Xavier Charest-Morin

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) 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.

Green fluorescent protein fused to peptide agonists of two dissimilar G protein-coupled receptors: novel ligands of the bradykinin B2 (rhodopsin family) receptor and parathyroid hormone PTH1 (secretin family) receptor

We hypothesized that peptide hormone sequences that stimulate and internalize G protein‐coupled receptors (GPCRs) could be prolonged with a functional protein cargo. To verify this, we have selected two widely different pairs of peptide hormones and GPCRs that nevertheless share agonist‐induced arrestin‐mediated internalization. For the parathyroid hormone (PTH) PTH1 receptor (PTH1R) and the bradykinin (BK) B2 receptor (B2R), we have designed fusion proteins of the agonists PTH1‐34 and maximakinin (MK, a BK homologue) with the enhanced green fluorescent protein (EGFP), thus producing candidate high molecular weight ligands. According to docking models of each hormone to its receptor, EGFP was fused either at the N‐terminus (MK) or C‐terminus (PTH1‐34) of the ligand; the last construction is also secretable due to inclusion of the preproinsulin signal peptide and has been produced as a conditioned medium. EGFP‐MK has been produced as a lysate of transfected cells. Using an enzyme‐linked immunosorbent assay (ELISA) for GFP, average concentrations of 1.5 and 1670 nmol/L, respectively, of ligand were found in these preparations. The functional properties and potential of these analogs for imaging receptor‐expressing cells were examined. Microscopic and cytofluorometric evidence of specific binding and internalization of both fusion proteins was obtained using recipient HEK 293a cells that expressed the cognate recombinant receptor. Endosomal colocalization studies were conducted (Rab5, Rab7, β‐arrestin1). Evidence of agonist signaling was obtained (expression of c‐Fos, cyclic AMP responsive element (CRE) reporter gene for PTH1‐34‐EGFP). The constructs PTH1‐34‐EGFP and EGFP‐MK represent bona fide agonists that support the feasibility of transporting protein cargoes inside cells using GPCRs.

Inhibitory effects of cytoskeleton disrupting drugs and GDP-locked Rab mutants on bradykinin B2 receptor cycling

The bradykinin (BK) B₂ receptor (B₂R) is G protein coupled and phosphorylated upon agonist stimulation; its endocytosis and recycling are documented. We assessed the effect of drugs that affect the cytoskeleton on B2R cycling. These drugs were targeted to tubulin (paclitaxel, or the novel combretastatin A-4 mimetic 3,4,5-trimethoxyphenyl-4-(2-oxoimidazolidin-1-yl)benzenesulfonate [IMZ-602]) and actin (cytochalasin D). Tubulin ligands did not alter agonist-induced receptor endocytosis, as shown using antibodies reactive with myc-tagged B₂Rs (microscopy, cytofluorometry), but rather reduced the progression of the ligand-receptor-β-arrestin complex from the cell periphery to the interior. The 3 fluorescent probes of this complex (B2R-green fluorescent protein [B2R-GFP], the fluorescent agonist fluorescein-5-thiocarbamoyl-D-Arg-[Hyp³, Igl⁵, Oic⁷, Igl⁸]-BK and β-arrestin2-GFP) were condensed in punctuate structures that remained close to the cell surface in the presence of IMZ-602. Cytochalasin D selectively inhibited the recycling of endocytosed B₂R-GFP (B₂R-GFP imaging, [³H]BK binding). Dominant negative (GDP-locked)-Rab5 and -Rab11 reproduced the effects of inhibitors of tubulin and actin, respectively, on the cycling of B₂R-GFP. GDP-locked-Rab4 also inhibited B₂R-GFP recycling to the cell surface. Consistent with the displacement of cargo along specific cytoskeletal elements, Rab5-associated progression of the endocytosed BK B₂R follows microtubules toward their (-) end, while its recycling progresses along actin fibers to the cell surface. However, tubulin ligands do not suppress the tested desensitization or resensitization mechanisms of the B₂R.

