Structure-activity relationship of GPR15L peptide analogues and investigation of their interaction with the GPR15 receptor.

The 57-mer full-length GPR15L(25-81) peptide has been identified as the principal endogenous agonist of the G protein-coupled receptor GPR15. Its main activity resides in the C-terminal 11-mer GPR15L(71-81), which has full efficacy but ~40-fold lower potency than the full-length peptide. Here, we systematically investigated the structure-activity relationship of GPR15L(71-81) by truncations/extensions, alanine-scanning, and N- and C-terminal capping. The synthesized peptide analogues were tested at GPR15 stably expressed in HEK293A cells using a homogenous time-resolved Förster resonance energy transfer-based Gi cAMP functional assay. We show that the C-terminal α carboxyl group and the residues Leu78 , Pro75 , Val74 , and Trp72 are critical for receptor interaction and contribute significantly to the peptide potency. Furthermore, we tested the ability of GPR15L(71-81), C-terminally amidated GPR15L(71-81), and GPR15L(25-81) to activate the three GPR15 receptor mutants in a bioluminescence resonance energy transfer-based G protein activation assay. The results demonstrate that the Lys192 and Glu272 residues in GPR15 are important for the potency of the GPR15L peptide. Overall, our study identifies critical residues in the peptide and receptor sequences for future drug design.

Effector membrane translocation biosensors reveal G protein and ßarrestin coupling profiles of 100 therapeutically relevant GPCRs.

The recognition that individual GPCRs can activate multiple signaling pathways has raised the possibility of developing drugs selectively targeting therapeutically relevant ones. This requires tools to determine which G proteins and ßarrestins are activated by a given receptor. Here, we present a set of BRET sensors monitoring the activation of the 12 G protein subtypes based on the translocation of their effectors to the plasma membrane (EMTA). Unlike most of the existing detection systems, EMTA does not require modification of receptors or G proteins (except for Gs). EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. Profiling of 100 therapeutically relevant human GPCRs resulted in 1500 pathway-specific concentration-response curves and revealed a great diversity of coupling profiles ranging from exquisite selectivity to broad promiscuity. Overall, this work describes unique resources for studying the complexities underlying GPCR signaling and pharmacology.

Structural insights into the lipid and ligand regulation of serotonin receptors.

Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter1,2 that activates the largest subtype family of G-protein-coupled receptors3. Drugs that target 5-HT1A, 5-HT1D, 5-HT1E and other 5-HT receptors are used to treat numerous disorders4. 5-HT receptors have high levels of basal activity and are subject to regulation by lipids, but the structural basis for the lipid regulation and basal activation of these receptors and the pan-agonism of 5-HT remains unclear. Here we report five structures of 5-HT receptor-G-protein complexes: 5-HT1A in the apo state, bound to 5-HT or bound to the antipsychotic drug aripiprazole; 5-HT1D bound to 5-HT; and 5-HT1E in complex with a 5-HT1E- and 5-HT1F-selective agonist, BRL-54443. Notably, the phospholipid phosphatidylinositol 4-phosphate is present at the G-protein-5-HT1A interface, and is able to increase 5-HT1A-mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterol molecules-particularly in the case of 5-HT1A, in which cholesterol molecules are directly involved in shaping the ligand-binding pocket that determines the specificity for aripiprazol. Within the ligand-binding pocket of apo-5-HT1A are structured water molecules that mimic 5-HT to activate the receptor. Together, our results address a long-standing question of how lipids and water molecules regulate G-protein-coupled receptors, reveal how 5-HT acts as a pan-agonist, and identify the determinants of drug recognition in 5-HT receptors.

Targeting the APP-Mint2 Protein-Protein Interaction with a Peptide-Based Inhibitor Reduces Amyloid-ß Formation.

There is an urgent need for novel therapeutic approaches to treat Alzheimer's disease (AD) with the ability to both alleviate the clinical symptoms and halt the progression of the disease. AD is characterized by the accumulation of amyloid-ß (Aß) peptides which are generated through the sequential proteolytic cleavage of the amyloid precursor protein (APP). Previous studies reported that Mint2, a neuronal adaptor protein binding both APP and the γ-secretase complex, affects APP processing and formation of pathogenic Aß. However, there have been contradicting results concerning whether Mint2 has a facilitative or suppressive effect on Aß generation. Herein, we deciphered the APP-Mint2 protein-protein interaction (PPI) via extensive probing of both backbone H-bond and side-chain interactions. We also developed a proteolytically stable, high-affinity peptide targeting the APP-Mint2 interaction. We found that both an APP binding-deficient Mint2 variant and a cell-permeable PPI inhibitor significantly reduced Aß42 levels in a neuronal in vitro model of AD. Together, these findings demonstrate a facilitative role of Mint2 in Aß formation, and the combination of genetic and pharmacological approaches suggests that targeting Mint2 is a promising therapeutic strategy to reduce pathogenic Aß levels.

