HR/MS-based lipidome analysis of rat brain modulated by tolcapone.

Lipids play key roles in the body, influencing cellular regulation, function, and signalling. Tolcapone, a potent catechol-O-methyltransferase (COMT) inhibitor described to enhance cognitive performance in healthy subjects, was previously shown to impact fatty acid ß-oxidation and oxidative phosphorylation. However, its impact on the brain lipidome remains unexplored. Hence, this study aimed to assess how tolcapone affects the lipidome of the rat pre-frontal cortex (PFC), a region of the brain highly relevant to tolcapone therapeutic effect, while evaluating its influence on operant behaviour. Tolcapone at 20 mg/kg was chronically administered to Wistar rats during a behavioural task and an untargeted liquid chromatography high-resolution mass spectrometry (LC-HR/MS) approach was employed to profile lipid species. The untargeted analysis identified 7227 features, of which only 33% underwent statistical analysis following data pre-processing. The results revealed an improved cognitive performance and a lipidome remodelling promoted by tolcapone. The lipidomic analysis showed 32 differentially expressed lipid species in tolcapone-treated animals (FC ≥ 1.2, p-value ≤ 0.1), and among these several triacylglycerols, cardiolipins and N-acylethanolamine (NAE 16:2) were found upregulated whereas fatty acids, hexosylceramides, and several phospholipids including phosphatidylcholines and phosphatidylethanolamines were downregulated. These preliminary findings shed light on tolcapone impact on lipid pathways within the brain. Although tolcapone improved cognitive performance and literature suggests the significance of lipids in cognition, this study did not conclusively establish that lipids directly drove or contributed to this outcome. Nevertheless, it underscores the importance of lipid modulation and encourages further exploration of tolcapone-associated mechanisms in the central nervous system (CNS).

Limiting Monoamines Degradation Increases L-DOPA Pro-Locomotor Action in Newborn Rats.

L-DOPA, the precursor of catecholamines, exerts a pro-locomotor action in several vertebrate species, including newborn rats. Here, we tested the hypothesis that decreasing the degradation of monoamines can promote the pro-locomotor action of a low, subthreshold dose of L-DOPA in five-day-old rats. The activity of the degrading pathways involving monoamine oxidases or catechol-O-methyltransferase was impaired by injecting nialamide or tolcapone, respectively. At this early post-natal stage, the capacity of the drugs to trigger locomotion was investigated by monitoring the air-stepping activity expressed by the animals suspended in a harness above the ground. We show that nialamide (100 mg/kg) or tolcapone (100 mg/kg), without effect on their own promotes maximal expression of air-stepping sequences in the presence of a sub-effective dose of L-DOPA (25 mg/kg). Tissue measurements of monoamines (dopamine, noradrenaline, serotonin and some of their metabolites) in the cervical and lumbar spinal cord confirmed the regional efficacy of each inhibitor toward their respective enzyme. Our experiments support the idea that the raise of monoamines boost L-DOPA's locomotor action. Considering that both inhibitors differently altered the spinal monoamines levels in response to L-DOPA, our data also suggest that maximal locomotor response can be reached with different monoamines environment.

The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injury.

Background: Evidence supports an important link between mitochondrial DNA (mtDNA) variation and adverse drug reactions such as idiosyncratic drug-induced liver injury (iDILI). Here, we describe the generation of HepG2-derived transmitochondrial cybrids, to investigate the impact of mtDNA variation on mitochondrial (dys)function and susceptibility to iDILI. This study created 10 cybrid cell lines, each containing distinct mitochondrial genotypes of haplogroup H or haplogroup J backgrounds. Methods: HepG2 cells were depleted of mtDNA to make rho zero cells, before the introduction of known mitochondrial genotypes using platelets from healthy volunteers (n=10), thus generating 10 transmitochondrial cybrid cell lines. The mitochondrial function of each was assessed at basal state and following treatment with compounds associated with iDILI; flutamide, 2-hydroxyflutamide, and tolcapone, and their less toxic counterparts bicalutamide and entacapone utilizing ATP assays and extracellular flux analysis. Results: Whilst only slight variations in basal mitochondrial function were observed between haplogroups H and J, haplogroup-specific responses were observed to the mitotoxic drugs. Haplogroup J showed increased susceptibility to inhibition by flutamide, 2-hydroxyflutamide, and tolcapone, via effects on selected mitochondrial complexes (I and II), and an uncoupling of the respiratory chain. Conclusions: This study demonstrates that HepG2 transmitochondrial cybrids can be created to contain the mitochondrial genotype of any individual of interest. This provides a practical and reproducible system to investigate the cellular consequences of variation in the mitochondrial genome, against a constant nuclear background. Additionally, the results show that inter-individual variation in mitochondrial haplogroup may be a factor in determining sensitivity to mitochondrial toxicants. Funding: This work was supported by the Centre for Drug Safety Science supported by the Medical Research Council, United Kingdom (Grant Number G0700654); and GlaxoSmithKline as part of an MRC-CASE studentship (grant number MR/L006758/1).

