Positive allosteric modulator activity of ginsenosides is restricted to P2X7 and P2X4 receptors

Positive allosteric modulator activity of ginsenosides is restricted to P2X7 and P2X4 receptors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Positive allosteric modulator activity of ginsenosides is restricted to P2X7 and P2X4 receptors Elizabeth Allum, Leanne Stokes This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8724351/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Positive allosteric modulators of P2X7 receptors hold promise as host-directed immune enhancers in intracellular infections. We have previously reported the use of protopanaxadiol ginsenosides as positive allosteric modulators of human, rat and mouse P2X7 receptors plus human P2X4 receptors, however, the effect of ginsenosides on other P2X receptors is unknown. Here, we screened a range of ginsenosides and related glycosides at hP2X1, hP2X2 and hP2X3 receptors expressed in HEK-293 cells utilising a membrane potential assay. Our results demonstrate the utility of the membrane potential assay across all P2X receptors and the lack of significant potentiation of ATP-induced responses at hP2X1, hP2X2 and hP2X3 receptors by ginsenosides and related glycosides. We report that S-Rg3 also acts as a positive allosteric modulator at hP2X4 receptors in addition to hP2X7 receptors. This information is important for determining the selectivity of ginsenoside positive allosteric modulators for P2X7 and P2X4. P2X receptor positive modulation ginsenoside glycoside Figures Figure 1 Figure 2 Introduction The seven subtypes of P2X receptors are structurally similar with a common trimeric structure [ 1 ]. Subtle differences exist in the orthosteric ligand binding site, giving rise to different affinities for ATP and differences in pharmacological activity of ATP analogues (e.g. BzATP, αβ-meATP) [ 2 ]. Allosteric modulators can interact with several sites on P2X receptors, identified through a number of x-ray crystallography and cryo-EM studies [ 3 – 7 ]. One negative allosteric modulator is located behind the orthosteric ATP site on P2X7 and an analogous site is found on P2X4 [ 3 – 6 ] whereas with P2X3, a negative allosteric site is located beneath the orthosteric site [ 7 ]. Several negative allosteric modulators for P2X receptors have entered clinical trials for a range of disorders [ 2 ], with gefapixant being the first clinically used medicine targeting a P2X receptor [ 8 ]. The discovery and characterisation of positive allosteric modulators of P2X receptors lags behind that for negative allosteric modulators. Over the last decade several have been documented, reviewed in [ 9 ] with a bias towards those acting at P2X7. Our group discovered a series of positive allosteric modulator compounds derived from Panax ginseng [ 10 ] and subsequently we have characterised their functional effects in macrophages [ 11 , 12 ] and explored a potential binding site in the central vestibule region [ 13 ]. We found that several ginsenoside compounds could also potentiate P2X4 responses [ 14 ], suggesting that this could be a conserved mechanism of action within the family. We have also previously reported a series of related glycosides with PAM activity at P2X7 [ 15 ]. This study aims to investigate whether ginsenosides could potentiate ATP responses at other P2X receptors, focusing on P2X1-3. To do this we wanted to use the same experimental assay system to minimise variation between assays therefore we employed the membrane potential assay used in several other studies [ 16 , 17 ]. Methods Cell culture HEK-293 cells were used to generate stable cell lines expressing human P2X receptors. Original transfections were performed using Lipofectamine 2000 (Fisher Scientific) and cells were subjected to geneticin (800 µg/ml) selection for 2–3 weeks followed by single cell cloning by limiting dilution in 96-well plates. Individual clones were selected for further study based on ATP-induced responses. Cell lines were maintained in DMEM:F12 media supplemented with 10% foetal bovine serum (PanBiotech), penicillin (100 U/ml) and streptomycin (100 µg/ml) (Fisher Scientific) and kept in a humidified incubator with 5% CO 2 . Stable P2X-expressing cells were kept under geneticin (HelloBio) selection at 400 µg/ml. Passaging was performed twice weekly using 0.25% Trypsin-EDTA (Fisher Scientific). Membrane potential measurements 96-well plates (catalogue number 10212811, ThermoFisher Scientific) were coated with 50 µg/mL Poly-D-Lysine (P6407, Sigma-Aldrich) and left for at least one hour before being washed with sterile water and allowed to dry. Cells were plated in 100 µL of complete DMEM/F-12 the day prior to experimentation into PDL-coated 96- well plates at 2–3 x 10 4 cells per well. Plated cells were kept in a humidified 5% CO 2 incubator at 37°C. The Membrane Potential Blue Kit (Molecular Devices) was reconstituted according to the manufacturer’s instructions using standard extracellular buffer (145 mM NaCl, 2 mM KCl, 2 mM CaCl 2 , 1mM MgCl 2 , 13 mM glucose, 10 mM HEPES, pH 7.3–7.4) and kept in 5ml aliquots frozen at -20˚C. This was then diluted 1 in 2 with the extracellular buffer immediately prior to use unless otherwise stated. Cell medium was removed from the 96-well plate and replaced with 100 µL of membrane potential blue assay buffer. Cells were incubated in this buffer for 30 minutes at 37°C in a humidified incubator with 5% CO 2 . Cells were then transferred to a FlexStation 3 plate reader (Molecular Devices) and warmed at 37°C for an additional 5 minutes prior to experimentation. Fluorescence measurements were acquired using SoftMax Pro v5.4 software (Molecular Devices). Fluorescence emission was measured at 565 nm for 120 seconds with an excitation wavelength of 525 nm (3 reads per well, PMT medium, interval 2 seconds). Agonists and screened compounds were made up at either 5x or 10x final concentrations in buffer and applied to cells after 20 seconds of read time at an injection rate of 2–4. For dual injection screening experiments conducted in membrane potential blue, compounds were injected at 20 seconds, followed by a 60 second interval before the injection of the agonist ATP at 80 seconds (allowing for 60 seconds of pre-treatment