Recovery of biological active catechol-O-methyltransferase isoforms from Q-sepharose.

The development of new catechol-O-methyltransferase inhibitors has led to an improvement in the treatment of Parkinson's disease. However, despite the fact that the soluble isoform has been extensively investigated, few studies have been published concerning membrane isoform chromatographic recovery and bioactivity levels. In this work, chromatographic profiles of both catechol-O-methyltransferase isoforms were compared using quaternary amine as a ligand to evaluate its activity levels and recovery rates. Results show that both proteins required different conditions for adsorption; the soluble isoform adsorption was performed at low ionic strength, while the membrane isoform required increasing linear salt gradient. However, the application of 0.5% Triton X-100 promoted membrane isoform adsorption even at low ionic strength. Indeed, chromatographic conditions of both isoforms became similar when detergents were applied. The developed methods also appear to be highly effective in bioactivity recovery, presenting rates of 107% for soluble protein and 67 and 91% for membrane isoform without and with detergents, respectively. The chromatographic strategies with and without detergents resulted in a 4.3- and sevenfold purification, respectively, corresponding to specific activity values of 331 and 496 nmol/h/mg. Thus, the use of Q-sepharose as anion exchanger was effective in the recovery of both enzymes, which is a requirement for further kinetic and pharmacological trials.

Assessment of COMT isolation by HIC using a dual salt system and low temperature.

Sodium citrate (SC) and low temperatures between 7 and 5 degrees C are effective in suppressing aggregation of proteins and may be beneficial to be included during a purification process. In this work, we analyzed the application of dual salt system, ammonium sulfate (AS) and SC on binding and elution conditions of recombinant hSCOMT on typical HIC sorbents. Specifically in butyl and octyl supports, the use of, respectively, 300 mM AS/200 mM SC and 25 mM AS/25 mM SC in the loading buffer resulted in complete binding of COMT. Elution was obtained by decreasing the ionic strength to 0 M of salt. For the delineate goal, it also favorably increased the support chain length while a consequent decrease in the dual ionic strength was observed for hSCOMT retention. In the presence of dual salt systems octyl media exhibited classic HIC behavior, good protein selectivity, an excellent purification factor and reduced denaturation effects of hSCOMT observed with higher salt concentrations. Also the inclusion of temperature control during the elution step appears to be advantageous for greater activity recovery without enzyme aggregation. In fact, these results could allow the prediction of most stabilizing conditions for this termolabile enzyme on the chromatographic stage, regarding salt types and therefore effectiveness to improve HIC selectivity and desirable purity on the target fractions.

Application of a fed-batch bioprocess for the heterologous production of hSCOMT in Escherichia coli.

In this paper, a fed-batch cultivation process in recombinant Escherichia coli BL21(DE3) bacteria, for the production of human soluble catechol-O-methyltransferase (hSCOMT), is presented. For the first time, a straightforward model is applied in a recombinant hSCOMT expression system and distinguishes an initial cell growth phase from a protein production phase upon induction. Specifically, the kinetic model predicts biomass, substrate, and product concentrations in the culture over time and was identified from a series of fed-batch experiments designed by testing several feed profiles. The main advantage of this model is that its parameters can be identified more reliably from distinct fed-batch strategies, such as glycerol pulses and exponential followed by constant substrate additions. Interestingly, with the limited amount of data available, the proposed model accomplishes satisfactorily the experimental results obtained for the three state variables, and no exhaustive process knowledge is required. The comparison of the measurement data obtained in a validation experiment with the model predictions showed the great extrapolation capability of the model presented, which could provide new complementary information for the COMT production system.

A new approach on the purification of recombinant human soluble catechol-O-methyltransferase from an Escherichia coli extract using hydrophobic interaction chromatography.

