Crystal Structure Of Bovine Rhodopsin Research Articles

Recent Advances in Structure, Function, and Pharmacology of Class A Lipid GPCRs: Opportunities and Challenges for Drug Discovery.

Great progress has been made over the past decade in understanding the structural, functional, and pharmacological diversity of lipid GPCRs. From the first determination of the crystal structure of bovine rhodopsin in 2000, much progress has been made in the field of GPCR structural biology. The extraordinary progress in structural biology and pharmacology of GPCRs, coupled with rapid advances in computational approaches to study receptor dynamics and receptor-ligand interactions, has broadened our comprehension of the structural and functional facets of the receptor family members and has helped usher in a modern age of structure-based drug design and development. First, we provide a primer on lipid mediators and lipid GPCRs and their role in physiology and diseases as well as their value as drug targets. Second, we summarize the current advancements in the understanding of structural features of lipid GPCRs, such as the structural variation of their extracellular domains, diversity of their orthosteric and allosteric ligand binding sites, and molecular mechanisms of ligand binding. Third, we close by collating the emerging paradigms and opportunities in targeting lipid GPCRs, including a brief discussion on current strategies, challenges, and the future outlook.

In Silico Investigations of Possible Therapeutic Peptides as Drugs for Anti-Inflammatory Responses

The chemokine called interleukin-8 (IL-8 or CXCL8) plays a central role in human immune response by binding and activating the receptor CXCR1 that belongs to the G-protein coupled receptor (GPCR) family. Upon binding of CXCL8, CXCR1 undergoes conformational change resulting in signal transduction.The purpose of this study was to determine the role of the fragments of the CXCL8 binding sites of CXCR1 as potential therapeutic targets for developing novel drugs against inflammatory diseases.Homology modeling studies were firstly performed to construct the three-dimensional structure of CXCR1 by taking the crystal structure of bovine rhodopsin (PDB code: 1U19) as the template with the sequence similarity up to 41.8 %. By the following molecular docking programs several potential peptides were selected and synthesized to validate the anti-inflammatory efficiency through flow cytometry analysis. The preferred complexes of CXCL8 binding CXCR1 from docking predictions were then embedded into POPC lipid bilayers for 50 ns MD simulation to investigate the binding interaction. Simulation results show that electrostatic interaction dominates the binding of CXCL8 and CXCR1, consistent with the previous experiments.Small peptide drugs are novel therapeutic candidates for inflammatory diseases. High-throughput, structure-based virtual screening combining MD simulations is an effective computer-based drug design method for discovering anti-inflammatory drug candidates.