Differential GLP-1R Binding and Activation by Peptide and Non-peptide Agonists.

Peptide drugs targeting class B1 G-protein-coupled receptors (GPCRs) can treat multiple diseases; however, there remains substantial interest in the development of orally delivered non-peptide drugs. Here, we reveal unexpected overlap between signaling and regulation of the glucagon-like peptide-1 (GLP-1) receptor by the non-peptide agonist PF 06882961 and GLP-1 that was not observed for another compound, CHU-128. Compounds from these patent series, including PF 06882961, are currently in clinical trials for treatment of type 2 diabetes. High-resolution cryoelectron microscopy (cryo-EM) structures reveal that the binding sites for PF 06882961 and GLP-1 substantially overlap, whereas CHU-128 adopts a unique binding mode with a more open receptor conformation at the extracellular face. Structural differences involving extensive water-mediated hydrogen bond networks could be correlated to functional data to understand how PF 06882961, but not CHU-128, can closely mimic the pharmacological properties of GLP-1. These findings will facilitate rational structure-based discovery of non-peptide agonists targeting class B GPCRs.

Novel approaches leading towards peptide GPCR de-orphanisation.

The discovery of novel ligands for orphan GPCRs has profoundly affected our understanding of human biology, opening new opportunities for research, and ultimately for therapeutic development. Accordingly, much effort has been directed towards the remaining orphan receptors, yet the rate of GPCR de-orphanisation has slowed in recent years. Here, we briefly review contemporary methodologies of de-orphanisation and then highlight our recent integrated computational and experimental approach for discovery of novel peptide ligands for orphan GPCRs. We identified putative endogenous peptide ligands and found peptide receptor sequence and structural characteristics present in selected orphan receptors. With comprehensive pharmacological screening using three complementary assays, we discovered novel pairings of 17 peptides with five different orphan GPCRs and revealed potential additional ligands for nine peptide GPCRs. These promising findings lay the foundation for future studies on these peptides and receptors to characterise their roles in human physiology and disease.

Discovery of Human Signaling Systems: Pairing Peptides to G Protein-Coupled Receptors.

The peptidergic system is the most abundant network of ligand-receptor-mediated signaling in humans. However, the physiological roles remain elusive for numerous peptides and more than 100 G protein-coupled receptors (GPCRs). Here we report the pairing of cognate peptides and receptors. Integrating comparative genomics across 313 species and bioinformatics on all protein sequences and structures of human class A GPCRs, we identify universal characteristics that uncover additional potential peptidergic signaling systems. Using three orthogonal biochemical assays, we pair 17 proposed endogenous ligands with five orphan GPCRs that are associated with diseases, including genetic, neoplastic, nervous and reproductive system disorders. We also identify additional peptides for nine receptors with recognized ligands and pathophysiological roles. This integrated computational and multifaceted experimental approach expands the peptide-GPCR network and opens the way for studies to elucidate the roles of these signaling systems in human physiology and disease. VIDEO ABSTRACT.

5-HT2C Receptor Structures Reveal the Structural Basis of GPCR Polypharmacology.

Drugs frequently require interactions with multiple targets-via a process known as polypharmacology-to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT2C receptor ligands or creating drugs with a defined polypharmacological profile, especially aimed at G protein-coupled receptors (GPCRs), remain extremely difficult. Here, we solved two structures of the 5-HT2C receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT2A-C receptor-selective inverse agonist ritanserin at resolutions of 3.0 Å and 2.7 Å, respectively. We analyzed their respective binding poses to provide mechanistic insights into their receptor recognition and opposing pharmacological actions. This study investigates the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs.

Structural Mapping of Adenosine Receptor Mutations: Ligand Binding and Signaling Mechanisms.

