Decreased left posterior insular activity during auditory language in autism.

BACKGROUND AND PURPOSE: Individuals with autism spectrum disorders often exhibit atypical language patterns, including delay of speech onset, literal speech interpretation, and poor recognition of social and emotional cues in speech. We acquired functional MR images during an auditory language task to evaluate systematic differences in language-network activation between control and high-functioning autistic populations. MATERIALS AND METHODS: Forty-one right-handed male subjects (26 high-functioning autistic subjects, 15 controls) were studied by using an auditory phrase-recognition task, and areas of differential activation between groups were identified. Hand preference, verbal intelligence quotient (IQ), age, and language-function testing were included as covariables in the analysis. RESULTS: Control and autistic subjects showed similar language-activation networks, with 2 notable differences. Control subjects showed significantly increased activation in the left posterior insula compared with autistic subjects (P < .05, false discovery rate), and autistic subjects showed increased bilaterality of receptive language compared with control subjects. Higher receptive-language scores on standardized testing were associated with greater activation of the posterior aspect of the left Wernicke area. A higher verbal IQ was associated with greater activation of the bilateral Broca area and involvement of the prefrontal cortex and lateral premotor cortex. CONCLUSIONS: Control subjects showed greater activation of the posterior insula during receptive language, which may correlate with impaired emotive processing of language in autism. Subjects with autism showed greater bilateral activation of receptive-language areas, which was out of proportion to the differences in hand preference in autism and control populations.

Targeted disruption of serine racemase affects glutamatergic neurotransmission and behavior.

A subset of glutamate receptors that are specifically sensitive to the glutamate analog N-methyl-D-aspartate (NMDA) are molecular coincidence detectors, necessary for activity-dependent processes of neurodevelopment and in sensory and cognitive functions. The activity of these receptors is modulated by the endogenous amino acid D-serine, but the extent to which D-serine is necessary for the normal development and function of the mammalian nervous system was previously unknown. Decreased signaling at NMDA receptors has been implicated in the pathophysiology of schizophrenia based on pharmacological evidence, and several human genes related to D-serine metabolism and glutamatergic neurotransmission have been implicated in the etiology of schizophrenia. Here we show that genetically modified mice lacking the ability to produce D-serine endogenously have profoundly altered glutamatergic neurotransmission, and relatively subtle but significant behavioral abnormalities that reflect hyperactivity and impaired spatial memory, and that are consistent with elevated anxiety.

Automated identification of Fos expression.

The concentration of Fos, a protein encoded by the immediate-early gene c-fos, provides a measure of synaptic activity that may not parallel the electrical activity of neurons. Such a measure is important for the difficult problem of identifying dynamic properties of neuronal circuitries activated by a variety of stimuli and behaviours. We employ two-stage statistical pattern recognition to identify cellular nuclei that express Fos in two-dimensional sections of rat forebrain after administration of antipsychotic drugs. In stage one, we distinguish dark-stained candidate nuclei from image background by a thresholding algorithm and record size and shape measurements of these objects. In stage two, we compare performance of linear and quadratic discriminants, nearest-neighbour and artificial neural network classifiers that employ functions of these measurements to label candidate objects as either Fos nuclei, two touching Fos nuclei or irrelevant background material. New images of neighbouring brain tissue serve as test sets to assess generalizability of the best derived classification rule, as determined by lowest cross-validation misclassification rate. Three experts, two internal and one external, compare manual and automated results for accuracy assessment. Analyses of a subset of images on two separate occasions provide quantitative measures of inter- and intra-expert consistency. We conclude that our automated procedure yields results that compare favourably with those of the experts and thus has potential to remove much of the tedium, subjectivity and irreproducibility of current Fos identification methods in digital microscopy.

Conformational preferences of the potent dopamine reuptake blocker BTCP and its analogs and their incorporation into a pharmacophore model.

