Human Y1 Receptor Research Articles

Controlling toxicity of Peptide-drug conjugates by different chemical linker structures.

The side effects of chemotherapy can be overcome by linking toxic agents to tumor-targeting peptides with cleavable linkers. Herein, this concept is demonstrated by addressing the human Y1 receptor (hY1 R), overexpressed in breast tumors, with analogues of the hY1 R-preferring [F(7) ,P(34) ]NPY. First, carboxytetramethylrhodamine was connected to [F(7) ,P(34) ]NPY by an amide, ester, disulfide, or enzymatic linkage. Live imaging revealed hY1 R-mediated delivery and allowed visualization of time-dependent intracellular release. Next, the fluorophore was replaced by the toxic agent methotrexate (MTX). In addition to linkage through the amide, ester, disulfide bond, or enzymatic cleavage site, a novel disulfide/ester linker was designed and coupled to [F(7) ,P(34) ]NPY by solid-phase peptide synthesis. Internalization studies showed hY1 R subtype selective uptake, and cell viability experiments demonstrated hY1 R-mediated toxicity that was clearly dependent on the linkage type. Fast release profiles for fluorophore-[F(7) ,P(34) ]NPY analogues correlated with high toxicities of MTX conjugates carrying the same linker types and emphasize the relevance of new structures connecting the toxophore and the carrier.

Receptor‐Mediated Uptake of Boron‐Rich Neuropeptide Y Analogues for Boron Neutron Capture Therapy

Peptidic ligands selectively targeting distinct G protein-coupled receptors that are highly expressed in tumor tissue represent a promising approach in drug delivery. Receptor-preferring analogues of neuropeptide Y (NPY) bind and activate the human Y1 receptor subtype (hY1 receptor), which is found in 90% of breast cancer tissue and in all breast-cancer-derived metastases. Herein, novel highly boron-loaded Y1 -receptor-preferring peptide analogues are described as smart shuttle systems for carbaboranes as (10) B-containing moieties. Various positions in the peptide were screened for their susceptibility to carbaborane modification, and the most promising positions were chosen to create a multi-carbaborane peptide containing 30 boron atoms per peptide with excellent activation and internalization patterns at the hY1 receptor. Boron uptake studies by inductively coupled plasma mass spectrometry revealed successful uptake of the multi-carbaborane peptide into hY1 -receptor-expressing cells, exceeding the required amount of 10(9) boron atoms per cell. This result demonstrates that the NPY/hY receptor system can act as an effective transport system for boron-containing moieties.

Peptide-templated acyl transfer: a chemical method for the labeling of membrane proteins on live cells.

The development of a method is described for the chemical labeling of proteins which occurs with high target specificity, proceeds within seconds to minutes, and offers a free choice of the reporter group. The method relies upon the use of peptide templates, which align a thioester and an N-terminal cysteinyl residue such that an acyl transfer reaction is facilitated at nanomolar concentrations. The protein of interest is N-terminally tagged with a 22 aa long Cys-E3 peptide (acceptor), which is capable of forming a coiled-coil with a reporter-armed K3 peptide (donor). This triggers the transfer of the reporter to the acceptor on the target protein. Because ligation of the two interacting peptides is avoided, the mass increase at the protein of interest is minimal. The method is exemplified by the rapid fluorescent labeling and fluorescence microscopic imaging of the human Y2 receptor on living cells.

Computational prediction of alanine scanning and ligand binding energetics in G-protein coupled receptors.

Site-directed mutagenesis combined with binding affinity measurements is widely used to probe the nature of ligand interactions with GPCRs. Such experiments, as well as structure-activity relationships for series of ligands, are usually interpreted with computationally derived models of ligand binding modes. However, systematic approaches for accurate calculations of the corresponding binding free energies are still lacking. Here, we report a computational strategy to quantitatively predict the effects of alanine scanning and ligand modifications based on molecular dynamics free energy simulations. A smooth stepwise scheme for free energy perturbation calculations is derived and applied to a series of thirteen alanine mutations of the human neuropeptide Y1 receptor and series of eight analogous antagonists. The robustness and accuracy of the method enables univocal interpretation of existing mutagenesis and binding data. We show how these calculations can be used to validate structural models and demonstrate their ability to discriminate against suboptimal ones.

