Quinoxaline-Based Anti-Schistosomal Compounds Have Potent Anti-Malarial Activity.

The human pathogens Plasmodium and Schistosoma are each responsible for over 200 million infections annually, being particularly problematic in low- and middle-income countries. There is a pressing need for new drug targets for these diseases, driven by emergence of drug-resistance in Plasmodium and the overall dearth of new drug targets for Schistosoma. Here, we explored the opportunity for pathogen-hopping by evaluating a series of quinoxaline-based anti-schistosomal compounds for activity against P. falciparum. We identified compounds with low nanomolar potency against 3D7 and multidrug-resistant strains. Evolution of resistance using a mutator P. falciparum line revealed a low propensity for resistance. Only one of the series, compound 22, yielded resistance mutations, including point mutations in a non-essential putative hydrolase pfqrp1, as well as copy-number amplification of a phospholipid-translocating ATPase, pfatp2, a potential target. Notably, independently generated CRISPR-edited mutants in pfqrp1 also showed resistance to compound 22 and a related analogue. Moreover, previous lines with pfatp2 copy-number variations were similarly less susceptible to challenge with the new compounds. Finally, we examined whether the predicted hydrolase activity of PfQRP1 underlies its mechanism of resistance, showing that both mutation of the putative catalytic triad and a more severe loss of function mutation elicited resistance. Collectively, we describe a compound series with potent activity against two important pathogens and their potential target in P. falciparum.

Small Molecule Ligands of the BET-like Bromodomain, SmBRD3, Affect Schistosoma mansoni Survival, Oviposition, and Development.

Schistosomiasis is a disease affecting >200 million people worldwide, but its treatment relies on a single agent, praziquantel. To investigate new avenues for schistosomiasis control, we have conducted the first systematic analysis of bromodomain-containing proteins (BCPs) in a causative species, Schistosoma mansoni. Having identified 29 putative bromodomains (BRDs) in 22 S. mansoni proteins, we selected SmBRD3, a tandem BRD-containing BCP that shows high similarity to the human bromodomain and extra terminal domain (BET) family, for further studies. Screening 697 small molecules identified the human BET BRD inhibitor I-BET726 as a ligand for SmBRD3. An X-ray crystal structure of I-BET726 bound to the second BRD of SmBRD3 [SmBRD3(2)] enabled rational design of a quinoline-based ligand (15) with an ITC Kd = 364 ± 26.3 nM for SmBRD3(2). The ethyl ester pro-drug of compound 15 (compound 22) shows substantial effects on sexually immature larval schistosomula, sexually mature adult worms, and snail-infective miracidia in ex vivo assays.

Chemical modulation of Schistosoma mansoni lysine specific demethylase 1 (SmLSD1) induces wide-scale biological and epigenomic changes.

Background: Schistosoma mansoni, a parasitic worm species responsible for the neglected tropical disease schistosomiasis, undergoes strict developmental regulation of gene expression that is carefully controlled by both genetic and epigenetic processes. As inhibition of S. mansoni epigenetic machinery components impairs key transitions throughout the parasite's digenetic lifecycle, a greater understanding of how epi-drugs affect molecular processes in schistosomes could lead to the development of new anthelmintics. Methods:   In vitro whole organism assays were used to assess the anti-schistosomal activity of 39 Homo sapiens Lysine Specific Demethylase 1 (HsLSD1) inhibitors on different parasite life cycle stages. Moreover, tissue-specific stains and genomic analysis shed light on the effect of these small molecules on the parasite biology. Results: Amongst this collection of small molecules, compound 33 was the most potent in reducing ex vivo viabilities of schistosomula, juveniles, miracidia and adults. At its sub-lethal concentration to adults (3.13 µM), compound 33 also significantly impacted oviposition, ovarian as well as vitellarian architecture and gonadal/neoblast stem cell proliferation. ATAC-seq analysis of adults demonstrated that compound 33 significantly affected chromatin structure (intragenic regions > intergenic regions), especially in genes differentially expressed in cell populations (e.g., germinal stem cells, hes2 + stem cell progeny, S1 cells and late female germinal cells) associated with these ex vivo phenotypes. KEGG analyses further highlighted that chromatin structure of genes associated with sugar metabolism as well as TGF-beta and Wnt signalling were also significantly perturbed by compound 33 treatment. Conclusions: This work confirms the importance of histone methylation in S. mansoni lifecycle transitions, suggesting that evaluation of LSD1 - targeting epi-drugs may facilitate the search for next-generation anti-schistosomal drugs. The ability of compound 33 to modulate chromatin structure as well as inhibit parasite survival, oviposition and stem cell proliferation warrants further investigations of this compound and its epigenetic target SmLSD1.

Modified Hederagenin Derivatives Demonstrate Ex Vivo Anthelmintic Activity against Fasciola hepatica.

Infection with Fasciola hepatica (liver fluke) causes fasciolosis (or fascioliasis) and poses a considerable economic as well as welfare burden to both the agricultural and animal health sectors. Here, we explore the ex vivo anthelmintic potential of synthetic derivatives of hederagenin, isolated in bulk from Hedera helix. Thirty-six compounds were initially screened against F. hepatica newly excysted juveniles (NEJs) of the Italian strain. Eleven of these compounds were active against NEJs and were selected for further study, using adult F. hepatica derived from a local abattoir (provenance unknown). From these eleven compounds, six demonstrated activity and were further assessed against immature liver flukes of the Italian strain. Subsequently, the most active compounds (n = 5) were further evaluated in ex vivo dose response experiments against adult Italian strain liver flukes. Overall, MC042 was identified as the most active molecule and the EC50 obtained from immature and adult liver fluke assays (at 24 h post co-culture) are estimated as 1.07 µM and 13.02 µM, respectively. When compared to the in vitro cytotoxicity of MDBK bovine cell line, MC042 demonstrated the highest anthelmintic selectivity (44.37 for immature and 3.64 for adult flukes). These data indicate that modified hederagenins display properties suitable for further investigations as candidate flukicides.

