Enzyme assemblies for nanotechnology applied to drug
metabolism Sheila Sadeghi, Ph.D.
Dept. of Life Sciences & Systems Biology University of Torino
www. biochemistry-scienze.unito.it/Home.html
Drug metabolising enzymes Proteins are intrinsically able to achieve specific
self-assembly in the nanoscale dimensions. Our group is developing new approaches to
wiring engineered enzymes to surfaces, building microchips for pharmaceutical exploitation.
Enzymes involved=Human liver monooxygenases
Most important in Phase I Membrane-bound (difficult to work with)
Cytochrome P450 FMO
2D623%
2B62%1A2
5%
2C9/1915%
2A62%
3A450%
2E13%
Relative Importance of P450s in Drug Metabolism
Cys466
2
P450 human
P450 human
h P450: Membrane-bound
h P450: Solubilised
P450 BM3: soluble, self-sufficient
FAD FMN
BMR
h P450-BMR: soluble, self-sufficient
P450 human
FAD FMN
BMR
FMN
D. vulgaris flavodoxin: soluble
FAD FMN
BMRBMP
P450
h P450-FLD: soluble
P450 human
FMN
Molecular Lego: assembling building blocks
3 Gilardi, G., Meharenna, Y.T., Tsotsou, G.E., Sadeghi, S.J., Fairhead, M., Giannini, S. Molecular Lego: Design of molecular assemblies of P450 enzymes for nanobiotechnology Biosensors and Bioelectronics, 17(1-2), (2002), 119-131
Chimeric protein expression
4
Steady state spectrophotometry
kDa
97
66
45
30
20
14
MW fusion
loop FLD P450
MW fusion
expressed in E.coli High yields (40mg/l) Reduced protein shows characteristic
450nm shift upon binding CO
DNA gel Protein gel
DNA Sequencing Dodhia, V.R., Fantuzzi, A. and Gilardi, G. Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego. J Biol Inorg Chem (2006), 11(7), 903-916
Au(111)withSAM:
BoundP450:
50 100 150 0
6
12
Hei
ght (
nm)
(nm) 3 6 9 12 0
15
30
Cou
nts
Height (nm)
55 110 165 0
6
12
Hei
ght (
nm)
(nm)
0 250 500
250
500 nm
0 200 400 600
400
200
600
nm
Histogramofthemolecularheights
Protein topography- Tapping mode AFM
5
Ferrero, V.E., Andolfi, L., Di Nardo, G., Sadeghi, S.J., Fantuzzi, A., Cannistraro, S. and Gilardi, G Protein and electrode engineering for the covalent immobilization of P450 BMP on gold, Anal. Chem., (2008), 80, 8438-8446.
Metabolism of cancer drugs- tamoxifen
0
1
2
3
2 4 6 8 10 12 14 16 18 20
Retention time (min)
Ab
so
rba
nc
e
TAM-NO
TAM
Au Electrode
TAMTAM-NO
SAM = dithio-bismaleimidoethane (DTME)
HPLC analysis
6
Sadeghi SJ, Meirinhos R, Catucci G, Dodhia VR, Di Nardo G and Gilardi G. Direct electrochemistry of drug metabolizing human flavin-containing monooxygenase: electrochemical turnover of bzd and tamoxifen, J Am Chem Soc. (2010), 132(2), 458-9.
Applications
(1) Drug discovery
(2) Drug-drug or drug-food interactions The adverse effects of drug-drug and drug-food
interactions are costly both in terms of human life and investment (withdrawal of drugs from the market).
(3) Personalised medicine
7
1. Drug discovery
8
Optical binding assay- fluorescent probes
9
Ferrero, V.E.V., Di Nardo, G., Catucci, C., Sadeghi, S.J. and Gilardi G. Fluorescence detection of ligand binding to labeled cytochrome P450 BM3, Dalton Transactions, (2011), 40, 1-8
10
Optical binding assay- fluorescent probes
Ferrero, V.E.V., Di Nardo, G., Catucci, C., Sadeghi, S.J. and Gilardi G. Fluorescence detection of ligand binding to labeled cytochrome P450 BM3, Dalton Transactions, (2011), 40, 1-8
P450 - microfluidic device
Fantuzzi A., Capria E., Mak L.H., Dodhia V.R., Sadeghi S.J., Collins S., Somers G., Huq E., Gilardi G. An electrochemical microfluidic platform for human P450 drug metabolism profiling Anal. Chem., (2010) 82, 10222-10227
Working electrode (WE)
Screen printed Counter electrode (CE) Reference electrode (RE)
Inlet nanoport
Outlet nanoport
O - ring
O - ring
Screws
Pogo - pin
CE -
WE holder
Cell
Working electrode (WE)
Screen printed Counter electrode (CE) Reference electrode (RE)
Inlet nanoport
Outlet nanoport
O - ring
O - ring
Screws
Pogo - pin
- RE holder
WE holder
Cell
polymethylmetacrylate
Evaporated gold on silicon working electrode
Screen printed carbon and Ag/AgCl inks on polytethylene terephthalate counter/reference electrodes
Exploded view of the microfluidic cell
12
Fantuzzi A., Capria E., Mak L.H., Dodhia V.R., Sadeghi S.J., Collins S., Somers G., Huq E., Gilardi G. An electrochemical microfluidic platform for human P450 drug metabolism profiling Anal. Chem., (2010) 82, 10222-10227
2. Drug/drug and food/drug interactions
14
Xenobiotics (food components or drugs) that induce or inhibit P450 enzymes can change the rate or extent of drug metabolism
Bottom line: a greater concentration of drug remains in the plasma
Drugs Removed from or Restricted in the U.S. Market Because of Drug Interactions
Terfenadine (Seldane) February 1998 Anti-histamine Interaction with drugs e.g. ketoconazole, erythromycin Interaction with food e.g. grapefruit juice
Mibefradil (Posicor) June 1998 Calcium channel blocker
Astemizole (Hismanal) July 1999 Anti-histamine
Grepafloxacin (Raxar) October 1999 Anti-bacterial
Cisapride (Propulsid) January 2000 Treatment of constipation
15
Drug-drug interactions
Ketoconazole
Cimetidine
Diclofenac
According to the FDA, IC50 represents the concentration of a drug that is required for 50% inhibition in vitro.
