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Small Molecules to Combat Infectious Disease (2015 to present)

Origin Story: Since 2003, we were doing small molecule screens with C. elegans with the goal of developing new small molecule tools for biological interrogation and to better understand small molecule accumulation in nematodes. However, the grant panels that funded our work wanted to see a deeper connection to human health before extending our funding. We had been thinking of re-focusing our pipeline to identify molecules that kill parasitic nematodes, so this was a perfect opportunity to pivot. As it turned out, adding this new pragmatic dimension to our work gave us renewed energy and excitement.

    Our first major publication on nematicides was our 2015 Nature Comm paper, which described the screening of over 67,000 small molecules for their ability to disrupt C. elegans development and viability. In this paper, we revealed 30 small molecule scaffolds that selectivity kill nematodes and drilled down on one of them (wact-11), which we found to be a nematode-selective inhibitor of complex II (succinate dehydrogenase) of the electron transport chain. Since then, we have worked to characterize the potential utility and mechanism of action of several of the remaining 29 scaffolds.

     Excitingly, our efforts to develop new nematicides led to the development of a novel technology that facilitates the identification of small molecules and targets against many different human parasites, pests and pathogens in a massively paralleled fashion. We call this technology PEXIL (described elsewhere on our website), which we are currently knee-deep in.

PJR, April 2023


Burns, A., and Roy, P.  2012.  To Kill a Mocking Worm: Strategies to Improve Caenorhabditis elegans as a Model System for use in Anthelmintic Discovery.  A chapter in the book, “Parasitic Helminths: Targets, Drugs and Vaccines”.  Eds. Caffrey, C. , Selzer, P., Wiley VCH, Publishers. Read the chapter here.


Burns, A.R.*, Luciani, G.M., Musso, G., Bagg, R., Yeo, M., Zhang, Y., Rajendran, L., Glavin, J., Hunter, R., Redman, E., Stasiuk, S., Schertzberg, M.,  McQuibban, G.A., Caffrey, C.R.,  Cutler, S., R., Tyers, M., Giaever, G., Nislow, C., Fraser, A.G., MacRae, C. A., Gilleard, J. and Roy, P.J.  2015. Caenorhabditis elegans is a useful model for Anthelmintic Discovery.  Nature Communications. 6:7485 | DOI: 10.1038/ncomms8485. Read the paper here.


Burns, A.R., Bagg, R., Yeo, M., Luciani, G.M., Schertzberg, M., Fraser, A.G., and Roy, P.J. 2017. The Novel Nematicide wact-86 Interacts with Aldicarb to Kill Nematodes.  PLoS Neglected Tropical Diseases. 11(4): e0005502. Read the paper here.


Knox, J., Joly. N., Linossi, E. M., Carmona-Negrón, J.A., Jura, N., Pintard, L., Zuercher, W., and Roy, P.J. 2021. A Survey of the Kinome Pharmacopeia Reveals Multiple Scaffolds and Targets for the Development of Novel Anthelmintics. Scientific Reports. 11:9161 Read the paper here.


Cammalleri, S. R., Knox, J., and Roy, P.J§. 2022. Culturing and Screening the Plant Parasitic Nematode Ditylenchus dipsaci. J. Vis. Exp. (179), e63438, doi:10.3791/63438. Read the paper here.


Harrington, S., Knox, J.J., Burns, A.R., Loon-Choo, K., Au, A., Kitner, M., Haeberli, C., Pyche, J., D’Amata, C., Kim, H-Y., Volpatti, J.R., Guiliani, M., Snider, J., Wong, V., Palmeira, B.M., Redman, E.M., Vaidya, A.S., Gilleard, J., Stagljar, I., Cutler, S.R., Kulke, D., Dowling, J.J., Yip, C.M., Keiser, J., Zasada, I., Lautens, M., and Roy, P.J. 2022. Egg-Laying and Locomotory Screens with C. elegans Yield a Nematode-Selective Small Molecule Stimulator of Neurotransmitter Release. Communications Biology, 2022. 5, 865. Read the paper here.


Harrington S.*, Pyche J.*, Burns A.R., Spalholz T., Ryan, K.T., Baker RJ, Ching J, Rufener, L., Lautens M., Kulke D., Vernudachi, A., Zamanian, M., Deuther-Conrad W., Brust P., and Roy P.J. 2023. Nemacol is a Small Molecule Inhibitor of C. elegans Vesicular Acetylcholine Transporter with Anthelmintic Potential. Nature Communications 14(1816). Read the paper here.


