[H]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 Transient Receptor Potential Vanilloid-1 (TRPV1) Receptor

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) receptormediated 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 ([H]A-778317; 29.3 Ci/mmol) and used to study recombinant human TRPV1 (hTRPV1) receptors expressed in Chinese ovary cells (CHO) cells. [H]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 [H]A-778317 to hTRPV1-expressing CHO cell membranes was reversible. The rank-order potency of TRPV1 receptor antagonists to inhibit binding of 2 nM [H]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 [H]A-778317 possesses favorable binding properties to facilitate further studies of hTRPV1 receptor pharmacology. The transient receptor potential vanilloid-1 (TRPV1) receptor is a nonselective cation channel, characterized by six transmembrane domains and intracellular N and C termini (Benham et al., 2002). The distribution of the TRPV1 receptor is widespread throughout the nervous system, showing highest levels of expression in sensory neurons of the dorsal root and trigeminal ganglia (Mezey et al., 2000; Sanchez et al., 2001). It is believed that nociceptive signaling of acute and chronic inflammatory pain is mediated in part by activation of the TRPV1 receptor (Honore et al., 2005). Both native and recombinant TRPV1 receptors are activated by diverse pain-producing stimuli, such as noxious heat ( 43°C), protons ( pH 6), capsaicin (CAP), and resiniferatoxin (RTX) (Szallasi and Blumberg, 1999; Caterina and Julius, 2001). Furthermore, several naturally occurring lipArticle, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.124305. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 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, [6,8H]isoquinolin-5-ylamine; dpm, disintegrations per minute. 0022-3565/07/3231-285–293$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 323, No. 1 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 124305/3260977 JPET 323:285–293, 2007 Printed in U.S.A. 285 http://jpet.aspetjournals.org/content/suppl/2007/07/27/jpet.107.124305.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jne 4, 2017 jpet.asjournals.org D ow nladed from ids have been described to have agonist activity at the TRPV1 receptor, including the endocannabinoid anandamide (Smart et al., 2000) and N-acyl-dopamine derivatives, such as N-arachidonoyl-dopamine (NADA) (Huang et al., 2002). Small molecule antagonists of the TRPV1 receptor have been identified from diverse structural classes (Correll and Palani, 2006) and reported to be effective in preclinical animal models of pain and hyperalgesia. These include the following: SB-452533 (Rami et al., 2004); AMG6880 (Doherty et al., 2005; Gavva et al., 2005); A-784168 (Cui et al., 2006); JNJ compound (Jetter et al., 2004); and A-425619 (El Kouhen et al., 2005) (Fig. 1). A-425619 has been shown to reduce nociception in rat models of postoperative pain and chronic inflammatory pain and also showed partial efficacy in a neuropathic pain model (Honore et al., 2005). A-425619 also potently blocked other modes of TRPV1 activation, including anandamide, NADA, acid, and heat with equivalent efficacy (El Kouhen et al., 2005). The relative efficacy of small molecule TRPV1 receptor antagonists to block these different modes of TRPV1 receptor activation could be an important factor in determining their antinociceptive activity in vivo (Gavva et al., 2005). In vitro binding assays have been developed previously for the TRPV1 receptor using the potent agonist radioligand [H]RTX (Szallasi and Blumberg, 1990; Szallasi et al., 1999) and also the antagonist radioligand [I]iodo-RTX (I-RTX) (Wahl et al., 2001). A potential problem with [H]RTX is that it is an agonist radioligand and may bind to sites on the TRPV1 protein that represent one mode of activation of the TRPV1 receptor only. This was later resolved upon the development of [I]I-RTX (an antagonist radioligand sharing the same pharmacophore as [H]RTX). [I]I-RTX has been reported to potently block activation of the TRPV1 receptor by CAP, acid, and heat in in vitro functional studies (Seabrook et al., 2002). However, both [H]RTX and [I]I-RTX exhibit a high degree of