Aryl sulfonamides containing tetralin allylic amines as potent and selective bradykinin B1 receptor antagonists

The bradykinin B1 receptor has been shown to mediate pain response and is rapidly induced upon injury. Blocking this receptor may provide a promising treatment for inflammation and pain. We previously reported tetralin benzyl amines as potent B1 antagonists. Here we describe the synthesis and SAR of B1 receptor antagonists with homobenzylic amines. The SAR of different linkers led to the discovery of tetralin allylic amines as potent and selective B1 receptor antagonists (hB1 IC₅₀ = 1.3 nM for compound 16). Some of these compounds showed modest oral bioavailability in rats.     

Alpha-hydroxy amides as a novel class of bradykinin B1 selective antagonists

Antagonism of the bradykinin B(1) receptor represents a potential treatment for chronic pain and inflammation. Novel antagonists incorporating alpha-hydroxy amides were designed that display low-nanomolar affinity for the human bradykinin B(1) receptor and good bioavailability in the rat and dog. In addition, these functionally active compounds show high passive permeability and low susceptibility to phosphoglycoprotein mediated efflux, predictive of good CNS exposure.     

Functional duality of kinin receptors in pathophysiology

Kinins are autacoid peptides and central neuromediators involved in cardiovascular regulation, inflammation and pain. Their effects are mediated by two transmembrane G-protein-coupled receptors denoted as B1 and B2. While the B2 receptor is constitutive, the B1 receptor is inducible and up-regulated in the presence of cytokines, endotoxins or during tissue injury. The B2 receptor is believed to play an important role in the beneficial effects of angiotensin-1 converting enzyme inhibitors used in the treatment of cardiovascular diseases, yet it is involved in the acute phase of inflammation and of somatic and visceral pain. Conversely, the B1 receptor participates in the chronic phase of these responses and is likely to play a strategic role in diseases with a strong immune component such as rheumatoid arthritis, multiple sclerosis, septic shock and diabetes. A dual function for the B1 receptor is also reported in some pathologies in which it can exert either a protective (multiple sclerosis and septic shock) or harmful (pain and inflammation) effect. Therefore, the use of antagonists for these receptors as clinical therapeutic agents requires a rigorous evaluation of the potential side effects.     

Potent bradykinin B1 receptor antagonists: 4-substituted phenyl cyclohexanes

Selective bradykinin (BK) B(1) receptor antagonists have been shown to be antinociceptive in animal models and could be novel therapeutic agents for the treatment of pain and inflammation. Elucidation of the structure-activity relationships of the biphenyl moiety of the lead compound 1 provided a potent new structural class of BK B(1) receptor antagonists.     

Acidification of rat TRPV1 alters the kinetics of capsaicin responses

Background

Spinal cord N-methyl-D-aspartate (NMDA) receptors are intimately involved in the development and maintenance of central sensitization. However, the mechanisms mediating the altered function of the NMDA receptors are not well understood. In this study the role of phosphorylation of NR1 splice variants and NR2 subunits was examined following hind paw inflammation in rats. We further examined the level of expression of these proteins following the injury.

Results

Lumbar spinal cord NR1 subunits were found to be phosphorylated on serine residues within two hours of the induction of hind paw inflammation with carrageenan. The enhanced NR1 serine phosphorylation reversed within six hours. No phosphorylation on NR1 threonine or tyrosine residues was observed. Likewise, no NR2 subunit phosphorylation was observed on serine, threonine or tyrosine residues. An analysis of NR1 and NR2 protein expression demonstrated no change in the levels of NR1 splice variants or NR2A following the inflammation. However, spinal cord NR2B expression was depressed by the hind paw inflammation. The expression of NR2B remained depressed for more than one week following initiation of the inflammation.

Conclusion

These data suggest that NR1 serine phosphorylation leads to an initial increase in NMDA receptor activity in the spinal cord following peripheral injury. The suppression of NR2B expression suggests compensation for the enhanced nociceptive activity. These data indicate that spinal cord NMDA receptors are highly dynamic in the development, maintenance and recovery from central sensitization following an injury. Thus, chronic pain therapies targeted to NMDA receptors should be designed for the exact configuration of NMDA receptor subunits and post-translational modifications present during specific stages of the disease.     

