Rhodopsin-based Model Research Articles

Mapping the binding pocket of a novel, high-affinity, slow dissociating tachykinin NK3 receptor antagonist: Biochemical and electrophysiological characterization

The NK3 receptor is a GPCR that is prominently expressed in limbic areas of the brain, many of which have been implicated in schizophrenia. Phase II clinical trials in schizophrenia with two selective NK3 antagonists (osanetant and talnetant) have demonstrated significant improvement in positive symptoms. The objective of this study was to characterize the properties of a novel dual NK2/NK3 antagonist, RO5328673. [3H]RO5328673 bound to a single saturable site on hNK2, hNK3 and gpNK3 with high-affinity. RO5328673 acted as an insurmountable antagonist at both human and guinea-pig NK3 receptors in the [3H]IP accumulation assay. In binding kinetic analyses, [3H]RO5328673 had fast association and dissociation rates at hNK2 while it had a fast association rate and a remarkably slow dissociation rate at gp and hNK3. In electrophysiological recordings of gp SNpc, RO5328673 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurons with an insurmountable mechanism of action. RO5328673 exhibited in-vivo activity in gerbils, robustly reversing the senktide-induced locomotor activity. The TM2 residue gpNK3-A1142.58 (threonine in all other species) was identified as the critical residue for the RO5328673's slower dissociation kinetics and stronger insurmountable mode of antagonism in the guinea-pig as compared to hNK3-T1392.58. Using site-directed mutagenesis, [3H]RO5328673 binding and rhodopsin-based modeling, the important molecular determinants of the RO5328673-binding pocket of hNK3 were determined. A comparison of the RO5328673-binding pocket with that of osanetant showed that two antagonists have similar contact sides on hNK3 binding crevice except for three mutations V95L1.42, Y247W5.38, V255I5.46, which behaved differently between interacting modes of two antagonists in hNK3.

Open conformation of adipokinetic hormone receptor from the malaria mosquito facilitates hormone binding

Insect flight requires rapid mobilization of energy reserves during flight, which is mediated and regulated by hormonal control via adipokinetic hormones. The structure of the G-protein receptors to which these hormones bind, are crucial in understanding many of the physiological processes in which they play a central role. To date no 3D structure of an insect G-protein coupled receptor (GPCR) is available. Here, the first models of the 3D structures of a GPCR from the malaria mosquito are presented. Homology modeling of the receptor identified from the genome of Anopheles gambiae was used to construct two models of the receptor. The 7 transmembrane helical bundles of these two models are based on the crystal structures of beta2-adrenergic receptor and rhodopsin. The flexible loop regions were modeled using high temperature simulated annealing and constrained molecular dynamic simulations. The two receptor models differ in a number of critical features, the most important of which is that the rhodopsin-based model has a ‘closed’ structure while the beta2-based structure is ‘open’. The ‘open’ conformation provides easy access of the hormone to the binding pocket. Docking calculations with the insect adipokinetic hormones, AKH-1 (pGlu-Leu-Thr-Phe-Thr-Pro-Ala-Trp-NH 2) from the malaria mosquito and Del-CC (pGlu-Lys-Asn-Phe-Ser-Pro-Asn-Trp-Gly-Asn-NH 2) from the blister beetle showed that while the binding motif of the two is similar, AKH-1 has more than 30 times higher affinity than Del-CC, which strongly suggests that the binding is specific, and that the correct binding site was identified. Using these models it is possible to design antagonists, which block the binding site and are thus species-specific insecticides.

Adenosine Receptor Modeling: What Does the A2A Crystal Structure Tell Us?

