N-terminal extended conjugates of the agonists and antagonists of both bradykinin receptor subtypes: Structure–activity relationship, cell imaging using ligands conjugated with fluorophores and prospect for functionally active cargoes

Peptide agonists and antagonists of both bradykinin (BK) B1 and B2 receptors (B1R, B2R) 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 ɛ-aminocaproyl (ɛ-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 (B1R: B-10376: CF-ɛ-ACA-Lys-Lys-[Hyp3, CpG5, d-Tic7, CpG8]des-Arg9-BK; B2R: B-10380: CF-ɛ-ACA-d-Arg-[Hyp3, Igl5, d-Igl7, Oic8]-BK and fluorescein-5-thiocarbamoyl (FTC)-B-9430) label the plasma membrane of cells expressing the cognate receptors. The B2R agonists CF-ɛ-ACA-BK, AF350-ɛ-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 B1R agonist B-10378 (CF-ɛ-ACA-Lys-des-Arg9-BK) is compatible with a particular form of agonist-induced receptor translocation. CF-ɛ-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.

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► Ligands of bradykinin B1 and B2 receptors have been extended at their N-terminus.
► A certain loss of affinity was observed, more so for agonists than antagonists.
► The full set of fluorescent agonist and antagonist ligands has been characterized.
► Angiotensin converting enzyme was imaged using a fluorescent bradykinin analog.
► Metal- or drug-containing conjugates show the prospect of functional cargoes.