We briefly review cysteine proteases (orthologs of mammalian cathepsins B, L, F, and C) that are expressed in flatworm and nematode parasites. Emphasis is placed on enzyme activities that have been functionally characterized, are associated with the parasite gut, and putatively contribute to degrading host proteins to absorbable nutrients [1–4]. Often, gut proteases are expressed as multigene families, as is the case with Fasciola [5] and Haemonchus [6], presumably expanding the range of substrates that can be degraded, not least during parasite migration through host tissues [5]. The application of the free-living planarian and Caenorhabditis elegans as investigative models for parasite cysteine proteases is discussed. Finally, because of their central nutritive contribution, targeting the component gut proteases with small-molecule chemical inhibitors and understanding their utility as vaccine candidates are active areas of research [7].

In September 2022, the Drug Discovery Unit at the University of Dundee, UK, organised an international meeting at the Wellcome Collection in London to explore the current clinical situation and challenges associated with treating schistosomiasis. The aim of this meeting was to discuss the need for new treatments in view of the clinical situation and to ascertain what the key requirements would be for any potential new anti-schistosomals. This information will be essential to inform ongoing drug discovery efforts for schistosomiasis. We also discussed the potential drug discovery pathway and associated criteria for progressing compounds to the clinic. To date, praziquantel (PZQ) is the only drug available to treat all species causing schistosomiasis, but it is often unable to completely clear parasites from an infected patient, partially due to its inactivity against juvenile worms. PZQ-mediated mass drug administration campaigns conducted in endemic areas (e.g., sub-Saharan Africa, where schistosomiasis is primarily prevalent) have contributed to reducing the burden of disease but will not eliminate the disease as a public health problem. The potential for Schistosoma to develop resistance towards PZQ, as the sole treatment available, could become a concern. Consequently, new anthelmintic medications are urgently needed, and this Perspective aims to capture some of the learnings from our discussions on the key criteria for new treatments.

Schistosoma flatworm parasites cause schistosomiasis, a chronic and debilitating disease of poverty in developing countries. Praziquantel is employed for treatment and disease control. However, its efficacy spectrum is incomplete (less active or inactive against immature stages of the parasite) and there is a concern of drug resistance. Thus, there is a need to identify new drugs and drug targets. We show that RNA interference (RNAi) of the Schistosoma mansoni ortholog of human polo-like kinase (huPLK)1 elicits a deleterious phenotypic alteration in post-infective larvae (schistosomula or somules). Phenotypic screening and analysis of schistosomula and adult S. mansoni with small molecule inhibitors of huPLK1 identified a number of potent anti-schistosomals. Among these was a GlaxoSmithKline (GSK) benzimidazole thiophene inhibitor that has completed Phase I clinical trials for treatment of solid tumor malignancies. We then obtained GSKs Published Kinase Inhibitor Sets (PKIS) 1 and 2, and phenotypically screened an expanded series of 38 benzimidazole thiophene PLK1 inhibitors. Computational analysis of controls and PLK1 inhibitor-treated populations of somules demonstrated a distinctive phenotype distribution. Using principal component analysis (PCA), the phenotypes exhibited by these populations were mapped, visualized and analyzed through projection to a low-dimensional space. The phenotype distribution was found to have a distinct shape and topology, which could be elicited using cluster analysis. A structure-activity relationship (SAR) was identified for the benzimidazole thiophenes that held for both somules and adult parasites. The most potent inhibitors produced marked phenotypic alterations at 1–2 μM within 1 h. Among these were compounds previously characterized as potent inhibitors of huPLK1 in cell assays. The reverse genetic and chemical SAR data support a continued investigation of SmPLK1 as a possible drug target and/or the prosecution of the benzimidazole thiophene chemotype as a source of novel anti-schistosomals. Just one drug is available to treat schistosomiasis, a parasitic disease that affects hundreds of millions of people in developing countries. In the search for new drugs and drug targets, therefore, we have been interested in the schistosome version of human polo-like kinase (huPLK)1, an enzyme with critical functions in cell division. We used RNA interference to knock down messenger RNA for the SmPLK1 –the Schistosoma mansoni parasite’s version of huPLK1. This interference caused disruptive changes in the morphology of the immature ‘somule’ stage of the parasite, indicating that SmPLK1 is an important protein for survival. We then purchased, or acquired from GlaxoSmithKline (GSK), various small chemical inhibitors of huPLK1 and tested these against both the somules and adult parasites in culture. Many of these inhibitors caused severe changes in the parasite and, for somules, the differences could be computationally mapped and distinguished from unexposed parasites. For the GSK inhibitors, we observed ‘somule-adult bioactivity clustering,’ that is, chemicals active against the adults were also active against somules. This suggests that certain chemical attributes in the inhibitors are being favoured. Interestingly, many of the GSK inhibitors most active against the parasite are also known to both potently inhibit huPLK1 and kill cancer cells. Overall, our data suggest that SmPLK1 is a possible drug target and that the GSK chemistries could form the basis for developing a new drug to treat schistosomiasis.

