Melanocortin-4 Receptor PLC Activation Is Modulated by an Interaction with the Monocarboxylate Transporter 8.

The melanocortin-4 receptor (MC4R) is a key player in the hypothalamic leptin-melanocortin pathway that regulates satiety and hunger. MC4R belongs to the G protein-coupled receptors (GPCRs), which are known to form heterodimers with other membrane proteins, potentially modulating receptor function or characteristics. Like MC4R, thyroid hormones (TH) are also essential for energy homeostasis control. TH transport across membranes is facilitated by the monocarboxylate transporter 8 (MCT8), which is also known to form heterodimers with GPCRs. Based on the finding in single-cell RNA-sequencing data that both proteins are simultaneously expressed in hypothalamic neurons, we investigated a putative interplay between MC4R and MCT8. We developed a novel staining protocol utilizing a fluorophore-labeled MC4R ligand and demonstrated a co-localization of MC4R and MCT8 in human brain tissue. Using in vitro assays such as BRET, IP1, and cAMP determination, we found that MCT8 modulates MC4R-mediated phospholipase C activation but not cAMP formation via a direct interaction, an effect that does not require a functional MCT8 as it was not altered by a specific MCT8 inhibitor. This suggests an extended functional spectrum of MCT8 as a GPCR signaling modulator and argues for the investigation of further GPCR-protein interactions with hitherto underrepresented physiological functions.

Development of a Potent and Selective G2A (GPR132) Agonist.

G protein-coupled receptor G2A was postulated to be a promising target for the development of new therapeutics in neuropathic pain, acute myeloid leukemia, and inflammation. However, there is still a lack of potent, selective, and drug-like G2A agonists to be used as a chemical tool or as the starting matter for the development of drugs. In this work, we present the discovery and structure-activity relationship elucidation of a new potent and selective G2A agonist scaffold. Systematic optimization resulted in (3-(pyridin-3-ylmethoxy)benzoyl)-d-phenylalanine (T-10418) exhibiting higher potency than the reference and natural ligand 9-HODE and high selectivity among G protein-coupled receptors. With its favorable activity, a clean selectivity profile, excellent solubility, and high metabolic stability, T-10418 qualifies as a pharmacological tool to investigate the effects of G2A activation.

Illuminating the neuropeptide Y4 receptor and its ligand pancreatic polypeptide from a structural, functional, and therapeutic perspective.

The neuropeptide Y4 receptor (Y4R), a rhodopsin-like G protein-coupled receptor (GPCR) and the hormone pancreatic polypeptide (PP) are members of the neuropeptide Y family consisting of four receptors (Y1R, Y2R, Y4R, Y5R) and three highly homologous peptide ligands (neuropeptide Y, peptide YY, PP). In this family, the Y4R is of particular interest as it is the only subtype with high affinity to PP over NPY. The Y4R, as a mediator of PP signaling, has a pivotal role in appetite regulation and energy homeostasis, offering potential avenues for the treatment of metabolic disorders such as obesity. PP as anorexigenic peptide is released postprandial from the pancreas in response to food intake, induces satiety signals and contributes to hamper excessive food intake. Moreover, this system was also described to be associated with different types of cancer: overexpression of Y4R have been found in human adenocarcinoma cells, while elevated levels of PP are related to the development of pancreatic endocrine tumors. The pharmacological relevance of the Y4R advanced the search for potent and selective ligands for this receptor subtype, which will be significantly progressed through the elucidation of the active state PP-Y4R cryo-EM structure. This review summarizes the development of novel PP-derived ligands, like Obinepitide as dual Y2R/Y4R agonist in clinical trials or UR-AK86c as small hexapeptide agonist with picomolar affinity, as well as the first allosteric modulators that selectively target the Y4R, e.g. VU0506013 as potent Y4R positive allosteric modulator or (S)-VU0637120 as allosteric antagonist. Here, we provide valuable insights into the complex physiological functions of the Y4R and PP and the pharmacological relevance of the system in appetite regulation to open up new avenues for the development of tool compounds for targeted therapies with potential applications in metabolic disorders.

