Multivariate chemogenomic screening prioritizes new macrofilaricidal leads

Abstract

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.

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

“…To explore the gross effects of cholinergic compounds on parasite health, we optimized a number of phenotypic readouts in Brugia adults and microfilariae [67]. Parasites were incubated in muscarinic and nicotinic compounds and the stage-specific effects of these chemical perturbations on worm motility, viability, and fecundity were measured using a customized imaging platform [68].…”

“…To quantify progeny, 50 μL aliquots were transferred to wells of a 96-well plate (Greiner), and each well was imaged with transmitted light at 2X with an ImageXpress Nano (Molecular Devices). Images of progeny were segmented as previously described [67], and segmented pixels were counted to infer output of progeny using a previously generated model [67].…”

“…Brugia malayi microfilariae motility and cell toxicity assays were performed as described [67,110]. Briefly, mf are purified using a PD-10 Desalting Column (VWR, cat# 95017-001) into culture media and titered to a concentration of 10 mf/μL.…”

“…Multivariate phenotyping of adult parasites was performed as described [67,109]. After receipt from the FR3, adult male and female B. pahangi parasites were manually sorted into 24-well plates filled with 750 µL of complete media (RPMI 1640 + 10% FBS + pen/strep) per well.…”

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

“…Multivariate phenotyping of Brugia spp. microfilariae and adults (including motility, viability, and fecundity) and Caenorhabditis elegans feeding and development assays are described elsewhere [16,17]. Schistosoma mansoni motility and fecundity assays are described below.…”