Structure of the human κ-opioid receptor in complex with JDTic.

Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and--in the case of κ-opioid receptor (κ-OR)--dysphoria and psychotomimesis. Here we report the crystal structure of the human κ-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 Å resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human κ-OR. Modelling of other important κ-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5'-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure-activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for κ-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human κ-OR.

Chemotype-selective modes of action of κ-opioid receptor agonists.

The crystal structures of opioid receptors provide a novel platform for inquiry into opioid receptor function. The molecular determinants for activation of the κ-opioid receptor (KOR) were studied using a combination of agonist docking, functional assays, and site-directed mutagenesis. Eighteen positions in the putative agonist binding site of KOR were selected and evaluated for their effects on receptor binding and activation by ligands representing four distinct chemotypes: the peptide dynorphin A(1-17), the arylacetamide U-69593, and the non-charged ligands salvinorin A and the octahydroisoquinolinone carboxamide 1xx. Minimally biased docking of the tested ligands into the antagonist-bound KOR structure generated distinct binding modes, which were then evaluated biochemically and pharmacologically. Our analysis identified two types of mutations: those that affect receptor function primarily via ligand binding and those that primarily affect function. The shared and differential mechanisms of agonist binding and activation in KOR are further discussed. Usually, mutations affecting function more than binding were located at the periphery of the binding site and did not interact strongly with the various ligands. Analysis of the crystal structure along with the present results provide fundamental insights into the activation mechanism of the KOR and suggest that "functional" residues, along with water molecules detected in the crystal structure, may be directly involved in transduction of the agonist binding event into structural changes at the conserved rotamer switches, thus leading to receptor activation.

9-Aminomethyl-9,10-dihydroanthracene (AMDA) analogs as structural probes for steric tolerance in 5-HT2A and H1 receptor binding sites.

Synthesis, radioligand binding and molecular modeling studies of several 9-aminomethyl-9,10-dihydroanthracene (AMDA) analogs were carried out to determine the extent of the steric tolerance associated with expansion of the tricyclic ring system and amine substitution at 5-HT(2A) and H(1) receptors. A mixture of (7,12-dihydrotetraphene-12-yl)methanamine and (6,11-dihydrotetracene-11-yl)methanamine in a 75-25% ratio was found to have an apparent K(i) of 10nM at the 5-HT(2A) receptor. A substantial binding affinity for (7,12-dihydrotetraphene-3-methoxy-12-yl)methanamine at the 5-HT(2A) receptor (K(i)=21 nM) was also observed. Interestingly, this compound was found to have 100-fold selectivity for 5-HT(2A) over the H(1) receptor (K(i)=2500 nM). N-Phenylalkyl-AMDA derivatives, in which the length of the alkyl chain varied from methylene to n-butylene, were found to have only weak affinity for both 5-HT(2A) and H(1) receptors (K(i)=223 to 964 nM). Our results show that large rigid annulated AMDA analogs can be sterically accommodated within the proposed 5-HT(2A) binding site.

Structure-based design, synthesis, and biochemical and pharmacological characterization of novel salvinorin A analogues as active state probes of the kappa-opioid receptor.

Salvinorin A, the most potent naturally occurring hallucinogen, has attracted an increasing amount of attention since the kappa-opioid receptor (KOR) was identified as its principal molecular target by us [Roth, B. L., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11934-11939]. Here we report the design, synthesis, and biochemical characterization of novel, irreversible, salvinorin A-derived ligands suitable as active state probes of the KOR. On the basis of prior substituted cysteine accessibility and molecular modeling studies, C315(7.38) was chosen as a potential anchoring point for covalent labeling of salvinorin A-derived ligands. Automated docking of a series of potential covalently bound ligands suggested that either a haloacetate moiety or other similar electrophilic groups could irreversibly bind with C315(7.38). 22-Thiocyanatosalvinorin A (RB-64) and 22-chlorosalvinorin A (RB-48) were both found to be extraordinarily potent and selective KOR agonists in vitro and in vivo. As predicted on the basis of molecular modeling studies, RB-64 induced wash-resistant inhibition of binding with a strict requirement for a free cysteine in or near the binding pocket. Mass spectrometry (MS) studies utilizing synthetic KOR peptides and RB-64 supported the hypothesis that the anchoring residue was C315(7.38) and suggested one biochemical mechanism for covalent binding. These studies provide direct evidence of the presence of a free cysteine in the agonist-bound state of the KOR and provide novel insights into the mechanism by which salvinorin A binds to and activates the KOR.

