title: Design of a high-affinity competitive antagonist of the vanilloid receptor selective for the calcium entry-linked receptor population.
abstract: We describe the synthesis and characterization of N-(4-chlorobenzyl) -N'-(4-hydroxy-3-iodo-5-methoxybenzyl)thiourea (IBTU), a novel antagonist of the vanilloid receptor 1 (TRPV1 or VR1). IBTU competitively inhibited 45Ca2+ uptake into CHO cells heterologously expressing rat TRPV1, whether induced by capsaicin or resiniferatoxin (Ki = 99 +/- 23 and 93 +/- 34 nM, respectively). IBTU was thus somewhat more potent (5-fold) than capsazepine. In contrast to its antagonism of vanilloid-induced calcium uptake, IBTU (30 microM) inhibited [3H]resiniferatoxin binding to TRPV1 by less than 10%. We hypothesize that these dramatically distinct potencies reflect different fractions of TRPV1 in this system: namely, a minor plasma membrane fraction controlling 45Ca2+ uptake, and the predominant intracellular fraction that dominates the [3H]resiniferatoxin binding measurements. Intracellular Ca2+ imaging supports this explanation. IBTU antagonized the elevation in intracellular Ca2+ in response to 50 nM capsaicin with an IC50 of 106 +/- 35 nM. Likewise, 600 nM IBTU was able to antagonize the elevation in intracellular Ca2+ in response to 100 pM resiniferatoxin in the presence of normal (1.8 mM) extracellular Ca2+, where the increase in intracellular calcium reflects calcium influx. In contrast, in the absence of extracellular Ca2+, where in this system resiniferatoxin induces a modest increase in calcium from intracellular stores, IBTU was unable to block the response to resiniferatoxin, although the TRPV1 antagonist 5-iodoresiniferatoxin was able to do so. In summary, IBTU is a novel, potent TRPV1 antagonist with marked selectivity between subpopulations of TRPV1 and may permit the function of these distinct pools to be explored and potentially exploited.
authors.affiliation: Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
authors.forename: Attila
authors.initials: A
authors.lastname: Tóth
authors.forename: Peter M
authors.initials: PM
authors.lastname: Blumberg
authors.forename: Zili
authors.initials: Z
authors.lastname: Chen
authors.forename: Alan P
authors.initials: AP
authors.lastname: Kozikowski
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002211:Capsaicin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D044169:Receptors, Calcium-Sensing / Q000378:metabolism*; D011955:Receptors, Drug / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D050916:TRPV Cation Channels; D013890:Thiourea / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000378:metabolism* / Q000494:pharmacology
chemical_list: C482496:N-(4-chlorobenzyl) -N'-(4-hydroxy-3-iodo-5-methoxybenzyl)thiourea; D044169:Receptors, Calcium-Sensing; D011955:Receptors, Drug; D050916:TRPV Cation Channels; C494484:TRPV1 receptor; D013890:Thiourea; C071423:capsazepine; D002211:Capsaicin; D002118:Calcium