Pharmacological effects of recombinant human tissue kallikrein on bradykinin B2 receptors

Tissue kallikrein (KLK‐1), a serine protease, initiates the release of bradykinin (BK)‐related peptides from low‐molecular weight kininogen. KLK‐1 and the BK B2 receptor (B2R) mediate beneficial effects on the progression of type 2 diabetes and renal disease, but the precise role of KLK‐1 independent of its kinin‐forming activity remains unclear. We used DM199, a recombinant form of human KLK‐1, along with the isolated human umbilical vein, a robust bioassay of the B2R, to address the previous claims that KLK‐1 directly binds to and activates the human B2R, with possible receptor cleavage. DM199 (1–10 nmol/L) contracted the isolated vein via the B2R, but in a tachyphylactic, kinin‐dependent manner, without desensitization of the tissue to exogenously added BK. In binding experiments with recombinant N‐terminally tagged myc‐B2Rs expressed in HEK 293a cells, DM199 displaced [3H]BK binding from the rabbit myc‐B2R, but not from the human or rat myc‐B2Rs. No evidence of myc‐B2R degradation by immunoblot analysis was apparent following treatment of these 3 myc‐B2R constructs with DM199 (30 min, ≤10 nmol/L). In HEK 293 cells stably expressing rabbit B2R‐GFP, DM199 (11–108 pmol/L) elicited signaling‐dependent endocytosis and reexpression, while a higher concentration (1.1 nmol/L) induced a partially irreversible endocytosis of the construct (microscopy), paralleled by the appearance of free GFP in cells (immunoblotting, indicative of incomplete receptor down‐regulation). The pharmacology of DM199 at relevant concentrations (<10 nmol/L) is essentially based on the activity of locally generated kinins. Binding to and mild down‐regulation of the B2R is possibly a species‐dependent idiosyncratic response to DM199.

Pharmacological evidence of bradykinin regeneration from extended sequences that behave as peptidase–activated B2 receptor agonists

While bradykinin (BK) is known to be degraded by angiotensin converting enzyme (ACE), we have recently discovered that Met-Lys-BK-Ser-Ser is paradoxically activated by ACE. We designed and evaluated additional “prodrug” peptides extended around the BK sequence as potential ligands that could be locally activated by vascular or blood plasma peptidases. BK regeneration was estimated using the contractility of the human umbilical vein as model of vascular functions mediated by endogenous B2 receptors (B2Rs) and the endocytosis of the fusion protein B2R-green fluorescent protein (B2R-GFP) expressed in Human Embryonic Kidney 293 cells. Of three BK sequences extended by a C-terminal dipeptide, BK-His-Leu had the most desirable profile, exhibiting little direct affinity for the receptor but a significant one for ACE (as shown by competition of [3H]BK binding to B2R-GFP or of [3H]enalaprilat to recombinant ACE, respectively). The potency of the contractile effect of this analog on the vein was reduced 18-fold by the ACE inhibitor enalaprilat, pharmacologically evidencing BK regeneration in situ. BK-Arg, a potential substrate of arginine carboxypeptidases, had a low affinity for B2Rs and its potency as a contractile agent was reduced 15-fold by tissue treatment with an inhibitor of these enzymes, Plummer’s inhibitor. B2R-GFP internalization in response to 100 nM of the extended peptides recapitulated these findings, as enalaprilat selectively inhibited the effect of BK-His-Leu and Plummer’s inhibitor, that of BK-Arg. The two peptidase inhibitors did not affect BK-induced effects in either assay. The novel C-terminally extended BKs had no or very little affinity for the kinin B1 receptor (competition of [3H]Lys-des-Arg9-BK binding). The feasibility of peptidase-activated B2R agonists is illustrated by C-terminal extensions of the BK sequence.

C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

The C–C chemokine receptor-7 (CCR7) is a G protein coupled receptor that has a role in leukocyte homing, but that is also expressed in aggressive tumor cells. Preclinical research supports that CCR7 is a valid target in oncology. In view of the increasing availability of therapeutic monoclonal antibodies that carry cytotoxic cargoes, we studied the feasibility of forcing intact cells to internalize known monoclonal antibodies by exploiting the cycle of endocytosis and recycling triggered by the CCR7 agonist CCL19. Firstly, an anti-CCR7 antibody (CD197; clone 150503) labeled surface recombinant CCR7 expressed in intact HEK 293a cells and the fluorescent antibody was internalized following CCL19 treatment. Secondly, a recombinant myc-tagged CCL19 construction was exploited along the anti-myc monoclonal antibody 4A6. The myc-tagged ligand was produced as a conditioned medium of transfected HEK 293a cells that contained the equivalent of 430 ng/ml of immunoreactive CCL19 (average value, ELISA determination). CCL19-myc, but not authentic CCL19, carried the fluorophore-labeled antibody 4A6 into other recipient cells that expressed recombinant CCR7 (microscopy, cytofluorometry). The immune complexes were apparent in endosomal structures, co-localized well with the small GTPase Rab5 and progressed toward Rab7-positive endosomes. A dominant negative form of Rab5 (GDP-locked) inhibited this endocytosis. Further, endosomes in CCL19-myc- or CCL19-stimulated cells were positive for β-arrestin2, but rarely for β-arrestin1. Following treatment with CCL19-myc and the 4A6 antibody, the melanoma cell line A375 that expresses endogenous CCR7 was specifically stained using a secondary peroxidase-conjugated antibody. Agonist-stimulated CCR7 can transport antibody-based cargoes, with possible therapeutic applications in oncology.

Autophagic flux inhibition and lysosomogenesis ensuing cellular capture and retention of the cationic drug quinacrine in murine models

The proton pump vacuolar (V)-ATPase is the driving force that mediates the concentration of cationic drugs (weak bases) in the late endosome-lysosome continuum; secondary cell reactions include the protracted transformation of enlarged vacuoles into autophagosomes. We used the inherently fluorescent tertiary amine quinacrine in murine models to further assess the accumulation and signaling associated with cation trapping. Primary fibroblasts concentrate quinacrine ∼5,000-fold from their culture medium (KM 9.8 µM; transport studies). The drug is present in perinuclear granules that are mostly positive for Rab7 and LAMP1 (microscopy). Both drug uptake and retention are extensively inhibited by treatments with the V-ATPase inhibitor bafilomycin A1. The H+ ionophore monensin also prevented quinacrine concentration by fibroblasts. However, inhibition of plasma membrane transporters or of the autophagic process with spautin-1 did not alter quinacrine transport parameters. Ancillary experiments did not support that low micromolar concentrations of quinacrine are substrates for organic cation transporters-1 to -3 or P-glycoprotein. The secondary autophagy induced by quinacrine in cells may derive from the accumulation of incompetent autophagolysosomes, as judged from the accumulation of p62/SQSTM1 and LC3 II (immunoblots). Accordingly, protracted lysosomogenesis is evidenced by increased expression of LAMP1 and LAMP2 in quinacrine-treated fibroblasts (48 h, immunoblots), a response that follows the nuclear translocation of the lysosomal genesis transcription factor TFEB and upregulation of LAMP1 and −2 mRNAs (24 h). Quinacrine administration to live mice evidenced variable distribution to various organs and heterogeneous accumulation within the lung (stereo-microscopy, extraction). Dose-dependent in vivo autophagic and lysosomal accumulation was observed in the lung (immunoblots). No evidence has been found for transport or extrusion mechanisms modulating the cellular uptake of micromolar quinacrine at the plasma membrane level. As shown in vitro and in vivo, V-ATPase-mediated cation sequestration is associated, above a certain threshold, to autophagic flux inhibition and feed-back lysosomogenesis.

Vasopeptidase-activated latent ligands of the histamine receptor-1.

Whether peptidases present in vascular cells can activate prodrugs active on vascular cells has been tested with 2 potential latent ligands of the histamine H1 receptor (H1R). First, a peptide consisting of the antihistamine cetirizine (CTZ) condensed at the N-terminus of ε-aminocaproyl-bradykinin (εACA-BK) was evaluated for an antihistamine activity that could be revealed by degradation of the peptide part of the molecule. CTZ-εACA-BK had a submicromolar affinity for the BK B2 receptor (B2R; IC50 of 590 nM, [(3)H]BK binding competition), but a non-negligible affinity for the human H1 receptor (H1R; IC50 of 11 μM for [(3)H]pyrilamine binding). In the human isolated umbilical vein, a system where both endogenous B2R and H1R mediate strong contractions, CTZ-εACA-BK exerted mild antagonist effects on histamine-induced contraction that were not modified by omapatrilat or by a B2R antagonist that prevents endocytosis of the BK conjugate. Cells expressing recombinant ACE or B2R incubated with CTZ-εACA-BK did not release a competitor of [(3)H]pyrilamine binding to H1Rs. Thus, there is no evidence that CTZ-εACA-BK can release free cetirizine in biological environments. The second prodrug was a blocked agonist, L-alanyl-histamine, potentially activated by aminopeptidase N (APN). This compound did not compete for [(3)H]pyrilamine binding to H1Rs. The human umbilical vein contractility assay responded to L-alanyl-histamine (EC50 54.7 μM), but the APN inhibitor amastatin massively (17-fold) reduced its apparent potency. Amastatin did not influence the potency of histamine as a contractile agent. One of the 2 tested latent H1R ligands, L-alanyl-histamine, supported the feasibility of pro-drug activation by vascular ectopeptidases.