Molecular mechanics calculations using MM3-92 and ab initio quantum mechanical calculations using SPARTAN 5.0 were performed on the structurally similar PCP and BTCP, in which only the latter has a cocaine-like pharmacological profile as a dopamine reuptake blocker. Calculations were also performed on BTCP analogs with a methyl group in various positions of the cyclohexane ring. The results for the cis-2-methyl compound, which retains good pharmacological activity, allowed us to determine that an aryl-axial conformer is the biologically active form for at least some of the compounds in this series. However, an aryl-equatorial conformer presents the identical pharmacophore, as shown by superposition of the two conformers. X-ray crystallographic structures were also obtained for BTCP and related compounds with a 2-methyl group on the cyclohexane ring, with reasonable agreement between the computational and experimental results. Superposition studies were performed with two rigid analogs of cocaine which illustrate the optimal orientations of the ammonium hydrogen for monoamine transporters. There is excellent agreement between a 'back-bridged' cocaine analog that is optimal as a dopamine reuptake blocker and the previously proposed biologically active conformer of methylphenidate. However, BTCP is found to be a better fit to the 'front-bridged' cocaine analog that is optimal for a serotonin reuptake blocker.

Slow-onset, long-duration 3-(3',4'-dichlorophenyl)-1-indanamine monoamine reuptake blockers as potential medications to treat cocaine abuse.

A series of 3-(3',4'-dichlorophenyl)-1-indanamine monoamine reuptake blockers have been synthesized in an effort to develop a compound that could be used as a maintenance therapy to treat cocaine abuse. Since the effects of cocaine on dopamine (DA) and serotonin (5HT) transporters are important components of its pharmacological activity, the focus was on nonselective inhibitors of monoamine transport. To reduce or eliminate the abuse potential of a DA reuptake blocker, the compounds were designed to be slow-onset, long-duration prodrugs whose N-demethylated metabolites would have increased activity over the parent compound with the ideal being a parent compound that has little or no activity. To achieve this, pairs of compounds with different groups on the amine nitrogen and with and without an additional N-methyl group were synthesized. All of the synthesized compounds were screened for binding and reuptake at the cloned human DA, 5HT, and norepinephrine (NE) transporters. As previously found, trans isomers are nonselective blockers of DA, 5HT, and NE reuptake, cis isomers with small N-alkyl groups are selective blockers of 5HT reuptake, and tertiary amines of the trans compounds are less potent than the corresponding N-demethylated secondary amines as blockers of DA reuptake. Larger N-alkyl groups in both the trans and cis series were found to reduce activity for the 5HT and NE transporters with less effect at DA transporters. Selected trans compounds were also screened for locomotor activity in mice and generalization to a cocaine-like profile in rats. With intraperitoneal administration, all of the trans isomers showed a slow onset of at least 20 min and an extremely long duration of action in the locomotor assays. Several of the trans compounds also fully generalized to a cocaine-like pharmacological profile. An initial lead compound, the N,N-dimethyl analogue trans-1b, was resolved into chirally pure enantiomers. Surprisingly, both enantiomers were found to have significant affinity for the DA transporter and to cause locomotor activation. This is in contrast to the N-methyl compound in which only the (+)-enantiomer had significant activity. The absolute configuration of the more active enantiomer was determined by X-ray crystallography to be 3R,1S.

Conformational analysis and a crystal structure of bupropion, an antidepressant with dopamine reuptake blocking activity.

A conformational analysis has been performed on the antidepressant bupropion using the MM3-92 program. In addition, the structure of the compound in the crystal state was obtained. There is good agreement between the computed global minimum and the structure observed by crystallography. The three-dimensional structure of the preferred conformer of bupropion is consistent with the three-dimensional structures of other dopamine reuptake blockers such as cocaine, CFT, and methylphenidate.

Activation of midline thalamic nuclei by antipsychotic drugs.

