Yasuhiro Mie

Electrochemically Driven Drug Metabolism by Membranes Containing Human Cytochrome P450

Rapid analyses of drug metabolism reactions by human cytochrome P450s (CYPs) that metabolize 95% of all current drugs are very important in drug development processes and effective therapies. Since CYPs need electrons to metabolize drugs, electrons supplied from electrodes to activate CYP molecules are expected to be very useful to develop rapid assay methods for CYP reactions. Although several studies on the direct electrochemistry of isolated and purified human CYPs have been reported, the use of microsomes (membranes) containing CYP is more suitable, because they are frequently used in drug research due to their easy preparation and low cost. Herein, we demonstrate electrons supplied from an electrode to microsomes containing CYP and CYP-reductase (CPR) on an electrode coated with hydrophobic thin films and observe electrochemically driven drug metabolisms by voltammetry for the first time. We tested the immobilization of microsomes on gold electrodes coated with several thiolates and found that microsomes immobilized on thin hydrophobic surfaces of aromatic compounds showed well-defined redox peaks. Furthermore, electrochemically driven drug metabolism reactions of CYP3A4 were clearly observed by voltammetry, and these reactions were inhibited by a CYP3A4 inhibitor, ketoconazole. We also found that metabolism reactions were facilitated in the presence of CPR.

Electrochemistry of monoazahemin reconstituted myoglobin at an indium oxide electrode

Abstract Monoazahemin reconstituted myoglobin was prepared and its electrochemical behavior was studied in comparison with native myoglobin. For both myoglobins well-defined voltammograms were clearly obtained at highly hydrophilic surfaces of indium oxide electrodes. Although monoazahemin showed a more positive redox potential than hemin (measured in methanol), monoazahemin reconstituted myoglobin showed a more negative redox potential than native myoglobin in a 50 mM bis-Tris buffer solution (pH 6.5), suggesting that for both native and reconstituted myoglobins the heme environment including proximal histidine as an axial ligand of the redox center plays an important role in determining the redox potential. Also, a unique electrochemical response of cyano-monoazahemin reconstituted myoglobin was demonstrated.

Electrochemistry of myoglobins reconstituted with azahemes and mesohemes

Abstract The iron complexes of α-azamesoporphyrin XIII and β, δ-diazamesoporphyrin III were incorporated into apomyoglobin to obtain mono- and di-azaheme reconstituted myoglobins and their electrochemical behavior was investigated. To understand precisely the effect of introduced nitrogen atom(s) to the porphyrin backbone on electrochemical behavior of reconstituted myoglobins, iron complexes of mesoporphyrin XIII and mesoporphyrin III as well as myoglobins reconstituted with these mesohemes were also prepared and their electrochemical properties were examined. Significant positive shifts in redox potential of azahemes compared with the corresponding mesoporphyrins were observed. However, the redox potentials of azaheme reconstituted myoglobins showed no or much less positive shift compared with those of mesoheme reconstituted myoglobins, suggesting the heme environment including axial ligands of the redox center would be more important than the structure of porphyrin ring itself for determining the redox potential of reconstituted myoglobins. Azaheme reconstituted myoglobins showed larger affinity with cyanide ion than the corresponding mesoheme reconstituted myoglobins. Electron transfer kinetics measured both on an electrode and by chemical reduction with dithionite correlated well to each other and depended on the structures of hemes. Azaheme reconstituted myoglobins showed larger electron transfer than the corresponding mesoheme reconstituted myoglobins, which would be explained in terms of the change in spin-state of the heme iron for azaheme reconstituted myoglobins. Also, electron transfer reaction at an In 2 O 3 electrode showed the largest kinetics at pH 6.5 for myoglobins having aquomet type redox centers.

