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NEUROPHARMACOLOGY

[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 Transient Receptor Potential Vanilloid-1 (TRPV1) Receptor

Departments of Neuroscience Research (B.R.B., R.E.K., T.R.N., C.-H.L., A.G., H.A.M., C.S.S., C.R.F., M.F.J., R.B.M., P.S.P.) and Radiochemistry (S.N.R., S.N.V., B.S.), Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois

Received for publication April 11, 2007
Accepted July 26, 2007.

	Abstract

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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 (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.

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).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Chemical structures of A-778317, A-778316, A-425619, A-784168, SB-452533, AMG6880, JNJ Compound, and CPZ.

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 ([³H]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 [³H]A-778317.

	Materials and Methods

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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-xanthen-9-yl]-2-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxoethyl]amino]-5-methyl-phenoxy]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 Ca²⁺, Mg²⁺, and 1 mg/ml D-glucose) (pH 7.4), phosphate-buffered saline (PBS) (without Ca²⁺ and Mg²⁺), Hanks' balanced salt solution (HBSS) (without Ca²⁺ and Mg²⁺), 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 [EC] ), pH 7.4, Trizma preset crystals, HEPES, and 2-morpholinoethanesulfonic acid were purchased from Sigma-Aldrich. 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).

[³H]A-778317 Synthesis and Purification. A mixture of 6,8-diiodo-isoquinolin-5-ylamine (8.72 mg, 0.022 mmol), 10% palladium on carbon (7.12 mg), triethylamine (75 µl), 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 [6,8-³H]isoquinolin-5-ylamine (crude 1) (47% product formation with R_f 0.45) (Fig. 2). Crude 1 was purified by solid-phase extraction using silica gel and 5% methanol in methylene chloride (>98% radiochemically pure). To synthesize [³H]A-778317, a mixture of purified 1 (100 mCi), 5-tert-butyl-1-isocyanato-indan (7.01 mg, 0.033 mmol), and 10 µl 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 R_f 0.3) (Fig. 2). The crude product was then applied to two preparative silica gel thin-layer chromatography plates (1500µ, 20 x 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 x 25 ml). Solvent was removed on a rotary evaporator to yield 36 mCi of [³H]A-778317 (>90% radiochemically pure). Additional and final purification of [³H]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 400-µl sample was injected onto a Phenomenex Luna C18 column (5µ, 4.6 x 250 mm; Phenomenex, Torrance, CA). [³H]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 [³H]A-778317 were collected at approximately 14.5 min. The above purification procedure was repeated four more times to process all of the [³H]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.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Synthesis of [3H]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 [GenBank] . 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 Ca²⁺ 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 µg/ml G418 sulfate. TRPV1 function remained stable over many cell passages.

Ca²⁺ Flux Assay. Cellular flux of Ca²⁺ was measured in hTRPV1-expressing CHO cells using the fluorescent Ca²⁺ 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 µM fluo-4 AM (in D-PBS, pH 7.4, containing Ca²⁺, Mg²⁺, and 1 mg/ml D-glucose) for 2 h at 25°C. To remove extracellular dye, cells were then washed five times with 200 µl of assay buffer (D-PBS, pH 7.4, containing Ca²⁺, Mg²⁺, 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 µl) were added to the cells at a delivery rate of 50 µl/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 µl. 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 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 µg/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 360-µm polyimide tip (Cell Microcontrols, Norfolk, VA), positioned 100 µm 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 µM 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-cm² 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-cm² 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 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-cm² 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 50-µg protein/90 µl. Membranes were stored at –80°C until use.