Catechol-O-methyltransferase (COMT) is a significant target in protein engineering due to its role not only in normal brain function but also to its possible involvement in some human disorders. In this work, a new approach was employed for the purification of recombinant human soluble COMT (hSCOMT) using hydrophobic interaction chromatography, as the main isolation method, from an Escherichia coli culture broth. A simplified overall process flow is proposed. Indeed, with an optimized heterologous expression system for recombinant hSCOMT production, such as E. coli, it was possible to produce and recover the active monomeric enzyme directly from the cell crude culture broth either by a freeze/thaw or ultrasonication lysis step. The recombinant enzyme present in the bacterial soluble fraction, exhibited similar affinity for epinephrine (K(m) 276 [215; 337] microM) and the methyl donor (S-adenosyl-L-methionine, SAMe) (K(m) 36 [30; 41]microM) as human SCOMT. After the precipitation step by 55% of ammonium sulphate, a HIC step on the butyl-sepharose resin was found to be highly effective in selectively eluting a range of contaminating key proteins present in the concentrate soluble extract. Consequently, the partially purified eluate from HIC could then be loaded and polished by gel filtration in order to increase the process efficiency. The final product appeared as a single band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The procedure resulted in a global 10.9-fold purification with a specific activity of 5500 nmol/h/mg of protein. The widespread applicability of the process, here described, to different COMT sources could make this protocol highly useful for all studies requiring purified and active COMT proteins.

'In vitro' amyloid fibril formation from transthyretin: the influence of ions and the amyloidogenicity of TTR variants.

The mechanisms of amyloid formation in Familial Amyloidotic Polyneuropathy (FAP) are unknown, as well as the factors determining the development of this pathology. To get some insights into this process, we have first tested a fluorimetric assay with thioflavine T, as a quantitative method for transthyretin (TTR) amyloid estimation, using amyloid isolated from post-mortem tissues of a FAP patient. Then production of amyloid fibrils from soluble TTR was achieved by acidification and optimized for protein concentration and pH. The effect of different ions such as metal and sulphate ions in the process of amyloid formation from wild type TTR was compared using a kinetic assay. Under the conditions tested sulphate diminishes the amount of amyloid formed from wild type TTR and in addition appears to promote aggregation of preexisting amyloid fibrils. The relative amyloidogenicity of three TTR variants, TTR Met30, TTR Pro55 and TTR Met119 respectively, was evaluated using a pH dependent assay. It was shown that the Pro55 variant is highly susceptible to amyloid formation as compared to the wild type protein; on the contrary, the Met119 variant is more resistant than the other TTR proteins towards precipitation into amyloid. These results are in agreement with the pathological conditions associated with these mutations. This type of assay has a wide application for testing the influence of other factors, such as therapeutical agents, on amyloid formation.

Crystallization and preliminary X-ray diffraction studies of a catechol-O-methyltransferase/inhibitor complex.

Inhibitors of the enzyme catechol-O-methyltransferase (COMT) are used as co-adjuvants in the therapy of Parkinson's disease. A recombinant form of the soluble cytosolic COMT from rat has been co-crystallized with a new potent inhibitor, BIA 8-176 [(3,4-dihydroxy-2-nitrophenyl)phenylmethanone], by the vapour-diffusion method using PEG 6K as precipitant. Crystals diffract to 1.6 A resolution on a synchrotron-radiation source and belong to the monoclinic space group P2(1), with unit-cell parameters a = 52.77, b = 79.63, c = 61.54 A, beta = 91.14 degrees.

Evaluation of Mut(S) and Mut⁺ Pichia pastoris strains for membrane-bound catechol-O-methyltransferase biosynthesis.

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is an enzyme that catalyzes the methylation of catechol substrates, and while structural and functional studies of its membrane-bound isoform (MBCOMT) are still hampered by low recombinant production, Pichia pastoris has been described as an attractive host for the production of correctly folded and inserted membrane proteins. Hence, in this work, MBCOMT biosynthesis was developed using P. pastoris X33 and KM71H cells in shake flasks containing a semidefined medium with different methanol concentrations. Moreover, after P. pastoris glass beads lysis, biologically and immunologically active hMBCOMT was found mainly in the solubilized membrane fraction whose kinetic parameters were identical to its correspondent native enzyme. In addition, mixed feeds of methanol and glycerol or sorbitol were also employed, and its levels quantified using liquid chromatography coupled to refractive index detection. Overall, for the first time, two P. pastoris strains with opposite phenotypes were applied for MBCOMT biosynthesis under the control of the strongly methanol-inducible alcohol oxidase (AOX) promoter. Moreover, this eukaryotic system seems to be a promising approach to deliver MBCOMT in high quantities from fermentor cultures with a lower cost-benefit due to the cheaper cultivation media coupled with the higher titers tipically achieved in biorreactors, when compared with previously reported mammallian cell cultures.

Pharmacological profile of opicapone, a third-generation nitrocatechol catechol-O-methyl transferase inhibitor, in the rat.

