Abstract
1-((R)-5-tert-Butyl-indan-1-yl)-3-isoquinolin-5-yl-urea (A-778317) is a novel, stereoselective, competitive antagonist that potently blocks transient receptor potential vanilloid-1 (TRPV1) receptor-mediated changes in intracellular calcium concentrations (pIC50 = 8.31 ± 0.13). The (S)-stereoisomer, 1-((S)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea (A-778316), is 6.8-fold less potent (pIC50 = 7.47 ± 0.07). A-778317 also potently blocks capsaicin and acid activation of native rat TRPV1 receptors in dorsal root ganglion neurons. A-778317 was tritiated ([3H]A-778317; 29.3 Ci/mmol) and used to study recombinant human TRPV1 (hTRPV1) receptors expressed in Chinese ovary cells (CHO) cells. [3H]A-778317 labeled a single class of binding sites in hTRPV1-expressing CHO cell membranes with high affinity (KD = 3.4 nM; Bmax = 4.0 pmol/mg protein). Specific binding of 2 nM [3H]A-778317 to hTRPV1-expressing CHO cell membranes was reversible. The rank-order potency of TRPV1 receptor antagonists to inhibit binding of 2 nM [3H]A-778317 correlated well with their functional potencies in blocking TRPV1 receptor activation. The present data demonstrate that A-778317 blocks polymodal activation of the TRPV1 receptor by binding to a single high-affinity binding site and that [3H]A-778317 possesses favorable binding properties to facilitate further studies of hTRPV1 receptor pharmacology.
Footnotes
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.124305.
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ABBREVIATIONS: TRPV1, transient receptor potential vanilloid-1; h, human; CHO, Chinese hamster ovary; DRG, dorsal root ganglion; D-PBS, Dulbecco's phosphate-buffered saline; HBSS, Hanks' balanced salt solution; DMEM, Dulbecco's modified Eagle's medium; A-778317, 1-((R)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea; A-778316, 1-((S)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea; A-425619, 1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea; A-784168, 1-[3-(trifluoromethyl)pyridin-2-yl]-N-[4-(trifluoromethylsulfonyl-)phenyl]-1,2,3,6-tetrahydropyridine-4-carboxamide; SB-452533, 1-(2-bromo-phenyl)-3-[2-(ethyl-m-tolyl-amino)-ethyl]-urea; AMG6880, (E)-3-(2-(piperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)-N-(quinolin-7-yl)acrylamide; JNJ compound, 1-[6-fluoro-1-(3-trifluoromethyl-benzyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-3-isoquinolin-5-yl-urea; CPZ, capsazepine; CAP, capsaicin; RTX, resiniferatoxin; I-RTX, iodo-RTX; NADA, N-arachidonoyl-dopamine; TFA, trifluoroacetic acid; nH, Hill slope; crude 1, [6,8-3H]isoquinolin-5-ylamine; dpm, disintegrations per minute.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received April 11, 2007.
- Accepted July 26, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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