Synthesis, structure-affinity relationships, and modeling of AMDA analogs at 5-HT2A and H1 receptors: structural factors contributing to selectivity.

Histamine H(1) and serotonin 5-HT(2A) receptors present in the CNS have been implicated in various neuropsychiatric disorders. 9-Aminomethyl-9,10-dihydroanthracene (AMDA), a conformationally constrained diarylalkyl amine derivative, has affinity for both of these receptors. A structure-affinity relationship (SAFIR) study was carried out studying the effects of N-methylation, varying the linker chain length and constraint of the aromatic rings on the binding affinities of the compounds with the 5-HT(2A) and H(1) receptors. Homology modeling of the 5-HT(2A) and H(1) receptors suggests that AMDA and its analogs, the parent of which is a 5-HT(2A) antagonist, can bind in a fashion analogous to that of classical H(1) antagonists whose ring systems are oriented toward the fifth and sixth transmembrane helices. The modeled orientation of the ligands are consistent with the reported site-directed mutagenesis data for 5-HT(2A) and H(1) receptors and provide a potential explanation for the selectivity of ligands acting at both receptors.

Methoxy-substituted 9-aminomethyl-9,10-dihydroanthracene (AMDA) derivatives exhibit differential binding affinities at the 5-HT(2A) receptor.

The effects of methoxy-substitution at the 1-, 2-, 3-, and 4-positions of 9-aminomethyl-9,10-dihydroanthracene (AMDA) on h5-HT(2A) receptor affinity were determined. Racemic mixtures of these compounds were found to show the following affinity trend: 3-MeO > 4-MeO > 1-MeO approximately 2-MeO. Comparison of the effects of these substitutions, with the aid of computational molecular modeling techniques, suggest that the various positional and stereochemical isomers of the methoxy-substituted AMDA compounds interact differently with the h5-HT(2A) receptor. It is predicted that for the compounds with higher affinities, the methoxy oxygen atom is able to interact with hydrogen bond-donating sidechains within alternative h5-HT(2A) receptor binding sites, whereas the lower-affinity isomers lack this ability.

Screening the receptorome to discover the molecular targets for plant-derived psychoactive compounds: a novel approach for CNS drug discovery.

Because psychoactive plants exert profound effects on human perception, emotion, and cognition, discovering the molecular mechanisms responsible for psychoactive plant actions will likely yield insights into the molecular underpinnings of human consciousness. Additionally, it is likely that elucidation of the molecular targets responsible for psychoactive drug actions will yield validated targets for CNS drug discovery. This review article focuses on an unbiased, discovery-based approach aimed at uncovering the molecular targets responsible for psychoactive drug actions wherein the main active ingredients of psychoactive plants are screened at the "receptorome" (that portion of the proteome encoding receptors). An overview of the receptorome is given and various in silico, public-domain resources are described. Newly developed tools for the in silico mining of data derived from the National Institute of Mental Health Psychoactive Drug Screening Program's (NIMH-PDSP) K(i) Database (K(i) DB) are described in detail. Additionally, three case studies aimed at discovering the molecular targets responsible for Hypericum perforatum, Salvia divinorum, and Ephedra sinica actions are presented. Finally, recommendations are made for future studies.

Single Amino Acid Variation Underlies Species-Specific Sensitivity to Amphibian Skin-Derived Opioid-like Peptides.

It has been suggested that the evolution of vertebrate opioid receptors (ORs) follow a vector of increased functionality. Here, we test this idea by comparing human and frog ORs. Interestingly, some of the most potent opioid peptides known have been isolated from amphibian skin secretions. Here we show that such peptides (dermorphin and deltorphin) are highly potent in the human receptors and inactive in frog ORs. The molecular basis for the insensitivity of the frog ORs to these peptides was studied using chimeras and molecular modeling. The insensitivity of the delta OR (DOR) to deltorphin was due to variation of a single amino acid, Trp7.35, which is a leucine in mammalian DORs. Notably, Trp7.35 is completely conserved in all known DOR sequences from lamprey, fish, and amphibians. The deltorphin-insensitive phenotype was verified in fish. Our results provide a molecular explanation for the species selectivity of skin-derived opioid peptides.

Application of ligand SAR, receptor modeling and receptor mutagenesis to the discovery and development of a new class of 5-HT(2A) ligands.