nonspecific binding, and additional treatment of membranes with the vanilloid binding agent 1-acid glycoprotein at 4°C is required to reduce the nonspecific binding (Szallasi et al., 1992; Szallasi and Blumberg, 1993; Wahl et al., 2001). A-778317 (Fig. 1) is one of the first chiral nonvanilloid TRPV1 antagonists reported (Gomtsyan et al., 2005). It is a competitive antagonist of CAP at the recombinant hTRPV1 receptor that shows stereospecific activity in blocking TRPV1 receptor-mediated changes in intracellular calcium concentrations. A tritiated form of A-778317 was synthesized with high specific activity ([H]A-778317; 29.3 Ci/mmol) and found to be a useful radioligand to study the recombinant hTRPV1 receptor in a heterologous expression system. The present study describes the synthesis, pharmacology, and binding properties of [H]A-778317. Materials and Methods CAP and capsazepine (CPZ) were purchased from Sigma-Aldrich (St. Louis, MO). Olvanil (N-9-Z-octadecenoyl-vanillamide) and NADA were purchased from Tocris Cookson, Inc. (Ellisville, MO). RTX and tinyatoxin were purchased from LKT Laboratories, Inc. (St. Paul, MN). A-778317, its enantiomer A-778316, A-425619, A-784168, SB-452533, AMG6880, and the JNJ compound (Jetter et al., 2004) were synthesized in-house (Abbott Laboratories, Abbott Park, IL) (Fig. 1). N-[4-[6-[(Acetyloxy)methoxy]-2,7-difluoro-3-oxo-3H-xanthen9-yl]-2-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxoethyl]glycine, (acetyloxy)methyl ester (fluo-4 AM) was purchased from Texas Fluorescence Laboratories, Inc. (Austin, TX). Ham’s F-12 nutrient mixture, Dulbecco’s phosphate-buffered saline (D-PBS) (with Ca , Mg , and 1 mg/ml D-glucose) (pH 7.4), phosphate-buffered saline (PBS) (without Ca and Mg ), Hanks’ balanced salt solution (HBSS) (without Ca and Mg ), 0.25% trypsin-EDTA, penicillin/ streptomycin, and Lipofectamine Plus transfection reagent were purchased from Invitrogen (Carlsbad, CA). Dulbecco’s modified Eagle’s medium (DMEM) (with 4.5 mg/ml D-glucose), L-glutamine, fetal bovine serum, bovine serum albumin (fraction V), collagenase/ dispase (EC 3.4.24.3), pH 7.4, Trizma preset crystals, HEPES, and 2-morpholinoethanesulfonic acid were purchased from SigmaAldrich. Collagenase B and nerve growth factor were purchased from Roche Diagnostics (Indianapolis, IN), and Geneticin (G418 sulfate) was purchased from Calbiochem-Novabiochem Corp. (San Diego, CA). CHO cells were obtained from American Type Culture Collection (Manassas, VA). [H]A-778317 Synthesis and Purification. A mixture of 6,8diiodo-isoquinolin-5-ylamine (8.72 mg, 0.022 mmol), 10% palladium on carbon (7.12 mg), triethylamine (75 l), and methanol (2 ml) was attached to a tritiation manifold and degassed by freeze-pump-thaw. Tritium gas (0.063 mmol, 3654 mCi) was introduced, and then the mixture was vigorously stirred for 2.5 h at 22–23°C. Excess of gas was removed to a charcoal trap cooled to 196°C. The catalyst was filtered, and the labile tritium from the filtrate was removed by three evaporations of methanol to obtain 505 mCi of crude [6,8-H]isoquinolin-5-ylamine (crude 1) (47% product formation with Rf 0.45) (Fig. 2). Crude 1 was purified by solid-phase extraction using silica Fig. 1. Chemical structures of A-778317, A-778316, A-425619, A-784168, SB-452533, AMG6880, JNJ Compound, and CPZ. 286 Bianchi et al. at A PE T Jornals on Jne 4, 2017 jpet.asjournals.org D ow nladed from gel and 5% methanol in methylene chloride ( 98% radiochemically pure). To synthesize [H]A-778317, a mixture of purified 1 (100 mCi), 5-tert-butyl-1-isocyanato-indan (7.01 mg, 0.033 mmol), and 10 l of toluene was stirred for 24 h at 22–23°C. Solvent was removed on a rotary evaporator to obtain crude product (54% product formation with Rf 0.3) (Fig. 2). The crude product was then applied to two preparative silica gel thin-layer chromatography plates (1500 , 20 20 cm, 5% methanol in methylene chloride with 0.1% ammonium hydroxide), and bands corresponding to the product were scraped and extracted with 5% methanol in methylene chloride with 0.1% ammonium hydroxide (4 25 ml). Solvent was removed on a rotary evaporator to yield 36 mCi of [H]A-778317 ( 90% radiochemically pure). Additional and final purification of [H]A-778317 was achieved with