Target-based discovery of a broad-spectrum flukicide.

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 because of a single amino acid change within the target of PZQ, a transient receptor potential ion channel in the melastatin family (TRPMPZQ), in Fasciola species. Here, we identify benzamidoquinazolinone analogs that are active against Fasciola TRPMPZQ. Structure-activity studies define an optimized ligand (BZQ) that caused protracted paralysis and tegumental damage to these liver flukes. BZQ also retained activity against Schistosoma mansoni comparable to PZQ and was active against TRPMPZQ orthologs in all profiled species of parasitic fluke. This broad-spectrum activity manifests as BZQ adopts a pose within the binding pocket of TRPMPZQ that is dependent on a ubiquitously conserved residue. BZQ therefore acts as a universal activator of trematode TRPMPZQ and a first-in-class, broad-spectrum flukicide.

Progress interrogating TRPMPZQ as the target of praziquantel.

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.

The anthelmintic meclonazepam activates a schistosome transient receptor potential channel.

Parasitic flatworms 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 flukes. 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 druggable target that could circumvent any target-based resistance emerging in response to current mass drug administration campaigns centered on PZQ.

Two-pore channel-2 and inositol trisphosphate receptors coordinate Ca2+ signals between lysosomes and the endoplasmic reticulum.

Lysosomes and the endoplasmic reticulum (ER) are Ca2+ stores mobilized by the second messengers NAADP and IP3, respectively. Here, we establish Ca2+ signals between the two sources as fundamental building blocks that couple local release to global changes in Ca2+. Cell-wide Ca2+ signals evoked by activation of endogenous NAADP-sensitive channels on lysosomes comprise both local and global components and exhibit a major dependence on ER Ca2+ despite their lysosomal origin. Knockout of ER IP3 receptor channels delays these signals, whereas expression of lysosomal TPC2 channels accelerates them. High-resolution Ca2+ imaging reveals elementary events upon TPC2 opening and signals coupled to IP3 receptors. Biasing TPC2 activation to a Ca2+-permeable state sensitizes local Ca2+ signals to IP3. This increases the potency of a physiological agonist to evoke global Ca2+ signals and activate a downstream target. Our data provide a conceptual framework to understand how Ca2+ release from physically separated stores is coordinated.

The Anthelmintic Activity of Praziquantel Analogs Correlates with Structure-Activity Relationships at TRPMPZQ Orthologs.

The anthelmintic drug praziquantel remains a key clinical therapy for treating various diseases caused by parasitic flatworms. The parasite target of praziquantel has remained undefined despite longstanding usage in the clinic, although a candidate ion channel target, named TRPMPZQ, has recently been identified. Intriguingly, certain praziquantel derivatives show different activities against different parasites: for example, some praziquantel analogs are considerably more active against cestodes than against schistosomes. Here we interrogate whether the different activities of praziquantel analogs against different parasites are also reflected by unique structure-activity relationships at the TRPMPZQ channels found in these different organisms. To do this, several praziquantel analogs were synthesized and functionally profiled against schistosome and cestode TRPMPZQ channels. Data demonstrate that structure-activity relationships are closely mirrored between parasites and their TRPMPZQ orthologs, providing further support for TRPMPZQ as the therapeutically relevant target of praziquantel.

Target-based discovery of a broad spectrum flukicide.

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 (TRPMPZQ), in Fasciola species. Here we identify benzamidoquinazolinone analogs that are active against Fasciola TRPMPZQ. 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 TRPMPZQ orthologs in all profiled species of parasitic fluke. This broad spectrum activity was manifest as BZQ adopts a pose within the binding pocket of TRPMPZQ dependent on a ubiquitously conserved residue. BZQ therefore acts as a universal activator of trematode TRPMPZQ and a first-in-class, broad spectrum flukicide.

Progesterone receptor membrane component 1 facilitates Ca2+ signal amplification between endosomes and the endoplasmic reticulum.

Membrane contact sites (MCSs) between endosomes and the endoplasmic reticulum (ER) are thought to act as specialized trigger zones for Ca2+ signaling, where local Ca2+ released via endolysosomal ion channels is amplified by ER Ca2+-sensitive Ca2+ channels into global Ca2+ signals. Such amplification is integral to the action of the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, functional regulators of inter-organellar Ca2+ crosstalk between endosomes and the ER remain poorly defined. Here, we identify progesterone receptor membrane component 1 (PGRMC1), an ER transmembrane protein that undergoes a unique heme-dependent dimerization, as an interactor of the endosomal two pore channel, TPC1. NAADP-dependent Ca2+ signals were potentiated by PGRMC1 overexpression through enhanced functional coupling between endosomal and ER Ca2+ stores and inhibited upon PGRMC1 knockdown. Point mutants in PGMRC1 or pharmacological manipulations that reduced its interaction with TPC1 were without effect. PGRMC1 therefore serves as a TPC1 interactor that regulates ER-endosomal coupling with functional implications for cellular Ca2+ dynamics and potentially the distribution of heme.

