N^ω-Carbamoylation of the Argininamide Moiety: An Avenue to Insurmountable NPY Y₁ Receptor Antagonists and a Radiolabeled Selective High-Affinity Molecular Tool ([³H]UR-MK299) with Extended Residence Time

Abstract

Analogues of the argininamide-type NPY Y1 receptor (Y1R) antagonist BIBP3226, bearing carbamoyl moieties at the guanidine group, revealed subnanomolar Ki values and caused depression of the maximal response to NPY (calcium assay) by up to 90% in a concentration- and time-dependent manner, suggesting insurmountable antagonism. To gain insight into the mechanism of binding of the synthesized compounds, a tritiated antagonist, (R)-N(α)-diphenylacetyl-N(ω)-[2-([2,3-(3)H]propionylamino)ethyl]aminocarbonyl-(4-hydroxybenzyl)arginin-amide ([(3)H]UR-MK299, [(3)H]38), was prepared. [(3)H]38 revealed a dissociation constant in the picomolar range (Kd 0.044 nM, SK-N-MC cells) and very high Y1R selectivity. Apart from superior affinity, a considerably lower target off-rate (t1/2 95 min) was characteristic of [(3)H]38 compared to that of the higher homologue containing a tetramethylene instead of an ethylene spacer (t1/2 3 min, Kd 2.0 nM). Y1R binding of [(3)H]38 was fully reversible and fully displaceable by nonpeptide antagonists and the agonist pNPY. Therefore, the insurmountable antagonism observed in the functional assay has to be attributed to the extended target-residence time, a phenomenon of relevance in drug research beyond the NPY receptor field.

Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor

“…This is supported by studies showing that some other carbamoylated argininamide-type Y 1 R antagonists containing longer carbamoyl chains, such as UR-MK136 (Extended Data Fig. 1j), bind to the receptor with a relatively high affinity 10 .…”

“…All binding experiments with [ 3 H]-UR-MK299 (synthesis described elsewhere 10 ) were performed at Sf9 membrane preparations in PP 96-well microplates (Greiner bio-one) at 23±1 °C using a sodium-containing, iso-osmotic HEPES buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM KCl, 2.5 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 1.2 mM Mg 2 SO 4 and 25 mM NaHCO 3 supplemented with 1% BSA) for competition binding studies with antagonists, and a sodium-free, hypo-osmotic HEPES buffer (25 mM HEPES, pH 7.4, 2.5 mM CaCl 2 and 1 mM MgCl 2 supplemented with 1% BSA) for competition binding studies with the agonist NPY (in the following, both buffers are referred to as ‘binding buffer’). Prior to competition binding experiments, K d values of [ 3 H]-UR-MK299 were determined by saturation binding using the respective binding buffer.…”

“…The first characterized NPY receptor Y 1 R is widely expressed in a variety of tissues and involved in regulation of many physiological functions, related to obesity 8 and cancer 9 . To better understand the ligand binding behavior of NPY receptors and provide a basis for drug discovery, we solved crystal structures of Y 1 R in complex with two structurally diverse antagonists, UR-MK299, an argininamide with high Y 1 R selectivity 10 , and BMS-193885, which displays anorectic activity in animal models 6 (Fig. 1 and Extended Data Table 1).…”

“…UR-MK299 was reported to exhibit high Y 1 R selectivity (Y 2 R: K i >3,000 nM, Y 4 R/Y 5 R: K i >10,000 nM) and specificity compared to two related neuropeptide FF (NPFF) receptors (NPFF 1 R: K i =1,000 nM; NPFF 2 R: K i >3,000 nM) 10 . Sequence alignment reveals that most of the key residues involved in UR-MK299 binding are conserved between Y 1 R, the other NPY receptors and the NPFFRs, except for F 4.60 , Q 5.46 , N 6.55 and F 6.58 (Extended Data Fig.…”

“…In Y 4 R, E 6.58 disturbs the F 6.54 /F 6.58 /F 7.35 hydrophobic patch and most likely mediates selectivity, supported by the F 6.58 E mutation in Y 1 R dramatically reducing binding affinity for BIBP3226 22 , which contains the same diphenylmethyl group as UR-MK299. Similarly, hydrophilic residues at key positions impede high-affinity binding at Y 5 R (T 6.58 ) and NPFF 2 R (S 6.58 ), while the hydrophobic pocket is preserved though with less bulk in NPFF 1 R (L 6.54 /I 6.58 /F 7.35 ), leading to moderate affinity of BIBP3226 ( K i =18 nM) 10 .…”