high performance liquid chromatography. The residue was dissolved in acetonitrile (1 ml) and water (1 ml) with 0.1% trifluoroacetic acid (TFA), and then a 400l sample was injected onto a Phenomenex Luna C18 column (5 , 4.6 250 mm; Phenomenex, Torrance, CA). [H]A-778317 was eluted off at a flow rate of approximately 4 ml/min, increasing the gradient mobile phase B from 5 to 95% over a 20-min period (mobile phase A 0.1% TFA/ water; mobile phase B 0.1% TFA/acetonitrile) and then held at 95% (mobile phase B) for 5 min. Peak elution was detected with an Agilent variable wavelength UV detector set at 215 nM and chemstation software (Agilent Technologies, Palo Alto, CA). The fractions containing [H]A-778317 were collected at approximately 14.5 min. The above purification procedure was repeated four more times to process all of the [H]A-778317, and then the fractions were combined and solvents were evaporated under vacuum. The end product was dissolved in 5 ml of ethanol ( 97% radiochemically pure). The specific activity was determined to be 29.3 Ci/mmol based on mass spectrometry by measuring the isotopic ratios compared with authentic A-778317. Cell Transfection and Culture. Human TRPV1 was cloned from ileum as described by Witte et al. (2002). Sequence analysis showed that the cDNA coded an amino acid sequence identical to that of GenBank accession no. AL136801. The pCIneo expression vector containing cDNA for the wild-type hTRPV1 receptor was introduced into CHO cells using the Lipofectamine Plus transfection protocol (Invitrogen). Single colonies surviving selection by G418 sulfate were screened for functional expression of the hTRPV1 receptor in response to CAP stimulation (100 nM) using the Ca flux assay. CHO cells were grown in Ham’s F-12 nutrient mixture containing 2 mM L-glutamine and 10% (v/v) fetal bovine serum and maintained in a 37°C incubator under a humidified 5% CO2 atmosphere. CHO cells stably expressing the hTRPV1 receptor were grown in the same medium supplemented with 300 g/ml G418 sulfate. TRPV1 function remained stable over many cell passages. Ca Flux Assay. Cellular flux of Ca was measured in hTRPV1-expressing CHO cells using the fluorescent Ca chelating dye fluo-4 AM. Cells were grown as a monolayer in black 96-well tissue culture plates (with clear bottoms) (Costar; Corning Life Sciences, Acton, MA). Before the start of the assay, the growth medium was removed and cells were preincubated with 2 M fluo-4 AM (in D-PBS, pH 7.4, containing Ca , Mg , and 1 mg/ml D-glucose) for 2 h at 25°C. To remove extracellular dye, cells were then washed five times with 200 l of assay buffer (D-PBS, pH 7.4, containing Ca , Mg , and 1 mg/ml D-glucose) using a MultiWash Advantage multiplate washer (model 8070-16; Tricontinent, Inc., Suffolk, UK). All compounds were dissolved in dimethyl sulfoxide (10 mM). Test compound plates were prepared using a Biomek 2000 robotic workstation, programmed to change pipette tips following each dilution. Compounds (50 l) were added to the cells at a delivery rate of 50 l/s. For determination of agonist activity, a single addition of the test compounds was made at the 10-s time point of the experimental run. For determination of antagonist activity, a second addition of the TRPV1 receptor agonist CAP (50 nM final concentration) was made 5 min after addition of the test compounds to challenge the TRPV1 receptor. Schild analyses of A-778317 were also double addition experiments where half-log concentration-effect curves of CAP were generated in the presence of five different concentrations of A-778317 (5, 20, 40, 320, and 1280 nM). Final assay volume was 200 l. Length of the experimental run was 5 min for single addition experiments and 10 min for double addition experiments. Changes in fluorescence were recorded in a fluorometric imaging plate reader ( excitation 488 nm, emission 540 nm; Molecular Devices, Sunnyvale, CA). The peak increase in fluorescence over baseline was calculated and expressed as a percentage of the maximal or control response to CAP. A four-parameter logistic Hill equation was then used to curve-fit the concentration-effect data and derive EC50 and IC50 values (GraphPad Software, Inc., San Diego, CA). DRG Neuronal Cultures. All experiments