Ligand-guided homology modeling drives identification of novel histamine H3 receptor ligands

In this study, we report a ligand-guided homology modeling approach allowing the analysis of relevant binding site residue conformations and the identification of two novel histamine H3 receptor ligands with binding affinity in the nanomolar range. The newly developed method is based on exploiting an essential charge interaction characteristic for aminergic G-protein coupled receptors for ranking 3D receptor models appropriate for the discovery of novel compounds through virtual screening

Using molecular dynamics for the refinement of atomistic models of GPCRs by homology modeling

Despite GPCRs sharing a common seven helix bundle, analysis of the diverse crystallographic structures available reveal specific features that might be relevant for ligand design. Despite the number of crystallographic structures of GPCRs steadily increasing, there are still challenges that hamper the availability of new structures. In the absence of a crystallographic structure, homology modeling remains one of the important techniques for constructing 3D models of proteins. In the present study we investigated the use of molecular dynamics simulations for the refinement of GPCRs models constructed by homology modeling. Specifically, we investigated the relevance of template selection, ligand inclusion as well as the length of the simulation on the quality of the GPCRs models constructed. For this purpose we chose the crystallographic structure of the rat muscarinic M3 receptor as reference and constructed diverse atomistic models by homology modeling, using different templates. Specifically, templates used in the present work include the human muscarinic M2; the more distant human histamine H1 and the even more distant bovine rhodopsin as shown in the GPCRs phylogenetic tree. We also investigated the use or not of a ligand in the refinement process. Hence, we conducted the refinement process of the M3 model using the M2 muscarinic as template with tiotropium or NMS docked in the orthosteric site and compared with the results obtained with a model refined without any ligand bound.Peer ReviewedPostprint (author's final draft

Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC) and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS). Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand

A constitutively active G protein-coupled acetylcholine receptor regulates motility of larval Schistosoma mansoni

The neuromuscular system of helminths controls a variety of essential biological processes and therefore represents a good source of novel drug targets. The neuroactive substance, acetylcholine controls movement of Schistosoma mansoni but the mode of action is poorly understood. Here, we present first evidence of a functional G protein-coupled acetylcholine receptor in S. mansoni, which we have named SmGAR. A bioinformatics analysis indicated that SmGAR belongs to a clade of invertebrate GAR-like receptors and is related to vertebrate muscarinic acetylcholine receptors. Functional expression studies in yeast showed that SmGAR is constitutively active but can be further activated by acetylcholine and, to a lesser extent, the cholinergic agonist, carbachol. Anti-cholinergic drugs, atropine and promethazine, were found to have inverse agonist activity towards SmGAR, causing a significant decrease in the receptor’s basal activity. An RNAi phenotypic assay revealed that suppression of SmGAR activity in early-stage larval schistosomulae leads to a drastic reduction in larval motility. In sum, our results provide the first molecular evidence that cholinergic GAR -like receptors are present in schistosomes and are required for proper motor control in the larvae. The results further identify SmGAR as a possible candidate for antiparasitic drug targeting

Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

When it is difficult to develop selective ligands within a family of related G-protein-coupled receptors (GPCRs), chemically engineered receptors activated solely by synthetic ligands (RASSLs) are useful alternatives for probing receptor function. In the present work, we explored whether a RASSL of the free fatty acid receptor 2 (FFA2) could be developed on the basis of pharmacological variation between species orthologs. For this, bovine FFA2 was characterized, revealing distinct ligand selectivity compared with human FFA2. Homology modeling and mutational analysis demonstrated a single mutation in human FFA2 of C4.57G resulted in a human FFA2 receptor with ligand selectivity similar to the bovine receptor. This was exploited to generate human FFA2-RASSL by the addition of a second mutation at a known orthosteric ligand interaction site, H6.55Q. The resulting FFA2-RASSL displayed a >100-fold loss of activity to endogenous ligands, while responding to the distinct ligand sorbic acid with pEC(50) values for inhibition of cAMP, 5.83 ± 0.11; Ca(2+) mobilization, 4.63 ± 0.05; ERK phosphorylation, 5.61 ± 0.06; and dynamic mass redistribution, 5.35 ± 0.06. This FFA2-RASSL will be useful in future studies on this receptor and demonstrates that exploitation of pharmacological variation between species orthologs is a powerful method to generate novel chemically engineered GPCRs

Multiple Residues in the Second Extracellular Loop Are Critical for M3 Muscarinic Acetylcholine Receptor Activation

Recent studies suggest that the second extracellular loop (o2 loop) of bovine rhodopsin and other class I G protein-coupled receptors (GPCRs) targeted by biogenic amine ligands folds deeply into the transmembrane receptor core where the binding of cis-retinal and biogenic amine ligands is known to occur. In the past, the potential role of the o2 loop in agonist-dependent activation of biogenic amine GPCRs has not been studied systematically. To address this issue, we used the M3 muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR, as a model system. Specifically, we subjected the o2 loop of the M3R to random mutagenesis and subsequently applied a novel yeast genetic screen to identity single amino acid substitutions that interfered with M3R function. This screen led to the recovery of about 20 mutant M3Rs containing single amino acid changes in the o2 loop that were inactive in yeast. In contrast, application of the same strategy to the extracellular N-terminal domain of the M3R did not yield any single point mutations that disrupted M3R function. Pharmacological characterization of many of the recovered mutant M3Rs in mammalian cells, complemented by site-directed mutagenesis studies, indicated that the presence of several o2 loop residues is important for efficient agonist-induced M3R activation. Besides the highly conserved Cys220 residue, Gln207, Gly211, Arg213, Gly218, Ile222, Phe224, Leu225, and Pro228 were found to be of particular functional importance. In general, mutational modification of these residues had little effect on agonist binding affinities. Our findings are therefore consistent with a model in which multiple o2 loop residues are involved in stabilizing the active state of the M3R. Given the high degree of structural homology found among all biogenic amine GPCRs, our findings should be of considerable general relevance

Structural and functional characterisation of neuronal Gq protein coupled receptors.