Burns, A.R.§, Ross, R., Kitner, M., Knox, J., Cooke, B., Volpatti, J., Vaidya, A. S., Puumala, E., Palmeira, B.M., Redman, E.M., Snider, J., Marwah, S., Chung, S., MacDonald, M.H., Tiefenbach, J., Hu, C., Xiao, Q., Finney, C.A.M., Krause, H.M., MacParland, S.A., Stagljar, I., Gilleard, J.S., Cowen, L.E., Meyer, S., Cutler, S.R., Dowling, J.J., Lautens, M., Zasada, I., Roy, P.J§. 2023. Selective Control of Parasitic Nematodes Using Bioactivated Nematicides. Nature  Read the paper here.


Knox, J., Burns, A.R., Cooke, B., Cammalleri, S. R., Kitner, M., Castelli, J.M.P., Puumala, E., Snider, J., Stagljar, I., Cowen, L.E., Zasada, I., and Roy, P.J. Cyproside-3 Selectively Kills Plant Parasitic Nematodes via P450 Bioactivation (manuscript in preparation).

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Our Early Chemical Genetics and Drug Screens (2005-2011)

Origin Story: In 2003, about a year after I started my lab, Sean Cutler dropped my office. Sean was a friend from the Botany Department at UofT (but later relocated to UC Riverside; sad for me- good for Sean). He was doing small molecule screens against the model plant, Arabidopsis thaliana, to look for molecules that might disrupt germination. He was curious to know how the bioactivity of his library of 10,000 small molecules might translate to an animal model system and asked me if I wanted to screen it against worms (C. elegans). I eagerly agreed to the proposition.

     My naïve dream was to identify a collection of small molecule tools, each of which would target a different core signalling pathway in the worm and yield the wide-variety of phenotypes that Sydney Brenner first described in his seminal 1974 Genetics paper. My hope was that the C. elegans community could then use these small molecule tools to further interrogate the signalling pathways that are highly conserved among animals. Disappointingly, we did not see the variety of phenotypes from our screen that I hoped for. Of the 308 hits that we found, only a handful of hits induced the dramatic plate phenotypes originally described by Sydney Brenner. No matter. We landed a cool Nature paper out of the screen and I was hooked on drugs (i.e., very excited about the idea that we could search for new small molecules that might have utility as tools to better understand the biology of the worm and animals in general).

     Below are the papers that show how, over time, we gradually learned how to best screen small molecules and characterize hits using C. elegans genetics.

PJR, April 2023


Kwok, T., Ricker, N., Fraser, R., Chan, A., Burns, A., Stanley, E.F., McCourt, P., Cutler, S., and Roy, P.J. 2006. A Small Molecule Screen in C. elegans Yields a New Calcium Channel Antagonist. Nature.  441, 91-95. Read the paper here.


Burns, A.R., Kwok, T.C.Y., Howard, A., Houston, E., Johanson, K., Chan, A., Cutler, S.R., McCourt, P., Roy, P.J. 2006.  High-throughput Screening of Small Molecules for Bioactivity and Target Identification in Caenorhabditis elegansNature Protocols. 1, 1906-1914. Read the paper here.


Kwok, T.C.Y.*, Hui, K.*, Kostelecki, W., Selman, G., Ricker, N., Feng, Z.P., and Roy, P.J. 2008.  A Screen for Dihydropyridine (DHP)-Resistant Worms Reveals a Mechanism for DHP-Blockage of Mammalian Calcium Channels. PLoS Genetics, 4(5):e1000067. Read the paper here.


Hui, K.*, Kwok, T.C.Y.*, Kostelecki, W., Leen, J., Roy, P.J., and Feng, Z.P. 2009.  Differential sensitivities of CaV1.2 IIS5-S6 mutants to 1,4-dihydropyridine analogs. European Journal of Pharmacology, 602, p255-261. Read the paper here.


Burns, A.R., Wallace, I.M., Wildenhain, J., Tyers, M., Giaever, G., Bader, G., Nislow, C., Cutler, S.R., and  Roy, P.J.  2010. A Predictive Model for Drug Bioaccumulation and Bioactivity in Caenorhabditis elegans. Nature Chemical Biology, 6, p549-557 (doi: 10.1038/nchembio.380). Read the paper here.


