Characterization of a selective antagonist of neuropeptide Y at the Y2 receptor. Synthesis and pharmacological evaluation of a Y2 antagonist.
Neuropeptide Y (NPY) is a potent inhibitor of neurotransmitter release through the Y2 receptor subtype. Specific antagonists for the Y2 receptors have not yet been described. Based on the concept of template-assembled synthetic proteins we have used a cyclic template molecule containing two beta-turn mimetics for covalent attachment of four COOH-terminal fragments RQRYNH2 (NPY 33-36), termed T4-[NPY(33-36)]4. This structurally defined template-assembled synthetic protein has been tested for binding using SK-N-MC and LN319 cell lines that express the Y1 and Y2 receptor, respectively. T4-[NPY(33-36)]4 binds to the Y2 receptor with high affinity (IC50 = 67.2 nM) and has poor binding to the Y1 receptor. This peptidomimetic tested on LN319 cells at concentrations up to 10 microM shows no inhibitory effect on forskolin-stimulated cAMP levels (IC50 for NPY = 2.5 nM). Furthermore, we used confocal microscopy to examine the NPY-induced increase in intracellular calcium in single LN319 cells. Preincubation of the cells with T4-[NPY(33-36)]4 shifted to the right the dose-response curves for intracellular mobilization of calcium induced by NPY at concentrations ranging from 0.1 nM to 10 microM. Finally, we assessed the competitive antagonistic properties of T4-[NPY(33-36)]4 at presynaptic peptidergic Y2 receptors modulating noradrenaline release. the compound T4-[NPY(33-36)]4 caused a marked shift to the right of the concentration-response curve of NPY 13-36, a Y2-selective fragment, yielding a pA2 value of 8.48. Thus, to our best knowledge, T4-[NPY(33-36)]4 represents the first potent and selective Y2 antagonist.
Grouzmann
E
E
Division d'Hypertension, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. Eric.Grouzmann@chuv.hospvd.ch
Buclin
T
T
Martire
M
M
Cannizzaro
C
C
Dörner
B
B
Razaname
A
A
Mutter
M
M
D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D009478:Neuropeptide Y / Q000037:antagonists & inhibitors*; D009638:Norepinephrine / Q000378:metabolism; D010456:Peptides, Cyclic / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology*; D017476:Receptors, Neuropeptide Y / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D009478:Neuropeptide Y; D010456:Peptides, Cyclic; D017476:Receptors, Neuropeptide Y; C105135:T(4)-(neuropeptide Y (33-36))4; C095889:neuropeptide Y2 receptor; D000242:Cyclic AMP; D002118:Calcium; D009638:Norepinephrine