For a long time, there have been no experimentally determined structural data for any adenosine receptor (AR) and the only approach available for making structure/function correlations about these proteins has been homology modeling. While the early attempts to model these receptors followed the crystallization of bacteriorhodopsin, the cryo-microscopy studies of bovine and frog rhodopsin, and the modeling of a Calpha-template for the TM helices in the rhodopsin family of GPCRs, the crystallization of bovine rhodopsin by Palczewski was of extreme importance as it first provided the crystal structure of an eukaryotic GPCR to be used as template for more realistic homology models. Since then, rhodopsin-based modeling became the routine approach to develop AR structural models that proved to be useful for interpretation of site-directed mutagenesis data and for molecular docking studies. The recently reported crystal structures of the adrenergic beta1 and beta2 receptors only partially confirmed the structural features showed by bovine rhodopsin, raising a question about which template would have been better for further modeling of ARs. Such question remained actually not-answered, due to the publication in late 2008 of the crystal structure of human adenosine A(2A) receptor (AA(2A)R). Since its publication, this structure has been used for ligands docking analysis and has provided a high similarity template for homology modeling of the other AR subtypes. Still, the AA(2A)R crystal structure allows to verify the hypotheses that were made on the basis of the previously reported homology modeling and molecular docking.

A computational study on cannabinoid receptors and potent bioactive cannabinoid ligands: homology modeling, docking, de novo drug design and molecular dynamics analysis

When X-ray structure of a ligand-bound receptor is not available, homology models of the protein of interest can be used to obtain the ligand-binding cavities. The steroelectronic properties of these cavities are directly related to the performed molecular model coordinates. Thus, the use of different template structures for homology may result in variation of ligand-binding modes. We have recently reported the MD simulations of a potent CB ligand at bovine rhodopsin-based CB1 and CB2 receptors (Durdagi et al., Bioorg Med Chem 16:7377-7387, 2008). In this present study, a homology modeling study based on the beta2-adrenergic receptor for both CB1 and CB2 receptors was performed, and the results were compared with rhodopsin-based models. In addition, the role of membrane bilayers to the adopted conformations of potent AMG3 CB ligand has been analyzed for receptor-free and membrane-associated receptor systems. The performed MD trajectory analysis results have shown that gauche conformations at the terminal segment of the alkyl side chain of AMG3 are not favored in solution. Different adopting dihedral angles defined between aromatic and dithiolane rings at the active sites of the CB1 and CB2 receptors, which are adapted lead to different alkyl side chain orientations and thus, may give clues to the medicinal chemists to synthesize more selective CB ligands. The binding sites of receptors derived by rhodopsin-based models have been regenerated using the beta2-adrenergic based template receptors. The re-obtained models confirmed the ligand-binding pockets that were derived based on rhodopsin.

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.

Molecular modeling of A1 and A2A adenosine receptors: Comparison of rhodopsin‐ and β2‐adrenergic‐based homology models through the docking studies

Adenosine receptors (ARs) are members of the superfamily of G protein-coupled receptors. The homology models of adenosine A1 and A2A receptors were constructed. The high-resolution X-ray structure of bovine rhodopsin and crystal structure of beta2-adrenergic receptor were used as templates. The binding sites of the A1 and A2A ARs were constructed by using data obtained from mutagenesis experiments as well as docking simulations of the respective AR antagonsists DPCPX and XAC. To compare rhodopsin- and beta2-adrenergic-based models, the binding mode of A1 (KW-3902, LUF-5437) and A2A (KW-6002, ZM-241385) ARs antagonists were also examined. The differences in the binding ability of both models were noted during the study. The beta2-adrenergic-based A2A AR model was much more capable to stabilize the ligand in the binding site cavity than the corresponding rhodopsin-based A2A AR model, however, such differences were not so clear in case of A1 AR models. It was suggested that for the A1 AR it is possible to use the crystal structure of rhodopsin as a template as well as beta2-adrenergic receptor, but for A2A AR, with the now available beta2-adrenergic receptor X-ray structure, docking studies should be avoided on the rhodopsin-based model. However, taking into account that the beta2AR shares about 31% of the residues with the AR in comparison to 21% in case of bRho, we suggest using beta2-adrenergic-based models for the A1 and A2A ARs for further in silico ligand screening also because of their generally better ability to stabilize ligands inside the binding pocket.