BACKGROUND AND PURPOSE: Catechol-O-methyltransferase (COMT) is an important target in the levodopa treatment of Parkinson's disease; however, the inhibitors available have problems, and not all patients benefit from their efficacy. Opicapone was developed to overcome those limitations. In this study, opicapone's pharmacological properties were evaluated as well as its potential cytotoxic effects. EXPERIMENTAL APPROACH: The pharmacodynamic effects of opicapone were explored by evaluating rat COMT activity and levodopa pharmacokinetics, in the periphery through microdialysis and in whole brain. The potential cytotoxicity risk of opicapone was explored in human hepatocytes by assessing cellular ATP content and mitochondrial membrane potential. KEY RESULTS: Opicapone inhibited rat peripheral COMT with ED50 values below 1.4 mg⋅kg(-1) up to 6 h post-administration. The effect was sustained over the first 8 h and by 24 h COMT had not returned to control values. A single administration of opicapone resulted in increased and sustained plasma levodopa levels with a concomitant reduction in 3-O-methyldopa from 2 h up to 24 h post-administration, while tolcapone produced significant effects only at 2 h post-administration. The effects of opicapone on brain catecholamines after levodopa administration were sustained up to 24 h post-administration. Opicapone was also the least potent compound in decreasing both the mitochondrial membrane potential and the ATP content in human primary hepatocytes after a 24 h incubation period. CONCLUSIONS AND IMPLICATIONS: Opicapone has a prolonged inhibitory effect on peripheral COMT, which extends the bioavailability of levodopa, without inducing toxicity. Thus, it exhibits some improved properties compared to the currently available COMT inhibitors.

Studies on the synthesis and secretion of transthyretin by the human hepatoma cell line Hep G2.

Transthyretin (TTR) is a circulatory protein which plays an important role in the transport of both thyroid hormone and retinol. Hep G2 cells, a human hepatoma-derived cell line, have been used extensively in studies of protein secretion by liver cells. The original description of this cell line indicated that this line, unlike primary hepatocytes, does not secrete TTR. We now report studies which reexamine the ability of Hep G2 cells to synthesize and secrete TTR. For this purpose, total RNA was isolated from Hep G2 cells grown on both uncoated and collagen-coated plastic plates and was examined for TTR expression by Northern blot analysis. TTR mRNA was found to be present in nearly equal amounts in Hep G2 cells cultured in either condition. When Hep G2 cells were cultured in [35S]methionine-containing medium, the cells were found both to synthesize and to secrete immunoprecipitable [35S]TTR. Hep G2 cells were found, by sensitive and specific radioimmunoassay, to contain 142 +/- 91 ng TTR/10(6) cells and to secrete TTR into the medium at a nearly constant rate for at least 24 h after medium change. Our data demonstrate that Hep G2 cells do synthesize and secrete TTR and suggest that this cell line might be useful for studies of the secretion of TTR.

Retrovirus-mediated gene transfer of transthyretin and transthyretin-methionine 30: a potential tool for the study of amyloidogenesis.

Familial amyloidotic polyneuropathy (FAP) is a genetic disease characterized by systemic amyloid deposition particularly in the peripheral nervous system. These deposits are composed mainly of a mutant form of the serum protein transthyretin (TTR) having a methionine for valine substitution at position 30-TTR Met 30. The factors involved in the formation of these deposits are unknown. The existence of animal models for FAP should allow elucidation of these factors. As one approach to the development of animal models for amyloidogenesis in FAP, we have constructed recombinant retrovirus vectors, carrying the full length human cDNA for either TTR or TTR Met 30 under the control of the Moloney murine leukemia virus (MoMLV) LTR element. After transfection of the packaging cell line, psi 2, viral stocks were used to infect a rat hepatoma cell line, H56, mouse fibroblast cell line, NIH3T3, and mouse primary fibroblasts. H56 cells efficiently secreted both TTR and TTR Met 30 as assessed by immunoprecipitation and ELISA, whereas NIH3T3 fibroblasts appeared not to release these proteins under the conditions tested. Primary fibroblasts secreted the mutant protein as assessed by ELISA. These genetically modified cells can be grafted into animals for in vivo study of amyloidogenesis, as well as be used in culture to investigate factors that might regulate the rate of amyloid deposition.

Synthesis, anticonvulsant properties and pharmacokinetic profile of novel 10,11-dihydro-10-oxo-5H-dibenz/b,f/azepine-5-carboxamide derivatives.