with the compound before the application of ATP). Fura-2 calcium measurements Cells were loaded with 100µL of loading buffer per well (low divalent extracellular buffer 145 mM NaCl, 2 mM KCl, 0.2 mM CaCl 2 , 13 mM glucose, 10 mM HEPES, pH 7.3–7.4) containing 250µM sulfinpyrazone and 2µM Fura-2-acetoxymethyl (fura-2AM) calcium indicator dye (Hello Bio, UK) and incubated for 45 minutes at 37°C with 5% CO 2 . This allows the membrane-permeant fura-2 AM ester to move into cells, after which it is cleaved by cellular esterases to reveal the active fura-2 Ca2 + indicator dye. After loading, the loading buffer was removed and replaced with 180µL per well of standard extracellular buffer. Compounds to be tested were made up at 10x final concentration and were injected 40 seconds into read time at a rate of 2–4. Intracellular Ca 2+ measurements were made using a Flexstation 3 Multimode Plate Reader (Molecular Devices) and data analysed using SoftMax Pro v5.4 software (Molecular Devices). Fluorescence emission was measured at 510 nm for 300 seconds (3 reads per well, PMT medium, interval 3.5 seconds) with dual excitation wavelengths of 340nm and 380nm to give a ratio between Ca 2+ -bound and Ca 2+ -free fura-2 respectively. Plotting and Statistical analysis Data from membrane potential experiments were plotted as area under curve (AUC) as % of control where response with each compound was normalised to the vehicle control. For fura-2 experiments, ratio data was analysed with zero baseline (3 points) and area under curve calculated from 0-300 seconds. Data from three independent experiments was collated and tested for statistical significance using a one-way ANOVA with Dunnett’s multiple comparison test (GraphPad Prism v9). P < 0.05 was taken as the minimum level of statistical significance. Dose-response curves were plotted by a nonlinear regression fit with variable slope using GraphPad Prism software version 9. Half-maximal responses are expressed as mean EC 50 values with standard deviation from three independent experiments. Results Firstly, we optimised the membrane potential blue assay for measuring P2X responses to the primary agonist ATP. A Flexstation 3 plate reader was used to measure ATP-induced responses and concentration-response curves were generated from area under the curve (AUC) data. We generated clonal stable cell lines for each of the human receptors (hP2X1, hP2X2a, hP2X3) in addition to our stable cell lines for hP2X4 and hP2X7 to reduce variability from transient transfections. ATP responses were measured in standard extracellular buffer containing 2 mM calcium and 1 mM magnesium (Fig. 1 ). We did not study hP2X5 or hP2X6 receptors as these are not known to exist as homomeric receptors in vivo [ 18 ]. EC 50 values for ATP were as follows; hP2X1 8.2 ± 2.4 µM, hP2X2a 17.6 ± 4.0 µM, hP2X3 2.5 ± 0.5 µM, hP2X4 0.8 ± 0.26 µM, and hP2X7 638.5 ± 354 µM (n = 3 independent experiments). A representative trace showing the typical response of each P2X receptor is shown in Fig. 1 C. We tested 9 ginsenoside chemicals on HEK-293 cells expressing hP2X receptors using the membrane potential blue assay and standard extracellular buffer to keep the experimental conditions consistent. An approximate EC 50 concentration of agonist was used for each individual P2X receptor, and a dual injection protocol was implemented allowing compound/vehicle injection 60 seconds before the agonist. All ginsenosides were tested at a final concentration of 10 µM and data in Fig. 1 is presented as % of control. We first confirmed that robust potentiation by protopanaxadiol ginsenosides was seen in HEK-hP2X7 using 200 µM ATP (as in previous studies) to induce ion channel activation. 20-S-Rg3, CK and F2 performed as the best PAMs at hP2X7 (Fig. 1 D) which agrees with our previous work where screening was performed using the YO-PRO-1 dye uptake assay [ 15 ]. For hP2X4 we have previously reported that ginsenosides CK, Rd, Rb1, and Rh2 displayed positive allosteric modulator activity using fura-2 calcium measurements [ 14 ]. This was confirmed in the membrane potential assay, and here we discovered that 20-S-Rg3 and F2 are also effective at potentiating the P2X4 receptor (Fig. 1 H). We tested the stereoisomers of Rg3 and Rh2 as we previously reported a striking difference in PAM activity at hP2X7 with only S-enantiomers being active [ 15 ]. R-enantiomers displayed some PAM activity at hP2X4 in these screening experiments, although this did not reach statistical significance (Fig. 1 H). For hP2X2a we observed increased ATP responses with CK, 20-S-Rg3, 20-R-Rg3, 20-S-Rh2 and Rb1 (Fig. 1 F) however, this was variable and not statistically significant. hP2X1 and hP2X3 did not show significant potentiation by any of the ginsenosides tested (Fig. 1 E, 1 G). Next, we assessed a series of glycosides with similar chemical structures to the ginsenosides. Most of the glycosides did not show robust potentiating activity at hP2X7 in this assay with the exception of stevenleaf (Fig. 2 A). Our previous study had demonstrated that gypenoside XLIX, XVII and daucosterol showed PAM activity at hP2X7 although this was seen as an increase in maximum response in the dye uptake assay rather than a shift in the ATP dose response curves [ 15 ]. For hP2X2a, there was a lot of variability and none of the glycosides showed a statistically significant increase compared to DMSO control (Fig. 2 B). For hP2X4 receptors several glycosides increased the EC 50 ATP responses including oleanolic acid and stevenleaf (Fig. 2 C). We further investigated selected ginsenosides at hP2X2a and hP2X4 using concentration response curves to ATP. Using the membrane potential assay, ginsenosides CK and Rb1 induced a minor shift in the ATP concentration response curve at hP2X2a receptors (Fig. 2 D). The average EC 50 for ATP was 12.62 ± 5.4 µM in these experiments and in the presence of ginsenosides CK and Rb1 this was 8.93 ± 4.2 µM and 8.52 ± 3.3 µM respectively. For hP2X4, a left-ward shift in the ATP concentration response curve was observed for ginsenoside CK and S-Rg3 (Fig. 2 E) with EC 50 values shifting from 975 ± 333 nM to 407 ± 302 nM with CK and to 563 ± 572 nM with S-Rg3. We then used a second assay to measure P2X2 and P2X4 responses, the fura-2 