Homology modeling of the human 5-HT1A, 5-HT2A, D1, and D2 receptors: model refinement with molecular dynamics simulations and docking evaluation

5-HT(1A) serotonin and D1 dopamine receptor agonists have been postulated to be able to improve negative and cognitive impairment symptoms of schizophrenia, while partial agonists and antagonists of the D2 and 5-HT(2A) receptors have been reported to be effective in reducing positive symptoms. There is therefore a need for well-defined homology models for the design of more selective antipsychotic agents, since no three-dimensional (3D) crystal structures of these receptors are currently available. In this study, homology models were built based on the high-resolution crystal structure of the β(2)-adrenergic receptor (2RH1) and further refined via molecular dynamics simulations in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer system with the GROMOS96 53A6 united atom force field. Docking evaluations with representative agonists and antagonists using AutoDock 4.2 revealed binding modes in agreement with experimentally determined site-directed mutagenesis data and significant correlations between the computed and experimental pK (i) values. The models are also able to distinguish between antipsychotic agents with different selectivities and binding affinities for the four receptors, as well as to differentiate active compounds from decoys. Hence, these human 5-HT(1A), 5-HT(2A), D1 and D2 receptor homology models are capable of predicting the activities of novel ligands, and can be used as 3D templates for antipsychotic drug design and discovery.     

Development of CoMFA, advance CoMFA and CoMSIA models in pyrroloquinazolines as thrombin receptor antagonist

3D QSAR studies namely CoMFA, advance CoMFA and CoMSIA have been carried out on a series of pyrroloquinazolines for their thrombin receptor antagonistic activity. The predicted activities by highly significant CoMFA (q2=0.66 and CoMSIA (q2=0.67) models were in good accordance with observed activities and the models may be useful for optimization of thrombin receptor antagonistic activity.     

Evidence of 1,25-dihydroxyvitamin D3-receptors in human digestive mucosa and carcinoma tissue biopsies taken at different levels of the digestive tract, in 152 patients

Epidemiological and experimental data suggest that dietary calcium and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) are protective against colorectal cancers, while their activity on colon mucosa still remains unknown. Since the presence of receptors is required for steroid action, specific 1,25-(OH)2D3 receptors were investigated in biopsies taken at different levels of the digestive tract from the oesophagus to the rectum and in pancreas. The total study involved biopsies from 152 patients. In 82% of the cases they were paired biopsies in adenocarcinoma tissue and in adjacent normal mucosa (NM). There were 120 operated on for colorectal adenocarcinoma (HCRA). 1,25-(OH)2D3 receptor was assayed in tissue extract by the dextran-coated charcoal (DCC) technique and also characterised by sucrose density gradient centrifugation. Scatchard analyses showed a single class of specific high affinity-low capacity sites binding for 1,25-(OH)2D3 with a Kd = 1.48 +/- 0.8 x 10(-10) M (n = 119). The sedimentation coefficient of the steroid receptor complex was approximately 3.2 S. The incidence of 1,25-(OH)2D3 receptors was significantly higher in NM (82.5%) than in HCRA (34.5%). In HCRA this incidence decreased from right colon (64.7%) to left colon (27.7%) and rectum (15%). All positive HCRA in left colon and rectum (16/76) were histologically well differentiated. The receptor content in NM and HCRA was in the same range: (median) 10-314 (58) and 13-175 (64) fmol/mg protein. These data suggest that 1,25-(OH)2D3 may modulate calcium transport in colon, as in the intestine. Also, loss of receptivity to 1,25-(OH)2D3 is observed as associated with malignant transformation of the human colorectal mucosa.     