The thalamus has been proposed as a site which may be involved in the production of the syndrome of schizophrenia and the response of schizophrenic symptoms to treatment. These studies test whether, consistent with this hypothesis, the activation of thalamic nuclei is a shared property of neuroleptic antipsychotic drugs. Rats were given single doses of the typical high and low potency neuroleptics haloperidol (1 mg/kg) and chlorpromazine (20 mg/kg), the atypical neuroleptics thiroridazine (20 mg/kg) and clozapine (20 mg/kg), the specific dopamine antagonist raclopride (3 mg/kg), the mixed dopamine/serotonin antagonist risperidone (3 mg/kg) or drug-free vehicle. Increased expression of Fos-like protein was utilized as a marker of cellular activation. All drugs tested, including typical and atypical antipsychotic agents, led to similar effects on the midline thalamic paraventricular, centromedian and rhomboid nuclei and the nucleus reuniens. These results suggest that midline thalamic nuclei may participate in neural circuits mediating some of the shared effects of antipsychotic drugs.

The incorporation of butyrophenones and related compounds into a pharmacophore for dopamine D2 antagonists.

This study is an attempt to incorporate the butyrophenones, an important class of nontricyclic antipsychotic drugs, into a previously proposed pharmacophore model of tricyclic dopamine D2 receptor antagonist ligands. Conformational energy calculations were performed using the MM3-92 program on spiperone, as a representative butyrophenone, and milenperone and R48455, as related compounds with more limited conformational freedom. Twenty seven conformers were evaluated for spiperone with MM3-92 calculations and nine of these were within 1.1 kcal/mole of the global minima indicating the flexibility of the compound. A conformational analysis of twenty crystal structures of butyrophenones was also performed and six distinct conformers were represented. All of the energy minimized conformers of spiperone were superimposed in a least squares sense onto loxapine as a relatively rigid, typical D2 antagonist and a pair of mirror image conformers, which are observed in one crystal structure of spiperone, were found to be the best fit. However, it was not possible to discriminate between these two conformers since they fit the pharmacophore model equally well. The para-fluoro and carbonyl group of the butyrophenones were found to correspond best to the oxygen and chlorine atoms of loxapine, respectively. The conformations of milenperone and R48455 were also consistent with the two putative biologically active forms of spiperone and the pharmacophore model. Conformational energy calculations were also performed on molindone, an antipsychotic drug in clinical use, which can be related to the butyrophenones since both have a carbonyl group adjacent to an aromatic ring. A putative biologically active form was proposed for molindone and this was related to the structure of piquindone, a rigid analog of molindone. All of the compounds were found to be entirely consistent with the pharmacophore model. However, as previously found, there is great variability in the distance between the ammonium nitrogen and the center of the relevant aromatic ring with the most extreme case in the present study being R48455 where the distance is 7.2 A. The results of the present study should also be relevant to the structures of novel, atypical antipsychotic drugs such as risperidone which appear to be analogs of the butyrophenones.

Conformational analysis of methylphenidate and its structural relationship to other dopamine reuptake blockers such as CFT.

PURPOSE: This work was performed 1) to determine the conformational preferences of the threo and erythro isomers of the dopamine reuptake blocker methylphenidate, 2) to determine the crystal conformation of the threo isomer, 3) to confirm the absolute configuration of the more active threo enantiomer, and 4) to incorporate the compound into a previously determined pharmacophore for dopamine reuptake blockers. METHODS: A conformational analysis was performed with the MM2-87 program, a crystal of the (-)-threo HCl salt was analyzed by x-ray crystallography, and the global minima of the (+)-threo isomer and the potent dopamine reuptake blocker CFT were superimposed. RESULTS: In the global minimum of the threo isomer, the carbonyl oxygen of the ester group is oriented toward the ammonium group as was also found in the crystal state. In the erythro isomer, the ester group prefers an extended conformation relative to the piperidine group. The absolute configuration of the biologically active (+)-threo enantiomer was confirmed to be R,R. The atomic sequence from the amine group through the ester group is identical in the active enantiomers of methylphenidate and CFT. CONCLUSIONS: The dopamine reuptake protein requires a precise orientation of the ammonium and ester groups but allows considerable leeway in the position of the phenyl ring. The pKa of the threo isomer is predicted to be higher than that of the erythro isomer.