A new era for GPCR research: structures, biology and drug discovery

Cells in a living organism must communicate with each other through continuously sending and receiving messages. G-protein coupled receptors (GPCRs) are the largest family of communicating molecules at the cell surface. They transmit diverse extracellular signals, ranging from light and small chemical hormones to large peptide and protein hormones, and as such they play crucial roles in numerous physiological and pathological processes. More importantly, GPCRs are the most successful class of drug targets that are relevant to many major diseases, including cancer, heart failure, and inflammatory diseases. Over 50% of currently used drugs are targeted to GPCRs. However, these drugs target only 50–60 GPCRs, leaving the majority of human GPCRs, exceeding 800, unexplored for drug discovery. Given the prominent roles of GPCRs in biology and their successful track records as drug targets, GPCRs have become a hot frontier in basic research of life science and therapeutic discovery of translational medicines. In this special issue, there are nine exciting reviews that cover a broad scope of GPCR structures, biology, diseases, and drug discovery. Among them, four reviews are dedicated to GPCR structures. Over the last few years, we have witnessed a revolution in Class A GPCR structural biology. Rhodopsin is the founding member of the GPCR family and its signaling mechanism is a paradigm for many other GPCRs. The crystal structure of bovine rhodopsin was first solved in 2000 and since then structures of rhodopsin have been solved in several functional states, including the inactivated dark state, partially active opsin, and the fully active state that is bound with a Gαt peptide. ZHOU, MELCHER, and XU review the signaling mechanisms gained from rhodopsin structures and compare these mechanisms to other members of Class A GPCRs, most notably β2-adrenergic receptor (β2AR)1. Class B and C GPCRs distinguished themselves from Class A GPCRs by having a large extracellular domain for binding of ligands. Compared to Class A GPCRs, structure determination for the full-length receptors of other families of GPCRs is lagging behind. However, structures for a half dozen of Class B and C GPCR extracellular domains (ECDs) in complex with their respective ligands have been determined. The structures and ligand binding mechanisms of Class B and Class C GPCRs will be reviewed by the XU and LIU groups, respectively2, 3. The rapid explosion of Class A GPCR structures is owed to technology development in various stages of structure determination; including protein expression, purification, crystallization, and X-ray diffraction, for which a thorough review is provided by ZHAO and WU4. Following the reviews of GPCR structures, five reviews are focused on GPCR biology, diseases, and drug discovery. β2AR is a prototype of GPCRs, and in parallel to rhodopsin, it has been serving as a model for studying other GPCRs. WOO and XIAO review the signaling mechanisms by β-adrenergic receptor subtypes that provide a basis for developing selective biased β2 agonists for the treatment of heart failure5. Moving from heart to immune systems, YE and SUN review the role of GPCRs in inflammation processes from chemotaxisis to transcriptional regulation of inflammation programs6. Lappano and Maggiolini provide a systematic review on the relationship of cancers with various subfamilies of GPCRs, which may serve as the basis for developing novel pharmacological interventions for cancers7. A large number of GPCRs are orphan receptors whose ligand remains unknown. Despite missing cognate ligands, many orphan GPCRs are known to play important physiological functions, mostly from genetic knockout studies. LIU and colleagues provide a thorough review on different class of orphan nuclear receptors, their functions, and possible strategies for identification of endogenous ligands8. Finally, as one of the most important drug targets, GPCRs are widely pursued by both academic and industrial research for drug discovery. ZHANG and XIE provide a comprehensive review on methods of GPCR drug discovery, including various ligand binding and cell-based functional assays, many of which are tool kits for basic research as well as drug discovery for GPCRs9. Followed the waves of technological advances and inter-disciplinary approaches in modern biological research, the field of GPCRs is evolving rapidly into a new phase of exciting progresses. With a rich history of biology and drug discovery, GPCR research is expected to continue to be a dominant source of innovative medicines for the 21st Century. We hope that the collection of these nine exciting reviews will provide a window into this new era of GPCR research and drug discovery.

Molecular docking of 2-(benzimidazol-2-ylthio)-N-phenylacetamide-derived small-molecule agonists of human formyl peptide receptor 1

Human N-formyl peptide receptor 1 (FPR1) is a G protein-coupled receptor (GPCR) involved in host defense and sensing cellular damage. Since structure-based ligand design for many GPCRs, including FPR1, is restricted by the lack of experimental three dimensional structures, homology modeling has been widely used to study GPCR-ligand binding. Indeed, receptor-ligand binding mode predictions can be derived from homology modeling with supporting ligand information. In the present work, we report comparative docking studies of 2-(benzimidazol-2-ylthio)-N-phenylacetamide derived FPR1 agonists, identified here and previously, with several known FPR1 peptide agonists in a FPR1 homology model that is based on the crystal structure of bovine rhodopsin. We found that the binding pocket of the most active molecules shares some common features with high affinity FPR1 peptide agonists, suggesting that they may bind to similar binding sites. Classification tree analysis led to the derivation of a good recognition model based on four amino acid descriptors for distinguishing FPR1 ligands from inactive analogs. Hence, the corresponding residues (Thr199, Arg201, Gly202, and Ala261) can be considered as markers of important areas in the ligand binding site. Concurrently, we identified several unique binding features of benzimidazole derivatives and showed that alkoxy-substituents of the benzimidazole ring are located within a FPR1 hole bounded by Thr199, Thr265, Ile268, and Leu271 or in a groove in the vicinity of Leu198, Arg201, Gly202, and Arg205. The understanding of these molecular features will likely prove beneficial in future design of novel FPR1 agonists based on the benzimidazole scaffold.

Comparative modeling of human kappa opioid receptor and docking analysis with the peptide YFa

The kappa opioid receptor belongs to the super family of G protein – coupled receptors that are of utmost significance in the development of potent analgesic drugs for the treatment of severe pain. An accurate evaluation of the ligand binding pathways into this receptor at molecular level may play a key role in the design of new molecules with more desirable properties and reduced side effects. In this study, homology model of the human kappa opioid receptor was developed by MODELLER using the X-ray crystal structure of bovine rhodopsin as template. Initial structure of the receptor was refined computationally with energy minimization and molecular dynamics simulation at 300 K in a pre-equilibrated phospholipid bilayer by GROMACS. The Met-enkaphalin-Arg-Phe based opioid peptide YFa (YGGFMKKKFMRF) designed and characterized by our laboratory was docked into the optimized model and the critical amino acids responsible for binding were identified. A number of low energy binding poses of YFa with the receptor were assessed after the molecular docking in which the peptide was observed to interact with the receptor's extracellular amino acids through hydrogen bonds. The human kappa opioid receptor model optimized in a phospholipid bilayer should provide a good starting point for further characterization of the binding modes of other opioid ligands. Furthermore, the biologically favorable molecular interactions between YFa and human kappa opioid receptor observed by our study might be able to justify the specificity of this peptide.