The present review describes our approach to the development of a structurally unique class of 5-HT(2A) ligands. On the basis of an abbreviated graphics model of a 5-HT(2A) serotonin receptor, it was hypothesized that introduction of an additional aromatic ring might enhance the affinity of phenylethylamine (an agent that lacks significant affinity for the 5-HT(2A) receptors). Continued work with such structures, and the continual refinement of graphics receptor models, ultimately led to the identification of AMDA (27, 5-HT(2A) K(i) = 20 nM). AMDA is a 5-HT(2A) antagonist that, unlike certain other tricyclic 5-HT(2A) antagonists, binds with very low affinity at dopamine D(2) receptors, the serotonin transporter, and the norepinephrine transporter. Comparative structure-affinity studies indicate that AMDA binds in a manner distinct from the tricyclic antagonists Graphics models were employed to identify possible modes of binding. This investigation illustrates the impact of a combination of classical medicinal chemistry, receptor modeling, and molecular biology on novel drug design.

Geometry-affinity relationships of the selective serotonin receptor ligand 9-(aminomethyl)-9,10-dihydroanthracene.

With the exception of its two aromatic rings and basic nitrogen atom, 9-(aminomethyl)-9,10-dihydroanthracene (AMDA; 1) is remarkably devoid of the pharmacophore features usually associated with high-affinity receptor ligands such as the heteroatom hydrogen bonding features of the endogenous ligand serotonin. AMDA does contain a phenylethylamine skeleton within a tricyclic ring system, and the presence of the second aromatic group is necessary for optimal receptor affinity. The structural requirements for the binding of AMDA at 5-HT(2A) receptors were investigated with respect to the geometric relationship between the two aromatic rings. It appears that the geometry of the AMDA parent is in the optimal range for fold angle between aromatic moieties. Evaluation of conformationally constrained derivatives of AMDA suggests that a chain extended trans, gauche form is most likely responsible for high affinity.

Spiro[9,10-dihydroanthracene]-9,3'-pyrrolidine-a structurally unique tetracyclic 5-HT2A receptor antagonist.

Structural elaboration of phenylethylamine to spiro[9,10-dihydroanthracene]-9,3'-pyrrolidine (SpAMDA) produces an agent with unexpectedly high affinity (K(i)=4 nM) at 5-HT(2A) receptors. It was shown that SpAMDA acts as a 5-HT(2A) receptor antagonist. The structure and molecular geometry of SpAMDA are not consistent with existing pharmacophore features, and a novel 5-HT(2A) antagonist pharmacophore model is proposed for the binding of aminomethyl-9,10-dihydroanthracene analogs. Thus, SpAMDA may be a structurally novel parent of a new class of 5-HT(2A) receptor antagonists that binds to the receptor in a unique fashion that is distinct from the binding topology of existing 5-HT(2A) receptor antagonists.

A homology modeling study toward the understanding of three-dimensional structure and putative pharmacological profile of the G-protein coupled receptor GPR55.

The orphan G-protein coupled receptor GPR55 was shown to bind to certain cannabinoid compounds which led to its initial classification as the third type of cannabinoid receptor. Later studies showed that lysophosphatidylinositol (LPI) also activated GPR55, in particular 2-arachidonoyl-LPI was proposed to be its endogenous ligand. However, the results of pharmacological studies regarding GPR55 have been quite inconsistent. Despite its contradictory pharmacological profile, GPR55 has been implicated in various disease states including inflammatory and neuropathic pain, metabolic bone diseases, and cancer. Herein, we report the ligand binding properties of GPR55 by applying homology modeling and automated docking algorithms in order to understand its pharmacological profile. The 3D homology model of GPR55 was built based on the adenosine A(2A) receptor crystal structure. Docking studies of several types of reported ligands were carried out afterwards. The results indicated that both hydrogen bonding and hydrophobic interactions contributed significantly for its ligand binding and the amino acid residue Lys80 seemed to be the anchor residue for receptor recognition. In addition, its putative agonist and antagonist appeared to recognize different domains of the receptor corresponding to their reported pharmacological activities.

CoMFA analyses of C-2 position salvinorin A analogs at the kappa-opioid receptor provides insights into epimer selectivity.