Illuminating the Path to Enhanced Bioimaging by Phosphole-based Fluorophores.

As the research of biological systems becomes increasingly complex, there is a growing demand for fluorophores with a diverse range of wavelengths. In this study, we introduce phosphole-based fluorophores that surpass existing options like dansyl chloride. The reactive S-Cl bond in chlorosulfonylimino-5-phenylphosphole derivatives allows rapid and direct coupling to peptides making the fluorophores easily introducible to peptides. This coupling process occurs under mild conditions, demonstrated for [F7 ,P34 ]-NPY and its shorter analogues. Peptides linked with our fluorophores exhibit similar receptor activation to the control peptide, while maintaining high stability and low toxicity, making them ideal biolabeling reagents. In fluorescence microscopy experiments, they can be easily visualized even at low concentrations, without suffering from the typical issue of bleaching. These phosphole-based fluorophores represent a significant leap forward in the field. Their versatility, ease of modification, superior performance, and applicability in biological labeling make them a promising choice for researchers seeking advanced tools to unravel the details of complex biological systems.

Development of Radiopharmaceuticals for NPY Receptor-5 (Y5) Nuclear Imaging in Tumors by Synthesis of Specific Agonists and Investigation of Their Binding Mode.

The neuropeptide-Y (NPY) family acts through four G protein-coupled receptor subtypes in humans, namely, Y1, Y2, Y4, and Y5. A growing body of evidence suggest the involvement of the NPY system in several cancers, notably the Y5 subtype, thus acting as a relevant target for the development of radiopharmaceuticals for imaging or targeted radionuclide therapy (TRT). Here, the [cPP(1-7),NPY(19-23),Ala31,Aib32,Gln34]hPP scaffold, further referred to as sY5ago, was modified with a DOTA chelator and radiolabeled with 68Ga and 111In and investigated in vitro and in vivo using the MCF-7 model. For in vivo studies, MCF-7 cells were orthotopically implanted in female nude mice and imaging with small animal positron emission tomography/computed tomography (µPET/CT) was performed. At the end of imaging, the mice were sacrificed. A scrambled version of sY5ago, which was also modified with a DOTA chelator, served as a negative control (DOTA-[Nle]sY5ago_scrambled). sY5ago and DOTA-sY5ago showed subnanomolar affinity toward the Y5 (0.9 ± 0.1 and 0.8 ± 0.1 nM, respectively) and a single binding site at the Y5 was identified. [68Ga]Ga-DOTA-sY5ago and [111In]In-DOTA-sY5ago were hydrophilic and showed high specific internalization (1.61 ± 0.75%/106 cells at 1 h) and moderate efflux (55% of total binding externalized at 45 min). On µPET/CT images, most of the signal was depicted in the kidneys and the liver. MCF-7 tumors were clearly visualized. On biodistribution studies, [68Ga]Ga-DOTA-sY5ago was eliminated by the kidneys (∼60 %ID/g). The kidney uptake is Y5-mediated. A specific uptake was also noted in the liver (5.09 ± 1.15 %ID/g vs 1.13 ± 0.21 %ID/g for [68Ga]Ga-DOTA-[Nle]sY5ago_scrambled, p < 0.05), the lungs (1.03 ± 0.34 %ID/g vs 0.20 %ID/g, p < 0.05), and the spleen (0.85 ± 0.09%ID/g vs 0.16 ± 0.16%ID/g, p < 0.05). In MCF-7 tumors, [68Ga]Ga-DOTA-sY5ago showed 12-fold higher uptake than [68Ga]Ga-DOTA-[Nle]sY5ago_scrambled (3.43 ± 2.32 vs 0.27 ± 0.15 %ID/g, respectively, p = 0.0008) at 1 h post-injection. Finally, a proof-of-principle tissular micro-imaging study on a human primary cancer sample showed weak binding of [111In]In-DOTA-sY5ago in prostatic intra-neoplasia and high binding in the ISUP1 lesion while normal prostate was free of signal.