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.

New Insights into the Binding Mode of Melanin Concentrating Hormone Receptor-1 Antagonists: Homology Modeling and Explicit Membrane Molecular Dynamics Simulation Study

Melanin concentrating hormone (MCH) is a cyclic 19-amino-acid peptide expressed mainly in the hypothalamus. It is involved in the control of feeding behavior, energy homeostasis, and body weight. Administration of MCH-R1 antagonists has been proved to reduce food intake and cause weight loss in animal models. In the present study, a homology model of the human MCH-R1 was constructed using the crystal structure of bovine rhodopsin (PDB: 1u19) as a template. Based on the observation that MCH-R1 can bind ligands of high chemical diversity, the initial model was subjected to an extensive ligand-supported refinement using antagonists of different chemotypes. The refinement process involved stepwise energy minimizations and molecular dynamics simulations. The refined model was inserted into a pre-equilibrated DPPC/TIP3P membrane system and then simulated for 20 ns in complex with structurally diverse antagonists. This protocol was able to explain the SAR of MCH-R1 antagonists with diverse chemical structures. Moreover, it reveals new insights into the critical recognition sites within the receptor. This work represents the first detailed study of molecular dynamics of MCH-R1 inserted into a membrane-aqueous environment.

Homology modeling of human CCR5 and analysis of its binding properties through molecular docking and molecular dynamics simulation

In this study, homology modeling, molecular docking and molecular dynamics simulation were performed to explore structural features and binding mechanism of some inhibitors of chemokine receptor type 5 (CCR5), and to construct a model for designing new CCR5 inhibitors for preventing HIV attachment to the host cell. A homology modeling procedure was employed to construct a 3D model of CCR5. For this procedure, the X-ray crystal structure of bovine rhodopsin (1F88A) at 2.80Å resolution was used as template. After inserting the constructed model into a hydrated lipid bilayer, a 20ns molecular dynamics (MD) simulation was performed on the whole system. After reaching the equilibrium, twenty-four CCR5 inhibitors were docked in the active site of the obtained model. The binding models of the investigated antagonists indicate the mechanism of binding of the studied compounds to the CCR5 obviously. Moreover, 3D pictures of inhibitor–protein complex provided precious data regarding the binding orientation of each antagonist into the active site of this protein. One additional 20ns MD simulation was performed on the initial structure of the CCR5–ligand 21 complex, resulted from the previous docking calculations, embedded in a hydrated POPE bilayer to explore the effects of the presence of lipid bilayer in the vicinity of CCR5–ligand complex. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes

Characterization of the β-d-Glucopyranoside Binding Site of the Human Bitter Taste Receptor hTAS2R16

G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited. To address the molecular basis of bitter tastant discrimination by the hTAS2Rs, we performed ligand docking simulation and functional analysis using a series of point mutants of hTAS2R16 to identify its binding sites. The docking simulation predicted two candidate binding structures for a salicin-hTAS2R16 complex, and at least seven amino acid residues in transmembrane 3 (TM3), TM5, and TM6 were shown to be involved in ligand recognition. We also identified the probable salicin-hTAS2R16 binding mode using a mutated receptor experiment. This study characterizes the molecular interaction between hTAS2R16 and β-d-glucopyranoside and will also facilitate rational design of bitter blockers.