The four adenosine receptors (ARs), A1, A2A, A2B, and A3, constitute a subfamily of G protein-coupled receptors (GPCRs) with exceptional foundations for structure-based ligand design. The vast amount of mutagenesis data, accumulated in the literature since the 1990s, has been recently supplemented with structural information, currently consisting of several inactive and active structures of the A2A and inactive conformations of the A1 ARs. We provide the first integrated view of the pharmacological, biochemical, and structural data available for this receptor family, by mapping onto the relevant crystal structures all site-directed mutagenesis data, curated and deposited at the GPCR database (available through http://www.gpcrdb.org). This analysis provides novel insights into ligand binding, allosteric modulation, and signaling of the AR family.

Structure-activity relationships and identification of optmized CC-chemokine receptor CCR1, 5, and 8 metal-ion chelators.

Chemokine receptors are involved in trafficking of leukocytes and represent targets for autoimmune conditions, inflammatory diseases, viral infections, and cancer. We recently published CCR1, CCR8, and CCR5 agonists and positive modulators based on a three metal-ion chelator series: 2,2'-bipyridine, 1,10-phenanthroline, and 2,2';6',2″-terpyridine. Here, we have performed an in-depth structure-activity relationship study and tested eight new optimized analogs. Using density functional theory calculations we demonstrate that the chelator zinc affinities depend on how electron-donating and -withdrawing substituents modulate the partial charges of chelating nitrogens. The zinc affinity was found to constitute the major factor for receptor potency, although the activity of some chelators deviate suggesting favorable or unfavorable interactions. Hydrophobic and halogen substituents are generally better accommodated in the receptors than polar groups. The new analog brominated terpyridine (29) resulted in the highest chelator potencies observed so far CCR1 (EC50: 0.49 µM) and CCR8 (EC50: 0.28 µM). Furthermore, we identified the first selective CCR5 agonist chelator, meta dithiomethylated bipyridine (23). The structure-activity relationships contribute to small-molecule drug development, and the novel chelators constitute valuable tools for studies of structural mechanisms for chemokine receptor activation.

Chemogenomics of allosteric binding sites in GPCRs.

Chemogenomic techniques connect the chemical and biological domains to establish ligand and target relationships not evident from the individual disciplines. Chemogenomics has been applied in lead generation, target classification, focused library design as well as selectivity and polypharmacology profiling. This review describes recent developments structured into ligand-, target- and combined chemogenomic techniques and applications to allosteric GPCR ligands. It also outlines relative strengths and limitations of these techniques and the impact of the increasing crystallographic data.

A community standard format for the representation of protein affinity reagents.

Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one on-line warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site.

Definition of the G protein-coupled receptor transmembrane bundle binding pocket and calculation of receptor similarities for drug design.

Recent advances in structural biology for G-protein-coupled receptors (GPCRs) have provided new opportunities to improve the definition of the transmembrane binding pocket. Here a reference set of 44 residue positions accessible for ligand binding was defined through detailed analysis of all currently available crystal structures. This was used to characterize pharmacological relationships of Family A/Rhodopsin family GPCRs, minimizing evolutionary influence from parts of the receptor that do not generally affect ligand binding. The resultant dendogram tended to group receptors according to endogenous ligand types, although it revealed subdivision of certain classes, notably peptide and lipid receptors. The transmembrane binding site reference set, particularly when coupled with a means of identifying the subset of ligand binding residues, provides a general paradigm for understanding the pharmacology/selectivity profile of ligands at Family A GPCRs. This has wide applicability to GPCR drug design problems across many disease areas.

Seven evolutionarily conserved human rhodopsin G protein-coupled receptors lacking close relatives.

We report seven new members of the superfamily of human G protein-coupled receptors (GPCRs) found by searches in the human genome databases, termed GPR100, GPR119, GPR120, GPR135, GPR136, GPR141, and GPR142. We also report 16 orthologues of these receptors in mouse, rat, fugu (pufferfish) and zebrafish. Phylogenetic analysis shows that these are additional members of the family of rhodopsin-type GPCRs. GPR100 shows similarity with the orphan receptor SALPR. Remarkably, the other receptors do not have any close relative among other known human rhodopsin-like GPCRs. Most of these orphan receptors are highly conserved through several vertebrate species and are present in single copies. Analysis of expressed sequence tag (EST) sequences indicated individual expression patterns, such as for GPR135, which was found in a wide variety of tissues including eye, brain, cervix, stomach and testis. Several ESTs for GPR141 were found in marrow and cancer cells, while the other receptors seem to have more restricted expression patterns.