New Boron Delivery Agents.

This proceeding article compiles current research on the development of boron delivery drugs for boron neutron capture therapy that was presented and discussed at the National Cancer Institute (NCI) Workshop on Neutron Capture Therapy that took place on April 20-22, 2022. The most used boron sources are icosahedral boron clusters attached to peptides, proteins (such as albumin), porphyrin derivatives, dendrimers, polymers, and nanoparticles, or encapsulated into liposomes. These boron clusters and/or carriers can be labeled with contrast agents allowing for the use of imaging techniques, such as PET, SPECT, and fluorescence, that enable quantification of tumor-localized boron and their use as theranostic agents.

Pinpointing the interaction site between semaphorin-3A and its inhibitory peptide.

Semaphorin-3A (Sema-3A) is a chemorepellant protein with various biological functions, including kidney development. It interacts with a protein complex consisting of the receptors neuropilin-1 (NRP-1) and plexin-A1. After acute kidney injury, Sema-3A is overexpressed and secreted, leading to a loss of kidney function. The development of peptide inhibitors is a promising approach to modulate the interaction of Sema-3A with its receptor NRP-1. Few interaction points between these binding partners are known. However, an immunoglobulin-like domain-derived peptide of Sema-3A has shown a positive effect on cell proliferation. To specify these interactions between the peptide inhibitor and the Sema-3A-NRP-1 system, the peptides were modified with the photoactivatable amino acids 4-benzoyl-l-phenylalanine or photo-l-leucine by solid-phase peptide synthesis. Activity was tested by an enzyme-linked immunosorbent-based binding assay, and crosslinking experiments were analyzed by Western blot and mass spectrometry, demonstrating a specific binding site of the peptide at Sema-3A. The observed signals for Sema-3A-peptide interaction were found in a defined area of the Sema domain, which was also demonstrated to be involved in NRP-1 binding. The presented data identified the interaction site for further development of therapeutic peptides to treat acute kidney injury by blocking the Sema-3A-NRP-1 interaction.

Receptor-specific recognition of NPY peptides revealed by structures of NPY receptors.

In response to three highly conserved neuropeptides, neuropeptide Y (NPY), peptide YY, and pancreatic polypeptide (PP), four G protein-coupled receptors mediate multiple essential physiological processes, such as food intake, vasoconstriction, sedation, and memory retention. Here, we report the structures of the human Y1, Y2, and Y4 receptors in complex with NPY or PP, and the Gi1 protein. These structures reveal distinct binding poses of the peptide upon coupling to different receptors, reflecting the importance of the conformational plasticity of the peptide in recognizing the NPY receptors. The N terminus of the peptide forms extensive interactions with the Y1 receptor, but not with the Y2 and Y4 receptors. Supported by mutagenesis and functional studies, subtype-specific interactions between the receptors and peptides were further observed. These findings provide insight into key factors that govern NPY signal recognition and transduction, and would enable development of selective drugs.

Chemerin - exploring a versatile adipokine.

Chemerin is a small chemotactic protein and a key player in initiating the early immune response. As an adipokine, chemerin is also involved in energy homeostasis and the regulation of reproductive functions. Secreted as inactive prochemerin, it relies on proteolytic activation by serine proteases to exert biological activity. Chemerin binds to three distinct G protein-coupled receptors (GPCR), namely chemokine-like receptor 1 (CMKLR1, recently named chemerin1), G protein-coupled receptor 1 (GPR1, recently named chemerin2), and CC-motif chemokine receptor-like 2 (CCRL2). Only CMKLR1 displays conventional G protein signaling, while GPR1 only recruits arrestin in response to ligand stimulation, and no CCRL2-mediated signaling events have been described to date. However, GPR1 undergoes constitutive endocytosis, making this receptor perfectly adapted as decoy receptor. Here, we discuss expression pattern, activation, and receptor binding of chemerin. Moreover, we review the current literature regarding the involvement of chemerin in cancer and several obesity-related diseases, as well as recent developments in therapeutic targeting of the chemerin system.

