Potential Modes of Interaction of 9-Aminomethyl-9,10-dihydroanthracene (AMDA) Derivatives with the 5-HT 2A 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-HT2A 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-HT2A 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-HT2A antagonist, bind in a fashion analogous to that for the sterically demanding antagonist DOB-like compounds. The failure of the F3406.52L 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-HT2A and D2 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 κ-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

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 receptor (KOR)-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 KOR 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, [3H]diprenorphine or [125I]6β-iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5α-epoxymorphinan ([125I]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 [125I]IOXY set (Model 1) and [3H]diprenorphine set (Model 2) gave q2 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 r2 = 0.833; Model 2 PSET r2 = 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 β-epimers (R-configuration) of protonated amines at the C-2 position have a higher affinity than the corresponding β-epimers (S-configuration)

Coronagraphic Wavefront Control for the ATLAST-9.2m Telescope

The Advanced Technology for Large Aperture Space Telescope (ATLAST) concept was assessed as one of the NASA Astrophysics Strategic Mission Concepts (ASMC) studies. Herein we discuss the 9.2-meter diameter segmented aperture version and its wavefront sensing and control (WFSC) with regards to coronagraphic detection and spectroscopic characterization of exoplanets. The WFSC would consist of at least two levels of sensing and control: (i) an outer coarser level of sensing and control to phase and control the segments and secondary mirror in a manner similar to the James Webb Space Telescope but operating at higher temporal bandwidth, and (ii) an inner, coronagraphic instrument based, fine level of sensing and control for both amplitude and wavefront errors operating at higher temporal bandwidths. The outer loop would control rigid-body actuators on the primary and secondary mirrors while the inner loop would control one or more segmented deformable mirror to suppress the starlight within the coronagraphic field-of view. Herein we discuss the visible nulling coronagraph (VNC) and the requirements it levies on wavefront sensing and control and show the results of closed-loop simulations to assess performance and evaluate the trade space of system level stability versus control bandwidth

Methoxy-substituted 9-aminomethyl-9,10-dihydroanthracene (AMDA) derivatives exhibit differential binding affinities at the 5-HT2A receptor

The effects of methoxy-substitution at the 1-, 2-, 3- and 4-positions of 9-aminomethyl-9,10-dihydroanthracene (AMDA) on h5-HT2A receptor affinity were determined. Racemic mixtures of these compounds were found to show the following affinity trend: 3-MeO > 4-MeO > 1-MeO ~ 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-HT2A 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-HT2A receptor binding sites, whereas the lower-affinity isomers lack this ability

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-HT2A and H1 receptors. A mixture of (7,12-dihydrotetraphene-12-yl)methanamine and (6,11-dihydrotetracene-11-yl)methanamine in a 75% to 25% ratio was found to have an apparent Ki of 10 nM at the 5-HT2A receptor. A substantial binding affinity for (7,12-dihydrotetraphene-3-methoxy-12-yl)methanamine at the 5-HT2A receptor (Ki = 21 nM) was also observed. Interestingly, this compound was found to have 100-fold selectivity for 5-HT2A over the H1 receptor (Ki = 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-HT2A and H1 receptors (Ki = 223 to 964 nM). Our results show that large rigid annulated AMDA analogs can be sterically accommodated within the proposed 5-HT2A binding site

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

Histamine H1 and serotonin 5-HT2A 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-HT2A and H1 receptors. Homology modeling of the 5-HT2A and H1 receptors suggests that AMDA and its analogs, the parent of which is a 5-HT2A antagonist, can bind in a fashion analogous to that of classical H1 antagonists whose ring systems are oriented towards the fifth and sixth transmembrane helices. The modeled orientation of the ligands are consistent with the reported site-directed mutagenesis data for 5-HT2A and H1 receptors and provide a potential explanation for the selectivity of ligands acting at both receptors

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

Structure-Based Design, Synthesis, and Biochemical and Pharmacological Characterization of Novel Salvinorin A Analogues as Active State Probes of the κ-Opioid Receptor

Salvinorin A, the most potent naturally occurring hallucinogen, has gained increasing attention since the κ-opioid receptor (KOR) was identified as its principal molecular target by us (Roth et al, PNAS, 2002). Here we report the design, synthesis and biochemical characterization of novel, irreversible, salvinorin A-derived ligands suitable as active state probes of the KOR. Based on prior substituted cysteine accessibility and molecular modeling studies, C3157.38 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 C3157.38. 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 based on 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 C3157.38 and suggested one biochemical mechanism for covalent binding. These studies provide direct evidence for the presence of a free cysteine in the agonist-bound state of KOR and provide novel insights into the mechanism by which salvinorin A binds to and activates KOR

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 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. Interestingly, the insensitivity of the delta opioid receptor (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