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