Convergent activation of two-pore channels mediated by the NAADP-binding proteins JPT2 and LSM12.

The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) evokes calcium ion (Ca2+) release from endosomes and lysosomes by activating two-pore channels (TPCs) on these organelles. Rather than directly binding to TPCs, NAADP associates with proteins that indirectly confer NAADP sensitivity to the TPC complex. We investigated whether and how the NAADP-binding proteins Jupiter microtubule-associated homolog 2 (JPT2) and like-Sm protein 12 (LSM12) contributed to NAADP-TPC-Ca2+ signaling in human cells. Biochemical and functional analyses revealed that recombinant JPT2 and LSM12 both bound to NAADP with high affinity and that endogenous JPT2 and LSM12 independently associated with TPC1 and TPC2. On the basis of knockout and rescue analyses, both NAADP-binding proteins were required to support NAADP-evoked Ca2+ signaling and contributed to endolysosomal trafficking of pseudotyped coronavirus particles. These data reveal that the NAADP-binding proteins JPT2 and LSM12 convergently regulate NAADP-evoked Ca2+ release and function through TPCs.

Convergent activation of Ca2+ permeability in two-pore channel 2 through distinct molecular routes.

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.

Metabolism of (R)-Praziquantel versus the Activation of a Parasite Transient Receptor Potential Melastatin Ion Channel.

Praziquantel (PZQ) is an essential anthelmintic drug recently established to be an activator of a Transient Receptor Potential Melastatin (TRPMPZQ ) ion channel in trematode worms. Bioinformatic, mutagenesis and drug metabolism work indicate that the cyclohexyl ring of PZQ is a key pharmacophore for activation of trematode TRPMPZQ , as well as serving as the primary site of oxidative metabolism which results in PZQ being a short-lived drug. Based on our recent findings, the hydrophobic cleft in schistosome TRPMPZQ defined by three hydrophobic residues surrounding the cyclohexyl ring has little tolerance for polarity. Here we evaluate the in vitro and in vivo activities of PZQ analogues with improved metabolic stability relative to the challenge of maintaining activity on the channel. Finally, an estimation of the respective contribution to the overall activity of both the parent and the main metabolite of PZQ in humans is reported.

Vitamin C transport in neurons and epithelia is regulated by secretory carrier-associated membrane protein-2 (SCAMP2).

The human sodium-dependent vitamin C transporter-1 (hSVCT1) is localized at the apical membrane domain of polarized intestinal and renal epithelial cells to mediate ascorbic acid (AA) uptake. Currently, little is known about the array of interacting proteins that aid hSVCT1 trafficking and functional expression at the cell surface. Here we used an affinity tagging ('One-STrEP') and proteomic approach to identify hSVCT1 interacting proteins, which resolved secretory carrier-associated membrane protein-2 (SCAMP2) as a novel accessary protein partner. SCAMP2 was validated as an accessory protein by co-immunoprecipitation with hSVCT1. Co-expression of hSVCT1 and SCAMP2 in HEK-293 cells revealed both proteins co-localized in intracellular structures and at the plasma membrane. Functionally, over-expression of SCAMP2 potentiated 14C-AA uptake, and reciprocally silencing endogenous SCAMP2 decreased 14C-AA uptake. Finally, knockdown of endogenous hSVCT1 or SCAMP2 impaired differentiation of human-induced pluripotent stem cells (hiPSCs) toward a neuronal fate. These results establish SCAMP2 as a novel hSVCT1 accessary protein partner that regulates AA uptake in absorptive epithelia and during neurogenesis.

Use the force, fluke: Ligand-independent gating of Schistosoma mansoni ion channel TRPMPZQ.

The parasitic flatworm ion channel, TRPMPZQ, is a non-selective cation channel that mediates Ca2+ entry and membrane depolarization when activated by the anthelmintic drug, praziquantel (PZQ). TRPMPZQ is conserved in all platyhelminth genomes scrutinized to date, with the sensitivity of TRPMPZQ in any particular flatworm correlating with the overall sensitivity of the worm to PZQ. Conservation of this channel suggests it plays a role in flatworm physiology, but the nature of the endogenous cues that activate this channel are currently unknown. Here, we demonstrate that TRPMPZQ is activated in a ligand-independent manner by membrane stretch, with the electrophysiological signature of channel opening events being identical whether evoked by negative pressure, or by PZQ. TRPMPZQ is therefore a multimodal ion channel gated by both physical and chemical cues. The mechanosensitivity of TRPMPZQ is one route for endogenous activation of this ion channel that holds relevance for schistosome physiology given the persistent pressures and mechanical cues experienced throughout the parasite life cycle.

