Bivalent Argininamide‐Type Neuropeptide Y Y₁ Antagonists Do Not Support the Hypothesis of Receptor Dimerisation

Abstract

Bivalent ligands are potential tools to investigate the dimerisation of G-protein-coupled receptors. Based on the (R)-argininamide BIBP 3226, a potent and selective neuropeptide Y Y(1) receptor (Y(1)R) antagonist, we prepared a series of bivalent Y(1)R ligands with a wide range of linker lengths (8-36 atoms). Exploiting the high eudismic ratio (>1000) of the parent compound, we synthesised sets of R,R-, R,S- and S,S-configured bivalent ligands to gain insight into the "bridging" of two Y(1)Rs by simultaneous interaction with both binding sites of a putative receptor dimer. Except for the S,S isomers, the bivalent ligands are high-affinity Y(1)R antagonists, as determined by Ca(2+) assays on HEL cells and radioligand competition assays on human Y(1)R-expressing SK-N-MC and MCF-7 cells. Whereas the R,R enantiomers are most potent, no marked differences were observed relative to the corresponding meso forms. The difference between R,R and R,S diastereomers was most pronounced (about sixfold) in the case of the Y(1)R antagonist containing a spacer of 20 atoms in length. Among the R,R enantiomers, linker length and structural diversity had little effect on Y(1)R affinity. Although the bivalent ligands preferentially bind to the Y(1)R, the selectivity toward human Y(2), Y(4), and Y(5) receptors was markedly lower than that of the monovalent argininamides. The results of this study neither support the presence of Y(1)R dimers nor the simultaneous occupation of both binding pockets by the twin compounds. However, as the interaction with Y(1)R dimers cannot be unequivocally ruled out, the preparation of a bivalent radioligand is suggested to determine the ligand-receptor stoichiometry. Aiming at such radiolabelled pharmacological tools, prototype twin compounds were synthesised, containing an N-propionylated amino-functionalised branched linker (K(i)> or =18 nM), a tritiated form of which can be easily prepared.

Red-fluorescent argininamide-type NPY Y1 receptor antagonists as pharmacological tools

“…By contrast, labelling of amine 41 using succinimidyl ester 52 afforded 66 in 24% yield (Scheme 7). Labelling of the bivalent Y 1 R antagonists 67-69 (synthesis described elsewhere) 8 with Py-1 afforded the fluorescent bivalent ligands 70-72 in satisfactory yields, but efforts to label the bivalent Y 1 R ligand 69 with either 51 (activation with EDC or CDI) or its succinimidyl ester 52 failed.…”

“…The protected intermediates were purified by column chromatography prior to Boc-or Cbz-deprotection yielding the amines. Compound 40 was prepared through amidation of the N x -(7-carboxyheptanoyl), N x0 -Boc, O-tert-butyl protected argininamide 39 (synthesis reported elsewhere) 8 with mono Boc protected diamine 22. The carboxy group of 39 was activated with N-(3-dimethylaminopropyl)-N 0 -ethylcarbodiimide hydrochloride (EDC).…”

“…5 The same bioisosteric approach was also shown to be a successful strategy to develop receptor subtype-selective radioligands, bivalent ligands and prodrugs. [6][7][8][9][10] In continuation of our work on fluorescent GPCR ligands the present study is aiming at pharmacological tools with improved physicochemical and optical properties to broaden the range of potential applications, for example, with respect to optical in vivo imaging. The Y 1 R was selected as a representative model of a peptidergic GPCR, for instance, as this receptor subtype was recently associated with diagnosis and treatment of tumours.…”

[3H]UR‐MK136: A Highly Potent and Selective Radioligand for Neuropeptide Y Y1 Receptors

“…Recently, we reported the bioisosteric substitution of a guanidine by an acylguanidine entity in argininamide-type neuropeptide Y (NPY) Y 1 receptor (Y 1 R) antagonists, such as BIBP 3226 [1] (1; Scheme 1), to be an effective strategy for the preparation of fluorescent [2,3] and bivalent [4,5] Y 1 R ligands. Moreover, acylation of the guanidine group in 1 with tritiated propionic acid yielded [ 3 H]UR-MK114 (2 b; Scheme 1), a highly potent (K d = 1.2 nm) and selective radiotracer for the Y 1 R. [6] Radioligand 2 b shows excellent long-term stability and low radiolysis when stored as the trifluoroacetic acid (TFA) salt in ethanol.…”

Effect of Linker Length and Composition on Heterobivalent Ligand‐Mediated Receptor Cross‐Talk between the A1 Adenosine and β2 Adrenergic Receptors

“…The effect of linker lengths in bivalent ligands that allow bridging of two receptors has been discussed by numerous authors 6. 7, 11–14 Reports by Portoghese and co‐workers of an optimal linker length that allows bridging active sites in opioid dimers4, 5 led us to consider similar linker lengths in our own bivalent designs. Furthermore, the effect of linker type had not been explored in our previous work.…”