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NEUROPHARMACOLOGY
Departments of Neuroscience (L.K., R.T., J.-C.L., J.J.S.T., N.R.G.) and Protein Sciences (B.H., X.B., J.T., H.K., F.M.), Amgen Inc., Thousand Oaks, California
Received May 17, 2006; accepted July 13, 2006.
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Abstract |
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5.7) and heat (>42°C), acting as an integrator of multiple noxious stimuli (Caterina et al., 1997
; Tominaga et al., 1998; Szallasi and Blumberg, 1999; Holzer, 2004; Szolcsanyi, 2004). TRPV1 expression is increased after inflammatory injury in rodents, and the increased level of TRPV1 protein, combined with the confluence of stimuli present in inflammatory injury states, has been proposed to reduce the threshold of activation of nociceptors that express TRPV1, resulting in hyperalgesia (Ji et al., 2002). In agreement with this finding, TRPV1 knockout mice display reduced thermal hypersensitivity following inflammatory tissue injury (Caterina et al., 2000; Davis et al., 2000).
Several competitive antagonists of TRPV1 that interact with the capsaicin/vanilloid-binding pocket have been reported to prevent activation of the channel by different stimuli (for review, see Szallasi and Appendino, 2004). These antagonists include those that inhibit hyperalgesia in models of inflammatory pain (AMG9810, Gavva et al., 2005a; A-425619, Honore et al., 2005; and BCTC, Valenzano et al., 2003), skin incision-induced pain (A-425619, Honore et al., 2005; AMG0347, Gavva et al., 2005b), and bone cancer pain (JNJ-17203212, Ghilardi et al., 2005), supporting a role for TRPV1 in clinical pain states.
Using chimeric domain swap analysis between capsaicin-sensitive TRPV1 and capsaicin-insensitive TRPV1 (Jordt and Julius, 2002; Gavva et al., 2004), it was shown that capsaicin/vanilloid sensitivity and [3H]-resiniferatoxin binding were transferable with the TM3/4 region, suggesting that the TM3/4 region constitutes the vanilloid binding pocket. Furthermore, mutagenesis studies within the TM3/4 region identified several key molecular determinants such as Tyr511, Ser512,Met/Leu547, and Thr550 that led to the proposed models of agonist and antagonist interactions with the vanilloid-binding pocket (Jordt and Julius, 2002; Chou et al., 2004; Gavva et al., 2004; Lee et al., 2005). Molecular determinants for proton activation have been reported to be present in the prepore loop, such as Glu600 and Glu648 (Jordt et al., 2000) and are different from those that constitute the vanilloid-binding pocket. Competitive antagonists of TRPV1, such as AMG6880, AMG7472, and BCTC that interact at the vanilloid-binding pocket, seem to lock the channel conformation in the closed state to block all modes of activation (Gavva et al., 2005c). Ruthenium red, a nonselective pore blocker, acts as an antagonist of all modes of TRPV1 activation via interaction with residues in the channel pore, such as Asp646 (Garcia-Martinez et al., 2000).
Antibodies are generally peripherally restricted, should be devoid of potential central side effects, and with their long plasma half-life might be better therapeutic agents for chronic disease conditions such as pain. Hence, we used the prepore loop peptide of TRPV1 as an antigen to generate antagonist antibodies. We used agonist-induced 45Ca2+ uptake assays and Chinese hamster ovary (CHO) cells expressing TRPV1 to show that rabbit anti-TRPV1 polyclonal but, unfortunately, not monoclonal, antibodies act as antagonists.