Radioligand Binding. Membranes were incubated with [³H]A-778317 and test compounds in a final reaction volume of 200 µl in 12 x 75-mm polypropylene round-base tubes (Sarstedt, Inc., Newton, NC). Working concentrations (2.22X) of [³H]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 10x final concentrations in distilled water. Assay incubations were initiated with the addition of membrane suspension (50 µg 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 µM A-425619. Saturation-binding isotherms were generated at concentrations of [³H]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 µM 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 (K_D) and number of binding sites (B_max). For ligand-competition 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 (K_i) of the test compound was calculated by the Cheng-Prusoff equation: K_i = IC₅₀/(1 + ([L]/K_D)) (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 (k_ob). 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 µM A-425619), and then the time course was curve-fit to a one-phase exponential decay equation to derive the off-rate (k_off). The on-rate (k_on) was calculated from the equation k_on = (k_ob – k_off)/[L].

	Results

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A-778317 Is a Potent Antagonist at the Recombinant Human TRPV1 Receptor. The potency of A-778317, as well as a number of TRPV1 receptor agonists and antagonists, was determined in hTRPV1-expressing CHO cells using a fluorometric imaging plate reader-based assay to measure changes in intracellular calcium concentrations (El Kouhen et al., 2005) (Table 1). All of the TRPV1 receptor agonists increased intracellular calcium concentrations in a concentration-dependent fashion. CAP was 50-fold more potent than NADA, a putative endogenous agonist (Huang et al., 2002), in activating TRPV1 receptors (pEC₅₀ = 7.54 ± 0.11 and 5.83 ± 0.10, respectively). CAP did not evoke a Ca²⁺ influx response in untransfected CHO cells (data not shown).

A-778317 fully blocked the ability of 50 nM CAP to activate TRPV1 receptors (pIC₅₀ = 8.31 ± 0.13) (Fig. 3A). A-778317 was approximately 6.8-fold more potent than its enantiomer A-778316 (pIC₅₀ = 7.47 ± 0.07) and 58-fold more potent than the prototypical TRPV1 receptor antagonist CPZ (pIC₅₀ = 6.55 ± 0.04) (Table 2). The nature of TRPV1 antagonism was determined by Schild analysis. The concentration-effect curve of CAP was shifted to the right in the presence of increasing concentrations of A-778317 (Fig. 3B), without affecting the maximal response, indicating that A-778317 is a competitive antagonist of CAP at the hTRPV1 receptor. A Schild plot with linear regression of dose ratios gave a straight line with a slope of 1.39 ± 0.20 and goodness of fit r² = 0.94 (Fig. 3C).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Antagonist profile of A-778317 in recombinant hTRPV1-expressing CHO cells. A, inhibition of CAP (50 nM)-evoked Ca2+ flux by A-778317, A-778316, and CPZ. Data are expressed as a percentage of the control response to CAP and are shown as means ± S.E.M. of three determinations. B, concentration-effect curves of CAP alone and together with 5, 20, 40, 320, and 1280 nM A-778317. Data are expressed as a percentage of the maximal response to CAP (in absence of antagonist). C, Schild plot of A-778317 with linear regression of dose ratios gave a straight line with a slope of 1.39 ± 0.20 and goodness of fit r2 = 0.94. All data are shown as means ± S.E.M. of three determinations.

A-778317 Is a Potent Antagonist at the Native Rat TRPV1 Receptor. Whole-cell patch-clamp recordings were made from rat DRG neurons voltage-clamped at –60 mV. Application of 1 µM CAP, the EC₅₀ value for CAP at recombinant TRPV1 using patch-clamp (Welch et al., 2000), to small to medium diameter DRG neurons (between 25 and 35 µm) evoked an inward current in a majority (70%) of the neurons tested. Preincubation of the cells with increasing concentrations of A-778317 (0.1–10 nM) resulted in a concentration-dependent reduction in the peak CAP response (pIC₅₀ = 8.92, n = 5–9) (Fig. 4, A and B). Application of 10 nM A-778317 reduced the peak current by 89.9 ± 1.6% (n = 9), and was completely reversible within 5 to 7 min following washout of the antagonist with control external solution (Fig. 4, C and D). In addition, application of 10 nM A-778317 blocked 91.5 ± 4.0% (n = 6) of the peak current activated by application of an acidic solution (pH 5.5) (Fig. 4, E and F).

