wrmXpress: A modular package for high-throughput image analysis of parasitic and free-living worms

Ryan

Gallo

et al. 2022

Preprint

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Development of direct acting macrofilaricides for the treatment of human filariases is hampered by limitations in screening throughput imposed by the parasite life cycle. Efforts to circumvent arduous screening of adult filariae include drug repurposing and high-throughput screens that target commensal bacteria. In vitro adult screens typically assess single phenotypes without prior enrichment for chemicals with antifilarial potential. We developed a multivariate screen that identified dozens of compounds with submicromolar macrofilaricidal activity, achieving a hit rate of >50% by leveraging abundantly accessible microfilariae. Adult assays were multiplexed to thoroughly characterize compound activity across relevant parasite fitness traits, including neuromuscular control, fecundity, metabolism, and viability. 17 compounds from a diverse chemogenomic library elicited strong effects on at least one adult trait, with differential potency against microfilariae and adults. Stage-specific drug effects may be crucial to limiting adverse events in endemic regions, and our screen identified five compounds with high potency against adults but low potency or slow-acting microfilaricidal effects, at least one of which acts through a novel mechanism. We show that the use of microfilariae in a primary screen outperforms model nematode developmental assays and virtual screening of protein structures inferred with deep-learning. These data provide new leads for drug development, and the high-content and multiplex assays set a new foundation for antifilarial discovery.

Section: Screening Of Brugia Malayi Microfilariaementioning

confidence: 99%

“…Images were analyzed with the worm size module of wrmXpress 53 . Segmented worms were computationally straightened, and the length of each worm was used to measure development.…”

Section: Screening Of C Elegansmentioning

confidence: 99%

Multivariate chemogenomic screening prioritizes new macrofilaricidal leads

Ryan

Gallo

et al. 2022

Preprint

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“…Plates are washed three times and worms are paralyzed and straightened with 50 mM sodium azide before images are acquired using an high-content imaging system. Fluorescence is quantified using a wrmXpress [68] pipeline.…”

Section: Establishing a High Throughput Image-based Feeding Assay For...mentioning

confidence: 99%

“…Three biological replicates from separate batches of parasite infection cohorts were assayed with four worms per treatment. Videos were analyzed with the optical flow (motility) module of wrmXpress [68]. Conditioned media from female worms from the initial overnight incubation in complete media and 24/48 hr treatment plates were transferred to 1.5 mL tubes and centrifuged for 5 min at 10,000 x g to pellet progeny.…”

Section: Adult Parasite Assaysmentioning

confidence: 99%

Pharmacological profiling of a Brugia malayi muscarinic acetylcholine receptor as a putative antiparasitic target

Gallo

Elmi

et al. 2022

Preprint

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The diversification of anthelmintic targets and mechanisms of action will help ensure the sustainable control of nematode infections in response to the growing threat of drug resistance. G protein-coupled receptors (GPCRs) are established drug targets in human medicine but remain unexploited as anthelmintic substrates despite their important roles in nematode neuromuscular and physiological processes. Bottlenecks in exploring the druggability of parasitic nematode GPCRs include a limited helminth genetic toolkit and difficulties establishing functional heterologous expression. In an effort to address some of these challenges, we profile the function and pharmacology of muscarinic acetylcholine receptors in the human parasite Brugia malayi, an etiological agent of human lymphatic filariasis. While acetylcholine-gated ion channels are intensely studied as targets of existing anthelmintics, comparatively little is known about metabotropic receptor contributions to parasite cholinergic signaling. Using multivariate phenotypic assays in microfilariae and adults, we show that nicotinic and muscarinic compounds disparately affect parasite fitness traits. We identify a putative G protein-linked acetylcholine receptor (Bma-GAR-3) that is highly expressed across intra-mammalian life stages and adapt spatial RNA in situ hybridization to map receptor transcripts to critical parasite tissues. Tissue-specific expression of Bma-gar-3 in Caenorhabditis elegans (body wall muscle, sensory neurons, and pharynx) enabled receptor deorphanization and pharmacological profiling in a nematode physiological context. Lastly, we developed an image-based feeding assay as a reporter of pharyngeal activity to facilitate GPCR screening in parasitized strains. We expect that these receptor characterization approaches and improved knowledge of GARs as putative drug targets will further advance the study of GPCR biology across medically important nematodes.

“…Worm movement is often used as an initial screening outcome, reasoning that dead worms will not exhibit movement. Past methods of quantitatively analyzing adult schistosome movement were manual and low throughput, but methods using optical flow algorithms have been developed for automated screening of parasite movement (30)(31)(32). These approaches allow for quantitative readouts not biased by subjective scoring, but on their own do not necessarily provide a readout of worm viability since paralyzed worms may still be alive and recover after drug treatment.…”

Section: In Vitro Measurement Of Drug Activity On Adult Schistosomesmentioning

confidence: 99%

Phenotypic profiling of macrocyclic lactones on parasitic Schistosoma flatworms

Ryan

Kamara

et al. 2022

Preprint

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Macrocyclic lactones are front-line therapies for parasitic roundworm infections, but there are no comprehensive studies on the activity of this drug class against parasitic flatworms. Ivermectin is well known to be inactive against flatworms. However, the structure-activity relationship of macrocyclic lactones may vary across phyla, and it is entirely possible other members of this drug class do in fact show antiparasitic activity on flatworms. For example, there are several reports hinting at the anti-schistosomal activity of doramectin and moxidectin. To explore this class further, we developed an automated imaging assay combined with measurement of lactate levels from worm media. This assay was applied to the screening of 21 macrocyclic lactones (avermectins, milbemycins and others such as spinosyns) against adult schistosomes. These in vitro assays identified several macrocyclic lactones (emamectin, milbemycin oxime, and the moxidectin metabolite 23-ketonemadectin) that caused contractile paralysis and lack of lactate production. Several of these were also active against miracidia, a juvenile life cycle stage of the parasite. Hits prioritized from these in vitro assays were administered to mice harboring patent schistosome infections. However, no reduction in worm burden was observed. Nevertheless, these data show the utility of a multiplexed in vitro screening platform to quantitatively assess drug action and prioritize hits in a chemical series for in vivo studies. While the prototypical macrocyclic lactone ivermectin displays minimal activity against adult Schistosoma mansoni, this family of compounds does contain schistocidal compounds which may serve as a starting point for development of new anti-flatworm chemotherapies.