Me-Talnetant and Osanetant Interact within Overlapping but Not Identical Binding Pockets in the Human Tachykinin Neurokinin 3 Receptor Transmembrane Domains

Recent clinical trials have indicated that neurokinin 3 receptor antagonists (S)-(+)-N-{{3-[1-benzoyl-3-(3,4-dichlorophenyl)-piperidin-3-yl]prop-1-yl}-4-phenylpiperidin-4-yl}-N-methylacetamine (SR142801; osanetant) and (S)-(-)-N-(alpha-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carboxamide (SB223412; talnetant) may treat symptoms of schizophrenia. Using site-directed mutagenesis, rhodopsin-based modeling, [(3)H](S)-(-)-N-(alpha-ethylbenzyl)-3-methoxy-2-phenylquinoline-4-carboxamide (Me-talnetant) and [(3)H]osanetant binding, and functional Schild analyses, we have demonstrated the important molecular determinants of neurokinin B (NKB), Me-talnetant, and osanetant binding pockets. The residues Asn138(2.57), Asn142(2.61), Leu232(45.49), Tyr315(6.51), Phe342(7.39), and Met346(7.43) were found to be crucial for the NKB binding site. We observed that the M134(2.53)A, V169(3.36)M, F342(7.39)M, and S341(7.38)I/F342(7.39)M mutations resulted in the complete loss of [(3)H]Metalnetant and [(3)H]osanetant binding affinities and also abolished their functional potencies in an NKB-evoked accumulation of [(3)H]inositol phosphates assay, whereas the mutations V95(1.42)A, N142(2.61)A, Y315(6.51)F, and M346(7.43)A behaved differently between the interacting modes of two antagonists. V95(1.42)A and M346(7.43)A significantly decreased the affinity and potency of Me-talnetant. Y315(6.51)F, although not affecting Me-talnetant, led to a significant decrease in affinity and potency of osanetant. The mutation N142(2.61)A, which abolished the potency and affinity of osanetant, led to a significant increase in the affinity and potency of Me-talnetant. The proposed docking mode was further validated using (S)-2-(3,5-bis-trifluoromethyl-phenyl)-N-[4-(4-fluoro-2-methyl-phenyl)-6-((S)-4-methanesulfonyl-3-methyl-piperazin-1-yl)-pyridin-3-yl]-N-methyl-isobutyramide (RO49085940), from another chemical class. It is noteworthy that the mutation F342(7.39)A caused an 80-fold gain of RO4908594 binding affinity, but the same mutation resulted in the complete loss of the affinity of Me-talnetant and partial loss of the affinity of osanetant. These observations show that the binding pocket of Me-talnetant and osanetant are overlapping, but not identical. Taken together, our data are consistent with the proposed docking modes where Me-talnetant reaches deeply into the pocket formed by transmembrane (TM)1, -2, and -7, whereas osanetant fills the pocket TM3, -5, and -6 with its phenyl-piperidine moiety.

Mechanism of Activation of a G Protein-coupled Receptor, the Human Cholecystokinin-2 Receptor

G protein-coupled receptors (GPCRs) represent a major focus in functional genomics programs and drug development research, but their important potential as drug targets contrasts with the still limited data available concerning their activation mechanism. Here, we investigated the activation mechanism of the cholecystokinin-2 receptor (CCK2R). The three-dimensional structure of inactive CCK2R was homology-modeled on the basis of crystal coordinates of inactive rhodopsin. Starting from the inactive CCK2R modeled structure, active CCK2R (namely cholecystokinin-occupied CCK2R) was modeled by means of steered molecular dynamics in a lipid bilayer and by using available data from other GPCRs, including rhodopsin. By comparing the modeled structures of the inactive and active CCK2R, we identified changes in the relative position of helices and networks of interacting residues, which were expected to stabilize either the active or inactive states of CCK2R. Using targeted molecular dynamics simulations capable of converting CCK2R from the inactive to the active state, we delineated structural changes at the atomic level. The activation mechanism involved significant movements of helices VI and V, a slight movement of helices IV and VII, and changes in the position of critical residues within or near the binding site. The mutation of key amino acids yielded inactive or constitutively active CCK2R mutants, supporting this proposed mechanism. Such progress in the refinement of the CCK2R binding site structure and in knowledge of CCK2R activation mechanisms will enable target-based optimization of nonpeptide ligands.