A series of novel derivatives of oxcarbazepine (5), 10,11-dihydro-10-oxo-5H-dibenz/b,f/azepine-5-carboxamide was synthesised and evaluated for their anticonvulsant activity and sodium channel blocking properties. The oxime 8 was found to be the most active compound from this series, displaying greater potency than its geometric isomer 9 and exhibiting also the highest protective index value. Importantly, the metabolic profile of 8 differs from the already established dibenz/b,f/azepine-5-carboxamide drugs such as 1 and 5 which undergo rapid and complete conversion in vivo to several biologically active metabolites. In contrast 8 is metabolised to only a very minor extent leading to the conclusion that the observed anti-convulsant effect is solely attributable to 8. It is concluded that 8 may be as effective as 1 and 5 at controlling seizures and that the low toxicity and consequently high protective index should provide the compound with an improved side-effect profile.

Brain and peripheral pharmacokinetics of levodopa in the cynomolgus monkey following administration of opicapone, a third generation nitrocatechol COMT inhibitor.

OBJECTIVE: The present study aimed at evaluating the effect of opicapone, a third generation nitrocatechol catechol-O-methyltransferase (COMT) inhibitor, on the systemic and central bioavailability of 3,4-dihydroxy-l-phenylalanine (levodopa) and related metabolites in the cynomolgus monkey. METHODS: Four monkeys, implanted with guiding cannulas for microdialysis probes, in the substantia nigra, dorsal striatum and prefrontal cortex, were randomized in two groups that received, in a crossover design, vehicle or 100 mg/kg opicapone for 14 days. Twenty-three hours after last administration of vehicle or opicapone, animals were challenged with levodopa/benserazide (12/3 mg/kg). Extracellular dialysate and blood samples were collected over 360 min (at 30 min intervals) for the assays of catecholamine and COMT activity. RESULTS: Opicapone increased levodopa systemic exposure by 2-fold not changing Cmax values and reduced both 3-O-methyldopa (3-OMD) exposure and Cmax values by 5-fold. These changes were accompanied by ∼76-84% reduction in erythrocyte COMT activity. In dorsal striatum and substantia nigra, opicapone increased levodopa exposure by 1.7- and 1.4-fold, respectively, reducing 3-OMD exposure by 5- and 7-fold respectively. DOPAC exposure was increased by 4-fold in the substantia nigra. In the prefrontal cortex, opicapone increased levodopa exposure and reduced 3-OMD levels by 2.3- and 2.4-fold, respectively. CONCLUSIONS: Opicapone behaved as long-acting COMT inhibitor that markedly increased systemic and central levodopa bioavailability. Opicapone is a strong candidate to fill the unmet need for COMT inhibitors that lead to more sustained levodopa levels in Parkinson's disease patients.

Development of fed-batch profiles for efficient biosynthesis of catechol-O-methyltransferase.

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) plays a crucial role in dopamine metabolism which has intimately linked this enzyme to some neurodegenerative diseases, such as Parkinson's disease. In recent years, in the attempt of developing new therapeutic strategies for Parkinson's disease, there has been a growing interest in the search for effective COMT inhibitors. In order to do so, large amounts of COMT in an active form are needed, and the best way to achieve this is by up-scaling its production through biotechnological processes. In this work, a fed-batch process for the biosynthesis of the soluble isoform of COMT in Escherichia coli is proposed. This final process was selected through the evaluation of the effect of different dissolved oxygen concentrations, carbon and nitrogen source concentrations and feeding profiles on enzymatic production and cell viability, while controlling various parameters (pH, temperature, starting time of the feeding and induction phases and carbon source concentration) during the process. After several batch and fed-batch experiments, a final specific COMT activity of 442.34 nmol/h/mg with approximately 80% of viable cells at the end of the fermentation were achieved. Overall, the results described herein provide a great improvement on hSCOMT production in recombinant bacteria and provide a new and viable option for the use of a fed-batch fermentation with a constant feeding profile to the large scale production of this enzyme.

Optimization of fermentation conditions for the production of human soluble catechol-O-methyltransferase by Escherichia coli using artificial neural network.

