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Abstract: Several studies suggest a pivotal role of amyloid beta (Aβ)1-42 and nitric oxide (NO) in the pathogenesis of Alzheimer’s disease. NO also possess central neuromodulatory properties. To study the soluble Aβ1-42 effects on dopamine concentrations in rat prefrontal cortex, microdialysis technique was used. We showed that i.c.v. injection or retrodialysis Aβ1-42 administration reduced basal and K+-stimulated dopamine levels, measured 2 and 48 h after peptide administration. Immunofluorescent experiments revealed that after 48 h from i.c.v. injection Aβ1-42 was no longer detectable in the ventricular space. We then evaluated the role of NO on Aβ1-42-induced reduction in dopamine concentrations. Subchronic l-arginine administration decreased basal dopamine levels, measured either 2 h after i.c.v. Aβ1-42 or on day 2 post-injection, whereas subchronic 7-nitroindazole administration increased basal dopamine concentrations, measured 2 h after i.c.v. Aβ1-42 injection, and decreased them when measured on day 2 post-Aβ1-42-injection. No dopaminergic response activity was observed after K+ stimulation in all groups. These results suggest that the dopaminergic system seems to be acutely vulnerable to soluble Aβ1-42 effects. Finally, the opposite role of NO occurring at different phases might be regarded as a possible link between Aβ1-42-induced effects and dopaminergic dysfunction. [Copyright &y& Elsevier]

The cytochrome P450 superfamily are heme-thiolate enzymes able to carry out monooxygenase reactions. Several studies have demonstrated the feasibility of using a soluble bacterial reductase from Bacillus megaterium, BMR, as an artificial electron transfer partner fused to the human P450 domain in a single polypeptide chain in an approach known as ‘molecular Lego’. The 3A4-BMR chimera has been deeply characterized biochemically for its activity, coupling efficiency, and flexibility by many different biophysical techniques leading to the conclusion that an extension of five glycines in the loop that connects the two domains improves all the catalytic parameters due to improved flexibility of the system. In this work, we extend the characterization of 3A4-BMR chimeras using differential scanning calorimetry to evaluate stabilizing role of BMR. We apply the ‘molecular Lego’ approach also to CYP19A1 (aromatase) and the data show that the activity of the chimeras is very low (<0.003 min−1) for all the constructs tested with a different linker loop length: ARO-BMR, ARO-BMR-3GLY, and ARO-BMR-5GLY. Nevertheless, the fusion to BMR shows a remarkable effect on thermal stability studied by differential scanning calorimetry as indicated by the increase in Tonset by 10 °C and the presence of a cooperative unfolding process driven by the BMR protein domain. Previously characterized 3A4-BMR constructs show the same behavior of ARO-BMR constructs in terms of thermal stabilization but a higher activity as a function of the loop length. A comparison of the ARO-BMR system to 3A4-BMR indicates that the design of each P450-BMR chimera should be carefully evaluated not only in terms of electron transfer, but also for the biophysical constraints that cannot always be overcome by chimerization.

Human aromatase is a member of the cytochrome P450 superfamily, involved in steroid hormones biosynthesis. In particular, it converts androgen into estrogens being therefore responsible for the correct sex steroids balance. Due to its capacity in producing estrogens it has also been considered as a promising target for breast cancer therapy. Two single-nucleotide polymorphisms (R264C and R264H) have been shown to alter aromatase activity and they have been associated to an increased or decreased risk for estrogen-dependent pathologies. Here, the effect of these mutations on the protein dynamics is investigated by UV/FTIR and time resolved fluorescence spectroscopy. H/D exchange rates were measured by FTIR for the three proteins in the ligand-free, substrate- and inhibitor-bound forms and the data indicate that the wild-type enzyme undergoes a conformational change leading to a more compact tertiary structure upon substrate or inhibitor binding. Indeed, the H/D exchange rates are decreased when a ligand is present. In the variants, the exchange rates in the ligand-free and -bound forms are similar, indicating that a structural change is lacking, despite the single amino acid substitution is located in the peripheral shell of the protein molecule. Moreover, the fluorescence lifetimes data show that the quenching effect on tryptophan-224 observed upon ligand binding in the wild-type, is absent in both variants. Since this residue is located in the catalytic pocket, these findings suggest that substrate entrance and/or retention in the active site is partially compromised in both mutants. A contact network analysis demonstrates that the protein structure is organized in two main clusters, whose connectivity is altered by ligand binding, especially in correspondence of helix-G, where the amino acid substitutions occur. Our findings demonstrate that SNPs resulting in mutations on aromatase surface modify the protein flexibility that is required for substrate binding and catalysis. The cluster analysis provides a rationale for such effect, suggesting helix G as a possible target for aromatase inhibition.