assay and repeated the concentration responses to ATP (Fig. 2 F and 2 G). Discussion This is the first study where multiple P2X receptor responses have been measured using a single assay for screening purposes. The FLIPR membrane potential assay was easy to use and has potential value in screening programs. We used the membrane potential blue assay kit from Molecular Devices, however the same kit is available in a red assay utilising a different quenching dye. Others have used the membrane potential assay for investigating P2X1 responses [ 19 ] or P2X7 responses [ 16 ]. EC 50 values for ATP at multiple hP2X receptors were similar to those stated in literature [ 20 ] and we used this information to select an EC 50 concentration for screening experiments. With P2X7 we have a wealth of evidence for positive allosteric modulation by ginsenosides [ 10 , 13 , 15 ] and we see similar effects for key ginsenosides (CK, F2, S-Rg3, S-Rh2 and Rd) in the membrane potential assay, validating its use for screening. With P2X4, we have previously reported that CK and Rd are effective positive allosteric modulators [ 14 ] and here we can report a new finding that S-Rg3 is a good positive modulator at hP2X4 with some potentiation also seen with F2 and S-Rh2. For the other P2X receptors, there were no statistically significant results from our screening suggesting that ginsenoside potentiation is restricted to just P2X7 and P2X4 receptors. There was a minor potentiation of hP2X2 receptors by CK, S-Rg3 and Rb1 (Fig. 1 ) which we followed up by performing complete concentration-response curves for ATP in the absence and presence of ginsenosides (Fig. 2 ). CK did induce a small shift in the EC50 for ATP which is not hugely dissimilar to the effect of progesterone, an accepted positive modulator for P2X2 [ 21 ]. Regardless, the effect on P2X7 and P2X4 is much greater than at P2X2 suggesting the following rank order of effect for ginsenosides: hP2X7 > hP2X4 > > hP2X2 > hP2X1>hP2X3. Screening of the glycosides did not yield any new discoveries. Finally, we considered how this data could enhance understanding of the potential ginsenoside binding site in the central vestibule region, previously postulated in 2019, but not yet validated [ 13 ]. There is some sequence similarity in the β2 and β14 strands with key residues identified by us (D318, L320, S60) somewhat conserved in P2X1-4 receptors [ 13 ]. If this is the correct location for the ginsenoside binding pocket, there must be other limiting factors preventing potentiation by ginsenosides. For P2X1 and P2X3 receptors, which undergo rapid desensitisation following agonist binding, the lateral portals and central vestibule may not be accessible for long enough to allow significant binding to occur. Further mutagenesis around the predicted pocket and structural studies are needed to understand where positive allosteric modulators bind. Declarations There are no competing interests to declare. For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising from this submission. This project was funded by a BBSRC DTP training grant (reference 2059870) to EA and LS. The work described in this article was supported by the COST Action CA21130 “P2X receptors as a therapeutic opportunity (PRESTO).” Acknowledgements This project was funded by a BBSRC DTP training grant (reference 2059870) to EA and LS. The work described in this article was supported by the COST Action CA21130 “P2X receptors as a therapeutic opportunity (PRESTO).” References Oken AC et al (2022) Molecular Pharmacology of P2X Receptors: Exploring Druggable Domains Revealed by Structural Biology. Frontiers in Pharmacology, pp 13–2022 Illes P et al (2021) Update of P2X receptor properties and their pharmacology: IUPHAR Review 30. Br J Pharmacol 178(3):489–514 Karasawa A, Kawate T (2016) Structural basis for subtype-specific inhibition of the P2X7 receptor. eLife 5:e22153 Oken AC et al (2024) P2X7 receptors exhibit at least three modes of allosteric antagonism. Sci Adv 10(40):eado5084 Shen C et al (2023) Structural insights into the allosteric inhibition of P2X4 receptors. Nat Commun 14(1):6437 Shi H et al (2025) Human P2X4 receptor gating is modulated by a stable cytoplasmic cap and a unique allosteric pocket. Sci Adv 11(3):eadr3315 Wang J et al (2018) Druggable negative allosteric site of P2X3 receptors. Proceedings of the National Academy of Sciences, 115(19): pp. 4939–4944 Matsumoto H et al (2025) Real-world usage and response to gefapixant in refractory chronic cough. ERJ Open Res, : p. 01037–2024 Stokes L et al (2020) To Inhibit or Enhance? Is There a Benefit to Positive Allosteric Modulation of P2X Receptors? Front Pharmacol, Volume 11–2020 Helliwell RM et al (2015) Selected ginsenosides of the protopanaxdiol series are novel positive allosteric modulators of P2X7 receptors. Br J Pharmacol 172(13):3326–3340 Bidula S et al (2019) Positive allosteric modulation of P2X7 promotes apoptotic cell death over lytic cell death responses in macrophages. Cell Death Dis 10(12):882 Dhuna K et al (2024) Ginsenosides enhance P2X7-dependent cytokine secretion from LPS-primed rodent macrophages. Purinergic Signalling 20(1):65–71 Bidula SM et al (2019) Mapping a novel positive allosteric modulator binding site in the central vestibule region of human P2X7. Sci Rep 9(1):3231 Dhuna K et al (2019) Ginsenosides Act As Positive Modulators of P2X4 Receptors. Mol Pharmacol 95(2):210–221 Piyasirananda W et al (2021) Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors. Mol Pharmacol 99(2):163–174 Zuanon M, Brancale A, Young MT (2025) Identification of New Human P2X7 Antagonists Using Ligand- and Structure-Based Virtual Screening. J Chem Inf Model 65(13):7143–7155 Beswick P et al (2019) A challenge finding P2X1 and P2X4 ligands. Neuropharmacology 157:107674 Saul A et al (2013) Heteromeric assembly of P2X subunits. Front Cell Neurosci, Volume 7–2013 Ruepp M-D et al (2015) A fluorescent approach for identifying P2X1 ligands. Neuropharmacology 98:13–21 North RA, Surprenant A (2000) Pharmacology of Cloned P2X Receptors. Annual Review of Pharmacology and Toxicology, 40(Volume 40, 2000): pp. 563–580 De Roo M, Boué-Grabot E, Schlichter R (2010) Selective potentiation of homomeric P2X2 ionotropic ATP receptors by a fast non-genomic