GPR17: Molecular modeling and dynamics studies of the 3-D structure and purinergic ligand binding features in comparison with P2Y receptors

<p>Abstract</p> <p>Background</p> <p>GPR17 is a G-protein-coupled receptor located at intermediate phylogenetic position between two distinct receptor families: the P2Y and CysLT receptors for extracellular nucleotides and cysteinyl-LTs, respectively. We previously showed that GPR17 can indeed respond to both classes of endogenous ligands and to synthetic compounds active at the above receptor families, thus representing the first fully characterized non-peptide "hybrid" GPCR. In a rat brain focal ischemia model, the selective <it>in vivo </it>knock down of GPR17 by anti-sense technology or P2Y/CysLT antagonists reduced progression of ischemic damage, thus highlighting GPR17 as a novel therapeutic target for stroke. Elucidation of the structure of GPR17 and of ligand binding mechanisms are the necessary steps to obtain selective and potent drugs for this new potential target. On this basis, a 3-D molecular model of GPR17 embedded in a solvated phospholipid bilayer and refined by molecular dynamics simulations has been the first aim of this study. To explore the binding mode of the "purinergic" component of the receptor, the endogenous agonist UDP and two P2Y receptor antagonists demonstrated to be active on GPR17 (MRS2179 and cangrelor) were then modeled on the receptor.</p> <p>Results</p> <p>Molecular dynamics simulations suggest that GPR17 nucleotide binding pocket is similar to that described for the other P2Y receptors, although only one of the three basic residues that have been typically involved in ligand recognition is conserved (Arg255). The binding pocket is enclosed between the helical bundle and covered at the top by EL2. Driving interactions are H-bonds and salt bridges between the 6.55 and 6.52 residues and the phosphate moieties of the ligands. An "accessory" binding site in a region formed by the EL2, EL3 and the Nt was also found.</p> <p>Conclusion</p> <p>Nucleotide binding to GPR17 occurs on the same receptor regions identified for already known P2Y receptors. Agonist/antagonist binding mode are similar, but not identical. An accessory external binding site could guide small ligands to the deeper principal binding site in a multi-step mechanism of activation. The nucleotide binding pocket appears to be unable to allocate the leukotrienic type ligands in the same effective way.</p

The Dual Behaviour of a GPCR Involved in Brain Damage an Repair: Forced Unbinding of the Receptor GPR17 Ligands from Wild Type and R255I Mutant Models Through a Computational Approach

GPR17 is a hybrid G-protein-coupled receptor activated by two unrelated ligand families, extracellular nucleotides and cysteinyl-leukotrienes, and involved in brain damage and repair. Its exploitment as a target for novel neuroreparative strategies depends on the elucidation of the molecular determinants driving binding of its ligands. We applied docking and molecular dynamics simulations to analyse the binding and the forced unbinding of two GPR17 ligands (the purinergic agonist UDP and the leukotriene receptor antagonist pranlukast) from both the wild-type receptor and a mutant model, where a basic residue hypothesized to be crucial for nucleotide binding had been mutated (R255I). Molecular dynamics suggested that GPR17 nucleotide binding pocket is enclosed between the helical bundle and EL2. The driving interaction involves R255 and the UDP phosphate moiety. Steered molecular dynamics experiments showed that the energy required to unbind UDP is higher for the wild-type receptor than for R255I. Three potential binding sites for pranlukast were found. In one of its preferential docking conformations, pranlukast tetrazole group is close to R255 and phenyl rings are placed into a subpocket highly conserved among GPCRs. Pulling forces developed to break polar and aromatic interactions of pranlukast were comparable. No differences between the wild-type receptor and the R255I receptor were found for the unbinding of pranlukast. These data suggest a crucial role for R255 in binding of nucleotides to GPR17. Aromatic interactions are instead likely to play a predominant role in the recognition of pranlukast, suggesting that two different binding subsites are present on GPR17