Effects of pharmacological and genetic regulation of COMT activity in alcohol use disorder: a randomized, placebo-controlled trial of tolcapone.

Alcohol Use Disorder (AUD) is characterized by loss of control over drinking. Behavioral control is mediated, in part, by cortical dopamine signaling. Inhibition of catechol-O-methyltransferase (COMT), the enzyme primarily responsible for cortical dopamine inactivation, may increase cortical dopamine, especially among individuals with genetically mediated lower dopaminergic tone, such as COMT rs4680 (val158met) val-allele homozygotes. This study was a randomized, placebo-controlled, pharmacogenetic trial of the COMT inhibitor tolcapone. Ninety non-treatment-seeking AUD individuals were prospectively genotyped for rs4680 and randomized to tolcapone (200 mg t.i.d.) or placebo for 8 days. At baseline and on day 7, peripheral COMT activity was assayed, and participants completed an fMRI alcohol cue-reactivity task; on day 8, they completed a bar-lab paradigm. Primary outcomes were: (1) natural drinking during the medication period; (2) alcohol self-administration in the bar lab; and (3) alcohol cue-elicited cortical (right inferior frontal gyrus [rIFG]) and ventral striatal activation. At baseline, the rs4680 val-allele had an additive effect on COMT activity. Tolcapone, relative to placebo, reduced COMT activity in all genotype groups. COMT genotype moderated tolcapone's effect on drinking during the medication period and in the bar lab, such that tolcapone, relative to placebo, reduced drinking only among val-allele homozygotes. Tolcapone did not affect cue-elicited ventral striatal activation but reduced rIFG activation; less rIFG activation on day 7 was associated with less drinking during the medication period. Taken together, these data suggest that COMT inhibition may reduce drinking specifically among individuals genetically predisposed to excessive COMT activity and potentially low cortical dopamine tone.ClinicalTrials.gov identifier: NCT02949934 https://clinicaltrials.gov/ct2/show/NCT02949934.

Ethanol concentration induces production of 3,4-dihydroxyphenylacetic acid and homovanillic acid in mouse brain through activation of monoamine oxidase pathway.

First, we aimed to investigate ex vivo the effects of ethanol (EtOH) on levels of norepinephrine (NE), dopamine (DA), serotonin (5-HT), and their metabolites in the frontal cortex, hippocampus, and striatum of Aldh2-knockout (Aldh2-KO) and wild-type (WT) mice. Animals were treated intraperitoneally with saline (control) or EtOH (1.0, 2.0, or 3.0 g/kg). Brain samples were collected 60 and 120 min after EtOH injection, and monoamines and their metabolites were measured by HPLC-ECD. We found in both WT and Aldh2-KO mice that 3.0 g/kg EtOH increased the levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and decreased the level of 3-methoxytyramine (3-MT). A 2.0 g/kg dose of EtOH also increased HVA, but there was not a consistent effect within the brain regions of Aldh2-KO and WT mice. There were inconsistent findings of genotype differences in the levels of DA, 5-HT, and their metabolites in the brain regions tested. None of the EtOH doses altered NE, DA, 5-HT, or 5-hydroxyindoleacetic acid contents in any of the brain regions studied. Second, we tested whether EtOH-induced increases in DOPAC and HVA are mediated by increased monoamine oxidase (MAO) or catechol-O-methyltransferase (COMT) activity. To test this, we used the MAO blocker clorgyline (2.0 and 4.0 mg/kg) and the COMT blocker tolcapone (15 and 30 mg/kg) alone or in combination with EtOH (3.0 g/kg). Clorgyline alone increased 3-MT and decreased DOPAC and HVA levels, whereas tolcapone alone increased DOPAC and decreased 3-MT and HVA levels. Surprisingly, the combination of EtOH with clorgyline (4.0 mg/kg) or tolcapone (30 mg/kg) further decreased 3-MT and increased DOPAC and HVA levels, an effect that reversed the inhibitor-induced decreases in HVA. These results suggest that a high concentration of EtOH can accelerate DA metabolism, as evidenced by the increase in DOPAC and HVA, and this effect is likely a consequence of increased degradation of DA by MAO.

