Human cytochromes P450 (CYP) constitute one of the most important and studied classes of
phase I drug metabolizing enzymes. A small group of 6-7 isoforms of the 57 identified until
now accounts for 90-95% of the metabolism of clinically used drugs and can contain mutations
(single nucleotide polymorphisms) that, when located in the coding regions, can lead to absent,
deficient or enhanced enzyme activity. Thanks to the development of pharmacogenetics and
later on pharmacogenomics, the presence of these single nucleotide polymorphisms (SNP) has
been associated to inter-individual and inter-ethnic variability in the response to several
important therapeutic agents. Due to this direct correlation between polymorphism and efficacy
of drug treatment, the pharmaceutical industry is particularly interested in developing new
analytical tools for the determination of the cytochrome P450 in vitro metabolism to correlate to
the in vivo situation. The use of amperometric sensing systems represents an interesting
alternative to the traditional methods.
In the present study an electrochemical characterisation of the human cytochrome P450 2C9 and
its two main allelic variants the CYP2C9*2 and CYP2C9*3 have been performed. Different
methods of immobilisation and electrodes have been used to investigate the conditions that are
more suitable to maintain and guarantee the active state and biocatalytic response of an
immobilised cytochrome P450.
The CYP2C9 and its two main allelic variants CYP2C9*2 and CYP2C9*3 have been inserted
into engineered constructs where the human cytochrome P450 gene is linked to artificial redox
chains to regulate the electron flow from the electrode surface to the haem. All the constructs
have been successfully expressed and purified in heterologous E.Coli cells, with the
CYP2C9FLD chimeras showing higher yields.
Preliminary spectroelectrochemical studies on semi-conductive and optically transparent tin-dioxide
allowed the combination of absorption spectroscopy and electrochemistry (cyclic
voltammetry) to ascertain the native state of these P450 enzymes once immobilised. The results
showed how the conversion from the active P450 form to the inactive P420 one can be achieved
by modifying the surface with polycations with slightly different chemical properties. A proper
characterisation of the two species was performed for the first time and, under certain
conditions, the inactive P420 species appeared to dominate the cyclic voltammogram (CV).
Similar findings have been observed when the CYP2C9 wild type have been electrochemically
characterised on DDAB and PDDA modified glassy carbon electrodes. Electrocatalysis in
presence of S-warfarin and FT-IR spectra of the CYP2C9/DDAB/GC electrodes revealed higher catalytic activity and maintenance of the native secondary structure of the enzyme.
Immobilisation of the CYP2C9FLD, CYP2C9*2FLD and CYP2C9*3FLD on DDAB modified
glassy carbon electrodes showed well defined redox couples on the oxygen-free cyclic
voltammograms (CVs) 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*3FLD and CYP2C9*2FLD compared to the wild type CYP2C9FLD
as it was expected from literature data. When the CYP2C9FLD, CYP2C9*2FLD and
CYP2C9*3FLD were immobilised on alkanethiol modified gold electrodes, the system was
tested as both amperometric sensor and catalyser. Parameters such as the apparent Michaelis–
Menten constant Km and the Vmax were determined and the metabolic profile of S-warfarin was
confirmed to be in line with literature findings.
The fundamental knowledge acquired was then transferred to a commercial prototype sensor for
testing the metabolic profile of known drugs. Encouraging results in line with the previous
findings were achieved.