Electrochemically driven drug metabolism via cytochrome P450 2C9 isozyme microsomes with cytochrome P450 reductase and indium tin oxide nanoparticle composites

Article abstract of DOI:10.1039/c2cc33575a

We describe herein an electrochemically driven drug metabolism strategy based on nanocomposites that integrate cyt P450 2C9 (CYP2C9) isozyme microsomes with cyt P450 reductase (CPR), indium tin oxide (ITO) nanoparticles and chitosan (CS). This novel bioel

Full text of DOI:10.1039/c2cc33575a

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Electrochemically driven drug metabolism via cytochrome P450 2C9
isozyme microsomes with cytochrome P450 reductase and indium tin
oxide nanoparticle compositesw
Xuan Xu, Wei Wei, Minghe Huang, Li Yao and Songqin Liu*
Received 17th May 2012, Accepted 9th June 2012
DOI: 10.1039/c2cc33575a
We describe herein an electrochemically driven drug metabolism
strategy based on nanocomposites that integrate cyt P450 2C9
(CYP2C9) isozyme microsomes with cyt P450 reductase (CPR),
indium tin oxide (ITO) nanoparticles and chitosan (CS). This
novel bioelectronic system enables monitoring of the drug
metabolism and enzyme inhibition.
ITO nanoparticles and CYP2C9/CPR-microsomes encapsulated
by CS on a glassy carbon electrode to electrochemically drive the
drug metabolism process.
TEM images reveal that the vesicle-like microsomes are
surrounded by amorphous ITO nanoparticles, whose average
diameter is ca. 10–40 nm (Supporting information Fig. S1w).
ITO nanoparticles in the film played an important role in
transferring electrons between the electrode and the active sites
of proteins in microsomes. This is verified by the following
electrochemical measurements.
Cytochrome P450s (cyt P450s) are a group of heme-containing
proteins that have benefited from the attention of scientists
in different research fields due to its role in the metabolism
of endogenous and exogenous compounds, including more
than 60% of all clinically used drugs.1 There are 11 major
drug-metabolizing cyt P450s expressed in the human liver.
Among these isoforms, cyt P450 2C9 (CYP2C9) is found to be
responsible for about 16% of therapeutically important
drugs.2
As shown in Fig. 1A, CYP2C9/CPR-microsome/ITO/CS/
GCE gave a couple of stable and well-defined redox peaks at
À0.382 and À0.404 V (curve c), while no redox peak was
observable for CYP2C9/CPR-microsome/CS/GCE (curve a)
nor ITO/CS/GCE (curve b). Obviously, the response was
attributed to the redox of the electroactive centers in the
immobilized proteins, which were the heme cofactor in
CYP2C9 and the flavin cofactor in CPR.10 This also confirmed
that the ITO nanoparticles were important for direct electron
transfer of proteins in the film. ITO nanoparticles with high
electrical conductivity over a broad potential window might
not only facilitate electron transport toward the electroactive
centers of proteins, but also provide a three-dimensional stage
that allows some of the restricted orientations to favor
the direct electron transfer between the immobilized protein
molecules and the conductor surface.11 Compared with the
A typically catalytic cycle of cyt P450s features delivery
of electrons from NADPH to cyt P450 reductase (CPR)
for subsequent delivery to cyt P450 heme.3 Attempts at
‘‘bioelectronic’’ initiation of cyt P450s catalysis have been
made by replacing the electron donation from expensive
NADPH with electrodes. The direct electrochemistry and
biocatalysis of pure cyt P450s have been studied in several
reports.4 Gilardi and coworkers found that the genetically
fused reductase domain of CPR and flavodoxin with CYP3A4
could improve the catalytic activity of CYP3A4.5 Rusling and
coworkers constructed a LBL film featuring excess human cyt
P450s and CPR microsomes to accurately mimic the natural
cyt P450s catalytic cycle at high catalytic turnover.6 In our
current work, CYP2C9 microsomes with CPR (CYP2C9/
CPR-microsomes) are used because the microsomes containing
CYP enzyme and CPR might be more easily electrochemically
activated along the same pathway utilized in vivo.7 By combining
the good conductivity of ITO nanoparticles8 with the excellent
biocompatibility of CS,9 we constructed a mixed film containing
Fig. 1 (A) CV curves obtained at CYP2C9/CPR-microsome/CS (a),
ITO/CS (b), CYP2C9/CPR-microsome/ITO/CS (c) and CYP2C9/
ITO/CS (d) modified GCE at a scan rate of 0.5 V sÀ1 in anaerobic
0.1 M pH 7.4 PBS. (B) CYP2C9/CPR-microsome/ITO/CS film in
electrochemical biocatalysis. Influence of increasing tolbutamide
concentrations on rotating disk voltammograms (1000 rpm) in 0.1 M
pH 7.4 PBS with saturated oxygen.
School of Chemistry and Chemical Engineering, Southeast University
Jiangning, Nanjing 211189, People’s Republic of China.
E-mail: liusq@seu.edu.cn; Fax: +86-25-52090618;
Tel: +86-25-52090613
w Electronic supplementary information (ESI) available: detailed
experimental description, characterization of the film. See DOI:
10.1039/c2cc33575a
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun.