View larger version (36K): [in this window] [in a new window]   Fig. 4. Antagonist profile of A-778317 in cultured rat DRG neurons. A, representative traces illustrating concentration-dependent inhibition by A-778317 (1 and 10 nM) of inward currents elicited by application of 1 µM CAP to small-diameter rat DRG neurons that were voltage-clamped at –60 mV. B, CAP-evoked currents were reduced in the presence of A-778317 in a concentration-dependent manner (pIC50 = 8.92; n = 5–9). C, representative trace showing that 10 nM A-778317 was completely reversible. D, time course illustrating that peak current amplitudes return to control levels within 5 to 7 min following the application of 10 nM A-778317 (n = 3). E, representative traces of inward currents evoked by application of an acidic solution (pH 5.5) in the presence (10 nM) and absence (control) of A-778317. F, average peak current evoked by application of pH 5.5 solution alone (386.6 ± 86.6 pA, n = 6) and following inhibition by 10 nM A-778317 (27.3 ± 14.8 pA, n = 6) in rat DRG neurons.

[³H]A-778317 Labels the Recombinant hTRPV1 Receptor with High Affinity. Preliminary binding studies with 2 nM [³H]A-778317 indicated a high percentage of nonspecific binding (60%) relative to total binding in a crude membrane preparation of hTRPV1-expressing CHO cells. Nonspecific binding was reduced significantly by 1) adding 1 mg/ml bovine serum albumin to the assay mixture and 2) replacing the crude membrane preparation with a P2 membrane fraction (50 µgof protein per assay tube). Under these assay conditions, specific binding of 2 nM [³H]A-778317 to the membranes comprised approximately 90% of the total binding. Manipulation of other assay parameters, including increasing the temperature to 37°C, raising the pH to 7.8, or adding Ca²⁺/Mg²⁺ to the assay mixture, did not significantly increase the percentage of specifically bound radioligand (data not shown); thus, subsequent assay parameters were kept at 25°C, pH 7.4, and Ca²⁺/Mg²⁺-free.

Specific binding of 2 nM [³H]A-778317 reached equilibrium within 30 min at 25°C (Fig. 5A). Analysis of the association kinetics indicated that the t_1/2 for association was 4.94 ± 0.54 min (n = 3). Dissociation binding experiments revealed that the specific binding of [³H]A-778317 was reversible and completely displaced by 3 µM A-425619 over a 60-min period at 25°C (Fig. 5B). The t_1/2 for dissociation was 14.3 ± 2.5 min (n = 3). The k_on and k_off values were 0.0459 ± 0.0069 nM^–1 min^–1 and 0.0504 ± 0.0077 min^–1, respectively (n = 3), yielding a K_D value (k_off/k_on) of 1.10 nM. Saturation binding experiments indicated that specific binding of [³H]A-778317 was saturable over a 12-point concentration range between 0.05 and 20 nM when conducted at binding equilibrium (60 min at 25°C) (Fig. 6A). An apparent K_D value of 3.39 ± 0.34 nM (n = 3) was derived from a curve-fit of the concentration-effect data to a one-site binding model. The apparent B_max was 4.02 ± 0.63 pmol/mg protein (n = 3). A Scatchard plot of the ratio of bound/free versus bound counts (expressed as picomole per milligram protein) fit a straight line with a goodness of fit r² = 0.9883 (Fig. 6B), indicating that [³H]A-778317 bound to a single class of binding sites. No specific [³H]A-778317 binding was detected in untransfected CHO cell membranes (data not shown).