The aim of this work was to optimize the temperature, pH and stirring rate of the production of human soluble catechol-O-methyltransferase (hSCOMT) in a batch Escherichia coli culture process. A central composite design (CCD) was firstly employed to design the experimental assays used in the evaluation of these operational parameters on the hSCOMT activity for a semi-defined and complex medium. Predictive artificial neural network (ANN) models of the hSCOMT activity as function of the combined effects of these variables was proposed based on this exploratory experiments performed for the two culture media. The regression coefficients (R(2)) for the final models were 0.980 and 0.983 for the semi-defined and complex medium, respectively. The ANN models predicted a maximum hSCOMT activity of 183.73 nmol/h, at 40 °C, pH 6.5 and stirring rate of 351 rpm, and 132.90 nmol/h, at 35 °C, pH 6.2 and stirring rate of 351 rpm, for semi-defined and complex medium, respectively. These results represent a 4-fold increase in total hSCOMT activity by comparison to the standard operational conditions used for this bioprocess at slight scale.

Analysis of hSCOMT adsorption in bioaffinity chromatography with immobilized amino acids: the influence of pH and ionic strength.

In the last years, chromatographic supports with amino acids as immobilized ligands (AAILs) were been used successfully for isolation of several biomolecules, such as proteins. In this context and based on specific properties of human soluble cathecol-O-methyltransferase (hSCOMT), we screened and analyzed the effect of experimental conditions, such as pH and ionic strength manipulation for hSCOMT adsorption, over six different AAIL commercial supports. Typically, the proteins adsorption on AAIL chromatographic supports is around their pI. While hSCOMT isoelectric point is around 5.5, this parameter leads us to design new adsorption strategies with several acid buffers for the chromatographic process. In terms of the ionic strength manipulation strategy, the results suggest that the AAILs-hSCOMT interaction is strongly affected by the intrinsic hSCOMT hydrophobic domains. On the other hand, the interaction mechanism of hSCOMT on amino acid resins appears to be highly dependent on the binding pH. Consequently the retention mechanism of the target enzyme on the AAILs can be as either in typical hydrophobic or ionic chromatographic supports, so long as selecting various mobile phases and separation conditions. In spite of these mixed-mode interactions and operation strategies, the elution of interferent's proteins from recombinant host can be achieved only with suitable adjusts in pH mobile phase set point. This lead to a new approach in biochromatographic COMT retention, while possess a higher specificity than other chromatographic methods reported in literature.

A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltransferase.

Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. While large amounts of purified proteins are required for pharmaceutical and crystallization attempts, there is an unmet need for the development of novel heterologous membrane protein overexpression systems. Specifically, we tested the application of Brevibacillus choshinensis cells for the biosynthesis of human membrane bound catechol-O-methyltransferase (hMBCOMT). In terms of the upstream stage moderate to high expression was obtained for complex media formulation with a value near 45 nmol/h/mg for hMBCOMT specific activity achieved at 20 h culture with 37°C and 250 rpm. Subsequently, the efficiency for reconstitution of hMBCOMT is markedly null in the presence of ionic detergents, such as sodium dodecyl sulphate (SDS). In general, for non-ionic and zwiterionic detergents, until a detergent critic micellar concentration (CMC) of 1.0 mM, hMBCOMT shows more biological activity at lower detergent concentrations while for detergent CMC higher than 1 mM, higher detergent concentrations seem to be ideal for hMBCOMT solubilization. Indeed, from the detergents tested, the non-ionic digitonin at 0.5% (w/v) appears to be the most suitable for hMBCOMT solubilization.

Comparative study on the interaction of recombinant human soluble catechol-O-methyltransferase on some hydrophobic adsorbents.

The main scope of this work is the evaluation and potential application of hydrophobic interaction chromatography in the isolation of recombinant human soluble catechol-O-methyltransferase (hSCOMT) from an Escherichia coli cell extract. Therefore, a comparative study on the interaction of recombinant hSCOMT with different hydrophobic adsorbents (butyl-, octyl-, phenyl- and epoxy-Sepharose), was developed. The four adsorbents were evaluated in terms of selectivity, recovery and fractionation of recombinant hSCOMT from its Escherichia coli-free culture broth. Our data shows that the adjustment of the ionic strength on the mobile phase and the type of hydrophobic ligand are the most useful factors for a complete binding of hSCOMT and a selective fractionation of contaminating proteins. The results of these studies demonstrate that, although epoxy-Sepharose is used as a last resort due to the high salt concentrations needed, hSCOMT bind to the other three resins at low concentrations of ammonium sulfate (< or = 0.6 M) and eluted just by decreasing the ionic strength on the eluent to 0 M, without loss of specific of activity. The stepwise gradient with 0.6, 0.2, 0.075 and 0 M of ammonium sulfate onto a butyl-Sepharose media was found to be the most effective in the isolation of hSCOMT, leading to an enzyme solution with a 3.9-fold increased in specific activity. Since biochemical and structural studies require significant quantities of the enzyme in an active form, the above described approach may give some insight into the optimization and development of new purification strategies of mammalian COMTs.