Bradykinin antagonists: new opportunities

The pro-inflammatory, pain producing, and cardiovascular effects of bradykinin B2 receptor activation are well characterized. Bradykinin B1 receptors also produce inflammation and pain. Therefore, antagonists are expected to be anti-inflammatory/analgesic drugs. Other exploitable clinical opportunities may exist. The newly discovered non-peptide B2 receptor antagonists and the equivalent B1 receptor pharmacological agents, which are in the pipeline, are suitable preclinical tools to properly evaluate potential utilities.     

缓激肽B1受体与疼痛

摘要：缓激肽B1受体（bradykinin 1 receptors,B1Rs）是与Gq蛋白相偶联的受体。正常状态下,B1R除了在神经系统中（如脊髓背角浅层和感觉神经节）有少数表达外,其他机体组织中几乎不存在。在炎症或者神经受损的情况下,脊髓背角浅层和感觉神经节B1R表达量大大上升,参与炎性疼痛和神经病理性疼痛的产生和维持。近年来的研究表明,B1R在糖尿病性神经病理疼痛的发病中起着重要的作用。阻断B1R能有效抑制糖尿病诱发的热痛觉过敏和冷觉及触觉超敏。此外,B1R和癌症痛的发生也有密切关系,所以,对B1R的研究可能会为治疗这些临床顽症提供新的靶点。关键词：缓激肽B1受体;炎性痛;神经病理性痛中图分类号：Q189;Q42;R338文献标志码：A     

Salak Plum Peel Extract as a Safe and Efficient Antioxidant Appraisal for Cosmetics

We searched in this study for novel agonists of transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and transient receptor potential cation channel, subfamily A, member 1 (TRPA1) in pepper, focusing attention on 19 compounds contained in black pepper. Almost all the compounds in HEK cells heterogeneously expressed TRPV1 or TRPA1, increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a concentration-dependent manner. Among these, piperine, isopiperine, isochavicine, piperanine, pipernonaline, dehydropipernonaline, retrofractamide C, piperolein A, and piperolein B relatively strongly activated TRPV1. The EC₅₀ values of these compounds for TRPV1 were 0.6–128 μM. Piperine, isopiperine, isochavicine, piperanine, piperolein A, piperolein B, and N-isobutyl-(2E,4E)-tetradeca-2,4-diamide also relatively strongly activated TRPA1, the EC₅₀ values of these compounds for TRPA1 were 7.8–148 μM. The Ca²⁺ responses of these compounds for TRPV1 and TRPA1 were significantly suppressed by co-applying each antagonist. We identified in this study new transient receptor potential (TRP) agonists present in black pepper and found that piperine, isopiperine, isochavicine, piperanine, piperolein A, and piperolein B activated both TRPV1 and TRPA1.     

Generation and characterization of a humanized bradykinin B1 receptor mouse

Antagonists of the B1 bradykinin receptor (B1R), encoded by the BDKRB1 gene, offer the promise of novel therapeutic agents for inflammatory and neuropathic pain. However, the in vivo characterization of the pharmacodynamics of B1R antagonists is hindered by the low level of B1R expression in healthy tissue and the profound species selectivity exhibited by many compounds for the B1R. To circumvent these issues we generated two genetically engineered rodent models. The first is a transgenic rat over-expressing the human B1R under the control of the neuronal-specific enolase promoter; we previously reported the utility of this model in assessing human B1R receptor occupancy in the central nervous system of the rat. The second model, reported here, utilized gene-targeting by homologous recombination to replace the genomic coding sequence for the endogenous mouse B1R with that of the human B1R. The mRNA expression profile of the humanized Bdkrb1 (hBkdrb1) allele is similar to that of the mouse Bdkrb1 (mBkdrb1) in the wild-type animal. Furthermore, in vitro assays indicate that tissues isolated from the humanized mouse possess pharmacological properties characteristic of the human B1R. Therefore, we have generated a humanized B1R mouse model that is suitable for testing the efficacy of human B1R-selective compounds.     

