Benzodiazepines represent a major class of drugs prescribed as treatments for anxiety, insomnia and seizures. They act by engaging GABAA 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 Ca2+ 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.

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.TRPMPZQ, 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.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2+-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 Ca2+ 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 Ca2+ 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.

The ionotropic portfolio of parasitic flatworms affords considerable opportunity for development of new anthelmintics. In this regard, transient receptor potential ion channels (TRP channels), cation channels responsive to various physiochemical cues, have emerged as promising druggable targets. This is based on the recent discovery that two members of a TRP channel subfamily (TRP melastatin or TRPM channels) are selectively activated by the clinical drug praziquantel (TRPMPZQ), or the anthelmintic benzodiazepine meclonazepam (TRPMMCLZ). Here, the efficacy of meclonazepam was investigated in a trematode (the liver fluke, Fasciola hepatica) and a cestode (Echinococcus multilocularis) model, in which an observed lack of meclonazepam sensitivity correlated with the lack of efficacy of meclonazepam on TRPMMCLZ in these different parasites. Such correlations support assignment of TRPMMCLZ as the meclonazepam target. Bioinformatic analysis of all available parasitic flatworm genomes allowed prediction of the meclonazepam binding pocket in over sixty different TRPMMCLZ orthologs. Mutagenesis and functional profiling analyses highlighted the importance of a key residue in the S4 transmembrane helix of TRPMMCLZ that impacts meclonazepam potency and efficacy. Variation of this residue and overall binding pocket architecture between different parasitic flatworm TRPMMCLZ orthologs restricts meclonazepam action to a subset of schistosome species.

Benzodiazepines are widely prescribed therapeutics whose actions are primarily attributed to the modulation of GABAA receptors. Here, we identify a previously unrecognized target of a subset of benzodiazepines-the cold- and menthol-sensitive ion channel TRPM8 (transient receptor potential melastatin 8). Clonazepam and related 7-nitrobenzodiazepines act as potent TRPM8 agonists, eliciting calcium influx and membrane depolarization in heterologous expression systems, native human cells, and trigeminal sensory neurons. Pharmacological inhibition, genetic knockdown, and gene deletion of TRPM8 abolish clonazepam-evoked responses, establishing TRPM8 as the relevant molecular target. Mechanistically, clonazepam shares pharmacological features with icilin, a canonical TRPM8 agonist, and engages conserved determinants of channel activation. These findings expand the pharmacology of benzodiazepines beyond GABAA receptors and suggest that TRPM8 activation may contribute to the clinical efficacy of clonazepam in burning mouth disorder and other sensory neuropathic conditions.

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 Ca2+ when activated by the calcium-mobilizing messenger NAADP, but largely Na+-selective when activated by the signaling lipid PI(3,5)P2. Co-stimulation increases Ca2+ 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)P2. 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 Ca2+ 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 Ca2+ permeability, endo-lysosomal integrity and disease. The selectivity of lysosomal TPC2 ion channels for either Ca²⁺ or Na⁺ depends on which agonists activate the channel. This article shows that this plasticity is governed by a distal allosteric network that allows TPC2 to discriminate between agonists and generate specific lysosomal ion fluxes. A distal allosteric network controls agonist-specific ion selectivity.

The anthelmintic praziquantel (PZQ) has been used for decades as the clinical therapy for schistosomiasis, and remains the only available drug. As a cheap and effective drug therapy for all human disease-causing Schistosoma species, usage of PZQ underpins mass drug administration strategies aimed at eliminating schistosomiasis as a public health problem by 2030. Concern over the potential emergence of resistance to PZQ is therefore warranted, as it would constitute a major threat to this approach. In terms of molecular adaptations conferring PZQ resistance, variation in the sequence and/or expression of the drug target is an obvious mechanism and should be a priority for surveillance efforts. The target of PZQ is a transient receptor potential ion channel, TRPMPZQ, which is established as a locus that regulates schistosome sensitivity to PZQ. Here, we describe the establishment of a community resource, ‘TRPtracker’, which coalesces data on TRPMPZQ natural variants together with measurements of individual TRPMPZQ variant sensitivity to PZQ assessed by profiling TRPMPZQ in a heterologous expression system. A compendium of laboratory-generated mutants in TRPMPZQ is also compiled in the TRPtracker database to delimit regions within TRPMPZQ that are critical for PZQ sensitivity. Aggregation of data from multiple research groups into TRPtracker catalogues which TRPMPZQ variants have been functionally profiled, where geographically these variants have been found, their frequency within populations, and their potential impact on PZQ sensitivity. The overall goal is to facilitate rapid community-wide exchange of data to monitor predicted variants of concern that are likely to be associated with decreased PZQ efficacy.