were carried out in accordance with the guidelines and the approval of the Institutional Animal Care and Use Committee (IACUC). DRG cultures were prepared according to previous studies (El Kouhen et al., 2005), with minor modifications. Sprague-Dawley rats (6–8-day-old; Charles River Laboratories International, Inc., Wilmington, MA.) were deeply anesthetized with CO2 and euthanized by decapitation. DRGs were rapidly removed and collected in HBSS. DRGs were transferred to a tube containing 0.1% collagenase/dispase and 0.1% collagenase B and allowed to incubate at 37°C for 1 h. After the incubation, the tissue was centrifuged at 600 rpm for 5 min, and the supernatant was removed, replaced with 0.25% trypsin-EDTA, and allowed to incubate at 37°C for an additional 30 min. The tissue was centrifuged and dissociated by trituration in DMEM, with sequential use of a Fig. 2. Synthesis of [H]A-778317. [H]A-778317, a Novel TRPV1 Receptor Radioligand 287 at A PE T Jornals on Jne 4, 2017 jpet.asjournals.org D ow nladed from plastic Pasteur pipette. Undisrupted tissue fragments were allowed to settle, and the supernatant was transferred to a new tube and centrifuged. The tissue pellet was resuspended in HBSS, triturated, layered over HBSS containing 2% fetal bovine serum, and centrifuged at 600 rpm for 5 min. The resulting pellet was resuspended in DMEM containing 100 U/ml penicillin, 100 g/ml streptomycin, 10% fetal bovine serum, and 100 ng/ml nerve growth factor. Cells were plated onto Biocoat poly-D-lysine coverslips (BD Biosciences, Bedford, MA). All experiments were conducted 24 to 48 h after plating. Whole-Cell Patch-Clamp Electrophysiology. DRG neurons plated on poly-D-lysine-coated coverslips were maintained at room temperature in an extracellular recording solution (pH 7.4, 325 mOsm) consisting of 155 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 12 mM glucose. For experiments involving application of acidic solution (pH 5.5), HEPES was replaced with 2-morpholinoethanesulfonic acid in the external solution. Patch-pipettes composed of borosilicate glass (1B150F-3; World Precision Instruments, Inc., Sarasota, FL) were pulled and fire-polished using a DMZ-Universal micropipette puller (Zeitz-Instruments, Martinsried, Germany). Pipettes (2–6 M ) were filled with an internal solution (pH 7.3, 295 mOsm) consisting of 122.5 mM potassium aspartate, 20 mM KCl, 1 mM MgCl2, 10 mM EGTA, 5 mM HEPES, and 2 mM ATP Mg. Standard whole-cell recording techniques were utilized for voltage-clamp studies using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). Coverslips plated with DRG neurons (20–48 h after dissociation) were placed in a perfusion chamber, and after establishment of whole-cell recording conditions, bath perfusion ( 2 ml/min) was initiated. Application of control bath solution through a MPRE8 multi-barrel application device with a common 360m polyimide tip (Cell Microcontrols, Norfolk, VA), positioned 100 m from the cell, was continued throughout the recording except during drug application. Each drug reservoir was connected to solenoid Teflon valves that were controlled by a ValveLink16 system (AutoMate Scientific, San Francisco, CA). Drugs were applied using rapid valve switching of the ValveLink system controlled by the data acquisition software pCLAMP9.0 (Axon Instruments). Activators (1 M CAP or pH 5.5) were applied for 5 s at 2-min intervals to individual cells until subsequent responses produced responses with similar amplitudes. At this point, A-778317 was preapplied for 60 to 90 s before coapplication with each activator of TRPV1. Peak amplitudes were measured and expressed as a percentage of the control response to activator alone. A nonlinear regression sigmoidal function (GraphPad Prism Software, Inc.) was then used to curve-fit the concentration-effect data and derive an IC50 value (maximal values were constrained to not exceed 100%, and minimal values were constrained not to go below 0%). Membrane Preparation. hTRPV1-expressing and untransfected (null) CHO cells were rinsed with ice-cold PBS and harvested from 150-cm flasks by manual scraping. The cells were pelleted by low-speed centrifugation (1000g for 10 min) at 4°C and then resuspended in ice-cold 10 mM HEPES