Homology modeling of dopamine d(2) and d(3) receptors: molecular dynamics refinement and docking evaluation

Dopamine (DA) receptors, a class of G-protein coupled receptors (GPCRs), have been targeted for drug development for the treatment of neurological, psychiatric and ocular disorders. The lack of structural information about GPCRs and their ligand complexes has prompted the development of homology models of these proteins aimed at structure-based drug design. Crystal structure of human dopamine D(3) (hD(3)) receptor has been recently solved. Based on the hD(3) receptor crystal structure we generated dopamine D(2) and D(3) receptor models and refined them with molecular dynamics (MD) protocol. Refined structures, obtained from the MD simulations in membrane environment, were subsequently used in molecular docking studies in order to investigate potential sites of interaction. The structure of hD(3) and hD(2L) receptors was differentiated by means of MD simulations and D(3) selective ligands were discriminated, in terms of binding energy, by docking calculation. Robust correlation of computed and experimental K(i) was obtained for hD(3) and hD(2L) receptor ligands. In conclusion, the present computational approach seems suitable to build and refine structure models of homologous dopamine receptors that may be of value for structure-based drug discovery of selective dopaminergic ligands.     

Biochemical characterization of positive cooperativity in the binding of 1 alpha, 25-dihydroxyvitamin D3 to its chick intestinal chromatin receptor

We have characterized a positive cooperativity mechanism in the binding of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) to its chick duodenum chromatin receptor. The Hill plot which can take account of the possibility of cooperativity resulted in a much better fitting of the experimental data than the Scatchard model (r = +0.998 versus r = -0.94). Concentrating the chromatin receptor preparation from 10 to 40% resulted in an increase of the Hill coefficient (nH) from 1.09 +/- 0.08 to 1.46 +/- 0.08 (S.D.). Increasing the temperature of incubation from 1 degree C to 40 degrees C resulted in a decrease of nH from 1.46 +/- 0.08 to 1.10 +/- 0.02 (S.D.). The calculation of the thermodynamics of the interaction of 1,25-(OH)2D3 with the second binding site of the receptor (from a Van't Hoff plot) showed that this process occurred spontaneously (delta G0 = -11.6 kcal X mol-1 at 1 degree C), was entropy-driven (delta S0 = +26 cal degree-1 mol-1), and was energy-requiring (delta H0 = -4.37 kcal X mol-1). The temperature controlled reversibility of the cooperativity demonstrates that this phenomenon is not an artifact. Finally, in a study of the rate of dissociation of [3H]1,25-(OH)2D3 from the duodenal receptor preparation, we have found two slopes (k-1 = 32 X 10(-3) min-1; k-2 = 3.2 X 10(-3) min-1); this suggests the existence of two species of receptor. These receptor species could result possibly from either a monomer-dimer system or from a conformational change of a monomer via site-site interactions. In conclusion, the positive cooperativity in the binding of 1,25-(OH)2D3 to the two binding sites of its intestinal receptor is an entropy-driven process and requires energy, is reversible with temperature, and has been shown to take place in concentrated chromatin aggregates.     

Effects of the Dopamine D2 Allosteric Modulator,PAOPA, on the Expression of GRK2, Arrestin-3, ERK1/2, and on Receptor Internalization

The activity of G protein-coupled receptors (GPCRs) is intricately regulated by a range of intracellular proteins, including G protein-coupled kinases (GRKs) and arrestins. Understanding the effects of ligands on these signaling pathways could provide insights into disease pathophysiologies and treatment. The dopamine D2 receptor is a GPCR strongly implicated in the pathophysiology of a range of neurological and neuropsychiatric disorders, particularly schizophrenia. Previous studies from our lab have shown the preclinical efficacy of a novel allosteric drug, 3(R)- [(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA), in attenuating schizophrenia-like behavioural abnormalities in rodent models of the disease. As an allosteric modulator, PAOPA binds to a site on the D2 receptor, which is distinct from the endogenous ligand-binding site, in order to modulate the binding of the D2 receptor ligand, dopamine. The exact signaling pathways affected by this allosteric modulator are currently unknown. The objectives of this study were to decipher the in vivo effects, in rats, of chronic PAOPA administration on D2 receptor regulatory and downstream molecules, including GRK2, arrestin-3 and extracellular receptor kinase (ERK) 1/2. Additionally, an in vitro cellular model was also used to study PAOPA’s effects on D2 receptor internalization. Results from western immunoblots showed that chronic PAOPA treatment increased the striatal expression of GRK2 by 41%, arrestin-3 by 34%, phospho-ERK1 by 51% and phospho-ERK2 by 36%. Results also showed that the addition of PAOPA to agonist treatment in cells increased D2 receptor internalization by 33%. This study provides the foundational evidence of putative signaling pathways, and changes in receptor localization, affected by treatment with PAOPA. It improves our understanding on the diverse mechanisms of action of allosteric modulators, while advancing PAOPA’s development into a novel drug for the improved treatment of schizophrenia.     