Development of selective agonists and antagonists of P2Y receptors

Although elucidation of the medicinal chemistry of agonists and antagonists of the P2Y receptors has lagged behind that of many other members of group A G protein-coupled receptors, detailed qualitative and quantitative structure–activity relationships (SARs) were recently constructed for several of the subtypes. Agonists selective for P2Y1, P2Y2, and P2Y6 receptors and nucleotide antagonists selective for P2Y1 and P2Y12 receptors are now known. Selective nonnucleotide antagonists were reported for P2Y1, P2Y2, P2Y6, P2Y11, P2Y12, and P2Y13 receptors. At the P2Y1 and P2Y12 receptors, nucleotide agonists (5′-diphosphate derivatives) were converted into antagonists of nanomolar affinity by altering the phosphate moieties, with a focus particularly on the ribose conformation and substitution pattern. Nucleotide analogues with conformationally constrained ribose-like rings were introduced as selective receptor probes for P2Y1 and P2Y6 receptors. Screening chemically diverse compound libraries has begun to yield new lead compounds for the development of P2Y receptor antagonists, such as competitive P2Y12 receptor antagonists with antithrombotic activity. Selective agonists for the P2Y4, P2Y11, and P2Y13 receptors and selective antagonists for P2Y4 and P2Y14 receptors have not yet been identified. The P2Y14 receptor appears to be the most restrictive of the class with respect to modification of the nucleobase, ribose, and phosphate moieties. The continuing process of ligand design for the P2Y receptors will aid in the identification of new clinical targets

Computing Highly Correlated Positions Using Mutual Information and Graph Theory for G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) are a superfamily of seven transmembrane-spanning proteins involved in a wide array of physiological functions and are the most common targets of pharmaceuticals. This study aims to identify a cohort or clique of positions that share high mutual information. Using a multiple sequence alignment of the transmembrane (TM) domains, we calculated the mutual information between all inter-TM pairs of aligned positions and ranked the pairs by mutual information. A mutual information graph was constructed with vertices that corresponded to TM positions and edges between vertices were drawn if the mutual information exceeded a threshold of statistical significance. Positions with high degree (i.e. had significant mutual information with a large number of other positions) were found to line a well defined inter-TM ligand binding cavity for class A as well as class C GPCRs. Although the natural ligands of class C receptors bind to their extracellular N-terminal domains, the possibility of modulating their activity through ligands that bind to their helical bundle has been reported. Such positions were not found for class B GPCRs, in agreement with the observation that there are not known ligands that bind within their TM helical bundle. All identified key positions formed a clique within the MI graph of interest. For a subset of class A receptors we also considered the alignment of a portion of the second extracellular loop, and found that the two positions adjacent to the conserved Cys that bridges the loop with the TM3 qualified as key positions. Our algorithm may be useful for localizing topologically conserved regions in other protein families

G-protein coupled receptors activation mechanism: from ligand binding to the transmission of the signal inside the cell

G-protein coupled receptors (GPCRs) are the largest family of pharmaceutical drug targets in the human genome and are modulated by a large variety of en- dogenous and synthetic ligands. GPCRs activation usually depends on agonist binding (except for receptors with basal activity), which stabilizes receptor con- formations and allow the requirement and activation of intracellular transducers. GPCRs are unique receptors and very well studied, since they play an important role in a great number of diseases. They interact with different type of ligands (such as light, peptides, proteins) and different partners in the intracellular part (such as G-proteins or β-arrestins). Based on homology and function GPCRs are divided in five classes: Class A or Rhodopsin, Class B1 or Secretin, Class B2 or Adhesion, Class C or Glutamate, Class F or Frizzled. What is still missing in the state of the art of these receptor, and in particular in Class A, is a global study on different binding cavities with divergent properties, with the aim to discover common binding characteristics, preserved during years of evolution. Gaining more knowledge on common features for ligand recognition shared among all the recep- tors may become crucial to deeply understand the mechanism used to transmit the signal into the cell. In the first step of this thesis we have used all the solved Class A receptors structures to analyze and find, if exist, a common way to transmit the signal inside the cell. We identified and validated ten positions shared between all the binding cavities and always involved in the interaction with ligands. We demonstrated that residues in these positions are conserved and have co-evolved together. In a second step, we used these positions to understand how ligands could be positioned in the binding cavities of three study cases: Muscarinic receptors, Kisspeptin receptors and the GPR3 receptor. We did not have any experimental information a priori. We used homology modeling and docking techniques for the first two cases, adding molecular dynamics simulations in the third case. All the predictions and suggestions from the computational point of view, turned out to be very successful. In particular for the GPR3 receptor we were able to identify and validate by alanine-scanning mutagenesis the role of three functionally relevant residues. The latter were correlated with the constitutive and agonist-stimulated adenylate cyclase activity of GPR3 receptor. Taken together, these results suggest an important role of computational structural biology and pave the way of strong collaborations between computational and experimental researches