The highly potent and kappa-opioid (KOP) receptor-selective hallucinogen Salvinorin A and selected analogs have been analyzed using the 3D quantitative structure-affinity relationship technique Comparative Molecular Field Analysis (CoMFA) in an effort to derive a statistically significant and predictive model of salvinorin affinity at the KOP receptor and to provide additional statistical support for the validity of previously proposed structure-based interaction models. Two CoMFA models of Salvinorin A analogs substituted at the C-2 position are presented. Separate models were developed based on the radioligand used in the kappa-opioid binding assay, [(3)H]diprenorphine or [(125)I]6 beta-iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5 alpha-epoxymorphinan ([(125)I]IOXY). For each dataset, three methods of alignment were employed: a receptor-docked alignment derived from the structure-based docking algorithm GOLD, another from the ligand-based alignment algorithm FlexS, and a rigid realignment of the poses from the receptor-docked alignment. The receptor-docked alignment produced statistically superior results compared to either the FlexS alignment or the realignment in both datasets. The [(125)I]IOXY set (Model 1) and [(3)H]diprenorphine set (Model 2) gave q(2) values of 0.592 and 0.620, respectively, using the receptor-docked alignment, and both models produced similar CoMFA contour maps that reflected the stereoelectronic features of the receptor model from which they were derived. Each model gave significantly predictive CoMFA statistics (Model 1 PSET r(2)=0.833; Model 2 PSET r(2)=0.813). Based on the CoMFA contour maps, a binding mode was proposed for amine-containing Salvinorin A analogs that provides a rationale for the observation that the beta-epimers (R-configuration) of protonated amines at the C-2 position have a higher affinity than the corresponding alpha-epimers (S-configuration).

Design, synthesis, and biological evaluation of 6alpha- and 6beta-N-heterocyclic substituted naltrexamine derivatives as mu opioid receptor selective antagonists.

Opioid receptor selective antagonists are important pharmacological probes in opioid receptor structural characterization and opioid agonist functional study. Thus far, a nonpeptidyl, highly selective and reversible mu opioid receptor (MOR) antagonist is unavailable. On the basis of our modeling studies, a series of novel naltrexamine derivatives have been designed and synthesized. Among them, two compounds were identified as leads based on the results of in vitro and in vivo assays. Both of them displayed high binding affinity for the MOR (K(i) = 0.37 and 0.55 nM). Compound 6 (NAP) showed over 700-fold selectivity for the MOR over the delta receptor (DOR) and more than 150-fold selectivity over the kappa receptor (KOR). Compound 9 (NAQ) showed over 200-fold selectivity for the MOR over the DOR and approximately 50-fold selectivity over the KOR. Thus these two novel ligands will serve as leads to further develop more potent and selective antagonists for the MOR.

Galpha-subunits differentially alter the conformation and agonist affinity of kappa-opioid receptors.

Although ligand-induced conformational changes in G protein-coupled receptors (GPCRs) are well-documented, there is little direct evidence for G protein-induced changes in GPCR conformation. To investigate this possibility, the effects of overexpressing Galpha-subunits (Galpha16 or Galphai2) with the kappa-opioid receptor (KOR) were examined. The changes in KOR conformation were subequently examined via the substituted cysteine accessibility method (SCAM) in transmembrane domains 6 (TM6) and 7 (TM7) and extracellular loop 2 (EL2). Significant conformational changes were observed on TM7, the extracellular portion of TM6, and EL2. Seven SCAM-sensitive residues (S3107.33, F3147.37, and I3167.39 to Y3207.43) on TM7 presented a cluster pattern when the KOR was exposed to baseline amounts of G protein, and additional residues became sensitive upon overexpression of various G proteins. In TM7, S3117.34 and N3267.49 were found to be sensitive in Galpha16-overexpressed cells and Y3137.36, N3227.45, S3237.46, and L3297.52 in Galphai2-overexpressed cells. In addition, the degree of sensitivity for various TM7 residues was augmented, especially in Galphai2-overexpressed cells. A similar phenomenon was also observed for residues in TM6 and EL2. In addition to an enhanced sensitivity of certain residues, our findings also indicated that a slight rotation was predicted to occur in the upper part of TM7 upon G protein overexpression. These relatively modest conformational changes engendered by G protein overexpression had both profound and differential effects on the abilities of agonists to bind to KOR. These data are significant because they demonstrate that Galpha-subunits differentially modulate the conformation and agonist affinity of a prototypical GPCR.

Potential modes of interaction of 9-aminomethyl-9,10-dihydroanthracene (AMDA) derivatives with the 5-HT2A receptor: a ligand structure-affinity relationship, receptor mutagenesis and receptor modeling investigation.

