John A. Schetz

Interactions of the Novel Antipsychotic Aripiprazole (OPC-14597) with Dopamine and Serotonin Receptor Subtypes

OPC-14597 {aripiprazole; 7-(4-(4-(2,3-dichlorophenyl)-1-piperazinyl)butyloxy)-3,4-dihydro-2(1H)-quinolinone} is a novel candidate antipsychotic that has high affinity for striatal dopamine D2-like receptors, but causes few extrapyramidal effects. These studies characterized the molecular pharmacology of OPC-14597, DM-1451 (its major rodent metabolite), and the related quinolinone derivative OPC-4392 at each of the cloned dopamine receptors, and at serotonin 5HT6 and 5HT7 receptors. All three compounds exhibited highest affinity for D2L and D2S receptors relative to the other cloned receptors examined. Both OPC-4392 and OPC-14597 demonstrated dual agonist/antagonist actions at D2L receptors, although the metabolite DM-1451 behaved as a pure antagonist. These data suggest that clinical atypicality can occur with drugs that exhibit selectivity for D2L/D2S rather than D3 or D4 receptors, and raise the possibility that the unusual profile of OPC-14597 in vivo (presynaptic agonist and postsynaptic antagonist) may reflect different functional consequences of this compound interacting with a single dopamine receptor subtype (D2) in distinct cellular locales.

A prototypical Sigma-1 receptor antagonist protects against brain ischemia.

Previous studies indicate that the Sigma-1 ligand 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) protects the brain from ischemia. Less clear is whether protection is mediated by agonism or antagonism of the Sigma-1 receptor, and whether drugs already in use for other indications and that interact with the Sigma-1 receptor might also prevent oxidative damage due to conditions such as cerebral ischemic stroke. The antipsychotic drug haloperidol is an antagonist of Sigma-1 receptors and in this study it potently protects against oxidative stress-related cell death in vitro at low concentrations. The protective potency of haloperidol and a number of other butyrophenone compounds positively correlate with their affinity for a cloned Sigma-1 receptor, and the protection is mimicked by a Sigma-1 receptor-selective antagonist (BD1063), but not an agonist (PRE-084). In vivo, an acute low dose (0.05 mg/kg s.c.) of haloperidol reduces by half the ischemic lesion volume induced by a transient middle cerebral artery occlusion. These in vitro and in vivo pre-clinical results suggest that a low dose of acutely administered haloperidol might have a novel application as a protective agent against ischemic cerebral stroke and other types of brain injury with an ischemic component.

Exploration of structure-antifouling relationships of capsaicin-like compounds that inhibit zebra mussel (Dreissena polymorpha) macrofouling.

Abstract Macrofouling of aquatic man-made structures by zebra mussels (Dreissena polymorpha) poses significant economic burdens on commercial freshwater shipping and facilities utilising raw water. The negative environmental impact of some current antifouling technologies has limited their use and prompted investigation of non-organometallic and non-oxidising antifoulants as possible environment-friendly alternatives. The plant-derived natural product capsaicin and 18 other compounds with one or more capsaicin-like structural features were tested for their potential to inhibit zebra mussel byssal attachment at a single high concentration of 30 μM. Of these, three compounds displaying the highest levels of attachment inhibition where selected for further concentration-response testing. This testing revealed that capsaicin (8-methyl-N-vanillyl-trans-6-nonenamide), N-vanillylnonanamide, and N-benzoylmonoethanolamine benzoate all inhibited byssal attachment with potency values (EC50) in the micromolar range. None of these compounds were lethal to adult specimens of the water flea, Daphnia magna, at concentrations that inhibited mussel byssal attachment.

Ligand Selectivity of D2 Dopamine Receptors Is Modulated by Changes in Local Dynamics Produced by Sodium Binding