Polymorphism is an important aspect in drug metabolism responsible for different individual response to drug dosage, often leading to adverse drug reactions. Here human CYP2C9 as well as its polymorphic variants CYP2C9*2 and CYP2C9*3 present in approximately 35% of the Caucasian population have been engineered by linking their gene to the one of D. vulgaris flavodoxin (FLD) that acts as regulator of the electron flow from the electrode surface to the haem. The redox properties of the immobilised proteins were investigated by cyclic voltammetry and electrocatalysis was measured in presence of the largely used anticoagulant drug S-warfarin, marker substrate for CYP2C9. Immobilisation of the CYP2C9-FLD, CYP2C9*2-FLD and CYP2C9*3-FLD on DDAB modified glassy carbon electrodes showed well defined redox couples on the oxygen-free cyclic voltammograms and mid-point potentials of all enzymes were calculated. Electrocatalysis in presence of substrate and quantification of the product formed showed lower catalytic activities for the CYP2C9*3-FLD (2.73 ± 1.07 min -1 ) and CYP2C9*2-FLD (12.42 ± 2.17 min -1 ) compared to the wild type CYP2C9-FLD (18.23 ± 1.29 min -1 ). These differences in activity among the CYP2C9 variants are in line with the reported literature data, and this set the basis for the use of the bio-electrode for the measurement of the different catalytic responses towards drugs very relevant in therapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The linker region of multi-domain enzymes has a very important role for the interconnection of different enzyme modules and for the efficiency of catalytic activity. This is particularly evident for artificial chimeric systems. We characterised an artificial self-sufficient enzyme developed by genetic fusion of the catalytic domain of cytochrome P450 3A4 and reductase domain of Bacillus megaterium BM3 (BMR). Here we report the direct electrochemistry of 3A4-BMR chimeras immobilised on glassy carbon electrodes and we investigated the effect of inter-domain loop length and immobilising environment flexibility on both redox properties and electrocatalysis. We observe that redox potential can be modulated by the linker length and the immobilising layer flexibility. In addition, enzyme inter-domain dynamics and environment flexibility also modulate 3A4-BMR turnover efficiency on electrode system. V max values are increased up to about 100% in the presence of testosterone and up to about 50% in presence of tamoxifen by decreasing immobilising film rigidity. The effect on 3A4-BMR V max values is dependent on inter-domain loop length with 3A4-5GLY-BMR chimera being the more affected. The underlying reason for these observations is the potential motion of the FMN domain that is the key to shuttle electrons from FAD to haem., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Chimerogenesis involving cytochromes P450 is a successful approach to generate catalytically self-sufficient enzymes. However, the connection between the different functional modules should allow a certain degree of flexibility in order to obtain functional and catalytically efficient proteins. We previously applied the molecular Lego approach to develop a chimeric P450 3A4 enzyme linked to the reductase domain of P450 BM3 (BMR). Three constructs were designed with the connecting loop containing no glycine, 3 glycine or 5 glycine residues and showed a different catalytic activity and coupling efficiency. Here we investigate how the linker affects the ability of P450 3A4 to bind substrates and inhibitors. We measure the electron transfer rates and the catalytic properties of the enzyme also in the presence of ketoconazole as inhibitor. The data show that the construct 3A4-5GLY-BMR with the longest loop better retains the binding ability and cooperativity for testosterone, compared to P450 3A4. In both 3A4-3GLY-BMR and 3A4-5GLY-BMR, the substrate induces an increase in the first electron transfer rate and a shorter lag phase related to a domain rearrangements, when compared to the construct without Gly. These data are consistent with docking results and secondary structure predictions showing a propensity to form helical structures in the loop of the 3A4-BMR and 3A4-3GLY-BMR. All three chimeras retain the ability to bind the inhibitor ketoconazole and show an IC 50 comparable with those reported for the wild type protein. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Human flavin-containing monooxygenase 3 (hFMO3) catalyses the oxygenation of a wide variety of compounds including drugs as well as dietary compounds. It is the major hepatic enzyme involved in the production of the N-oxide of trimethylamine (TMAO) and clinical studies have uncovered a striking correlation between plasma TMAO concentration and cardiovascular disease. Certain mutations within the hFMO3 gene cause defective trimethylamine (TMA) N-oxygenation leading to trimethylaminuria (TMAU) also known as fish-odour syndrome. In this paper, the inactivation mechanism of a TMAU-causing polymorphic variant, N61S, is investigated. Transient kinetic experiments show that this variant has a > 170-fold lower NADPH binding affinity than the wild type. Thermodynamic and spectroscopic experiments reveal that the poor NADP + binding affinity accelerates the C4a-hydroperoxyFAD intermediate decay, responsible for an unfavourable oxygen transfer to the substrate. Steady-state kinetic experiments show significantly decreased N61S catalytic activity towards other substrates; methimazole, benzydamine and tamoxifen. The in vitro data are corroborated by in silico data where compared to the wild type enzyme, a hydrogen bond required for the stabilisation of the flavin intermediate is lacking. Taken together, the data presented reveal the molecular basis for the loss of function observed in N61S mutant.