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Materials and Methods |
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). Stable expression of human TRPV1 was achieved by transfection of AM1-D CHO cells (Hu et al., 2001) with a pDSR
21 expression construct. Transfection was performed using LF2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Briefly, 4 x 106 AM1-D CHO cells were plated 24 h prior to transfection in 100-mm-diameter plastic Petri dishes (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) in 10 ml of Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 5% fetal bovine serum, 1x penicillin-streptomycin and glutamine (Invitrogen), nonessential amino acids (Invitrogen), sodium pyruvate, and hypoxanthine thymidine (Invitrogen). Approximately 30 µg of each pDSR21-human TRPV1 plasmid DNA was linearized using the restriction enzyme PvuI (New England Biolabs, Ipswich, MA) and diluted in 2 ml of Opti-MEM I (Invitrogen). The diluted DNAs were mixed with 75 µl of LF2000 diluted in 2 ml of Opti-MEM I, and the mixture was incubated for 20 min at room temperature. The DNA/LF2000 mixture was added to the cells and incubated overnight for transfection. The following day, fresh growth medium was added and cells were cultured for 48 h, then plated in selection medium, containing DMEM supplemented with 10% dialyzed fetal bovine serum, 1x penicillin-streptomycin and glutamine, nonessential amino acids (Invitrogen), and sodium pyruvate, in 1:10 dilution. Approximately 2 weeks after transfection, surviving cells were plated one cell per well into five 96-well plates, using limited dilution techniques. CHO cells stably expressing rat TRPV1 were maintained in DMEM supplemented with 10% dialyzed fetal bovine serum, 800 µg/ml genetecin, penicillin, streptomycin, L-glutamine, and nonessential amino acids. HEK293 cells transiently transfected with human TRPV1 cDNA using Fugene were used for some of the whole-cell ELISA experiments. Generation of Rabbit Polyclonal Antibodies to Prepore Loop of Rat TRPV1. Synthetic peptide corresponding to Glu600 to Pro623 (EDGKNNSLPMESTPHKCRGSACKP) was coupled to KLH and used to generate polyclonal antibodies following the standard procedures. A peptide affinity column was made by covalently coupling 1.76 mg of the synthetic antigen peptide to 2 ml of Pierce SulfoLink Coupling Gel (Pierce Chemical, Rockford, IL). Ten milliliters of serum diluted with 20 ml of PBS was applied to the column by gravity flow. The column was washed with 20 ml of PBS, and then the antibodies were eluted with 3 x 4.0 ml of Pierce ImmunoPure IgG Elution Buffer. Each fraction was neutralized with 160 µl of 1 M Tris, pH 9.2, buffer, concentrated in a 20-ml VivaSpin 10-kDa cut-off unit, and dialyzed against PBS.
Generation of Rabbit Monoclonal Antibodies to Prepore Loop of Human TRPV1. Monoclonal antibodies to prepore loop of human TRPV1 (Thr598LIEDGKND SLPSESTSH RWRGPACRP PDSSYNSLY STCys636) were generated by Epitomics (Burlingame, CA) as a contract service, following the published protocols (Spieker-Polet et al., 1995). Conditioned media (45-48 ml) from hybridomas was mixed batch-wise overnight with 50 µl of Protein A-Sepharose FF from GE Healthcare (Piscataway, NJ). The gel was transferred to 0.5-ml SpinX filter tubes, and the gel was washed (5 x 250 µl of PBS) and eluted (3 x 75 µl of Pierce IgG ImmunoPure Elution Buffer) by spinning in an Eppendorf 5415 centrifuge. Each fraction was neutralized with 3 µl of 1 M Tris, pH 9.2.
Whole-Cell ELISA. CHO cells expressing TRPV1 and parental CHO cells were grown to confluence in 96-well plates at 37°C. ELISA was done at room temperature. Growth medium was aspirated, and cells were incubated with blocking buffer (2% milk in PBS with Ca2+ and Mg2+) for 1 h. Blocking buffer was removed, and cells were incubated with purified antibodies (at 5 µg/ml in blocking buffer) or conditioned media (at a 1:1 ratio in blocking buffer) for 1 h. Cells were washed three times with wash buffer (PBS supplemented with 0.05% Tween) and then incubated with a secondary biotinylated antibody in blocking buffer at a 1:2000 dilution (catalog no. BA-1000; Vector Laboratories, Burlingame, CA) for 1 h. After three more washes, a Eu3+-streptavidin-based DELFIA detection system (catalog no. 1244; PerkinElmer Life and Analytical Sciences, Boston, MA) was used to measure time resolved fluorescence on a 96-well plate reader (Victor; PerkinElmer Life and Analytical Sciences).