Dopaminergic (4aR,10bS)-cis- and (4aS,10bS)-trans-octahydrobenzo[F]quinolines have similar pharmacophores.

The structures and absolute configurations of two N-phenethyl substituted cis- and trans-octahydrobenzo[f]quinolines were determined by X-ray crystallography. The absolute configurations of the enantiomers that have high affinity for dopaminergic receptors were found to be (4aR,10bS) and (4aS,10bS) for the (-)-cis- and (-)-trans-8,9-dihydroxy substituted compounds. This is consistent with previous results for a dopamine agonist pharmacophore. MM2-87 calculations for a cis isomer, which has two alternative chair conformations of the piperidine ring, indicated that the preferred conformer is the same as that observed in the crystal structure. Superposition of the more active cis and trans enantiomers showed that the three dimensional orientations of the phenyl ring and the ammonium group are similar in the two geometrical isomers. The cis isomer, however, has steric bulk out of the plane of the molecule and this appears to result in a loss of agonist efficacy. The addition of the N-phenethyl group to the 7-OH and 7,8-diOH cis compounds, however, appears to be sufficient to restore high affinity for dopaminergic receptors unlike previously synthesized cis compounds. These cis compounds, however, appear to be mixed agonist/antagonists or antagonists on functional assays of dopaminergic activity.

Absolute configurations and conformations of the opioid agonist and antagonist enantiomers of picenadol.

The absolute configurations of the enantiomers of the opioid picenadol [cis-1,3-dimethyl-4-propyl-4-(3-hydroxyphenyl)piperidine; cis-3-methyl, 4-propyl] have been determined by an X-ray crystallographic study of the chloride salt of the (+)-enantiomer. The agonist (+)-enantiomer and the antagonist (-)-enantiomer were found to have the 3R,4R and 3S,4S absolute configurations, respectively. The conformational properties of the enantiomers were also examined with MM2-87 calculations. There was good agreement between the computed global minimum and the crystallographic structure with the phenyl ring approximately bisecting the piperidine ring by both methods. This orientation of the phenyl ring differs from that of related opioids such as the phenylmorphans, prodines, meperidine, and ketobemidone in which the phenyl ring tends to eclipse one edge of the piperidine ring. Because the phenyl ring bisects the piperidine ring in picenadol, there is little difference in the three-dimensional orientations of the phenyl rings of the two enantiomers when one superimposes the piperidine rings. The agonist (+)-enantiomer is ambiguous with respect to an opioid ligand model, which suggests that agonist activity requires a specific range of dihedral angles for the phenyl ring. While the global minimum of the agonist is not consistent with the model, a second conformer that is only 1.2 kcal/mol above the global minimum is consistent. An alternative explanation is that agonist or antagonist activity is solely due to the presence of the 3-methyl group on the different edges of the piperidine ring.(ABSTRACT TRUNCATED AT 250 WORDS)

Homology modeling of the dopamine D2 receptor and its testing by docking of agonists and tricyclic antagonists.

We present the first model of dopamine D2 receptor transmembrane helices constructed directly from the bacteriorhodopsin (bR) coordinates derived from two-dimensional electron diffraction experiments. We have tested this model by its ability to accommodate rigid agonist and semirigid antagonist molecules which were docked into the putative binding pocket with stabilizing interactions. The model is consistent with structure-activity relationships of agonists and antagonists that interact with the receptor. It also illuminates data on a Na+ site for regulation of receptor function. The plausibility of the model is increased by its consistency with many mutagenesis studies on G protein-coupled receptors. Further, this model provides a basis to suggest testable molecular mechanisms for changes in the D2 conformational states for high- and low-affinity binding and signal transduction. Changes in the conformational state of the receptor are hypothesized to be due partly to movement of helix 7. In contrast to the model presented here, other published models were built using ideal helical structures or following the sense of the bacteriorhodopsin structure rather than the actual available coordinates. The presented model for the dopamine G protein-coupled receptor can be reconciled with the recent rhodopsin projection structure (Schertler, G. F. X.; Villa, C.; Henderson, R. Projection Structure of Rhodopsin.