The effect of temperature on the analysis of metanephrine for catechol-O-methyltransferase activity assay by HPLC with electrochemical detection.

The enzyme catechol-O-methyltransferase (COMT) plays an important role in the metabolism of catechol estrogens and degradation of the catecholamine neurotransmitters, such as epinephrine. Several analytical methods, mainly high-performance liquid chromatography with electrochemical amperometric detection, have been reported for the analysis of catecholamines and their metabolites in biological fluids. In this paper we report the relevance of controlling temperature in calibration procedures of metanephrine, an O-methylated product of catechol-O-methyltransferase, using epinephrine as substrate. The results at higher temperatures show shorter retention times of metanephrine, no undue band-broadening and increased electro signals. This study also showed that, despite different temperatures leading to similarly specific activities of recombinant human COMT as expected, there are additional advantages in flow analytical methods where good sensitivity, efficiency and selectivity is required, mainly in tissues with low levels of COMT activity.

Human metabolism of nebicapone (BIA 3-202), a novel catechol-o-methyltransferase inhibitor: characterization of in vitro glucuronidation.

Nebicapone (BIA 3-202; 1-[3,4-dihydroxy-5-nitrophenyl]-2-phenylethanone), a novel catechol-O-methyltransferase inhibitor, is mainly metabolized by glucuronidation. The purpose of this study was to characterize the major plasma metabolites of nebicapone following p.o. administration of nebicapone to healthy volunteers, and to determine the human UDP-glucuronosyltransferase (UGT) enzymes involved in nebicapone glucuronidation. Plasma samples were collected as part of a clinical trial at different time points postdose and were analyzed for nebicapone and its metabolites using a validated method consisting of a solid-phase extraction, followed by high-performance liquid chromatography/mass spectrometry detection. The primary metabolic pathways of nebicapone in humans involve mainly 3-O-glucuronidation, the major early metabolite, and 3-O-methylation, the predominant late metabolite. Of the nine commercially available recombinant UGT enzymes studied (UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15), only UGT1A9 exhibited high nebicapone glucuronosyltransferase specific activity (24.3 +/- 1.3 nmol/mg protein/min). UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B7, and UGT2B15 exhibited low activity (0.1-1.1 nmol/mg protein/min), and UGT1A1 and UGT1A3 showed extremely low activities (less than 0.03 nmol/mg protein/min). The results show that nebicapone is mainly glucuronidated in humans and that multiple UGT enzymes are involved in this reaction.

Comparative study of ortho- and meta-nitrated inhibitors of catechol-O-methyltransferase: interactions with the active site and regioselectivity of O-methylation.

In this work, we present a comparative case study of "ortho-" and "meta-nitrated" catecholic inhibitors of catechol-O-methyltransferase (COMT), with regard to their interaction with the catalytic site of the enzyme and the in vitro regioselective formation of their mono-O-methyl ether metabolites. In particular, the effects of altering the attachment position of the inhibitors' side-chain substituent, within the classic nitrocatechol pharmacophore, were investigated. For this purpose, we compared two simple regioisomeric nitrocatechol-type inhibitors of COMT, BIA 3-228 and BIA 8-176, which contain the benzoyl substituent attached at the meta and ortho positions, respectively, relative to the nitro group. The two compounds were slowly O-methylated by COMT in vitro, but the particular substitution pattern of each compound was shown to have a profound impact on the regioselectivity of their O-methylation. To provide a plausible interpretation of these results, a comprehensive analysis of the protein-inhibitor interactions and of the relative chemical susceptibility to O-methylation of the catechol hydroxyl groups was performed by means of docking simulations and ab initio molecular orbital calculations. The major structural and chemical factors that determine the enzyme regioselectivity of O-methylation were identified, and the X-ray structure of the complex of COMT with S-adenosyl-l-methionine and BIA 8-176 is herein disclosed. This is the first reported structure of the soluble form of COMT complexed with a nitrocatecholic inhibitor having a bulky substituent group in adjacent position (ortho) to the nitro group. Structural and dynamic aspects of this complex are analyzed and discussed, in the context of the present study.