45Ca2+ Uptake Assay. Two days prior to the assay, cells were seeded in Cytostar 96-well plates (GE Healthcare) at a density of 20,000 cells/well. The activation of TRPV1 was followed as a function of cellular uptake of radioactive calcium (45Ca2+; MP Biomedicals, Irvine, CA). All the antagonist 45Ca2+ uptake assays had a final 45Ca2+ concentration of 10 µCi/ml.
Antagonist Assay of Capsaicin Activation. Purified antibodies were preincubated with TRPV1-expressing CHO cells for 2 h at 37°C prior to addition of 45Ca2+ and capsaicin (final concentration, 0.5 µM) in F12 media and then incubated for an additional 2 min at room temperature.
Antagonist Assay of Proton Activation. Purified antibodies were preincubated with TRPV1-expressing CHO cells for 2 h at 37°C prior to addition of 45Ca2+ in F12 media supplemented with 30 mM HEPES, 30 mM MES, and 0.1 mg/ml BSA adjusted to pH 4.1 with HCl (final assay pH 5 or 5.5) and then incubated for an additional 2 min.
Antagonist Assay of Heat Activation. Purified antibodies were preincubated with TRPV1-expressing CHO cells for 2 h at 37°C prior to addition of 45Ca2+ in F12 media supplemented with 30 mM HEPES and then incubated for an additional 5 min on a heat block to reach a final temperature of 45°C.
Assay Termination and Data Analysis. Immediately after the 2-min incubation with agonist, assay plates were washed two times with PBS and 0.1 mg/ml BSA using an ELX405 plate washer (Bio-Tek Instruments, Winooski, VT) immediately after the functional assay. Radioactivity was measured using a MicroBeta Jet (PerkinElmer Life and Analytical Sciences). Data were analyzed using GraphPad Prism 4.01 (GraphPad Software Inc., San Diego, CA).
Reagents. All cell culture reagents were purchased from Invitrogen.
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We hypothesized that if Ab-156H inhibition of TRPV1 activation by protons occur simply by occluding the residues from protonation, this antibody will not block capsaicin or anandamide activation that occurs through vanilloid-binding pocket located in the TM3/4 region. To test this hypothesis, we evaluated the effect of Ab-156H antibody on capsaicin- and anandamide-induced 45Ca2+ uptake by CHO cells expressing rat TRPV1 (Fig. 2C). Ab-156H inhibited both capsaicin- and anandamide-induced 45Ca2+ uptake in a concentration-dependent manner with maximum inhibition of approximately 55% at the highest concentration tested (1 µM), suggesting that Ab-156H may partially lock the channel conformation in the closed state. In agreement with this, Ab-156H partially inhibited (approximately 55%) heat-induced 45Ca2+ uptake in to CHO cells expressing rat TRPV1 (Fig. 2D).
Since protons directly activate TRPV1 at pH 5.7 and potentiate capsaicin activation at pH 6.0 (Jordt et al., 2000; Ryu et al., 2003), we were curious to see if Ab-156H could block proton-potentiated capsaicin activation. We evaluated the effect of two concentrations of Ab-156H (10 and 100 µg/ml) against pH 5.5 and capsaicin (0.1 µM) at pH 7.2 and 6.0 (Fig. 2E). Compared with activation at pH 7.2, capsaicin at pH 6.0 caused a 3-fold higher net 45Ca2+ uptake into CHO cells expressing TRPV1, indicating potentiation by pH 6.0. Ab-156H blocks both nonpotentiated and pH 6.0 potentiated capsaicin activation (Fig. 2E). It seems that Ab-156H blocks pH 6.0 potentiated capsaicin activation better than capsaicin activation at pH 7.2, suggesting that Ab-156H blocks not only the direct activation of TRPV1 by protons but also potentiation of agonists by protons as well.
