (0.197 mg, 1.05 mmol, 1.5 equiv.) according to General Procedure B to give Sx (161.9 mg, 0.40 mmol, 58%).
2-(4-(difluoromethoxy)phenyl)-5-ethynylpyridine (Sx): Sx (162.0 mg, 0.4 mmol, 1 equiv.) was dissolved in THF (1.3 mL, 0.3 M) and cooled to 0 °C. TBAF (0.8 mL, 0.8 mmol, 1 M in THF, 2 equiv.) was added dropwise. The reaction was allowed to stir from 0 °C to room temperature for 16 hours. H2O was added and the aqueous phase was extracted with EtOAc. The combined organics was washed with brine, dried with MgSO4, and concentrated in vacuo. The crude mixture was purified by flash column chromatography, eluting with a 5 to 10% (v/v) EtOAc:pentanes gradient to give Sx (76.5 mg, 0.31 mmol, 78%).
2-(4-(difluoromethoxy)phenyl)-5-(prop-1-yn-1-yl)pyridine (xx): Sx (77.0 mg, 0.31 mmol, 1 equiv.) was dissolved in THF (1.0 mL, 0.3 M) and cooled to -78 °C. nBuLi (0.15 mL, 0.37 mmol, 2.5 M in hexane, 1.2 equiv.) was added dropwise. The mixture was stirred at -78 °C for 1 hour. Iodomethane (0.03 mL, 0.47 mmol, 1.5 equiv.) was added dropwise. The reaction was allowed to stir from -78 °C to room temperature for 16 hours. The reaction was quenched with saturated NH4Cl(aq) solution and extracted with EtOAc. The combined organics was washed with brine, dried with MgSO4, and concentrated in vacuo. The crude mixture was purified by flash column chromatography, eluting with a 2.5 to 5% (v/v) EtOAc:pentanes gradient to give xx (28.3 mg, 0.11 mmol, 35%).
(6-(4-(difluoromethoxy)phenyl)pyridin-3-yl)methanamine [nementin-12-17, YHK01- 061, 098, 108, 112, 119, 120, 123Cr]
(6-bromopyridin-3-yl)methanol (Sx): 6-bromonicotinaldehyde (1.302 g, 7 mmol, 1 equiv.) was dissolved in MeOH (7 mL, 1 M). NaBH4 (397.0 mg, 10.5 mmol, 1.5 equiv.) was added in three portions. The reaction was stirred at room temperature for 5 hours. The mixture was then cooled to 0 °C and 1 M HCl was added dropwise until bubbling seized, followed with dilution of the mixture with H2O. The aqueous phase was extracted with EtOAc. The combined organics was washed with brine, dried with MgSO4, and concentrated in vacuo. The crude mixture was purified by flash column chromatography, eluting with a 50 to 60% (v/v) EtOAc:pentanes gradient to give Sx (1.2859 g, 6.84 mmol, 98%).
2-bromo-5-(((tert-butyldimethylsilyl)oxy)methyl)pyridine (Sx): Sx (884.0 mg, 4.7 mmol, 1 equiv.) was dissolved in DMF (4.7 mL, 1 M) and cooled to 0 °C. TBSCl (1.4160 g, 9.4 mmol, 2 equiv.) was added followed by imidazole (960.0 mg, 14.1 mmol, 3 equiv.). The reaction was allowed to stir from 0 °C to room temperature for 16 hours. H2O was added to the mixture and the aqueous phase was extracted with EtOAc. The combined organics was washed with brine, dried with MgSO4, and concentrated in vacuo. The crude mixture was purified by flash column chromatography, eluting with a 1 to 2% (v/v) EtOAc:pentanes gradient to give xx (1.0952 g, 3.63 mmol, 77%).
5-(((tert-butyldimethylsilyl)oxy)methyl)-2-(4-(difluoromethoxy)phenyl)pyridine (Sx): Suzuki reaction was carried out to couple Sx (1.0900 g, 3.63 mmol, 1 equiv.) and (4-(difluoromethoxy)phenyl)boronic acid (1.0200 g, 5.45 mmol, 1.5 equiv.) according to General Procedure B to give Sx (751.6 mg, 1.98 mmol, 54%).
(6-(4-(difluoromethoxy)phenyl)pyridin-3-yl)methanol (Sx): Sx (751.6 mg, 1.98 mmol, 1 equiv.) was dissolved in THF (6.6 mL, 0.3 M) and cooled to 0 °C. TBAF (2.38 mL, 2.38 mmol, 1 M in THF, 1.2 equiv.) was added dropwise. The reaction was allowed to stir from 0 °C to room temperature for 16 hours. H2O was added to the mixture and the aqueous phase was extracted with EtOAc. The combined organics was washed with brine, dried with MgSO4, and concentrated in vacuo. The crude mixture was purified by flash column chromatography, eluting with a 50 to 60% (v/v) EtOAc:pentanes gradient to give Sx (363.0 mg, 1.45 mmol, 73%).