Catechol-O-methyltransferase activity does not influence emotional processing in men.

BACKGROUND: Catechol-O-methyltransferase (COMT) regulates cortical dopaminergic transmission and prefrontal-dependent cognitive function. However, its role in other cognitive processes, including emotional processing, is relatively unexplored. We therefore investigated the separate and interactive influences of COMT inhibition and Val158Met (rs4680) genotype on performance on an emotional test battery. METHODS: We recruited 74 healthy men homozygous for the functional COMT Val158Met polymorphism. Volunteers were administered either a single 200 mg dose of the brain-penetrant COMT inhibitor tolcapone or placebo in a double-blind, randomised manner. Emotional processing was assessed using the emotional test battery, and mood was rated using visual analogue scales and the Profile of Mood States (POMS) questionnaire across the test day. RESULTS: There were no main or interactive effects of Val158Met genotype or tolcapone on any of the emotional processing measures or mood ratings. CONCLUSIONS: Our findings suggest that, at least in healthy adult men, COMT has little or no effect on emotional processing or mood. These findings contrast with several neuroimaging studies that suggest that COMT modulates neural activity during emotional processing. Thus, further studies are required to understand how COMT impacts on the relationship between behavioural output and neural activity during emotional processing. Nevertheless, our data suggest that novel COMT inhibitors under development for treating cognitive dysfunction are unlikely to have acute off target effects on emotional behaviours.

Cortical dopamine reduces the impact of motivational biases governing automated behaviour.

Motivations shape our behaviour: the promise of reward invigorates, while in the face of punishment, we hold back. Abnormalities of motivational processing are implicated in clinical disorders characterised by excessive habits and loss of top-down control, notably substance and behavioural addictions. Striatal and frontal dopamine have been hypothesised to play complementary roles in the respective generation and control of these motivational biases. However, while dopaminergic interventions have indeed been found to modulate motivational biases, these previous pharmacological studies used regionally non-selective pharmacological agents. Here, we tested the hypothesis that frontal dopamine controls the balance between Pavlovian, bias-driven automated responding and instrumentally learned action values. Specifically, we examined whether selective enhancement of cortical dopamine either (i) enables adaptive suppression of Pavlovian control when biases are maladaptive; or (ii) non-specifically modulates the degree of bias-driven automated responding. Healthy individuals (n = 35) received the catechol-o-methyltransferase (COMT) inhibitor tolcapone in a randomised, double-blind, placebo-controlled cross-over design, and completed a motivational Go NoGo task known to elicit motivational biases. In support of hypothesis (ii), tolcapone globally decreased motivational bias. Specifically, tolcapone improved performance on trials where the bias was unhelpful, but impaired performance in bias-congruent conditions. These results indicate a non-selective role for cortical dopamine in the regulation of motivational processes underpinning top-down control over automated behaviour. The findings have direct relevance to understanding neurobiological mechanisms underpinning addiction and obsessive-compulsive disorders, as well as highlighting a potential trans-diagnostic novel mechanism to address such symptoms.

Enhancing dopamine tone modulates global and local cortical perfusion as a function of COMT val158met genotype.

The cognitive effects of pharmacologically enhancing cortical dopamine (DA) tone are variable across healthy human adults. It has been postulated that individual differences in drug responses are linked to baseline cortical DA activity according to an inverted-U-shaped function. To better understand the effect of divergent starting points along this curve on DA drug responses, researchers have leveraged a common polymorphism (rs4680) in the gene encoding the enzyme catechol-O-methyltransferase (COMT) that gives rise to greater (Met allele) or lesser (Val allele) extracellular levels of cortical DA. Here we examined the extent to which changes in resting cortical perfusion following the administration of two mechanistically-distinct dopaminergic drugs vary by COMT genotype, and thereby track predictions of the inverted-U model. Using arterial spin labeling (ASL) and a double-blind, within-subject design, perfusion was measured in 75 healthy, genotyped participants once each after administration of tolcapone (a COMT inhibitor), bromocriptine (a DA D2/3 agonist), and placebo. COMT genotype and drug interacted such that COMT Val homozygotes exhibited increased prefusion in response to both drugs, whereas Met homozygotes did not. Additionally, tolcapone-related perfusion changes in the right inferior frontal gyrus correlated with altered performance on a task of executive function. No comparable effects were found for a genetic polymorphism (rs1800497) affecting striatal DA system function. Together, these results indicate that both the directionality and magnitude of drug-induced perfusion change provide meaningful information about individual differences in response to enhanced cortical DA tone.