Structure-Activity Relationships of GPR120 Agonists Based on a Docking Simulation

GPR120 is a G protein-coupled receptor expressed preferentially in the intestinal tract and adipose tissue, that has been implicated in mediating free fatty acid-stimulated glucagon-like peptide-1 (GLP-1) secretion. To develop GPR120-specific agonists, a series of compounds (denoted as NCG compounds) derived from a peroxisome proliferator-activated receptor γ agonist were synthesized, and their structure-activity relationships as GPR120 agonists were explored. To examine the agonistic activities of these newly synthesized NCG compounds, and of compounds already shown to have GPR120 agonistic activity (grifolic acid and MEDICA16), we conducted docking simulation in a GPR120 homology model that was developed on the basis of a photoactivated model derived from the crystal structure of bovine rhodopsin. We calculated the hydrogen bonding energies between the compounds and the GPR120 model. These energies correlated well with the GPR120 agonistic activity of the compounds (R(2) = 0.73). NCG21, the NCG compound with the lowest calculated hydrogen bonding energy, showed the most potent extracellular signal-regulated kinase (ERK) activation in a cloned GPR120 system. Furthermore, NCG21 potently activated ERK, intracellular calcium responses and GLP-1 secretion in murine enteroendocrine STC-1 cells that express GPR120 endogenously. Moreover, administration of NCG21 into the mouse colon caused an increase in plasma GLP-1 levels. Taken together, our present study showed that a docking simulation using a GPR120 homology model might be useful to predict the agonistic activity of compounds.

Molecular modeling aided design of nicotinic acid receptor GPR109A agonists

A homology model of the nicotinic acid receptor GPR109A was constructed based on the X-ray crystal structure of bovine rhodopsin. An HTS hit was docked into the homology model. Characterization of the binding pocket by a grid-based surface calculation of the docking model suggested that a larger hydrophobic body plus a polar tail would improve interaction between the ligand and the receptor. The designed compounds were synthesized, and showed significantly improved binding affinity and activation of GPR109A.

Ligand-Specific Contribution of the N Terminus and E2-Loop to Pharmacological Properties of the Histamine H1-Receptor

There are species differences between human histamine H(1) receptor (hH(1)R) and guinea pig (gp) histamine H(1) receptor (gpH(1)R) for phenylhistamines and histaprodifens. Several studies showed participation of the second extracellular loop (E2-loop) in ligand binding for some G protein-coupled receptors (GPCRs). Because there are large species differences in the amino acid sequence between hH(1)R and gpH(1)R for the N terminus and E2-loop, we generated chimeric hH(1)Rs with gp E2-loop (h(gpE2)H(1)R) and gp N terminus and gp E2-loop (h(gpNgpE2)H(1)R). hH(1)R, gpH(1)R, and chimeras were expressed in Sf9 insect cells. [(3)H]Mepyramine binding assays and steady-state GTPase assays were performed. In the series hH(1)R > h(gpE2)H(1)R > h(gpNgpE2)H(1)R, we observed a significant decrease in potency of histamine 1 in the GTPase assay. For phenoprodifen 5 and the chiral phenoprodifens 6R and 6S, a significant decrease in affinity and potency was found in the series hH(1)R > h(gpE2)H(1)R > h(gpNgpE2)H(1)R. In addition, we constructed new active-state H(1)R models based on the crystal structure of the human beta(2)-adrenergic receptor (hbeta(2)AR). Compared with the H(1)R active-state models based on the crystal structure of bovine rhodopsin, the E2-loop differs in its contact to the ligand bound in the binding pocket. In the bovine rhodopsin-based model, the backbone carbonyl of Lys187 (gpH(1)R) interacts with large histaprodifens in the binding pocket, but in the hbeta(2)AR-based model, Lys187 (gpH(1)R) is located distantly from the binding pocket. In conclusion, the differences in N terminus and E2-loop between hH(1)R and gpH(1)R exert an influence on affinity and/or potency for histamine and phenoprodifens 5, 6R, and 6S.

GPC Receptors and Not Ligands Decide the Binding Mode in Neuropeptide Y Multireceptor/Multiligand System

Many G protein-coupled receptors belong to families of different receptor subtypes, which are recognized by a variety of distinct ligands. To study such a multireceptor/multiligand system, we investigated the Y-receptor family. This family consists of four G protein-coupled Y receptors in humans (hY 1R, hY 2R, hY 4R, and hY 5R) and is activated by the so-called NPY hormone family, which itself consists of three native peptide ligands named neuropeptide Y (NPY), pancreatic polypeptide (PP), and peptide YY (PYY). The hY 5R shows high affinity for all ligands, although for PP binding, the affinity is slightly decreased. As a rational explanation, we suggest that Tyr (27) is lost as a contact point between PP and the hY 5R in contrast to NPY or PYY. Furthermore, several important residues for ligand binding were identified by the first extensive mutagenesis study of the hY 5R. Using a complementary mutagenesis approach, we were able to discover a novel interaction point between hY 5R and NPY. The interaction between NPY(Arg (25)) and hY 5R(Asp (2.68)) as well as between NPY(Arg (33)) and hY 5R(Asp (6.59)) is maintained in the binding of PYY and PP to hY 5R but different to the PP-hY 4R and NPY-hY 1R contact points. Therefore, we provide evidence that the receptor subtype and not the pre-orientated conformation of the ligand at the membrane decides the binding mode. Furthermore, the first hY 5R model was set up on the basis of the crystal structure of bovine rhodopsin. We can show that most of the residues identified to be critical for ligand binding are located within the now postulated binding pocket.

