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Identification of novel modulators of a schistosome transient receptor potential channel targeted by praziquantel

['Evgeny G. Chulkov', 'Department Of Cell Biology', 'Neurobiology', 'Anatomy', 'Medical College Of Wisconsin', 'Milwaukee', 'Wisconsin', 'United States Of America', 'Emery Smith', 'Department Of Molecular Medicine']

Date: 2021-12

Given the worldwide burden of neglected tropical diseases, there is ongoing need to develop novel anthelmintic agents to strengthen the pipeline of drugs to combat these burdensome infections. Many diseases caused by parasitic flatworms are treated using the anthelmintic drug praziquantel (PZQ), employed for decades as the key clinical agent to treat schistosomiasis. PZQ activates a flatworm transient receptor potential (TRP) channel within the melastatin family (TRPM PZQ ) to mediate sustained Ca 2+ influx and worm paralysis. As a druggable target present in many parasitic flatworms, TRPM PZQ is a promising target for a target-based screening campaign with the goal of discovering novel regulators of this channel complex. Here, we have optimized methods to miniaturize a Ca 2+ -based reporter assay for Schistosoma mansoni TRPM PZQ (Sm.TRPM PZQ ) activity enabling a high throughput screening (HTS) approach. This methodology will enable further HTS efforts against Sm.TRPM PZQ as well as other flatworm ion channels. A pilot screen of ~16,000 compounds yielded a novel activator of Sm.TRPM PZQ , and numerous potential blockers. The new activator of Sm.TRPM PZQ represented a distinct chemotype to PZQ, but is a known chemical entity previously identified by phenotypic screening. The fact that a compound prioritized from a phenotypic screening campaign is revealed to act, like PZQ, as an Sm.TRPM PZQ agonist underscores the validity of TRPM PZQ as a druggable target for antischistosomal ligands.

The drug praziquantel is used to treat diseases caused by parasitic flatworms. Praziquantel is an old drug, and there is a need to identify novel treatments that retain desirable features and improve weaknesses in the mode of PZQ action. One way to do this is to identify new drugs that exploit vulnerabilities in the same drug target but work in slightly differently ways. Here, we have optimized high throughput screening methods to pharmacologically profile a parasitic flatworm ion channel targeted by PZQ. We have identified several new chemical structures that interact with this channel complex. These ligands provide new opportunity for developing tools to manipulate flatworm biology and potentially new trajectories for anthelmintic drug development.

Funding: This work was supported by NIH R01-AI145871 and R01-AI155405 (to JSM), NIH F31-AI145091 (to NAY) and the Marcus Family (to JSM). This work was also supported in part by a NIH S10 instrument award (1S10OD025282-01) that provided Scripps Research with the FLIPR Tetra system integrated into HTS operations (to TPS,LS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Chulkov et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we executed a pilot screen of ~16,000 compounds against Sm.TRPM PZQ which identified numerous antagonists as well as a single novel activator of Sm.TRPM PZQ . Interestingly this Sm.TRPM PZQ activator is a known chemical entity previously prioritized from a phenotypic screen of schistosome worms. These data underscore first, the feasibility of a high throughput screening (HTS) approach for flatworm TRP channels, and second convergence of target- and phenotype-based screening approaches on the same ligand here revealed to act as a Sm.TRPM PZQ agonist.

To characterize flatworm TRP channel pharmacology, it would be helpful to establish methods for screening individual channels against diverse drug libraries. Sm.TRPM PZQ is a good candidate for optimizing such an approach given it is a targeted by PZQ, and mediates a large, sustained Ca 2+ signal in heterologously expressing cells. In collaboration with the Molecular Screening Center at Scripps Research in Florida, we optimized target-based screening approaches for Sm.TRPM PZQ with the goal of discovering other ligands that engage this ion channel complex [ 18 ]. The hope is that by optimizing high-throughput screening (HTS) methods for TRPM PZQ , new chemotypes distinct from PZQ can be identified including ligands that act at different sites on the channel relative to the transmembrane PZQ-binding pocket [ 9 ]. These could include allosteric modulators, or ligands that interact with the pore-forming domain (S5-S6). Such ‘hits’ could then be further iterated and evaluated as leads for anthelmintic development.

