Nutlin-3a-aa: Improving the Bioactivity of a p53/MDM2 Interaction Inhibitor by Introducing a Solvent-Exposed Methylene Group.

Nutlin-3a is a reversible inhibitor of the p53/MDM2 interaction. We have synthesized the derivative Nutlin-3a-aa bearing an additional exocyclic methylene group in the piperazinone moiety. Nutlin-3a-aa is more active than Nutlin-3a against purified wild-type MDM2, and is more effective at increasing p53 levels and releasing transcription of p53 target genes from MDM2-induced repression. X-ray analysis of wild-type MDM2-bound Nutlin-3a-aa indicated that the orientation of its modified piperazinone ring was altered in comparison to the piperazinone ring of MDM2-bound Nutlin-3a, with the exocyclic methylene group of Nutlin-3a-aa pointing away from the protein surface. Our data point to the introduction of exocyclic methylene groups as a useful approach by which to tailor the conformation of bioactive molecules for improved biological activity.

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.

Structural investigations of cell-free expressed G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are of great pharmaceutical interest and about 35% of the commercial drugs target these proteins. Still there is huge potential left in finding molecules that target new GPCRs or that modulate GPCRs differentially. For a rational drug design, it is important to understand the structure, binding and activation of the protein of interest. Structural investigations of GPCRs remain challenging, although huge progress has been made in the last 20 years, especially in the generation of crystal structures of GPCRs. This is mostly caused by issues with the expression yield, purity or labeling. Cell-free protein synthesis (CFPS) is an efficient alternative for recombinant expression systems that can potentially address many of these problems. In this article the use of CFPS for structural investigations of GPCRs is reviewed. We compare different CFPS systems, including the cellular basis and reaction configurations, and strategies for an efficient solubilization. Next, we highlight recent advances in the structural investigation of cell-free expressed GPCRs, with special emphasis on the role of photo-crosslinking approaches to investigate ligand binding sites on GPCRs.

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.

Structure-Activity Relationship Study of the High-Affinity Neuropeptide Y4 Receptor Positive Allosteric Modulator VU0506013.

Positive allosteric modulators targeting the Y4 receptor (Y4R), a G protein-coupled receptor (GPCR) involved in the regulation of satiety, offer great potential in anti-obesity research. In this study, we selected 603 compounds by using quantitative structure-activity relationship (QSAR) models and tested them in high-throughput screening (HTS). Here, the novel positive allosteric modulator (PAM) VU0506013 was identified, which exhibits nanomolar affinity and pronounced selectivity toward the Y4R in engineered cell lines and mouse descending colon mucosa natively expressing the Y4R. Based on this lead structure, we conducted a systematic SAR study in two regions of the scaffold and presented a series of 27 analogues with modifications in the N- and C-terminal heterocycles of the molecule to obtain insight into functionally relevant positions. By mutagenesis and computational docking, we present a potential binding mode of VU0506013 in the transmembrane core of the Y4R. VU0506013 presents a promising scaffold for developing in vivo tools to move toward anti-obesity drug research focused on the Y4R.

LRP1 is the cell-surface endocytosis receptor for vaspin in adipocytes.

Vaspin is a serine protease inhibitor that protects against adipose tissue inflammation and insulin resistance, two key drivers of adipocyte dysfunction and metabolic disorders in obesity. Inhibition of target proteases such as KLK7 has been shown to reduce adipose tissue inflammation in obesity, while vaspin binding to cell surface GRP78 has been linked to reduced obesity-induced ER stress and insulin resistance in the liver. However, the molecular mechanisms by which vaspin directly affects cellular processes in adipocytes remain unknown. Using fluorescently labeled vaspin, we found that vaspin is rapidly internalized by mouse and human adipocytes, but less efficiently by endothelial, kidney, liver, and neuronal cells. Internalization occurs by active, clathrin-mediated endocytosis, which is dependent on vaspin binding to the LRP1 receptor, rather than GRP78 as previously thought. This was demonstrated by competition experiments and RNAi-mediated knock-down in adipocytes and by rescuing vaspin internalization in LRP1-deficient Pea13 cells after transfection with a functional LRP1 minireceptor. Vaspin internalization is further increased in mature adipocytes after insulin-stimulated translocation of LRP1. Although vaspin has nanomolar affinity for LRP1 clusters II-IV, binding to cell surface heparan sulfates is required for efficient LRP1-mediated internalization. Native, but not cleaved vaspin, and also vaspin polymers are efficiently endocytosed, and ultimately targeted for lysosomal degradation. Our study provides mechanistic insight into the uptake and degradation of vaspin in adipocytes, thereby broadening our understanding of its functional repertoire. We hypothesize the vaspin-LRP1 axis to be an important mediator of vaspin effects not only in adipose tissue but also in other LRP1-expressing cells.