Pancreatic Polypeptide Is Recognized by Two Hydrophobic Domains of the Human Y4 Receptor Binding Pocket

Structural characterization of the human Y4 receptor (hY4R) interaction with human pancreatic polypeptide (hPP) is crucial, not only for understanding its biological function but also for testing treatment strategies for obesity that target this interaction. Here, the interaction of receptor mutants with pancreatic polypeptide analogs was studied through double-cycle mutagenesis. To guide mutagenesis and interpret results, a three-dimensional comparative model of the hY4R-hPP complex was constructed based on all available class A G protein-coupled receptor crystal structures and refined using experimental data. Our study reveals that residues of the hPP and the hY4R form a complex network consisting of ionic interactions, hydrophobic interactions, and hydrogen binding. Residues Tyr(2.64), Asp(2.68), Asn(6.55), Asn(7.32), and Phe(7.35) of Y4R are found to be important in receptor activation by hPP. Specifically, Tyr(2.64) interacts with Tyr(27) of hPP through hydrophobic contacts. Asn(7.32) is affected by modifications on position Arg(33) of hPP, suggesting a hydrogen bond between these two residues. Likewise, we find that Phe(7.35) is affected by modifications of hPP at positions 33 and 36, indicating interactions between these three amino acids. Taken together, we demonstrate that the top of transmembrane helix 2 (TM2) and the top of transmembrane helices 6 and 7 (TM6-TM7) form the core of the peptide binding pocket. These findings will contribute to the rational design of ligands that bind the receptor more effectively to produce an enhanced agonistic or antagonistic effect.

Preparation and Characterization of Albumin Conjugates of a Truncated Peptide YY Analogue for Half-Life Extension

Recombinant human serum albumin (HSA) conjugates of a 15-amino-acid truncated peptide YY (PYY) analogue were prepared using three heterobifunctional linkers [succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC), 6-maleimidohexanoic acid N-hydroxysuccinimide ester (MHS), and N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (GMBS)] in 2 synthetic steps involving (1) reaction of succinimidyl ester on linker with ε-amine of Lys2 on the peptide and (2) reaction of maleimide on peptide linker with free thiol of Cysteine 34 (Cys34) on albumin. In-process controls using ESI LC-MS were used to follow reactions and identify reaction products. Proteolytic digests of the conjugate revealed that peptide conjugation occurs at Cys34 on HSA. Conjugates were assayed in cell-based assays to determine potency at the human Y2-receptor, and selectivity at the human Y1-, Y4-, and Y5-receptors using a calcium flux assay. All three conjugates assayed were selective agonists of the Y2-receptor, and displayed nanomolar potencies. MCC and MH conjugates were selected for acute PK/PD studies in DIO mice. Significant reduction in food intake was observed with the MH conjugate, which lasted for 24 h at the 10 mg (or 4 μmol)/kg dose. While the MCC conjugate exhibited greater potency in vitro, it was slightly less effective than the MH conjugate in vivo with respect to reduction in food intake. Both conjugates were significantly less active than the peptide coupled to a 30 kDa PEG. The observed T1/2 (8-9 h) for both conjugates was significantly lower than that observed for the PEGylated peptide (∼25 h). These results suggest that, as compared with the unmodified and PEGylated peptide, the extended circulation half-life of albumin conjugates is mediated through uptake and recirculation by FcRn, and allometric scaling methods are necessary to account for interspecies variation in pharmacokinetic properties.