Virtual Screening of FDA-Approved Drugs against Triose Phosphate Isomerase from Entamoeba histolytica and Giardia lamblia Identifies Inhibitors of Their Trophozoite Growth Phase.

Infectious diseases caused by intestinal protozoan, such as Entamoeba histolytica (E. histolytica) and Giardia lamblia (G. lamblia) are a worldwide public health issue. They affect more than 70 million people every year. They colonize intestines causing primarily diarrhea; nevertheless, these infections can lead to more serious complications. The treatment of choice, metronidazole, is in doubt due to adverse effects and resistance. Therefore, there is a need for new compounds against these parasites. In this work, a structure-based virtual screening of FDA-approved drugs was performed to identify compounds with antiprotozoal activity. The glycolytic enzyme triosephosphate isomerase, present in both E. histolytica and G. lamblia, was used as the drug target. The compounds with the best average docking score on both structures were selected for the in vitro evaluation. Three compounds, chlorhexidine, tolcapone, and imatinib, were capable of inhibit growth on G. lamblia trophozoites (0.05-4.935 µg/mL), while folic acid showed activity against E. histolytica (0.186 µg/mL) and G. lamblia (5.342 µg/mL).

Dopaminergic modulation of regional cerebral blood flow: An arterial spin labelling study of genetic and pharmacological manipulation of COMT activity.

Dopamine has direct and complex vasoactive effects on cerebral circulation. Catechol-O-methyltransferase (COMT) regulates cortical dopamine, and its activity can be influenced both genetically and pharmacologically. COMT activity influences the functional connectivity of the PFC at rest, as well as its activity during task performance, determined using blood oxygen level-dependent (BOLD) fMRI. However, its effects on cerebral perfusion have been relatively unexplored. Here, 76 healthy males, homozygous for the functional COMT Val158Met polymorphism, were administered either the COMT inhibitor tolcapone or placebo in a double-blind, randomised design. We then assessed regional cerebral blood flow at rest using pulsed arterial spin labelling. Perfusion was affected by both genotype and drug. COMT genotype affected frontal regions (Val158 > Met158), whilst tolcapone influenced parietal and temporal regions (placebo > tolcapone). There was no genotype by drug interaction. Our data demonstrate that lower COMT activity is associated with lower cerebral blood flow, although the regions affected differ between those affected by genotype compared with those altered by acute pharmacological inhibition. The results extend the evidence for dopaminergic modulation of cerebral blood flow. Our findings also highlight the importance of considering vascular effects in functional neuroimaging studies, and of exercising caution in ascribing group differences in BOLD signal solely to altered neuronal activity if information about regional perfusion is not available.

Tolcapone, a potent aggregation inhibitor for the treatment of familial leptomeningeal amyloidosis.

Hereditary transthyretin amyloidosis (ATTR) is a disease characterized by the extracellular deposition of transthyretin (TTR) amyloid fibrils. Highly destabilizing TTR mutations cause leptomeningeal amyloidosis, a rare, but fatal, disorder in which TTR aggregates in the brain. The disease remains intractable, since liver transplantation, the reference therapy for systemic ATTR, does not stop mutant TTR production in the brain. In addition, despite current pharmacological strategies have shown to be effective against in vivo TTR aggregation by stabilizing the tetramer native structure and precluding its dissociation, they display low brain permeability. Recently, we have repurposed tolcapone as a molecule to treat systemic ATTR. Crystal structures and biophysical analysis converge to demonstrate that tolcapone binds with high affinity and specificity to three unstable leptomeningeal TTR variants, stabilizing them and, consequently, inhibiting their aggregation. Because tolcapone is an FDA-approved drug that crosses the blood-brain barrier, our results suggest that it can translate into a first disease-modifying therapy for leptomeningeal amyloidosis. DATABASES: PDB codes for A25T-TTR, V30G-TTR, and Y114C-TTR bound to tolcapone are 6TXV, 6TXW, and 6XTK, respectively.