action of progesterone. Neuropharmacology 58(3):569–577 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 16 Feb, 2026 Reviews received at journal 14 Feb, 2026 Reviews received at journal 13 Feb, 2026 Reviewers agreed at journal 04 Feb, 2026 Reviewers agreed at journal 02 Feb, 2026 Reviewers invited by journal 02 Feb, 2026 Editor assigned by journal 31 Jan, 2026 Submission checks completed at journal 30 Jan, 2026 First submitted to journal 28 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8724351","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":584919003,"identity":"ddd55a15-066b-4904-b957-7beec5ddb5e3","order_by":0,"name":"Elizabeth Allum","email":"","orcid":"","institution":"University of East Anglia","correspondingAuthor":false,"prefix":"","firstName":"Elizabeth","middleName":"","lastName":"Allum","suffix":""},{"id":584919004,"identity":"7ec70bd3-c234-4d47-8d1d-b4a6b3c820d4","order_by":1,"name":"Leanne Stokes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACPjB5gIGBHy7EQ0ALGwMDYwNIi2QDyVoMDhCvhf35gw9nbOyNbyQf/MBQY8dgcOYAIS08ho0zbqQlbruRlizBcCyZweBsA0EtjM08Hw4nmN3IMZBgYDvAYHCesMMeArX8tzeekf/5B8M/orQwGDbz3DjAuEEih02Cse0AEQ5j5jGcOeNMcuKMM8/MLBL7knkkCXmfn739wYcPx+zs+duTH9/48M1Oju9MAgGXMSNzEgjHyigYBaNgFIwCYgAA/l9BCI9+zuYAAAAASUVORK5CYII=","orcid":"","institution":"University of East Anglia","correspondingAuthor":true,"prefix":"","firstName":"Leanne","middleName":"","lastName":"Stokes","suffix":""}],"badges":[],"createdAt":"2026-01-28 18:23:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8724351/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8724351/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101843972,"identity":"8ff4f75e-1343-4d6e-87b4-108368c5f6df","added_by":"auto","created_at":"2026-02-04 08:58:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":262077,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMembrane potential assay responses for human P2X receptors. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eConcentration response curves for ATP are shown for \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(A)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e P2X1 and P2X3, \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(B)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e P2X2a, P2X4 and P2X7. \u0026nbsp;Responses were baseline corrected and AUC was calculated using Softmax Pro v5.4 software. Data is from 3 independent experiments performed with triplicate wells. Curves are fit using a four-parameter non-linear regression in GraphPad Prism. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC-H\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e)\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eAll\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eginsenosides were screened at 10 µM using a dual injection protocol (modulator added 60 seconds before agonist). ATP was used at approximate EC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e concentrations for each receptor; \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(D)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e hP2X7 200 µM, \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(E)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e hP2X1 10 µM \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(F)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e hP2X2a 10 µM \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(G)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e hP2X3 10 µM \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(H)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e hP2X4 300nM.\u0026nbsp; Responses were baseline corrected and AUC calculated using Softmax Pro v5.4 software then normalised to the vehicle control. Data is from 3-4 independent experiments performed with triplicate wells\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8724351/v1/0f37a39c926bbfe5d08316b4.png"},{"id":101843971,"identity":"85a6a383-edef-48ed-940a-e498d85d5f2d","added_by":"auto","created_at":"2026-02-04 08:58:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":230013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eScreening glycosides for positive modulation at human P2X receptors. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eAll\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eglycosides were screened at 10 µM using a dual injection protocol (modulator added 60 seconds before agonist). ATP was used at approximate EC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e concentrations for each receptor; (A) hP2X7 200 µM, (B) hP2X2a 10 µM (C) hP2X4 300nM.\u0026nbsp; Responses were baseline corrected and AUC calculated using Softmax Pro v5.4 software then normalised to the vehicle control. Data is from 3-4 independent experiments performed with triplicate wells. Concentration response curves were generated for ATP at (D) hP2X2a and E) hP2X4 in membrane potential blue assay and (F) hP2X2a and (G) hP2X4 in a fura-2 assay.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8724351/v1/0059c9e53308c710fec07825.png"},{"id":101843998,"identity":"885375b3-2641-4151-8dbe-b4e49f8f85de","added_by":"auto","created_at":"2026-02-04 08:58:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":902115,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8724351/v1/db8d416c-d28b-4a9b-a832-9854b872ce3b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Positive allosteric modulator activity of ginsenosides is restricted to P2X7 and P2X4 receptors","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe seven subtypes of P2X receptors are structurally similar with a common trimeric structure [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Subtle differences exist in the orthosteric ligand binding site, giving rise to different affinities for ATP and differences in pharmacological activity of ATP analogues (e.g. BzATP, αβ-meATP) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Allosteric modulators can interact with several sites on P2X receptors, identified through a number of x-ray crystallography and cryo-EM studies [\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. One negative allosteric modulator is located behind the orthosteric ATP site on P2X7 and an analogous site is found on