Peptide backbone conformation affects the substrate preference of protein arginine methyltransferase I.

Asymmetric dimethylation of arginine side chains is a common post-translational modification of eukaryotic proteins, which serves mostly to regulate protein-protein interactions. The modification is catalyzed by type I protein arginine methyltransferases, PRMT1 being the predominant member of the family. Determinants of substrate specificity of these enzymes are poorly understood. The Nuclear poly(A) binding protein 1 (PABPN1) is methylated by PRMT1 at 13 arginine residues located in RXR sequences in the protein's C-terminal domain. We have identified a preferred site for PRMT1-catalyzed methylation in PABPN1 and in a corresponding synthetic peptide. Variants of these substrates were analyzed by steady-state kinetic analysis and mass spectrometry. The data indicate that initial methylation is directed toward the preferred arginine residue by an N-terminally adjacent proline. Enhanced methylation upon peptide cyclization suggests that induction of a reverse turn structure is the basis for the ability of the respective proline residue to enable preferred methylation of the neighboring arginine residue, and this notion is supported by far-UV circular dichroism spectroscopy. We suggest that the formation of a reverse turn facilitates the access of arginine side chains to the active sites of PRMT1, which are located in the central cavity of a doughnut-shaped PRMT1 homodimer.

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.

Cleavage of the vaspin N-terminus releases cell-penetrating peptides that affect early stages of adipogenesis and inhibit lipolysis in mature adipocytes.

Vaspin expression and function is related to metabolic disorders and comorbidities of obesity. In various cellular and animal models of obesity, diabetes and atherosclerosis vaspin has shown beneficial, protective and/or compensatory action. While testing proteases for inhibition by vaspin, we noticed specific cleavage within the vaspin N-terminus and sequence analysis predicted cell-penetrating activity for the released peptides. These findings raised the question whether these proteolytic peptides exhibit biological activity.We synthesized various N-terminal vaspin peptides to investigate cell-penetrating activity and analyse uptake mechanisms. Focusing on adipocytes, we performed microarray analysis and functional assays to elucidate biological activities of the vaspin-derived peptide, which is released by KLK7 cleavage (vaspin residues 21-30; VaspinN). Our study provides first evidence that proteolytic processing of the vaspin N-terminus releases cell-penetrating and bioactive peptides with effects on adipocyte biology. The VaspinN peptide increased preadipocyte proliferation, interfered with clonal expansion during the early stage of adipogenesis and blunted adrenergic cAMP-signalling, downstream lipolysis as well as insulin signalling in mature adipocytes.Protease-mediated release of functional N-terminal peptides presents an additional facet of vaspin action. Future studies will address the mechanisms underlying the biological activities and clarify, if vaspin-derived peptides may have potential as therapeutic agents for the treatment of metabolic diseases.

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).

Agonist induced receptor internalization of neuropeptide Y receptor subtypes depends on third intracellular loop and C-terminus.

Agonist stimulation of G-protein coupled receptors (GPCRs) results in the redistribution of the receptor from the cell surface into intracellular compartments through the process of endocytosis. Monitoring ligand-mediated internalization of GPCRs in living cells has become experimentally accessible by applying fluorescent reagents and fluorescence microscopy. By using cell lines that transiently, stably or endogenously express the human Y receptor (hYR) subtypes hY(1)R, hY(2)R, hY(4)R and hY(5)R and differently fluorescently tagged receptor proteins we were able to unravel further details concerning the internalization behavior of this multi-receptor/multi-ligand system. For the first time we could show that also the hY(2)R is internalized with a rate which is comparable to the hY(1)R and the hY(4)R. In contrast, the hY(5)R was internalized much slower and the rate remained unaffected by co-expression with other hYR subtypes. Furthermore receptor subtype co-expressing cells and selectively binding peptides revealed a receptor subtype selective internalization. By using novel hY(5)/hY(2) receptor chimera the receptor subtype dependent differences in hY receptor internalization could be identified on a molecular level.

Cell-Free Expression and Photo-Crosslinking of the Human Neuropeptide Y2 Receptor.

