QSAR features for inhibitors of mitochondrial QSAR features for inhibitors of mitochondrial

bioenergetics.bioenergetics.

Anatoly A. StarkovAnatoly A. Starkov

HH++HH++

Oxygen

C-III

SDH

C

C-IV

C-I

FMN

IM

NADH

NAD+

Succinate

Fumarate

Water

e

e

e

ee

e

e

CoQ CoQ

Fuel Fuel Supply Supply SystemSystem

Electron transfer in the respiratory chainElectron transfer in the respiratory chain

NADH Oxygen, CoQH2

e

HH++ p.m.f. = p.m.f. = + + pHpH

1. What is “uncoupling”?

2. What are “uncouplers”?

3. What are the mechanisms of uncoupling?

4. How much uncoupling is toxic?

5. Is a class-independent QSAR model for uncouplers

possible? What descriptors should be selected?

6. What models should be used to test the uncouplers?

A. UNCOUPLING.

Classical definitions:

Uncoupling of oxidative phosphorylation is a process de-coupling oxygen consumption from ATP production.

Uncouplers:

1.Stimulate resting respiration. 2.Decrease ATP yield (P:O ratio).3.Activate latent ATPase.

any energy-dissipating process competing for energy with routinemitochondrial functions, thus inducing a metabolically futile wasting of energy.

Wallace KB, Starkov AA. Mitochondrial targets of drug toxicity. Annu Rev Pharmacol Toxicol. 2000;40:353-88.

UNCOUPLING:.

Respiratorychain

H+

H+

AH

AH A-

A-

H+

H+

IM

Matrix

A-

AHRespiratory

chain

H+

H+

AH

AH A-

H+

H+

IM

Matrix

pH

HA2-

AH

A-

Proton shuttling by lipophilic weak acids.

substituted phenols trifluoromethylbenzimidazolessalicylanilidescarbonylcyanide phenylhydrazones

-

+

-

+

pH

1. 2.

Blaikie FH, Brown SE, Samuelsson LM, Brand MD, Smith RA, Murphy MP. Targeting dinitrophenol to mitochondria: limitations to the development of a self-limiting mitochondrial protonophore. Biosci Rep. 2006 Jun;26(3):231-43.

Terada H. Uncouplers of oxidative phosphorylation. Environ Health Perspect. 1990 Jul;87:213-8.

[uncoupler], M

Sta

te 4

, nm

ol O

2/m

in/m

gState 3 respiration rate

[Uncoupler] max

Respiratorychain

H+

H+

RN+

RN+ RN

RN

H+

H+

IM

Matrix-

+

pH

A-

Respiratorychain

H+

H+

RNA-H+

RNA-H+ RN

H+

H+

IM

Matrix

pH

A-

RN

-

+

Proton shuttling by lipophilic weak bases and ion pairs.

amine local anesthetics

3. 4.

Respiratorychain

H+

H+

AH

AH A-

A-

H+

H+

IM

Matrix

P

Protein –mediated uncoupling by non-permeating anions and protein modifying reagents.

P: ATP/ADP translocator, Glutamate transporter

Long-chain fatty acids, SDS, 2,4-DNP

Respiratorychain

H+

H+H+

H+

IM

Matrix

P

P: Uncoupling Protein 1 (UCP1), anion carriers, membrane-active peptides, Permeability transition Pore (mPTP).

Long-chain fatty acids, SH-modifying reagents.

pH

pH

-

+

-

+

5. 6.

Respiratorychain

H+

H+2H+

2H+

IM

Matrix

EU

pH

-

+

Ca2+

Ca2+

U: Ca2+ uniporter.

E: Ca2+ ionophores.

7.

Ion cycling.

(Variant : U=valinomycin, Ca2+ =K+, E=nigericine)

RCRC

H+

H+

Ca2+

Ca2+

2H+

IM

MatrixMatrix

pH

Ca2+

Ca2+

2H+

UU

EE

precipitateprecipitate

++CypDCypD

++++

Fuel Fuel Supply Supply SystemSystem

PTPPTP

CytosolCytosolCaCa2+ 2+ signalsignal

ER storageER storage

++

1. Normal Ca1. Normal Ca2+2+ signaling: signaling:

Uncoupling due to the permeability transition pore (mPTP).8.

RCRC

H+

H+

Ca2+

Ca2+

2H+

IM

MatrixMatrix

pH

Ca2+

Ca2+

2H+

UU

EE

precipitateprecipitate

++CypDCypD

++++

Fuel Fuel Supply Supply SystemSystem

PTPPTP

CytosolCytosol

CaCa2+ 2+ floodingflooding ER storageER storage

++

2. Pathological Ca2. Pathological Ca2+2+ flooding opens mPTP: flooding opens mPTP:

McLaughlin SG, Dilger JP. Transport of protons across membranes by weak acids. Physiol Rev. 1980 Jul;60(3):825-63.

Classical efficient uncoupler: 4<pKa<7.2, 3<logP<8

McLaughlin SG, Dilger JP. Transport of protons across membranes by weak acids. Physiol Rev. 1980 Jul;60(3):825-63.

Steps: Mechanism type descriptorsAcquire H+ 1-5, 7 pKaAdsorb to the membrane 1-5, 7, (6) D(water-membrane) (partition coefficient)Partition into the membrane 1-7, (6) K,K’(surface-core) (species partition coefficient)Cross the membrane 1-5, 7 k,k’(species) (translocation rate constant)Release H+ inside matrix 1-5, 7 pKaCross the membrane 1-5, 7 k’(species) (translocation rate constant)Acquire H+ 1-5, 7 pKa’

Information on the surrounding: pH out and in, lipid phase volume, lipid phase(s) dielectric constants and viscosity, gradient of the electrical membrane potential across the membrane, total amount of a compound.