P2X4 [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] whereas with P2X3, a negative allosteric site is located beneath the orthosteric site [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Several negative allosteric modulators for P2X receptors have entered clinical trials for a range of disorders [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], with gefapixant being the first clinically used medicine targeting a P2X receptor [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe discovery and characterisation of positive allosteric modulators of P2X receptors lags behind that for negative allosteric modulators. Over the last decade several have been documented, reviewed in [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] with a bias towards those acting at P2X7. Our group discovered a series of positive allosteric modulator compounds derived from Panax ginseng [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and subsequently we have characterised their functional effects in macrophages [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and explored a potential binding site in the central vestibule region [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. We found that several ginsenoside compounds could also potentiate P2X4 responses [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], suggesting that this could be a conserved mechanism of action within the family. We have also previously reported a series of related glycosides with PAM activity at P2X7 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This study aims to investigate whether ginsenosides could potentiate ATP responses at other P2X receptors, focusing on P2X1-3. To do this we wanted to use the same experimental assay system to minimise variation between assays therefore we employed the membrane potential assay used in several other studies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHEK-293 cells were used to generate stable cell lines expressing human P2X receptors. Original transfections were performed using Lipofectamine 2000 (Fisher Scientific) and cells were subjected to geneticin (800 \u0026micro;g/ml) selection for 2\u0026ndash;3 weeks followed by single cell cloning by limiting dilution in 96-well plates. Individual clones were selected for further study based on ATP-induced responses. Cell lines were maintained in DMEM:F12 media supplemented with 10% foetal bovine serum (PanBiotech), penicillin (100 U/ml) and streptomycin (100 \u0026micro;g/ml) (Fisher Scientific) and kept in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. Stable P2X-expressing cells were kept under geneticin (HelloBio) selection at 400 \u0026micro;g/ml. Passaging was performed twice weekly using 0.25% Trypsin-EDTA (Fisher Scientific).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMembrane potential measurements\u003c/h3\u003e\n\u003cp\u003e96-well plates (catalogue number 10212811, ThermoFisher Scientific) were coated with 50 \u0026micro;g/mL Poly-D-Lysine (P6407, Sigma-Aldrich) and left for at least one hour before being washed with sterile water and allowed to dry. Cells were plated in 100 \u0026micro;L of complete DMEM/F-12 the day prior to experimentation into PDL-coated 96- well plates at 2\u0026ndash;3 x 10\u003csup\u003e4\u003c/sup\u003e cells per well. Plated cells were kept in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C. The Membrane Potential Blue Kit (Molecular Devices) was reconstituted according to the manufacturer\u0026rsquo;s instructions using standard extracellular buffer (145 mM NaCl, 2 mM KCl, 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 1mM MgCl\u003csub\u003e2\u003c/sub\u003e, 13 mM glucose, 10 mM HEPES, pH 7.3\u0026ndash;7.4) and kept in 5ml aliquots frozen at -20˚C. This was then diluted 1 in 2 with the extracellular buffer immediately prior to use unless otherwise stated. Cell medium was removed from the 96-well plate and replaced with 100 \u0026micro;L of membrane potential blue assay buffer. Cells were incubated in this buffer for 30 minutes at 37\u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were then transferred to a FlexStation 3 plate reader (Molecular Devices) and warmed at 37\u0026deg;C for an additional 5 minutes prior to experimentation. Fluorescence measurements were acquired using SoftMax Pro v5.4 software (Molecular Devices). Fluorescence emission was measured at 565 nm for 120 seconds with an excitation wavelength of 525 nm (3 reads per well, PMT medium, interval 2 seconds). Agonists and screened compounds were made up at either 5x or 10x final concentrations in buffer and applied to cells after 20 seconds of read time at an injection rate of 2\u0026ndash;4. For dual injection screening experiments conducted in membrane potential blue, compounds were injected at 20 seconds, followed by a 60 second interval before the injection of the agonist ATP at 80 seconds (allowing for 60 seconds of pre-treatment with the compound before the application of ATP).\u003c/p\u003e\n\u003ch3\u003eFura-2 calcium measurements\u003c/h3\u003e\n\u003cp\u003eCells were loaded with 100\u0026micro;L of loading buffer per well (low divalent extracellular buffer 145 mM NaCl, 2 mM KCl, 0.2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 13 mM glucose, 10 mM HEPES, pH 7.3\u0026ndash;7.4) containing 250\u0026micro;M sulfinpyrazone and 2\u0026micro;M Fura-2-acetoxymethyl (fura-2AM) calcium indicator dye (Hello Bio, UK) and incubated for 45 minutes at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. This allows the membrane-permeant fura-2 AM ester to move into cells, after which it is cleaved by cellular esterases to reveal the active fura-2 Ca2\u0026thinsp;+\u0026thinsp;indicator dye. After loading, the loading buffer was removed and replaced with 180\u0026micro;L per well of standard extracellular buffer. Compounds to be tested were made up at 10x final concentration and were injected 40 seconds into read time at a rate of 2\u0026ndash;4. Intracellular Ca\u003csup\u003e2+\u003c/sup\u003e measurements were made using a Flexstation 3 Multimode Plate Reader (Molecular Devices) and data analysed using SoftMax Pro v5.4 software (Molecular Devices). Fluorescence emission was measured at 510 nm for 300 seconds (3 reads per well, PMT medium, interval 3.5 seconds) with dual excitation wavelengths of 340nm and 380nm to give a ratio between Ca\u003csup\u003e2+\u003c/sup\u003e-bound and Ca\u003csup\u003e2+\u003c/sup\u003e-free fura-2 respectively.