Homology Modeling of the Serotonin 5-HT1A Receptor Using Automated Docking of Bioactive Compounds with Defined Geometry

This paper describes a rhodopsin-based model of 5-HT(1A) serotonin receptor. The flexibility of the receptor was considered by using large number of models for ligand dockings. Rearrangements of the heptahelical bundle were introduced, which resulted in the improvement of correlation between computational results and experimental data. The model was validated by automated docking of conformationally restricted arylpiperazines. Specific interactions, responsible for the recognition of arylpiperazine derivatives, were identified. An ionic bond was formed between the protonated amine of ligands and Asp3.32. The aromatic moiety and its substituents specifically interacted with Phe6.52 and Ser5.42, respectively, while the carbonyl groups of imide part of ligands formed hydrogen bonds with Asn7.39 and Tyr7.43. The model reproduced the binding affinity of the test group of ligands (correlation r = 0.8 between pK(i) and docking score). It also gave the enrichment in virtual screening-like experiment (100 compounds), in which 34 high-affinity compounds were found among 50 top-scored ligands.

Synthesis, Biological Evaluation, and Molecular Modeling of Ribose-Modified Adenosine Analogues as Adenosine Receptor Agonists

A number of 3'-C-methyl analogues of selective adenosine receptor agonists such as CPA, CHA, CCPA, 2'-Me-CCPA, NECA, and IB-MECA was synthesized to further investigate the subdomain of the receptor that binds the ribose moiety of the ligands. Affinity data at A(1), A(2A), and A(3) receptors in bovine brain membranes showed that the 3'-C-modification in adenosine resulted in a decrease of the affinity at all three receptor subtypes. When this modification was combined with N(6)-substitution with groups that induce high potency and selectivity at A(1) receptor, the affinity and selectivity were increased. However, all 3'-C-methyl derivatives proved to be very less active than the corresponding 2'-C-methyl analogues. The most active compound was found to be 3'-Me-CPA which displayed a K(i) value of 0.35 microM at A(1) receptor and a selectivity for A(1) vs A(2A) and A(3) receptors higher than 28-fold. 2'-Me-CCPA was confirmed to be the most selective, high affinity agonist so far known also at human A(1) receptor with a K(i) value of 3.3 nM and 2903- and 341-fold selective vs human A(2A) and A(3) receptors, respectively. In functional assay, 3'-Me-CPA, 3'-Me-CCPA, and 2-Cl-3'-Me-IB-MECA inhibited forskolin-stimulated adenylyl cyclase activity with IC(50) values ranging from 0.3 to 4.9 microM, acting as full agonists. A rhodopsin-based model of the bovine A(1)AR was built to rationalize the higher affinity and selectivity of 2'-C-methyl derivatives of N(6)-substituted-adenosine compared to that of 3'-C-methyl analogues. In the docking exploration, it was found that 2'-Me-CCPA was able to form a number of interactions with several polar residues in the transmembrane helices TM-3, TM-6, and TM-7 of bA(1)AR which were not preserved in the molecular dynamics simulation of 3'-Me-CCPA/bA(1)AR complex.

Combined Target-Based and Ligand-Based Drug Design Approach as a Tool To Define a Novel 3D-Pharmacophore Model of Human A3Adenosine Receptor Antagonists: Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine Derivatives as a Key Study

A combined target-based and ligand-based drug design approach has been carried out to define a novel pharmacophore model of the human A(3) receptor antagonists. High throughput molecular docking and comparative molecular field analysis (CoMFA) have been used in tandem to assemble a new target based pharmacophore model. In parallel, to provide more accurate information about the putative binding site of these A(3) inhibitors, a rhodopsin-based model of the human A(3) receptor was built and a novel Y-shape binding motif has been proposed. Docking-based structure superimposition has been used to perform a quantitative study of the structure-activity relationships for binding of these pyrazolo-triazolo-pyrimidines to adenosine A(3) receptor using CoMFA. Both steric and the electrostatic contour plots obtained from the CoMFA analysis nicely fit on the hypothetical binding site obtained by molecular docking. On the basis of the combined hypothesis, we have designed, synthesized, and tested 17 new derivatives. Consistently, the predicted K(i) values were very close to the experimental values.