Chiral resolution of 1,3-dimethyl-4-phenylpiperidine derivatives using high-performance liquid chromatography with a chiral stationary phase.

A number of racemic 1,3-dimethyl-4-phenylpiperidines which serve as intermediates in the synthesis of opioid analgesics have been resolved on two commercially available high-performance liquid chromatography columns containing cellulose-based chiral stationary phases: Chiralcel OD and Chiralcel OJ. The resolution results were complementary between the two columns. Also, the polarity of substituents appears to play an important role on the ability of the Chiralcel OD column to resolve pairs of enantiomers.

Conformational analysis of the opioid phenylmorphan and its 9 alpha-methyl analogue in solution using high-resolution nuclear magnetic resonance spectroscopy.

The solution conformations of the opioid phenylmorphan (5-m-hydroxyphenyl-2-methylmorphan) and its 9 alpha-methyl analogue were studied using one- and two-dimensional high resolution NMR techniques. The NMR spectra were analyzed by interpreting the phase-sensitive 2-D COSY and double quantum filtered COSY spectra, 1H-1H vicinal coupling constants, and nuclear Overhauser effects in the phase-sensitive 2-D NOESY spectra. The results show that, for both compounds, a chair-chair conformation of the cyclohexane and piperidine rings is exclusively preferred with some distortion of the rings from perfectly staggered chairs. For phenylmorphans, the phenyl ring is oriented to fit into the cleft formed by the cyclohexane and piperidine rings. Thus, for the (+)-enantiomer, the phenyl group assumes the same orientation with regard to the piperidine ring as morphine consistent with the morphine-like properties of the compound. For the 9 alpha-methyl analogue, the plane of the phenyl ring essentially bisects the piperidine ring to which it is attached and is outside of the required range of opioid agonists. This is consistent with the atypical properties of the two enantiomers. The NMR results are compared to the conformations of (-)-phenylmorphan and the (+)-9 alpha-methyl analogue in the crystal state and to the results of molecular mechanics (MM2) studies.

Biologically active conformers of phenothiazines and thioxanthenes. Further evidence for a ligand model of dopamine D2 receptor antagonists.

Conformational analyses have been performed on several phenothiazine and thioxanthene dopamine antagonists using the MM2-87 program and parameter set. The compounds that were examined are thioridazine (2), methotrimeprazine (3), cis- and trans-chlorprothixene, and a piperidylidene derivative of chlorprothixene. In addition, (+)-2 and (-)-3 were determined by X-ray crystallography to have the R absolute configuration. The above compounds were superimposed onto loxapine, which was used as a template for the previously proposed dopamine D2 receptor ligand model. The conformational properties and receptor affinities of these compounds were found to be entirely consistent with the ligand model. For example, a conformer of (+)-R-2 that is consistent with the ligand model is lower in energy than a consistent conformer for (-)-S-2, which agrees with the higher D2 receptor affinity of the former. Similarly, in agreement with the much higher affinity of (-)-R-3 relative to (+)-S-3, only the former contains a low energy conformer consistent with the ligand model. The ligand model is also consistent with the greater potency of cis-thioxanthenes over the trans isomers. These results emphasize the importance of the correct orientation of the ammonium hydrogen for high affinity at the D2 receptor. The pharmacophore for D2 receptor ligands is compared with a recently proposed pharmacophore for D1 ligands.

Enantiomeric conformers of the opioid agonist ketobemidone HCl in the crystal state.