View larger version (8K): [in this window] [in a new window]   Fig. 5. Binding kinetics of [3H]A-778317 in membranes prepared from recombinant hTRPV1-expressing CHO cells. A, representative association time course for 2 nM [3H]A-778317. Nonspecific binding was defined with 3 µM A-425619. Specific bound counts (dpm) were calculated by subtracting nonspecific from total bound counts and expressed as a percentage of the maximal specific bound counts (% bound) observed over 60 min at 25°C. B, representative dissociation time course. Dissociation of [3H]A-778317 was initiated by the addition of 3 µM A-425619 and carried out over a 60-min period at 25°C. The binding observed at the 60-min time point was taken to represent nonspecific binding. Specific bound counts (dpm) were calculated by subtracting nonspecific from total bound counts and expressed as a percentage of the maximal specific bound counts (% bound).

View larger version (15K): [in this window] [in a new window]   Fig. 6. Specific binding of [3H]A-778317 in membranes prepared from recombinant hTRPV1-expressing CHO cells was saturable. A, representative saturation binding curves. Total bound counts (dpm) (filled circles) and nonspecific bound counts (dpm) (open circles) are shown for 12 different radioligand concentrations (between 0.05 and 20 nM) after a 60-min incubation at 25°C. Nonspecific binding was defined with 3 µM A-425619. Specific bound counts (dpm) (filled squares) were calculated by subtracting nonspecific from total bound counts. B, corresponding Scatchard plot of saturation binding data.

Both TRPV1 receptor agonists and antagonists inhibited the specific binding of 2 nM [³H]A-778317 in a concentration-dependent fashion (Fig. 7, A and B; Table 3). The most potent inhibitor was A-778317 (pK_i = 8.13 ± 0.02; n_H = 0.881). Its functionally weaker enantiomer A-778316 was approximately 15-fold less potent than A-778317 (pK_i = 6.95 ± 0.08; n_H = 1.08). The rank-order potency of the TRPV1 receptor antagonists was A-778317 > AMG6880 > A-425619 JNJ compound > A-784168 > A-778316 > SB-452533 > CPZ. The binding potencies of the TRPV1 receptor antagonists (expressed as pK_i) were positively correlated with their functional potencies in the Ca²⁺ flux assay (expressed as pIC₅₀). A linear regression curve-fit of the data (Fig. 7C) had a slope less than 1 (0.67 ± 0.14) and goodness of fit r² = 0.79, P < 0.05.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Competition for 2 nM [3H]A-778317 in hTRPV1-expressing CHO cell membranes by various TRPV1 receptor agonists (A) and antagonists (B). Dose-inhibition curves for RTX, tinyatoxin, and olvanil were carried out to a maximal concentration of 10 µM, whereas CAP and NADA were tested to maximal concentrations of 300 and 30 µM, respectively. All of the TRPV1 receptor antagonists (except A-778317 itself) were tested to a maximal concentration of 10 µM. Time of incubation was 60 min at 25°C, and nonspecific binding was defined with 3 µM A-425619. Specific bound counts (dpm) were calculated by subtracting nonspecific from total bound counts and expressed as a percentage of the specific bound counts (% bound) observed in the absence of agonist/antagonist. Data are shown as means ± S.E.M. for three or more determinations. Rank-order binding potencies of the TRPV1 receptor antagonists (expressed as pKi) correlated well with their functional potencies in the Ca2+ flux assay (expressed as pIC50). A plot of the binding pKi of the TRPV1 receptor antagonists against their pIC50 in the Ca2+ flux assay fit a straight line (C) with a slope less than 1 (0.67 ± 0.14) and goodness of fit r2 = 0.79.