Application of the rat thymus receptor in a specific, sensitive and simplified assay of 1,25-dihydroxyvitamin D in blood serum

A simple method has been employed to prepare crude nuclear extract from rat thymus, using hypertonic buffer after previous treatment with hypotonic buffer. The preparation is free from serum vitamin D-binding protein and contains a 3.7 S receptor molecule, which specifically binds 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3). The receptor is of high affinity (KD = 0.85 X 10(-11) M at O degrees C) and low capacity (260-460 fmol/g tissue). The Scatchard analysis of ligand binding results in a concave downward curve. The Hill analysis of the same data gives good linear fitting (r = +0.971) with the Hill coefficient nH = 1.63. These facts indicate positive cooperativity between two ligand binding sites of the rat thymus 1,25-(OH)2D3 receptor. The preparation was used in a competitive protein binding assay of 1,25-(OH)2D in serum extracts, purified on Sep-Pak C18 followed by silica Sep-Pak cartridges. The method was sensitive to 0.5 pg/tube (2.0 ng/l) when 1 ml of serum was extracted. Intra- and interassay coefficients of variation were 9% and 14%, respectively. The serum 1,25-(OH)2D concentration estimated in 33 children (mean age 6.5 +/- 3 years) was 46.6 +/- 18.4 ng/l (mean +/- S.D.).     

24R,25-dihydroxyvitamin D3 regulates 1,25-dihydroxyvitamin D3 binding to its chick intestinal receptor

We have studied the binding of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] to its crude chromatin chick intestinal receptor in the absence or presence of a ten-fold excess of 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] for each concentration of [3H]-1,25(OH)2D3 studied. We have found a significant shift to the right in the binding of 1,25(OH)2D3 to its receptor in the presence of this excess of 24R,25(OH)2D3. As a result, the affinity was found to be significantly reduced, the apparent dissociation constants varied from 0.97 +/- 0.09 (n = 5) to 1.36 +/- 0.04 nM (p less than 0.01). This reduction was related to a significant decrease in the positive cooperativity for the apparent Hill coefficient from nH = 1.49 +/- 0.06 to nH = 1.26 +/- 0.06 (p less than 0.03) in the binding of 1,25(OH)2D3 to its receptor. There was no significant change in the capacity of the receptor (189 +/- 11 compared to 200 +/- 9 fmoles/mg protein). These results suggest that the intestinal 1,25(OH)2D3 receptor must also have a binding recognition site for 24R,25(OH)2D3 which is postulated to play a regulatory role in the 1,25(OH)2D3 receptor's ligand binding properties.     

On the relationship of anthranilic derivatives structure and the FXR (Farnesoid X receptor) agonist activity