\u003c/p\u003e\n\u003ch3\u003ePlotting and Statistical analysis\u003c/h3\u003e\n\u003cp\u003eData from membrane potential experiments were plotted as area under curve (AUC) as % of control where response with each compound was normalised to the vehicle control. For fura-2 experiments, ratio data was analysed with zero baseline (3 points) and area under curve calculated from 0-300 seconds. Data from three independent experiments was collated and tested for statistical significance using a one-way ANOVA with Dunnett\u0026rsquo;s multiple comparison test (GraphPad Prism v9). P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was taken as the minimum level of statistical significance. Dose-response curves were plotted by a nonlinear regression fit with variable slope using GraphPad Prism software version 9. Half-maximal responses are expressed as mean EC\u003csub\u003e50\u003c/sub\u003e values with standard deviation from three independent experiments.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFirstly, we optimised the membrane potential blue assay for measuring P2X responses to the primary agonist ATP. A Flexstation 3 plate reader was used to measure ATP-induced responses and concentration-response curves were generated from area under the curve (AUC) data. We generated clonal stable cell lines for each of the human receptors (hP2X1, hP2X2a, hP2X3) in addition to our stable cell lines for hP2X4 and hP2X7 to reduce variability from transient transfections. ATP responses were measured in standard extracellular buffer containing 2 mM calcium and 1 mM magnesium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We did not study hP2X5 or hP2X6 receptors as these are not known to exist as homomeric receptors \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. EC\u003csub\u003e50\u003c/sub\u003e values for ATP were as follows; hP2X1 8.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 \u0026micro;M, hP2X2a 17.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0 \u0026micro;M, hP2X3 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026micro;M, hP2X4 0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 \u0026micro;M, and hP2X7 638.5\u0026thinsp;\u0026plusmn;\u0026thinsp;354 \u0026micro;M (n\u0026thinsp;=\u0026thinsp;3 independent experiments). A representative trace showing the typical response of each P2X receptor is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC.\u003c/p\u003e \u003cp\u003eWe tested 9 ginsenoside chemicals on HEK-293 cells expressing hP2X receptors using the membrane potential blue assay and standard extracellular buffer to keep the experimental conditions consistent. An approximate EC\u003csub\u003e50\u003c/sub\u003e concentration of agonist was used for each individual P2X receptor, and a dual injection protocol was implemented allowing compound/vehicle injection 60 seconds before the agonist. All ginsenosides were tested at a final concentration of 10 \u0026micro;M and data in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e is presented as % of control. We first confirmed that robust potentiation by protopanaxadiol ginsenosides was seen in HEK-hP2X7 using 200 \u0026micro;M ATP (as in previous studies) to induce ion channel activation. 20-S-Rg3, CK and F2 performed as the best PAMs at hP2X7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) which agrees with our previous work where screening was performed using the YO-PRO-1 dye uptake assay [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor hP2X4 we have previously reported that ginsenosides CK, Rd, Rb1, and Rh2 displayed positive allosteric modulator activity using fura-2 calcium measurements [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This was confirmed in the membrane potential assay, and here we discovered that 20-S-Rg3 and F2 are also effective at potentiating the P2X4 receptor (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). We tested the stereoisomers of Rg3 and Rh2 as we previously reported a striking difference in PAM activity at hP2X7 with only S-enantiomers being active [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. R-enantiomers displayed some PAM activity at hP2X4 in these screening experiments, although this did not reach statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). For hP2X2a we observed increased ATP responses with CK, 20-S-Rg3, 20-R-Rg3, 20-S-Rh2 and Rb1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) however, this was variable and not statistically significant. hP2X1 and hP2X3 did not show significant potentiation by any of the ginsenosides tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we assessed a series of glycosides with similar chemical structures to the ginsenosides. Most of the glycosides did not show robust potentiating activity at hP2X7 in this assay with the exception of stevenleaf (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Our previous study had demonstrated that gypenoside XLIX, XVII and daucosterol showed PAM activity at hP2X7 although this was seen as an increase in maximum response in the dye uptake assay rather than a shift in the ATP dose response curves [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. For hP2X2a, there was a lot of variability and none of the glycosides showed a statistically significant increase compared to DMSO control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). For hP2X4 receptors several glycosides increased the EC\u003csub\u003e50\u003c/sub\u003e ATP responses including oleanolic acid and stevenleaf (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eWe further investigated selected ginsenosides at hP2X2a and hP2X4 using concentration response curves to ATP. Using the membrane potential assay, ginsenosides CK and Rb1 induced a minor shift in the ATP concentration response curve at hP2X2a receptors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The average EC\u003csub\u003e50\u003c/sub\u003e for ATP was 12.62\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4 \u0026micro;M in these experiments and in the presence