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Kinins are the small and fragile hydrophilic peptides related to bradykinin (BK) and derived from circulating kininogens via the action of kallikreins. Kinins bind to the preformed and widely distributed B 2 receptor (B 2 R) and to the inducible B 1 receptor (B 1 R). B 2 Rs and B 1 Rs are related G protein coupled receptors that possess natural agonist ligands of nanomolar affinity (BK and Lys BK for B 2 Rs, Lys-des-Arg 9 -BK for B 1 R). Decades of structure-activity exploration have resulted in the production of peptide analogs that are antagonists, one of which is clinically used (the B 2 R antagonist icatibant), and also non-peptide ligands for both receptor subtypes. The modification of kinin receptor ligands has made them resistant to extracellular or endosomal peptidases and/or produced bifunctional ligands, defined as agonist or antagonist peptide ligands conjugated with a chemical fluorophore (emitting in the whole spectrum, from the infrared to the ultraviolet), a drug-like moiety, an epitope, an isotope chelator/carrier, a cleavable sequence (thus forming a pro-drug) and even a fused protein. Dual molecular targets for specific modified peptides may be a source of side effects or of medically exploitable benefits. Biotechnological protein ligands for either receptor subtype have been produced: they are enhanced green fluorescent protein or the engineered peroxidase APEX2 fused to an agonist kinin sequence at their C-terminal terminus. Antibodies endowed with pharmacological actions (agonist, antagonist) at B 2 R have been reported, though not monoclonal antibodies. These findings define classes of alternative ligands of the kinin receptor of potential therapeutic and diagnostic value., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Based on the known fact that the parathyroid hormone (PTH) might be extended at its C-terminus with biotechnological protein cargoes, a vector directing the secretion of PTH1-84 C-terminally fused with the antigenic epitope myc (PTH-myc) was exploited. The functional properties and potential of this analog for imaging PTH1R-expressing cells were examined. The PTH-myc construct was recombinantly produced as a conditioned medium (CM) of transfected HEK 293a cells (typical concentrations of 187nM estimated with ELISAs for PTH). PTH-myc CM induced cyclic AMP formations (10min), with a minor loss of potency relative to authentic PTH1-84, and c-Fos expression (1-3h). Treatment of recipient HEK 293a cells transiently expressing PTH1R with PTH-myc CM (supplemented with a fluorescent monoclonal anti-myc tag antibody, either 4A6 or 9E10) allowed the labeling of endosomal structures positive for Rab5 and/or for β-arrestin1 (microscopy, cytofluorometry). Authentic PTH was inactive in this respect, ruling out a non-specific form of endocytosis like pinocytosis. Using a horseradish peroxidase-conjugated secondary antibody, the endocytosis of the PTH-myc-based antibody complex by endogenous PTH1R was evidenced in MG-63 osteoblastoid cells. The secreted construct PTH-myc represents a bona fide agonist that supports the feasibility of transporting cargoes of considerable molecular weight inside cells using arrestin and Rab5-mediated PTH1R endocytosis. PTH-myc is also transported into cells that express PTH1R at a physiological level. Such tagged peptide hormones may be part of a cancer chemotherapy scheme exploiting a modular cytotoxic secondary antibody and the receptor repertoire expressed in a given tumor., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Peptide agonists and antagonists of both bradykinin (BK) B(1) and B(2) receptors (B(1)R, B(2)R) are known to tolerate to a certain level N-terminal sequence extensions. Using this strategy, we produced and characterized the full set of fluorescent ligands by extending both agonists and antagonist peptides at both receptor subtypes with 5(6)-carboxyfluorescein (CF) and the ε-aminocaproyl (ε-ACA) optional spacer. Alternatively, kinin receptor ligands were extended with another carboxylic acid cargo (chlorambucil, biotinyl, pentafluorocinnamoyl, AlexaFluor-350 (AF350), ferrocenoyl, cetirizine) or with fluorescein isothiocyanate. N-terminal extension always reduced receptor affinity, more importantly for bulkier substituents and more so for the agonist version compared to the antagonist. This loss was generally alleviated by the presence of the spacer