An evaluation of automated in silico ligand docking of amino acid ligands to Family C G-protein coupled receptors

doi:10.1016/j.bmc.2005.10.052

Bioorganic & Medicinal Chemistry

Volume 14, Issue 6, 15 March 2006, Pages 2032-2039

https://doi.org/10.1016/j.bmc.2005.10.052 Get rights and content

Abstract

Family C G-protein coupled receptors (GPCRs) consist of the metabotropic glutamate receptors (mGluRs), the calcium-sensing receptor (CaSR), the T1R taste receptors, the GABA_B receptor, the V2R pheromone receptors, and several chemosensory receptors. A common feature of Family C receptors is the presence of an amino acid binding pocket. The objective of this study was to evaluate the ability of the automatic docking program FlexX to predict the favored amino acid ligand at several Family C GPCRs. The docking process was optimized using the crystal structure of mGluR1 and the 20 amino acids were docked into homology models of the CaSR, the 5.24 chemosensory receptor, and the GPRC6A amino acid receptor. Under optimized docking conditions, glutamate was docked in the binding pocket of mGluR1 with a root mean square deviation of 1.56 angstroms from the co-crystallized glutamate structure and was ranked as the best ligand with a significantly better FlexX score compared to all other amino acids. Ligand docking to a homology model of the 5.24 receptor gave generally correct predictions of the favored amino acids, while the results obtained with models of GPRC6A and the CaSR showed that some of the favored amino acids at these receptors were correctly predicted, while a few other top scoring amino acids appeared to be false positives. We conclude that with certain caveats, FlexX can be successfully used to predict preferred ligands at Family C GPCRs.

Graphical abstract

Amino acids docked into the binding pocket of mGluR1 and scored by FlexX.

Introduction

Family C G-protein coupled receptors (GPCRs) possess large extracellular ligand binding domains which are homologous to the bacteria periplasmic binding proteins.¹ Most Family C receptors possess a conserved binding motif recognizing l-amino acids. Family C receptors include the metabotropic glutamate receptors (mGluRs) which regulate neurotransmission and are therapeutic targets for a number of neurological and psychiatric diseases.¹ Another member of this receptor family, the calcium-sensing receptor (CaSR), plays a critical role in regulating calcium homeostasis in the body. Inactivating and activating mutations in the CaSR have been identified in inherited human hypercalcemic and hypocalcemic disorders, respectively.² Aromatic amino acids can enhance the response of the CaSR to calcium,³ presumably through an amino acid binding pocket analogous to that of the glutamate binding pocket in the mGluRs.4, 5 Other Family C receptors such as the T1R1/T1R3 heteromeric taste receptor can sense most amino acids,⁶ while arginine and lysine are the most potent amino acid ligands for the fish 5.24 chemosensory receptor.⁷ A presumed mammalian homolog of the fish 5.24 receptor, GPRC6A, has been cloned and also shown to be activated by amino acids with preference for positively charged amino acids.8, 9

The availability of crystal structure of the ligand binding domain of mGluR1¹⁰ provides an excellent template to study the molecular determinants of ligand and drug binding to other Family C GPCRs. Based on extensive medicinal chemistry and pharmacophore studies, and molecular modeling and mutagenesis studies, the molecular determinants for glutamate and drug binding to mGluRs have been identified.11, 12, 13, 14 Although the mGluRs share several highly conserved residues in the binding pocket, subtle differences at other sites within pocket confer ligand and drug selectivity. The mode of glutamate binding in the mGluRs has shed light on ligand docking at other Family C GPCRs. For example, the 5.24 receptor was found to bind lysine and arginine in a manner similar to the mode of glutamate binding in the mGluRs.15, 16

Structure-based drug design has shown promise in reducing the time and money spent in the drug discovery process.17, 18 Automatic docking using computer programs to identify small molecule receptor ligands plays an increasingly important role in the drug discovery process.¹⁹ Although homology models are not as good as experimentally determined structures for docking applications, the use of homology models in ligand screening is gaining momentum.²⁰ The effective use of homology models in drug screening usually requires greater than 50% sequence identity between the target and the template.²¹ Therefore, the lower sequence identities among members of the Family C GPCRs to the only available template mGluR1 (36–42% for mGluRs, except for 69% for mGluR5, and 20–30% for non-mGluR Family C receptors) have resulted in the use of primarily manual docking of ligands and drugs.12, 13, 15, 16 Recently, however, several studies employing automated ligand docking and screening using homology models of mGluRs have been reported.22, 23, 24

Here we report our experience in using FlexX²⁵ to dock amino acids in homology models of the CaSR, the 5.24 receptor, and GPRC6A. We evaluated the ability of this program to correctly predict the rank order of amino acids at each of these receptors. By comparing the in silico docking score with experimental data from the literature, we found that the FlexX scoring functions can be used to discriminate and predict ligand selectivity.