Detecting ligand interactions with G protein-coupled receptors in real-time on living cells

G protein-coupled receptors (GPCRs) are a large group of receptors of great biological and clinical relevance. Despite this, the tools for a detailed analysis of ligand–GPCR interactions are limited. The aim of this paper was to demonstrate how ligand binding to GPCRs can be followed in real-time on living cells. This was conducted using two model systems, the radiolabeled porcine peptide YY (pPYY) interacting with transfected human Y2 receptor (hY2R) and the bombesin antagonist RM26 binding to the naturally expressed gastrin-releasing peptide receptor (GRPR). By following the interaction over time, the affinity and kinetic properties such as association and dissociation rate were obtained. Additionally, data were analyzed using the Interaction Map method, which can evaluate a real-time binding curve and present the number of parallel interactions contributing to the curve. It was found that pPYY binds very slowly with an estimated time to equilibrium of approximately 12h. This may be problematic in standard end-point assays where equilibrium is required. The RM26 binding showed signs of heterogeneity, observed as two parallel interactions with unique kinetic properties. In conclusion, measuring binding in real-time using living cells opens up for a better understanding of ligand interactions with GPCRs.

Mutagenesis and Computational Modeling of Human G-Protein-Coupled Receptor Y2 for Neuropeptide Y and Peptide YY

Neuropeptide Y and peptide YY receptor type 2 (Y2) is involved in appetite regulation and several other physiological processes. We have investigated the structure of the human Y2 receptor. Computational modeling of receptor-agonist interactions was used as a guide to design a series of receptor mutants, followed by binding assays using full-length and truncated peptide agonists and the Y2-specific antagonist BIIE0246. Our model suggested a hydrogen bond network among highly conserved residues Thr2.61, Gln3.32, and His7.39, which could play roles in ligand binding and/or receptor structure. In addition, the C-terminus of the peptide could make contact with residues Tyr5.38 and Leu6.51. Mutagenesis of all these positions, followed by binding assays, provides experimental support for our computational model: most of the mutants for the residues forming the proposed hydrogen bond network displayed reduced peptide agonist affinities as well as reduced hPYY3-36 potency in a functional assay. The Ala and Leu mutants of Gln3.32 and His7.39 disrupted membrane expression of the receptor. Combined with the modeling, the experimental results support roles for these hydrogen bond network residues in peptide binding as well as receptor architecture. The reduced agonist affinity for mutants of Tyr5.38 and Leu6.51 supports their role in a binding pocket surrounding the invariant tyrosine at position 36 of the peptide ligands. The results for antagonist BIIE0246 suggest several differences in interactions compared to those of the peptides. Our results lead to a new structural model for NPY family receptors and peptide binding.

Manipulating Y receptor subtype activation of short neuropeptide Y analogs by introducing carbaboranes

Short selective neuropeptide Y (NPY) analogs are highly attractive because of their facile synthesis. Based on the reduced-size NPY analog [Pro30, Nle31, Bpa32, Leu34]NPY 28–36 position 32 was identified as a key position to alter the preferential activation pattern of the human neuropeptide Y receptors (hYRs). By replacing benzoylphenylalanine (Bpa) by a biphenylalanine (Bip) the photostability was first improved while the biological activity was maintained. SAR-studies showed that both aromatic rings have a high influence on the preferential hYR subtype activation. Interestingly, replacement of Bpa32 by a strongly hydrophobic moiety changed the hYR subtype preference of the analog. Whereas the parent compound is able to activate the human neuropeptide Y1 receptor (hY1R) subtype, the introduction of an Nε-ortho-carbaboranyl propionic acid modified lysine resulted in a loss of activity at the hY1R but in an increased activity at both the hY2R and the hY4R. However, subsequent receptor internalization studies with this novel analog revealed that receptor internalization can neither be triggered at the hY2R nor at the hY4R suggesting a biased ligand. Surprisingly, investigations by 1H NMR spectroscopy revealed structural changes in the side chains of residues Pro30 and Leu34 which nicely correlates with the shift from hY1R/hY4R to hY2R/hY4R activation preference. Thus, position 32 has been identified to switch the bioactive conformation and subsequently influences receptor subtype activation behavior.