5-(bromomethyl)-2-(4-(difluoromethoxy)phenyl)pyridine (Sx): Sx (363.0 mg, 1.45 mmol, 1 equiv.) was dissolved in DCM (16.11 mL, 0.09 M). PPh3 (438.0 mg, 1.67 mmol, 1.15 equiv.) followed by NBS (297.2 mg, 1.67 mmol, 1.15 equiv.) were added. The reaction was stirred at room temperature for 3.5 hours. H2O was added to the mixture and the aqueous phase was extracted with EtOAc. The combined organics was washed with brine, dried with MgSO4, and concentrated in vacuo. The crude mixture was purified by flash column chromatography, eluting with a 15% (v/v) EtOAc:pentanes to give Sx (349.5 mg, 1.11 mmol, 77%).
2-((6-(4-(difluoromethoxy)phenyl)pyridin-3-yl)methyl)isoindoline-1,3-dione (Sx): Sx (349.5 mg, 1.11 mmol, 1 equiv.) was dissolved in DMF (3.1 mL, 0.36 M). Phthalimide potassium salt (438.0 mg, 1.67 mmol, 1.15 equiv.) was added. The reaction was stirred at room temperature for 24 hours. H2O was added to the mixture and the aqueous phase was extracted with EtOAc. The combined organics was washed with brine, dried with MgSO4, and concentrated in vacuo. The crude mixture was purified by flash column chromatography, eluting with a 20 to 25% (v/v) EtOAc:pentanes gradient to give Sx (262.2 mg, 0.69 mmol, 62%).
(6-(4-(difluoromethoxy)phenyl)pyridin-3-yl)methanamine (xx): Sx (262.2 mg, 0.69 mmol, 1 equiv.) was dissolved in MeOH (6.9 mL, 0.1 M). Hydrazine hydrate (0.26 mL,
3.56 mmol, 50-60%, 5 equiv.) was added. The reaction was refluxed at 70 °C for 4 hours. The mixture was cooled to room temperature and filtered through Celite, eluting with MeOH. The filtrate was concentrated in vacuo. H2O (7 mL) was added, followed by 1 M KOH (1.75 mL). The aqueous phase was extracted with DCM. The combined organics was dried with MgSO4 and then concentrated in vacuo to give xx (131.4 mg, 0.53 mmol, 76%).
2-(4-(difluoromethoxy)phenyl)-6-propylpyridine [nementin-12-20(F), YHK01-034C2F1]
xx was synthesized according to General Procedure A, B, and C using 6-bromopicolinaldehyde, ethyltriphenylphosphonium bromide, and (4- (difluoromethoxy)phenyl)boronic acid with an overall yield of 18%.
2-(4-(difluoromethoxy)phenyl)-4-propylpyridine [nementin-12-21(G), YHK01-152F1]
xx was synthesized according to General Procedure A, B, and C using 2- bromoisonicotinaldehyde, ethyltriphenylphosphonium bromide, and (4- (difluoromethoxy)phenyl)boronic acid with an overall yield of 9%.
2-(4-(difluoromethoxy)phenyl)-3-propylpyridine [nementin-12-22(H), YHK01-014F1]
xx was synthesized according to General Procedure A, B, and C using 2-bromonicotinaldehyde, ethyltriphenylphosphonium bromide, and (4-(difluoromethoxy)phenyl)boronic acid with an overall yield of 6%.
2-(3-(difluoromethoxy)phenyl)-5-propylpyridine [nementin-12-23(I), YHK01-036F1]
xx was synthesized according to General Procedure A, B, and C using 6-bromonicotinaldehyde, ethyltriphenylphosphonium bromide, and (3-(difluoromethoxy)phenyl)boronic acid with an overall yield of 21%.
2-(4-(difluoromethoxy)phenyl)-5-propylpyrimidine [nementin-12-25, KLC11-041F1]
Pd(OAc)2 (3.4 mg, 0.015 mmol, 5 mol%), PPh3 (7.9 mg, 0.03 mmol, 10 mol%), K2CO3 (124.4 mg, 0.9 mmol, 3 equiv.) and (4- (difluoromethoxy)phenyl)boronic acid (84.6 mg, 0.45 mmol, 1.5 equiv.) were added to a flamed-dried flask. The mixture was purged with nitrogen for 5 minutes. Dioxane (0.55 mL, 0.55 M) and H2O (0.14 mL, 2.18 M) were added, followed by 2-chloro-5-propylpyrimidine (0.04 mL, 0.3 mmol, 1 equiv.). The reaction was then refluxed at 100 °C for 16 hours. H2O was added to the mixture and the aqueous phase was extracted with EtOAc. The combined organics was washed with brine, dried with MgSO4, and concentrated in vacuo. The crude mixture was purified by flash column chromatography, eluting with a 2.5 to 5% (v/v) EtOAc:pentanes gradient to give xx (68.5 mg, 0.26 mmol, 86%).