Nonpeptide antagonists for kinin receptors

Kinins are a family of small peptides acting as mediators of inflammation and pain in the peripheral and central nervous system. The two main 'kinins' in mammals are the nonapeptide bradykinin (BK, Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) and the decapeptide kallidin (KD, [Lys0]-BK, Lys1-Arg2-Pro3-Pro4-Gly5-Phe6-Ser7-Pro8-Phe9- Arg10). Their biological actions are mediated by two distinct receptors, termed B1 and B2. Kinin B and B2 receptor antagonists may be useful drugs endowed with analgesic and anti-inflammatory properties, with potential use in asthma, allergic rhinitis and other diseases. The first nonpeptide kinin B2 receptor antagonist, WIN 64338, was reported in 1993. Despite its low selectivity, the compound provided a reference for pharmacological and modeling studies. Several quinoline and imidazo[1,2-a]pyridine derivatives have been shown by Fujisawa to possess high affinity and selectivity for kinin B2 receptors. Among them, FR 173657 displayed excellent in vitro and in vivo antagonistic activity, while FR 190997 emerged as the first nonpeptide agonist for B2 receptor. Two structurally related Fournier compounds were recently published. Other kinin B2 receptor ligands were obtained by rational design, through library screening or from natural sources. The only example of a nonpeptide kinin B1 receptor ligand has been reported in a patent by Sanofi.     

Identification of small molecule antagonists of the human mas-related gene-X1 receptor

The recently identified mas-related-gene (MRG) family of receptors, located primarily in sensory neurons of the dorsal root ganglion, has been implicated in the perception of pain. Thus, antagonists of this class of receptors have been postulated to be useful analgesics. Toward this end, we developed a cell-based beta-lactamase (BLA) reporter gene assay to identify small molecule antagonists of the human MRG-X1 receptor from a library of compounds. Single-cell clones expressing functional receptors were selected using the BLA reporter gene technology. The EC50 for the MRG agonist peptide, BAM15, appeared to be comparable between the BLA assay and the intracellular Ca2+ transient assays in these cells. Ultra high-throughput screening of approximately 1 million compounds in a 1.8-microl cell-based BLA reporter gene assay was conducted in a 3456-well plate format. Compounds exhibiting potential antagonist profile in the BLA assay were confirmed in the second messenger Ca2+ transient assay. A cell-based receptor trafficking assay was used to further validate the mechanism of action of these compounds. Several classes of compounds, particularly the 2,3-disubstituted azabicyclo-octanes, appear to be relatively potent antagonists at the human MRG-X1 receptors, as confirmed by the receptor trafficking assay and radioligand binding studies. Furthermore, the structure-activity relationship reveals that within this class of compounds, the diphenylmethyl moiety is constant at the 2-substituent, whereas the 3-substituent is directly correlated with the antagonist activity of the compound.     

Altered neutrophil homeostasis in kinin B1 receptor-deficient mice

The kallikrein-kinin system is activated during inflammation and plays a major role in the inflammatory process. One of the main mechanisms of kinin action includes the modulation of neutrophil function employing both receptors for kinins, B1 and B2. In this report we show by the use of B1 receptor-deficient mice that neutrophil migration in inflamed tissues is dependent on kinin B1 receptors. However, there is no change in circulating leukocyte number and composition after genetic ablation of this receptor. Furthermore, apoptosis of neutrophils necessary for the resolution of persistent inflammatory processes is impaired in mice lacking the B1 receptor. We also show that this receptor is expressed on neutrophils, thus it may be directly involved in the induction of apoptosis in these cells after prolonged activation at inflamed sites. In conclusion, our data show that the kinin B1 receptor modulates migration and the life span of neutrophils at sites of inflammation and may be therefore an important drug target in the therapy of inflammatory diseases.     

The C-terminal Src Inhibitory Kinase (Csk)-mediated Tyrosine Phosphorylation Is a Novel Molecular Mechanism to Limit P2X3 Receptor Function in Mouse Sensory Neurons