of ginsenosides CK and Rb1 this was 8.93\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2 \u0026micro;M and 8.52\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 \u0026micro;M respectively. For hP2X4, a left-ward shift in the ATP concentration response curve was observed for ginsenoside CK and S-Rg3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) with EC\u003csub\u003e50\u003c/sub\u003e values shifting from 975\u0026thinsp;\u0026plusmn;\u0026thinsp;333 nM to 407\u0026thinsp;\u0026plusmn;\u0026thinsp;302 nM with CK and to 563\u0026thinsp;\u0026plusmn;\u0026thinsp;572 nM with S-Rg3. We then used a second assay to measure P2X2 and P2X4 responses, the fura-2 assay and repeated the concentration responses to ATP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis is the first study where multiple P2X receptor responses have been measured using a single assay for screening purposes. The FLIPR membrane potential assay was easy to use and has potential value in screening programs. We used the membrane potential blue assay kit from Molecular Devices, however the same kit is available in a red assay utilising a different quenching dye. Others have used the membrane potential assay for investigating P2X1 responses [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] or P2X7 responses [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. EC\u003csub\u003e50\u003c/sub\u003e values for ATP at multiple hP2X receptors were similar to those stated in literature [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and we used this information to select an EC\u003csub\u003e50\u003c/sub\u003e concentration for screening experiments. With P2X7 we have a wealth of evidence for positive allosteric modulation by ginsenosides [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and we see similar effects for key ginsenosides (CK, F2, S-Rg3, S-Rh2 and Rd) in the membrane potential assay, validating its use for screening. With P2X4, we have previously reported that CK and Rd are effective positive allosteric modulators [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and here we can report a new finding that S-Rg3 is a good positive modulator at hP2X4 with some potentiation also seen with F2 and S-Rh2. For the other P2X receptors, there were no statistically significant results from our screening suggesting that ginsenoside potentiation is restricted to just P2X7 and P2X4 receptors.\u003c/p\u003e \u003cp\u003eThere was a minor potentiation of hP2X2 receptors by CK, S-Rg3 and Rb1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) which we followed up by performing complete concentration-response curves for ATP in the absence and presence of ginsenosides (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). CK did induce a small shift in the EC50 for ATP which is not hugely dissimilar to the effect of progesterone, an accepted positive modulator for P2X2 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Regardless, the effect on P2X7 and P2X4 is much greater than at P2X2 suggesting the following rank order of effect for ginsenosides: hP2X7\u0026thinsp;\u0026gt;\u0026thinsp;hP2X4\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;hP2X2\u0026thinsp;\u0026gt;\u0026thinsp;hP2X1\u0026gt;hP2X3. Screening of the glycosides did not yield any new discoveries.\u003c/p\u003e \u003cp\u003eFinally, we considered how this data could enhance understanding of the potential ginsenoside binding site in the central vestibule region, previously postulated in 2019, but not yet validated [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. There is some sequence similarity in the β2 and β14 strands with key residues identified by us (D318, L320, S60) somewhat conserved in P2X1-4 receptors [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. If this is the correct location for the ginsenoside binding pocket, there must be other limiting factors preventing potentiation by ginsenosides. For P2X1 and P2X3 receptors, which undergo rapid desensitisation following agonist binding, the lateral portals and central vestibule may not be accessible for long enough to allow significant binding to occur. Further mutagenesis around the predicted pocket and structural studies are needed to understand where positive allosteric modulators bind.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThere are no competing interests to declare.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising from this submission.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;This project was funded by a BBSRC DTP training grant (reference 2059870) to EA and LS. The work described in this article was supported by the COST Action CA21130 \u0026ldquo;P2X receptors as a therapeutic opportunity (PRESTO).\u0026rdquo;\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was funded by a BBSRC DTP training grant (reference 2059870) to EA and LS. The work described in this article was supported by the COST Action CA21130 \u0026ldquo;P2X receptors as a therapeutic opportunity (PRESTO).\u0026rdquo;\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOken AC et al (2022) Molecular Pharmacology of P2X Receptors: Exploring Druggable Domains Revealed by Structural Biology. Frontiers in Pharmacology, pp 13\u0026ndash;2022\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIlles P et al (2021) Update of P2X receptor properties and their pharmacology: IUPHAR Review 30. Br J Pharmacol 178(3):489\u0026ndash;514\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarasawa A, Kawate T (2016) Structural basis for subtype-specific inhibition of the P2X7 receptor. eLife 5:e22153\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOken AC et al (2024) P2X7 receptors exhibit at least three modes of allosteric antagonism. Sci Adv 10(40):eado5084\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen C et al (2023) Structural insights into the allosteric inhibition of P2X4 receptors. Nat Commun 14(1):6437\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi H et al (2025) Human P2X4 receptor gating is modulated by a stable cytoplasmic cap and a unique allosteric pocket. Sci Adv 11(3):eadr3315\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J et al (2018) \u003cem\u003eDruggable negative allosteric site of P2X3 receptors.