Orthogonal Peptide-Templated Labeling Elucidates Lateral ETA R/ETB R Proximity and Reveals Altered Downstream Signaling.

Fine-tuning of G protein-coupled receptor (GPCR) signaling is important to maintain cellular homeostasis. Recent studies demonstrated that lateral GPCR interactions in the cell membrane can impact signaling profiles. Here, we report on a one-step labeling method of multiple membrane-embedded GPCRs. Based on short peptide tags, complementary probes transfer the cargo (e. g. a fluorescent dye) by an acyl transfer reaction with high spatial and temporal resolution within 5 min. We applied this approach to four receptors of the cardiovascular system: the endothelin receptor A and B (ETA R and ETB R), angiotensin II receptor type 1, and apelin. Wild type-like G protein activation after N-terminal modification was demonstrated for all receptor species. Using FRET-competent dyes, a constitutive proximity between hetero-receptors was limited to ETA R/ETB R. Further, we demonstrate, that ETA R expression regulates the signaling of co-expressed ETB R. Our orthogonal peptide-templated labeling of different GPCRs provides novel insight into the regulation of GPCR signaling.

Binding of Natural Peptide Ligands to the Neuropeptide Y5 Receptor.

The binding mode of natural peptide ligands to the Y5 G protein-coupled receptor (Y5 R), an attractive therapeutic target for the treatment of obesity, is largely unknown. Here, we apply complementary biochemical and computational approaches, including scanning of the receptor surface with a genetically encoded crosslinker, Ala-scanning of the ligand and double-cycle mutagenesis, to map interactions in the ligand-receptor interface and build a structural model of the NPY-Y5 R complex guided by the experimental data. In the model, the carboxyl (C)-terminus of bound NPY is placed close to the extracellular loop (ECL) 3, whereas the characteristic α-helical segment of the ligand drapes over ECL1 and is tethered towards ECL2 by a hydrophobic cluster. We further show that the other two natural ligands of Y5 R, peptide YY (PYY) and pancreatic polypeptide (PP) dock to the receptor in a similar pose.

Arrestin-dependent internalization of rhodopsin-like G protein-coupled receptors.

The internalization of G protein-coupled receptors (GPCRs) is an important mechanism regulating the signal strength and limiting the opportunity of receptor activation. Based on the importance of GPCRs, the detailed knowledge about the regulation of signal transduction is crucial. Here, current knowledge about the agonist-induced, arrestin-dependent internalization process of rhodopsin-like GPCRs is reviewed. Arrestins are conserved molecules that act as key players within the internalization process of many GPCRs. Based on highly conserved structural characteristics within the rhodopsin-like GPCRs, the identification of arrestin interaction sites in model systems can be compared and used for the investigation of internalization processes of other receptors. The increasing understanding of this essential regulation mechanism of receptors can be used for drug development targeting rhodopsin-like GPCRs. Here, we focus on the neuropeptide Y receptor family, as these receptors transmit various physiological processes such as food intake, energy homeostasis, and regulation of emotional behavior, and are further involved in pathophysiological processes like cancer, obesity and mood disorders. Hence, this receptor family represents an interesting target for the development of novel therapeutics requiring the understanding of the regulatory mechanisms influencing receptor mediated signaling.

Neuropeptide Y as a risk factor for cardiorenal disease and cognitive dysfunction in chronic kidney disease: translational opportunities and challenges.