buffer, pH 7.8, containing 0.32 M sucrose (1.5 ml per 150-cm flask). The cells were homogenized in a glass-Teflon vessel (motor-driven, 10 up-and-down strokes), and the homogenate was centrifuged at low speed (1000g for 10 min) at 4°C. The resulting pellet (P1) was then re-homogenized (in 1⁄2 volume) and centrifuged again at low speed. The supernatants from the two low-speed spins were pooled together and centrifuged at 20,000g for 60 min to pellet the membranes (P2). The resulting P2 membrane fraction was resuspended in ice-cold 50 mM Tris HCl buffer, pH 7.4 (0.25 ml per 150-cm flask), by homogenization. Protein concentration was determined with a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Volume was adjusted with additional ice-cold 50 mM Tris HCl buffer, pH 7.4, to obtain 50g protein/90 l. Membranes were stored at 80°C until use. Radioligand Binding. Membranes were incubated with [H]A778317 and test compounds in a final reaction volume of 200 l in 12 75-mm polypropylene round-base tubes (Sarstedt, Inc., Newton, NC). Working concentrations (2.22X) of [H]A-778317 were prepared in 50 mM Tris HCl buffer, pH 7.4, containing 2.22 mg/ml bovine serum albumin. All compounds were dissolved in dimethyl sulfoxide (10 mM) and diluted to 10 final concentrations in distilled water. Assay incubations were initiated with the addition of membrane suspension (50 g of protein in 50 mM Tris HCl buffer, pH 7.4). Final concentration of bovine serum albumin was 1 mg/ml. For both ligand-competition and saturation binding experiments, incubations were carried out for 60 min at 25°C, and nonspecific binding was defined with 3 M A-425619. Saturation-binding isotherms were generated at concentrations of [H]A-778317 between 0.05 and 20 nM. The final radioligand concentration was 2 nM in association, dissociation, and ligand-competition binding experiments. To ascertain association kinetics, incubations were carried out at 25°C and terminated at different times (1, 2.5, 5, 7.5, 10, 20, 30, 45, and 60 min). To ascertain dissociation kinetics, equilibrium was established (60-min incubation at 25°C), and then 3 M A-425619 was added to inhibit binding over different times (1, 2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 min). All incubations were terminated by rapid filtration over Whatman glass fiber filter paper (GF/B) (fired) using a 48-well Brandel cell harvester (model M-48; Brandel, Inc., Gaithersburg, MD). Filters were washed three times with ice-cold 50 mM Tris HCl buffer, pH 7.4, and transferred to scintillation vials (mini Poly-Q vials; Beckman Coulter, Fullerton, CA). Ecolume LSC (MP Biomedicals, Inc., Aurora, OH) was added to each vial, and bound tritium radioactivity (dpm) was counted in a Beckman LS6500 scintillation counter. Data analysis was accomplished using Prism (GraphPad Software, Inc.). The specific bound counts (dpm) from the saturation binding experiments were converted to picomole per milligram protein, and then concentration-effect data were curve-fit to a one-site binding (hyperbola) equation to derive the dissociation constant of the radioligand (KD) and number of binding sites (Bmax). For ligandcompetition binding experiments, the specific bound counts (dpm) were expressed as a percentage of the maximal binding observed in the absence of test compound, and then the concentration-effect data were curve-fit to a four-parameter logistic Hill equation to derive the potency (IC50) of the test compound. The equilibrium dissociation constant (Ki) of the test compound was calculated by the ChengPrusoff equation: Ki IC50/(1 ([L]/KD)) (Cheng and Prusoff, 1973). For association binding experiments, the specific bound counts (dpm) were expressed as a percentage of the maximal binding observed at equilibrium, and then the time course was curve-fit to a one-phase exponential association equation to derive the observed on-rate (kob). For dissociation binding experiments, the specific bound counts (dpm) were expressed as a percentage of the maximal binding observed at equilibrium (before addition of 3 M A-425619), and then the time course was curve-fit to a one-phase exponential decay equation to derive the off-rate (koff). The on-rate (kon) was calculated from the equation kon (kob koff)/[L].