16
Sadeghi et al.. Drug-drug interactions and cooperative effects detected in electrochemically driven human cytochrome P450 3A4. Bioelectrochemistry (2012).
Drug-food interactions
0
20
40
60
80
100
resid
ual activity o
fC
YP
3A
4-B
MR
0 5 10 15 20 30 40 60 70 80 100 120 150 170 200 300
[curcumin] / _M
0
20
40
60
80
100
% r
esid
ual a
ctiv
ity o
f C
YP
3A4-
BM
R
0 10 20 50 200 500 1000
[resveratrol] / _M
0
20
40
60
80
100
% a
ttivi
t r
esid
ua d
el
CYP
3A4-
BMR
0 0.1 0.3 0.5 1 2 5
S1
% succo di pompelmo
Drug = erythromycin Food = grapefruit juice
red wine curry powder
17
Res
idua
l Act
ivity
%
0
100
80
60
40
20
log[resve]
0 1 2 3 4 5
%re
sid
ual a
ctiv
ity 0
20
40
60
80
100
Res
idua
l Act
ivity
%
0
100
80
60
40
20
log[Resveratrol] 0 1 2 3 4 5
IC50 = 250M IC50 = 208M
Res
idua
l Act
ivity
%
0
100
80
60
40
20
log[Curcumin] 0 0.5 1 1.5 2 2.5 3
log[Curcumin] 0 0.5 1 1.5 2 2.5 3
Res
idua
l Act
ivity
%
0
100
80
60
40
20
IC50 = 31M IC50 = 24M
B B
C C
0 0.1 1 10 100
Res
idua
l Act
ivity
%
Grapefruit juice %
0
100
80
60
40
20
Grapefruit juice %
X Data
0 1 2 3 4
Y D
ata
0
20
40
60
80
100
Col 1 vs Col 2
x column vs y column
0 0.1 1 10 100
Res
idua
l Act
ivity
%
0
100
80
60
40
20
IC50 = 0.5% IC50 = 0.7%
A A
log[Resveratrol] 0 1 2 3 4 5
18
3. Personalised medicine
Genotype identification
Amperometric metabolic profiling
Personalized dosage Drug Dose 100mg 250mg 50mg 100mg
Polymorphic variants amongst us
Panicco, P., Dodhia, V.R., Fantuzzi, A. and Gilardi, G. "First enzyme-based amperometric platform to determine the polymorphic response in drug metabolism by cytochromes P450". Anal. Chem. (2011), 83, 2179-2186
I359
R144
R296
T107
S486
2C9.1 2C9.2 (R144C) 35% caucasian
warfarin, KM increased, kcat decreased
2C9.3 (I359L) 35% caucasian
warfarin, KM increased, kcat decreased
2D6.1 2D6.2 (R296C, S486T) 22-34% caucasian
bufuralol, KM increased, kcat unchanged
2D6.17 (T107I, R296C, S486T) 34% black Africans
bufuralol, KM increased, kcat decreased
21
C
urre
nt /
A
[drug] / M
POTENTIOSTAT PC
300 uL well with 3 electrodes
immobilised P450
contact wires
KM, kcat, CLint
ELECTROCHEMICAL ARRAY
22
Panicco, P., Dodhia, V.R., Fantuzzi, A. and Gilardi, G. "First enzyme-based amperometric platform to determine the polymorphic response in drug metabolism by cytochromes P450". Anal. Chem. (2011), 83, 2179-2186
A B
Electrochemical titration of drugs in the array
23
in vitro vs in vivo
KM S-warfarin [ 2C9.1 < 2C9.2 < 2C9.3 ]
kcat S-warfarin [ 2C9.1 > 2C9.2 > 2C9.3 ]
KM bufuralol [ 2D6.1 < 2D6.2 < 2D6.17 ]
kcat bufuralol [ 2D6.1 2D6.2 > 2D6.17 ]
24 Panicco, P., Dodhia, V.R., Fantuzzi, A. and Gilardi, G. "First enzyme-based amperometric platform to determine the polymorphic response in drug metabolism by cytochromes P450". Anal. Chem. (2011), 83, 2179-2186
25
In conclusion. Our group is mainly involved in protein engineering approaches
for assembling of different enzymes or their domains for drug metabolism
Relevance is mainly directed to pharmaceutical companies interested in:
High throughput assays for drug discovery Platform for drug-drug and food-drug interactions Personalised medicine
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