Inactivation-Resistant Agonist of the Bradykinin B2 Receptor Derived from the Peptide Antagonist B-9430 (D-Arg-[Hyp,Igl,D-Igl,Oic]-bradykinin): Pharmacologic Profile and Effective Induction of Receptor Degradation

The bradykinin B2 receptor is a heptahelical receptor regulated by a cycle of phosphorylation, endocytosis, and extensive recycling at the cell surface following agonist stimulation. B-9430 (D-Arg-[Hyp,Igl,D-Igl,Oic]-bradykinin) is a second generation peptide antagonist found to be competitive at the human B2 receptor and insurmountable at the rabbit B2 receptor (contractility assays, isolated human umbilical and rabbit jugular veins). Two isomers of this peptide were prepared: B-10344 (D-Arg[Hyp,Igl,Oic,D-Igl]-bradykinin; inverted sequence Oic, D-Igl) and B-9972 (D-Arg-[Hyp,Igl,Oic,Igl]-bradykinin); they are lowand high-potency agonists, respectively, in vascular preparations. The potency gap between bradykinin and B-9972 is narrow in contractility assays, despite the fact that B-9972 affinity is 7-fold inferior at the rabbit B2 receptor (radioligand binding competition assay). The effects of agonists on receptors were compared using two chimerical constructions based on rabbit B2 receptors: conjugate of the B2 receptor with green fluorescent protein (B2R-GFP) and the N-terminally tagged conjugate of the myc epitope with the B2 receptor. Imaging and immunoblotting showed that B-9972 induced a persistent endocytosis of cell surface B2 receptors in human embryonic kidney 293 cells with slow receptor degradation (weak after 3 h of treatment, important at 12 h) and B2R-GFP desensitization ([H]bradykinin endocytosis and extracellular signal-regulated kinase 1/2 phosphorylation assays). Bradykinin was not active in this respect but when combined with captopril, induced some degradation. B-9430 reduced the endocytosis and degradation of B2 receptors by the agonists. The results illustrate the agonist-antagonist transition in B2 receptor peptide ligands with a constrained C-terminal structure, the importance of species in their pharmacological profile, and the possibility of selectively degrading receptors using a peptidase-resistant agonist. The blood-derived peptide bradykinin and its widely expressed B2 receptor subtype constitute together an excellent example of a cycling system of ligand-G protein-coupled receptor (GPCR) complex with agonist-induced receptor phosphorylation by GPCR kinases (Blaukat et al., 2001), receptorThis study was supported by the Canadian Institutes of Health Research (Operating Grant MOP-14077 to F.M. and Canada Graduate Scholarships Doctoral Award to G.M.). M.-T.B. and L.G. contributed equally to this work. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.123422. ABBREVIATIONS: GPCR, G protein-coupled receptor; B2R-GFP, conjugate of the B2 receptor with green fluorescent protein; ACE, angiotensin I-converting enzyme; HEK, human embryonic kidney; B-9430, D-Arg-[Hyp,Igl,D-Igl,Oic]-bradykinin; B-9972, D-Arg-[Hyp,Igl,Oic,Igl]-bradykinin; B-10344, D-Arg-[Hyp,Igl,Oic,D-Igl]-bradykinin; myc-B2R, conjugate of the myc epitope with the B2 receptor; Hoe 140, D-Arg[Hyp ,Thi, D-Tic,Oic]-bradykinin; LF16-0687, 1-[[2,4-dichloro-3-[(2,4-dimethylquinolin-8-yl)oxy]methyl]phenyl]sulfonyl]-N-[3-[[4-(aminoiminomethyl]-phenyl]carbonylamino]propyl]-2(S)-pyrrolidinecarboxamide, mesylate salt; bradyzide, (2S)-1-[4-(4-benzhydrylthiosemicarbazido)-3-nitrobenzenesulfonyl]-pyrrolidine-2-carboxylic acid {2-(2-dimethylaminoethyl)methylamino]ethyl}amide; compound 11, 2-{(2R)-1-[(3,4-dichlorophenyl) sulfonyl]-3oxo-1,2,3,4-tetrahydroquinoxalin-2-yl}-N-{2-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]ethyl}acetamide; PCR, polymerase chain reaction; FBS, fetal bovine serum; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; EGF, epidermal growth factor; BK, bradykinin; ANOVA, analysis of variance; NPC 17731, D-Arg[Hyp,D-HypE(trans-propyl),Oic]-bradykinin; B-9870, (CU201) dimer of B-9430 linked at their N terminus by suberidimyl. 