The effects of 3-position substitution of 9-aminomethyl-9,10-dihydroanthracene (AMDA) on 5-HT 2A receptor affinity were determined and compared to a parallel series of DOB-like 1-(2,5-dimethoxyphenyl)-2-aminopropanes substituted at the 4-position. The results were interpreted within the context of 5-HT 2A receptor models that suggest that members of the DOB-like series can bind to the receptor in two distinct modes that correlate with the compounds' functional activity. Automated ligand docking and molecular dynamics suggest that all of the AMDA derivatives, the parent of which is a 5-HT 2A antagonist, bind in a fashion analogous to that for the sterically demanding antagonist DOB-like compounds. The failure of the F340 (6.52)L mutation to adversely affect the affinity of AMDA and the 3-bromo derivative is consistent with the proposed modes of orientation. Evaluation of ligand-receptor complex models suggest that a valine/threonine exchange between the 5-HT 2A and D 2 receptors may be the origin of selectivity for AMDA and two substituted derivatives.

Differential helical orientations among related G protein-coupled receptors provide a novel mechanism for selectivity. Studies with salvinorin A and the kappa-opioid receptor.

Salvinorin A, the active component of the hallucinogenic sage Salvia divinorum, is an apparently selective and highly potent kappa-opioid receptor (KOR) agonist. Salvinorin A is unique among ligands for peptidergic G protein-coupled receptors in being nonnitrogenous and lipid-like in character. To examine the molecular basis for the subtype-selective binding of salvinorin A, we utilized an integrated approach using chimeric opioid receptors, site-directed mutagenesis, the substituted cysteine accessibility method, and molecular modeling and dynamics studies. We discovered that helix 2 is required for salvinorin A binding to KOR and that two residues (Val-108(2.53) and Val-118(2.63)) confer subtype selectivity. Intriguingly, molecular modeling studies predicted that these loci exhibit an indirect effect on salvinorin A binding, presumably through rotation of helix 2. Significantly, and in agreement with our in silico predictions, substituted cysteine accessibility method analysis of helix 2 comparing KOR and the delta-opioid receptor, which has negligible affinity for salvinorin A, revealed that residues known to be important for salvinorin A binding exhibit a differential pattern of water accessibility. These findings imply that differences in the helical orientation of helix 2 are critical for the selectivity of salvinorin A binding to KOR and provide a structurally novel basis for ligand selectivity.

Identification of the molecular mechanisms by which the diterpenoid salvinorin A binds to kappa-opioid receptors.

Salvinorin A is a naturally occurring hallucinogenic diterpenoid from the plant Salvia divinorumthat selectively and potently activates kappa-opioid receptors (KORs). Salvinorin A is unique in that it is the only known lipid-like molecule that selectively and potently activates a G-protein coupled receptor (GPCR), which has as its endogenous agonist a peptide; salvinorin A is also the only known non-nitrogenous opioid receptor agonist. In this paper, we identify key residues in KORs responsible for the high binding affinity and agonist efficacy of salvinorin A. Surprisingly, we discovered that salvinorin A was stabilized in the binding pocket by interactions with tyrosine residues in helix 7 (Tyr313 and Tyr320) and helix 2 (Tyr119). Intriguingly, activation of KORs by salvinorin A required interactions with the helix 7 tyrosines Tyr312, Tyr313, and Tyr320 and with Tyr139 in helix 3. In contrast, the prototypical nitrogenous KOR agonist U69593 and the endogenous peptidergic agonist dynorphin A (1-13) showed differential requirements for these three residues for binding and activation. We also employed a novel approach, whereby we examined the effects of cysteine-substitution mutagenesis on the binding of salvinorin A and an analogue with a free sulfhydryl group, 2-thiosalvinorin B. We discovered that residues predicted to be in close proximity, especially Tyr313, to the free thiol of 2-thiosalvinorin B when mutated to Cys showed enhanced affinity for 2-thiosalvinorin B. When these findings are taken together, they imply that the diterpenoid salvinorin A utilizes unique residues within a commonly shared binding pocket to selectively activate KORs.

Possible differences in modes of agonist and antagonist binding at human 5-HT6 receptors.

A graphics model of the human 5-HT6 receptor was constructed and automated docking studies were performed. The model suggests that 5-HT6 antagonist arylsulfonyltryptamines might bind differently than that of the agonist serotonin. Furthermore, the model explains many of the empirical results from our previous structure-affinity studies.

Ring substituted analogues of 5-aminomethyl-10,11-dihydro-dibenzo[a,d]cycloheptene (AMDH): potential modes of binding to the 5-HT(2A) receptor.

The synthesis and 5-HT(2A) receptor affinities of 2-substituted-5-aminomethyl-10,11-dihydrodibenzo[a,d]cycloheptene (AMDH) derivatives are described. Comparison of the effects of substitution on affinities allowed assignment of potential binding modes in comparison with DOB-like agonists/antagonists and 3-substituted 1-(aminomethyl)-9,10-dihydroanthracene structures.