We have uncovered a significant allosteric response of the D2 dopamine receptor to physiologically relevant concentrations of sodium (140 mM), characterized by a sodium-enhanced binding affinity for a D4-selective class of agonists and antagonists. This enhancement is significantly more pronounced in a D2-V2.61(91)F mutant and cannot be mimicked by an equivalent concentration of the sodium replacement cation N-methyl-d-glucamine. This phenomenon was explored computationally at the molecular level by analyzing the effect of sodium binding on the dynamic properties of D2 receptor model constructs. Normal mode analysis identified one mode (M19), which is involved in the open/closed motions of the binding cleft as being particularly sensitive to the sodium effect. To examine the consequences for D2 receptor ligand recognition, one of the ligands, L-745,870 [3-{[4-(4-chlorophenyl) piperazin-1-yl]-methyl}-1H-pyrrolo[2,3-b]pyridine or CPPMA, chlorophenylpiperazinyl methylazaindole], was docked into conformers along the M19 trajectory. Structurally and pharmacologically well established ligand-receptor interactions, including the ionic interaction with D3.32(114) and interactions between the ligand aryl moieties and V2.61(91)F, were achieved only in “open” phase conformers. The docking of (-)-raclopride [3,5-dichloro-N-(1-ethylpyrrolidin-2-ylmethyl)-2-hydroxy-6-methoxybenzamide] suggests that the same binding cleft changes in response to sodium-binding perturbation account as well for the enhancements in binding affinity for substituted benzamides in the wild-type D2 receptor. Our findings demonstrate how key interactions can be modulated by occupancy at an allosteric site and are consistent with a mechanism in which sodium binding enhances the affinity of selected ligands through dynamic changes that increase accessibility of substituted benzamides and 1,4-DAP ligands to the orthosteric site and accessibility of 1,4-DAPs to V2.61(91)F.

Dopamine Receptor Microdomains Involved in Molecular Recognition and the Regulation of Drug Affinity and Function

A cationic protonatable amine moiety on dopaminergic ligands forms a high affinity reinforced ionic bond with an anionic aspartic acid at position 3.32 of dopamine receptors. When present, catechol hydroxyls of the ligands form hydrogen bonds with serines at position 5.42, 5.43, and 5.46, and this network of hydrogen bonds serves to orient ligands in the binding-site crevice and increase their binding affinity. A steric clash between aromatic moieties of the ligands and aromatic amino acids of the receptor (e.g., H6.55, F6.52 or F6.51 and W6.48) is likely to be propagated in domino-like fashion along the length of TM6, which is believed to trigger activation of the receptor. Specifically, it is the change in the conformation of W6.48 from an orientation perpendicular to the plane of the lipid membrane to one that is parallel that is believed to result in activation. Molecular determinants that mediate the D4/D2-selectivity of many extremely D4-selective 1,4-DAP ligands, include a nonconserved cluster of bulky amino acids at the TM2/TM3 interface (positions 2.61, 3.28 and 3.29).

Differential subcellular distribution of rat brain dopamine receptors and subtype-specific redistribution induced by cocaine

We investigated the subcellular distribution of dopamine D(1), D(2) and D(5) receptor subtypes in rat frontal cortex, and examined whether psychostimulant-induced elevation of synaptic dopamine could alter the receptor distribution. Differential detergent solubilization and density gradient centrifugation were used to separate various subcellular fractions, followed by semi-quantitative determination of the relative abundance of specific receptor proteins in each fraction. D(1) receptors were predominantly localized to detergent-resistant membranes, and a portion of these receptors also floated on sucrose gradients. These properties are characteristic of proteins found in lipid rafts and caveolae. D(2) receptors exhibited variable distribution between cytoplasmic, detergent-soluble and detergent-resistant membrane fractions, yet were not present in buoyant membranes. Most D(5) receptor immunoreactivity was distributed into the cytoplasmic fraction, failing to sediment at forces up to 300,000g, while the remainder was localized to detergent-soluble membranes in cortex. D(5) receptors were undetectable in detergent-resistant fractions or raft-like subdomains. Following daily cocaine administration for seven days, a significant portion of D(1) receptors translocated from detergent-resistant membranes to detergent-soluble membranes and the cytoplasmic fraction. The distributions of D(5) and D(2) receptor subtypes were not significantly altered by cocaine treatment. These data imply that D(5) receptors are predominantly cytoplasmic, D(2) receptors are diffusely distributed within the cell, whereas D(1) receptors are mostly localized to lipid rafts within the rat frontal cortex. Dopamine receptor subtype localization is susceptible to modulation by pharmacological manipulations that elevate synaptic dopamine, however the functional implications of such drug-induced receptor warrant further investigation.

Transmembrane Segment Five Serines of the D4 Dopamine Receptor Uniquely Influence the Interactions of Dopamine, Norepinephrine, and Ro10-4548

