Practical use of JC-1 in a Drug Discovery setting

Per O.G. ArkhammarBioImage A/SMrkhj Bygade 28 DK-2860 Sborg Denmark

Molecular Devices 4th International Cell Analysis Products Users MeetingMay 30 June 2, 2000

Cationic carbocyanine potentiomentric dye JC-1: single excitiation, dual emission.JC-1 J-aggregate

Energised

527 nm

590 nm

Cationic carbocyanine potentiomentric dye JC-1: single excitiation, dual emission.488 nm

Energised

Depolarised

Pre-treatment

50 nM FCCP

Red:Green ratio corrects for bleach effects 4.5 and increases sensitivity to VmitJC-1 emission4 3.5 3

515 - 545 nm

2.5 2 1.5 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13

575 - 625 nm14 15 16

1.9

Ratio4 nM 20 nM 52 nM 100 nM Oligomycin 132 nM

JC-1 (R/G) ratio

1.7 1.5 1.3 1.1 0.9 0.7 0.5 0.3 0 1 2 3 4 5 6 7 8 9

Progressive uncoupling with FCCP

10 11 12 13

14 15 16

Minutes

Mitochondrial Respiratory chain:proton pumps and leaks, electron flowsIM space P-phase H+REV ERSIBLE

Matrix (N -phase) INADH/NAD +-280 mV

X

Rotenone Antimycin A

eII IIISucc/Fum-30 mV

UQ

H+REV ERSIBLE

XCyt c

H+ IV H+REV ERSIBLE

X

? O 2/H2O+390 mV

CNOligomycin

F 0F1 ATPase H+ Uncoupler

XATP

From: Nicholls & Ferguson, 1992

Mitochondrial sensitisation with Oligomycin and Antimycin AFCCP dose response in A10 cells(n=7) 14000

JC-1 Green Fluorescence (+SD)

12000 10000 8000 6000 4000 2000 0 0.1 1 10 100 FCCP Concentration (nM) Inhibited Control

JC-1 green signal dose responses for some metabolic toxins

Control cells

Inhibition with Oligomycin and Antimycin A for 20 minutes

JC-1 green signal dose responses for some metabolic toxins

Iodoacetic acid Ionomycin Oligomycin Nigericin Valinomycin Sodium Azide Rotenone KCN Atractyloside Dinitrophenol

Glucose metabolism

D-glucoseglucokinase

G6P F6P

D-glucose

phosphoglucoseisomerase

phosphofructokinase

F 1,6 DPaldolase

Iodoacetic acidG3PDH

GA 3 P

1,3 DP G

DHA P

triosephosphate isomerase

PGK PGM

PE P

pyruvatekinase

PyruvateLDH

Ionomycin

Nigericin Dinitrophenol FCCP

Lactate

TCAAtractyloside

ATPelectron transport

NADH FADH2 KCN

ATP

Sodium Azide Valinomycin Oligomycin Rotenone

JC-1 green signal dose responses for some metabolic toxins

Iodoacetic acid Ionomycin Oligomycin Nigericin Valinomycin Sodium Azide Rotenone KCN Atractyloside Dinitrophenol

JC-1 green signal dose responses for some metabolic toxins

Dinitrophenol4500 4000 3500 Inhib Control

Fluorescence

3000 2500 2000 1500 1000 500 0 0.1 1 10 100 1000 Concentration (M)

JC-1 green signal dose responses for some metabolic toxins

Nigericin5000 4500 4000 Inhib Clean

Fluorescence

3500 3000 2500 2000 1500 1000 500 0 0.01

0.1

1

10

100

Concentration (M)

JC-1 as counterscreen for unwanted metabolic effectsJC-1 comparison of two primary hits(n=5) 6000

JC-1 fluorescence (mean+SD)

5000 4000 3000Compound 1C

2000 1000 0 0.01

Compound 2C Compound 1I Compound 2I

0.1

1 Concentration (M)

10

100

JC-1 screen of LOPAC library(640 compounds) 50 45 40 35

Frequency

30 25 20 15 10 5 0 -30000 -10000 10000 30000 50000 70000 90000 110000 JC-1 G-R (AUC) Value

6%

Cationic carbocyanine potentiomentric dye JC-1: single excitiation, dual emission.JC-1 J-aggregate

Energised

527 nm

590 nm

JC-1 screen of LOPAC library(640 compounds) 50 45 40 35

Frequency

30 25 20 15 10 5 0 -30000 -10000 10000 30000 50000 70000 90000 110000 JC-1 G-R (AUC) Value

3%

JC-1 and AB comparison

JC-1 21 hits } 4 common AB 23 hits

Hits0

80

160

240

320

400

480

560

640

Compound ID

Practical use of JC-1 in a Drug Discovery setting Primary screening: Uncoupling agents Compounds that energize mitochondria Compounds that protect mitochondria Apoptosis MDR assessment

Secondary / counterscreening: Targets / events regulated by cellular metabolism ion channels ion pumps intracellular calcium changes secretion glycolysis, Krebs cycle

Practical use of JC-1 in a Drug Discovery setting

Acknowledgements:BioImage A/S: Dr. Bob Terry Dr. Morten Heide Dr. Kurt Scudder Molecular Devices: Dr. Frank Hafner

Molecular Devices 4th International Cell Analysis Products Users MeetingMay 30 June 2, 2000