0022-3565/07/3232-534–546$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 323, No. 2 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 123422/3267592 JPET 323:534–546, 2007 Printed in U.S.A. 534 at A PE T Jornals on July 7, 2017 jpet.asjournals.org D ow nladed from mediated ligand endocytosis and degradation (Munoz and Leeb-Lundberg, 1992), and, subsequently, very extensive receptor recycling at the cell surface upon agonist washout (Blaukat et al., 1996). In previous studies, a green fluorescent protein conjugate of the rabbit B2 receptor, B2R-GFP, has been shown to be internalized and extensively recycled when the agonist was degraded in the extracellular space (evidence based on immunoblotting using anti-GFP tag antibodies, imaging, and bradykinin concentration measurements; Bachvarov et al., 2001; Houle et al., 2003). Thus, bradykinin clearance, mainly by angiotensin I-converting enzyme (ACE) present in the serum-containing culture medium of HEK 293 cells expressing B2R-GFP, was sufficient for the recycling of the receptor construction at the cell surface in 60 min (Bachvarov et al., 2001). Pharmacological ACE inhibition with captopril or the use of a carboxypeptidaseand ACE-resistant bradykinin analog, [Phe (CH2NH)Arg ]bradykinin (Drapeau et al., 1988), led to a prolonged period (3 h) of B2R-GFP endocytosis in HEK 293 cells, but without degradation (down-regulation) (Bachvarov et al., 2001; Marceau et al., 2002; Houle and Marceau, 2003). Multiple cycles of receptor endocytosis and recycling may be suspected in these experiments. Bradykinin in the presence of captopril or the bradykinin C-protected analog may not be resistant to the multiple degradation pathways operating in endosomes (Munoz and Leeb-Lundberg, 1992). In recent experiments from another laboratory, the essentially complete recycling of human B2R-GFP was attributed to both a high level of surface expression and to a structural effect of GFP that favored receptor recycling over degradation, relative to wild-type human B2 receptors for which bradykinin-induced degradation was significant in less than 2 h (Kalatskaya et al., 2006). B-9430 (structure in Fig. 1) is a structurally constrained and sequence-related bradykinin antagonist with high affinity at the B2 receptor (Stewart et al., 1996). The drug has been widely exploited in studies attempting to define a role for the kallikrein-kinin system in physiology and pathology (Pan et al., 2001; Uknis et al., 2001; Leeb-Lundberg et al., 2005). Interestingly, an exact isomer of B-9430, B-9972 (Fig. 1; Oic,Igl sequence instead of D-Igl,Oic), is reportedly an agonist or a partial agonist possessing a large intrinsic activity (Bironaite et al., 2004; Taraseviciene-Stewart et al., 2005). The transition from an agonist to an antagonist peptide at the B2 receptor may be dependent on the spatial orientation of the C-terminal sequence (Vavrek and Stewart, 1985) and structural constraint in the backbone is not incompatible with the agonist status, as shown with B-9972. This peptide integrates several substitutions that should make it resistant to multiple peptidases/proteases, including aminopeptidases (Fig. 1). In a rare attempt to evaluate therapeutic actions of bradykinin receptor agonists, B-9972 was found to alleviate experimental pulmonary hypertension and its cardiac complications via the classical vasodilator effect mediated by endothelial B2 receptors (Taraseviciene-Stewart et al., 2005). The present work had three general aims: firstly, to extend the pharmacological characterization of B-9972 at the human and rabbit receptors, including some recombinant receptor systems. To further define the effect of the peptide C-terminal stereochemistry on receptor function, an intermediate isomer, B-10344, has been synthesized and evaluated. The second aim was to verify the hypothetical effect of the C-terminal receptor fusion with GFP on the recycling of the rabbit B2 receptor. A construction that included an N-terminal antigenic tag, myc-B2R, has been produced and evaluated for that purpose. Finally, the cycling of traceable forms of the rabbit B2 receptor in HEK 293 cells, B2R-GFP and the mycB2R construction, have been examined as a function of the agonist structure. As a peptide ligand with a predicted extended resistance to intracellular degradation, notably if compared with [Phe (CH2NH)Arg ]bradykinin, B-9972 might exert distinctive effects on receptor cycling. Long-term