Knockdown of a G protein-coupled receptor through efficient peptide-mediated siRNA delivery

In recent years, therapeutic applications of siRNAs have come into the focus of pharmaceutical research owing to their potential to specifically regulate gene expression. However, oligonucleotides have to overcome a series of extracellular and intracellular barriers which is why delivery systems helping to overcome these barriers are desperately needed. A promising approach to transport nucleic acids beyond cellular membranes is the use of cell-penetrating peptides (CPPs), which are able to autonomously cross the plasma membrane. Recently, we synthesized branched derivatives of truncated human calcitonin (hCT) and identified them as efficient vehicles for non-covalent gene delivery. Here we describe two novel branched hCT-derivatives that are optimized for efficient intracellular delivery of siRNA by conjugation with either a fatty acid or an endosomolytic peptide sequence. As target we chose the human NPY Y1 receptor (NPY1R), which belongs to the family of G protein-coupled receptors and thus constitutes a model for complex therapeutic targets related to various disorders. For instance, knockdown of Y1 receptor expression offers a potential therapy for osteoporosis. We present a read-out system that allows for the quantitation of the induced knockdown of receptor expression on the protein as well as on the mRNA level. As a result of this study, we could show that the herein presented cell-penetrating peptides effectively transport siRNA into HEK-293 cells without inducing cytotoxicity and that the knockdown rates are comparable to those obtained by lipofection.

Biosynthesis and Spectroscopic Characterization of 2‐TM Fragments Encompassing the Sequence of a Human GPCR, the Y4 Receptor

This paper presents a divide-and-conquer approach towards obtaining solution structures of G protein-coupled receptors. The human Y4 receptor was dissected into two to three transmembrane helix fragments, which were individually studied by solution NMR. We systematically compared various biosynthetic routes for the expression of the fragments in Escherichia coli and discuss purification strategies. In particular, we have compared the production of transmembrane (TM) fragments as inclusion bodies by using the ΔTrp leader sequence, with membrane-directed expression by using Mistic as the fusion partner, and developed methods for enzymatic cleavage. In addition, direct expression of two-TM fragments into inclusion bodies is a successful route in some cases. With the exception of TM13, we could produce all fragments in isotope-labeled form in quantities sufficient for NMR studies. Almost complete backbone resonance assignment was obtained for the first two helices, as well as for helices 5 and 7, and a high degree was obtained for TM6, while conformational exchange processes resulted in the disappearance of many signals from TM4. In addition, complete assignments were obtained for all residues of the N-terminal domain, as well as the extracellular and cytosolic loops (with the exception of an undecapeptide segment in the second extracellular loop, EC2) and for the complete cytosolic C-terminal tail. In total, backbone resonances of 78 % of all residues were assigned for the Y4 receptor. Predictions of secondary structure based on backbone chemical shifts indicate that most residues from the TM regions adopt helical conformations, with exception of those around polar residues or prolines. However, the domain boundaries differ slightly from those predicted for homology models. We suggest that the obtained chemical shifts might be useful in assigning the full-length receptor.

Identification of positions in the human neuropeptide Y/peptide YY receptor Y2 that contribute to pharmacological differences between receptor subtypes

The members of the neuropeptide Y (NPY) family are key players in food-intake regulation. In humans this family consists of NPY, peptide YY (PYY) and pancreatic polypeptide (PP) which interact with distinct preference for the four receptors showing very low sequence identity, i.e. Y1, Y2, Y4 and Y5. The binding of similar peptides to these divergent receptors makes them highly interesting for mutagenesis studies. We present here a site-directed mutagenesis study of four amino acid positions in the human Y2 receptor. T(3.40) was selected based on sequence alignments both between subtypes and between species and G(2.68), L(4.60) and Q(6.55) also on previous binding studies of the corresponding positions in the Y1 receptor. The mutated receptors were characterized pharmacologically with the peptide agonists NPY, PYY, PYY(3-36), NPY(13-36) and the non-peptide antagonist BIIE0246. Interestingly, the affinity of NPY and PYY(3-36) increased for the mutants T(3.40)I and Q(6.55)A. Increased affinity was also observed for PYY to Q(6.55)A. PYY(3-36) displayed decreased affinity for G(2.68)N and L(4.60)A whereas binding of NPY(13-36) was unaffected by all mutations. The antagonist BIIE0246 showed decreased affinity for T(3.40)I, L(4.60)A and Q(6.55)A. Although all positions investigated were found important for interaction with at least one of the tested ligands the corresponding positions in hY1 seem to be of greater importance for ligand binding. Furthermore these data indicate that binding of the agonists and the antagonist differs in their points of interaction. The increase in the binding affinity observed may reflect an indirect effect caused by a conformational change of the receptor. These findings will help to improve the structural models of the human NPY receptors.