Conserved serines of transmembrane segment (TM) five (TM5) are critical for the interactions of endogenous catecholamines with α1- and α2-adrenergic, β2-adrenergic, and D1, D2, and D3 dopamine receptors. The unique high-affinity interaction of the D4 dopamine receptor subtype with both norepinephrine and dopamine, and the fact that TM5 serine interactions have never been studied for this receptor subtype, led us to investigate the interactions of ligands with D4 receptor TM5 serines. Serine-to-alanine mutations at positions 5.42 and 5.46 drastically decreased affinities of dopamine and norepinephrine for the D4 receptor. The D4-S5.43A receptor mutant had substantially reduced affinity for norepinephrine, but a modest loss of affinity for dopamine. In functional assays of cAMP accumulation, norephinephrine was unable to activate any of the mutant receptors, even though the agonist quinpirole displayed wild-type functional properties for all of them. Dopamine was unable to activate the S5.46A mutant and had reduced potency for the S5.43A mutant and reduced potency and efficacy for the S5.42A mutant. In contrast, Ro10-4548 [RAC-2′-2-hydroxy-3-4-(4-hydroxy-2-methoxyphenyl)-1-piperazinyl-propoxy-acetanilide], a catechol-like antagonist of the wild-type receptor unexpectedly functions as an agonist of the S5.43A mutant. Other noncatechol ligands had similar properties for mutant and wild-type receptors. This is the first example of a dopamine receptor point mutation selectively changing the receptors interaction with a specific antagonist to that of an agonist, and together with other data, provides evidence, supported by molecular modeling, that catecholamine-type agonism is induced by different ligand-specific configurations of intermolecular H-bonds with the TM5 conserved serines.

Design, synthesis, radiolabeling and in vivo evaluation of carbon-11 labeled N-[2-[4-(3-cyanopyridin-2-yl)piperazin-1-yl]ethyl]-3-methoxybenzamide, a potential Positron Emission Tomography tracer for the dopamine D4 receptors

Here we describe the design, synthesis, and evaluation of physicochemical and pharmacological properties of D(4) dopamine receptor ligands related to N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide (2). Structural features were incorporated to increase affinity for the target receptor, to improve selectivity over D(2) and σ(1) receptors, to enable labeling with carbon-11 or fluorine-18, and to adjust lipophilicity within the range considered optimal for brain penetration and low nonspecific binding. Compounds 7 and 13 showed the overall best characteristics: nanomolar affinity for the D(4) receptor, >100-fold selectivity over D(2) and D(3) dopamine receptors, 5-HT(1A), 5-HT(2A), and 5-HT(2C) serotonin receptors and σ(1) receptors, and log P = 2.37-2.55. Following intraperitoneal administration in mice, both compounds rapidly entered the central nervous system. The methoxy of N-[2-[4-(3-cyanopyridin-2-yl)piperazin-1-yl]ethyl]-3-methoxybenzamide (7) was radiolabeled with carbon-11 and subjected to PET analysis in non-human primate. [(11)C]7 time-dependently accumulated to saturation in the posterior eye in the region of the retina, a tissue containing a high density of D(4) receptors.

Synthesis and structure-affinity relationships of novel small molecule natural product derivatives capable of discriminating between serotonin 5-HT1A, 5-HT2A, 5-HT2C receptor subtypes

Efforts to develop ligands that distinguish between clinically relevant 5-HT2A and 5-HT2C serotonin receptor subtypes have been challenging, because their sequences have high homology. Previous studies reported that a novel aplysinopsin belonging to a chemical class of natural products isolated from a marine sponge was selective for the 5-HT2C over the 5-HT2A receptor subtype. Our goal was to explore the 5-HT2A/2C receptor structure-affinity relationships of derivatives based on the aplysinopsin natural product pharmacophore. Twenty aplysinopsin derivatives were synthesized, purified and tested for their affinities for cloned human serotonin 5-HT1A, 5-HT2A, and 5-HT2C receptor subtypes. Four compounds in this series had >30-fold selectivity for 5-HT2A or 5-HT2C receptors. The compound (E)-5-((5,6-dichloro-1H-indol-3-yl)methylene)-2-imino-1,3-dimethylimidazolidin-4-one (UNT-TWU-22, 16) had approximately 2100-fold selectivity for the serotonin 5-HT2C receptor subtype: an affinity for 5-HT2C equal to 46 nM and no detectable affinity for the 5-HT1A or 5-HT2A receptor subtypes. The two most important factors controlling 5-HT2A or 5-HT2C receptor subtype selectivity were the combined R1,R3-alkylation of the imidazolidinone ring and the type and number of halogens on the indole ring of the aplysinopsin pharmacophore.

Protein Expression in the Drosophila Schneider 2 Cell System

The Schneider‐2 (S2) Drosophila cell line is well suited for the stable overexpression of recombinant proteins using plasmid‐based protein expression vectors. Following drug selection, a polyclonal S2 cell line can be induced to express on the order of 2 to 100 pmol/mg membrane protein for G‐coupled protein receptors, 4000 to 100,000 sites/cell for other membrane receptors and 3 to 35 mg/liter for soluble and secreted proteins.