Both G protein coupled receptor 10 (GPR10) and the muscarinic IVh receptor are members of family A of the G protein coupled receptor superfamily. GPR10 is the human orthologue of a former orphan receptor for which prolactin releasing peptides (PrRP) have been identified as the cognate ligands. In contrast, the N/h receptor represents a well-established receptor for which up until the discovery of AC-42 (4-(4-butyl-1-piperidinyl)-1-(2-methylphenyl)-1-butanone) it has been difficult to identify a selective agonist. Little is known of the pharmacology of PrRPs and AC-42 at GPR10 and the human Mi (hMi) receptor, respectively. Similarly, the molecular nature of their respective binding sites is as yet unknown. In the studies in this thesis, the interaction of PrRPs with GPR10 and of AC-42 (and other ectopic agonists) with the hMi receptor have been extensively characterised using a range of pharmacological techniques in both recombinant cell lines stably and transiently expressing the receptor(s) of interest and native tissue preparations. Furthermore, the molecular interactions between ligand and receptor have been probed using homology modelling, site-directed mutagenesis (SDM) and pharmacological evaluation. The results generated reveal that PrRPs are high affinity, potent agonists at GPR10 that cause the receptor to activate effector systems that are known to couple to Gq/n proteins. Radioligand binding studies suggest both high and low affinity sites for binding. In addition, homology modelling and SDM have been used to reveal key interactions of the C-terminal region of PrRP with transmembrane domain (TM) 6 (D302) and TM7 (Q317) and extracellular loop (ECL) 2 (E213). Selective activation of the Mi receptor can be achieved using AC-42 and other novel ligands. Using functional calcium mobilisation assays, inositol phosphate assays and radioligand binding studies it has been possible to demonstrate that this class of compounds interacts with the receptor in an allosteric manner. SDM studies also suggest that residues distinct from the orthosteric binding site form part of the binding site for AC-42 and related compounds. These studies provide the first extensive pharmacological analysis characterising the interaction of PrRP and GPR10 and identify the signal transduction cascade activated by this former orphan receptor. Furthermore, SDM studies have partly elucidated the molecular nature of the PrRP binding site. Both pharmacological and SDM based examination of the muscarinic Mi receptor have revealed a unique allosteric method of activation by AC-42 and a novel class of allosteric agonists

Effect of the ligand in the Modeling the 3d structure of the m3 muscarinic receptor

There are still many question open to completely understand the structure-activity relationships of G-protein coupled receptors. Issues like the actual mapping of the binding site of different subtypes, as well as the mechanism of activation are poorly understood [1]. Accordingly, further studies on the structure-activity relationships are necessary. In this regard, only a few 3D structures are available from X-ray diffraction studies. Computational studies can complement this information through the construction of reliable 3D models. The goal of the present work is to evaluate the effect of using different ligands to obtain reliable models of the three-dimensional structure of a G-protein coupled receptor using a specific template. Specifically, we have constructed in the present work a three dimensional model of the M3 muscarinic receptor by homology modelling, using the X-ray structure of M2 muscarinic acetylcholine receptor as template and the sequence analyses of muscarinic acetylcholine receptor family. Furthermore, we have studied the effect of the ligand used in the modelling process. For this purpose three models of the receptor were built, including one without ligand and two models with the selective antagonists tiotropium and N-Methylscopolamine, respectively docked into the orthosteric binding site. The constructed models were refined using molecular dynamic calculations to analyze the effect of ligand refinement process and to derive significant conformational information. Based on the analysis of the refined models done through the calculations of RMSD, RMSF, visualization of the structures and comparison between the refined models and the crystal structure of M3 muscarinic receptor, the addition of a ligand in construction of homology model (and the subsequent refinement process) stabilize the structure. Furthermore, the similarities in the structure conformations of both refined models of M3 muscarinic-ligand complexes and the crystal structure of the M3 muscarinic receptors, suggest that the methodology used in this study can be used in prediction of 3D structure prediction GPCR in the absence of crystal structures

The therapeutic potential of allosteric ligands for free fatty acid sensitive GPCRs

G protein coupled receptors (GPCRs) are the most historically successful therapeutic targets. Despite this success there are many important aspects of GPCR pharmacology and function that have yet to be exploited to their full therapeutic potential. One in particular that has been gaining attention in recent times is that of GPCR ligands that bind to allosteric sites on the receptor distinct from the orthosteric site of the endogenous ligand. As therapeutics, allosteric ligands possess many theoretical advantages over their orthosteric counterparts, including more complex modes of action, improved safety, more physiologically appropriate responses, better target selectivity, and reduced likelihood of desensitisation and tachyphylaxis. Despite these advantages, the development of allosteric ligands is often difficult from a medicinal chemistry standpoint due to the more complex challenge of identifying allosteric leads and their often flat or confusing SAR. The present review will consider the advantages and challenges associated with allosteric GPCR ligands, and examine how the particular properties of these ligands may be exploited to uncover the therapeutic potential for free fatty acid sensitive GPCRs