Rhodopsin-based Homology Model Research Articles

Carbon chain shape selectivity by the mouse olfactory receptor OR-I7.

The rodent OR-I7 is an olfactory receptor exemplar activated by aliphatic aldehydes such as octanal. Normal alkanals shorter than heptanal bind OR-I7 without activating it and hence function as antagonists in vitro. We report a series of aldehydes designed to probe the structural requirements for aliphatic ligand chains too short to meet the minimum approximate 6.9 Å length requirement for receptor activation. Experiments using recombinant mouse OR-I7 expressed in heterologous cells show that in the context of short aldehyde antagonists, OR-I7 prefers binding aliphatic chains without branches, though a single methyl on carbon-3 is permitted. The receptor can accommodate a surprisingly large number of carbons (e.g. ten in adamantyl) as long as the carbons are part of a conformationally constrained ring system. A rhodopsin-based homology model of mouse OR-I7 docked with the new antagonists suggests that small alkyl branches on the alkyl chain sterically interfere with the hydrophobic residues lining the binding site, but branch carbons can be accommodated when tied back into a compact ring system like the adamantyl and bicyclo[2.2.2]octyl systems.

Impact of the CXCR4 structure on docking-based virtual screening of HIV entry inhibitors

Herein we analyze in depth the receptor-based virtual screening (VS) performance of the five recent crystallized CXCR4 structures along with a CXCR4 rhodopsin-based homology model. All CXCR4 Protein Data Bank (PDB) structures are co-crystallized with a small organic antagonist except structure 3OE0, which is co-crystallized with a cyclic peptide analog. Evaluation of the CXCR4 models was done by retrospective docking-based VS using a test set of 248 known CXCR4 inhibitors from 4 different chemotype families and 4696 different presumed inactives. The performance of the docking protocol using the five different protein structures was assessed in terms of pose prediction and hits detection using 12 different docking scoring functions and a scoring function with rescoring. Results show that 3OE6 structure achieves the highest docking-based performance with an average area under the curve (aAUC) of 0.84 and an average enrichment factor (aEF) of 11.7 at 1% of decoys screened. CXCR4 rhodopsin-like homology model performs comparable to the crystallized structures in the 1% of database screened. Moreover, a detailed analysis of the retrospective docking results using the CXCR4 homology model in Discovery Studio allows us to hypothesize the existence of multiple binding sub-sites in CXCR4 binding pocket.

Role of the Transmembrane Domain 4/Extracellular Loop 2 Junction of the Human Gonadotropin-releasing Hormone Receptor in Ligand Binding and Receptor Conformational Selection

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.

Molecular Modeling Studies on the Human Neuropeptide S Receptor and Its Antagonists

Neuropeptide S (NPS) is a 20-residue peptide of great interest due to its potential involvement in several biological processes such as arousal, anxiety, and food intake. The NPS receptor belongs to the rhodopsin-like G-protein-coupled receptor superfamily, and several polymorphisms and isoforms of this receptor are associated with asthma, allergies, and bronchial hyper-responsiveness, in particular the Asn 107 Ile mutation. Limited structural information is available for this peptide-receptor system, particularly regarding the NPS receptor structure, its nonpeptide ligands, and the molecular aspects of agonist and antagonist binding processes. In this work, rhodopsin-based homology models of the NPS receptor and its Asn 107 Ile variant were built and refined in a membrane bilayer model, and binding modes for nonpeptide antagonists were simulated. This study provides the first structural study of the human NPS receptor, and the results provide a starting point for further characterization of the binding modes of its antagonists, and for the rational design of new NPS receptor ligands.

Human A3 Adenosine Receptor as Versatile G Protein-Coupled Receptor Example to Validate the Receptor Homology Modeling Technology

The development of ligands for the A(3) adenosine receptor (AR) has been directed mainly by traditional medicinal chemistry, but the influence of structure-based approaches is increasing. Rhodopsin-based homology modeling had been used for many years to obtain three-dimensional models of the A(3)AR, and different A(3)AR models have been published describing the hypothetical interactions with known A(3)AR ligands having different chemical scaffolds. The recently published structure of the human A(2A)AR provides a new template for GPCR modeling, however even use of the A(2A)AR as a template for modeling other AR subtypes is still imprecise. The models compared here are based on bovine rhodopsin, the human beta(2)-adrenergic receptor, and the A(2A)AR as templates. The sequence of the human A(3)AR contains only one cysteine residue (Cys166) in the second extracellular loop (EL2), which putatively forms a conserved disulfide bridge with the respective cysteine residues of TM3 (Cys83). Homology models of the A(3)AR have been helpful in providing structural hypotheses for the design of new ligands. Site-directed mutagenesis of the A(3)AR shows an important role in ligand recognition for specific residues in TM3, TM6 and TM7.