On sensory neurons, sensitization of P2X₃ receptors gated by extracellular ATP contributes to chronic pain. We explored the possibility that receptor sensitization may arise from down-regulation of an intracellular signal negatively controlling receptor function. In view of the structural modeling between the Src region phosphorylated by the C-terminal Src inhibitory kinase (Csk) and the intracellular C terminus domain of the P2X₃ receptor, we investigated how Csk might regulate receptor activity. Using HEK cells and the in vitro kinase assay, we observed that Csk directly phosphorylated the tyrosine 393 residue of the P2X₃ receptor and strongly inhibited receptor currents. On mouse trigeminal sensory neurons, the role of Csk was tightly controlled by the extracellular level of nerve growth factor, a known algogen. Furthermore, silencing endogenous Csk in HEK or trigeminal cells potentiated P2X₃ receptor responses, confirming constitutive Csk-mediated inhibition. The present study provides the first demonstration of an original molecular mechanism responsible for negative control over P2X₃ receptor function and outlines a potential new target for trigeminal pain suppression.ATP-activated P2X₃ receptors are expressed almost exclusively by mammalian sensory neurons to play an important role in the transduction of painful stimuli to the central nervous system (1). Activation of P2X₃ receptors by ATP released during acute and chronic pain is thought to send nociceptive signals to central pain-related networks (2). In view of the multitude of environmental stimuli normally reaching sensory terminals, the question then arises how inappropriate activation of P2X₃ receptors is normally prevented. This process may contribute to suppression of continuous pain sensation in conjunction with central synaptic inhibition.The molecular pathways triggered by algogenic substances and responsible for modulating P2X₃ receptor structure and function remain incompletely understood. This topic is of particular interest because it can provide original clues for novel approaches related to treat pain. The nerve growth factor, NGF,² is one of the most powerful endogenous substances which elicit pain and inflammation via the tyrosine kinase receptor TrkA (3). This neurotrophin stimulates an intracellular cascade that elicits PKC-dependent P2X₃ receptor phosphorylation with ensuing facilitation of receptor currents. Conversely, suppression of NGF signaling powerfully down-regulates P2X₃ receptor function (4). These observations are consistent with the raised NGF levels in acute or inflammatory pain conditions (3). The molecular mechanisms underlying these effects remain, however, unclear.A dynamic balance between tyrosine phosphorylation and dephosphorylation is a major factor controlling the activity of many neurotransmitter receptors (5). TrkA stimulation activates intracellular signaling including Src tyrosine kinases (6) that, in neurons, are important modulators of ligand-gated receptors like nicotinic (7), NMDA receptors (8), and TRPV1 receptors (9). All these receptors are involved in mediating various types of pain in the spinal cord and sensory ganglia. There is, however, no available data on the role of tyrosine phosphorylation on P2X₃ receptor function.The fundamental regulator of Src signaling is the C-terminal Src kinase (Csk) that blocks it via tyrosine phosphorylation (Tyr-527, Refs. 10, 11). We explored whether tyrosine phosphorylation might regulate P2X₃ receptors of sensory neurons by focusing on the P2X₃ C-terminal domain Tyr-393 residue, which is included in a region with significant similarity with the Csk-phosphorylating region of Src. Our data demonstrate that Csk activation induced an increased tyrosine (Tyr-393 residue) P2X₃ receptor phosphorylation with decreased receptor function, observed both in mouse trigeminal sensory neurons as well as a cell expression system. We, thus, propose that Csk-mediated P2X₃ receptor inhibition is a novel mechanism to limit overactivation of P2X₃ receptors.     

Chromenones as potent bradykinin B1 antagonists

A series of fused 6,6-bicyclic chromenones was investigated for activity against the bradykinin B1 receptor. SAR studies based on a pharmacophore model revealed compounds with high affinity for both human and rabbit B1. These compounds demonstrated favorable pharmacokinetic properties and 5-chlorochromenone 15 was efficacious in a carrageenan-induced mechanical hyperalgesia model for chronic pain.     

Juxtamembranous Region of the Amino Terminus of the Family B G Protein-coupled Calcitonin Receptor Plays a Critical Role in Small-molecule Agonist Action

