title: Structure-based identification and characterisation of structurally novel human P2X7 receptor antagonists.
abstract: The P2X7 receptor (P2X7R) plays an important role in diverse conditions associated with tissue damage and inflammation, meaning that the human P2X7R (hP2X7R) is an attractive therapeutic target. The crystal structures of the zebrafish P2X4R in the closed and ATP-bound open states provide an unprecedented opportunity for structure-guided identification of new ligands. The present study performed virtual screening of ∼100,000 structurally diverse compounds against the ATP-binding pocket in the hP2X7R. This identified three compounds (C23, C40 and C60) out of 73 top-ranked compounds by testing against hP2X7R-mediated Ca(2+) responses. These compounds were further characterised using Ca(2+) imaging, patch-clamp current recording, YO-PRO-1 uptake and propidium iodide cell death assays. All three compounds inhibited BzATP-induced Ca(2+) responses concentration-dependently with IC50s of 5.1±0.3μM, 4.8±0.8μM and 3.2±0.2μM, respectively. C23 and C40 inhibited BzATP-induced currents in a reversible and concentration-dependent manner, with IC50s of 0.35±0.3μM and 1.2±0.1μM, respectively, but surprisingly C60 did not affect BzATP-induced currents up to 100μM. They suppressed BzATP-induced YO-PRO-1 uptake with IC50s of 1.8±0.9μM, 1.0±0.1μM and 0.8±0.2μM, respectively. Furthermore, these three compounds strongly protected against ATP-induced cell death. Among them, C40 and C60 exhibited strong specificity towards the hP2X7R over the hP2X4R and rP2X3R. In conclusion, our study reports the identification of three novel hP2X7R antagonists with micromolar potency for the first time using a structure-based approach, including the first P2X7R antagonist with preferential inhibition of large pore formation.
authors.affiliation: School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, UK.
authors.forename: Emily A
authors.initials: EA
authors.lastname: Caseley
authors.affiliation: School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, UK.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Muench
authors.affiliation: School of Chemistry, University of Leeds, UK.
authors.forename: Colin W
authors.initials: CW
authors.lastname: Fishwick
authors.affiliation: School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, UK. Electronic address: l.h.jiang@leeds.ac.uk.
authors.forename: Lin-Hua
authors.initials: LH
authors.lastname: Jiang
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000577:Amides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D020013:Calcium Signaling / Q000187:drug effects; D057809:HEK293 Cells; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007211:Indoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D008024:Ligands; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D018408:Patch-Clamp Techniques; D058920:Purinergic P2X Receptor Antagonists / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D058477:Receptors, Purinergic P2X3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058848:Receptors, Purinergic P2X4 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058486:Receptors, Purinergic P2X7 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D059010:Single-Cell Analysis; D054852:Small Molecule Libraries; D013329:Structure-Activity Relationship; D013876:Thiophenes / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000577:Amides; D000894:Anti-Inflammatory Agents, Non-Steroidal; D007211:Indoles; D008024:Ligands; C548226:P2RX4 protein, human; C546468:P2RX7 protein, human; C546410:P2rx3 protein, rat; D058920:Purinergic P2X Receptor Antagonists; D058477:Receptors, Purinergic P2X3; D058848:Receptors, Purinergic P2X4; D058486:Receptors, Purinergic P2X7; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D013876:Thiophenes; C000618335:ZINC09315614; C000618334:ZINC58368839; C000618333:ZINC67825876; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate
keywords: Ca(2+) imaging; Patch-clamp recording; Virtual screening; YO-PRO-1 dye uptake; hP2X7R antagonists