Farnesoid X receptor (FXR) is a nuclear receptor related to lipid and glucose homeostasis and is considered an important molecular target to treatment of metabolic diseases as diabetes, dyslipidemia, and liver cancer. Nowadays, there are several FXR agonists reported in the literature and some of it in clinical trials for liver disorders. Herein, a compound series was employed to generate QSAR models to better understand the structural basis for FXR activation by anthranilic acid derivatives (AADs). Furthermore, here we evaluate the inclusion of the standard deviation (SD) of EC₅₀ values in QSAR models quality. Comparison between the use of experimental variance plus average values in model construction with the standard method of model generation that considers only the average values was performed. 2D and 3D QSAR models based on the AAD data set including SD values showed similar molecular interpretation maps and quality (Q²_LOO, Q²_(F2), and Q²_(F3)), when compared to models based only on average values. SD-based models revealed more accurate predictions for the set of test compounds, with lower mean absolute error indices as well as more residuals near zero. Additionally, the visual interpretation of different QSAR approaches agrees with experimental data, highlighting key elements for understanding the biological activity of AADs. The approach using standard deviation values may offer new possibilities for generating more accurate QSAR models based on available experimental data.     

Combined 3D QSAR and molecular docking studies to reveal novel cannabinoid ligands with optimum binding activity

The combination of NMR spectroscopy and molecular modeling studies provided the putative bioactive conformation for the analgesic cannabinoid (CB) ligand (−)-2-(6a,7,10,10a-tetrahydro-6,6,9-trimethylhydroxy-6H-dibenzo[b,d]pyranyl)-2-hexyl 1,3-dithiolane which served as a template in reported three-dimensional quantitative structure–activity relationship (3D QSAR) studies [Durdagi et al., J. Med. Chem. 2007, 50, 2875]. The reported 3D models of the CB1 receptor allowed us to construct a new 3D QSAR model based on theoretical calculations and molecular docking studies. Statistical comparison of the constructed two 3D QSAR studies showed the improvement of the new model. In addition, the new model can explain more effectively the experimental data and thus it can serve more efficiently in the rational drug design of pharmacologically optimized CB analogues.     

Functional characterization of the novel antipsychotic iloperidone at human D2, D3, alpha 2C, 5-HT6, and 5-HT1A receptors

Iloperidone has demonstrated an interesting monoamine receptor profile in radioligand binding studies, with nanomolar affinity for certain noradrenaline, dopamine, and serotonin receptors. In this study, the agonist/antagonist activity of iloperidone was determined in cell lines expressing recombinant human D(2A), D(3), alpha(2C), 5-HT(1A), or 5-HT(6) receptors. With the exception of 5-HT(6) receptors, these receptors are negatively coupled to cyclase. Thus, after stimulation with forskolin, the agonists dopamine (at D(2A) and D(3)), noradrenaline (at alpha(2C)), or 8-OH-DPAT (at 5-HT(1A)) induced a reduction in cAMP accumulation. Conversely, activation of the 5-HT(6) receptor by 5-HT led to an increase in cAMP accumulation. Iloperidone alone was devoid of significant agonist activity but inhibited the agonist response in all 5 cell lines in a surmountable and concentration-dependent fashion. Iloperidone was most potent at D(3) receptors (pK(B) 8.59 +/- 0.20; n = 6), followed by alpha(2C) (pK(B) 7.83 +/- 0.06; n = 15), 5-HT(1A) (pK(B) 7.69 +/- 0.18; n = 10), D(2A) (pK(B) 7.53 +/- 0.04; n = 11) and 5-HT(6) (pK(B) 7.11 +/- 0.08; n = 11) receptors.     

Synthesis, dopaminergic profile, and molecular dynamics calculations of N-aralkyl substituted 2-aminoindans