A Chimeric GPCR Model Mimicking the Ligand Binding Site of the Human Y1 Receptor Studied by NMR Spectroscopy

This paper describes the development of a structural mimetic for the extracellular loops of a human GPCR. The three extracellular loops were grafted onto the scaffold of the E.coli outer membrane beta-barrel protein OmpA

Alpha-Trinositol: a functional (non-receptor) neuropeptide Y antagonist in vasculature.

Neuropeptide Y is a sympathetic co-neurotransmitter released with noradrenaline upon sympathetic nerve stimulation. This study describes the ability of a synthetic inositol phosphate, alpha-trinositol(D-myo-inositol 1,2,6-triphosphate; PP 56) to antagonize vasoconstrictor responses to neuropeptide Y in-vitro as well as in-vivo. In human and guinea-pig isolated arteries alpha-trinositol potently (10 nM to 1 microM extracellular concentration) suppressed the constriction evoked by neuropeptide Y alone, the potentiation by neuropeptide Y of noradrenaline-evoked constriction, and the neuropeptide Y-induced inhibition of relaxation. Moreover, in the pithed (areflexive) rat, a non-adrenergic portion of the pressor response to preganglionic sympathetic nerve stimulation was sensitive to alpha-trinositol. As studied in the recently cloned human (vascular-type) Y1 receptor, the action of alpha-trinositol does not occur through antagonism at the neuropeptide Y recognition site nor does it induce allosteric changes of this receptor. However, we found alpha-trinositol to inhibit the rise in intracellular Ca2+ as well as inositol triphosphate concentrations induced by neuropeptide Y. It is, therefore, proposed that alpha-trinositol represents a non-receptor, but yet selective antagonist of neuropeptide Y in vasculature, opening up the possibility to investigate involvement of neuropeptide Y in sympathetic blood pressure control and in cardiovascular disorders.

G protein-coupled receptors function as logic gates for nanoparticle binding and cell uptake

More selective interactions of nanoparticles with cells would substantially increase their potential for diagnostic and therapeutic applications. Thus, it would not only be highly desirable that nanoparticles can be addressed to any cell with high target specificity and affinity, but that we could unequivocally define whether they rest immobilized on the cell surface as a diagnostic tag, or if they are internalized to serve as a delivery vehicle for drugs. To date no class of targets is known that would allow direction of nanoparticle interactions with cells alternatively into one of these mutually exclusive events. Using MCF-7 breast cancer cells expressing the human Y(1)-receptor, we demonstrate that G protein-coupled receptors provide us with this option. We show that quantum dots carrying a surface-immobilized antagonist remain with nanomolar affinity on the cell surface, and particles carrying an agonist are internalized upon receptor binding. The receptor functions like a logic "and-gate" that grants cell access only to those particles that carry a receptor ligand "and" where the ligand is an agonist. We found that agonist- and antagonist-modified nanoparticles bind to several receptor molecules at a time. This multiligand binding leads to five orders of magnitude increased-receptor affinities, compared with free ligand, in displacement studies. More than 800 G protein-coupled receptors in humans provide us with the paramount advantage that targeting of a plethora of cells is possible, and that switching from cell recognition to cell uptake is simply a matter of nanoparticle surface modification with the appropriate choice of ligand type.