Reliance on just one drug to treat the prevalent tropical disease, schistosomiasis, spurs the search for new drugs and drug targets. Inhibitors of human cyclic nucleotide phosphodiesterases (huPDEs), including PDE4, are under development as novel drugs to treat a range of chronic indications including asthma, chronic obstructive pulmonary disease and Alzheimer’s disease. One class of huPDE4 inhibitors that has yielded marketed drugs is the benzoxaboroles (Anacor Pharmaceuticals). A phenotypic screen involving Schistosoma mansoni and 1,085 benzoxaboroles identified a subset of huPDE4 inhibitors that induced parasite hypermotility and degeneration. To uncover the putative schistosome PDE4 target, we characterized four PDE4 sequences (SmPDE4A-D) in the parasite’s genome and transcriptome, and cloned and recombinantly expressed the catalytic domain of SmPDE4A. Among a set of benzoxaboroles and catechol inhibitors that differentially inhibit huPDE4, a relationship between the inhibition of SmPDE4A, and parasite hypermotility and degeneration, was measured. To validate SmPDE4A as the benzoxaborole molecular target, we first generated Caenorhabditis elegans lines that express a cDNA for smpde4a on a pde4(ce268) mutant (hypermotile) background: the smpde4a transgene restored mutant worm motility to that of the wild type. We then showed that benzoxaborole inhibitors of SmPDE4A that induce hypermotility in the schistosome also elicit a hypermotile response in the C. elegans lines that express the smpde4a transgene, thereby confirming SmPDE4A as the relevant target. The orthogonal chemical, biological and genetic strategies employed identify SmPDE4A’s contribution to parasite motility and degeneration, and its potential as a drug target. Transgenic C. elegans is highlighted as a potential screening tool to optimize small molecule chemistries to flatworm molecular drug targets. Just one drug, praziquantel (PZQ), is available to treat schistosomiasis, a flatworm disease that infects over 240 million people, mainly in Africa. With the expanding distribution of PZQ, and the associated threat of drug resistance, new drugs and drug targets are needed. We screened Schistosoma mansoni worms with over 1,000 benzoxaborole chemical molecules from Anacor Pharmaceuticals to identify a subset of human cyclic nucleotide phosphodiesterase 4 (huPDE4) inhibitors that cause parasite hypermotility and degeneration. We identified four PDE4 genes in the genome of the parasite and recombinantly expressed one of them (SmPDE4A) in bacteria. This enzyme was then used to uncover a relationship between the degree of enzyme inhibition, and the generation of parasite hypermotility and degeneration. To understand whether the SmPDE4A enzyme is the target of the benzoxaboroles in the parasite, we incorporated the coding DNA for SmPDE4A into the model nematode Caenorhabditis elegans that lacked its own functional PDE4 and, as a consequence, was hypermotile. These ‘transgenic’ worms displayed normal motility which could be increased by applying the most potent huPDE4 benzoxaborole inhibitors. In summary, the chemical, biological and genetic approaches taken identify SmPDE4A as a potential drug target for treating schistosomiasis. The potential value of C. elegans as a tool to test and optimize therapeutic chemistries for a flatworm disease is also highlighted.

Schistosomiasis is a chronic and painful disease of poverty caused by the flatworm parasite Schistosoma. Drug discovery for antischistosomal compounds predominantly employs in vitro whole organism (phenotypic) screens against two developmental stages of Schistosoma mansoni, post-infective larvae (somules) and adults. We generated two rule books and associated scoring systems to normalize 3898 phenotypic data points to enable machine learning. The data were used to generate eight Bayesian machine learning models with the Assay Central software according to parasite’s developmental stage and experimental time point (≤24, 48, 72, and >72 h). The models helped predict 56 active and nonactive compounds from commercial compound libraries for testing. When these were screened against S. mansoni in vitro, the prediction accuracy for active and inactives was 61% and 56% for somules and adults, respectively; also, hit rates were 48% and 34%, respectively, far exceeding the typical 1–2% hit rate for traditional high throughput screens.