Wallace, I.M., Urbanus, M.L., Luciani, G.M., Arora, K., Heisler, L.E., Lee, W., Proctor, M., Burns, A.R., St. Onge, R.P., Roy, P.J., Bader, G.D., Nislow, C., and Giaever, G. 2011. Compound prioritization methods increase rates of chemical probe discovery in model organisms. Chem Biol. 18, p1273-1283. Read the paper here.


Luciani, G.M., Magomedova, L., Puckrin, R., Urbanus, M., L., Wallace, I.M., Giaever, G., Nislow, C., Cummins, C.L.­, and  Roy, P.J. 2011. Dafadine Inhibits DAF-9 to Promote Dauer-Formation and Longevity of Caenorhabditis elegansNature Chemical Biology 7, 891-893.  doi: 10.1038/nchembio.698. Read the paper here.

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Small Molecule Crystal Formation in C. elegans (2019 to present) 

Origin Story: Since our very first small molecule screens, we found that some molecules elicited a ‘dark pharynx’ phenotype in which dark material accumulated near the lumen of the pharynx. We ignored this phenotype for over a decade until 2015, when it piqued the interest of a new graduate student, Muntasir Kamal. Together, we found that the darkening of the pharynx was caused by the crystallization of the small molecules themselves on the non-luminal side of pharynx cuticle. As debunked in our 2019 Nat Comm paper, the dark pharynx phenotype is not the result of the animal simply eating precipitated small molecule in the media. We found that as the crystals grow, they puncture the underlying plasma membrane and ultimately grow to such a size that causes the arrest and death of young larvae.

     To better understand why small molecules crystallize within the pharynx cuticle, we felt it necessary to investigate how the pharynx cuticle is constructed. This lead to the creation of blueprint for cuticle construction and the discovery of a plethora of low-complexity proteins that are added to the developing cuticle in temporal waves. Through forward genetic screens for mutants that resist crystal formation, we identified a key phospholipid synthesis pathway and the PGP-14 p-glycoprotein pump that establish a lipid barrier within the cuticle that inadvertently acts as a sink for the accumulation of hydrophobic small molecules. PGP-14 is homologous to human ABCB4, which is mutated in a childhood liver disease called PFIC3. In on-going studies, we have been exploring the utility of the pgp-14 mutant and its resistance to crystal-induced death as a potential model for ABCB4 deficiency. The study of small molecule crystal formation has led to inspiring biological insights and continues to do so.

PJR, April 2023


Kamal, M.*, Tokmakjian, L.*, Knox, J.J.*, Mastrangelo, P., Ji, J., Cai, H., Wojciechowski, J.W., Hughes, M.P., Takács, K., Chu, X., Pei, J., Grolmusz, V., Kotulska, M., Forman-Kay, J.D., and Roy, P.J. 2022. A Spatiotemporal Reconstruction of the C. elegans Pharyngeal Cuticle Reveals a Structure Rich in Phase-Separating Proteins. ELife. Oct 19;11:e79396. doi: 10.7554/eLife.79396. Read the paper here.


Roy, PJ. 2022. Temporal Regulation of Gene Expression in Post-Mitotic Cells is Revealed from a Synchronized Population of C. elegans Larvae. MicroPubl Biol. Jun 10;2022:10.17912/micropub.biology.000587. Read the paper here.


Kamal, M., Moshiri, H., Magomedova, L., Han, D., Nguyen, K.C.Q., Yeo, M., Knox, J., Bagg, R., Won, A.M., Szlapa, K., Yip, C., Cummins, C.L., Hall, D.H., and Roy, P.J. 2019. The Marginal Cells of the Caenorhabditis elegans Pharynx Scavenge Cholesterol and Other Hydrophobic Small Molecules. Nature Communications. 10, 1-16. Read the paper here.


Kamal, M., Tokmakjian, L., Knox, J., Han, D., Magomedova, L., Moshiri, H., Nguyen, K.C.Q., Hall, D.H., Cummins, C.L., and Roy, P.J. The ABCB4 Homolog PGP-14 Establishes a Lipid Permeability Barrier within the C. elegans Pharyngeal Cuticle. (In review).


Kamal, M.*, Knox, J.J.*, Han, D., Burns, A.R., Nguyen, K.C.Q., Hall, D.H., and Roy, P.J. Amyloidophilic Small Molecules Disrupt the Pharyngeal Cuticle of the Nematode Caenorhabditis elegans. Manuscript in preparation. Read the pre-print here.