The rank-order potency of the TRPV1 receptor agonists was RTX > tinyatoxin > olvanil > CAP > NADA. RTX inhibited binding of [³H]A-778317 potently (pK_i = 7.19 ± 0.23). The putative endovanilloid NADA was a weak inhibitor of binding. Poor aqueous solubility of NADA precluded testing at concentrations greater than 100 µM, so that only an estimate of its potency could be derived in the binding assay (pK_i < 5.2). Nevertheless, these data agree with its low-micromolar potency in the Ca²⁺ flux assay (pEC₅₀ = 5.83 ± 0.10) (Table 1). In contrast, CAP was also found to be a weak inhibitor of [³H]A-778317 binding (pK_i = 4.70 ± 0.33).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
TRPV1 is a ligand-gated, nonselective cation channel. The vanilloid CPZ was the first small molecule competitive antagonist to be reported (Walpole et al., 1994). However, CPZ exhibits weak potency at the rat (Szallasi et al., 1999) and human (El Kouhen et al., 2005) TRPV1 receptors and is limited as a tool by its actions at other receptors besides TRPV1 (Docherty et al., 1997; Liu and Simon, 1997). More recently, a number of structurally novel nonvanilloid TRPV1 antagonists have been described (Gavva et al., 2005, Jetter et al., 2004; Gomtsyan et al., 2005, Rami et al., 2004, Doherty et al., 2005). A-425619 is one such compound that potently blocks TRPV1 receptor activation by capsaicin, acid, and heat (El Kouhen et al., 2005; Gomtsyan et al., 2005) and effectively reduces inflammatory pain in animal models (Honore et al., 2005). Further structure-activity studies of A-425619 led to the preparation of the (R)- and (S)-stereoisomers, A-778317 and A-778316. The (R)-stereoisomer (A-778317) was 6.8-fold more potent than the (S)-enantiomer in blocking TRPV1 receptor-mediated changes in intracellular calcium concentrations. Furthermore, A-778317 demonstrates a high degree of specificity compared to its activity at other cell surface receptors and ion channels. Based on the properties described above, we developed a radioligand binding assay.

For a radioligand to have widespread application, it should be competitive with a number of different structural types of receptor ligands. [³H]A-778317 competed with two other related isoquinolinyl urea series [i.e., A-425619 (El Kouhen et al., 2005) and a JNJ compound (Jetter et al., 2004)], a 2-bromophenyl urea (SB-452533) (Rami et al., 2004), an N-aryl cinnamide (AMG6880) (Doherty et al., 2005; Gavva et al., 2005), and a highly lipophilic ligand from a series of tetrahydopyridines (A-784168) (Cui et al., 2006). All were found to completely inhibit binding at or near the highest concentrations tested, and a linear correlation was observed between functional inhibition of the calcium flux assay and competition binding across the different structural classes.

Evaluation of a number of TRPV1 agonists and antagonists revealed that, with the exception of CAP, agonists and antagonists of the TRPV1 receptor inhibited [³H]A-778317 binding to hTRPV1-expressing CHO cell membranes with potencies in general agreement with those observed in a Ca²⁺ flux assay for the TRPV1 receptor. In contrast, CAP showed significant potency differences in the radioligand binding (pK_i = 4.7 ± 0.33) and functional (pEC₅₀ = 7.54 ± 0.11) assays. This observation is consistent with previous studies that have reported discrepancies between binding and functional (⁴⁵Ca uptake) potencies for CAP in both rat DRG neurons and hTRPV1-expressing CHO and human embryonic kidney 293 cells (Acs et al., 1996; Szallasi et al., 1999). Although the mechanistic basis for this discrepancy remains unclear, several possibilities may contribute to these findings. Whereas A-778317, A-415619, and other TRPV1 antagonists (Gavva et al., 2005) are functionally competitive blockers of capsaicin's ability to activate TRPV1, these ligands may not share overlapping binding sites. This hypothesis is also supported by data showing that these antagonists can also fully block TRPV1 receptor activation by acid and heat (El Kouhen et al., 2005; Gavva et al., 2005; Neelands et al., 2005). It is also likely that cell preparation and type of assay may also account for differences between binding and functional data. In this regard, a previous study from our laboratory has demonstrated that the host cell expression system influences the pharmacology of TRPV1 (Bianchi et al., 2006). Furthermore, the functional potency of CAP for activating TRPV1 is assay-dependent with high potency (pEC₅₀ = 7.54 ± 0.11) in the Ca²⁺ flux assay and much lower affinity in whole-cell patch-clamp studies (EC₅₀ = 640 nM) (Neelands et al., 2005).