Analysis of efficacy of chiral adrenergic agonists

The origin of terms, affinity, intrinsic activity (or efficacy) and spare receptors has been reviewed. The Easson-Stedman theory (1933) in relation to the activation of adrenoceptors by agonists proved to be useful in the analysis of affinity and efficacy. Eudismic ratios of agonists provided critical information about the receptor-mediated activation. The evidence from circular dichroism spectroscopy with a fluorescent-tagged adrenoceptor agonist indicates a stereoselective interaction with the receptor. Thus, the simplest definition of efficacy may include the rate of change of the specific conformation of the receptor by the agonist, leading to the organized response. The functional groups of the potent enantiomer are postulated to interact in a "preferred" sequence with the receptor. The 7TM GPCR protein crystal structure of bovine rhodopsin was used as a model to construct the agonist interacting amino acid residues for alpha(1A)- and beta1-adrenoceptors. It was observed that both --+NH3 group and chiral --OH group of (-)-epinephrine interact with Asp106 TM III of alpha 1A-adrenoceptor. Similar interactions were observed for (+)-epinephrine but critical differences were observed. Enantiomers of epinephrine and oxymetazoline were also docked in the position at beta1-adrenoceptor to elucidate the conformational changes. Some unique information has emerged about the activation of adrenoceptors by agonists. The differences in the pharmacological efficacy of the enantiomers compare favorably with the dynamics of conformational changes by the agonist at alpha1A- and beta1 adrenoceptors.

Understanding the structural and functional differences between mouse thyrotropin‐releasing hormone receptors 1 and 2

Multiple computational methods have been employed in a comparative study of thyrotropin-releasing hormone receptors 1 and 2 (TRH-R1 and TRH-R2) to explore the structural bases for the different functional properties of these G protein-coupled receptors. Three-dimensional models of both murine TRH receptors have been built and optimized by means of homology modeling based on the crystal structure of bovine rhodopsin, molecular dynamics simulations, and energy minimizations in a membrane-aqueous environment. The comparison between the two models showed a correlation between the higher flexibility and higher basal activity of TRH-R2 versus the lesser flexibility and lower basal activity of TRH-R1 and supported the involvement of the highly conserved W6.48 in the signaling process. A correlation between the level of basal activity and conformational changes of TM5 was detected also. Comparison between models of the wild type receptors and their W6.48A mutants, which have reversed basal activities compared with their respective wild types, further supported these correlations. A flexible molecular docking procedure revealed that TRH establishes a direct interaction with W6.48 in TRH-R2 but not in TRH-R1. We designed and performed new mutagenesis experiments that strongly supported these observations.

Improved model building and assessment of the Calcium‐sensing receptor transmembrane domain

A three-dimensional model of the human Calcium-sensing receptor (CaSR) seven transmembrane domain was built via a novel sequence alignment method based on the conserved contacts in proteins using the crystal structure of bovine rhodopsin as the template. This model was tested by docking NPS 2143, the first identified allosteric antagonist of CaSR. In our model, Glu837 plays a critical role in anchoring the protonated nitrogen atom and hydroxy oxygen atom of NPS 2143. The phenyl moiety of the ligand contacts residues Phe668, Pro672, and Ile841. The naphthalene moiety is surrounded by several hydrophobic residues, including Phe684, Phe688, and Phe821. Our model appears to be consistent with all six residues that have been demonstrated to be critical for NPS 2143 binding, in contrast with existing homology models based on traditional sequence alignment of CaSR to rhodopsin. This provides validation of our sequence alignment method and the use of the rhodopsin backbone as the initial structure in homology modeling of other G protein-coupled receptors that are not members of the rhodopsin family.