Synthesis, screening, and molecular modeling of new potent and selective antagonists at the alpha 1d adrenergic receptor.

In the present study, more than 75 compounds structurally related to BMY 7378 have been designed and synthesized. Structural variations of each part of the reference molecule have been introduced, obtaining highly selective ligands for the alpha(1d) adrenergic receptor. The molecular determinants for selectivity at this receptor are essentially held by the phenyl substituent in the phenylpiperazine moiety. The integration of an extensive SAR analysis with docking simulations using the rhodopsin-based models of the three alpha(1)-AR subtypes and of the 5-HT(1A) receptor provides significant insights into the characterization of the receptor binding sites as well as into the molecular determinants of ligand selectivity at the alpha(1d)-AR and the 5-HT(1A) receptors. The results of multiple copies simultaneous search (MCSS) on the substituted phenylpiperazines together with those of manual docking of compounds BMY 7378 and 69 into the putative binding sites of the alpha(1a)-AR, alpha(1b)-AR, alpha(1d)-AR, and the 5-HT(1A) receptors suggest that the phenylpiperazine moiety would dock into a site formed by amino acids in helices 3, 4, 5, 6 and extracellular loop 2 (E2), whereas the spirocyclic ring of the ligand docks into a site formed by amino acids of helices 1, 2, 3, and 7. This docking mode is consistent with the SAR data produced in this work. Furthermore, the binding site of the imide moiety does not allow for the simultaneous involvement of the two carbonyl oxygen atoms in H-bonding interactions, consistent with the SAR data, in particular with the results obtained with the lactam derivative 128. The results of docking simulations also suggest that the second and third extracellular loops may act as selectivity filters for the substituted phenylpiperazines. The most potent and selective compounds for alpha(1d) adrenergic receptor, i.e., 69 (Rec 26D/038) and 128 (Rec 26D/073), are characterized by the presence of the 2,5-dichlorophenylpiperazine moiety.

Engineering of A3 Adenosine and P2Y Nucleotide Receptors and Their Ligands.

Modification of the ribose moiety of nucleotides and nucleosides has provided new insights into structural and conformational requirements for ligands at P2Y nucleotide receptors and at adenosine receptors (ARs). Methanocarba derivatives (containing a rigid bicyclic ring system in place of ribose) of adenosine, ATP, ADP, UTP, UDP, and other receptor agonist analogs were synthesized. Biological evaluation led to the conclusion that in general the Northern (N)-conformation was favored over the Southern (S)-conformation of the pseudoribose moiety at A1 and A3 ARs and at P2Y1, P2Y2, P2Y4, or P2Y11 receptors, but not P2Y6 receptors. At the hA3 AR a new full agonist, MRS1898, the (N)-methanocarba equivalent of CI-IB-MECA (2-chloro-N 6-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine), had a Ki value of 1.9 nM in binding to the hA3 AR expressed in CHO cells. Functional assays confirmed the selectivity of MRS1898, although CI-IB-MECA was even more functionally selective for human A3 vs. hA1 and hA2A ARs. Thirty μM MRS1898 did not induce apoptosis in HL-60 cells, suggesting that some of the proapoptotic effects of CI-IB-MECA may be nonreceptor-mediated. Manipulation of the sequence of A3 ARs through site-directed mutagenesis has led to pharmacologically unique constructs: constitutively active receptors and "neoceptors." Such engineered receptors may later prove to have potential for cardioprotection through gene transfer. Effects of single amino acid replacement were interpreted using a rhodopsin-based model of ligand-A3 receptor interactions, leading to the proposal that a movement of the conserved W243 in TM6 may be involved in AR activation.