2-(4-(difluoromethoxy)phenyl)-5-isobutylpyridine [nementin-12-27, YHK01-118F1]
xx was synthesized according to General Procedure A, B, and C using 6-bromonicotinaldehyde, isopropyltriphenylphosphonium iodide, and (4- (difluoromethoxy)phenyl)boronic acid with an overall yield of 23%.
5-butyl-2-(4-(difluoromethoxy)phenyl)pyridine [nementin-12-27, YHK01-118F1]
xx was synthesized according to General Procedure A, B, and C using 6-bromonicotinaldehyde, propyltriphenylphosphonium bromide, and (4- (difluoromethoxy)phenyl)boronic acid with an overall yield of 32%.
5-butyl-2-(4-methoxyphenyl)pyridine [nementin-12-28, YHK01-121F1]
xx was synthesized according to General Procedure A, B, and C using 6-bromonicotinaldehyde, ethyltriphenylphosphonium bromide, and (4- methoxyphenyl)boronic acid with an overall yield of 41%.
4-(5-butylpyridin-2-yl)phenol [nementin-12-29, YHK01-153F1]
xx was synthesized according to General Procedure A, B, and C using 6-bromonicotinaldehyde, ethyltriphenylphosphonium bromide, and (4- (benzyloxy)phenyl)boronic acid with an overall yield of 31%.
2-(4-(2,2-difluoroethoxy)phenyl)-5-propylpyridine. [nementin-12-30, YHK01-168F1]
xx (21.3 mg, 0.1 mmol) was dissolved in DMF (0.33 ml, 0.3 M) and cooled to 0 °C. NaH (6.0 mg, 0.15 mmol, 60%, 1.5 equiv.) was added and the mixture was stirred at 0 °C for 30 minutes. 2- bromo-1,1-difluoroethane (0.02 mL, 0.15 mmol, 1.5 equiv.) was added dropwise and the reaction mixture was stirred from 0 °C to room temperature overnight. H2O was added and the aqueous phase was extracted with EtOAc. The combined organics was washed with brine, dried with MgSO4, and concentrated in vacuo. The crude mixture was purified by flash column chromatography, eluting with 5 to 10% (v/v) EtOAc:pentanes to give xx (17.6 mg, 0.063 mmol, 63%).
Movie S1
Example of typical wild type Caenorhabditis elegans locomotion. Wild type C. elegans (strain N2) are swimming on solid media containing only 1% DMSO solvent control that is incorporated into the agar. Animals have been swimming on the plate for 80 minutes at the time the movie was made.
Movie S2
Example of compound-induced convulsions. Wild type C. elegans (strain N2) are shown after swimming on solid media containing 60 µM Nementin-1 for 80 minutes.
Movie S3
Example of compound-induced coiling. Wild type C. elegans (strain N2) are shown after swimming on solid media containing 60 µM wact-45 for 80 minutes.
Movie S4
Example of compound-induced shaking. Wild type C. elegans (strain N2) are shown after swimming on solid media containing 60 µM Nementin-1 for 80 minutes. At the 18 second mark of the movie, C. elegans (strain N2) are shown after swimming on solid media containing 60 µM wact-203 for 80 minutes. At the 21 second mark of the movie, wild type Rhabditophanes diutinus animals are shown after swimming on solid media containing 60 µM Nementin-1 for 170 minutes.
Movie S5
Example of compound-induced jerky-unc phenotype. Wild-type C. elegans (strain N2) are shown after swimming on solid media containing 60 µM wact-45 for 80 minutes.
Movie S6
Example of compound-induced reversal-defective phenotype. Wild type C. elegans (strain N2) are shown after swimming on solid media containing 60 µM wact-38 for 80 minutes.
Data S1. (Separate file)
Egg-laying rates with and without a stimulatory cocktail.
Data S2. (Separate file)
The Wactive Library egg-laying screen data.
Data S3. (Separate file)
The locomotory survey of the egl-modulators.
Data S4. (Separate file)
Nematode and Counter-Screen Bioassay Data.
Acknowledgements
We are grateful for the strains given to us by the Caenorhabditis Genetics Center (University of Minnesota), Marie-Anne Félix (IBENS, Paris), Ken Miller (Oklahoma Medical Research Foundation), and Mei Zhen and Wesley Hung (Lunenfeld- Tanenbaum Research Institute, Toronto). Many thanks to Dr. Doug Colwell and Dawn Gray at the Lethbridge and Agri-Food Canada Research Centre for supplying faeces from infected calves for Cooperia oncophora egg collection.