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literature, the midpoint potential of CYP2C9/CPR-microsomes
(À0.393 V vs. SCE) was more positive than pure CYP2C9
(À0.46 V vs. SCE) bonded to a gold electrode,12 but more
negative than CYP2C9/flavodoxin (À0.33 V vs. SCE) covalently
linked to polycrystalline gold substrate.2b
To further confirm the electrode reaction involving electron
transfer of proteins, voltammetry with CYP2C9/ITO/CS/
GCE was carried out in the same condition. The film with
pure CYP2C9 also displayed a couple of well-defined redox
peaks located at À0.356 and À0.401 V (curve d in Fig. 1A).
The reduction peak potential was almost the same as that of
CYP2C9/CPR-microsomes, while the oxidation peak potential
shifted to a more positive value. This suggested that the presence
of CPR led to easier oxidation of the CYP2C9 in the film. Similar
results were obtained for PDDA(/PSS/cyt P450 1A2)4, whose
midpoint potential of redox peaks was also more positive than
that of PDDA/PSS(/cyt P450 1A2/msCRP)6.6 This might
confirm that some electrons were transferred to CPR sites in
CYP2C9/CPR-microsomes but not directly to CYP2C9,
which was close to the natural pathway.6
Fig. 2 (A) Current–time curve for CYP2C9/CPR-microsome/ITO/
CS/GCE with successive addition of tolbutamide (indicated by arrows
for marked concentrations) in 0.1 M pH 7.4 PBS with saturated
oxygen at an applied potential of À0.48 V vs. SCE. (B) Calibration
plots illustrating the electrode response to tolbutamide addition.
Insert: double-reciprocal plot of catalytic current and the concen-
tration of tolbutamide.
Thus, a conclusion could be drawn that the change of cathodic
current in the presence of the drug mainly came from the
metabolism of tolbutamide driven by CYP2C9/CPR-microsome/
ITO/CS/GCE.
Integrations of the CVs revealed the amount of electroactive
proteins in the film was 4.623 nmol cmÀ2, which was only a
small part of the total cyt P450s enzymes immobilized. This
was possibly due to the inactivation of membrane enzymes or
the wrong orientation for electron transfer.4c With an increase
of scan rates from 0.1 to 0.5 V sÀ1, the anodic and cathodic
peak potentials of CYP2C9/CPR-microsome/ITO/CS/GCE
exhibited a small shift and the peak currents increased linearly,
characteristic of a surface-controlled process (Supporting
information Fig. S2w). The heterogeneous electron transfer
Plotting the values of catalytic current at À0.48 V as a
function of the concentration of the added substrate showed a
hyperbolic behavior that could be fitted with a Michaelis–
Menten model (Fig. 2B). According to the meaning of the
Michaelis–Menten equation,16 the apparent Michaelis–Menten
constant Kampp was calculated to be 202.84 mM. This was in good
agreement with previous reports, where tolbutamide hydroxylation
exhibited simple Michaelis–Menten kinetics with Kampp values
ranging from 60 to 400 mM in human liver microsomes.17 Such
a wide range of Kampp values came from the differences in the
source of the P450 enzymes or the conditions of assay.16,18
To further confirm this electrochemically driven drug
metabolism strategy, electrolysis was done by CYP2C9/CPR-
microsome/ITO/CS/GCE in the presence of 600 mM tolbutamide
at an applied potential of À0.48 V (vs. SCE) for 1 h. The
formation of 4-hydroxytolbutamide in the above electro-
chemically driven drug metabolism process was then measured
by high performance liquid chromatography-mass spectrometry
(HPLC-MS) and electrospray ionization-mass spectrometry
(ESI-MS). The typical chromatogram of the tolbutamide-
containing solution showed two peaks at 4.706 and 2.829 min
(Supporting information Fig. S4Aw), whereas only one peak at
4.71 min was observed for the pure tolbutamide solution (data