Analysis of the activation mechanism of the guinea-pig Histamine H1-receptor

The Histamine H(1)-receptor (H1R), belonging to the amine receptor-class of family A of the G-protein coupled receptors (GPCRs) gets activated by agonists. The consequence is a conformational change of the receptor, which may involve the binding-pocket. So, for a good prediction of the binding-mode of an agonist, it is necessary to have knowledge about these conformational changes. Meanwhile some experimental data about the structural changes of GPCRs during activation exist. Based on homology modeling of the guinea-pig H1R (gpH1R), using the crystal structure of bovine rhodopsin as template, we performed several MD simulations with distance restraints in order to get an inactive and an active structure of the gpH1R. The calculations led to a Phe6.44/Trp6.48/Phe6.52-switch and linearization of the proline kinked transmembrane helix VI during receptor activation. Our calculations showed that the Trp6.48/Phe6.52-switch induces a conformational change in Phe6.44, which slides between transmembrane helices III and VI. Additionally we observed a hydrogen bond interaction of Ser3.39 with Asn7.45 in the inactive gpH1R, but because of a counterclockwise rotation of transmembrane helix III Ser3.39 establishes a water-mediated hydrogen bond to Asp2.50 in the active gpH1R. Additionally we simulated a possible mechanism for receptor activation with a modified LigPath-algorithm.

Binding mode analysis and enrichment studies on homology models of the human histamine H4 receptor

Ligand-supported homology models of the human histamine H4 receptor (hH4R) were developed based on the crystal structure of bovine rhodopsin and different known H4 ligands (histamine, OUP-16, JNJ7777120). Enrichment tests were performed to analyze whether our hH4R models can select known actives from random decoys. The impact of receptor conformation and the effect of different sets of random decoys, docking methods (FlexX, FlexX-Pharm) and scoring functions (FlexX-Score, D-Score, PMF-Score, G-Score, ChemScore) were investigated. We found that two agonists (histamine and OUP-16) form complementary interactions with Asp94 (3.32), Glu182 (5.46) and Thr323 (6.55), whereas JNJ7777120 interacts with Asp94 (3.32) and Glu182 (5.46) only. These results suggest a role of Thr323 (6.55) in ligand binding and presumably also in receptor activation. The models optimized in the presence of an agonist (histamine) and an antagonist (JNJ7777120) were compared in more detail. We conclude that the ligand used in the model building process can significantly influence the efficacy of virtual screening.

Generation of a homology model of the human histamine H3 receptor for ligand docking and pharmacophore-based screening

The human histamine H(3) receptor (hH(3)R) is a G-protein coupled receptor (GPCR), which modulates the release of various neurotransmitters in the central and peripheral nervous system and therefore is a potential target in the therapy of numerous diseases. Although ligands addressing this receptor are already known, the discovery of alternative lead structures represents an important goal in drug design. The goal of this work was to study the hH(3)R and its antagonists by means of molecular modelling tools. For this purpose, a strategy was pursued in which a homology model of the hH(3)R based on the crystal structure of bovine rhodopsin was generated and refined by molecular dynamics simulations in a dipalmitoylphosphatidylcholine (DPPC)/water membrane mimic before the resulting binding pocket was used for high-throughput docking using the program GOLD. Alternatively, a pharmacophore-based procedure was carried out where the alleged bioactive conformations of three different potent hH(3)R antagonists were used as templates for the generation of pharmacophore models. A pharmacophore-based screening was then carried out using the program Catalyst. Based upon a database of 418 validated hH(3)R antagonists both strategies could be validated in respect of their performance. Seven hits obtained during this screening procedure were commercially purchased, and experimentally tested in a [(3)H]N(alpha)-methylhistamine binding assay. The compounds tested showed affinities at hH(3)R with K ( i ) values ranging from 0.079 to 6.3 muM.

Critical Role for the Second Extracellular Loop in the Binding of Both Orthosteric and Allosteric G Protein-coupled Receptor Ligands

The second extracellular (E2) loop of G protein-coupled receptors (GPCRs) plays an essential but poorly understood role in the binding of non-peptidic small molecules. We have utilized both orthosteric ligands and allosteric modulators of the M2 muscarinic acetylcholine receptor, a prototypical Family A GPCR, to probe possible E2 loop binding dynamics. We developed a homology model based on the crystal structure of bovine rhodopsin and predicted novel cysteine substitutions that should dramatically reduce E2 loop flexibility via disulfide bond formation and significantly inhibit the binding of both types of ligands. This prediction was validated experimentally using radioligand binding, dissociation kinetics, and cell-based functional assays. The results argue for a flexible "gatekeeper" role of the E2 loop in the binding of both allosteric and orthosteric GPCR ligands.