Jonathan S. Marchant

Praziquantel (PZQ) is an essential medicine used to treat schistosomiasis and other infectious diseases caused by parasitic flatworms. The parasite target of PZQ is a transient receptor potential ion channel of the melastatin subfamily, TRPM _PZQ . As little is currently known about the native role of TRPM _PZQ in schistosomes, we developed and validated TRPM _PZQ specific antibodies to immunolocalize TRPM _PZQ across various S. mansoni life cycle stages (adult worms, juvenile worms, miracidia and cercariae). TRPM _PZQ was expressed in muscle, nervous tissues, as well as various sensory structures on the surface of the parasite across all these life cycle stages. In adult male worms, TRPM _PZQ positive neurons innervated the oral and ventral suckers as well as sensory papillae covering the head of the worm. TRPM _PZQ was also present in papillae throughout the body, and within tubercles. In a S. mansoni strain selected in the laboratory for resistance to PZQ, reduced expression and immunostaining of TRPM _PZQ was apparent. In immature worms, which can recover from PZQ exposure, TRPM _PZQ expression was predominantly found within the musculature compared with the neuronal localization evident in adult worms. A developmental transition of TRPM _PZQ from muscle to nervous tissues during schistosome maturation was also supported from single cell RNAseq data. Because muscle buffers Ca ²⁺ transients more effectively than neurons, the transition of TRPM _PZQ away from muscle during schistosome maturation may increase the sensitivity of adult male worms to PZQ.

Schistosomiasis, a neglected tropical disease caused by infection with parasitic blood flukes of the genus Schistosoma , affects more than 250 million people worldwide. Currently, patients rely on a single medicine, praziquantel (PZQ), discovered half a century ago. To combat drug resistance and reach elimination of the schistosome parasite, alternative drugs to PZQ are required that treat and prevent disease. Here, we describe a drug candidate, M5493, with potent activity against multiple parasite life cycle stages. The target is a parasite transient receptor potential ion channel, distinct from the target of PZQ. To circumvent its limited bioavailability, the active agent was converted to prodrug M5493, which displayed robust activity against the schistosome parasite at doses as low as 5 milligrams per kilogram in mice infected with Schistosoma mansoni . Unlike the benzodiazepine meclonazepam, which displays clinical antihelminthic activity through the same target, M5493 showed no sedating side effects given that it did not engage GABA _A receptors. Overall, this compound has the potential to provide single-dose treatment, broad-spectrum efficacy against schistosome parasites at different life cycle stages, and transmission blocking activity.

Ion channels possess selectivity filters that are hardwired to ensure the selective passage of ions. Lysosomal two-pore channels are unusual as they are able to switch their cation selectivity in an agonist-specific manner, allowing differential control of organellar activity. TPC2 is permeable to Ca ²⁺ when activated by the calcium-mobilizing messenger NAADP, but largely Na ⁺ -selective when activated by the signaling lipid PI(3,5)P ₂ . Co-stimulation increases Ca ²⁺ but not Na ⁺ permeability; however, the molecular basis for these specificity switches is not well understood. Here we show that mutation of TPC2 residues within the distal cytosolic linker, which connects the first voltage-sensing-like domain to the pore, rendered TPC2 largely unable to discriminate its agonists and highly calcium-permeable, even in the presence of PI(3,5)P ₂ . This mutation induced a co-activated-like state by disrupting a network of residues that connects the linker to the activation gate. Such deregulated agonist action increased lysosomal Ca ²⁺ flux and compromised locomotion and viability when expressed in C. elegans . A proximal disease-linked mutation perturbed agonist action in a similar way both in vitro and in vivo. Biased signaling through TPC2 thus proceeds through molecular determinants that are remote from the selectivity filter, affecting Ca ²⁺ permeability, endo-lysosomal integrity and disease.

Mass drug administration (MDA) with praziquantel is the cornerstone of schistosomiasis control and elimination efforts. To assess how long-term MDA affects parasite populations, we analyzed whole-genome sequence data from 570 Schistosoma mansoni samples and the closely related Schistosoma rodhaini across eight countries, combining new parasite material with publicly available sequence data. We observed a broad-scale genetic structure between countries alongside evidence of extensive long-distance transmission. Functional profiling of the recently identified transient receptor potential melastatin ion channel, Sm. TRPM _PZQ , revealed four naturally occurring variants associated with reduced praziquantel sensitivity, indicating standing variation for resistance. Analyses of parasite infrapopulations collected from people pre– and post–praziquantel treatment further identified instances of treatment failure, supporting the potential for praziquantel resistance. As schistosomiasis is targeted for elimination as a public health problem, with interruption of transmission in selected regions by 2030, our study provides a comprehensive genomic framework for endemic populations, highlights an approach to detect potential resistance, and endorses the development of precision surveillance and adaptive treatment strategies.

