A NOVEL BIOLUMINESCENT PROBE FOR NON-INVASIVE QUANTIFICATIONOF MITOCHONDRIAL MEMBRANE POTENTIAL

1

Main function of mitochondria is ATP synthesis

Electrochemical proton gradient is the driving force for ATP synthesis 2

Electron transport chain (ETC)

3Nicholls, D. G. & Ferguson, S. J. Bioenergetics. Fourth edition / edn, (Academic Press, Elsevier, 2013).

Energy of proton gradient:∆𝐺 = 𝐹∆𝜓 RT ln 𝐻𝐻 = 𝐹∆𝜓 2.3RT Δ𝑝𝐻Δψ – electric potential, MMP

4Figures are adapted from Cell Metabolism (2015) 22, 204-206Mol. Cell Bio. (2005) 6, 318-327

Intracellular signaling• Change of gene expression

profile• Adaptation to stress• Apoptosis

Biosynthetic function• Long chain fatty acids• Branched chain amino acids• Nucleotide biosynthesis

ATP synthesis is not the only role of mitochondria in a cell

5

Monitoring of mitochondria function is important to understand progression of these pathologies

Mitochondrial dysfunction is involved in many processes

MMP dysregulation is implicated in :• Aging• Alzheimer disease• Parkinson’s disease• Diabetes mellitus 2 type• Cardiomyopathy• Ischemia

Figure is adapted from Cell (2013) 153, 1194-1217

Mitochondrial membrane potential (MMP) is one of the key parameters that characterizes function of mitochondria

Quantification of MMP relies on fluorescent dyes

Cytosol

30-60 mV

Mitochondrion

180-200 mV

O

O

O

NN

TMRM

O

O

O

NN

TMRM

Accumulation

Decrease of MMP willlead to decrease offluorescent signal.

6

Intensity of the signal isproportional to the amount ofTMRM that was accumulated

∆𝜓 = 𝑅𝑇𝑧𝐹 ln 𝑇𝑀𝑅𝑀𝑇𝑀𝑅𝑀

Drawbacks of fluorescent dyes for measuring MMP

• None of them are suitable to be used in living animals (mammals)

Methods, (2008), 46, 304-3117

Measurement of MMP in vivo is extremely important

8

• Aging• Alzheimer disease• Parkinson’s disease• Diabetes mellitus 2 type• Cardiomyopathy• Ischemia

Impossible to create relevant disease model in cell culture

Strategy for non-invasive imaging of mitochondrial membrane potential (MMP)

Bio-orthogonal reaction

+

Bioluminescent imaging

MMP

in a context of various diseases

9

PPh2R'N3

Phosphine Azide PhP

Ph

NR'

N2

Aza-ylide

+

H2O

PPh2

OR'NH2+

AminePhosphine oxide

The Staudinger reaction

Staudinger, H.; Meyer, J. Helv. Chim. Acta 1919, 2, 635.

Hermann Staudinger

The Staudinger ligation – the first bioorthogonal reaction

Science (2000) 287, 2007

Using the Staudinger ligation to activate luciferin

O

OCH3

PPh

Ph

+ N3 R'

N2O

OCH3

PPh

PhN R'

Aza-ylide

CH3OH

O

N

PPhPh

R'H2O

O

NH

PPh

PhO

R'

phosphine

Ligation product

S

N

S

N CO2H

OR

R = H light

R = not H no light

Nat Chem Biol (2005) 1, 13

Using TPP cation to deliver reagents to mitochondria

M. Murphy, Biochimica et Biophysica Acta, 1777 (2008), 1028-1031Edward J. Gane, et al. Liver international (2010), 1019-1026

Cytosol

P X

30-60 mV

3-10 X

100-500 X Mitochondrion

180-200 mV

TPP

13

- MitoQ (CoQ10)

In Phase 2 was proved to protect against liver damageduring hepatitis C

O

O

O

O H

6-10

Design of mitochondria-activated luciferin (MAL) probe

14

MMP DEPENDENT ACCUMULATION MMP DEPENDENT RATE OF LUCIFERIN RELEASE

How to make luciferin uncaging MMP dependent?1. Take two reagents that release luciferin upon reaction

2. Target them to mitochondria

15Potentiation of reaction rate – 105 fold Only mitochondrial uncaging is observed

The bioluminescent signal intensity depends on MMP

Reaction rate enhancement

16

𝑅 = 𝑇𝑃𝑃𝑇𝑃𝑃 ≈ 𝐴𝑧𝑖𝑑𝑜𝑇𝑃𝑃𝐴𝑧𝑖𝑑𝑜𝑇𝑃𝑃 = 𝑇𝑃𝑃𝐶𝐿𝑇𝑃𝑃𝐶𝐿 = 10 ∆. ,𝜐𝜐 = 𝑘 𝐴𝑧𝑖𝑑𝑜𝑇𝑃𝑃 𝑇𝑃𝑃𝐶𝐿𝑘 𝐴𝑧𝑖𝑑𝑜𝑇𝑃𝑃 𝑇𝑃𝑃𝐶𝐿 = 𝑅 𝐴𝑧𝑖𝑑𝑜𝑇𝑃𝑃 𝑇𝑃𝑃𝐶𝐿𝐴𝑧𝑖𝑑𝑜𝑇𝑃𝑃 𝑇𝑃𝑃𝐶𝐿 = 𝑅 = 10 ∆. = 10 ∆. ,

The bioluminescent signal intensity depends on MMP

17

Intact mitochondria Depolarized mitochondria

High rate of luciferin releaseHigh signal

Low rate of luciferin releaseLow signal

FCCP, valinomycin

Valinomycin

Nigericin

FCCPNigericin

Hyperpolarized mitochondria

Very high rate of releaseExtremely high signal

18

HT-1080 luc2 cells

With MAL depolarization is detectable down to 0.5 uM

Mitochondria activated luciferin (MAL) TMRM

Detection of valinomycin induced depolarization in cells

O

O

O

NN

vs

19

HT-1080 luc2 cells

Mitochondria activated luciferin-3 (MAL3) TMRM

Detection of valinomycin induced depolarization in cells

O

O

O

NN

vs

Tota

l pho

ton

flux

[p/(c

m2 *s

r)]

RFU

Response of MAL3 assay is more non-linear

20

HT-1080 luc2 cells

Mitochondria activated luciferin-3 (MAL3) TMRM

Detection of hyperpolarization in cells

O

O

O

NN

vs

21

Monitoring mitochondrial potential in vivo