Human liver cytochrome P450 3A4 is the main enzyme involved in drug metabolism. This makes it an attractive target for biocatalytic applications, such as the synthesis of pharmaceuticals and drug metabolites. However, its poor solubility, stability and low coupling have limited its application in the biotechnological context. We previously demonstrated that the solubility of P450 3A4 can be increased by creating fusion proteins between the reductase from Bacillus megaterium BM3 (BMR) and the N-terminally modified P450 3A4 (3A4-BMR). In this work, we aim at increasing stability and coupling efficiency by varying the length of the loop connecting the two domains to allow higher inter-domain flexibility, optimizing the interaction between the domains. Starting from the construct 3A4-BMR containing the short linker Pro-Ser-Arg, two constructs were generated by introducing a 3 and 5 glycine hinge (3A4-3GLY-BMR and 3A4-5GLY-BMR). The three fusion proteins show the typical absorbance at 450 nm of the reduced heme-CO adduct as well as the correct incorporation of the FAD and FMN cofactors. Each of the three chimeric proteins were more stable than P450 3A4 alone. Moreover, the 3A4-BMR-3-GLY enzyme showed the highest NADPH oxidation rate in line with the most positive reduction potential. On the other hand, the 3A4-BMR-5-GLY fusion protein showed a V max increased by 2-fold as well as a higher coupling efficiency when compared to 3A4-BMR in the hydroxylation of the marker substrate testosterone. This protein also showed the highest rate value of cytochrome c reduction when this external electron acceptor is used to intercept electrons from BMR to P450. The data suggest that the flexibility and the interaction between domains in the chimeric proteins is a key parameter to improve turnover and coupling efficiency. These findings provide important guidelines in engineering catalytically self-sufficient human P450 for applications in biocatalysis.