Ab-156H Acts as a Full Antagonist of Proton Activation of Both Capsaicin-Sensitive and -Insensitive Rat TRPV1 Channels. We have shown previously that agonists and competitive antagonists require Thr550 for their actions at TRPV1. Mutation of Thr550 to Ile550 (T550I) renders rat TRPV1 insensitive to capsaicin and anandamide as well as to antagonists such as AMG9810 and BCTC (Gavva et al., 2004, 2005a). The ability of protons to activate the TRPV1 channel was not altered by the T550I mutation. To investigate whether the T550I mutation affects the ability of Ab-156H to block proton activation, we measured proton-induced 45Ca2+ uptake by HEK293 cells transiently expressing either wild-type rat TRPV1 or the T550I mutant in the presence of increasing concentrations of Ab-156H (Fig. 3). As expected, control rabbit polyclonal antibodies did not affect proton activation of either wild-type rat TRPV1 or the T550I mutant. AMG9810, a small-molecule antagonist, inhibited only wild-type rat TRPV1 (IC50 value, 265.8 ± 118.3 nM) but not the T500I mutant (IC50 value, >40,000 nM), in agreement with predicted loss of AMG9810 binding to the T550I mutant. In contrast, Ab-156H inhibited proton activation of both wild-type rat TRPV1 and its T550I mutant with IC50 values of 23.9 ± 4.9 and 52.4 ± 7.6 nM, respectively, demonstrating that disruption of vanilloid binding pocket conformation does not affect Ab-156H binding or antagonism of rat TRPV1.
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Rabbit Monoclonal Antibodies to the Human TRPV1 Prepore Peptide Bind to TRPV1 but Are Ineffective at Blocking Channel Activation. Because rabbit polyclonal antibodies to the rat TRPV1 prepore loop act as antagonists, we sought to generate monoclonal antagonist antibodies to human TRPV1. We used the entire prepore region of human TRPV1 (Thr598LIEDGKND SLPSESTSH RWRGPACRP PDSSYNSLY STCys636) as an antigen to generate antibodies. First, we evaluated preimmune and immune serum from the rabbit immunized with the Thr598-Cys636 peptide in protonand capsaicin-induced 45Ca2+ uptake assays with or without either antigen peptide (Thr598-Cys636) or an unrelated peptide (Fig. 4). Preimmune serum combined with 10 µM of either antigen peptide or an unrelated peptide did not significantly inhibit either pH 5 or capsaicin activation of human TRPV1 (Fig. 4, A and B). However, immune serum to Thr598-Cys636 peptide combined with 10 µM of an unrelated peptide showed complete inhibition of both pH 5 and capsaicin activation. This inhibition was prevented by the antigen peptide (Thr598-Cys636) in a concentration-dependent manner (Fig. 4, A and B), indicating specific antagonism of TRPV1 activation by the antiserum.
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) through a contract service provided by Epitomics. Conditioned media were generated using immortalized clonal cell lines and purified monoclonal IgGs were evaluated in a whole-cell ELISA for specific binding to human TRPV1 (Fig. 5A). Both the conditioned media and the purified IgGs gave positive signals in whole-cell ELISA, with CHO cells expressing human TRPV1 but not with parental CHO cells (Fig. 5A). Twenty-six purified monoclonal IgGs were identified as specific binders to cells expressing human TRPV1 and were evaluated for antagonism of proton and capsaicin activation in 45Ca2+ uptake assays.
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None of the twenty-six rabbit monoclonal IgGs that are selective binders of human TRPV1 blocked either proton or capsaicin activation in 45Ca2+ uptake assays (Fig. 5, B and C, respectively). The concentration range of the purified antibodies used in 45Ca2+ assay was 88.5 to 266.8 µg/ml. A small-molecule antagonist, AMG9810, was used as a positive control in the assay (IC50 values at proton and capsaicin activation were 70.60 ± 7.43 and 19.0 ± 2.7 nM, respectively). We also generated a number of mouse and fully human monoclonal antibodies to the prepore loop region of human TRPV1. The majority of these monoclonal antibodies showed selective binding to CHO cells expressing TRPV1 (but not to parental CHO cells); however, none were able to block either proton or capsaicin activation of TRPV1 (data not shown).
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Discussion |
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), and the current studies with polyclonal antagonist antibody (Ab-156H) demonstrate that the channel conformation can be stabilized partially in the closed state by binding to a region out side of the capsaicin-binding pocket.
