Received for publication April 12, 2007.
Revised July 26, 2007.
Accepted for publication July 26, 2007.

[³H]A-778317 [1-((R)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea]: A Novel, Stereoselective, High-Affinity Antagonist is a Useful Radioligand For The Human TRPV1 Receptor

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, A-778316, is 6.8-fold less potent (pIC₅₀ = 7.47 ± 0.07). A-778317 also potently blocks capsaicin (CAP) and acid activation of native rat TRPV1 receptors in dorsal root ganglion (DRG) neurons. A-778317 was tritiated ([³H]A-778317; 29.3 Ci/mmol) and utilized to study recombinant human TRPV1 (hTRPV1) receptors expressed in CHO cells. ([³H]A-778317 labeled a single class of binding sites in hTRPV1-expressing CHO cell membranes with high affinity (K_D = 3.4 nM; B_max = 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.

Key words: capsaicin, capsazepine, radioligand, resiniferatoxin, tinyatoxin, transient receptor potential vanilloid-1