Melanokortīnu un purīnu receptoru funkcionalitāte un ģenētika

Darba mērķis bija pētīt funkcionāli raksturīgās pazīmes, kas ir būtiskas receptoru aktivācijā divās GPCR apakšgrupās ar strukturāli atšķirīgiem dabīgo ligandu veidiem, lietojot raugu ekspresijas sistēmu, un pētīt melanokortīnu un purīnu nozīmību multifaktoriālajās slimībās ar ģenētiskās asociācijas metodēm. MC4R 126. pozīcijas un P2Y12R 181., 256., 265. un 280 pozīciju randomās bibliotēkas analīze parādīja, šo pozīciju nozīmību receptora signāla pārnesē. Iegūts arī 3D modelis P2Y12R liganda piesaistes raksturošanai. Ģenētiskā saistība netika atrasta starp P2RY1 lokusu un miokarda infarktu, kā arī ar citiem saistītiem fenotipiem. Būtisku gēnā MC4R nesinonīmo variantu ietekmi uz funkcionalitāti uzrādīja nomaiņas: S127L un oriģināli atklātā - V166I. rs17782313 gēnā MC4R nebija saistīts ar dzīvībai bīstamu aptaukošanos un citiem fenotipiem. Atslēgasvārdi: melanokortīnu receptori, purīnu receptori, aptaukošanās ģenētika, sirdsslimību ģenētika.The aim of the study was to explore functional characteristics that are important for receptor activation of two GPCR subgroups with structurally different natural ligand types using yeast expression system and investigate the implication of melanocortin and purine receptors in multifactorial conditions by genetic association approach. Functional analysis of positions 181, 256, 265 and 280 of P2Y12R and 126 of MC4R randomized libraries proved importance of these residues in receptor signalling. 3D model was obtained, that characterised P2Y12R ligand binding pocket. SNPs of P2RY1 locus were not associated with myocardial infarction and related phenotypes. rs17782313 of MC4R was not associated with morbid obesity and other traits. S127L and originally discovered V166I MC4R variants had significant effect on functional activity of MC4R. Keywords: melanocortin receptors, purinergic receptors, obesity genetics, heart disease genetics

Comparative Sequence and Structural Analyses of G-Protein-Coupled Receptor Crystal Structures and Implications for Molecular Models

BACKGROUND:Up until recently the only available experimental (high resolution) structure of a G-protein-coupled receptor (GPCR) was that of bovine rhodopsin. In the past few years the determination of GPCR structures has accelerated with three new receptors, as well as squid rhodopsin, being successfully crystallized. All share a common molecular architecture of seven transmembrane helices and can therefore serve as templates for building molecular models of homologous GPCRs. However, despite the common general architecture of these structures key differences do exist between them. The choice of which experimental GPCR structure(s) to use for building a comparative model of a particular GPCR is unclear and without detailed structural and sequence analyses, could be arbitrary. The aim of this study is therefore to perform a systematic and detailed analysis of sequence-structure relationships of known GPCR structures. METHODOLOGY:We analyzed in detail conserved and unique sequence motifs and structural features in experimentally-determined GPCR structures. Deeper insight into specific and important structural features of GPCRs as well as valuable information for template selection has been gained. Using key features a workflow has been formulated for identifying the most appropriate template(s) for building homology models of GPCRs of unknown structure. This workflow was applied to a set of 14 human family A GPCRs suggesting for each the most appropriate template(s) for building a comparative molecular model. CONCLUSIONS:The available crystal structures represent only a subset of all possible structural variation in family A GPCRs. Some GPCRs have structural features that are distributed over different crystal structures or which are not present in the templates suggesting that homology models should be built using multiple templates. This study provides a systematic analysis of GPCR crystal structures and a consistent method for identifying suitable templates for GPCR homology modelling that will help to produce more reliable three-dimensional models

Agonist-Promoted Regulation of the P2Y1 Receptor: Quantification of Native and Recombinant Receptors with the Novel Radioligand [32P]MRS2500