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The Search for a Novel Guidance Cue (2005-2016)

Origin Story: When I started my lab in 2002, I wanted to continue my search for novel components required for guided cell and axon migration. I knew that nematodes have an unusual architecture; their body wall muscles extend membrane to the axonal tracks along the dorsal and ventral nerve cords. These muscle membrane extensions are called muscle arms. Ed Hedgecock and colleagues previously demonstrated that in mutants with misguided axons, the muscle arms still extend to the motor axons that are situated in errant lateral positions. This was compelling evidence that indicated that the motor axons are likely secreting a cue that interacts with receptors on the extending muscle arms.

     In 2002, I started our ‘muscle arm’ project to identify the cue and downstream signalling elements that guide muscle arms, thinking that these components might be employed in other animals to guide migrations. Using genetics, we hit upon several new and highly conserved gene products that are key in guiding muscle arms to their targets. We ultimately identified the secreted guidance cue, which we called MADD-4, in 2011. It is a conserved protein with immunoglobulin and thrombospondin repeats and is orthologous to human ADAMTSL1 and ADAMTSL3, both of which are implicated in human disease, including microcephaly (which, in an amazing coincidence, was the cause of death of my infant brother). Whether or not the ADAMTSLs guide migrations in humans remains an open question.

     A second amazing finding that came out of this work was MADD-2, which functions downstream of the presumptive UNC-40/EVA-1 co-receptor complex. MADD-2 is not only required for muscle arm guidance towards the ventral and dorsal nerve cords, but is needed for the migrations of many other cell types towards the worm midline (i.e., the dorsal and ventral apexes). The MADD-2 human ortholog is called MID1, which is mutated in a human disease called Opitz Syndrome. Amazingly, kids with Opitz Syndrome have numerous midline defects that are likely the result of neural crest cell migration defects, which is strikingly similar to the mutant phenotypes seen in worms.

     Anyway, below is the list of publications that came from our work. Having accomplished what we set out to do, we concluded this project with our last muscle arm paper published in 2016.

PJR, April 2023


Dixon, S.J., and Roy, P.J. 2005.  Muscle arm development in Caenorhabditis elegansDevelopment. 132, 3079-3092. Read the paper here.


Dixon, S.J., Alexander, M., Ricker, N., Fernandes, R., Roy, P.J. 2006. FGF Signaling Negatively Regulates Muscle Membrane Extension in C. elegans. Development.133, 1263-1275. Read the paper here.


Dixon, S.J.*, Alexander, M.*, Chan, K.K., Roy, P.J. 2008. Insulin-like Signaling Negatively Regulates Muscle Arm Extension through DAF-12 in Caenorhabditis elegans. Developmental Biology, 318, p153-161. Read the paper here.


Alexander, M.*, Chan, K.K.*, Byrne, A.*, Selman, G., Lee, T., Ono, J., Wong, E., Puckrin, R., Dixon, S.J., and Roy, P.J.  2009. An UNC-40 Pathway that Directs Postsynaptic Membrane Extension in Caenorhabditis elegans. Development, 136, 911-922. Read the paper here.


Alexander, M., Selman, G., Seetharaman, A., Chan, K.K., D’Souza, S.A., Byrne, A.B., and Roy, P.J. 2010. MADD-2, a Homologue of the Opitz Syndrome Protein MID1, Regulates Guidance to the Midline through UNC-40 in Caenorhabditis elegans. Developmental Cell, 18, p961–972. Read the paper here.


Seetharaman, A., Selman, G., Puckrin, R., Barbier, L., Wong, E., D’Souza, S.A., and Roy, P.J.  2011. MADD-4 is a Secreted Cue Required for Midline-Oriented Guidance in Caenorhabditis elegans. Developmental Cell 21, p669-680.

Read the paper here. 

Morf, M.K., Rimann, I, Alexander, M., Roy, P.J., and Hajnal, A.  2013. The Caenorhabditis elegans homolog of the Opitz syndrome gene, madd-2/Mid1, regulates anchor cell invasion.  Developmental Biology, 374, 108-114.  Read the paper here.


