Comparison of responses to TRPM8 ligands in wild type and TRPM8 KO mice.

<p>(<b>A-C</b>) Responses to the TRPM8 agonists (A) menthol (300μM), (B) icilin (50μM) and (C) WS-12 (50μM) in wild type (WT, black) and TRPM8 knockout mice (TRPM8 KO, purple) when applied during the sustained phase of a KPSS-evoked contraction (shown by black circle). (<b>D</b>) Similar assay for (<i>S</i>)-PZQ (50μM) evoked relaxation during KPSS-evoked contraction in wild type (WT, black) and TRPM8 knockout mice (TRPM8 KO, purple). (<b>E</b>) Cumulative dataset measuring (<i>S</i>)-PZQ (50μM) evoked relaxation from experiments such as shown in (D). Data represent mean±s.e.m. from averaged measurements from mesenteric vessel strips from n≥3 mice.</p

±PZQ causes relaxation of precontracted mesenteric arteries.

<p>(<b>A</b>) Changes in tension measured in mouse mesenteric artery vessel strips evoked by KPSS (left, arrow), ±PZQ (circle, 100μM) added at basal tone (middle) and KPSS (arrow) followed by addition of ±PZQ to a contracted vessel (purple circle and trace, right). Solution exchanges to physiological saline shown as ‘w’ (wash). (<b>B</b>) Dose-response relationship quantifying peak relaxation evoked by indicated concentrations of ±PZQ. (<b>C</b>) Effect of repeated additions of ±PZQ (10μM, black circles) without solution exchange on contractile tone in a vessel contracted by solution exchange to KPSS (arrow).</p

Stereoselective activation of hTRPM8 by (<i>S</i>)-PZQ.

<p>(<b>A</b>) Representative fluorescence traces from cells loaded with fluo-4-AM in a HEK293 cell line following addition of menthol (300μM), or ±PZQ (100μM) followed by ACh (100μM). (<b>B</b>) Pseudocolored confocal images from the field of view are displayed following addition of vehicle (DMSO, 0.05%), (<i>R</i>)-PZQ or (<i>S</i>)-PZQ (50μM), and ACh (100μM) in untransfected HEK293 cells (top) or cells transfected with hTRPM8 (bottom three panels). (<b>C</b>) Representative fluorescence traces from cells loaded with fluo-4-AM in a HEK293 cell line transfected with hTRPM8 following addition of menthol (300μM), or ±PZQ (100μM), or (<i>R</i>)-PZQ or (<i>S</i>)-PZQ (50μM), followed by ACh (100μM). (<b>D</b>) Cumulative measurements of peak fluorescence ratio (F/F₀, where ‘F’ represents fluorescence at peak and ‘F₀’ represents fluorescence at time = 0) from Ca²⁺ imaging experiments under indicated conditions. AMTB (10μM) was added to cells 30min before addition of agonists. Data represent representing population mean±s.e.m. (≥20 cells) from n≥3 independent transfections. (<b>E</b>) Dose response relationship for (<i>S</i>)-PZQ evoked Ca²⁺ mobilization in TRPM8 expressing cells.</p

Interrogating a TRP channel panel for praziquantel activity.

<p>(<b>A</b>) Graphical schematic of results from primary screen measuring activation (EC₅₀, left) or inhibition (IC₅₀, right) of 17 individual human TRP channels to ±PZQ, (<i>R</i>)-PZQ and (<i>S</i>)-PZQ. EC₅₀ and IC₅₀ values are color encoded as per legend key, where increasing warm coloration represents higher resolved potency in assays. Responsive hTRPs are identified with asterisks (*). (<b>B-E</b>) Individual dose-response curves for activation of (B) hTRPA1, (C) hTRPC3, (D) hTRPC7 and (E) hTRPM8 by ±PZQ (open purple squares), (<i>R</i>)-PZQ (red squares) and (<i>S</i>)-PZQ (blue squares).</p