Abstract:The human Y receptor (YR) 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 (tert-butylphenoxycyclohexanol), a novel and selective YR positive allosteric modulator that potentiates YR activation in G-protein signaling and arrestin3 recruitment experiment… Show more
“…In the arr3 recruitment studies, the Y 4 R was C-terminally tagged to Renilla luciferase 8 (Rluc8) and cloned in a pcDNA3 vector. Arr3 was N-terminally fused to venus fluorescent protein and cloned in a pcDNA3 plasmid. , The sequence of all constructs was confirmed by DNA sequencing.…”
Section: Methodsmentioning
confidence: 99%
“…Aiming at the development of small-molecule probes to selectively target the human Y 4 R, approximately 77 000 compounds were tested by high-throughput screening (HTS). We recently reported on the discovery of Y 4 R positive allosteric modulators (PAM) from this experiment. , In comparison to PAMs and agonists, the hit rate of negative allosteric modulators (NAM) and antagonists was very low (0.022%, Table S1, Supporting Information), reflecting the challenges in the development of Y 4 R antagonists. ,, …”
Human neuropeptide Y receptors (Y 1 R, Y 2 R, Y 4 R, and Y 5 R) belong to the superfamily of G protein-coupled receptors and play an important role in the regulation of food intake and energy metabolism. We identified and characterized the first selective Y 4 R allosteric antagonist (S)-VU0637120, an important step toward validating Y receptors as therapeutic targets for metabolic diseases. To obtain insight into the antagonistic mechanism of (S)-VU0637120, we conducted a variety of in vitro, ex vivo, and in silico studies. These studies revealed that (S)-VU0637120 selectively inhibits native Y 4 R function and binds in an allosteric site located below the binding pocket of the endogenous ligand pancreatic polypeptide in the core of the Y 4 R transmembrane domains. Taken together, our studies provide a first-of-its-kind tool for probing Y 4 R function and improve the general understanding of allosteric modulation, ultimately contributing to the rational development of allosteric modulators for peptide-activated G protein-coupled receptors (GPCRs).
“…In the arr3 recruitment studies, the Y 4 R was C-terminally tagged to Renilla luciferase 8 (Rluc8) and cloned in a pcDNA3 vector. Arr3 was N-terminally fused to venus fluorescent protein and cloned in a pcDNA3 plasmid. , The sequence of all constructs was confirmed by DNA sequencing.…”
Section: Methodsmentioning
confidence: 99%
“…Aiming at the development of small-molecule probes to selectively target the human Y 4 R, approximately 77 000 compounds were tested by high-throughput screening (HTS). We recently reported on the discovery of Y 4 R positive allosteric modulators (PAM) from this experiment. , In comparison to PAMs and agonists, the hit rate of negative allosteric modulators (NAM) and antagonists was very low (0.022%, Table S1, Supporting Information), reflecting the challenges in the development of Y 4 R antagonists. ,, …”
Human neuropeptide Y receptors (Y 1 R, Y 2 R, Y 4 R, and Y 5 R) belong to the superfamily of G protein-coupled receptors and play an important role in the regulation of food intake and energy metabolism. We identified and characterized the first selective Y 4 R allosteric antagonist (S)-VU0637120, an important step toward validating Y receptors as therapeutic targets for metabolic diseases. To obtain insight into the antagonistic mechanism of (S)-VU0637120, we conducted a variety of in vitro, ex vivo, and in silico studies. These studies revealed that (S)-VU0637120 selectively inhibits native Y 4 R function and binds in an allosteric site located below the binding pocket of the endogenous ligand pancreatic polypeptide in the core of the Y 4 R transmembrane domains. Taken together, our studies provide a first-of-its-kind tool for probing Y 4 R function and improve the general understanding of allosteric modulation, ultimately contributing to the rational development of allosteric modulators for peptide-activated G protein-coupled receptors (GPCRs).