The identification of a transient receptor potential ion channel of the melastatin subfamily activated by praziquantel (TRPMPZQ) 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 TRPMPZQ (Sm.TRPMPZQ), as well as a schistosome TRPM channel activated by meclonazepam (Sm.TRPMMCLZ), and TRPMPZQ from Fasciola hepatica (Fh.TRPMPZQ) and Echinococcus granulosus (Eg.TRPMPZQ), 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.TRPMPZQ showing a similar activation profile to PZQ. Like PZQ, they did not activate Fh.TRPMPZQ or Sm.TRPMMCLZ at the tested concentrations but activated Eg.TRPMPZQ with similar potencies to Sm.TRPMPZQ. Molecular modeling studies suggest that the PZQ binding site on Sm.TRPMPZQ 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.

This chapter describes work that led to the identification of the target of praziquantel (PZQ) in parasitic flatworms. The molecular target of this drug is an ion channel of the transient receptor potential family, named TRPMPZQ. TRPMPZQ is activated by PZQ to cause a rapid depolarization of excitable cells in schistosomes, and the properties of this recently discovered ion channel align well with all known facets of PZQ action. The discovery of this elusive target has rapidly enabled progress to illuminate how PZQ works, understand scenarios in which PZQ is poorly effective, and unlock a new phase of target-based discovery to realize new anthelmintic chemotypes and useful tools for probing parasitic flatworm biology.

The drug praziquantel (PZQ) has been used for decades to treat clinical and veterinary infections caused by parasitic flatworms. Although PZQ is efficacious against many different types of flukes and tapeworms, PZQ activity is lower against certain types of parasites, including pseudophyllidean cestodes. The target of PZQ is a parasitic flatworm transient receptor potential ion channel (TRPMPZQ), and interrogation of this target affords the opportunity to understand why PZQ efficacy varies between different parasites and how target-based design strategies could help deliver new analogs with improved efficacy against currently hard-to-treat diseases. In this study, we consider natural amino acid variation within cestode TRPMPZQ binding pockets to design thioamide derivatives of PZQ with greater efficacy at pseudophyllidean cestode TRPMPZQ. Target-based design across parasite TRPMPZQ orthologues, as well as at other TRPM paralogues in this ion channel family, provides an opportunity to expand and improve on the current anthelmintic toolbox.

Diseases caused by parasitic flatworms impart a considerable healthcare burden worldwide. Many of these diseases – for example, the parasitic blood fluke infection, schistosomiasis – are treated with the drug praziquantel (PZQ). However, PZQ is ineffective against disease caused by liver flukes from the genus Fasciola . This is due to a single amino acid change within the target of PZQ, a transient receptor potential ion channel (TRPM PZQ ), in Fasciola species. Here we identify benzamidoquinazolinone analogs that are active against Fasciola TRPM PZQ . Structure-activity studies define an optimized ligand (BZQ) that caused protracted paralysis and damage to the protective tegument of these liver flukes. BZQ also retained activity against Schistosoma mansoni comparable to PZQ and was active against TRPM PZQ orthologs in all profiled species of parasitic fluke. This broad spectrum activity was manifest as BZQ adopts a pose within the binding pocket of TRPM PZQ dependent on a ubiquitously conserved residue. BZQ therefore acts as a universal activator of trematode TRPM PZQ and a first-in-class, broad spectrum flukicide.

Parasitic fl atworms cause various clinical and veterinary infections that impart a huge burden worldwide. The most clinically impactful infection is schistosomiasis, a neglected tropical disease caused by parasitic blood fl ukes. Schistosomiasis is treated with praziquantel (PZQ), an old drug introduced over 40 years ago. New drugs are urgently needed, as while PZQ is broadly effective it suffers from several limitations including poor efficacy against juvenile worms, which may prevent it from being completely curative. An old compound that retains efficacy against juvenile worms is the benzodiazepine meclonazepam (MCLZ). However, host side effects caused by benzodiazepines preclude development of MCLZ as a drug and MCLZ lacks an identified parasite target to catalyze rational drug design for engineering out human host activity. Here, we identify a transient receptor potential ion channel of the melastatin subfamily, named TRPMMCLZ, as a parasite target of MCLZ. MCLZ potently activates Schistosoma mansoni TRPMMCLZ through engagement of a binding pocket within the voltage-sensor-like domain of the ion channel to cause worm paralysis, tissue depolarization, and surface damage. TRPMMCLZ reproduces all known features of MCLZ action on schistosomes, including a lower activity versus Schistosoma japonicum, which is explained by a polymorphism within this voltage-sensor-like domain-binding pocket. TRPMMCLZ is distinct from the TRP channel targeted by PZQ (TRPMPZQ), with both anthelmintic chemotypes targeting unique parasite TRPM paralogs. This advances TRPMMCLZ as a novel drug- gable target that could circumvent any target-based resistance emerging in response to current mass drug administration campaigns centered on PZQ.