Tissue and interspecies comparison of catechol-O-methyltransferase mediated catalysis of 6-O-methylation of esculetin to scopoletin and its inhibition by entacapone and tolcapone.

Catechol-O-methyltransferase (COMT) methylates both endogenous and exogenous catechol compounds to inactive and safe metabolites. We first optimised conditions for a convenient and sensitive continuous fluorescence-based 6-O-methylation assay of esculetin, which we used for investigating the COMT activity in human, mouse, rat, dog, rabbit, and sheep liver cytosols and microsomes and in ten different rat tissues. Furthermore, we compared the inhibition potencies and mechanisms of two clinically used COMT inhibitors, entacapone and tolcapone, in these species. In most tissues, the COMT activity was at least three times higher in cytosol than in microsomes. In the rat, the highest COMT activity was found in the liver, followed by kidney, ileum, thymus, spleen, lung, pancreas, heart, brain, and finally, skeletal muscle. Entacapone and tolcapone were characterised as highly potent mixed type tight-binding inhibitors. The competitive inhibition type dominated over the uncompetitive inhibition with entacapone, whereas uncompetitive inhibition dominated with tolcapone. Rats, dogs, pigs, and sheep are high COMT activity species, in contrast to humans, mice, and rabbits; COMT activity is highest in the liver. Both entacapone and tolcapone are potent COMT inhibitors, but their inhibition mechanisms differ.

Blinded potency comparison of transthyretin kinetic stabilisers by subunit exchange in human plasma.

Transthyretin (TTR) tetramer dissociation is rate limiting for aggregation and subunit exchange. Slowing of TTR tetramer dissociation via kinetic stabiliser binding slows cardiomyopathy progression. Quadruplicate subunit exchange comparisons of the drug candidate AG10, and the drugs tolcapone, diflunisal, and tafamidis were carried out at 1, 5, 10, 20 and 30 µM concentrations in 4 distinct pooled wild type TTR (TTRwt) human plasma samples. These experiments reveal that the concentration dependence of the efficacy of each compound at inhibiting TTR dissociation was primarily determined by the ratio between the stabiliser's dissociation constants from TTR and albumin, which competes with TTR to bind kinetic stabilisers. The best stabilisers, tafamidis (80 mg QD), AG10 (800 mg BID), and tolcapone (3 x 100 mg over 12 h), exhibit very similar kinetic stabilisation at the plasma concentrations resulting from these doses. At a 10 µM plasma concentration, AG10 is slightly more potent as a kinetic stabiliser vs. tolcapone and tafamidis (which are similar), which are substantially more potent than diflunisal. Dissociation of TTR can be limited to 10% of its normal rate at concentrations of 5.7 µM AG10, 10.3 µM tolcapone, 12.0 µM tafamidis, and 188 µM diflunisal. The potency similarities revealed by our study suggest that differences in safety, adsorption and metabolism, pharmacokinetics, and tissue distribution become important for kinetic stabiliser clinical use decisions.

The catechol-O-methyltransferase inhibitor tolcapone modulates alcohol consumption and impulsive choice in alcohol use disorder.

RATIONALE: Individuals suffering from alcohol use disorder (AUD) demonstrate difficulty with decision-making and impulsivity that may be associated with impaired frontal cortical function. Therapeutics that enhance frontal dopamine tone could decrease impulsivity and in turn reduce alcohol consumption in individuals with AUD. OBJECTIVES: To determine if the catechol-O-methyltransferase (COMT) inhibitor tolcapone can attenuate alcohol consumption in individuals with AUD and whether this attenuation correlates with tolcapone-induced changes in laboratory-based decision-making tasks. METHODS: We used daily self-report and a novel group laboratory bar task to assess the effects of randomized double-blind crossover administration of tolcapone (100 mg TID for 5 days) on alcohol consumption and laboratory tasks assessing impulsivity in 55 non-treatment-seeking subjects with AUD. RESULTS: Tolcapone significantly reduced self-reported alcohol consumption (t (54) = 2.05, p = 0.045). The effects of tolcapone on drinking significantly correlated with changes in impulsive decision-making, such that subjects with the greatest decrease in impulsive choice on tolcapone also reported the greatest decrease in alcohol consumption (r (45) = 0.40, p = 0.0053). We did not see effects of tolcapone on laboratory bar consumption. Adverse event (AE) reporting was low, with no significant difference in frequency or severity of AEs on tolcapone versus placebo. CONCLUSIONS: These data demonstrate that COMT inhibitors such as tolcapone may be useful therapeutics for AUD. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02740582.