“…The receptor activation, selectivity, and internalization were investigated to ensure that the attachment of the cleavable linker and tesa did not alter the behavior of NPY 1 R-preferring carrier peptide [F 7 , P 34 ]-NPY. The activation of the human Y-receptors was tested using Ca 2+ -flux assays in COS-7 cells stably expressing one specific Y-receptor subtype (NPY 1/2/4/5 R) and chimeric G protein (Δ6Gα qi4-myr ), opening the Ca 2+ channels upon receptor activation (Figure 4A-D, Table 3) [49,65]. Figure 4 Receptor activation and internalization of peptides.…”
Section: Resultsmentioning
confidence: 99%
“…Signal transduction Ca 2+ -flux assays were performed as previously described [49]. Briefly, COS-7 cells stably expressing NPY 1/2/4/5 R and chimeric G protein (Δ6Gα qi4*myr ) were seeded into black 96-well plates and grown for 24 h. The cells were incubated with 0.01% (v/v) Pluronic Acid F-127 and 2.4 μM Fluo2-AM in assay buffer (HBSS, 1.25 mM Probenecid, and 20 mM HEPES) at 37 °C for 60 min.…”
ObjectivePPARα/γ dual agonists have been in clinical development for the treatment of metabolic diseases including type 2 diabetes and dyslipidemia. However, severe adverse side effects led to complications in clinical trials. As most of the beneficial effects rely on the compound activity in adipocytes, the selective targeting of this cell type is a cutting-edge strategy to develop safe anti-diabetic drugs. The goal of this study was to strengthen the adipocyte-specific uptake of the PPARα/γ agonist tesaglitazar via NPY1R-mediated internalization.MethodsNPY1R-preferring peptide tesaglitazar-[F7, P34]-NPY (tesa-NPY) was synthesized by a combination of automated SPPS and manual couplings. Following molecular and functional analyses for proof of concept, cell culture experiments were conducted to monitor the effects on adipogenesis. Mice treated with peptide drug conjugates or vehicle either by gavage or intraperitoneal injection were characterized phenotypically and metabolically. Histological analysis and transcriptional profiling of the adipose tissue were performed.ResultsIn vitro studies revealed that the tesaglitazar-[F7, P34]-NPY conjugate selectively activates PPARγ in NPY1R-expressing cells and enhances adipocyte differentiation and adiponectin expression in adipocyte precursor cells. In vivo studies using db/db mice demonstrated that the anti-diabetic activity of the peptide conjugate is as efficient as that of systemically administered tesaglitazar. Additionally, tesa-NPY induces adipocyte differentiation in vivo.ConclusionsThe use of the tesaglitazar-[F7, P34]-NPY conjugate is a promising strategy to apply the beneficial PPARα/γ effects in adipocytes while potentially omitting adverse effects in other tissues.
“…Bioluminescence resonance energy transfer:H EK293 cells were transiently transfected with hY 1 R_eYFP_pVitro2 and arrestin-3_ RLuc8_pcDNA3 or hY 4 R_RLuc8_pcDNA3 and arrestin-3_Venus_ pcDNA3 and seeded into poly-d-lysine-coated black 96-well plates. [24] Transfection medium was DMEM (high glucose)/Ham's F12 (1:1, v/v)w ithout FBS. After 16 h, medium was removed and assay buffer (100 mL, 25 mm HEPES in HBSS, pH 7.3) was added per well.…”
The human Y receptor is overexpressed in breast tumour cells and is, therefore, a valuable target for site-selective drug delivery. The well-established hY R-selective ligand [Phe7,Pro34]NPY has been used to couple to drugs but its length of 36 amino acids also implies complex synthesis and high production costs. Therefore, shorter ligands are desirable. However, truncated versions of neuropeptide Y (NPY) usually result in reduced binding, antagonists, or only partial G-protein-biased agonists. Herein, we report on a nonamer peptide derived from the C terminus of NPY that is modified by a carbaborane, which is able to activate hY R fully in terms of G-protein activation but also arrestin recruitment and internalisation. We provide evidence that this unique behaviour is due to the bulky nature of the carbaborane cluster, which might address a specific second binding pocket in hY R that is crucial for arrestin recruitment. Thus, this study helps in deciphering ligand-induced onset of different pathways in hY R mediated signalling.
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