Since Ab-156H was more potent at blocking proton activation than activation by chemical ligands or heat, it is possible that Ab-156H inhibition of proton activation occurs by blocking protonation of the residues responsible for activation in addition to locking or stabilizing the channel in the closed state. We hypothesized that if Ab-156H inhibition of protonation occurs simply by occluding the residues from protonation, it would not block capsaicin or anandamide activation. Since Ab-156H acted as a partial antagonist of capsaicin, anandamide, and heat activation of TRPV1, we propose that Ab-156H partially locks or stabilizes the channel conformation in the closed or nonconducting state. Since Ab-156H binds to the prepore region in the vicinity of the pore, we cannot completely exclude the possibility that Ab-156H acts as a pore blocker of TRPV1.
We previously postulated that TRPV1 antagonists acting through the capsaicin-binding pocket fall into two distinct categories (Gavva et al., 2005c): class A compounds block channel activation both by capsaicin and protons, whereas class B compounds selectively abolish capsaicin activation. The results presented here indicate that the rabbit anti-rat TRPV1 polyclonal antibody (Ab-156H) binding at a site outside of the capsaicin-binding pocket acts as a full antagonist of proton activation and a partial antagonist of capsaicin, anandamide and heat activation, thus representing a class A antagonist with an unusual profile. This indicates the complexity of mechanisms for antagonism of TRPV1 channel.
Polyclonal antibodies that antagonize the function of ion channels and G protein-coupled receptors reported in the literature include antibodies to the prepore regions of Kv1.2, Kv3.1 (Zhou et al., 1998), TRP channels (TRPC1, TRPC5; Xu et al., 2005), antibodies to the second extracellular loop of the 5-hydroxytryptamine 4 receptor (Salle et al., 2001), and antibodies to the N-terminal peptide region of protease-activated receptor 2 (Kelso et al., 2006). Monoclonal antibodies to the second extracellular loop of M2 muscarinic receptor were reported to act as partial agonists (Elies et al., 1998). Interestingly, antagonist antibodies generated against Kv1.2, Kv3.1, TRPC1, and TRPC5 all have been reported to block 50 to 60% of channel activation. Since Ab-156H blocks >75% proton activation, it is possible that Ab-156H blocks the channel activation both by partially stabilizing the channel conformation in the closed or nonconducting state and also by directly blocking protonation as well. In agreement with antibodies to ion channels acting as modulators of function, it has been found that antisera from patients with Lambert-Eaton myasthenic syndrome inhibit calcium channel currents (Flink and Atchison, 2003, and refs. therein). However, there are no reports in the literature indicating the existence of autoantibodies to TRPV1 that can cause insensitivity or reduced sensitivity to inflammatory or neuropathic pain.
It is noteworthy that almost all TRPV1 antagonists reported thus far seem to bind at the capsaicin site and thus are competitive antagonists of capsaicin activation. Compounds that block TRPV1 activation via interaction at a site outside of the capsaicin-binding pocket include arginine-rich peptides such as R4W2 (Himmel et al., 2002; Planells-Cases et al., 2002), trialkylglycine-based compounds such as DD161515
[GenBank]
and DD191515
[GenBank]
(Garcia-Martinez et al., 2002), and acylpolyamine toxins derived from funnel web spider venom (Kitaguchi and Swartz, 2005). In vivo efficacy of peptide, antibody, and toxin blockers of TRPV1 has not been demonstrated in models of pain except for a report by Kamei et al. (2001) that polyclonal antibodies to TRPV1 (Neuromics, Bloomington, MN) attenuated thermal hyperalgesia in a diabetic pain model in mice. However, no information is available regarding the antigen peptide or potency at blocking different modes of TRPV1 activation for this antibody. Since the two TRPV1 polyclonal antibodies sold by Neuromics were generated against either an intracellular N or C terminus regions of TRPV1, it is not clear how these antibodies acted as antagonists in vivo.