The Family B G protein-coupled calcitonin receptor is an important drug target. The aim of this work was to elucidate the molecular mechanism of action of small-molecule agonist ligands acting at this receptor, comparing it with the action mechanism of the receptor''s natural peptide ligand. cAMP responses to four non-peptidyl ligands and calcitonin were studied in COS-1 cells expressing wild-type and chimeric calcitonin-secretin receptors. All compounds were full agonists at the calcitonin receptor with no activity at the secretin receptor. Only chimeric constructs including the calcitonin receptor amino terminus exhibited responses to any of these ligands. We progressively truncated this domain and tested constructs for cAMP responses. Although calcitonin was able to activate the calcitonin receptor fully with the first 58 residues absent, its potency was 3 orders of magnitude lower than that at the wild-type receptor. After truncation of 114 residues, there was no response to calcitonin. In contrast, small-molecule ligands were fully active at receptors having up to 149 amino-terminal residues absent. Those compounds finally became inactive after truncation of 153 residues. Deletion and/or alanine replacement of the region of the calcitonin receptor between residues 150 and 153 resulted in marked reduction in cAMP responses to these compounds, with some compound-specific differences observed, supporting a critical role for this region. Binding studies further supported distinct sites of action of small molecules relative to that of calcitonin. These findings focus attention on the potential importance of the juxtamembranous region of the amino terminus of the Family B calcitonin receptor for agonist drug action.Calcitonin (CT),³ a 32-amino acid peptide secreted by the thyroid gland in response to elevations in blood calcium levels, acts on bone and kidney to maintain calcium homeostasis (1, 2). CT is widely used therapeutically for the treatment of bone-related disorders such as osteoporosis, hypercalcemia of malignancy, and Paget disease (1, 2). This peptide must be administered parenterally, whereas small-molecule agonist ligands that can be administered orally have substantial clinical advantage, particularly for long-term treatment.CT acts on the CT receptor, a Family B G protein-coupled receptor (GPCR). Members of this family include receptors for secretin, vasoactive intestinal polypeptide, parathyroid hormone, corticotrophin-releasing factor, glucagon, glucagon-like peptide 1, CT, and CT gene-related peptide (3), with each having a long extracellular amino-terminal domain containing six conserved cysteine residues that form three intradomain disulfide bonds. This region has been shown to play a critical role in natural peptide ligand binding and receptor activation (4–11).Development of small-molecule ligands for Family B GPCRs represents an area of great interest. For the CT receptor, several such ligands have been developed (12–14). However, the mechanisms of their actions at their receptors remain unclear. Most recently, synthesis of a series of pyrazolopyridine CT receptor ligands described as partial agonists has been reported (15). Here, we explore the structural basis for their action at the CT receptor. Using CT-secretin receptor chimeric analysis, the amino terminus of the CT receptor was identified as a critical region for the action of these small-molecule ligands but with determinants that are distinct from those interacting with the natural ligand, CT. Truncation, deletion, and alanine replacement mutations of the CT receptor identified that three specific receptor residues, Tyr¹⁵⁰, Leu¹⁵¹, and Ile¹⁵³, within the juxtamembranous region of the amino-terminal domain of the CT receptor, play important roles in the actions of these small-molecule agonists. This study represents the first identification of this region as being critical for the action of small-molecule drugs acting at Family B GPCRs.     

Blockade of Bradykinin receptors worsens the dystrophic phenotype of <Emphasis Type="Italic">mdx</Emphasis> mice: differential effects for B1 and B2 receptors

The Kallikrein Kinin System (KKS) is a vasoactive peptide system with known functions in the maintenance of tissue homeostasis, renal function and blood pressure. The main effector peptide of KKS is Bradykinin (BK). This ligand has two receptors: a constitutive B2 receptor (B2R), which has been suggested to have anti-fibrotic effects in renal and cardiac models of fibrosis; and the inducible B1 receptor (B1R), whose expression is induced by damage and inflammation. Inflammation and fibrosis are hallmarks of Duchenne muscular dystrophy (DMD), therefore we hypothesized that the KKS may play a role in this disease. To evaluate this hypothesis we used the mdx mouse a model for DMD. We blocked the endogenous activity of the KKS by treating mdx mice with B2R antagonist (HOE-140) or B1R antagonist (DesArgLeu⁸BK (DALBK)) for four weeks. Both antagonists increased damage, fibrosis, TGF-β and Smad-dependent signaling, CTGF/CCN-2 levels as well as the number of CD68 positive inflammatory cells. B2R blockade also reduced isolated muscle contraction force. These results indicate that the endogenous KKS has a protective role in the dystrophic muscle. The KKS may be a new target for future therapies to reduce inflammation and fibrosis in dystrophic muscle.     

Update on peripheral mechanisms of pain: beyond prostaglandins and cytokines

The peripheral nociceptor is an important target of pain therapy because many pathological conditions such as inflammation excite and sensitize peripheral nociceptors. Numerous ion channels and receptors for inflammatory mediators were identified in nociceptors that are involved in neuronal excitation and sensitization, and new targets, beyond prostaglandins and cytokines, emerged for pain therapy. This review addresses mechanisms of nociception and focuses on molecules that are currently favored as new targets in drug development or that are already targeted by new compounds at the stage of clinical trials - namely the transient receptor potential V1 receptor, nerve growth factor, and voltage-gated sodium channels - or both.     