Electrophysiological characterization of a schistosome transient receptor potential channel activated by praziquantel.

Ion channels have proved to be productive targets for anthelmintic chemotherapy. One example is the recent discovery of a parasitic flatworm ion channel targeted by praziquantel (PZQ), the main clinical therapy used for treatment of schistosomiasis. The ion channel activated by PZQ - a transient receptor potential ion channel of the melastatin subfamily, named TRPMPZQ - is a Ca2+-permeable ion channel expressed in all parasitic flatworms that are PZQ-sensitive. However, little is currently known about the electrophysiological properties of this target that mediates the deleterious action of PZQ on many trematodes and cestodes. Here, we provide a detailed biophysical characterization of the properties of Schistosoma mansoni TRPMPZQ channel (Sm.TRPMPZQ) in response to PZQ. Single channel electrophysiological analysis demonstrated that Sm.TRPMPZQ when activated by PZQ is a non-selective, large conductance, voltage-insensitive cation channel that displays distinct properties from human TRPM paralogs. Sm.TRPMPZQ is Ca2+-permeable but does not require Ca2+ for channel gating in response to PZQ. TRPMPZQ from Schistosoma japonicum (Sj.TRPMPZQ) and Schistosoma haematobium (Sh.TRPMPZQ) displayed similar characteristics. Profiling Sm.TRPMPZQ responsiveness to PZQ has established a biophysical signature for this channel that will aid future investigation of endogenous TRPMPZQ activity, as well as analyses of endogenous and exogenous regulators of this novel, druggable antiparasitic target.

Molecular and cellular basis of praziquantel action in the cardiovascular system.

The anthelmintic drug praziquantel (PZQ) causes contraction of parasitic schistosomes as well as constriction of blood vessels within the mesenteric vasculature of the host where the adult blood flukes reside. The contractile action of PZQ on the vasculature is mediated by the activation of host serotonergic 5-HT2B receptors (5-HT2BRs). However, the molecular basis for PZQ interaction with these targets and the location of these 5-HT2B receptors in the vessel wall have not been experimentally defined. Evaluation of a PZQ docking pose within the 5-HT2BR orthosteric site, using both Ca2+ reporter and bioluminescence resonance energy transfer (BRET) assays, identified residues F3406.51 and F3416.52 (transmembrane helix 6, TM6) as well as L209EL2 (extracellular loop 2) as critical for PZQ-mediated agonist activity. A key determinant of PZQ selectivity for the 5-HT2B receptor over the 5-HT2A/2C receptors was determined by M2185.39 in transmembrane helix 5 (TM5) of the orthosteric site. Mutation of this residue to valine (M218V), as found in 5-HT2A and 5-HT2C, decreased PZQ agonist activity, whereas the reciprocal mutation (V215M) in 5-HT2C increased PZQ activity. Two-photon imaging in intact mesenteric arterial strips visualized PZQ-evoked Ca2+ transients within the smooth muscle cells of the vessel wall. PZQ also triggered cytoplasmic Ca2+ signals in arterial smooth muscle cells in primary culture that were isolated from mesenteric blood vessels. These data define the molecular basis for PZQ action on 5-HT2B receptors localized in vascular smooth muscle.

Natural variation in the binding pocket of a parasitic flatworm TRPM channel resolves the basis for praziquantel sensitivity.

The drug praziquantel (PZQ) is the key clinical therapy for treating schistosomiasis and other infections caused by parasitic flatworms. A schistosome target for PZQ was recently identified- a transient receptor potential ion channel in the melastatin subfamily (TRPMPZQ)-however, little is known about the properties of TRPMPZQ in other parasitic flatworms. Here, TRPMPZQ orthologs were scrutinized from all currently available parasitic flatworm genomes. TRPMPZQ is present in all parasitic flatworms, and the consensus PZQ binding site was well conserved. Functional profiling of trematode, cestode, and a free-living flatworm TRPMPZQ ortholog revealed differing sensitives (~300-fold) of these TRPMPZQ channels toward PZQ, which matched the varied sensitivities of these different flatworms to PZQ. Three loci of variation were defined across the parasitic flatworm TRPMPZQ pocketome with the identity of an acidic residue in the TRP domain acting as a gatekeeper residue impacting PZQ residency within the TRPMPZQ ligand binding pocket. In trematodes and cyclophyllidean cestodes, which display high sensitivity to PZQ, this TRP domain residue is an aspartic acid which is permissive for potent activation by PZQ. However, the presence of a glutamic acid residue found in other parasitic and free-living flatworm TRPMPZQ was associated with lower sensitivity to PZQ. The definition of these different binding pocket architectures explains why PZQ shows high therapeutic effectiveness against specific fluke and tapeworm infections and will help the development of better tailored therapies toward other parasitic infections of humans, livestock, and fish.

Endo-Lysosomal Two-Pore Channels and Their Protein Partners.