Calcium ions (Ca2+) play a vital role as intracellular messengers, regulating essential cellular processes. Nicotinic acid adenine dinucleotide phosphate (NAADP) serves as a potent second messenger, responsible for releasing Ca2+ in both mammals and echinoderms. Despite identification of two human NAADP receptor proteins, their counterparts in sea urchins remain elusive. Sea urchin NAADP binding proteins are important due to their unique identities and NAADP binding properties which may illuminate new signaling modalities in other species. Consequently, the development of new photoactive and clickable NAADP analogs with specificity for binding targets in sea urchin egg homogenates is a priority. We designed and synthesized diazirine-AIOC-NAADP, a photoactive and "clickable" NAADP analog, to specifically label and identify sea urchin NAADP receptors. This analog, synthesized using a chemo-enzymatic approach, induced Ca2+ release from sea urchin egg homogenates at low-micromolar concentrations. The ability of diazirine-AIOC-NAADP to mobilize Ca2+ in cultured human cells was investigated by microinjection of the probe into U2OS cells. Microinjected NAADP elicited a robust Ca2+ release, but even 6000-fold higher concentrations of diazirine-AIOC-NAADP were unable to release Ca2+. Our results indicate that our new probe is specifically recognized at low concentration by sea urchin egg NAADP receptors but not by the NAADP receptors in a human cultured cell line.

Infections caused by parasitic flatworms impart a significant disease burden. This is well exemplified by the neglected tropical disease schistosomiasis, which afflicts millions of people worldwide. The anti-schistosomal activity of various chemotypes has been known for decades, but the parasite targets of many of these remain undefined. Until recently, this included the current clinical therapy, praziquantel (PZQ). However, the tempo of target discovery has recently gathered pace, with discoveries of schistosome targets for praziquantel (PZQ) and the anthelmintic benzodiazepine, meclonazepam (MCLZ). This steady patter of target illumination has also revealed a pattern in that both PZQ and MCLZ target members of the same ion channel subgroup—transient receptor potential ion channels of the melastatin family (TRPM channels). PZQ activates one member of this family (TRPMPZQ) and MCLZ activates a different channel (TRPMMCLZ). Here, similarities and differences between these two new targets are discussed. These data highlight the need for further study of TRPM channels in parasitic flatworms given their vulnerability to chemotherapeutic attack.

The drug praziquantel (PZQ) has served as the long-standing drug therapy for treatment of infections caused by parasitic flatworms. These encompass diseases caused by parasitic blood, lung, and liver flukes, as well as various tapeworm infections. Despite a history of clinical usage spanning over 4 decades, the parasite target of PZQ has long resisted identification. However, a flatworm transient receptor potential ion channel from the melastatin subfamily (TRPMPZQ) was recently identified as a target for PZQ action. Here, recent experimental progress interrogating TRPMPZQ is evaluated, encompassing biochemical, pharmacological, genetic, and comparative phylogenetic data that highlight the properties of this ion channel. Various lines of evidence that support TRPMPZQ being the therapeutic target of PZQ are presented, together with additional priorities for further research into the mechanism of action of this important clinical drug.

Mass-drug administration (MDA) of human populations using praziquantel monotherapy has become the primary strategy for controlling and potentially eliminating the major neglected tropical disease schistosomiasis. To understand how long-term MDA impacts schistosome populations, we analysed whole-genome sequence data of 570 Schistosoma mansoni samples (and the closely related outgroup species, S. rodhaini) from eight countries incorporating both publicly-available sequence data and new parasite material. This revealed broad-scale genetic structure across countries but with extensive transmission over hundreds of kilometres. We characterised variation across the transient receptor potential melastatin ion channel, TRPMPZQ, a target of praziquantel, which has recently been found to influence praziquantel susceptibility. Functional profiling of TRPMPZQ variants found in endemic populations identified four mutations that reduced channel sensitivity to praziquantel, indicating standing variation for resistance. Analysis of parasite infrapopulations sampled from individuals pre- and post-treatment identified instances of treatment failure, further indicative of potential praziquantel resistance. As schistosomiasis is targeted for elimination as a public health problem by 2030 in all currently endemic countries, and even interruption of transmission in selected African regions, we provide an in-depth genomic characterisation of endemic populations and an approach to identify emerging praziquantel resistance alleles.

TPC2 is a pathophysiologically relevant lysosomal ion channel that is activated directly by the phosphoinositide PI(3,5)P2 and indirectly by the calcium ion (Ca2+)-mobilizing molecule NAADP through accessory proteins that associate with the channel. TPC2 toggles between PI(3,5)P2-induced, sodium ion (Na+)-selective and NAADP-induced, Ca2+-permeable states in response to these cues. To address the molecular basis of polymodal gating and ion-selectivity switching, we investigated the mechanism by which NAADP and its synthetic functional agonist, TPC2-A1-N, induced Ca2+ release through TPC2 in human cells. Whereas NAADP required the NAADP-binding proteins JPT2 and LSM12 to evoke endogenous calcium ion signals, TPC2-A1-N did not. Residues in TPC2 that bind to PI(3,5)P2 were required for channel activation by NAADP but not for activation by TPC2-A1-N. The cryptic voltage-sensing region of TPC2 was required for the actions of TPC2-A1-N and PI(3,5)P2 but not for those of NAADP. These data mechanistically distinguish natural and synthetic agonist action at TPC2 despite convergent effects on Ca2+ permeability and delineate a route for pharmacologically correcting impaired NAADP-evoked Ca2+ signals.