Section snippets

Homology modeling

Homology models of the CaSR, GPRC6A, and the 5.24 receptor were constructed and refined in parallel. The amino acids aligned in the binding pockets are shown in Table 1. The residues in the binding pocket that interact with the glycine moiety (the α-carboxyl group and α-amino group of the bound amino acid ligand) are highly conserved in Family C receptors.1, 16 In contrast, the amino acids that interact with the side chains of the bound amino acid ligands are not conserved and are responsible

Optimization of the docking parameters

Among the ligand–receptor docking programs available, FlexX,²⁵ Dock,²⁶ Gold,³¹ and Glide³² are the most widely used. The performances of these programs have varied depending on the system investigated, and the intrinsic physicochemical properties of the ligand–protein interactions involved.32, 33, 34 Thus, optimization of the individual docking system and its parameters is important in drug and ligand docking studies. In the present study, the optimization of FlexX in Sybyl was performed by

Molecular modeling

Homology models of the fish 5.24 receptor, the mouse CaSR, and mouse GPRC6A were generated using the X-ray crystal structure of the extracellular domain of rat mGluR1 as the template (PDB coordinates, 1EWK¹⁰) and version 6.0 of the MODELER program³⁸ to generate the models. The sequences of the receptors were aligned using ClustalX and Sybyl 6.9 (Tripos Inc., St. Louis, MO) was used to view, analyze, and manipulate the structure. The preferred highest affinity ligands (arginine for fish 5.24

Acknowledgments

We thank Dr. L. P. Kotra for insightful comments on the manuscript. This work was supported by grants from the Canadian Institutes for Health Research, and the Natural Sciences and Engineering Research Council of Canada, and by the resources of the Molecular Design and Information Technology Centre in the Faculty of Pharmacy, University of Toronto.

References and notes (45)

J.P. Pin et al.
Pharmacol. Ther.
(2003)
E.M. Brown
Am. J. Med.
(1999)
Z. Zhang et al.
J. Biol. Chem.
(2002)
H.C. Mun et al.
J. Biol. Chem.
(2005)
D.J. Speca et al.
Neuron
(1999)
S.J. Conway et al.
Bioorg. Med. Chem. Lett.
(2001)
E. Rosemond et al.
J. Biol. Chem.
(2002)
D. Kuang et al.
J. Biol. Chem.
(2003)
A.C. Anderson
Chem. Biol.
(2003)
A. Hillisch et al.
Drug Discov. Today
(2004)

M.B. Hermit et al.

J. Biol. Chem.

(2004)

M. Rarey et al.

J. Mol. Biol.

(1996)

I.D. Kuntz et al.

J. Mol. Biol.

(1982)

C. Silve et al.

J. Biol. Chem.

(2005)

G. Jones et al.

J. Mol. Biol.

(1997)

A. Sali et al.

J. Mol. Biol.

(1993)

D.A. Pearlman et al.

Comput. Phys. Commun.

(1995)

J.-P. Ryckaert et al.

J Comput. Phys.

(1977)

A.D. Conigrave et al.

Proc. Natl. Acad. Sci. U.S.A.

(2000)

X. Li et al.

Proc. Natl. Acad. Sci. U.S.A.

(2002)

P. Wellendorph et al.

Mol. Pharmacol.

(2005)

D. Kuang et al.

J. Neurochem.

(2005)

Cited by (19)