Glucose oxidase (GOD) was immobilized on glassy carbon electrodes in the presence of graphene oxide (GO) as a model system for the interaction between GO and biological molecules. Lyotropic properties of didodecyldimethylammonium bromide (DDAB) were used to stabilize the enzymatic layer on the electrode surface resulting in a markedly improved electrochemical response of the immobilized GOD. Transmission electron microscopy images of the GO with DDAB confirmed the distribution of the GO in a two-dimensional manner as a foil-like material. Although it is known that glassy carbon surfaces are not ideal for hydrogen peroxide detection, successful chronoamperometric titrations of the GOD in the presence of GO with β-d-glucose were performed on glassy carbon electrodes, whereas no current response was detected upon β-d-glucose addition in the absence of GO. The GOD-DDAB-GO system displayed a high turnover efficiency and substrate affinity as a glucose biosensor. The simplicity and ease of the electrode preparation procedure of this GO/DDAB system make it a good candidate for immobilizing other biomolecules for fabrication of amperometric biosensors., (© 2015 International Union of Biochemistry and Molecular Biology, Inc.)

The [FeFe]-hydrogenase CpHydA from Clostridium perfringens was immobilized by adsorption on anatase TiO2 electrodes for clean hydrogen production. The immobilized enzyme proved to perform direct electron transfer to and from the electrode surface and catalyses both H2 oxidation (H2 uptake) and H2 production (H2 evolution) with a current density for H2 evolution of about 2 mA cm(-1). The TiO2/CpHydA bioelectrode remained active for several days upon storage and when a reducing potential was set, H2 evolution occurred with a mean Faradaic efficiency of 98%. The high turnover frequency of H2 production and the tight coupling of electron transfer, resulting in a Faradaic efficiency close to 100%, support the exploitation of the novel TiO2/CpHydA stationary electrode as a powerful device for H2 production., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Antimicrobial resistance is a global issue currently resulting in the deaths of hundreds of thousands of people a year worldwide. Data present in the literature illustrate the emergence of many bacterial species that display resistance to known antibiotics; Acinetobacter spp. are a good example of this. We report here that Acinetobacter radioresistens has a Baeyer-Villiger monooxygenase (Ar-BVMO) with 100% amino acid sequence identity to the ethionamide monooxygenase of multidrug-resistant (MDR) Acinetobacter baumannii. Both enzymes are only distantly phylogenetically related to other canonical bacterial BVMO proteins. Ar-BVMO not only is capable of oxidizing two anticancer drugs metabolized by human FMO3, danusertib and tozasertib, but also can oxidize other synthetic drugs, such as imipenem. The latter is a member of the carbapenems, a clinically important antibiotic family used in the treatment of MDR bacterial infections. Susceptibility tests performed by the Kirby-Bauer disk diffusion method demonstrate that imipenem-sensitive Escherichia coli BL21 cells overexpressing Ar-BVMO become resistant to this antibiotic. An agar disk diffusion assay proved that when imipenem reacts with Ar-BVMO, it loses its antibiotic property. Moreover, an NADPH consumption assay with the purified Ar-BVMO demonstrates that this antibiotic is indeed a substrate, and its product is identified by liquid chromatography-mass spectrometry to be a Baeyer-Villiger (BV) oxidation product of the carbonyl moiety of the β-lactam ring. This is the first report of an antibiotic-inactivating BVMO enzyme that, while mediating its usual BV oxidation, also operates by an unprecedented mechanism of carbapenem resistance., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

This work reports for the first time the direct electron transfer of the Canis familiaris cytochrome P450 2D15 on glassy carbon electrodes to provide an analytical tool as an alternative to P450 animal testing in the drug discovery process. Cytochrome P450 2D15, that corresponds to the human homologue P450 2D6, was recombinantly expressed in Escherichia coli and entrapped on glassy carbon electrodes (GC) either with the cationic polymer polydiallyldimethylammonium chloride (PDDA) or in the presence of gold nanoparticles (AuNPs). Reversible electrochemical signals of P450 2D15 were observed with calculated midpoint potentials (E1/2) of −191 ± 5 and −233 ± 4 mV vs. Ag/AgCl for GC/PDDA/2D15 and GC/AuNPs/2D15, respectively. These experiments were then followed by the electro-catalytic activity of the immobilized enzyme in the presence of metoprolol. The latter drug is a beta-blocker used for the treatment of hypertension and is a specific marker of the human P450 2D6 activity. Electrocatalysis data showed that only in the presence of AuNps the expected α-hydroxy-metoprolol product was present as shown by HPLC. The successful immobilization of the electroactive C. familiaris cytochrome P450 2D15 on electrode surfaces addresses the ever increasing demand of developing alternative in vitromethods for amore detailed study of animal P450 enzymes' metabolism, reducing the number of animals sacrificed in preclinical tests.