We recently discovered that PZQ activates a Ca 2+ -permeable ion channel in Schistosoma mansoni that belongs to the melastatin family of transient receptor potential (TRP) channels (christened Sm.TRPM PZQ [ 7 , 8 ]). PZQ also acts a potent activator of TRPM PZQ in other PZQ-sensitive parasites [ 9 ]. TRP channels, which act as non-selective cation channels, are appealing targets for anthelmintic drug discovery owing both to their important physiological roles in sensory physiology as well as their druggability [ 10 – 15 ]. However, little is currently known about the pharmacology of flatworm TRP channels. Efforts to profile these channels will be important for validating tools to selectively manipulate worm physiology, as well as for anthelmintic development [ 13 – 15 ]. Insight to date suggests schistosome TRP channels, like other flatworm targets [ 16 ], exhibit a different pharmacological profile compared with their closest mammalian counterparts. For example, Schistosoma mansoni TRPA1 responds to capsaicin, a human TRPV ligand [ 17 ]. Sm.TRPM PZQ , which harbors a TRPM8-like binding pocket in the voltage-sensor like domain (transmembrane helices S1-S4, [ 9 ]), is not activated by the human TRPM8 agonists menthol and icilin [ 7 ]. Customization of these TRP channels to parasite-specific functions may underpin this divergence and specialization.

Over a billion people worldwide require chemotherapy for neglected tropical diseases (NTDs, [ 1 ]). Schistosomiasis, a disease caused by infection by parasitic flatworms known as schistosomes, is one of several NTDs targeted for elimination as a public health problem in the World Health Organization 2021–2030 NTD road map [ 1 ]. Schistosomiasis, as well as several other parasitic flatworm infections [ 2 ], are treated using the anthelmintic drug praziquantel (PZQ). PZQ has remained an effective treatment for schistosomiasis over four decades of clinical use [ 3 ], underpinning recent mass drug administration (MDA) campaigns aimed at decreasing infections and morbidity in vulnerable populations. Alternatives to PZQ are however needed. PZQ has several features that could be improved and the threat of drug resistance, potentially accelerated by the rollout of MDA initiatives, persists [ 4 – 6 ].

Results

Activation of Sm.TRPM PZQ heterologously expressed in HEK293 cells was resolved by following changes in fluorescence emission of a synthetic Ca2+ indicator over time [7]. In cells, transiently expressing Sm.TRPM PZQ and seeded into a 96-well plate, addition of ±PZQ (3 μM) caused a rapid, dose-dependent increase in fluorescence (Fig 1A, [7]). Addition of higher concentrations of PZQ (≤30 μM) to untransfected HEK293 cells failed to elicit any change in basal fluorescence (Fig 1A, [7]).

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TIFF original image Download: Fig 1. Optimization of assay conditions for monitoring Sm.TRPM PZQ activity. (A) Representative fluorescence trace showing the kinetics of the Ca2+ signal resulting from addition of PZQ (3 μM) to HEK293 cells transiently expressing Sm.TRPM PZQ (closed symbols), or addition of a higher concentration of PZQ (30 μM) to untransfected HEK293 cells (open symbols). (B) Concentration response curves of PZQ-stimulated Ca2+ signals in Sm.TRPM PZQ expressing HEK293 cells resolved at various densities of cells per well (c/w) measured in suspension in 1,536 well plates at room temperature. Each point on each curve represents the average of responses from 16 replicates which were also run in 2 separate experiments to achieve average and standard deviation. https://doi.org/10.1371/journal.pntd.0009898.g001

In order to support a large scale HTS, we trialed various conditions to support miniaturization of this basic reporter assay into smaller volumes necessary for screening in 1536-well format. Miniaturization of the screening assay was aided by the large amplitude of the Sm.TRPM PZQ –dependent Ca2+ transient (change of fluorescence/basal fluorescence, ΔF/F = 12.3±2.1 for PZQ signals at Sm.TRPM PZQ versus ΔF/F = 4.5±1.5 for ATP signals through endogenous receptors as measured by confocal Ca2+ imaging [7]) and the non-desensitizing nature of the PZQ-evoked Ca2+ signal, which was sustained over several minutes (Fig 1A). Experimental performance was compared (i) using various densities of Sm.TRPM PZQ -transfected cells assayed in suspension, (ii) at different temperatures (room temperature (RT) versus 37°C), and (iii) using either freshly transfected cells, or thawed stocks of previously frozen transfected cells. Each condition was trialed in a 1,536 well format using the fluorescent dye calcium-5 (K d for Ca2+ = 390 nM). Assay performance under these screening conditions was compared by calculating the Z’ factor (Z’), a widely used indicator of HTS assay robustness [19], as well as by monitoring the dynamic range of the assay (signal[F max ]:basal[F basal ], S:B). Z’ values over 0.5 are considered to be a prerequisite for an excellent HTS.