and modulated by the species of origin for the receptor. We report and review the pharmacological properties of these N-terminally extended peptides and the use of fluorophore-conjugated ligands in imaging of cell receptors and of angiotensin converting enzyme (ACE) in intact cells. Antagonists (B(1)R: B-10376: CF-ε-ACA-Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK; B(2)R: B-10380: CF-ε-ACA-D-Arg-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]-BK and fluorescein-5-thiocarbamoyl (FTC)-B-9430) label the plasma membrane of cells expressing the cognate receptors. The B(2)R agonists CF-ε-ACA-BK, AF350-ε-ACA-BK and FTC-B-9972 are found in endosomes and model the endosomal degradation of BK in a complementary manner. The uneven surface fluorescence associated to the B(1)R agonist B-10378 (CF-ε-ACA-Lys-des-Arg(9)-BK) is compatible with a particular form of agonist-induced receptor translocation. CF-ε-ACA-BK binds to the carboxydipeptidase ACE with an affinity identical to that of BK. Metal- or drug-containing cargoes further show the prospect of ligands that confer special signaling to kinin receptors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Angiotensin converting enzyme (ACE) is a drug target and an effective bradykinin (BK)-inactivating ectopeptidase. We exploited a recently described [(3)H]enalaprilat binding assay to quantify the full dynamic range of ACE expression in intact human umbilical vein endothelial cells (HUVECs) stimulated with known or novel modulators of ACE expression. Further, the affinities for ACE of a set of physiological substrates were determined using the same assay. BK has the highest affinity (K(i) 525 nM) among known substrates to displace [(3)H]enalaprilat binding from ACE. Tumor necrosis factor (TNF)-alpha repressed the expression of ACE in HUVECs while phorbol 12-myristate 13-acetate (PMA) upregulated it in 24h (approximately 12-fold dynamic range by [(3)H]enalaprilat binding, corroborated by ACE immunoblotting). Intermediate levels of ACE expression were seen in cells stimulated with both PMA and a cytokine. In contrast, high glucose, insulin or EGF failed to affect ACE expression. The effect of TNF-alpha was abated by etanercept, the IKK2 inhibitor TPCA-1, or a p38 inhibitor while that of PMA was reduced by inhibitors of PKC isoforms sensitive to phorbol esters and calcium. The short-term PKC- and MEK1-dependent increase of c-Fos expression was best correlated to PMA-induced ACE upregulation. The [(3)H]enalaprilat binding assay applied to HUVECs supports that ACE is a particularly active kininase and that endothelial ACE expression is dynamically and specifically regulated. This has potential importance in inflammatory diseases and diabetes., (Copyright 2010 Elsevier Inc. All rights reserved.)

B-9430 (d-Arg-[Hyp3, Igl5, D-Igl7, Oic8]-bradykinin), where Hyp is trans-4-hydroxyproline, Igl is alpha-(2-indanyl)glycine and Oic is (3as, 7as)-octahydroindol-2-yl-carbonyl is a high affinity bradykinin B2 receptor antagonist with effects extended to the B1 receptors at high concentrations. The N-terminus of B-9430 has been extended with d-biotinyl (B-10330) or 5(6)-carboxyfluorescein-epsilon-aminocaproyl (B-10380) to derive fluorescent receptor probes. The pharmacological profile of B-10380 was similar to that of B-9430 with a minor loss of potency (a competitive antagonist of bradykinin at the B2 receptors of the human isolated umbilical vein, pA2 6.83; an insurmountable antagonist at the B2 receptors in the rabbit jugular vein; a weak competitive antagonist of the B1 receptors in the rabbit aorta, pA2 5.95). B-10330 and B-10380 displaced the binding of [3H]bradykinin from rabbit B2 receptors with a potency slightly inferior to that of B-9430 (larger gap at the rat B2 receptor). Treatment with B-10330 and fluorescent streptavidin did not support imaging of recombinant B2 receptors. However, the plasma membrane of HEK 293a cells that transiently expressed recombinant rabbit B2 receptors, but not B1 receptors, was labeled with 5-50 nM B-10380 (epifluorescence microscopy). B-10380 staining was not observed in nontransfected cells and was abolished by co-treating receptor-expressing cells with a nonpeptide antagonist. The N-terminal extension of a potent peptide antagonist of the bradykinin B2 receptor with a fluorophore produced a fluorescent probe suitable for live cell imaging and other applications at the expense of a minor loss of affinity.