not shown). Thus, the peak at 2.829 min was attributed to the
product of 4-hydroxytolbutamide, which increased the polarity
of tolbutamide and moved fast in a polar mobile phase. The
formation of the metabolized product of 4-hydroxytolbutamide
was also confirmed by ESI-MS (Supporting information
Fig. S4Bw). After electrolysis, two new MS peaks at 287.3
and 309 m/z were observed corresponding to the molecular ion
and molecular plus sodium ion of 4-hydroxytolbutamide.
These peaks could not be observed in the ESI-MS spectrum
of pure tolbutamide whose MS peak of the molecular ion was
at 271.1 m/z. All these results demonstrated that the successful
electrode-driven metabolism process generated 4-hydroxytol-
butamide from tolbutamide.
rate constant was 12.3 sÀ1 at the scan rate of 0.5 V sÀ1 13 In the
.
presence of dissolved oxygen, the shape of the CV curves for
the direct electron transfer of CYP2C9/CPR-microsome/ITO/
CS changed dramatically with an increased reduction peak
current and a decreased oxidation peak current (Support
information Fig. S3w), suggesting a typical electrocatalytic
oxygen reduction process based on the fast binding of oxygen
to the reduced ferrous cytochrome P450.14
To evaluate the concept of electrochemically driven drug
metabolism by CYP2C9/CPR-microsomes, tolbutamide was
chosen as a model which can be metabolized by CYP2C9
to yield 4-hydroxytolbutamide.15 CYP2C9/CPR-microsome/
ITO/CS gave an increase in cathodic current beginning at
around À0.5 V with increasing tolbutamide concentrations
in the voltammograms recorded at a rotating disk electrode
(Fig. 1B). This is characteristic of the catalytic process in
which electrons are consumed as follows:
RH + O2 + 2H+ + 2eÀ - ROH +H2O
(1)
Fig. 2A displays current–time curve for the CYP2C9/
CPR-microsome/ITO/CS/GCE upon successive addition of
tolbutamide at À0.48 V (vs. SCE). When tolbutamide was
added into the buffer solution, the reduction current rose
steeply and reached a stable value within less than 5 s. For
control experiments, an equivalent aliquot (10 mL) of other
reagents (ethanol, PBS) was also injected into the solution,
generating no response. Besides this, no signal was detected on
ITO/CS/GCE upon the addition of tolbutamide (data not shown).
Sulfaphenazole is identified as
a strong, competitive
and very selective inhibitor of CYP2C9 for its high affinity
c
Chem. Commun.
This journal is The Royal Society of Chemistry 2012

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Basic Research Program of China (No.2010CB732400), National
Natural Science Foundation of China (Grant Nos. 21175021),
the Key Program (BK2010059) from the Natural Science
Foundation of Jiangsu province, Foundation for Excellent
Doctoral Dissertation from Southeast University (YBJJ1112)
and Scholarship Award for Excellent Doctoral Student
granted by Ministry of Education.
Notes and references
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In summary, a new bioelectronic system is successfully
constructed based on nanocomposites that integrate CYP2C9/
CPR-microsomes, ITO nanoparticles and CS. The metabolite
produced in the system is determined to be the same as in vivo.
Inhibition experiments further confirm the bioactivity of CYP2C9
in the film. This system may have potential for applications in
drug discovery and development by monitoring substrate
metabolism and enzyme inhibition. Other applications include
efficient biosensors for toxicity analysis, bioreactors for chemical
synthesis and design of biological fuel cells.21
We gratefully acknowledge the Key Program (21035002) from
the National Natural Science Foundation of China, the National
21 A. Ramanavicius and A. Ramanaviciene, Fuel Cells, 2009, 9,
25–36.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun.