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Fortin, Jean-Philippe.

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Fortin

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Jean-Philippe.

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Faculté de médecine, Université Laval

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Voici les éléments 1 - 6 sur 6
  • PublicationAccès libre
    A tagged parathyroid hormone derivative as a carrier of antibody cargoes transported by the G protein coupled PTH1 receptor
    (ScienceDirect, 2014-08-12) Fortin, Jean-Philippe.; Allaeys, Isabelle; Poubelle, Patrice; Marceau, François; Lodge, Robert; Charest-Morin, Xavier
    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 187 nM estimated with ELISAs for PTH). PTH-myc CM induced cyclic AMP formations (10 min), with a minor loss of potency relative to authentic PTH1–84, and c-Fos expression (1–3 h). 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.
  • PublicationAccès libre
    Stabilité moléculaire du récepteur B1 des kinines et de ses ligands
    (2006) Fortin, Jean-Philippe.; Marceau, François
    Le récepteur Bj (RBi) des kinines émerge présentement comme une cible thérapeutique de choix pour le traitement de l'inflammation et de la douleur inflammatoire. Bien que les mécanismes régissant l'expression du RBi en conditions pathologiques soient relativement bien documentés au niveau transcriptionnel, peu de données sont disponibles sur sa régulation post-traductionnelle. Les travaux présentés dans cette thèse, principalement basés sur des modèles cellulaires exprimant des RBi recombinants ou naturels, de même que sur des essais de contractilité exploitant des vaisseaux sanguins isolés, visaient à améliorer notre connaissance du RBi et de ses ligands à l'échelle moléculaire. La première partie des études traite des processus enzymatiques qui affectent négativement la puissance de divers ligands peptidiques du RBi dans l'aorte de lapin. L'identification de l'aminopeptidase N exprimée par les cellules musculaires lisses en tant que voie d'inactivation majeure des ligands peptidiques du RBi, a mené à la synthèse de nouveaux antagonistes résistants au métabolisme de cette enzyme. Un prototype d'une nouvelle classe de médicaments inhibant à la fois le RBi et l'aminopeptidase N a aussi été découvert. Le deuxième volet des travaux a démontré que le RBi exprimé à la surface cellulaire est instable et rapidement internalisé en absence de stimulation par l'agoniste. De plus, la démonstration que de nouveaux antagonistes non peptidiques du RBi peuvent agir comme chaperons pharmacologiques en optimisant la maturation du récepteur, a permis de mettre en lumière l'existence d'une forme hautement glycosylée et sensible à l'agoniste du RBi. De façon globale, ces travaux ont mis en évidence l'instabilité moléculaire du RBi et de ses ligands. Les études de pharmacologie moléculaire présentées dans cette Thèse soulèvent aussi plusieurs questions, et ouvrent la voie à de nouvelles investigations sur la régulation du RBi des kinines dans un modèle cellulaire humain.
  • PublicationAccès libre
    Bradykinin receptors : agonists, antagonists, expression, signaling, and adaptation to sustained stimulation
    (Elsevier Science, 2020-02-25) Bachelard, Hélène; Bawolak, Marie-Thérèse; Fortin, Jean-Philippe.; Marceau, François; Morissette, Guillaume; Charest-Morin, Xavier; Bouthillier, Johanne
    Bradykinin-related peptides, the kinins, are blood-derived peptides that stimulate 2 G protein–coupled receptors, the B1 and B2 receptors (B1R, B2R). The pharmacologic and molecular identities of these 2 receptor subtypes will be succinctly reviewed herein, with emphasis on drug development, receptor expression, signaling, and adaptation to persistent stimulation. Peptide and non-peptide antagonists and fluorescent ligands have been produced for each receptor. The B2R is widely and constitutively expressed in mammalian tissues, whereas the B1R is mostly inducible under the effect of cytokines during infection and immunopathology. The B2R is temporarily desensitized by a cycle of phosphorylation/endocytosis followed by recycling, whereas the nonphosphorylable B1R is relatively resistant to desensitization and translocated to caveolae on activation. Both receptor subtypes, mainly coupled to protein G Gq, phospholipase C and calcium signaling, mediate the vascular aspects of inflammation (vasodilation, edema formation). On this basis, icatibant, a peptide antagonist of the B2R, is approved in the management of hereditary angioedema attacks. This disease is the therapeutic showcase of the kallikrein-kinin system, with an orally bioavailable B2R antagonist under development, as well as other agents that inhibit the kinin forming protease, plasma kallikrein. Other clinical applications are still elusive despite the maturity of the medicinal chemistry efforts applied to kinin receptors.
  • PublicationAccès libre
    Vascular smooth muscle contractility assays for inflammatory and immunological mediators
    (Elsevier, 2010-09-08) Audet, Ritchie.; Deblois, Denis; Drapeau, Guy; Sabourin, Thierry; Bawolak, Marie-Thérèse; Koumbadinga, Gérémy Abdull; Fortin, Jean-Philippe.; Levesque, Luc; Marceau, François; Petitclerc, Éric; Morissette, Guillaume; Larrivée, Jean-François.; Godin, Denis; Gera, Lajos; Houle, Steeve.; Bouthillier, Johanne
    The blood vessels are one of the important target tissues for the mediators of inflammation and allergy; further cytokines affect them in a number of ways. We review the use of the isolated blood vessel mounted in organ baths as an important source of pharmacological information. While its use in the bioassay of vasoactive substances tends to be replaced with modern analytical techniques, contractility assays are effective to evaluate novel synthetic drugs, generating robust potency and selectivity data about agonists, partial agonists and competitive or insurmountable antagonists. For instance, the human umbilical vein has been used extensively to characterize ligands of the bradykinin B2 receptors. Isolated vascular segments are live tissues that are intensely reactive, notably with the regulated expression of gene products relevant for inflammation (e.g., the kinin B1 receptor and inducible nitric oxide synthase). Further, isolated vessels can be adapted as assays of unconventional proteins (cytokines such as interleukin-1, proteases of physiopathological importance, complement-derived anaphylatoxins and recombinant hemoglobin) and to the gene knockout technology. The well known cross-talks between different cell types, e.g., endothelium-muscle and nerve terminal-muscle, can be extended (smooth muscle cell interaction with resident or infiltrating leukocytes and tumor cells). Drug metabolism and distribution problems can be modeled in a useful manner using the organ bath technology, which, for all these reasons, opens a window on an intermediate level of complexity relative to cellular and molecular pharmacology on one hand, and in vivo studies on the other.
  • PublicationAccès libre
    Bifunctional ligands of the bradykinin B2 and B1 receptors : an exercise in peptide hormone plasticity
    (Elsevier, 2018-05-23) Bachelard, Hélène; Roy, Caroline; Bawolak, Marie-Thérèse; Fortin, Jean-Philippe.; Marceau, François; Morissette, Guillaume; Lajos, Gera; Charest-Morin, Xavier
    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 B2 receptor (B2R) and to the inducible B1 receptor (B1R). B2Rs and B1Rs are related G protein coupled receptors that possess natural agonist ligands of nanomolar affinity (BK and Lys BK for B2Rs, Lys-des-Arg9-BK for B1R). Decades of structure-activity exploration have resulted in the production of peptide analogs that are antagonists, one of which is clinically used (the B2R 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 B2R have been reported, though not monoclonal antibodies. These findings define classes of alternative ligands of the kinin receptor of potential therapeutic and diagnostic value.
  • PublicationAccès libre
    Bradykinin receptors : agonists, antagonists, expression, signaling and adaptation to sustained stimulation
    (2013-12-01) Fortin, Sébastien; Sabourin, Thierry; Roy, Caroline; Bawolak, Marie-Thérèse; Koumbadinga, Gérémy Abdull; Fortin, Jean-Philippe.; Marceau, François; Morissette, Guillaume; Lodge, Robert; C. Gaudreault, René.; Houle, Steeve.; Charest-Morin, Xavier; Bouthillier, Johanne; Gera, Lajos
    Bradykinin-related peptides, the kinins, are blood-derived peptides that stimulate 2 G protein–coupled receptors, the B1 and B2 receptors (B1R, B2R). The pharmacologic and molecular identities of these 2 receptor subtypes will be succinctly reviewed, with emphasis on drug development, receptor expression, signaling, and adaptation to persistent stimulation. Peptide and nonpeptide antagonists and fluorescent ligands have been produced for each receptor. The B2R is widely and constitutively expressed in mammalian tissues, whereas the B1R is mostly inducible under the effect of cytokines during infection and immunopathology. Both receptor subtypes mediate the vascular aspects of inflammation (vasodilation, edema formation). On this basis, icatibant, a peptide antagonist of the B2R, is approved in the management of hereditary angioedema attacks. Other clinical applications are still elusive despite the maturity of the medicinal chemistry efforts applied to kinin receptors. While both receptor subtypes are mainly coupled to the Gq protein and related second messengers, the B2R is temporarily desensitized by a cycle of phosphorylation/endocytosis followed by recycling, whereas the nonphosphorylable B1R is relatively resistant to desensitization and translocated to caveolae on activation.