Effects of Dopaminergic Drugs on Cognitive Control Processes Vary by Genotype.

Dopamine (DA) has been implicated in modulating multiple cognitive control processes, including the robust maintenance of task sets and memoranda in the face of distractors (cognitive stability) and, conversely, the ability to switch task sets or update the contents of working memory when it is advantageous to do so (cognitive flexibility). In humans, the limited specificity of available pharmacological probes has posed a challenge for understanding the mechanisms by which DA, acting on multiple receptor families across the PFC and striatum, differentially influences these cognitive processes. Using a within-subject, placebo-controlled design, we contrasted the impact of two mechanistically distinct DA drugs, tolcapone (an inhibitor of catechol-O-methyltransferase [COMT], a catecholamine inactivator) and bromocriptine (a DA agonist with preferential affinity for the D2 receptor), on the maintenance and switching of task rules. Given previous work demonstrating that drug effects on behavior are dependent on baseline DA tone, participants were stratified according to genetic polymorphisms associated with cortical (COMT Val158Met) and striatal (Taq1A) DA system function. Our results were partially consistent with an inverted-U-shaped relationship between tolcapone and robust rule maintenance (interaction with COMT genotype) and between bromocriptine and cued rule switching (interaction with Taq1A genotype). However, when task instructions were ambiguous, a third relationship emerged to explain drug effects on spontaneous task switching (interaction of COMT genotype and bromocriptine). Together, this pattern of results suggests that the effects of DA drugs vary not only as a function of the DA system component upon which they act but also on subtle differences in task demands and context.

Transthyretin stabilization activity of the catechol-O-methyltransferase inhibitor tolcapone (SOM0226) in hereditary ATTR amyloidosis patients and asymptomatic carriers: proof-of-concept study.

Objective: To assess the transthyretin (TTR) stabilization activity of tolcapone (SOM0226) in patients with hereditary ATTR amyloidosis, asymptomatic carriers and healthy volunteers. Methods: A phase IIa proof-of-concept trial included two phases separated by a 6-week washout period. Phase A: single 200 mg dose of tolcapone; phase B: three 100 mg doses taken at 4 h intervals. The primary efficacy variable was TTR stabilization. Results: Seventeen subjects were included (wild type, n = 6; mutation TTR Val30Met, n = 11). TTR stabilization was observed in all participants. Two hours after dosing, 82% of participants in phase A and 93% of those in phase B reached a TTR stabilization value of at least 20%. In phase A, there was an increase of 52% in TTR stabilization vs baseline values 2 h after dosing, which decreased to 22.9% at 8 h. In phase B, there was a significant increase of 38.8% in TTR stabilization 2 h after the first 100 mg dose. This difference was maintained after 10 h and decreased after 24 h. No serious adverse events were observed. Conclusions: The ability of tolcapone for stabilizing TTR supports further development and repositioning of the drug for the treatment of ATTR amyloidosis. EudraCT trial number: 2014-001586-27 ClinicalTrials.gov Identifier: NCT02191826.

Coupling S-adenosylmethionine-dependent methylation to growth: Design and uses.

We present a selection design that couples S-adenosylmethionine-dependent methylation to growth. We demonstrate its use in improving the enzyme activities of not only N-type and O-type methyltransferases by 2-fold but also an acetyltransferase of another enzyme category when linked to a methylation pathway in Escherichia coli using adaptive laboratory evolution. We also demonstrate its application for drug discovery using a catechol O-methyltransferase and its inhibitors entacapone and tolcapone. Implementation of this design in Saccharomyces cerevisiae is also demonstrated.

Pharmacodynamic evaluation of novel Catechol-O-methyltransferase inhibitors.