Using ChEMBL to Complement Schistosome Drug Discovery.

Schistosomiasis is one of the most important neglected tropical diseases. Until an effective vaccine is registered for use, the cornerstone of schistosomiasis control remains chemotherapy with praziquantel. The sustainability of this strategy is at substantial risk due to the possibility of praziquantel insensitive/resistant schistosomes developing. Considerable time and effort could be saved in the schistosome drug discovery pipeline if available functional genomics, bioinformatics, cheminformatics and phenotypic resources are systematically leveraged. Our approach, described here, outlines how schistosome-specific resources/methodologies, coupled to the open-access drug discovery database ChEMBL, can be cooperatively used to accelerate early-stage, schistosome drug discovery efforts. Our process identified seven compounds (fimepinostat, trichostatin A, NVP-BEP800, luminespib, epoxomicin, CGP60474 and staurosporine) with ex vivo anti-schistosomula potencies in the sub-micromolar range. Three of those compounds (epoxomicin, CGP60474 and staurosporine) also demonstrated potent and fast-acting ex vivo effects on adult schistosomes and completely inhibited egg production. ChEMBL toxicity data were also leveraged to provide further support for progressing CGP60474 (as well as luminespib and TAE684) as a novel anti-schistosomal compound. As very few compounds are currently at the advanced stages of the anti-schistosomal pipeline, our approaches highlight a strategy by which new chemical matter can be identified and quickly progressed through preclinical development.

Perspective on Schistosomiasis Drug Discovery: Highlights from a Schistosomiasis Drug Discovery Workshop at Wellcome Collection, London, September 2022.

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.

Flukicidal effects of abietane diterpenoid derived analogues against the food borne pathogen Fasciola hepatica.

Control of liver fluke infections remains a significant challenge in the livestock sector due to widespread distribution of drug resistant parasite populations. In particular, increasing prevalence and economic losses due to infection with Fasciola hepatica is a direct result of drug resistance to the gold standard flukicide, triclabendazole. Sustainable control of this significant zoonotic pathogen, therefore, urgently requires the identification of new anthelmintics. Plants represent a source of molecules with potential flukicidal effects and, amongst their secondary metabolites, the diterpenoid abietic acids can be isolated in large quantities. In this study, nineteen (19) chemically modified abietic acid analogues (MC_X) were first evaluated for their anthelmintic activities against F. hepatica newly excysted juveniles (NEJs, from the laboratory-derived Italian strain); from this, 6 analogues were secondly evaluated for their anthelmintic activities against adult wild strain flukes. One analogue, MC010, was progressed further against 8-week immature- and 12-week mature Italian strain flukes. Here, MC010 demonstrated moderate activity against both of these intra-mammalian fluke stages (with an adult fluke EC50 = 12.97 µM at 72 h post culture). Overt mammalian cell toxicity of MC010 was inferred from the Madin-Darby bovine kidney (MDBK) cell line (CC50 = 17.52 µM at 24 h post culture) and demonstrated that medicinal chemistry improvements are necessary before abietic acid analogues could be considered as potential anthelmintics against liver fluke pathogens.

Identification of anti-schistosomal, anthelmintic and anti-parasitic compounds curated and text-mined from the scientific literature.

More than a billion people are infected with parasitic worms, including nematodes, such as hookworms, and flatworms, such as blood flukes. Few drugs are available to treat worm infections, but high-throughput screening approaches hold promise to identify novel drug candidates. One problem for researchers who find an interesting 'hit' from a high-throughput screen is to identify whether that compound, or a similar compound has previously been published as having anthelmintic or anti-parasitic activity. Here, we present (i) data sets of 2,828 anthelmintic compounds, and 1,269 specific anti-schistosomal compounds, manually curated from scientific papers and books, and (ii) a data set of 24,335 potential anthelmintic and anti-parasitic compounds identified by text-mining PubMed abstracts. We provide their structures in simplified molecular-input line-entry system (SMILES) format so that researchers can easily compare 'hits' from their screens to these anthelmintic compounds and anti-parasitic compounds and find previous literature on them to support/halt their progression in drug discovery pipelines.

Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production.

α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni's α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p<0.05; α-NAGAL > α-GAL), which was consistent with smp_089290's female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in α-GAL activities were observed in the same extracts. Despite this, decreases in α-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds.

Structural Investigations on Novel Non-Nucleoside Inhibitors of Human Norovirus Polymerase.

Human norovirus is the first cause of foodborne disease worldwide, leading to extensive outbreaks of acute gastroenteritis, and causing around 200,000 children to die annually in developing countries. No specific vaccines or antiviral agents are currently available, with therapeutic options limited to supportive care to prevent dehydration. The infection can become severe and lead to life-threatening complications in young children, the elderly and immunocompromised individuals, leading to a clear need for antiviral agents, to be used as treatments and as prophylactic measures in case of outbreaks. Due to the key role played by the viral RNA-dependent RNA polymerase (RdRp) in the virus life cycle, this enzyme is a promising target for antiviral drug discovery. In previous studies, following in silico investigations, we identified different small-molecule inhibitors of this enzyme. In this study, we rationally modified five identified scaffolds, to further explore structure-activity relationships, and to enhance binding to the RdRp. The newly designed compounds were synthesized according to multiple-step synthetic routes and evaluated for their inhibition of the enzyme in vitro. New inhibitors with low micromolar inhibitory activity of the RdRp were identified, which provide a promising basis for further hit-to-lead optimization.