In Vivo Effects of Bradykinin B2 Receptor Agonists with Varying Susceptibility to Peptidases.

We reported evidence of bradykinin (BK) regeneration from C-terminal extended BK sequences that behave as peptidase-activated B2 receptor (B2R) agonists. Further to these in vitro studies, we carried out in vivo experiments to verify hemodynamic effects of BK analogs exhibiting variable susceptibility toward vascular and blood plasma peptidases. Rats were anesthetized and instrumented to record blood pressure and heart rate responses to bolus intravenous (i.v.) injection of increasing doses of BK, B-9972 (D-Arg-[Hyp3,Igl5,Oic7,Igl8]-BK), BK-Arg, BK-His-Leu or BK-Ala-Pro, in the absence or presence of specific inhibitors. In some experiments, pulsed Doppler flow probes measured hindquarter Doppler shift in response to i.v. injections of kinins. BK caused rapid, transient and dose-related hypotensive effects. These effects were potentiated ∼15-fold by the angiotensin converting enzyme (ACE) inhibitor, enalaprilat, but extensively inhibited by icatibant (a B2R antagonist) and not influenced by the Arg-carboxypeptidase (CP) inhibitor (Plummer’s inhibitor). The hypotensive responses elicited by the peptidase-resistant B2R agonist, B-9972, were not affected by enalaprilat, but were inhibited by icatibant. The hypotensive responses to BK-Arg were abolished by pre-treatment with either the Arg-CP inhibitor or icatibant, pharmacologically evidencing BK regeneration. The hypotensive effects of BK-His-Leu and BK-Ala-Pro, previously reported as ACE-activated substrates, were abolished by icatibant, but not by enalaprilat. In vivo regeneration of BK from these two C-terminally extended analogs with no affinity for the B2R must follow alternative cleavage rules involving unidentified carboxypeptidase(s) when ACE is blocked. The transient hypotensive responses to BK and three tested analogs coincided with concomitant vasodilation (increased Doppler shift signal). Together, these results provide in vivo evidence that interesting hypotensive and vasodilator effects can be extracted from prodrug peptides that behave as peptidase-activated B2R agonists.

Biotechnological Fluorescent Ligands of the Bradykinin B1 Receptor: Protein Ligands for a Peptide Receptor.

The bradykinin (BK) B1 receptor (B1R) is a peculiar G protein coupled receptor that is strongly regulated to the point of being inducible in immunopathology. Limited clinical evidence suggests that its expression in peripheral blood mononuclear cells is a biomarker of active inflammatory states. In an effort to develop a novel imaging/diagnostic tool, we report the rational design and testing of a fusion protein that is a ligand of the human B1R but not likely to label peptidases. This ligand is composed of a fluorescent protein (FP) (enhanced green FP [EGFP] or mCherry) prolonged at its N-terminus by a spacer peptide and a classical peptide agonist or antagonist (des-Arg9-BK, [Leu8]des-Arg9-BK, respectively). The design of the spacer-ligand joint peptide was validated by a competition assay for [3H]Lys-des-Arg9-BK binding to the human B1R applied to 4 synthetic peptides of 18 or 19 residues. The labeling of B1R-expressing cells with EGFP or mCherry fused with 7 of such peptides was performed in parallel (microscopy). Both assays indicated that the best design was FP-(Asn-Gly)n-Lys-des-Arg9-BK; n = 15 was superior to n = 5, suggesting benefits from minimizing steric hindrance between the FP and the receptor. Cell labeling concerned mostly plasma membranes and was inhibited by a B1R antagonist. EGFP-(Asn-Gly)15-Lys-des-Arg9-BK competed for the binding of [3H]Lys-des-Arg9-BK to human recombinant B1R, being only 10-fold less potent than the unlabeled form of Lys-des-Arg9-BK to do so. The fusion protein did not label HEK 293a cells expressing recombinant human BK B2 receptors or angiotensin converting enzyme. This study identifies a modular C-terminal sequence that can be adapted to protein cargoes, conferring high affinity for the BK B1R, with possible applications in diagnostic cytofluorometry, histology and drug delivery (e.g., in oncology).