Currently, peripheral COMT inhibitors have an important role in the treatment of Parkinson's disease, and central COMT inhibitors have a potential role in the treatment of various neuropsychiatric disorders, such as attention deficit hyperactivity disorder. Adverse reactions, low bioavailability and short elimination half-lives have prompted the development of new selective COMT inhibitors. The objective of this study was to evaluate the pharmacodynamic properties of novel tight-binding COMT inhibitors (NC, NE, NDE, NCAPE, CNCAFBn, CNCAPE, NCAFBn, CNCAPA, CNCABA and CNCAHA) and compared to standard inhibitors tolcapone and entacapone. The activity of soluble (S) and membrane bound (MB) COMT from rat liver and brain was assessed in the presence of varying concentrations of each inhibitor. NE and NC behaved most potently against liver S-COMT, and CNCAPE was the most potent inhibitor against brain MB-COMT. The cytotoxicity of tolcapone and CNCAPE in human neuroblastoma SK-N-SH cells and human liver adenocarcinoma SK-HEP-1 cells was also assessed. At lower concentrations, CNCAPE did not reduce cell viability, suggesting that CNCAPE may have a potential therapeutic role as a centrally active COMT inhibitor.

COMT Inhibition Alters Cue-Evoked Oscillatory Dynamics during Alcohol Drinking in the Rat.

Alterations in the corticostriatal system have been implicated in numerous substance use disorders, including alcohol use disorder (AUD). Adaptations in this neural system are associated with enhanced drug-seeking behaviors following exposure to cues predicting drug availability. Therefore, understanding how potential treatments alter neural activity in this system could lead to more refined and effective approaches for AUD. Local field potentials (LFPs) were acquired simultaneously in the prefrontal cortex (PFC) and nucleus accumbens (NA) of both alcohol preferring (P) and Wistar rats engaged in a Pavlovian conditioning paradigm wherein a light cue signaled the availability of ethanol (EtOH). On test days, the catechol-o-methyl-transferase (COMT) inhibitor tolcapone was administered prior to conditioning. Stimulus-evoked voltage changes were observed following the presentation of the EtOH cue in both strains and were most pronounced in the PFC of P rats. Phase analyses of LFPs in the θ band (5-11 Hz) revealed that PFC-NA synchrony was reduced in P rats relative to Wistars but was robustly increased during drinking. Presentation of the cue resulted in a larger phase reset in the PFC of P rats but not Wistars, an effect that was attenuated by tolcapone. Additionally, tolcapone reduced cued EtOH intake in P rat but not Wistars. These results suggest a link between corticostriatal synchrony and genetic risk for excessive drinking. Moreover, inhibition of COMT within these systems may result in reduced attribution of salience to reward paired stimuli via modulation of stimulus-evoked changes to cortical oscillations in genetically susceptible populations.

Dopamine, time perception, and future time perspective.

RATIONALE: Impairment in time perception, a critical component of decision-making, represents a risk factor for psychiatric conditions including substance abuse. A therapeutic that ameliorates this impairment could be advantageous in the treatment of impulsivity and decision-making disorders. OBJECTIVES: Here we hypothesize that the catechol-O-methyltransferase (COMT) inhibitor tolcapone, which increases dopamine tone in frontal cortex (Ceravolo et al Synapse 43:201-207, 2002), improves time perception, with predictive behavioral, genetic, and neurobiological components. METHODS: Subjects (n = 66) completed a duration estimation task and other behavioral testing in each of two sessions after receiving a single oral dose of tolcapone (200 mg) or placebo in randomized, double-blind, counterbalanced, crossover fashion. Resting state fMRI data were obtained in a subset of subjects (n = 40). Subjects were also genotyped for the COMT (rs4680) polymorphism. RESULTS: Time perception was significantly improved across four proximal time points ranging from 5 to 60 s (T(524) = 2.04, p = 0.042). The degree of this improvement positively correlated with subjective measures of stress, depression, and alcohol consumption and was most robust in carriers of the COMT Val158 allele. Using seed regions defined by a previous meta-analysis (Wiener et al Neuroimage 49:1728-1740, 2010), we found not only that a connection from right inferior frontal gyrus (RIFG) to right putamen decreases in strength on tolcapone versus placebo (p < 0.05, corrected), but also that the strength of this decrease correlates inversely with the increase in duration estimation on tolcapone versus placebo (r = - 0.37, p = 0.02). CONCLUSIONS: Compressed time perception can be ameliorated by administration of tolcapone. Additional studies should be conducted to determine whether COMT inhibitors may be effective in treating decision-making disorders and addictive behaviors.