Study on the anti-inflammatory activity of the porcine bone collagen peptides prepared by ultrasound-assisted enzymatic hydrolysis
2023, Ultrasonics Sonochemistry
In this study, the effects of ultrasound-assisted enzymatic hydrolysis on the extraction of anti-inflammatory peptides from porcine bone collagen were investigated. The results showed that ultrasound treatment increased the content of α-helix while decreased β-chain and random coil, promoted generation of small molecular peptides. Ultrasound-assisted enzymatic hydrolysis improved the peptide content, enhanced ABTS⁺ radical scavenging and ferrous ion chelating ability than non-ultrasound group. At the ultrasonic power of 450 W (20 min), peptides possessed significant anti-inflammatory activity, where the releasing of interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) was all suppressed in lipopolysaccharide (LPS) induced RAW264.7 cells. After the analysis with LC-MS/MS, eight peptides with potential anti-inflammatory activities were selected by the PeptideRanker and molecular docking. In general, the ultrasound-assisted enzymatic hydrolysis was an effective strategy to extract the bioactive peptides from porcine bone, and the inflammatory regulation capacity of bone collagen sourced peptides was firstly demonstrated.
Allosteric modulation of the calcium-sensing receptor by γ-glutamyl peptides: Inhibition of pth secretion, suppression of intracellular cAMP levels, and a common mechanism of action with L-amino acids
2011, Journal of Biological Chemistry
Citation Excerpt :
Chimeric receptor analysis previously demonstrated that the binding sites for l-amino acids (14) and glutathione (21) both localize to the receptor VFT domain. To investigate whether these two classes of positive allosteric modulator might bind at a common or overlapping site and/or act via a common mechanism, as suggested by molecular modeling analysis (18), we took advantage of a previously identified double mutant of the CaR VFT domain (T145A/S170T) that selectively disables l-amino acid sensing with little or no effect on Cao2+-sensing (15). This mutant disabled the effects of l-Phe as well as S-methylglutathione, on Ca2+i mobilization (Figs. 5–8) as well as suppression of forskolin-elevated cAMP levels in CaR-expressing HEK-293 cells (Fig. 9).
γ-Glutamyl peptides were identified previously as novel positive allosteric modulators of Ca²⁺_o-dependent intracellular Ca²⁺ mobilization in HEK-293 cells that bind in the calcium-sensing receptor VFT domain. In the current study, we investigated whether γ-glutamyl-tripeptides including γ-Glu-Cys-Gly (glutathione) and its analogs S-methylglutathione and S-propylglutathione, or dipeptides including γ-Glu-Ala and γ-Glu-Cys are positive allosteric modulators of Ca²⁺_o-dependent Ca²⁺_i mobilization and PTH secretion from normal human parathyroid cells as well as Ca²⁺_o-dependent suppression of intracellular cAMP levels in calcium-sensing receptor (CaR)-expressing HEK-293 cells. In addition, we compared the effects of the potent γ-glutamyl peptide S-methylglutathione, and the amino acid l-Phe on HEK-293 cells that stably expressed either the wild-type CaR or the double mutant T145A/S170T, which exhibits selectively impaired responses to l-amino acids. We find that γ-glutamyl peptides are potent positive allosteric modulators of the CaR that promote Ca²⁺_o-dependent Ca²⁺_i mobilization, suppress intracellular cAMP levels and inhibit PTH secretion from normal human parathyroid cells. Furthermore, we find that the double mutant T145A/S170T exhibits markedly impaired Ca²⁺_i mobilization and cAMP suppression responses to S-methylglutathione as well as l-Phe indicating that γ-glutamyl peptides and l-amino acids activate the CaR via a common mechanism.
Broad-spectrum amino acid-sensing class C G-protein coupled receptors: Molecular mechanisms, physiological significance and options for drug development
2010, Pharmacology and Therapeutics
In this article, we consider the molecular mechanisms that underlie broad-spectrum amino acid sensing by a discrete subgroup of class C G-protein-coupled receptors that includes the calcium-sensing receptor, GPRC6A and heterodimers composed of two closely related receptor subunits, T1R₁ and T1R₃. We consider their physiological significance highlighting their diverse spectrum of cellular responses and the phenotypes of global and conditional knock-out mice. In addition, we consider strategies for the development of new drugs that target these receptors.
Evidence for the presence of GPRC6A receptors in rat mesenteric arteries
2008, Cell Calcium