Novel potent and highly selective human A 3 adenosine receptor antagonists belonging to the 4-amido-2-arylpyrazolo[3,4- c]quinoline series: Molecular docking analysis and pharmacological studies

The study of novel 2-arylpyrazolo[3,4- c]quinolin-4-(hetero)arylamides, designed as human (h) A 3 adenosine receptor antagonists, is reported. The new derivatives are endowed with nanomolar hA 3 receptor affinity and high selectivity versus hA 1, hA 2A and hA 2B receptors. Among the (hetero)aroyl residues introduced on the 4-amino group, the 2-furyl and 4-pyridyl rings turned out to be the most beneficial for hA 3 affinity ( K i = 3.4 and 5.0 nM, respectively). An intensive molecular docking study to a rhodopsin-based homology model of the hA 3 receptor was carried out to obtain a ‘structure-based pharmacophore model’ that proved to be helpful for the interpretation of the observed affinities of the new hA 3 pyrazoloquinoline antagonists.

Delineation of Agonist Binding to the Human Histamine H4 Receptor Using Mutational Analysis, Homology Modeling, and ab Initio Calculations

A three-dimensional homology model of the human histamine H 4 receptor was developed to investigate the binding mode of a series of structurally diverse H 4-agonists, i.e. histamine, clozapine, and the recently described selective, nonimidazole agonist VUF 8430. Mutagenesis studies and docking of these ligands in a rhodopsin-based homology model revealed two essential points of interactions in the binding pocket, i.e. Asp3.32 and Glu5.46 (Ballesteros-Weinstein numbering system). It is postulated that Asp3.32 interacts in its anionic state, whereas Glu5.46 interacts in its neutral form. The hypothesis was tested with the point mutations D3.32N and E5.46Q. For the D3.32N no binding affinity toward any of the ligands could be detected. This is in sharp contrast to the E5.46Q mutant, which discriminates between various ligands. The affinity of histamine-like ligands was decreased approximately a 1000-fold, whereas the affinity of all other ligands remained virtually unchanged. The proposed model for agonist binding as well as ab initio calculations for histamine and VUF 8430 explain the observed differences in binding to the H 4R mutants. These studies provide a molecular understanding for the action of a variety of H 4 receptor-ligands. The resulting H 4 receptor model will be the basis for the development of new H 4 receptor-ligands.

On the applicability of GPCR homology models to computer-aided drug discovery: a comparison between in silico and crystal structures of the beta2-adrenergic receptor.

The publication of the crystal structure of the beta2-adrenergic receptor (beta2-AR) proved that G protein-coupled receptors (GPCRs) share a structurally conserved rhodopsin-like 7TM core. Here, to probe to which extent realistic GPCR structures can be recreated through modeling, carazolol was docked at two rhodopsin-based homology models of the human beta 2-AR. The first featured a rhodopsin-like second extracellular loop, which interfered with ligand docking and with the orientation of several residues in the binding pocket. The second featured a second extracellular loop built completely de novo, which afforded a more accurate model of the binding pocket and a better docking of the ligand. Furthermore, incorporating available biochemical and computational data to the model by correcting the conformation of a single residue lining the binding pocket --Phe290(6.52)--, resulted in significantly improved docking poses. These results support the applicability of GPCR modeling to the design of site-directed mutagenesis experiments and to drug discovery.

Functional Analysis of Transmembrane Domain 2 of the M1 Muscarinic Acetylcholine Receptor

Ala substitution scanning mutagenesis has been used to probe the functional role of amino acids in transmembrane (TM) domain 2 of the M1 muscarinic acetylcholine receptor, and of the highly conserved Asn43 in TM1. The mutation of Asn43, Asn61, and Leu64 caused an enhanced ACh affinity phenotype. Interpreted using a rhodopsin-based homology model, these results suggest the presence of a network of specific contacts between this group of residues and Pro415 and Tyr418 in the highly conserved NPXXY motif in TM7 that exhibit a similar mutagenic phenotype. These contacts may be rearranged or broken when ACh binds. D71A, like N414A, was devoid of signaling activity. We suggest that formation of a direct hydrogen bond between the highly conserved side chains of Asp71 and Asn414 may be a critical feature stabilizing the activated state of the M1 receptor. Mutation of Leu67, Ala70, and Ile74 also reduced the signaling efficacy of the ACh-receptor complex. The side chains of these residues are modeled as an extended surface that may help to orient and insulate the proposed hydrogen bond between Asp71 and Asn414. Mutation of Leu72, Gly75, and Met79 in the outer half of TM2 primarily reduced the expression of functional receptor binding sites. These residues may mediate contacts with TM1 and TM7 that are preserved throughout the receptor activation cycle. Thermal inactivation measurements confirmed that a reduction in structural stability followed the mutation of Met79 as well as Asp71.