Human flavin-containing monooxygenase 3 (hFMO3), a membrane-bound hepatic protein, belonging to the second most important class of phase-1 drug-metabolizing enzymes, was immobilized in its active form on graphene oxide (GO) for enhanced electrochemical response. To improve protein stabilization and to ensure the electrocatalytic activity of the immobilized enzyme, didodecyldimethylammonium bromide (DDAB) was used to mimic lipid layers of biological membranes and acted as an interface between GO nanomaterial and the hFMO3 biocomponent. Grazing angle attenuated total reflectance Fourier transform infrared (GATR-FT-IR) experiments confirmed the preservation of the protein secondary structure and fold. Electrochemical characterization of the immobilized enzyme with GO and DDAB on glassy carbon electrodes was carried out by cyclic voltammetry, where several parameters including redox potential, electron transfer rate, and surface coverage were determined. This system's biotechnological application in drug screening was successfully demonstrated by the N-oxidation of two therapeutic drugs, benzydamine (nonsteroidal anti-inflammatory) and tamoxifen (antiestrogenic widely used in breast cancer therapy and chemoprevention), by the immobilized enzyme.

A gene coding for a class VII cytochrome P450 monooxygenase (CYP116B5) was identified from Acinetobacter radioresistens S13 growing on media with medium (C14, C16) and long (C24, C36) chain alkanes as the sole energy source. Phylogenetic analysis of its N- and C-terminal domains suggests an evolutionary model involving a plasmid-mediated horizontal gene transfer from the donor Rhodococcus jostii RHA1 to the receiving A. radioresistens S13. This event was followed by fusion and integration of the new gene in A. radioresistens chromosome. Heterologous expression of CYP116B5 in Escherichia coli BL21, together with the A. radioresistens Baeyer-Villiger monooxygenase, allowed the recombinant bacteria to grow on long- and medium-chain alkanes, showing that CYP116B5 is involved in the first step of terminal oxidation of medium-chain alkanes overlapping AlkB and in the first step of sub-terminal oxidation of long-chain alkanes. It was also demonstrated that CYP116B5 is a self-sufficient cytochrome P450 consisting of a heme domain (aa 1-392) involved in the oxidation step of n-alkanes degradation, and its reductase domain (aa 444-758) comprising the NADPH-, FMN- and [2Fe2S]-binding sites. To our knowledge, CYP116B5 is the first member of this class to have its natural substrate and function identified., (© 2014 John Wiley & Sons Ltd.)

Inhibition of human cytochrome P450 2A6 has been demonstrated to play an important role in nicotine metabolism and consequent smoking habits. Here, the "molecular Lego" approach was used to achieve the first reported electrochemical signal of human CYP2A6 and to improve its catalytic efficiency on electrode surfaces. The enzyme was fused at the genetic level to flavodoxin from Desulfovibrio vulgaris (FLD) to create the chimeric CYP2A6-FLD. Electrochemical characterization by cyclic voltammetry shows clearly defined redox transitions of the haem domain in both CYP2A6 and CYP2A6-FLD. Electrocatalysis experiments using coumarin as substrate followed by fluorimetric quantification of the product were performed with immobilized CYP2A6 and CYP2A6-FLD. Comparison of the kinetic parameters showed that coumarin catalysis was carried out with a higher efficiency by the immobilized CYP2A6-FLD, with a calculated kcat value significantly higher (P < 0.005) than that of CYP2A6, whereas the affinity for the substrate (KM) remained unaltered. The chimeric system was also successfully used to demonstrate the inhibition of the electrochemical activity of the immobilized CYP2A6-FLD, toward both coumarin and nicotine substrates, by tranylcypromine, a potent and selective CYP2A6 inhibitor. This work shows that CYP2A6 turnover efficiency is improved when the protein is linked to the FLD redox module, and this strategy can be utilized for the development of new clinically relevant biotechnological approaches suitable for deciphering the metabolic implications of CYP2A6 polymorphism and for the screening of CYP2A6 substrates and inhibitors.