Identification by Site-directed Mutagenesis of Residues Involved in Ligand Recognition and Activation of the Human A3 Adenosine Receptor

Ligand recognition has been extensively explored in G protein-coupled A(1), A(2A), and A(2B) adenosine receptors but not in the A(3) receptor, which is cerebroprotective and cardioprotective. We mutated several residues of the human A(3) adenosine receptor within transmembrane domains 3 and 6 and the second extracellular loop, which have been predicted by previous molecular modeling to be involved in the ligand recognition, including His(95), Trp(243), Leu(244), Ser(247), Asn(250), and Lys(152). The N250A mutant receptor lost the ability to bind both radiolabeled agonist and antagonist. The H95A mutation significantly reduced affinity of both agonists and antagonists. In contrast, the K152A (EL2), W243A (6.48), and W243F (6.48) mutations did not significantly affect the agonist binding but decreased antagonist affinity by approximately 3-38-fold, suggesting that these residues were critical for the high affinity of A(3) adenosine receptor antagonists. Activation of phospholipase C by wild type (WT) and mutant receptors was measured. The A(3) agonist 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine stimulated phosphoinositide turnover in the WT but failed to evoke a response in cells expressing W243A and W243F mutant receptors, in which agonist binding was less sensitive to guanosine 5'-gamma-thiotriphosphate than in WT. Thus, although not important for agonist binding, Trp(243) was critical for receptor activation. The results were interpreted using a rhodopsin-based model of ligand-A(3) receptor interactions.

3-Aryl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones: a new class of selective A1 adenosine receptor antagonists.

Radioligand binding assays using bovine cortical membrane preparations and biochemical in vitro studies revealed that various 3-aryl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one (ATBI) derivatives, previously reported by us as ligands of the central benzodiazepine receptor (BzR) (Primofiore, G.; et al. J. Med. Chem. 2000, 43, 96-102), behaved as antagonists at the A1 adenosine receptor (A1AR). Alkylation of the nitrogen at position 10 of the triazinobenzimidazole nucleus conferred selectivity for the A1AR vs the BzR. The most potent ligand of the ATBI series (10-methyl-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one 12) displayed a Ki value of 63 nM at the A1AR without binding appreciably to the adenosine A2A and A3 nor to the benzodiazepine receptor. Pharmacophore-based modeling studies in which 12 was compared against a set of well-established A1AR antagonists suggested that three hydrogen bonding sites (HB1 acceptor, HB2 and HB3 donors) and three lipophilic pockets (L1, L2, and L3) might be available to antagonists within the A1AR binding cleft. According to the proposed pharmacophore scheme, the lead compound 12 engages interactions with the HB2 site (via the N2 nitrogen) as well as with the L2 and L3 sites (through the pendant and the fused benzene rings). The results of these studies prompted the replacement of the methyl with more lipophilic groups at the 10-position (to fill the putative L1 lipophilic pocket) as a strategy to improve A1AR affinity. Among the new compounds synthesized and tested, the 3,10-diphenyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one (23) was characterized by a Ki value of 18 nM which represents a 3.5-fold gain of A1AR affinity compared with the lead 12. A rhodopsin-based model of the bovine adenosine A1AR was built to highlight the binding mode of 23 and two well-known A1AR antagonists (III and VII) and to guide future lead optimization projects. In our docking simulations, 23 receives a hydrogen bond (via the N1 nitrogen) from the side chain of Asn247 (corresponding to the HB1 and HB2 sites) and fills the L1, L2, and L3 lipophilic pockets with the 10-phenyl, 3-phenyl, and fused benzene rings, respectively.

Synthesis, CoMFA analysis, and receptor docking of 3,5-diacyl-2, 4-dialkylpyridine derivatives as selective A3 adenosine receptor antagonists.

