High Affinity Antagonists of the Vanilloid Receptor YUN WANG, 1 TAMAS SZABO, JACQUELINE D. WELTER, ATTILA TOTH, RICHARD TRAN, JIYOUN LEE, SANG UK KANG, YOUNG-GER SUH, PETER M. BLUMBERG, and JEEWOO LEE National Cancer Institute, Bethesda, Maryland (Y.W., T.S., J.D.W., A.T., R.T., P.M.B.); and College of Pharmacy, Seoul National University, Seoul, Korea (Ji.L., S.U.K., Y.-G.S., Je.L.) Received March 12, 2002; accepted July 16, 2002 This article is available online at http://molpharm.aspetjournals.org ABSTRACT The vanilloid receptor VR1 has attracted great interest as a sensory transducer for capsaicin, protons, and heat, and as a therapeutic target. Here we characterize two novel VR1 antagonists, KJM429 [N-(4-tert-butylbenzyl)-N-[4-(methyl- sulfonylamino)benzyl]thiourea] and JYL1421 [ N-(4- tert - butylbenzyl)- N -[3-fluoro-4-(methylsulfonylamino)benzyl]- thiourea], with enhanced activity compared with capsazepine on rat VR1 expressed in Chinese hamster ovary (CHO) cells. JYL1421, the more potent of the two novel antagonists, inhibited [ 3 H]resiniferatoxin binding to rVR1 with an affinity of 53.5  6.5 nM and antagonized capsaicin- induced calcium uptake with an EC 50 of 9.2  1.6 nM, reflecting 25- and 60-fold greater potencies than capsa- zepine. Both JYL1421 and KJM429 antagonized RTX as well as capsaicin and their mechanism was competitive. The responses to JYL1421 and KJM429 differed for calcium uptake by rVR1 induced by heat or pH. JYL1421 antagonized the response to both pH 6.0 and 5.5, whereas KJM429 antagonized at pH 6.0 but was an agonist at lower pH (5.5). For heat, JYL1421 fully antagonized and KJM429 partially antagonized. Capsazepine showed only weak antagonism for both pH and heat. Responses of rVR1 to different acti- vators could thus be differentially affected by different li- gands. In cultured dorsal root ganglion neurons, JYL1421 and KJM429 likewise behaved as antagonists for capsaicin, confirming that the antagonism is not limited to heterologous expression systems. Finally, JYL1421 and KJM429 had little or no effect on ATP-induced calcium uptake in CHO cells lacking rVR1, unlike capsazepine. We conclude that JYL1421 is a competitive antagonist of rVR1, blocking re- sponse to all three of the agonists (capsaicin, heat, and protons) with enhanced potency relative to capsazepine. A vanilloid receptor (VR1) that is activated by capsaicin, low pH, and temperatures higher than 42°C has been cloned from rat dorsal root ganglia (Caterina et al., 1997; Tominaga et al., 1998). It is a nonselective cation channel, with high permeability for divalent cations, expressed on unmyelinated pain-sensing nerve fibers (C-fibers) and small A fibers in the dorsal root, trigeminal, and nodose ganglia. Initially, activa- tion of VR1 by pungent agonists such as capsaicin leads to excitation of primary sensory neurons gating nociceptive in- puts to the central nervous system. Subsequently, these fi- bers may become desensitized/defunctionalized, and this de- sensitization forms a basis for the therapeutic use of VR1 agonists (Szallasi and Blumberg, 1999). Potential therapeu- tic applications include detrusor hyperreflexia, postherpetic neuralgia, diabetic neuropathy, cluster headache, osteoar- thritis, and pruritus (Rains and Bryson, 1995; Kim and Chancellor, 2000). The exciting potential therapeutic applications for va- nilloids have motivated efforts to identify or design novel derivatives with improved properties (Walpole et al., 1993a,b,c; Wrigglesworth et al., 1996). An important advance was the identification of resiniferatoxin (RTX), a diterpene related to the phorbol esters, as an ultrapotent capsaicin analog (Szallasi and Blumberg, 1989). RTX demonstrated that orders of magnitude of additional affinity for VR1 could be captured through appropriate chemistry. Furthermore, because RTX was much more potent than capsaicin for de- sensitization, whereas it was only modestly more potent for inducing acute pain, as determined in the eye wiping assay, the behavior of RTX strongly suggested that these different biological endpoints could be dissociated, at least in part. This was important, because desensitization is a therapeutic goal, whereas the induction of acute pain is the limiting This research was partly supported by a Fund 2000 grant from the Korea Research Foundation. 1 Present address: Neuroscience Research Institute, Peking University, 100083, Beijing, People’s Republic of China. ABBREVIATIONS: RTX, resiniferatoxin; HEK, human embryonic kidney; CHO, Chinese hamster ovary; rVR1, cloned rat vanilloid receptor subtype-1; CHO/rVR1 cell, Chinese Hamster Ovary cells transfected with cloned rat vanilloid receptor subtype-1; DRG, dorsal root ganglion; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; JYL1421, N-(4-tert-butylbenzyl)-N-[3-fluoro-4-(methylsulfonylamino)ben- zyl]thiourea; KJM429, N-(4-tert-butylbenzyl)-N-[4-(methylsulfonylamino)benzyl]thiourea; DPBS, Dulbecco’s phosphate-buffered saline; PBS, phosphate-buffered saline; CPZ, capsazepine; MES, 2-[N-Morpholino]ethanesulfonic acid. 0026-895X/02/6204-947–956 MOLECULAR PHARMACOLOGY Vol. 62, No. 4 U.S. Government work not protected by U.S. copyright 1729/1014115 Mol Pharmacol 62:947–956, 2002 Printed in U.S.A. 947