To evaluate the feasibility of generating monoclonal antagonist antibodies to TRPV1, we used the entire prepore region of human TRPV1 as an antigen in two species, rabbits and mice. Monoclonal antibodies generated in both rabbits and mice selectively bound to TRPV1-expressing cells; however, they did not block channel activation by either protons or capsaicin, suggesting that monoclonal antibodies were not able to lock or stabilize the channel conformation in the closed or nonconducting state. Studies with Ab-156H suggest that polyclonal antibodies may stabilize or lock the channel conformation by binding to the entire 23-amino acid prepore region (Glu600-Pro623) of rat TRPV1, resulting in partial to full antagonism of activation by different stimuli. Monoclonal antibodies may only bind smaller epitope(s) of the 39-amino acid prepore region of human TRPV1 (Thr598-Cys636) and, hence, may not lock or stabilize the channel in the closed state and are ineffective against all modes of activation. Although it is recognized that antibody binding and function can be influenced by factors such as temperature, pH, and salt concentration (Lipman et al., 2005), it is unlikely that this can explain the differences between monoclonal and polyclonal antibodies because these bound to the channel but blocked activation differentially in the functional assays. Since no rabbit, mouse, or fully human monoclonal antibodies generated against the prepore region of human TRPV1 (Thr598-Cys636) were effective at blocking channel activation, we hypothesize that it may not be possible to lock the channel conformation through high-affinity binders to small epitopes in this region. Interestingly, our attempts to mix several monoclonal antibodies to mimic a polyclonal antibody did not result in antagonism of either proton or capsaicin activation of human TRPV1 (data not shown). Since it is not known to which epitope each monoclonal antibody binds within the prepore region (Thr598-Cys636), we are not sure if a mix of monoclonal antibodies would represent a true polyclonal antibody. No monoclonal antagonist antibodies to ion channels have been identified to date. It remains to be seen if monoclonal antibodies targeting prepore regions in ion channels can act as antagonists.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: VR1/TRPV1, transient receptor potential vanilloid type 1; AMG9810, (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)acrylamide; A-425619, 1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea; BCTC, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahy-dropyrazine-1(2H)-carboxamide; AMG0347, (E)-N-(7-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl)-3-(2-(piperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)acrylamide; JNJ-17203212, 4-(3-(trifluoromethyl)pyridin-2-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)piperazine-1-carboxamide; TM3/4, transmembrane 3-4 region; AMG6880, (2E)-3-[2-piperidin-1-yl-6-(trifluoromethyl)pyridin-3-yl]-N-quinolin-7-ylacrylamide; AMG7472, 5-chloro-6-{(3R)-3-methyl-4-[6-(trifluoromethyl)-4-(3,4,5-trifluorophenyl)-1H-benzimidazol-2-yl]piperazin-1-yl}pyridin-3-yl)methanol; CHO, Chinese hamster ovary; HEK, human embryonic kidney; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; MES, 4-morpholineethane-sulfonic acid; DD161515 [GenBank] , N-[2-(2-(N-methylpyrrolidinyl)ethyl]glycyl]-[N-[2,4-dichlorophenethyl]glycyl]-N-(2,4-dichlorophenethyl)glycinamide; DD191515 [GenBank] , [N-[3-(N,N-diethylamino)propyl]glycyl]-[N-[2,4-dichlorophenethyl]glycyl]-N-(2,4-dichlorophenethyl)glycinamide.
Address correspondence to: Dr. Narender R. Gavva, Department of Neuroscience, Amgen Inc., MS-29-2-B, Thousand Oaks, CA 91320-1799. E-mail: ngavva{at}amgen.com
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References |
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Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, Koltzenburg M, Basbaum AI, and Julius D (2000) Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science (Wash DC) 288: 306-313.
Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, and Julius D (1997) The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature (Lond) 389: 816-824.[CrossRef][Medline]
Chou MZ, Mtui T, Gao YD, Kohler M, and Middleton RE (2004) Resiniferatoxin binds to the capsaicin receptor (TRPV1) near the extracellular side of the S4 transmembrane domain. Biochemistry 43: 2501-2511.[CrossRef][Medline]
Davis JB, Gray J, Gunthorpe MJ, Hatcher JP, Davey PT, Overend P, Harries MH, Latcham J, Clapham C, Atkinson K, et al. (2000) Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature (Lond) 405: 183-187.[CrossRef][Medline]
Elies R, Fu LX, Eftekhari P, Wallukat G, Schulze W, Granier C, Hjalmarson A, and Hoebeke J (1998) Immunochemical and functional characterization of an agonist-like monoclonal antibody against the M2 acetylcholine receptor. Eur J Biochem 251: 659-666.[Medline]
Flink MT and Atchison WD (2003) Ca2+ channels as targets of neurological disease: Lambert-Eaton Syndrome and other Ca2+ channelopathies. J Bioenerg Biomembr 35: 697-718.[CrossRef][Medline]
Garcia-Martinez C, Humet M, Planells-Cases R, Gomis A, Caprini M, Viana F, De La Pena E, Sanchez-Baeza F, Carbonell T, De Felipe C, et al. (2002) Attentuation of thermal nociception and hyperalgesia by VR1 blockers. Proc Natl Acad Sci USA 99: 2374-2379.
Garcia-Martinez C, Morenilla-Palao C, Planells-Cases R, Merino JM, and Ferrer-Montiel A (2000) Identification of an aspartic residue in the P-loop of the vanilloid receptor that modulates pore properties. J Biol Chem 275: 32552-32558.
Gavva NR, Bannon AW, Tamir R, Wang Q, Zhu D, Le A, Youngblood B, Kuang R, Deng H, Wang J, et al. (2005b) Identification and biological evaluation of AMG0347 [(E)-N-(7-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl)-3-(2-(piperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)acrylamide], a potent vanilloid receptor 1 antagonist, in Society for Neuroscience Annual Meeting; 2005 Nov 12-16; Washington, DC. Abstract 292, pp 16. Society for Neuroscience, Washington, DC.
Gavva NR, Klionsky L, Qu Y, Shi L, Tamir R, Edenson S, Zhang TJ, Viswanadhan VN, Toth A, Pearce LV, et al. (2004) Molecular determinants of vanilloid sensitivity in TRPV1. J Biol Chem 279: 20283-20295.
Gavva NR, Tamir R, Klionsky L, Norman MH, Louis J-C, Wild KD, and Treanor JJS (2005c) Proton activation does not alter antagonist interaction with the capsaicin-binding pocket of TRPV1. Mol Pharm 68: 1524-1533.
Gavva NR, Tamir R, Qu Y, Klionsky L, Zhang TJ, Immke D, Wang J, Zhu D, Vanderah TW, Porreca F, et al. (2005a) AMG9810, (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide, a novel vanilloid receptor 1 (TRPV1) antagonist with antihyperalgesic properties. J Pharmacol Exp Ther 313: 474-484.
Ghilardi JR, Rohrich H, Lindsay TH, Sevcik MA, Schwei MJ, Kubota K, Halvorson KG, Poblete J, Chaplan SR, Dubin AE, et al. (2005) Selective blockade of the capsaicin receptor TRPV1 attenuates bone cancer pain. J Neurosci 25: 3126-3131.
Himmel HM, Kiss T, Borvendeg SJ, Gillen C, and Illes P (2002) The arginine-rich hexapeptide R4W2 is a stereoselective antagonist at the vanilloid receptor 1: a Ca2+ imaging study in adult rat dorsal root ganglion neurons. J Pharmacol Exp Ther 301: 981-986.
Holzer P (2004) TRPV1 and the gut: from a tasty receptor for a painful vanilloid to a key player in hyperalgesia. Eur J Pharmacol 500: 231-241.[CrossRef][Medline]
Honore P, Wismer CT, Mikusa JP, Zhu CZ, Zhong C, Gauvin DM, Gomtsyan A, El Kouhen R, Lee CH, Marsh K, et al. (2005) A-425619, a novel TRPV1 receptor antagonist, relieves pathophysiological pain associated with inflammation and tissue injury in rats. J Pharmacol Exp Ther 314: 410-421.
Hu S-F, Gudas JM, and Brankow DW (2001) inventors, Amgen, Inc., assignee. Overexpressing cyclin D1 in a eukaryotic cell line. U.S. patent 6,210,924. 2001 Apr 3.