Benzodiazepines represent a major class of drugs prescribed as treatments for anxiety, insomnia and seizures. They act by engaging GABA _A receptors in the central nervous system to depress neuronal excitability. Here, the polypharmacology of benzodiazepines was explored by studying their engagement of human TRP channels revealing clonazepam acts as a robust and selective activator of the TRPM8 ion channel. Clonazepam-evoked Ca ²⁺ signals were observed in cells expressing human TRPM8 channels, and in mouse trigeminal neurons. These responses were completely blocked by pharmacological or genetic inhibition of TRPM8 function. This discovery likely explains why clonazepam is an effective treatment for the painful oral condition known as burning mouth disorder, where local activation of TRPM8 channels in sensory neurons mitigates the painful symptoms of this disease.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca ²⁺ -releasing second messenger that activates two-pore channels (TPCs) on endosomes and lysosomes. Rather than binding TPCs directly, NAADP acts through cytoplasmic NAADP-binding proteins (NAADP-BPs) which are essential for endolysosomal Ca ²⁺ release. Here we characterized the properties of two recombinant, purified NAADP-BPs: Jupiter Microtubule Associated Homolog 2 (JPT2) and like-Sm protein 12 (LSM12). In contrast to LSM12, JPT2 is predicted to be an intrinsically disordered protein, a feature confirmed by circular dichroism and NMR spectroscopy. Under conditions of low Na ⁺ concentration or molecular crowding, JPT2 underwent phase separation, as demonstrated by multiple orthogonal approaches. JPT2 condensates displayed liquid-like behavior and efficiently recruited LSM12, a novel fluorescent NAADP analog, as well as tubulin. JPT2 condensates also interacted with polymerized microtubules and lysosomes isolated from human cell lines. These findings reveal an unexpected capability of NAADP-BPs to undergo phase separation, and segregate with components needed for NAADP-dependent Ca ²⁺ release. We speculate that these signaling condensates dictate cellular NAADP sensitivity, desensitization of NAADP responses, as well as NAADP targeting to TPCs at membrane contact sites between acidic organelles and the endoplasmic reticulum. Graphical

The identification of a transient receptor potential ion channel of the melastatin subfamily activated by praziquantel (TRPM_PZQ) has opened new opportunities for target-based schistosomiasis drug discovery. In this study, eight new 1H-1,2,3-triazole derivatives of praziquantel (PZQ), and their synthetic intermediates, were prepared and evaluated for their schistosomicidal activity on schistosomula, juvenile, and adult Schistosoma mansoni. Their ability to activate schistosome wild-type (WT) and mutant TRPM_PZQ (Sm.TRPM_PZQ), as well as a schistosome TRPM channel activated by meclonazepam (Sm.TRPM_MCLZ), and TRPM_PZQ from Fasciola hepatica (Fh.TRPM_PZQ) and Echinococcus granulosus (Eg.TRPM_PZQ), was also assessed. Initial screening of schistosomula identified six compounds significantly affecting parasite motility/morphology at 25-50 μM. Compounds 3, 4, and 5e were active against juveniles by two orthogonal methods. All compounds impaired adult worm motility, with 4 being the most potent in males (EC₅₀: 1.3-2.3 μM) and 5e being the most potent in females (EC₅₀: 3.1-3.9 μM). Compound 5e showed the highest selectivity indexes (75 for females and 155 for males) when compared with the HepG2 human cell line. Compounds 2, 3, 4, and 5e activated WT (EC₅₀: 0.9-13.5 μM), and mutant Sm.TRPM_PZQ showing a similar activation profile to PZQ. Like PZQ, they did not activate Fh.TRPM_PZQ or Sm.TRPM_MCLZ at the tested concentrations but activated Eg.TRPM_PZQ with similar potencies to Sm.TRPM_PZQ. Molecular modeling studies suggest that the PZQ binding site on Sm.TRPM_PZQ may accommodate extended substituents on position 9 of the pyrazinoisoquinoline ring due to a conformational flexibility of the Y1517 side chain. This feature could be explored to design new PZQ analogues with improved drug metabolism and pharmacokinetic properties.