\u003c/em\u003e Proceedings of the National Academy of Sciences, 115(19): pp. 4939\u0026ndash;4944\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsumoto H et al (2025) Real-world usage and response to gefapixant in refractory chronic cough. ERJ Open Res, : p. 01037\u0026ndash;2024\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStokes L et al (2020) To Inhibit or Enhance? Is There a Benefit to Positive Allosteric Modulation of P2X Receptors? Front Pharmacol, Volume 11\u0026ndash;2020\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHelliwell RM et al (2015) Selected ginsenosides of the protopanaxdiol series are novel positive allosteric modulators of P2X7 receptors. Br J Pharmacol 172(13):3326\u0026ndash;3340\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBidula S et al (2019) Positive allosteric modulation of P2X7 promotes apoptotic cell death over lytic cell death responses in macrophages. Cell Death Dis 10(12):882\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDhuna K et al (2024) Ginsenosides enhance P2X7-dependent cytokine secretion from LPS-primed rodent macrophages. Purinergic Signalling 20(1):65\u0026ndash;71\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBidula SM et al (2019) Mapping a novel positive allosteric modulator binding site in the central vestibule region of human P2X7. Sci Rep 9(1):3231\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDhuna K et al (2019) Ginsenosides Act As Positive Modulators of P2X4 Receptors. Mol Pharmacol 95(2):210\u0026ndash;221\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePiyasirananda W et al (2021) Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors. Mol Pharmacol 99(2):163\u0026ndash;174\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZuanon M, Brancale A, Young MT (2025) Identification of New Human P2X7 Antagonists Using Ligand- and Structure-Based Virtual Screening. J Chem Inf Model 65(13):7143\u0026ndash;7155\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeswick P et al (2019) A challenge finding P2X1 and P2X4 ligands. Neuropharmacology 157:107674\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaul A et al (2013) Heteromeric assembly of P2X subunits. Front Cell Neurosci, Volume 7\u0026ndash;2013\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuepp M-D et al (2015) A fluorescent approach for identifying P2X1 ligands. Neuropharmacology 98:13\u0026ndash;21\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNorth RA, Surprenant A (2000) \u003cem\u003ePharmacology of Cloned P2X Receptors.\u003c/em\u003e Annual Review of Pharmacology and Toxicology, 40(Volume 40, 2000): pp. 563\u0026ndash;580\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Roo M, Bou\u0026eacute;-Grabot E, Schlichter R (2010) Selective potentiation of homomeric P2X2 ionotropic ATP receptors by a fast non-genomic action of progesterone. Neuropharmacology 58(3):569\u0026ndash;577\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"purinergic-signalling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pusi","sideBox":"Learn more about [Purinergic Signalling](http://link.springer.com/journal/11302)","snPcode":"11302","submissionUrl":"https://submission.nature.com/new-submission/11302/3","title":"Purinergic Signalling","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"P2X receptor, positive modulation, ginsenoside, glycoside","lastPublishedDoi":"10.21203/rs.3.rs-8724351/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8724351/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePositive allosteric modulators of P2X7 receptors hold promise as host-directed immune enhancers in intracellular infections. We have previously reported the use of protopanaxadiol ginsenosides as positive allosteric modulators of human, rat and mouse P2X7 receptors plus human P2X4 receptors, however, the effect of ginsenosides on other P2X receptors is unknown. Here, we screened a range of ginsenosides and related glycosides at hP2X1, hP2X2 and hP2X3 receptors expressed in HEK-293 cells utilising a membrane potential assay. Our results demonstrate the utility of the membrane potential assay across all P2X receptors and the lack of significant potentiation of ATP-induced responses at hP2X1, hP2X2 and hP2X3 receptors by ginsenosides and related glycosides. We report that S-Rg3 also acts as a positive allosteric modulator at hP2X4 receptors in addition to hP2X7 receptors. This information is important for determining the selectivity of ginsenoside positive allosteric modulators for P2X7 and P2X4.\u003c/p\u003e","manuscriptTitle":"Positive allosteric modulator activity of ginsenosides is restricted to P2X7 and P2X4 receptors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-04 08:58:37","doi":"10.21203/rs.3.rs-8724351/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-16T10:31:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-14T18:29:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-13T12:24:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172443233840178680517929828898419844741","date":"2026-02-04T07:50:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"141728477256726182842515531879657814066","date":"2026-02-02T19:32:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-02T18:22:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-31T05:05:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-30T08:40:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Purinergic Signalling","date":"2026-01-28T18:06:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"purinergic-signalling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pusi","sideBox":"Learn more about [Purinergic Signalling](http://link.springer.com/journal/11302)","snPcode":"11302","submissionUrl":"https://submission.nature.com/new-submission/11302/3","title":"Purinergic Signalling","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d31b161c-5da9-44af-993c-13229cf15b5e","owner":[],"postedDate":"February 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T09:40:36+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-04 08:58:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8724351","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8724351","identity":"rs-8724351","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}