Phenotypic classification of mutants: a tool for understanding ligand binding and activation of muscarinic acetylcholine receptors

GPCRs (G-protein-coupled receptors) such as the M(1) muscarinic receptor have so far proved recalcitrant to direct structure determination. Nevertheless systematic mutagenesis, particularly alanine scanning, has advanced our understanding of their structure-function relationships. GPCRs exhibit multiple conformational states with different affinities for and abilities to activate their cognate G-proteins. Ligand binding alters these conformational equilibria, thus promoting or inhibiting signalling. Alanine-scanning mutagenesis probes the relative contributions of a particular amino acid side chain to the stability of the ground and activated states of the receptor and its complexes. These determine the phenotype of the mutant receptor. Classification of the phenotypes suggests functional roles for particular amino acid side chains, allowing us to group them accordingly. From a rhodopsin-based homology model of the M(1) mAChR, a coherent view emerges of how these clusters of residues function in ligand anchoring, transduction of binding energy, global structural stabilization and selective stabilization of the ground state or the activated state of the receptor. We can identify differences in ligand-binding modes, and suggest inter- and intra-molecular interactions that are weakened or broken, or formed or intensified during acetylcholine-induced activation. In due course, we may be able to extend these insights to activation by unconventional agonists.

Molecular modeling of the human P2Y2 receptor and design of a selective agonist, 2'-amino-2'-deoxy-2-thiouridine 5'-triphosphate.

A rhodopsin-based homology model of the nucleotide-activated human P2Y2 receptor, including loops, termini, and phospholipids, was optimized with the Monte Carlo multiple minimum conformational search routine. Docked uridine 5'-triphosphate (UTP) formed a nucleobase pi-pi complex with conserved Phe3.32. Selectivity-enhancing 2'-amino-2'-deoxy substitution interacted through pi-hydrogen-bonding with aromatic Phe6.51 and Tyr3.33. A "sequential ligand composition" approach for docking the flexible dinucleotide agonist Up4U demonstrated a shift of conserved cationic Arg3.29 from the UTP gamma position to the delta position of Up4U and Up4 ribose. Synthesized nucleotides were tested as agonists at human P2Y receptors expressed in 1321N1 astrocytoma cells. 2'-Amino and 2-thio modifications were synergized to enhance potency and selectivity; compound 8 (EC50 = 8 nM) was 300-fold P2Y2-selective versus P2Y4. 2'-Amine acetylation reduced potency, and trifluoroacetylation produced intermediate potency. 5-Amino nucleobase substitution did not enhance P2Y2 potency through a predicted hydrophilic interaction possibly because of destabilization of the receptor-favored Northern conformation of ribose. This detailed view of P2Y2 receptor recognition suggests mutations for model validation.

Molecular dynamics simulation of the P2Y 14 receptor. Ligand docking and identification of a putative binding site of the distal hexose moiety

A rhodopsin-based homology model of the P2Y 14 receptor was inserted into a phospholipid bilayer and refined by molecular dynamics (MD) simulation. The binding modes of several known agonists, namely UDP-glucose and its analogues, were proposed using automatic molecular docking combined with Monte Carlo Multiple Minimum calculations. Compared to other P2Y receptors, the P2Y 14 receptor has an atypical binding mode of the nucleobase, ribose, and phosphate moieties. The diphosphate moiety interacts with only one cationic residue, namely Lys171 of EL2, while in other P2Y receptor subtypes three Arg or Lys residues interact with the phosphate chain. Two other conserved cationic residues, namely Arg253 (6.55) and Lys277 (7.35) of the P2Y 14 receptor together with two anionic residues (Glu166 and Glu174, located in EL2), are likely involved in interactions with the distal hexose moiety.

