Identification of a Cyanine-Dye Labeled Peptidic Ligand for Y₁R and Y₄R, Based upon the Neuropeptide Y C-Terminal Analogue, BVD-15

Abstract

Traceable truncated Neuropeptide Y (NPY) analogues with Y 1 receptor (Y 1 R) affinity and selectivity are highly desirable tools in studying receptor location, regulation, and biological functions. A range of fluorescently labeled analogues of a reported Y 1 R/Y 4 R preferring ligand BVD-15 have been prepared and evaluated using high content imaging techniques. One peptide, [Lys 2 (sCy5), Arg 4 ]BVD-15, was characterized as an Y 1 R antagonist with a pK D of 7.2 measured by saturation analysis using fluorescent imaging. The peptide showed 8-fold lower affinity for Y 4 R (pK D = 6.2) and was a partial agonist at this receptor. The suitability of [Lys 2 (sCy5), Arg 4 ]BVD-15 for Y 1 R and Y 4 R competition binding experiments was also demonstrated in intact cells. The nature of the label was shown to be critical with replacement of sCy5 by the more hydrophobic Cy5.5 resulting in a switch from Y 1 R antagonist to Y 1 R partial agonist.

Heterodimeric Analogues of the Potent Y1R Antagonist 1229U91, Lacking One of the Pharmacophoric C-Terminal Structures, Retain Potent Y1R Affinity and Show Improved Selectivity over Y4R

“…Indeed, the Lys derivative analogue 11c demonstrated preserved Y 4 R activity and an enhanced maximal response compared to 1229U91, accompanied by a loss of Y 1 R:Y 4 R selectivity compared to 1229U91 (Table 2). The activity of 11c is similar, in these systems, to dimeric pentapeptide BVD74-D which represents the lead truncated synthetic ligand at the Y 4 R. [32][33][34] Our data indicate that the larger ring size represented by the 11a (Dab), 11b (Orn) and 11c…”

“…Understanding the SAR of 1229U91 at both receptors better may therefore provide a route to new Y 4 R ligands, for which there is a limited pharmacological toolbox. For example, the state of play for Y 4 agonists, which are of interest in promoting satiety 2 , is represented by dimeric or modified C tail fragments of NPY such as BVD74-D, [33][34][35] or Tyr 32 βCpe ]-NPY 32-36 , 36 with a single report of a small molecule allosteric modulator 37 . Y 4 antagonists to date have moderate affinity and tend to lack selectivity over the Y 1 receptor, with the exception of the dimeric argininamide compound, UR-MEK388, which was > 20 fold selective.…”

Red-Emitting Dibenzodiazepinone Derivatives as Fluorescent Dualsteric Probes for the Muscarinic Acetylcholine M2 Receptor

“…Fluorescence arbitrary values from high-content imaging fluorescence images were obtained using a previously described procedure. 13,27…”

“…For this reason, the pharmacophore (ligand) and the fluorophore are often held apart by a linker or spacer moiety. [6][7][8] There are numerous reports on fluorescent probes for GPCRs, for instance for neuropeptide Y, [9][10][11][12][13][14] histamine, [15][16][17][18][19] opioid, [20][21][22] dopamine, 23,24 neurotensin, [25][26][27][28] and adenosine 29 receptors.…”

The Peptide PnPP-19, a Spider Toxin Derivative, Activates μ-Opioid Receptors and Modulates Calcium Channels

“…Images (4 central sites/well) were acquired automatically on the IX Ultra confocal plate reader, using 405 nm/488 nm laser lines for H33342 and complemented YFP excitation, respectively. Data was analyzed by the use of MetaXpress software (version 5.3, Sunnyvale, CA, USA, 2013) as described by Liu and co-authors [ 38 ] and normalized by 10 μM of DAMGO (100%).…”