Discovery of Small-Molecule Modulators of the Human Y₄ Receptor

<div><p>The human neuropeptide Y₄ receptor (Y₄R) and its native ligand, pancreatic polypeptide, are critically involved in the regulation of human metabolism by signaling satiety and regulating food intake, as well as increasing energy expenditure. Thus, this receptor represents a putative target for treatment of obesity. With respect to new approaches to treat complex metabolic disorders, especially in multi-receptor systems, small molecule allosteric modulators have been in the focus of research in the last years. However, no positive allosteric modulators or agonists of the Y₄R have been described so far. In this study, small molecule compounds derived from the Niclosamide scaffold were identified by high-throughput screening to increase Y₄R activity. Compounds were characterized for their potency and their effects at the human Y₄R and as well as their selectivity towards Y₁R, Y₂R and Y₅R. These compounds provide a structure-activity relationship profile around this common scaffold and lay the groundwork for hit-to-lead optimization and characterization of positive allosteric modulators of the Y₄R.</p></div

YR subtype selectivity of Y₄R PAMs.

<p>Effect of 30 μM compound on the pEC₅₀ of Y-receptor agonists in COS-7 cells stable expressing a Y receptor subtype and the chimeric G-protein G_α6qi4myr. Receptors were stimulated with their native ligands (Y₁R, Y₂R, Y₅R: NPY; Y₄R: PP). For Y-axis values, positive modulation represents an increase in the apparent potency (pEC₅₀) of the native agonist and negative modulation represents a decrease in the apparent potency of the native agonist. Data represent the mean ± SEM of at least two independent experiments (for full concentration-response curves see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157146#pone.0157146.s002" target="_blank">S2 Fig</a>) (*p < .05, ***p < .001 Bonferroni).</p

Distinct positions of the Niclosamide scaffold were shown to be relevant for Y₄R PAM activity and YR selectivity.

<p>Substitutions in the benzoyl ring are important for Y₄R potency (green), and offer a potential modification site (grey). Modifications in the aniline ring engender selectivity towards Y₁R / Y₅R subtype (red).</p

Y₄R PAM activity of Niclosamide-like compounds.

<p>Potency of the Y₄R PAMs was investigated with an inositol phosphate accumulation assay through potentiation of a PP EC₂₀ response. Data have been normalized to the maximum IP accumulation caused by the Y₄R native ligand PP. Data represent the mean ± SEM of three independent experiments performed in duplicate.</p

Validation of Y₄R PAM activity and subtype selectivity of initial Ca²⁺-flux-based screen hit compounds in an inositol phosphate accumulation assay.

<p>(A) Compound structures. (B) Effect of 10 μM compound on submaximal YR activation by 1 nM ligand, which represents EC₂₀-EC₆₀ (Y_1,2,5R: NPY; Y₄R: PP). Data represent the mean ± SEM of two independent experiments each performed in quadruplicate (*** p ≤ .001 Bonferroni).</p

Structures of Y₄R PAMs identified by HTS and inactive control compounds chosen for further characterization of Y₄R PAM activity and YR subtype selectivity.

<p>Structures of Y₄R PAMs identified by HTS and inactive control compounds chosen for further characterization of Y₄R PAM activity and YR subtype selectivity.</p

Identification and Characterization of the First Selective Y₄ Receptor Positive Allosteric Modulator

The human Y₄ receptor (Y₄R) and its cognate ligand, pancreatic polypeptide (PP), are involved in the regulation of energy expenditure, satiety, and food intake. This system represents a potential target for the treatment of metabolic diseases and has been extensively investigated and validated in vivo. Here, we present the compound tBPC (<i>tert</i>-butylphenoxy­cyclohexanol), a novel and selective Y₄R positive allosteric modulator that potentiates Y₄R activation in G-protein signaling and arrestin3 recruitment experiments. The compound has no effect on the binding of the orthosteric ligands, implying its allosteric mode of action at the Y₄R and evidence for a purely efficacy-driven positive allosteric modulation. Finally, the ability of tBPC to selectively potentiate Y₄R agonism initiated by PP was confirmed in mouse descending colon mucosa preparations expressing native Y₄R, demonstrating Y₄R positive allosteric modulation in vitro