Assays in the 1536 well format demonstrated that increasing the cell count per well resulted in increased signal and dynamic range (Fig 1B), as well as a decrease in the measured EC 50 (from 135±15 nM at 1000 cells/well, to 79±2 nM at 4000 cells/well, Table 1). The temperature at which responses were recorded (RT versus 37°C) did not change the sensitivity of Sm.TRPM PZQ under these assay conditions (Table 1). Optimal conditions for executing the assay were selected as 4000 cells/well in suspension at room temperature, where assay performance exceeded needed parameters (Table 1). Similar assay performance was achieved using either fresh cells (EC 50 = 79±2 nM, Z’ = 0.82±0.03, S:B = 10.05±0.08, Table 1) or thawed cells from previously frozen stocks (EC 50 = 90±10 nM, Z’ = 0.86±0.02, S:B = 10.55±0.08) under the same assay conditions. Therefore, to further minimize assay variation, pilot screens were all performed using a single batch of transfected cells prepared in bulk and then frozen. This facilitated execution of screens and removed transfection efficiency as an experimental variable. Data from all of these trials are summarized in Table 1.

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TIFF original image Download: Table 1. Assay metrics under specified conditions. EC 50 , Z’ and S:B were determined for Sm.TRPM PZQ activation by PZQ at indicated cell densities and temperatures. Conditions in bold text indicate the final conditions selected for the screen. For each condition, responses were found to be positively cooperative (Hill coefficients range of 1.5–2). https://doi.org/10.1371/journal.pntd.0009898.t001

After optimizing conditions for miniaturization into a 1,536 well format, we proceeded to execute a screening campaign against a total of 15,984 compounds. The screened libraries comprised LOPAC 1280 (1280 compounds), the Pathogen Box (400 compounds) and a subset of the in-house Scripps Drug Discovery Library, which included the Maybridge Hitfinder library. The screening pipeline consisted of (i) a primary, fixed concentration screen (5 μM) performed in triplicate in both ‘agonist’ and ‘antagonist’ mode, followed by (ii) titration and finally (iii) counter-screening assays of all putative ‘hits’. For the primary screen, following compound addition and completion of the ‘agonist’ (AG) mode read of 3 minutes, the ‘antagonist’ (ANT) mode commenced by PZQ addition (at an EC 80 concentration) to wells that contain either compounds or DMSO (‘low control’). The output was compared to the response of wells with DMSO without PZQ stimulation (‘high control’). A summary of the workflow and observed compound attrition through each of these steps is shown schematically in Fig 2. Single point scatterplots from all compounds tested in the primary screen are shown in Fig 3.

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TIFF original image Download: Fig 2. Sm.TRPM PZQ screening pipeline. All HTS assays were performed in 1,536 well format. A fixed concentration (nominally 5 μM) primary screen was followed by titration assays and counter-screening in untransfected HEK cells. All assays were performed in triplicate in both agonist (AG) and antagonist (ANT) mode. From the original 15,934 compounds (cmpds) screened, the pipeline yielded 3 putative agonist ‘hits’ (two hits were identified as PZQ present as a ligand in the screened libraries) and 32 putative antagonist ‘hits’. Selected compounds were validated using electrophysiology and by monitoring contraction of schistosome worms ex vivo. https://doi.org/10.1371/journal.pntd.0009898.g002

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TIFF original image Download: Fig 3. Primary HTS assay data. Scatterplot of all 15,934 compounds tested in (A) agonist mode and (B) antagonist mode. Each dot graphed represents the activity of a well containing test compound (black, sample field) or controls (red high control; green, low control). Arrows (right) indicated the high control (red), low control (green) and the sample field (black, datapoints between the calculated cutoffs (red lines) and high control). For the agonist screen, high control reflected responses to PZQ (10 μM, red) and the low control represented responses to DMSO (green). For the antagonist screen, the high control reflected responses to DMSO (red) and the low control represented responses to submaximal PZQ (500 nM, green). The response to a maximal PZQ concentration (EC MAX , 10 μM) is also indicated (magenta). The EC 80 stimulation is qualified as a percentage of the response to the high control PZQ EC 100 (‘EC MAX ’). All plates are assessed for robustness with Z’ >0.5 and EC Stim between 70–95%. https://doi.org/10.1371/journal.pntd.0009898.g003

Assay performance in the primary agonist screen met quality thresholds (Z’ = 0.71±0.04, S:B = 11.40±0.04) and the observed sensitivity to PZQ was consistent with the prior assay optimization trials (EC 50 for PZQ = 101±3 nM). Assay performance in the primary antagonist screen also met quality thresholds (Z’ = 0.72±0.04, S:B = 9.4±0.4). Hit cut-offs for both screening modes was determined using an interval-based algorithm [20,21]. The sample field in the agonist screen was taken as a percentage response (‘hit-cutoff’ >16%) which identified 181 putative ‘hits’ for further progression. The initial sample field in the antagonist screen was calculated as a ‘hit-cutoff’ >36%, which identified 188 putative ‘hits’ for further evaluation.