3,5-Diacyl-2,4-dialkyl-6-phenylpyridine derivatives have been found to be selective antagonists at both human and rat A3 adenosine receptors (Li et al. J. Med. Chem. 1998, 41, 3186-3201). In the present study, ring-constrained, fluoro, hydroxy, and other derivatives in this series have been synthesized and tested for affinity at adenosine receptors in radioligand binding assays. Ki values at recombinant human and rat A3 adenosine receptors were determined using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyladenosine). Selectivity for A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors, and structure-activity relationships at various positions of the pyridine ring (the 3- and 5-acyl substituents and the 2- and 4-alkyl substituents) were probed. At the 5-position inclusion of a beta-fluoroethyl (7) or a gamma-fluoropropyl ester (26) was favorable for human A3 receptor affinity, resulting in Ki values of 4.2 and 9.7 nM, respectively, while the pentafluoropropyl analogue was clearly less potent at human A3 receptors. At the 2-, 3-, and 4-positions, fluoro or hydroxy substitution failed to enhance potency and selectivity at human A3 receptors. Several analogues were nearly equipotent at rat and human A3 receptors. To further define the pharmacophore conformationally, a lactam, a lactone, and thiolactones were tested in adenosine receptor binding. The most potent analogue in this group was compound 34, in which a thiolactone was formed between 3- and 4-positions and which had a Ki value of 248 nM at human A3 receptors. Using affinity data and a general pharmacophore model for A3 adenosine receptor antagonists recently proposed, we applied comparative molecular field analysis (CoMFA) to obtain a three-dimensional quantitative structure-activity relationship for pyridine derivatives, having good predictability (r2pred = 0.873) for compounds in the test set. A rhodopsin-based model of the human A3 receptor was built, and the pyridine reference ligand 2,3,4, 5-tetraethyl-6-phenyl-pyridine-3-thiocarboxylate-5-carboxylate (MRS 1476) was docked in the putative ligand binding site. Interactions between receptor transmembrane domains and the steric and the electrostatic contour plots obtained from the CoMFA analysis were analyzed.

Mutagenesis of Human Mel1aMelatonin Receptor Expressed in Yeast Reveals Domains Important for Receptor Function

A yeast functional colorimetric assay was employed to test the effects of site-directed point mutations on the function of the human Mel1amelatonin receptor. Seven mutants were created in transmembrane domains III, V, and VII of the receptor to test the rhodopsin-based model of melatonin recognition. Two mutants in transmembrane domains III and VI were created to investigate the mechanisms of G protein activation in the melatonin receptor. Mutations in transmembrane domain V either potentiated agonist efficiencies (H195A) or totally abolished all responses to tested compounds (V192T+H195A). Mutation N124A in the conserved NRY motif in the end of transmembrane domain III seriously impaired receptor activation. Several mutants were found to have decreased ability to activate functional responses, reflecting the importance of these residues for receptor function. These data also suggest that activation of the receptor involves interaction of the 5-methoxy group of melatonin with the conserved histidine H195 in transmembrane domain V.

A rhodopsin-based model for melatonin recognition at its G protein-coupled receptor

The recent elucidation of the primary structures of different melatonin receptors as well as the deduction of the secondary structure of rhodopsin has allowed us to construct a model for melatonin recognition at its G protein-coupled receptor. To achieve this, we have used the quantum mechanics method Austin model 1 to fully optimize the structures of melatonin and several analogs. We also synthesized three compounds and used the three-dimensional analysis comparative molecular field analysis (CoMFA) to generate a model for the structure-activity relationships of melatonin and 27 melatonin-like compounds. This model predicted with good accuracy the affinities of the synthesized compounds for the melatonin receptor. We propose that recognition of the functional moieties of melatonin occurs through specific interaction of these moieties with fully conserved amino acid residues present in transmembrane helices V, VI and VII of the melatonin receptor. These residues are not found in other members of the G protein-coupled receptor family. The rhodopsin-based model can explain the importance of some structural features of melatonin and related active compounds.