Neuropeptide Y (NPY) is a 36-amino-acid peptide member of a family also including peptide YY and pancreatic polypeptide, which are all ligands to Gi/Go coupled receptors. NPY regulates several fundamental biologic functions including appetite/satiety, sex and reproduction, learning and memory, cardiovascular and renal function and immune functions. The mesenteric circulation is a major source of NPY in the blood in man and this peptide is considered a key regulator of gut-brain cross talk. A progressive increase in circulating NPY accompanies the progression of chronic kidney disease (CKD) toward kidney failure and NPY robustly predicts cardiovascular events in this population. Furthermore, NPY is suspected as a possible player in accelerated cognitive function decline and dementia in patients with CKD and in dialysis patients. In theory, interfering with the NPY system has relevant potential for the treatment of diverse diseases from cardiovascular and renal diseases to diseases of the central nervous system. Pharmaceutical formulations for effective drug delivery and cost, as well as the complexity of diseases potentially addressable by NPY/NPY antagonists, have been a problem until now. This in part explains the slow progress of knowledge about the NPY system in the clinical arena. There is now renewed research interest in the NPY system in psychopharmacology and in pharmacology in general and new studies and a new breed of clinical trials may eventually bring the expected benefits in human health with drugs interfering with this system.

Cyclic Derivatives of the Chemerin C-Terminus as Metabolically Stable Agonists at the Chemokine-like Receptor 1 for Cancer Treatment.

Chemerin is a small chemotactic protein and a modulator of the innate immune system. Its activity is mainly mediated by the chemokine-like receptor 1 (CMKLR1), a receptor expressed by natural killer cells, dendritic cells, and macrophages. Downregulation of chemerin is part of the immune evasion strategy exploited by several cancer types, including melanoma, breast cancer, and hepatocellular carcinoma. Administration of chemerin can potentially counteract these effects, but synthetically accessible, metabolically stable analogs are required. Other tumors display overexpression of CMKLR1, offering a potential entry point for targeted delivery of chemotherapeutics. Here, we present cyclic derivatives of the chemerin C-terminus (chemerin-9), the minimal activation sequence of chemerin. Chemerin-9 derivatives that were cyclized through positions four and nine retained activity while displaying full stability in blood plasma for more than 24 h. Therefore, these peptides could be used as a drug shuttle system to target cancer cells as demonstrated here by methotrexate conjugates.

Ligand-binding and -scavenging of the chemerin receptor GPR1.

Tight regulation of cytokines is essential for the initiation and resolution of inflammation. Chemerin, a mediator of innate immunity, mainly acts on chemokine-like receptor 1 (CMKLR1) to induce the migration of macrophages and dendritic cells. The role of the second chemerin receptor, G protein-coupled receptor 1 (GPR1), is still unclear. Here we demonstrate that GPR1 shows ligand-induced arrestin3 recruitment and internalization. The chemerin C-terminus triggers this activation by folding into a loop structure, binding to aromatic residues in the extracellular loops of GPR1. While this overall binding mode is shared between GPR1 and CMKLR1, differences in their respective extracellular loop 2 allowed for the design of the first GPR1-selective peptide. However, our results suggest that ligand-induced arrestin recruitment is not the only mode of action of GPR1. This receptor also displays constitutive internalization, which allows GPR1 to internalize inactive peptides efficiently by an activation-independent pathway. Our results demonstrate that GPR1 takes a dual role in regulating chemerin activity: as a signaling receptor for arrestin-based signaling on one hand, and as a scavenging receptor with broader ligand specificity on the other.

Development of a ghrelin receptor inverse agonist for positron emission tomography.

Imaging of Ghrelin receptors in vivo provides unique potential to gain deeper understanding on Ghrelin and its receptors in health and disease, in particular, in cancer. Ghrelin, an octanoylated 28-mer peptide hormone activates the constitutively active growth hormone secretagogue receptor type 1a (GHS-R1a) with nanomolar activity. We developed novel compounds, derived from the potent inverse agonist K-(D-1-Nal)-FwLL-NH2 but structurally varied by lysine conjugation with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), palmitic acid and/or diethylene glycol (PEG2) to allow radiolabeling and improve pharmacokinetics, respectively. All compounds were tested for receptor binding, potency and efficacy in vitro, for biodistribution and -kinetics in rats and in preclinical prostate cancer models on mice. Radiolabeling with Cu-64 and Ga-68 was successfully achieved. The Cu-64- or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiotracer were specifically accumulated by the GHS-R1a in xenotransplanted human prostate tumor models (PC-3, DU-145) in mice. The tumors were clearly delineated by PET. The radiotracer uptake was also partially blocked by K-(D-1-Nal)-FwLL-NH2 in stomach and thyroid. The presence of the GHS-R1a was also confirmed by immunohistology. In the arterial rat blood plasma, only the original compounds were found. The Cu-64 or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiolabeled inverse agonists turned out to be potent and safe. Due to their easy synthesis, high affinity, medium potency, metabolic stability, and the suitable pharmacokinetic profiles, they are excellent tools for imaging and quantitation of GHS-R1a expression in normal and cancer tissues by PET. These compounds can be used as novel biomarkers of the Ghrelin system in precision medicine.