A total of 368 compounds were then advanced to titration screening from the primary screen ‘agonist’ and ‘antagonist’ assays (1 compound was not commercially available). Each of these 368 compounds was profiled as a 10-point dose-response analysis run in triplicate against untransfected HEK cells (‘counterscreen’) as well as HEK cells expressing Sm.TRPM PZQ . Assay performance in these titration assays met required specifications (S1 Table). After curve-fitting, compounds displaying an EC 50 >5 μM (agonist) or IC 50 >5 μM (antagonist) were considered ‘inactive’ and not studied further. The surviving ‘hits’ that progressed through this pipeline comprised 3 potential agonists and 32 potential antagonists. Full details of these hits and associated assay data are provided in S2 and S3 Tables.

All three agonist hits were evaluated in further detail. Two of the agonist hits were identified from the Pathogen Box, and one from LOPAC1280. The primary screening data and titration analysis from the plates containing these compounds were extracted from the screening dataset. Two of these hits were identified as PZQ (red symbols, Fig 4A) as both the Pathogen Box and LOPAC1280 libraries contained PZQ as a test ligand. A third agonist ‘hit’ (christened ‘AG1’, blue circle), from the Pathogen Box, elicited strong activation of Sm.TRPM PZQ (B max = 93.9±5.6%) in the primary screen (Fig 4A). AG1 is a known chemical entity (3-(3,4-diimethoxyphenyl)-6-(3-(propan-2-yl)phenyl)-[1,2,4]triazolo[4,3-a]pyridine, MMV688313; Fig 4A) and represents a chemotype distinct from PZQ. Addition of AG1 (100 μM) resulted in a sustained cytoplasmic Ca2+ signal in cells expressing Sm.TRPM PZQ resembling the action of PZQ (compare Fig 4B with Fig 1A). AG1 action was also assessed against a Sm.TRPM PZQ channel mutant (Sm.TRPM PZQ [R1514A]) in the fourth transmembrane spanning helix (TM4) that blocks PZQ action by ablating interactions necessary to shape the PZQ binding pocket [9]. As expected, PZQ did not elevate Ca2+ in cells expressing Sm.TRPM PZQ [R1514A] (Fig 4B). However, this binding site mutant also ablated AG1 activity (Fig 4B), suggesting that AG1 also acts as an orthosteric ligand. Concentration-response curve analysis revealed AG1 acted as a full agonist of Sm.TRPM PZQ (EC 50 = 1.6±0.3 μM) in Ca2+ flux assays, with no activity observed on counter-screening in naïve HEK cells (Fig 4C). The potency of AG1 was lower than observed with either sample of PZQ (EC 50 s of 177±21 nM, 619±225 nM) present in two libraries screened under identical conditions (Fig 4C). Both ligands were then re-sourced for validation assays and activities were re-assessed from independently procured samples (PZQ, EC 50 = 406 nM; AG1, EC 50 = 9.2 μM). Finally, the action of AG1 was studied against adult schistosome worms isolated from infected mice. Addition of AG1 to schistosome worms ex vivo evoked a sustained contraction (Fig 4D), although the kinetics of onset of the AG1-evoked contraction were slower than observed with PZQ. Collectively, these data identify AG1, a distinct chemical entity from PZQ, as a novel Sm.TRPM PZQ agonist.