TRPV1 can be activated by vanilloids and bioactive lipids, protons, or heat (Ferrer-Montiel et al., 2004). Although protons and heat are thought to act at different sites in the protein, antagonists competitive with CAP can block all three modes of activation by an as yet undefined mechanism, presumably allosteric in nature (Bianchi et al., 2006, El Kouhen et al., 2005). This site has yet to be resolved by conventional means using either a high-affinity cross-linkable ligand and subsequent peptide mapping or the resolution of a TRPV1 x-ray crystallographic structure. Consequently, molecular biological studies using domain swaps and site-directed mutagenesis comprise our current models. Using domain swaps between human and rat TRPV1, the region encompassed by the second through fourth transmembrane-spanning domains were demonstrated to be key in capsaicin binding (Jordt and Julius, 2002). Mutagenesis studies have further defined the importance of tyrosine 511 and serine 512 in the third intracellular loop, as well as isoleucine 514 and valine 518 in the third transmembrane-spanning domain and methionine 547 in the fourth transmembrane-spanning domain (Jordt and Julius, 2002; Phillips et al., 2004; Gavva et al., 2005; Sutton et al., 2005; Johnson et al., 2006). In this report, CAP was less potent in competition binding assays for tritiated A-778317 than olvanil. The structural difference between olvanil and CAP is an 18versus 10-carbon lipid tail. Consistent with a role for a lipid component, a recent report of ultrapotent capsaicinoid TRPV1 agonists shows that the most potent agonists had more lipophilic tails, e.g., phenylacetylrinvanil (EC₅₀ = 90 pM, Appendino et al., 2005). Because A-778317 competes with capsaicin in functional calcium flux assays (Fig. 3), it is possible that the actual binding site of the ligand is deeper in the transmembrane domains as suggested for RTX (Chou et al., 2004).

Development of a useful, equilibrium binding assay for TRPV1 has been confounded by availability of potent and selective antagonists. as well as the propensity for lipophilic toxins, such as RTX and I-RTX, to exhibit high nonspecific binding and nonsaturable binding properties (Szallasi et al., 1992; Wahl et al., 2001). Radiolabeled A-778317 offers several advantages over the use of either [³H]RTX or [¹²⁵I]I-RTX. The binding protocol is relatively simple, using a rapid filtration step, and is accompanied by lower nonspecific binding levels than the previously described binding assays (Szallasi et al., 1992; Szallasi and Blumberg, 1993; Wahl et al., 2001).

A-778317 represents one of the first in a new class of TRPV1 radioligands. Although the competition with other TRPV1 antagonists and agonists was not examined, the 3-methylisoquinoline derivative of A-425619 was tritiated and used as a probe of the ligand binding site (compound A in Sutton et al., 2005; Johnson et al., 2006). This compound also binds potently to human TRPV1 (K_d = 6.2 nM) and was sensitive to the methionine 547 to leucine change near the extracellular side of fourth transmembrane-spanning domain (Johnson et al., 2006).

The present data demonstrate that A-778317 is a stereoselective high-affinity antagonist radioligand for the TRPV1 receptor. [³H]A-778317 proved to be a useful radioligand to study the recombinant hTRPV1 receptor in a heterologous expression system, possessing high affinity for the hTRPV1 receptor with minimal nonspecific binding. These qualities may provide a unique tool to further investigate the biology of TRPV1.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.124305.

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; n_H, Hill slope; crude 1, [6,8-³H]isoquinolin-5-ylamine; dpm, disintegrations per minute.

The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

Address correspondence to: Pamela S. Puttfarcken, Abbott Laboratories, R4PM, AP9A/2, 100 Abbott Park Rd, Abbott Park, IL 60064-6123. E-mail: pamela.puttfarcken{at}abbott.com

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