Effect of rapid heme rotation on electrochemistry of myoglobin

Myoglobins (Mbs) reconstituted with rotatable octamethylheme and non-rotatable etioheme were prepared and their electrochemical behavior was studied. The redox potential of octamethylheme reconstituted Mb (OMe- Mb), of which heme pops around iron-histidine (F8 -His) bond, shifted negatively by ca. 30 mV compared with non-rotatable etioheme reconstituted Mb (Etio-Mb). On the other hand, the redox potentials of octamethylheme and etioheme themselves were very similar to each other. Due to the similarity of the distal histidine side of the heme of these two reconstituted Mbs, the shifts of the redox potential would be attributable to the drastic change of the orientation of proximal histidine imidazole ring to the heme plane by heme rotation. The dissociation rate constant of cyanide ion from the ferrous heme iron (II) for OMe-Mb form at 5°C and pH 7.5 was three times faster than that of Etio-Mb. The electron transfer kinetics of these Mbs showed that the heme rotation causes faster electron transfer rates in both electrode reaction and chemical reduction in solution with dithionite. The obtained heterogeneous electron transfer rates constants at an In 2 O 3 electrode and first-order rate constants of the chemical reduction were 12(±0.5) × 10 -4 cm s -1 , 9.8(± 1.0) s -1 for OMe-Mb and 6.0(± 0.5) × 10 -4 cm s -1 , 4.5(± 1.0) s -1 for Etio-Mb under the present experimental conditions.

Electrochemical properties of interstrand cross-linked DNA duplexes labeled with Nile blue.

DNA molecules have attracted considerable attention as functional materials in various fields such as electrochemical sensors with redox-labeled DNA. However, the recently developed interstrand cross-link (ICL) technique for double-stranded DNA can adequately modify the electronic properties inside the duplex. Hence, the electrochemical investigation of ICL-DNA helps us to understand the electron transfer of redox-labeled DNA at an electrode surface, which would develop useful sensors. In this study, the first insight into this matter is presented. We prepared 17-mer DNA duplexes incorporating Nile blue (NB-DNA) at one end as a redox marker and a disulfide tether at the other end for immobilization onto an electrode. The duplexes were covalently cross-linked by bifunctional cross-linkers that utilize either a propyl or naphthalene residue to replace a base pair. Their electrochemical responses at the electrode surface were compared to evaluate the effect of the ICL on the electron-transfer reactions of the redox-labeled DNA duplexes. A direct transfer of electrons between NB and the electrode was observed for a standard DNA, as previously reported, whereas interstrand cross-linked DNA (CL-DNA) strands showed a decrease in the direct electron-transfer pathway. This is expected to result from constraining the elastic bending/flexibility of the duplex caused by the covalent cross-links. Interestingly, the CL-DNA incorporating naphthalene residues exhibited additional voltammetric peaks derived from DNA-mediated electron transfer (through base π stacking), which was not observed in the mismatched CL-DNA. The present results indicate that the ICL significantly affects electron transfer in the redox-labeled DNA at the electrode and can be an important determinant for electrochemical signaling in addition to its role in stabilizing the duplex structure.

Function Control of Anti-microRNA Oligonucleotides Using Interstrand Cross-Linked Duplexes

MicroRNA (miRNA)-guided argonaute (Ago) controls gene expression upon binding to the 3′ UTR of mRNA. The miRNA function can be competitively inhibited by single-stranded anti-miRNA oligonucleotides (AMOs). In this study, we constructed a novel type of AMO flanked by interstrand cross-linked 2′-O-methylated RNA duplexes (CLs) that confer a stable helical conformation. Compared with other structured AMOs, AMO flanked by CLs at the 5′ and 3′ termini exhibited much higher inhibitory activity in cells. Anti-miRNA activity, nuclease resistance, and miRNA modification pattern distinctly differed according to the CL-connected positions in AMOs. Moreover, we found that the 3′-side CL improves nuclease resistance, whereas the 5′-side CL contributes to stable binding with miRNA in Ago upon interaction with the 3′ part of miRNA. These structure-function relationship analyses of AMOs provide important insights into the function control of Ago-miRNA complexes, which will be useful for basic miRNA research as well as for determining therapeutic applications of AMO.

Modification of Thiols onto Single Crystal Surfaces of Gold Electrodes for Metalloportein Electrochemistry

Adsorption behavior of pyridine thiols (PySH) and corresponding disulfides (PySSPy) onto gold single crystal surfaces has been examined to understand in detail the suitable surface structures of the modified electrodes for cytochrome c electrochemistry. On the Au(111) surface PySH was rather easily decomposed with the C-S bond cleavage during modification of thiols from an ethanolic PySH solution, while PySSPy was more stable under the same conditions. Very interestingly, cytochrome c electrochemistry depended very much on the modifier structure at single crystal surfaces.