It has been recently proposed that the second extracellular loop of the human bradykinin (BK) B1 receptor (B1R) contains a conserved HExxH motif also present in peptidases possessing a Zn2+ prosthetic group, such as angiotensin converting enzyme (ACE), and that ACE inhibitors directly activate B1R signaling in endothelial cells. However, the binding of ACE inhibitors to the B1Rs has never been directly evaluated. Information about binding of a radiolabeled inhibitor to natural or recombinant ACE in intact cells (physiologic ionic composition) was also collected. We used the tritiated form of an ACE inhibitor previously proposed to activate the B1R, enalaprilat, to address these questions using recombinant human B1Rs and naturally expressed or recombinant ACE. [3H]Lys-des-Arg9-BK bound to the human recombinant B1Rs with high affinity (KD 0.35 nM) in HEK 293a cells. [3H]Enalaprilat (0.25-10 nM) did not bind to cells expressing recombinant human B1R, but bound with a subnanomolar affinity to recombinant ACE or to naturally expressed ACE in human umbilical vein endothelial cells. The radioligand was further validated using a binding competition assay that involved unlabeled ACE inhibitors or their prodrug forms in endothelial cells. Membranes of HEK 293a cells that expressed B1Rs did not hydrolyze hippuryl-glycylglycine (an ACE substrate). Enalaprilat did not stimulate calcium signaling in HEK 293a cells that expressed B1Rs. A typical ACE inhibitor did not bind to nor stimulate the human B1Rs; nevertheless, several other indirect mechanisms could connect ACE inhibition to B1R stimulation in vivo.

Large species differences have been previously observed in the pharmacology of bradykinin (BK) B2 receptor antagonists. We investigated the effect of two novel non-peptide antagonists, compound 9 (a benzodiazepine peptidomimetic related to icatibant) and the thiosemicarbazide bradyzide on the rabbit B2 receptor (contractility of the jugular vein, competition of [3H]BK binding to a B2 receptor-green fluorescent protein (B2R-GFP) conjugate, subcellular distribution of B2R-GFP). While compound 9 is about 9000-fold less potent than icatibant, it shares with the latter peptide drug a selective, insurmountable and largely irreversible antagonist behavior against BK and the capacity to translocate B2R-GFP from the membrane into the cells. Bradyzide, reportedly very potent at rodent B2 receptors, was a competitive and reversible antagonist of moderate potency at the rabbit B2 receptor (contractility pA2 6.84, binding competition IC50 5 nM). The C-terminal region of icatibant, reproduced by compound 9, is likely to be important in the non-equilibrium behavior of icatibant. Bradyzide, a non-peptide antagonist developed on different structural grounds, is competitive at the rabbit B2 receptor.

The purpose of the present review is to describe the place of endogenous kinins, mainly bradykinin (BK) and des-Arg(9)-BK in the kallikrein-kininogen-kinin system, to review and compare the different analytical methods reported for the assessment of endogenous kinins, to explain the difficulties and the pitfalls for their quantifications in biologic samples and finally to see how the results obtained by these methods could complement and extend the pharmacological evidence of their pathophysiological role.