Two-pore channels are ion channels expressed on acidic organelles such as the various vesicles that constitute the endo-lysosomal system. They are permeable to Ca2+ and Na+ and activated by the second messenger NAADP as well as the phosphoinositide, PI(3,5)P2 and/or voltage. Here, we review the proteins that interact with these channels including recently identified NAADP receptors.

Quantal leaps in understanding Ca2+ signaling: A "Taylored" approach.

On 2 September 2022, about 85 scientists gathered in person at Queens' College in Cambridge, UK, for a scientific meeting to celebrate the career of Colin W. Taylor of Cambridge University upon his retirement. The meeting was organized by the authors, who are all former graduate students in the Taylor laboratory, which has been at the forefront of Ca2+ signaling for more than 30 years.

Synthesis and biological evaluation of novel photo-clickable adenosine and cyclic ADP-ribose analogs: 8-N3-2'-O-propargyladenosine and 8-N3-2'-O-propargyl-cADPR.

A photo-clickable analog of adenosine was devised and synthesized in which the photoactive functional group (8-azidoadenosine) and the click moiety (2'-O-propargyl-ether) were compactly combined within the structure of the adenosine nucleoside itself. We synthesized 8-N3-2'-O-propargyl adenosine in four steps starting from adenosine. This photo-clickable adenosine was 5'-phosphorylated and coupled to nicotinamide mononucleotide to form the NAD analog 8-N3-2'-O-propargyl-NAD. This NAD analog was recognized by Aplysia californica ADP-ribosyl cyclase and enzymatically cyclized producing 8-N3-2'-O-propargyl cyclic ADP-ribose. Photo-clickable cyclic-ADP-ribose analog was envisioned as a probe to label cyclic ADP-ribose binding proteins. The monofunctional 8-N3-cADPR has previously been shown to be an antagonist of cADPR-induced calcium release [T.F. Walseth et. al., J. Biol. Chem (1993) 268, 26686-26691]. 2'-O-propargyl-cADPR was recognized as an agonist which elicited Ca2+ release when added at low concentration to sea urchin egg homogenates. The bifunctional 8-N3-2'-O-propargyl cyclic ADP-ribose did not elicit Ca2+ release at low concentration or impact cyclic ADP-ribose mediated Ca2+ release either when added to sea urchin egg homogenates or when microinjected into cultured human U2OS cells. The photo-clickable adenosine will none-the-less be a useful scaffold for synthesizing photo-clickable probes for identifying proteins that interact with a variety of adenosine nucleotides.

Implementation of an Active Screening Program for SARS-CoV2 - Experience at an Academic Medical Center.

BACKGROUND: This study documents the experience of an academic medical center implementing SARS-CoV2 screening of asymptomatic research personnel to support the "return-to-work" initiative and donor cadavers to support in-person student education. METHODS: Testing was performed on samples received June 1, 2020 (for the cadaver program) and July 20, 2020 (for the personnel screening program) through September 30, 2021. Data were evaluated to document the number of cases and the positivity rate. RESULTS: Approximately 3000 specimens were tested across both programs, with an overall positivity rate of 2.5% and 3.6% in the personnel and cadaver screening programs, respectively. DISCUSSION: This screening program serves as an example of institutional investment in the safety of its faculty, staff, and students alike to address specific needs of a global pandemic.

Electrophysiology of Endolysosomal Two-Pore Channels: A Current Account.

Two-pore channels TPC1 and TPC2 are ubiquitously expressed pathophysiologically relevant proteins that reside on endolysosomal vesicles. Here, we review the electrophysiology of these channels. Direct macroscopic recordings of recombinant TPCs expressed in enlarged lysosomes in mammalian cells or vacuoles in plants and yeast demonstrate gating by the Ca2+-mobilizing messenger NAADP and/or the lipid PI(3,5)P2. TPC currents are regulated by H+, Ca2+, and Mg2+ (luminal and/or cytosolic), as well as protein kinases, and they are impacted by single-nucleotide polymorphisms linked to pigmentation. Bisbenzylisoquinoline alkaloids, flavonoids, and several approved drugs demonstrably block channel activity. Endogenous TPC currents have been recorded from a number of primary cell types and cell lines. Many of the properties of endolysosomal TPCs are recapitulated upon rerouting channels to the cell surface, allowing more facile recording through conventional electrophysiological means. Single-channel analyses have provided high-resolution insight into both monovalent and divalent permeability. The discovery of small-molecule activators of TPC2 that toggle the ion selectivity from a Ca2+-permeable (NAADP-like) state to a Na+-selective (PI(3,5)P2-like) state explains discrepancies in the literature relating to the permeability of TPCs. Identification of binding proteins that confer NAADP-sensitive currents confirm that indirect, remote gating likely underpins the inconsistent observations of channel activation by NAADP.