A crystal of the potent opioid agonist ketobemidone [1-methyl-4-(3-hydroxyphenyl)-4-propionylipiperidine] HCl was analyzed by X-ray crystallography. The crystal was monoclinic, space group P2(1)/n with four molecules in the unit cell. In agreement with MM2 calculations (J. Med. Chem. 25:1127-1133, 1982), the crystal contains mirror image conformers in which the phenyl ring is equatorial to the piperidine ring. The conformers are enantiomers since they are not superimposable. One conformer is predicted to be responsible for the typical morphine-like activity of the compound since it closely matches the preferred conformer of the morphine-like (+)-phenylmorphan whereas the other conformer resembles the preferred conformers of (+)-beta-prodine and (-)-phenylmorphan which have atypical opioid properties and/or structure-activity relationships. The importance of considering the conformational enantiomers of a nonchiral receptor ligand in centrosymmetric crystal structures is emphasized.

Conformational preferences of the kappa-selective opioid agonist U50488. A combined molecular mechanics and nuclear magnetic resonance study.

The conformational preferences of the kappa-selective opioid agonist U50488 have been studied using MM2-87 calculations and nuclear magnetic resonance (NMR) spectroscopy. The calculations were performed for the protonated form with a dielectric constant of 80 and the unprotonated form with dielectric constants of 1.5 and 80. A systematic search found 72 stable conformers with certain consistent conformational preferences for some of the important dihedral angles. The preferred conformers proved to be compact structures stabilized by intramolecular attractive van der Waals interactions, though at least some of these appear to be electrostatically unfavorable. The conformation of U50488 was also examined in aqueous solution using one-dimensional (1D) and two-dimensional (2D) high-resolution 1H NMR techniques such as the interpretation of 1H-1H vicinal coupling constants, 1D and 2D nuclear Overhauser effect (NOE) experiments, and 2D correlated spectroscopy (COSY) experiments. Five crystallographic conformations were examined as well. There was generally good agreement between all three methods of conformational analysis. There appeared to be a reasonable geometrical agreement between the relatively rigid kappa-agonist (-)-ketazocine and a gauche conformer of U50488. The proposed pharmacophore is also consistent with other kappa-selective analogs of U50488 including one in which the peptide bond is incorporated into a lactam ring. The low affinity of U50488 for mu-receptors was attributed to its cyclohexane ring which occupies space not present in the nonselective (-)-ketazocine.

Phenylmorphans and analogues: opioid receptor subtype selectivity and effect of conformation on activity.

The morphine-like (+)-phenylmorphan, the atypical (-)-enantiomer, and some analogues have been tested in receptor binding assays selective for opioid mu 1, mu 2, delta, kappa 1, and kappa 3 receptors. The affinities of all of the compounds except one, including the atypical (-)-phenylmorphan, were greatest for mu 1 and mu 2 receptors. The only exception was the (+)-9 alpha-methyl analogue which had slightly greater affinity for the kappa 1 receptor. The selective receptor binding assays provide evidence that opioids in which the phenyl ring is constrained to be equatorial on the piperidine ring can have considerable affinity for mu receptors. In addition, dose-response curves were determined for (+)- and (-)-phenylmorphan using the mouse tail-flick assay with the (+)-enantiomer found to be about 7 times more potent. Pretreatment with the selective opioid antagonists beta-FNA (mu 1 and mu 2), naloxonazine (mu 1), nor-BNI (kappa 1), and naltrindole (delta) suggests that the antinociceptive activity of both enantiomers is mediated through mu receptors. The pretreatment with naloxonazine, which attenuated the antinociceptive effect, shows that both (+)- and (-)-phenylmorphan are mu 1 agonists while intrathecal administration shows that both are mu 2 agonists. Conformational energy calculations on the compounds were also performed using the MM2-87 program. Consistent with previous conformational results for the phenylmorphans (J. Med. Chem. 1984, 27, 1234-1237), the most potent antinociceptive compounds preferred a particular orientation of the phenyl ring.