Defining the nucleotide binding sites of P2Y receptors using rhodopsin-based homology modeling

Ongoing efforts to model P2Y receptors for extracellular nucleotides, i.e., endogenous ADP, ATP, UDP, UTP, and UDP-glucose, were summarized and correlated for the eight known subtypes. The rhodopsin-based homology modeling of the P2Y receptors is supported by a growing body of site-directed mutagenesis data, mainly for P2Y(1) receptors. By comparing molecular models of the P2Y receptors, it was concluded that nucleotide binding could occur in the upper part of the helical bundle, with the ribose moiety accommodated between transmembrane domain (TM) 3 and TM7. The nucleobase was oriented towards TM1, TM2, and TM7, in the direction of the extracellular side of the receptor. The phosphate chain was oriented towards TM6, in the direction of the extracellular loops (ELs), and was coordinated by three critical cationic residues. In particular, in the P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors the nucleotide ligands had very similar positions. ADP in the P2Y(12) receptor was located deeper inside the receptor in comparison to other subtypes, and the uridine moiety of UDP-glucose in the P2Y(14) receptor was located even deeper and shifted toward TM7. In general, these findings are in agreement with the proposed binding site of small molecules to other class A GPCRs.

Action of Nucleosides and Nucleotides at 7 Transmembrane-Spanning Receptors

Ribose ring-constrained nucleosides and nucleotides to act at cell-surface purine recesptors have been designed and synthesized. At the P2Y1 nucleotide receptor and the A3 adenosine receptor (AR) the North envelope conformation of ribose is highly preferred. We have applied mutagenesis and rhodopsin-based homology modeling to the study of purine receptors and used the structural insights gained to assist in the design of novel ligands. Two subgroups of P2Y receptors have been defined, containing different sets of cationic residues for coordinating the phosphate groups. Modeling/mutagenesis of adenosine receptors has focused on determinants of intrinsic efficacy in adenosine derivatives and on a conserved Trp residue (6.48) which is involved in the activation process. The clinical use of adenosine agonists as cytoprotective agents has been limited by the widespread occurrence of ARs, thus, leading to undesirable side effects of exogenously administered adenosine derivatives. In order to overcome the inherent nonselectivity of activating the native receptors, we have introduced the concept of neoceptors. By this strategy, intended for eventual use in gene therapy, the putative ligand binding site of a G protein-coupled receptor is reengineered for activation by synthetic agonists (neoligands) built to have a structural complementarity. Using a rational design process we have identified neoceptor-neoligand pairs which are pharmacologically orthogonal with respect to the native species.

CCR5 interactions with the variable 3 loop of gp120

The G-protein coupled receptor CCR5 functions pathologically as the primary co-receptor for macrophage tropic (R5) strains of HIV-1. The interactions responsible for co-receptor activity are unknown. Molecular-dynamics simulations of the extracellular and adjacent transmembrane domains of CCR5 were performed with explicit solvation utilizing a rhodopsin-based homology model. The functional unit of co-receptor binding was constructed via docking and molecular-dynamics simulation of CCR5 and the variable 3 loop of gp120, which is a dominant determinant of co-receptor utilization. The variable 3 loop was demonstrated to interact primarily with the amino terminus and the second extracellular loop of CCR5, providing novel structural information regarding the co-receptor-binding site. Alanine mutants that alter chemokine binding and co-receptor activity were examined. Molecular-dynamics simulations with and without the variable 3 loop of gp120 were able to rationalize the activities of these mutants successfully, providing support for the proposed model. Based on these results, the global complex of CCR5, gp120 including the V3 loop and CD4, was investigated. The utilization of computational analysis, in combination with molecular biological data, provides a powerful approach for understanding the use of CCR5 as a co-receptor by HIV-1.

Novel Strategies for the Design of New Potent and Selective Human A3 Receptor Antagonists: An Update

A computer-aided approach has been developed in order to understand the molecular pharmacology of human A3R, and specifically, to lead to the discovery and structural refinement of new, potent and selective human A3R antagonists. This review focuses on our combined target-based and ligand-based drug design strategy, recently applied to provide more accurate information about the recognition mode on human A3R of some pyrazolotriazolopyrimidine and triazoloquinoxalinone analogs. The 3D rhodopsin-based homology model of human A3R has represented the starting point of our approach. A high throughput molecular docking method on the considered antagonists has allowed us to generate a receptor-based pharmacophore model. A novel "Y-shaped" pharmacophore binding motif has been proposed for both pyrazolotriazolopyrimidine and triazoloquinoxalinone derivatives. Moreover, related receptor-based 3D-QSAR analysis has been carried out to provide a suitable tool for prediction of the antagonists binding affinity on human A3R.