Functional Characterization of the In Vitro Folded Human Y1 Receptor in Lipid Environment

We describe the recombinant production of the human Y(1) receptor from inclusion bodies of E. coli cultures. The in vitro refolding was carried out in the presence of lipids from bovine brain extracts. Y(1) receptors in brain lipids compete for cellular receptors in competitive binding experiments.

Prokaryotic expression, in vitro folding, and molecular pharmacological characterization of the neuropeptide Y receptor type 2

G protein-coupled receptors (GPCRs) are a class of membrane proteins that represent a major target for pharmacological developments. However, there is still little knowledge about GPCR structure and dynamics since high-level expression and characterization of active GPCRs in vitro is extremely complicated. Here, we describe the recombinant expression and functional folding of the human Y(2) receptor from inclusion bodies of E. coli cultures. Milligram protein quantities were produced using high density fermentation and isolated in a single step purification with a yield of over 20 mg/L culture. Extensive studies were carried out on in vitro refolding and stabilization of the isolated receptor in detergent solution. The specific binding of the ligand, the 36 residue neuropeptide Y (NPY), to the recombinant Y(2) receptors in micellar form was shown by several radioligand affinity assays. In competition experiments, an IC(50) value in low nanomolar range could be determined. Further, a K(D) value of 1.9 nM was determined from a saturation assay, where NPY was titrated to the recombinant Y(2) receptors.

Cloning and characterization of rabbit neuropeptide Y receptor subtypes

Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) are structurally related peptides that have numerous functions in both neural and endocrine signaling. These effects are mediated by the NPY receptor family and five members of this family have been cloned in mammals. To better characterize these receptor subtypes, we cloned and expressed the Y1, Y2, Y4 and Y5 receptor subtypes from the rabbit. Comparison of these sequences with human orthologs revealed that the Y1, Y2 and Y5 receptors have generally strong amino-acid sequence conservation, with 91–96% identity, while Y4 receptor showed relatively weak similarity with 82% identity, as with other species. Particularly in the transmembrane regions, Y1, Y2, and Y5 receptor subtypes showed remarkable conservation, with 98–99% amino acid identity. Competitive binding studies by NPY-family peptides and analogs showed that Y1, Y2 and Y5 receptors had similar pharmacological profiles between the respective rabbit and human receptor subtypes. Interestingly, all the tested peptides had a greater affinity for rabbit Y4 receptor than human Y4 receptor. These results suggest that rabbit and human Y1, Y2 and Y5 receptor subtypes are well conserved, whereas Y4 receptors are less well conserved.

Discovery of substituted 2,4,4-triarylimidazoline derivatives as potent and selective neuropeptide Y Y5 receptor antagonists

Novel imidazoline derivatives were discovered to be potent neuropeptide Y Y5 receptor antagonists. High-throughput screening of Merck sample collections against the human Y5 receptor resulted in the identification of 2,4,4-triphenylimidazoline ( 1), which had an IC 50 of 54 nM. Subsequent optimization led to the identification of several potent derivatives.

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR

The human Y4 receptor, a class A G-protein coupled receptor (GPCR) primarily targeted by the pancreatic polypeptide (PP), is involved in a large number of physiologically important functions. This paper investigates a Y4 receptor fragment (N-TM1-TM2) comprising the N-terminal domain, the first two transmembrane (TM) helices and the first extracellular loop followed by a (His)(6) tag, and addresses synthetic problems encountered when recombinantly producing such fragments from GPCRs in Escherichia coli. Rigorous purification and usage of the optimized detergent mixture 28 mM dodecylphosphocholine (DPC)/118 mM% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) resulted in high quality TROSY spectra indicating protein conformational homogeneity. Almost complete assignment of the backbone, including all TM residue resonances was obtained. Data on internal backbone dynamics revealed a high secondary structure content for N-TM1-TM2. Secondary chemical shifts and sequential amide proton nuclear Overhauser effects defined the TM helices. Interestingly, the properties of the N-terminal domain of this large fragment are highly similar to those determined on the isolated N-terminal domain in the presence of DPC micelles.