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TIFF original image Download: Fig 4. Identification of new chemotypes active at Sm.TRPM PZQ . (A) Primary screen of Sm.TRPM PZQ in agonist mode measuring peak Ca2+ signal amplitude in response to 1,678 compounds (LOPAC 1280 , Pathogen Box) tested at 5μM final concentration. Structures of ‘hits’ (PZQ, red; AG1, blue) are shown. (B) Kinetics of a response of wild-type Sm.TRPM PZQ to AG1 (30μM, closed circles), or the binding pocket mutant Sm.TRPM PZQ [R1514A] to AG1 (open circles). (C) Analysis of the three primary screen hits via full concentration response curves for PZQ (compounds #5, #1287) and AG1 (compound #1552) in HEK293 cells transiently transfected with Sm.TRPM PZQ (solid symbols) or untransfected controls (open). Data represent mean±sd of triplicate samples. (D) Images of adult schistosome worms with single frame image (red) overlayed with maximum intensity projection (green) of a time-lapse series to illustrate worm movement and effects of PZQ (500 nM) and AG1 (1 μM) on worm motion 15 mins after treatment. Graph shows effects of drugs on worm mobility after a 15 min and 180 min exposure (*, p<0.01). Data are analyzed from n≥3 independent infections. (E) Structure of a putative Sm.TRPM PZQ blocker (ANT1) from HTS screening activities. (F) Concentration-dependent blockade of Sm.TRPM PZQ dependent Ca2+ signals (evoked by 500 nM PZQ) by increasing concentrations of ANT1. (G) Images of adult schistosome worms with single frame image (red) overlayed with maximum intensity projection of a time-lapse series to illustrate worm movement (green) and the effect of PZQ (250 nM) as well as PZQ in the presence of ANT1 (50μM). Data are captured after 24 hours of incubation. Inset, quantitative analysis of data from n = 3 independent infections. https://doi.org/10.1371/journal.pntd.0009898.g004

Screening in antagonist mode identified several 32 potential blockers of Sm.TRPM PZQ (S2 Table). To begin analysis of this larger set of ligands, we investigated that action of one of the more potent blockers, the compound ANT1 (1-(9H-fluoren-9-yl)-4-(5-methyl-3-phenyl-1,2-oxazole-4-carbonyl)piperazine, Fig 4E). ANT1 blocked PZQ-evoked Ca2+ signals (IC 50 of 1.3±0.3 μM) mediated by Sm.TRPM PZQ (Fig 4F). When applied to intact worms ANT1 did not change worm motility, but at high concentrations (50μM) enhanced recovery of schistosome worms incubated in the continued presence of PZQ compared to worms treated with PZQ alone (Fig 4G).

To validate ANT1 using an orthogonal assay, ANT1 action versus PZQ and AG1 was assessed using an electrophysiological approach. Single channel recordings of Sm.TRPM PZQ activity were made in Ca2+-free buffer following addition of either PZQ or AG1 (10 μM) to a cell-free patch in ‘inside-out’ recording mode. Both PZQ and AG1 evoked step-wise openings of Sm.TRPM PZQ (Fig 5A) with channel activation defined by linear I-V relationship in response to both agonists (Fig 5B). The mean slope single-channel conductance for PZQ- and AG1- activated Sm.TRPM PZQ was 76 ± 8 and 61 ± 4 pS respectively (mean ± SD). Addition of PZQ, or AG1, evoked sustained, non-desensitizing Sm.TRPM PZQ currents, which were blocked by subsequent addition of ANT1 in a time-dependent manner (Fig 5C). Analysis of single channel open probability (P open ) from these records demonstrated ANT1 decreased P open at Sm.TRPM PZQ channels activated by either agonist (Fig 5D). After ANT1 application, brief channel openings persisted in the presence of PZQ or AG1 that were not seen in the presence of PZQ after addition of the pore blocker La3+ [7]. This observation is again suggestive of a competitive interplay between ANT1 and the channel activators, PZQ or AG1, within the orthosteric binding pocket of Sm.TRPM PZQ .

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TIFF original image Download: Fig 5. Electrophysiological analysis of new Sm.TRPM PZQ chemotypes. (A) Sm.TRPM PZQ channel fluctuations evoked by PZQ (red) or AG1 (blue) both applied at 10 μM compared to vehicle responses (grey, 0.1% DMSO). Recordings were made in Ca2+-free solution at a clamping potential of +60mV in an inside-out configuration. DMSO and the test drug (PZQ or AG1) were added sequentially to the same patch. (B) Current (I)-voltage (V) relationship of Sm.TRPM PZQ activated with PZQ (10 μM, red) or AG1 (10 μM, blue). (C) Effect of ANT1 (top and middle, 50 μM) or La3+ (bottom, 10 mM) addition on Sm.TRPM PZQ activity evoked by PZQ (10 μM) or AG1 (10 μM) measured in cell-free mode at +40mV using an inside-out configuration. (D) Measurements of single channel open probability (P open ) under the indicated conditions. Welch’s t-test: PZQ vs ‘PZQ+ANT1’, p = 0.002; AG1 vs ‘AG1+ANT1’, p = 0.004. https://doi.org/10.1371/journal.pntd.0009898.g005

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