The P2Y family of G-protein coupled receptors are activated by adenine and uridine di- and triphosphate nucleotides and nucleotide sugars and are implicated in a wide range of important human physiologies. Difficulty studying these receptors and in their successful manipulation as therapeutic targets has historically derived from a lack of available pharmacological tools that discriminate among members of the P2Y receptor family. The studies described here focus on the P2Y1 receptor, a key mediator of ADP-induced platelet aggregation. Based on the structure of the recently synthesized, high-affinity P2Y1 receptorselective antagonist, 2-iodo-N6-methyl-(N)-methanocarba-2 -deoxyadenosine-3´,5´- bisphosphate (MRS2500), we undertook the development of a high-specific radioactivity radioligand for the P2Y1 receptor, suitable for the study of endogenous receptors in mammalian tissues and cell lines. Using an enzymatic phosphorylation reaction, we successfully generated [32P]MRS2500 with a specific activity of 9120 Ci/mmol. The selectivity and affinity of [32P]MRS2500 for the P2Y1 receptor were confirmed in radioligand binding assays with Sf9 insect cell membranes overexpressing recombinant P2Y1 receptors. The utility of [32P]MRS2500 for the study of endogenous P2Y1 receptors was examined using washed human platelets and membranes prepared from various tissues of the adult rat. We applied this high-specific radioactivity radioligand to observe surface expression of P2Y1 receptor binding sites in human platelets and MDCK(II) epithelial cells following incubation with P2Y1 receptor agonists. In human platelets, the rapid, agonist-promoted desensitization of the P2Y1 receptor observed after incubation with the selective agonist (N)- methanocarba-2-methylthioadenosine-diphosphate (MRS2365) also occurs for the Gqcoupled 5-HT2A receptor after incubation of platelets with 5-hydroxytryptamine (5-HT). The rapid, agonist-promoted desensitization of the P2Y1 receptor of platelets was accompanied by a modest decrease (< 20%) in the number of surface [32P]MRS2500 binding sites and only a partial recovery of P2Y1 receptor responsiveness after removal of the selective agonist MRS2365. Platelets, therefore, appear to employ a unique mechanism for prolonged termination of P2Y1 receptor signaling in which desensitized receptors are maintained at the cell surface, unable to respond to subsequent agonist stimulation. In intact MDCK(II) cells overexpressing recombinant P2Y1 receptors, incubation with 2MeSADP was followed by a 50% loss of surface [32P]MRS2500 binding sites that was agonist-concentration dependent and required the formation of clathrin-coated pits. Mutagenesis studies indicated that this rapid, agonist-promoted loss of surface binding sites requires two serine residues, Ser352 and Ser354, in the receptor carboxyl terminus. The findings presented here indicate that P2Y1 receptor cell surface expression is regulated in an agonist-dependent manner that differs in at least two cell types and suggests an important role for phosphorylation in agonist-dependent desensitization and internalization of the P2Y1 receptor

Developing a recombinant model of the P2Y1 and P2Y11 receptor interactions mediating relaxation in gut smooth muscle

ATP and ADP mediate gut smooth muscle relaxation through two receptors, P2Y1 and P2Y11. This project aims to investigate the interaction between these two receptors by developing a recombinant model of the P2Y receptors expressed in gut smooth muscle cells (SMCs) by transfecting the human P2Y11 receptor cDNA into CHO-K1 cells, which express an endogenous P2Y1 receptor. Individual clonal cell lines expressing different densities of hP2Y11 were isolated from this stably-transfected CHO-K1:P2Y11 pool and characterized. A clone expressing a “high” density of hP2Y11 (13) and a clone expressing a “low” density of hP2Y11 (6) were selected for further study. Control 1321N1 cell lines expressing each receptor in isolation (1321N1-hP2Y1 and 1321N1-hP2Y11) were used for comparison purposes. The potency (EC50) of eight different nucleotide agonists was determined in calcium assays in the co-expressing cell lines. ADP and 2meSATP responses were biphasic in clone 13 but monophasic in clone 6. To investigate the nature of the two sites of the biphasic curves in clone 13, the effect of MRS 2179, NF 340 and Reactive Red on agonist responses was determined. MRS 2179 antagonized the high affinity site of the biphasic ADP and 2meSATP responses in clone 13 without affecting the low affinity site. NF 340 had no effect on agonist responses in clone 13. Reactive Red antagonized both sites of the biphasic curves in clone 13. These data suggest that the high-affinity site of the biphasic ADP and 2meSATP responses in clone 13 corresponds to P2Y1. The low-affinity site of the 2meSATP curve is most likely P2Y11. The low-affinity site the ADP response displays both P2Y1 and P2Y11-like. The novel ADP site, therefore, is elicited by differences in the expression level of P2Y11 and may correspond to a P2Y1:hP2Y11 receptor heteromer or a macromolecular complex containing both P2Y1 and P2Y11