Brain dopaminergic system has a crucial role in the etiology of several neuropsychiatric disorders, including Parkinson's disease, depression, and schizophrenia. Several dopaminergic drugs are used to treat these pathologies, but many problems are attributed to these therapies. Within this context, the search for new more efficient dopaminergic agents with less adverse effects represents a vast research field. The aim of the present study was to synthesize N-[2-(4,5-dihydroxyphenyl)-methyl-ethyl]-4,5-dihydroxy-2-aminoindan hydrobromide (3), planned to be a dopamine ligand, and to evaluate its dopaminergic action profile. This compound was assayed as a diastereoisomeric mixture in two experimental models: stereotyped behavior (gnaw) and renal urinary response, after central administration. The pharmacological results showed that compound 3 significantly blocked the apomorphine-induced stereotypy and dopamine-induced diuresis and natriuresis in rats. Thus, compound 3 demonstrated an inhibitory effect on dopaminergic-induced behavior and renal action. N-[2-(-Methyl-ethyl)]-4,5-dihydroxy-2-aminoindan hydrobromide (4) was previously reported as an inotropic agent, and in the present work it was also re-evaluated as a diastereoisomeric mixture for its possible central action on the behavior parameters such as stereotypy and dopamine-induced diuresis and natriuresis in rats. Our results indicate that compound 4 produces an agonistic response, possibly through dopaminergic mechanisms. To better understand the experimental results we performed molecular dynamics simulations of two complexes: compound 3/D(2)DAR (dopamine receptor) and compound 4/D(2)DAR. The differential binding mode obtained for these complexes could explain the antagonist and agonist activity obtained for compounds 3 and 4, respectively.     

CCK(B)/gastrin receptor mediates synergistic stimulation of DNA synthesis and cyclin D1, D3, and E expression in Swiss 3T3 cells.

In order to develop a model system for identifying signaling pathways and cell cycle events involved in gastrin-mediated mitogenesis, we have used high efficiency retroviral-mediated transfection of cholecystokinin (CCK)(B)/gastrin receptor into Swiss 3T3 cells. The retrovirally-transfected CCK(B)/gastrin receptor binds 125I-CCK-8 with high affinity (Kd = 1.1 nM) and is functionally coupled to intracellular signaling pathways including rapid and transient increase in Ca2+ fluxes, protein kinase C-dependent protein kinase D activation, and MEK-dependent ERK1/2 activation. In the presence of insulin, CCK-8 or gastrin induced a 66.5 +/- 8.8-fold (mean +/- SEM, n = 24 in eight independent experiments) increase in cellular DNA synthesis, reaching a level similar to that achieved by stimulation with a saturating concentration of fresh serum, and much greater than the response to each agonist added alone. CCK-8 also induced a striking increase in the expression of cyclins D1, D3, and E and hyperphosphorylation of Rb acting synergistically with insulin. Similar effects were observed when CCK(B)/gastrin receptor was activated in the presence of EGF or bombesin. Our results demonstrate that activation of CCK(B)/gastrin receptor retrovirally-transfected into Swiss 3T3 induces a potent synergistic effect on DNA synthesis, accumulation of cyclins D1, D3, and E and hyperphosphorylation of Rb in combination with insulin, EGF, or bombesin. Thus, the CCK(B)/gastrin receptor transfected into Swiss 3T3 cells provides a novel model system to elucidate mitogenic signal transduction pathways and cell cycle events activated via this receptor.     

Dopamine-D2S receptor inhibition of calcium influx,adenylyl cyclase,and mitogen-activated protein kinase in pituitary cells: distinct Galpha and Gbetagamma requirements

The G protein specificity of multiple signaling pathways of the dopamine-D2S (short form) receptor was investigated in GH4ZR7 lactotroph cells. Activation of the dopamine-D2S receptor inhibited forskolin-induced cAMP production, reduced BayK8644- activated calcium influx, and blocked TRH-mediated p42/p44 MAPK phosphorylation. These actions were blocked by pretreatment with pertussis toxin (PTX), indicating mediation by G(i/o) proteins. D2S stimulation also decreased TRH-induced MAPK/ERK kinase phosphorylation. TRH induced c-Raf but not B-Raf activation, and the D2S receptor inhibited both TRH-induced c-Raf and basal B-Raf kinase activity. After PTX treatment, D2S receptor signaling was rescued in cells stably transfected with individual PTX-insensitive Galpha mutants. Inhibition of adenylyl cyclase was partly rescued by Galpha(i)2 or Galpha(i)3, but Galpha(o) alone completely reconstituted D2S-mediated inhibition of BayK8644-induced L-type calcium channel activation. Galpha(o) and Galpha(i)3 were the main components involved in D2S-mediated p42/44 MAPK inhibition. In cells transfected with the carboxyl-terminal domain of G protein receptor kinase to inhibit Gbetagamma signaling, only D2S-mediated inhibition of calcium influx was blocked, but not inhibition of adenylyl cyclase or MAPK. These results indicate that the dopamine-D2S receptor couples to distinct G(i/o) proteins, depending on the pathway addressed, and suggest a novel Galpha(i)3/Galpha(o)-dependent inhibition of MAPK mediated by c-Raf and B-Raf-dependent inhibition of MAPK/ERK kinase.     