Identification of multiple allosteric sites on the M 1 muscarinic acetylcholine receptor

Staurosporine and four staurosporine derivatives were docked on the rhodopsin-based homology model of the M 1 muscarinic acetylcholine receptor in order to localize the possible allosteric sites of this receptor. It was found that there were three major allosteric sites, two of which are located at the extracellular face of the receptor, and one in the intracellular domain of the receptor. In the present study, the localization of these binding sites is described for the first time. The present study confirms the existence of multiple allosteric sites on the M 1 muscarinic receptor, and lays the ground for further experimental and computational analysis to better understand how muscarinic receptors are modulated via their allosteric sites. These findings will also help to design and develop novel drugs acting as allosteric modulators of the M 1 receptor, which can be used in the treatment of the Alzheimer’s disease.

Structural Determinants of Pharmacological Specificity Between D1and D2Dopamine Receptors

To test the hypothesis that pharmacological differentiation between D(1) and D(2) dopamine receptors results from interactions of selective ligands with nonconserved residues lining the binding pocket, we mutated amino acid residues in the D(2) receptor to the corresponding aligned residues in the D(1) receptor and vice versa and expressed the receptors in human embryonic kidney 293 cells. Determinations of the affinity of the 14 mutant D(2) receptors and 11 mutant D(1) receptors for D(1)- and D(2)-selective antagonists, and rhodopsin-based homology models of the two receptors, identified two residues whose direct interactions with certain ligands probably contribute to ligand selectivity. The D(1) receptor mutant W99(3.28)F showed dramatically increased affinity for several D(2)-selective antagonists, particularly spiperone (225-fold), whereas the D(2) receptor mutant Y417(7.43)W had greatly decreased affinity for benzamide ligands such as raclopride (200-fold) and sulpiride (125-fold). The binding of the D(1)-selective ligand R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) was unaffected, indicating that SCH23390 makes little contact with these ancillary pocket residues. Mutation of A/V(5.39) caused modest but consistent and reciprocal changes in affinity of the receptors for D(1) and D(2)-selective ligands, perhaps reflecting altered packing of the interface of helices 5 and 6. We also obtained some evidence that residues in the second extracellular loop contribute to ligand binding. We conclude that additional determinants of D(1)/D(2) receptor-selective binding are located either in that loop or in the transmembrane helices but, like residue 5.39, indirectly influence the interactions of selective ligands with conserved residues by altering the shape of the primary and ancillary binding pockets.

2- and 8-alkynyl-9-ethyladenines: Synthesis and biological activity at human and rat adenosine receptors

The synthesis of a series of 9-ethyladenine derivatives bearing alkynyl chains in 2- or 8-position was undertaken, based on the observation that replacement of the sugar moiety in adenosine derivatives with alkyl groups led to adenosine receptor antagonists. All the synthesized compounds were tested for their affinity at human and rat A1, A2A, and A3 adenosine receptors in binding assays; the activity at the human A2B receptor was determined in adenylyl cyclase experiments. Biological data showed that the 2-alkynyl derivatives possess good affinity and are slightly selective for the human A2A receptor. The same compounds tested on the rat A1 and A2A subtypes showed in general lower affinity for both receptors. On the other hand, the affinity of the 8-alkynyl derivatives at the human A1, A2A, and A2B receptors proved to be lower than that of the corresponding 2-alkynyl derivatives. On the contrary, the affinity of the same compounds for the human A3 receptor was improved, resulting in A3 selectivity. As in the case of the 2-alkynyl-substituted compounds, the 8-alkynyl derivatives showed decreased affinity for rat receptors. However, it is worthwhile to note that the 8-phenylethynyl-9-ethyladenine was the most active compound of the two series (Ki in the nanomolar range) at both the human and rat A3 subtype. Docking experiments of the 2- and 8-phenylethynyl-9-ethyladenines, at a rhodopsin-based homology model, gave a rational explanation of the preference of the human A3 receptor for the 8-substituted compound.

Structure-based Drug Discovery Using GPCR Homology Modeling: Successful Virtual Screening for Antagonists of the Alpha1A Adrenergic Receptor

In this paper, we describe homology modeling of the alpha1A receptor based on the X-ray structure of bovine rhodopsin. The protein model has been generated by applying ligand-supported homology modeling, using mutational and ligand SAR data to guide the protein modeling procedure. We performed a virtual screening of the company's compound collection to test how well this model is suited to identify alpha1A antagonists. We applied a hierarchical virtual screening procedure guided by 2D filters and three-dimensional pharmacophore models. The ca. 23,000 filtered compounds were docked into the alpha1A homology model with GOLD and scored with PMF. From the top-ranked compounds, 80 diverse compounds were tested in a radioligand displacement assay. 37 compounds revealed K(i) values better than 10 microM; the most active compound binds with 1.4 nM to the alpha1A receptor. Our findings suggest that rhodopsin-based homology models may be used as the structural basis for GPCR lead finding and compound optimization.