An amperometric biosensor with human CYP3A4 as a novel drug screening tool.
We developed a biosensor based on the redox properties of human CYP3A4 to directly monitor electron transfer to the heme protein. Enzyme films were assembled on gold electrodes by alternate adsorption of a CYP3A4 layer on top of a polycation layer. Direct, reversible electron transfer between the electrode and CYP3A4 was observed with voltammetry under anaerobic conditions. In the presence of oxygen, the oxidation peak of the hemoprotein disappeared, and the reduction peak increased 2- to 3-fold. Addition of CYP3A4 substrates (verapamil, midazolam, quinidine, and progesterone) to the oxygenated solution caused a concentration-dependent increase in the reduction current in cyclic voltammetric and amperometric experiments. Product analyses after electrolysis with the enzyme film showed catalytic activity of the biosensor depending on substrate concentration, its inhibition by ketoconazole, and a minor contribution of H(2)O(2) to the catalytic cycle. These results suggest that electron exchange between the electrode and the immobilized CYP3A4 occurred, and that metabolic activity of the enzyme was maintained. Thus, important requirements for the application of human CYP biosensors in order to identify drugs or drug candidates as substrates or inhibitors to the attached enzyme are fulfilled.
Joseph
Shiba
S
Institute for Pharmacology, Clinical Pharmacology, University of Köln, Köln, Germany.
Rusling
James F
JF
Lvov
Yuri M
YM
Friedberg
Thomas
T
Fuhr
Uwe
U
D015374:Biosensing Techniques / Q000379:methods*; D002384:Catalysis; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004563:Electrochemistry; D004800:Enzymes, Immobilized; D006801:Humans; D011073:Polyamines / Q000378:metabolism*; D000071228:Polyelectrolytes
D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System; D004800:Enzymes, Immobilized; D011073:Polyamines; D000071228:Polyelectrolytes; C104464:CYP3A protein, human; C510163:CYP3A4 protein, human