Ji RR, Samad TA, Jin SX, Schmoll R, and Woolf CJ (2002) p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron 36: 57-68.[CrossRef][Medline]
Jordt SE and Julius D (2002) Molecular basis for species-specific sensitivity to "hot" chili peppers. Cell 108: 421-430.[CrossRef][Medline]
Jordt SE, Tominaga M, and Julius D (2000) Acid potentiation of the capsaicin receptor determined by a key extracellular site. Proc Natl Acad Sci USA 97: 8134-8139.
Kamei J, Zushida K, Morita K, Sasaki M, and Tanaka S (2001) Role of vanilloid VR1 receptor in thermal allodynia and hyperalgesia in diabetic mice. Eur J Pharmacol 422: 83-86.[CrossRef][Medline]
Kelso EB, Lockhart JC, Hembrough T, Dunning L, Plevin R, Hollenberg MD, Sommerhoff CP, McLean JS, and Ferrell WR (2006) Therapeutic promise of PAR2 antagonism in joint inflammation. J Pharmacol Exp Ther 316: 1017-1024.
Kitaguchi T and Swartz KJ (2005) An inhibitor of TRPV1 channels isolated from funnel web spider venom. Biochemistry 44: 15544-15549.[CrossRef][Medline]
Lee J, Jin MK, Kang SU, Kim SY, Lee J, Shin M, Hwang J, Cho S, Choi YS, Choi HK, et al. (2005) Analysis of structure-activity relationships for the "B-region" of N-(4-t-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]-thiourea analogues as TRPV1 antagonists. Bioorg Med Chem Lett 15: 4143-4150.[CrossRef][Medline]
Lipman NS, Jackson LR, Trudel LJ, and Weis-Garcia F (2005) Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR J 46: 258-268.[Medline]
Planells-Cases R, Aracil A, Merino JM, Gallar J, Perez-Paya E, Belmonte C, Gonzalez-Ros JM, and Ferrer-Montiel AV (2002) Arginine-rich peptides are blockers of VR-1 channels with analgesic activity. FEBS Lett 481: 131-136.
Ryu S, Liu B, and Qin F (2003) Low pH potentiates both capsaicin binding and channel gating of VR1 receptors. J Gen Physiol 122: 45-61.
Salle L, Eftekhari P, Aupart M, Cosnay P, Hoebeke J, and Argibay JA (2001) Inhibitory activity of antibodies against the human atrial 5-HT4 receptor. J Mol Cell Cardiol 33: 405-417.[CrossRef][Medline]
Spieker-Polet H, Sethupathi P, Yam P, and Knight KL (1995) Rabbit monoclonal antibodies: generating a fusion partner to produce rabbit-rabbit hybridomas. Proc Natl Acad Sci USA 92: 9348-9352.
Szallasi A and Appendino G (2004) Vanilloid receptor TRPV1 antagonists as the next generation of painkillers. Are we putting the cart before the horse? J Med Chem 47: 2717-2723.[CrossRef][Medline]
Szallasi A and Blumberg PM (1999) Vanilloid (capsaicin) receptors and mechanisms. Pharmacol Rev 51: 159-212.
Szolcsanyi J (2004) Forty years in capsaicin research for sensory pharmacology and physiology. Neuropeptides 38: 377-384.[CrossRef][Medline]
Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann BE, Basbaum AI, and Julius D (1998) The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 21: 531-543.[CrossRef][Medline]
Valenzano KJ, Grant ER, Wu G, Hachicha M, Schmid L, Tafesse L, Sun Q, Rotshteyn Y, Francis J, Limberis JT, et al. (2003) BCTC (N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carbox-amide), a novel, orally-effective vanilloid receptor 1 antagonist with analgesic properties: I. In vitro characterization and pharmacokinetic properties. J Pharmacol Exp Ther 306: 377-386.
Xu SZ, Zeng F, Lei M, Li J, Gao B, Xiong C, Sivaprasadarao A, and Beech DJ (2005) Generation of functional ion-channel tools by E3 targeting. Nat Biotechnol 23: 1289-1293.[CrossRef][Medline]
Zhou BY, Ma W, and Huang XY (1998) Specific antibodies to the external vestibule of voltage-gated potassium channels block current. J Gen Physiol 111: 555-563.
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