Human aromatase (CYP19A1) is a steroidogenic cytochrome P450 converting androgens into estrogens. No ligand-free crystal structure of the enzyme is available to date. The crystal structure in complex with the substrate androstenedione and the steroidal inhibitor exemestane shows a very compact conformation of the enzyme, leaving unanswered questions on the conformational changes that must occur to allow access of the ligand to the active site. As H/D exchange kinetics followed by FTIR spectroscopy can provide information on the conformational changes in proteins where solvent accessibility is affected, here the amide I region was used to measure the exchange rates of the different elements of the secondary structure for aromatase in the ligand-free form and in the presence of the substrate androstenedione and the inhibitor anastrozole. Biphasic exponential functions were found to fit the H/D exchange data collected as a function of time. Two exchange rates were assigned to two populations of protons present in different flexible regions of the protein. The addition of the substrate androstenedione and the inhibitor anastrozole lowers the H/D exchange rates of the α-helices of the enzyme when compared to the ligand-free form. Furthermore, the presence of the inhibitor anastrozole lowers exchange rate constant (k1) for β-sheets from 0.22±0.06 min(-1) for the inhibitor-bound enzyme to 0.12±0.02 min(-1) for the free protein. Dynamics effects localised in helix F were studied by time resolved fluorescence. The data demonstrate that the fluorescence lifetime component associated to Trp224 emission undergoes a shift toward longer lifetimes (from ≈5.0 to ≈5.5 ns) when the substrate or the inhibitor are present, suggesting slower dynamics in the presence of ligands. Together the results are consistent with different degrees of flexibility of the access channel and therefore different conformations adopted by the enzyme in the free, substrate- and inhibitor-bound forms.