On the specificity of vitamin D compounds binding to chick pig intestinal 1,25-dihydroxyvitamin D3 receptor

The binding activity of four vitamin D metabolites and/or analogs for the intestinal 1,25-dihydroxyvitamin D3 receptor was evaluated after incubation at 25 degrees C for 1 h or at 0-4 degrees C for 18 h. The incubation conditions, which had no effect on the binding of 1,25-dihydroxyvitamin D3, had a dramatic effect on the binding of 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3 and a small but reproducible effect on 24,25-dihydroxyvitamin D3 binding to receptor. Affinities 10- to 20-fold higher were obtained for 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, and affinities 3-fold higher were obtained for 24,25-dihydroxyvitamin D3 at the 0-4 degrees C/18-h incubation. A comparison of intestinal receptor from chick and pig with nine vitamin D compounds showed no major differences between the two species. The relative affinity of the vitamin D analogs to compete with tritiated 1,25-dihydroxyvitamin D3 for the receptor in pig nuclear extract, expressed as ratios of the molar concentration required for 50% binding of the tritiated 1,25-dihydroxyvitamin D3 compared to nonradioactive 1,25-dihydroxyvitamin D3, are as follows: 1,25-dihydroxyvitamin D3 (1) = 1,25-dihydroxyvitamin D2 = 24-homo-1,25-dihydroxyvitamin D3 greater than 1,24,25-trihydroxyvitamin D3 (4) greater than 25-hydroxyvitamin D3 (21) = 10-oxo-19-nor-25-hydroxyvitamin D3 = 1 alpha-hydroxyvitamin D3 (37) greater than 24,25-dihydroxyvitamin D2 (257) much much greater than vitamin D3 (greater than 10(6)).     

Decorin, a novel player in the insulin-like growth factor system

Decorin is a multifunctional proteoglycan that is expressed by sprouting endothelial cells. Its expression supports capillary formation and cell survival. Previously, it was shown that some effects of decorin are mediated by protein kinase B and the cyclin-dependent kinase inhibitor, p21. However, the cell surface receptor responsible for these effects was unknown. We demonstrate that decorin binds to the insulin-like growth factor-I (IGF-I) receptor on endothelial cells with an affinity in the nanomolar range (K(D) = 18 nm), which is comparable with IGF-I (K(D) = 1.2 nm). Furthermore, decorin can bind IGF-I itself, but with a lower affinity (K(D) = 190 nm) than classical IGF-I-binding proteins. Decorin addition causes IGF-I receptor phosphorylation and activation, which is followed by receptor down-regulation. These effects are caused by the core protein of decorin, and the binding region could be mapped to the N terminus of the molecule. The physiological relevance of the decorin/IGF-I receptor interaction was corroborated in two animal models (e.g. inflammatory angiogenesis in the cornea and unilateral ureteral obstruction). In both models the IGF-I receptor was up-regulated in decorin-deficient mice compared with controls and the up-regulation could not compensate the decorin deficiency in the disease models. These data indicate that decorin is an important player in the IGF system and its loss cannot fully be compensated in different types of diseases.     