Cyclic Analogues of the Chemerin C-Terminus Mimic a Loop Conformation Essential for Activating the Chemokine-like Receptor 1.

The chemokine-like receptor 1 (CMKLR1) is a promising target for treating autoinflammatory diseases, cancer, and reproductive disorders. However, the interaction between CMKLR1 and its protein-ligand chemerin remains uncharacterized, and no drugs targeting this interaction have passed clinical trials. Here, we identify the binding mode of chemerin-9, the C-terminus of chemerin, at the receptor by combining complementary mutagenesis with structure-based modeling. Incorporating our experimental data, we present a detailed model of this binding site, including experimentally confirmed pairwise interactions for the most critical ligand residues: Chemerin-9 residue F8 binds to a hydrophobic pocket in CMKLR1 formed by the extracellular loop (ECL) 2, while F6 interacts with Y2.68, suggesting a turn-like structure. On the basis of this model, we created the first cyclic peptide with nanomolar activity, confirming the overall binding conformation. This constrained agonist mimics the loop conformation adopted by the natural ligand and can serve as a lead compound for future drug design.

Structural basis for ligand recognition of the neuropeptide Y Y2 receptor.

The human neuropeptide Y (NPY) Y2 receptor (Y2R) plays essential roles in food intake, bone formation and mood regulation, and has been considered an important drug target for obesity and anxiety. However, development of drugs targeting Y2R remains challenging with no success in clinical application yet. Here, we report the crystal structure of Y2R bound to a selective antagonist JNJ-31020028 at 2.8 Å resolution. The structure reveals molecular details of the ligand-binding mode of Y2R. Combined with mutagenesis studies, the Y2R structure provides insights into key factors that define antagonistic activity of diverse antagonists. Comparison with the previously determined antagonist-bound Y1R structures identified receptor-ligand interactions that play different roles in modulating receptor activation and mediating ligand selectivity. These findings deepen our understanding about molecular mechanisms of ligand recognition and subtype specificity of NPY receptors, and would enable structure-based drug design.

Highly Selective Y4 Receptor Antagonist Binds in an Allosteric Binding Pocket.

Human neuropeptide Y receptors (Y1R, Y2R, Y4R, and Y5R) 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 Y4R 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 Y4R function and binds in an allosteric site located below the binding pocket of the endogenous ligand pancreatic polypeptide in the core of the Y4R transmembrane domains. Taken together, our studies provide a first-of-its-kind tool for probing Y4R 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).

Strategies for Site-Specific Labeling of Receptor Proteins on the Surfaces of Living Cells by Using Genetically Encoded Peptide Tags.

Fluorescence microscopy imaging enables receptor proteins to be investigated within their biological context. A key challenge is to site-specifically incorporate reporter moieties into proteins without interfering with biological functions or cellular networks. Small peptide tags offer the opportunity to combine inducible labeling with small tag sizes that avoid receptor perturbation. Herein, we review the current state of live-cell labeling of peptide-tagged cell-surface proteins. Considering their importance as targets in medicinal chemistry, we focus on membrane receptors such as G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). We discuss peptide tags that i) are subject to enzyme-mediated modification reactions, ii) guide the complementation of reporter proteins, iii) form coiled-coil complexes, and iv) interact with metal complexes. Given our own contributions in the field, we place emphasis on peptide-templated labeling chemistry.