Immunolocalization and expression of kinin B1R and B2R receptors in human inflammatory bowel disease

Bradykinin is a mediator of inflammation, responsible for pain, vasodilation, and capillary permeability. Bradykinin receptor 1 (B(1)R) and bradykinin receptor 2 (B(2)R) are G protein-coupled receptors that mediate kinin effects. The latter is constitutive and rapidly desensitized; the former is induced by inflammatory cytokines and resistant to densensitization. The distribution of bradykinin receptors in human intestinal tissue was studied in patients with inflammatory bowel disease (IBD), namely ulcerative colitis (UC) and Crohn's disease (CD). Both B(2)R and B(1)R proteins are expressed in the epithelial cells of normal and IBD intestines. B(1)R protein is visualized in macrophages at the center of granulomas in CD. B(2)R protein is normally present in the apexes of enterocytes in the basal area and intracellularly in inflammatory tissue. In contrast, B(1)R protein is found in the basal area of enterocytes in normal intestine but in the apical portion of enterocytes in inflamed tissue. B(1)R protein is significantly increased in both active UC and CD intestines compared with controls. In patients with active UC, B(1)R mRNA is significantly higher than B(2)R mRNA. However, in inactive UC patients, the B(1)R and B(2)R mRNA did not differ significantly. Thus bradykinin receptors in IBD may reflect intestinal inflammation. Increased B(1)R gene and protein expression in active IBD provides a structural basis of the important role of bradykinin in chronic inflammation.     

Modeling the binding modes of stilbene analogs to cyclooxygenase-2: a molecular docking study

Stilbene analogs are a new class of anti-inflammatory compounds that effectively inhibit COX-2, which is the major target in the treatment of inflammation and pain. In this study, docking simulations were conducted using AutoDock 4 software that focused on the binding of this class of compounds to COX-2 protein. Our aim was to better understand the structural and chemical features responsible for the recognition mechanism of these compounds, and to explore their binding modes of interaction at the active site by comparing them with COX-2 co-crystallized with SC-558. The docking results allowed us to provide a plausible explanation for the different binding affinities observed experimentally. These results show that important conserved residues, in particular Arg513, Phe518, Trp387, Leu352, Leu531 and Arg120, could be essential for the binding of the ligands to COX-2 protein. The quality of the docking model was estimated based on the binding energies of the studied compounds. A good correlation was obtained between experimental logAr values and the predicted binding energies of the studied compounds.     

Identification and pharmacological characterization of domains involved in binding of CGRP receptor antagonists to the calcitonin-like receptor

The calcitonin-like receptor (CLR) and the calcitonin receptor (CTR) interact with receptor activity-modifying protein 1 (RAMP1) at the cell surface to form heterodimeric receptor complexes. CLR and CTR are members of the class II (family B) G-protein-coupled receptors (GPCR) and bind calcitonin gene-related peptide (CGRP) with similar affinities when coexpressed with RAMP1. The observation that various nonpeptide CGRP receptor antagonists display a higher affinity for the CLR/RAMP1 complex than for CTR/RAMP1 provided an opportunity to investigate the molecular determinants of the differential receptor affinities of these antagonists. A chimeric receptor approach was utilized to identify key domains within CLR responsible for conferring high-affinity antagonist binding. Initial chimera experiments implicated distinct regions within CLR as responsible for the affinities of structurally diverse CGRP receptor antagonists. Dissection of these key regions implicated amino acids 37-63 located in the amino terminus of CLR as responsible for the high-affinity interaction of one structural class, while transmembrane domain (TM) 7 was responsible for the interaction of a second class of antagonist. A unique binding interaction in the amino terminus of CLR is consistent with the observation that these compounds also interact with the extracellular region of RAMP1 and could suggest the formation of a binding pocket between the two proteins. Conversely, a compound which interacted with TM7 did not display a similar RAMP1 dependence, suggesting an allosteric mechanism of antagonism. Collectively, these data provide insight into two alternative mechanisms of antagonism for this unique heterodimeric receptor complex.