The vitamin D receptor is necessary for 1alpha,25-dihydroxyvitamin D(3) to suppress experimental autoimmune encephalomyelitis in mice

The active metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D(3), suppresses autoimmune disease in several animal models including experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. The molecular mechanism of this immunosuppression is at present unknown. While 1alpha,25-dihydroxyvitamin D(3) is believed to function through a single vitamin D receptor, there are reports of other vitamin D receptors as well as a "nongenomic" mode of action. We have prepared the EAE model possessing the vitamin D receptor null mutation and determined if 1alpha,25-dihydroxyvitamin D(3) can suppress this disease in the absence of a functional vitamin D receptor. Vitamin D receptor null mice develop EAE although the incidence rate is one-half that of wild-type controls. The administration of 1alpha,25-dihydroxyvitamin D(3) had no significant effect on the incidence of EAE in the vitamin D receptor null mice, while it completely blocked EAE in the wild-type mice. We conclude that 1alpha,25-dihydroxyvitamin D(3) functions to suppress EAE through the well-known VDR and not through an undiscovered receptor or through a "nongenomic" mechanism.     

Synthesis, receptor binding and functional studies of mesoridazine stereoisomers

The four stereoisomers of mesoridazine were synthesized and evaluated in D2, 5-HT1A, 5-HT2A, 5-HT2C, D1, and D3 receptor binding and functional assays. Two isomers demonstrated potent D2 receptor binding (Ki < 3 nM) and functional antagonism (IC50 < or = 10 nM) activities. These two isomers also showed moderate affinity for the 5-HT2A and D3 receptors. A third isomer was devoid of significant D2 receptor binding, but did have moderate affinity for the 5-HT2A and D3 receptors. The fourth isomer demonstrated poor affinity for all the receptors tested. Most significantly, the stereochemistry of the sulfoxide moiety played a dominant role in the observed structure-activity relationship (SAR).     

Molecular modeling and 3D-QSAR studies of indolomorphinan derivatives as kappa opioid antagonists

Molecular modeling and 3D-QSAR studies were performed on 31 indolomorphinan derivatives to evaluate their antagonistic behaviors on kappa opioid receptor and provide information for further modification of this kind of compounds. Best predictions were obtained with CoMFA standard model (q2 = 0.693, N = 4, r2 = 0.900) and CoMSIA combined model (q2 = 0.617, N = 4, r2 = 0.904). Both models were further validated by an external test set of eight compounds with satisfactory predictions: r2 = 0.607 for CoMFA and r2 = 0.701 for CoMSIA. In addition, the 3D structure of human kappa opioid receptor was constructed based on the crystal structure of bovine rhodopsin, and the CoMSIA contour plots were then mapped into the structural model of kappa opioid receptor-GNTI complex to identify key residues, which might account for kappa antagonist potency and selectivity. The roles of nonconserved Glu297 and conserved Lys227 of human kappa opioid receptor were then discussed.     

Molecular modeling of the second extracellular loop of G-protein coupled receptors and its implication on structure-based virtual screening

The current study describes the validation of high-throughput modeling procedures for the construction of the second extracellular loop (ecl2) of all nonolfactory human G Protein-coupled receptors. Our modeling flowchart is based on the alignment of essential residues determining the particular ecl2 fold observed in the bovine rhodopsin (bRho) crystal structure. For a set of GPCR targets, the dopamine D2 receptor (DRD2), adenosine A3 receptor (AA3R), and the thromboxane A2 receptor (TA2R), the implications of including ecl2 atomic coordinates is evaluated in terms of structure-based virtual screening accuracy: the suitability of the 3D models to distinguish between known antagonists and randomly chosen decoys using automated docking approaches. The virtual screening results of different models describing increasingly exhaustive receptor representations (seven helices only, seven helices and ecl2 loop, full model) have been compared. Explicit modeling of the ecl2 loop was found to be important in only one of three test cases whereas a loopless model was shown to be accurate enough in the two other receptors. An exhaustive comparison of ecl2 loops of 365 receptors to that of bRho suggests that explicit ecl2 loop modeling should be reserved to receptors where loop building can be guided by experimental restraints.