Chan, K.*, Seetharaman, A.*, Bagg, R., Selman, G., Zhang, Y., Kim, J., and Roy, P.J. 2014. EVA-1 Functions as an UNC-40 Co-Receptor to Enhance Attraction to the MADD-4 Guidance Cue in Caenorhabditis elegansPLoS Genetics 10(8):e1004521. doi: 10.1371/journal.pgen.1004521. Read the paper here.


Chan, K.*, Seetharaman, A.*, Selman, G.*, and Roy, P.J. 2015.  Immunoprecipitation of Proteins in Caenorhabditis elegans. Bioprotocol 5(7). Read the paper here.


D’Souza, S.A., Rajendran, L., Bagg, R., Barbier, L., and Roy, P.J. 2016. The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans. PLoS Genetics. 12(4): e1006010. Read the paper here.

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Stray Dogs

Our collection of papers that do not fit into the categories listed above, as well as our collaborative works.


Loll-Krippleber, R., Sajtovich, V.A., Ferguson, M.W., Ho, B., Burns, A.R., Payliss, B.J., Bellissimo, J., Peters, S., Roy, P.J., Wyatt, H.D.M., and Brown, G.W. 2022. Development of a yeast whole-cell biocatalyst for MHET conversion into terephthalic acid and ethylene glycol. Microbial Cell Factories 21, 280. Read the paper here.


Murareanu, B.M., Antao, N.V., Zhao, W., Dubuffet, A., El Alaoui, H., Knox, J., Ekiert, D.C., Bhabha, G., Roy, P.J., and A. W. Reinke. High-throughput small molecule screen identifies inhibitors of microsporidia invasion and proliferation in C. elegans. Nature Communications 13(1):5653. doi: 10.1038/s41467-022-33400-y. Read the paper here.


Volpatti, J., Endo, Y., Knox, J., Groom, L., Brennan, S., Noche, R., Noche, R., Zuercher, W., Roy, P., Dirksen, R. T., and Dowling, J.J. 2020. Identification of drug modifiers for RYR1 related myopathy using a multi-species discovery pipeline. eLife 2020;9: e57481 DOI: 10.7554/eLife.57481. Read the paper here.


Otten, C., Knox, J., Boulday, G., Eymery, M., Haniszewski, M., Neuenschwander,M., Radetzki, S., Vogt, I, Hähn, T.,  De Luca, C., Cardoso, C., Hamad, S., Gil, C., I., Roy, P., Albiges-Rizo, C., Faurobert, C., von Kries, J.P., Campillos, M., Tournier-Lasserve, E., Derry, W.B., and Abdelilah-Seyfried, S. 2018. Systematic pharmacological screens uncover novel pathways involved in cerebral cavernous malformations. EMBO Mol Medicine, 10(10). pii: e9155. doi: 10.15252/emmm.201809155. Read the paper here.


Tharmalingam, S., Burns, A. R., Roy, P.J., and Hampson, D.R.  2012.  Orthosteric and Allosteric Drug Binding Sites in the C. elegans mgl-2 Metabotropic Glutamate Receptor. Neuropharmacology 63, p667-674. Read the paper here.


Wang, W.§, Sun, Y., Dixon, S., Alexander, M., and Roy, P.  2009. An Automated Micropositioning System for Investigating C. elegans Locomotive Behavior. JALA. 14 (5), p269-276. doi:10.1016/j.jala.2008.12.006. Read the paper here.


Arnoldo, A.,  Curak, J., Kittanakom, S., Chevelev, I., Lee, V.T., Sahebol-Amri, M., Koscik, B., Ljuma, L., Roy, P.J., Bedalov, A., Giaever, G., Nislow, C., Merrill, R. A., Lory, S., and Stagljar, I. 2008. Identification of Small Molecule Inhibitors of Pseudomonas aeruginosa Exoenzyme S Using a Yeast Phenotypic Screen PLoS Genetics, 4(2): e1000005. Read the paper here.


Von Stetina, S. E., Watson, J.D., Fox, R.M., Olszewski, K.L., Roy, P.J., Miller, D.M. III. 2007. Cell-specific microarray profiling experiments reveal a comprehensive picture of gene expression in the C. elegans nervous system. Genome Biology, 8, R135. Read the paper here.


Byrne, A., Weirauch, M., Wong, V., Koeva, M., Dixon, S.J., Stuart, J., and Roy, P.J.  2007. A Global Analysis of Genetic Interactions in Caenorhabditis elegans. Journal of Biology, 6(3):8. Read the paper here.

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