Identification of Bioaminergic Lead Compounds to Treat Parasitic Blood Flukes (Schistosoma Spp.) through High Throughput Screening Natural Product Libraries

Abstract

Parasitic blood flukes (Schistosoma species of flatworms) infect over 200 million people worldwide, making them the second most costly parasite behind malaria. Despite this, the disease is currently treated with one broad spectrum drug, praziquantel, which has been in use for nearly 40 years. Concerns over reports of resistance in the field and the fact that the drug is ineffective against recently acquired infections highlight the need for research into new therapies to treat this disease. Towards this end, bioaminergic G‐protein coupled receptors (GPCRs) are attractive targets. In addition to the proven druggable track record of GPCRs, cAMP‐coupled bioaminergic signalling pathways control flatworm neuromuscular function and development. However, there are few reports of successful heterologous expression of flatworm GPCRs and no large scale drug screening studies. Therefore, we have recombinantly expressed a Schistosoma serotonin receptor required for parasite movement and developed an assay for high throughput screening of natural product libraries to identify anti‐schistosomal lead compounds.Homologs of a schistosome serotonin receptor were cloned for the three major species of Schistosoma parasites that account for human infections [S. mansoni (Sm.5HTR), S. haematobium (Sh.5HTR) and S. japonicum (Sj.5HTR)]. These receptors were stability expressed in mammalian HEK293 cells alongside a bioluminescent cAMP reporter to permit drug screening in 384 well format (Z′ score = 0.8, 6% coefficient of variation). Natural product libraries (>4000 compounds) were screened against this receptor at a fixed concentration (10μM). Agonist and antagonist primary hits were counter screened to exclude compounds with activity against endogenous mammalian GPCRs present in the HEK293 cell line, or those that compromised cell viability. Several classes of ligands were notable within the validated set of 29 hits ‐ benzylisoquinolines (10 compounds) and indoles (8 compounds). Further exploration of these chemical series identified potent schistosome 5HTR agonists (EC50 < 100nM) and antagonists (pKi ≤ 7). Structure activity relationships highlight key residues required for the efficacy of these pharmacophores at the schistosome receptor. For example, within the ergot‐alkaloid series of compounds, modification of the 2C position of the ergoline ring or saturation of the 9C–10C bond is crucial for agonist/antagonist activity. The most potent compounds were then screened against adult S. mansoni blood flukes; 5HTR agonists caused a dramatic hypermobilty in both male and female worms, while 5HTR antagonists paralyzed worms even in the presence of supra‐maximal concentrations of exogenous serotonin.These data represent the first reported high throughput screen of a flatworm GPCR, providing much needed leads for advancing in the pipeline for new anti‐schistosomal therapies. Additionally, this study evidences a workflow that may be adopted to rapidly screen other bioaminergic schistosome drug targets.Support or Funding InformationSupported by NIH (R21AI25821)