In order to develop in vitro methods as an alternative to P450 animal testing in the drug discovery process, two main requisites are necessary: 1) gathering of data on animal homologues of the human P450 enzymes, currently very limited, and 2) bypassing the requirement for both the P450 reductase and the expensive cofactor NADPH. In this work, P450 2C20 from Macaca fascicularis, homologue of the human P450 2C8 has been taken as a model system to develop such an alternative in vitro method by two different approaches. In the first approach called "molecular Lego", a soluble self-sufficient chimera was generated by fusing the P450 2C20 domain with the reductase domain of cytochrome P450 BM3 from Bacillus megaterium (P450 2C20/BMR). In the second approach, the need for the redox partner and also NADPH were both obviated by the direct immobilization of the P450 2C20 on glassy carbon and gold electrodes. Both systems were then compared to those obtained from the reconstituted P450 2C20 monooxygenase in presence of the human P450 reductase and NADPH using paclitaxel and amodiaquine, two typical drug substrates of the human P450 2C8. The K(M) values calculated for the 2C20 and 2C20/BMR in solution and for 2C20 immobilized on electrodes modified with gold nanoparticles were 1.9 ± 0.2, 5.9 ± 2.3, 3.0 ± 0.5 μM for paclitaxel and 1.2 ± 0.2, 1.6±0.2 and 1.4 ± 0.2 μM for amodiaquine, respectively. The data obtained not only show that the engineering of M. fascicularis did not affect its catalytic properties but also are consistent with K(M) values measured for the microsomal human P450 2C8 and therefore show the feasibility of developing alternative in vitro animal tests., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Nanosized particles of gold are widely used as advanced materials for enzyme catalysis investigations. In some bioanalytical methods these nanoparticles can be exploited to increase the sensitivity by enhancing electron transfer to the biological component i.e. redox enzymes such as drug metabolizing enzymes., Methods: In this work, we describe the characterization of human flavin-containing monooxygenase 3 (hFMO3) in a nanoelectrode system based on AuNPs stabilized with didodecyldimethylammonium bromide (DDAB) on glassy carbon electrodes. Once confirmed by FTIR spectroscopy that in the presence of DDAB-AuNPs the structural integrity of hFMO3 is preserved, the influence of AuNPs on the electrochemistry of the enzyme was studied by cyclic voltammetry and square wave voltammetry., Results: Our results show that AuNPs improve the electrochemical performance of hFMO3 on glassy carbon electrodes by enhancing the electron transfer rate and the current signal-to-noise ratio. Moreover, the electrocatalytic activity of hFMO3-DDAB-AuNP electrodes which was investigated in the presence of two well known substrates, benzydamine and sulindac sulfide, resulted in K(M) values of 52μM and 27μM, with V(max) of 8nmolmin(-1)mg(-1) and 4nmolmin(-1)mg(-1), respectively, which are in agreement with data obtained with the microsomal enzyme., Conclusions: The immobilization of hFMO3 protein in DDAB stabilized AuNP electrodes improves the bioelectrochemical performance of this important phase I drug metabolizing enzyme., General Significance: This bio-analytical method can be considered as a promising advance in the development of new techniques suitable for the screening of novel hFMO3 metabolized pharmaceuticals., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Human flavin-containing monooxygenases are the second most important class of drug-metabolizing enzymes after cytochromes P450. Here we report a simple but functional and stable enzyme-electrode system based on a glassy carbon (GC) electrode with human flavin-containing monooxygenase isoform 3 (hFMO3) entrapped in a gel cross-linked with bovine serum albumin (BSA) by glutaraldehyde. The enzymatic electrochemical responsiveness is characterised by using well-known substrates: trimethylamine (TMA), ammonia (NH(3)), triethylamine (TEA), and benzydamine (BZD). The apparent Michaelis-Menten constant (K'(M)) and apparent maximum current (I'(max)) are calculated by fitting the current signal to the Michaelis-Menten equation for each substrate. The enzyme-electrode has good characteristics: the calculated sensitivity was 40.9 +/- 0.5 mA mol(-1) L cm(-2) for TMA, 43.3 +/- 0.1 mA mol(-1) L cm(-2) for NH(3), 45.2 +/- 2.2 mA mol(-1) L cm(-2) for TEA, and 39.3 +/- 0.6 mA mol(-1) L cm(-2) for BZD. The stability was constant for 3 days and the inter-electrode reproducibility was 12.5%. This is a novel electrochemical tool that can be used to investigate new potential drugs against the catalytic activity of hFMO3.

Ganstigmine (CHF2819) is a novel, orally active acetylcholinesterase inhibitor that induces a stimulation of brain cholinergic transmission. In vivo studies show that, in rat prefrontal cortex, extracellular acetylcholine (ACh) concentrations are significantly increased either after local (1 and 10 microM) or oral (1.5 and 3 mg/kg) administration. Moreover, repeated oral treatment (six consecutive days; 3 mg/kg) with ganstigmine significantly increases basal extracellular concentrations of ACh in rat prefrontal cortex. Then, acute ganstigmine administration induces a significant increase in extracellular ACh concentrations (actual values) with respect to the last sample in ganstigmine-treated rats. Concentrations of serotonin (5-HT) and noradrenaline (NA) are not affected by any oral dose of ganstigmine (1.5 and 3 mg/kg) used. Moreover, levels of dopamine (DA) and metabolites are not modified either. Basal extracellular concentrations of 5-HT, NA, DA and metabolites are not affected by repeated (six consecutive days) ganstigmine treatment (3 mg/kg). Furthermore, there is no effect of the challenge dose of ganstigmine (3 mg/kg) on 5-HT, NA, DA and metabolites levels. Finally, ganstigmine reverses the scopolamine-induced deficits of habituation and non-spatial working memory in rats. Taken together, these findings suggest that ganstigmine appears to be a suitable candidate for the treatment of the cholinergic deficit in patients with Alzheimer's disease.