G protein-coupled receptors (GPCRs) represent a large family of different proteins, which are involved in physiological processes throughout the entire body. Furthermore, they represent important drug targets. For rational drug design, it is important to get further insights into the binding mode of endogenous ligands as well as of therapeutic agents at the respective target receptors. However, structural investigations usually require homogenous, solubilized and functional receptors, which is still challenging. Cell-free expression methods have emerged in the last years and many different proteins are successfully expressed, including hydrophobic membrane proteins like GPCRs. In this work, an Escherichia coli based cell-free expression system was used to express the neuropeptide Y2 receptor (Y2R) for structural investigations. This GPCR was expressed in two different variants, a C-terminal enhanced green fluorescent fusion protein and a cysteine deficient variant. In order to obtain soluble receptors, the expression was performed in the presence of mild detergents, either Brij-35 or Brij-58, which led to high amounts of soluble receptor. Furthermore, the influence of temperature, pH value and additives on protein expression and solubilization was tested. For functional and structural investigations, the receptors were expressed at 37°C, pH 7.4 in the presence of 1 mM oxidized and 5 mM reduced glutathione. The expressed receptors were purified by ligand affinity chromatography and functionality of Y2R_cysteine_deficient was verified by a homogenous binding assay. Finally, photo-crosslinking studies were performed between cell-free expressed Y2R_cysteine_deficient and a neuropeptide Y (NPY) analog bearing the photoactive, unnatural amino acid p-benzoyl-phenylalanine at position 27 and biotin at position 22 for purification. After enzymatic digestion, fragments of crosslinked receptor were identified by mass spectrometry. Our findings demonstrate that, in contrast to Y1R, NPY position 27 remains flexible when bound to Y2R. These results are in agreement with the suggested binding mode of NPY at Y2R.

Molecular recognition of the NPY hormone family by their receptors.

Many G-protein-coupled receptors belong to families of different receptor subtypes, which are recognized by a variety of distinct ligands. We summarize the current state of the art of the multireceptor/multiligand system of the so-called Y-receptor family. This family consists of four G-protein-coupled Y receptors in humans (hY(1), hY(2), hY(4), and hY(5)) and is activated by the so-called neuropeptide Y hormone family, which consists of three native peptide ligands named neuropeptide Y, pancreatic polypeptide, and peptide YY. We recently reported that one conserved aspartate residue in the third extracellular loop is essential for ligand binding in all four Y receptors, but binds the endogenous ligands in a different mode by interacting with different ligand arginine residues. By combining peptide synthesis to obtain chemically modified neuropeptide Y, peptide YY, and pancreatic polypeptide analogs, receptor mutagenesis, and receptor chimeras, we could trace binding and signaling to a molecular level. The data on the variation of the ligands and an overview of the currently known mutagenesis data are summarized and specific models for the binding mode of the three ligands in all four receptors are provided.

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 (tert-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.

A Deep Hydrophobic Binding Cavity is the Main Interaction for Different Y2 R Antagonists.

The neuropeptide Y2 receptor (Y2 R) is involved in various pathophysiological processes such as epilepsy, mood disorders, angiogenesis, and tumor growth. Therefore, the Y2 R is an interesting target for drug development. A detailed understanding of the binding pocket could facilitate the development of highly selective antagonists to study the role of Y2 R in vitro and in vivo. In this study, several residues crucial to the interaction of BIIE0246 and SF-11 derivatives with Y2 R were investigated by signal transduction assays. Using the experimental results as constraints, the antagonists were docked into a comparative structural model of the Y2 R. Despite differences in size and structure, all three antagonists display a similar binding site, including a deep hydrophobic cavity formed by transmembrane helices (TM) 4, 5, and 6, as well as a hydrophobic patch at the top of TM2 and 7. Additionally, we suggest that the antagonists block Q3.32 , a position that has been shown to be crucial for binding of the amidated C terminus of NPY and thus for receptor activation.

Discovery of Small-Molecule Modulators of the Human Y4 Receptor.

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.

Neuropeptide Y receptors: how to get subtype selectivity.

The neuropeptide Y (NPY) system is a multireceptor/multiligand system consisting of four receptors in humans (hY(1), hY(2), hY(4), hY(5)) and three agonists (NPY, PYY, PP) that activate these receptors with different potency. The relevance of this system in diseases like obesity or cancer, and the different role that each receptor plays influencing different biological processes makes this system suitable for the design of subtype selectivity studies. In this review we focus on the latest findings within the NPY system, we summarize recent mutagenesis studies, structure activity relationship studies, receptor chimera, and selective ligands focusing also on the binding mode of the native agonists.