In this study, the presence of GPRC6A receptors in rat mesenteric artery was investigated. In artery homogenates, GPRC6A mRNA was detected and Western blotting showed the presence of GPRC6A protein. Immunohistochemical studies revealed GPRC6A in both endothelial cells and myocytes. In whole vessel segments, the GPRC6A activators, 300 μM l-ornithine and 100 μM Al³⁺, induced endothelium-dependent myocyte hyperpolarizations sensitive to 10 μM TRAM-34, a blocker of intermediate conductance, Ca²⁺-sensitive K⁺ channels (IK_Ca). Activation of IK_Ca with calindol (300 nM; a positive allosteric Ca²⁺-sensing receptor – CaR – modulator) was inhibited by 500 nM ouabain (inhibition of rat type 2 and type 3 Na⁺/K⁺-ATPases) but unaffected by 30 μM Ba²⁺ (blockade of inwardly rectifying K⁺ channels). Neither l-ornithine nor Al³⁺ activated CaRs heterologously expressed in CHO or HEK293 cells. In the presence of 300 μM l-ornithine or 100 μM Al³⁺, myocyte hyperpolarizations to calindol were potentiated whereas this potentiation and hyperpolarizations to l-ornithine were lost following incubation with an anti-GPRC6A antibody. It is concluded that GPRC6A receptors are present on mesenteric artery endothelial cells and myocytes and that their activation selectively opens IK_Ca channels. This triggers a ouabain-sensitive myocyte hyperpolarization suggesting a close functional relationship between GPRC6A, the IK_Ca channel and type 2 and/or type 3 Na⁺/K⁺-ATPases.
Extracellular calcium-sensing receptors in fishes
2008, Comparative Biochemistry and Physiology - A Molecular and Integrative Physiology
The extracellular calcium-sensing receptor (CaSR) in fishes, like the CaSRs of tetrapod vertebrates, is a dimeric seven transmembrane, G protein-coupled receptor. The receptor is expressed on the plasma membranes of a variety of tissues and cells where it functions as a sensor of extracellular calcium concentration ([Ca²⁺]_o) in the physiological range. In the context of systemic calcium homeostasis, CaSR expressed in endocrine tissues that secrete calciotropic and other hormones (pituitary gland and corpuscles of Stannius) may play a central role in global integrative signaling, whereas receptor expressed in ion-transporting tissues (kidney, intestine, gills, and elasmobranch rectal gland) may have local direct effects on monovalent and divalent ion transport that are independent of endocrine signaling. In fishes, specifically, CaSR expression at the body surface (at the gills and olfactory tissues, for example) may permit direct sensing of environmental Ca²⁺ and Mg²⁺ concentrations, especially in the marine environment. Additionally, CaSRs may have other widespread and diverse roles in extracellular Ca²⁺ sensing related both to organismal calcium homeostasis and to intercellular Ca²⁺ signaling. As a consequence of the broad spectrum of recognized ligands, including polyvalent cations and amino acids, and of binding site shielding by monovalent cations, additional receptor functionalities related to salinity and nutrient detection are proposed for CaSRs. CaSR expression in the gastrointestinal tract may be multifunctional as a sensor for polyvalent cations and amino acids. Structural and phylogenetic analyses reveal strongly conserved features among CaSRs, and suggest that calcium sensing by mammalian parathyroid gland-type CaSR proteins may be restricted to chordates. Comparative functional and genomic studies that include piscine CaSRs can be useful model systems for testing existing hypotheses regarding receptor function, and will shed light on the evolutionary developmental history of calcium homeostasis in the vertebrates.
Broad-spectrum l-amino acid sensing by class 3 G-protein-coupled receptors
2006, Trends in Endocrinology and Metabolism
The sensing of nutrients is essential to the control of growth and metabolism. Although the sensing mechanisms responsible for the detection and coordination of metabolic responses to some nutrients, most notably glucose, are well understood, the molecular basis of amino acid sensing by cells and tissues is only now emerging. In this article, we consider evidence that some members of G-protein-coupled receptor class 3 are broad-spectrum amino acid sensors that couple changes in extracellular amino acid levels to the activation of intracellular signaling pathways. In particular, we consider both the molecular basis of specific and broad-spectrum amino acid sensing by different members of class 3 and the physiological significance of broad spectrum amino acid sensing by the extracellular calcium-sensing receptor, heterodimeric taste receptors and the recently ‘deorphanized’ receptor GPRC6A and its goldfish homolog, the 5.24 chemoreceptor.

View all citing articles on Scopus

View full text

An evaluation of automated in silico ligand docking of amino acid ligands to Family C G-protein coupled receptors

Abstract

Graphical abstract

Introduction

Section snippets

Homology modeling

Optimization of the docking parameters

Molecular modeling

Acknowledgments

Pharmacol. Ther.

Am. J. Med.

J. Biol. Chem.

J. Biol. Chem.

Neuron

Bioorg. Med. Chem. Lett.

J. Biol. Chem.

J. Biol. Chem.

Chem. Biol.

Drug Discov. Today

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

Comput. Phys. Commun.

J Comput. Phys.

Proc. Natl. Acad. Sci. U.S.A.

Proc. Natl. Acad. Sci. U.S.A.

Mol. Pharmacol.

J. Neurochem.