Minimum reasonable set of parameters to consider:

Classical: 4<pKa<7.2, 3<logP<8

Spycher S, Smejtek P, Netzeva TI, Escher BI. Toward a class-independent quantitative structure--activity relationship model for uncouplers of oxidative phosphorylation. Chem Res Toxicol. 2008 Apr;21(4):911-27.

Spycher S, Smejtek P, Netzeva TI, Escher BI. Toward a class-independent quantitative structure--activity relationship model for uncouplers of oxidative phosphorylation. Chem Res Toxicol. 2008 Apr;21(4):911-27.

McLaughlin SG, Dilger JP. Transport of protons across membranes by weak acids. Physiol Rev. 1980 Jul;60(3):825-63.

Black lipid membranes as a model to test the intrinsic efficiency of uncouplers:

Ilivicky J, Casida JE. Uncoupling action of 2,4-dinitrophenols, 2-trifluoromethylbenzimidazoles and certain other pesticide chemicals upon mitochondria from different sources and its relation to toxicity. Biochem Pharmacol. 1969 Jun;18(6):1389-401.

Isolated mammalian mitochondria as a model to test the toxicity of uncouplers

1. What do they do? – inhibit electron transport thereby suppressing H+ generation and stimulating ROS production.

2. How many are known? – a few hundreds of natural compounds and a gazillion of synthetic chemicals.

3. Are there some common chemical features in these compounds? – yes and no.

4. Is their MOA similar? – yes and no.

5. Is a class-independent QSAR model for the RC inhibitors possible? – Perhaps, but not there yet.

6. Why it is so? – insufficient knowledge of RC complexes and their structural diversity.

7. What models should be used to test the RC inhibitors? – isolated mammalian mitochondria.

B. Inhibitors of the respiratory chain complexes.

Oxygen

C-III

SDH C-I

FMN

C

C-IVIM

NADH

NAD+

Water

e

ee

e

e

CoQ CoQ

ROSROSROSROS ROSROSROSROS ROSROS

Succinate

Fumarate

e

e

Fuel Fuel Supply Supply SystemSystem

Qi site

Qo site

blow

bhighe

e

e

e

eeISP

Cyt.c1Cyt.c

Myxothiazol

Antimycin

Stigmatellin

Qi

Qo QH2

Q

Matrix side-

+

IM

AQH2

CoQ:Cytochrome c reductase (RC Complex III)

Antimycin A

Myxothiazol

Stigmatellin

Classical inhibitors of CoQ:Cytochrome c reductase (RC Complex III)

N3FMN

N1bN4N5N7

N2

N6aN6b

N1a

Complexity of mammalian NADH:CoQ reductase (RC Complex I)

Schuler F, Casida JE. The insecticide target in the PSST subunit of complex I. Pest Manag Sci. 2001 Oct;57(10):932-40

PSST subunit of Complex I is a common target for many and various inhibitors.

Inhibitor binding site

Different classes of the Q site Complex I inhibitors.

Degli Esposti M. Inhibitors of NADH-ubiquinone reductase: an overview. Biochim Biophys Acta. 1998 May 6;1364(2):222-35.

Future developments toward QSAR model of mitochondrial poisons:

1. Create a realistic biophysical model of the inner mitochondrial membrane;

2. Obtain more detailed information on the molecular structure of mitochondrial proteins targeted by toxins;

3. Create a unified database of mitochondrial toxins and analyze it toward both their molecular properties and the mechanisms of intrinsic activity;

4. Create a good team of researchers with proper expertise (and funding) to develop and validate QSAR models in a relevant biological model (isolated mitochondria) under physiologically meaningful conditions.

[O2]=0

Coupled respiration

ADP

ADP

Mito

O2 co

nsum

ed

50 n

mol

O2

1 min

State 4

State 4’

State 3

ADP

[O2]=0

10

0 n

mo

l AT

P

1 min

ADP

[ATP]

(

~2

0 m

V)

V state 3

V state 4,4’

ADP:O

50 n

mol

O2

1 min

ADP

[O2]=0

2,4-DNP

MitoO

2 co

nsum

ed

Uncoupled respiration

ADP

[O2]=0

10

0 n

mo

l AT

P

1 min

ADP

[ATP]

(

~2

0 m

V)

2,4-DNP

V(u) state 3

V(u) state 4,4’

ADP:O(u)

<

>

=

UncoupledCoupled

Uncoupling: less ATP for the same O2 and substrates

V state 3

V state 4,4’

ADP:O

V(u) state 3

V(u) state 4,4’

ADP:O(u)

Ilivicky J, Casida JE. Uncoupling action of 2,4-dinitrophenols, 2-trifluoromethylbenzimidazoles and certain other pesticide chemicals upon mitochondria from different sources and its relation to toxicity. Biochem Pharmacol. 1969 Jun;18(6):1389-401.

ROS production is regulated by ROS production is regulated by

mV100 110 120 130 140 150 160 170 180

H2O

2pr

oduc

tion

, pm

olx m

in-1

x m

g-1 100

90

80

70

60

50

40

30 State 3

Sta

te 3

-Ketoglutarate…+ ADP

Glutamate + malate…+ ADP

mV100 110 120 130 140 150 160 170 180

mV100 110 120 130 140 150 160 170 180

H2O

2pr

oduc

tion

, pm

olx m

in-1

x m

g-1 100

90

80

70

60

50

40

30H2O

2pr

oduc

tion

, pm

olx m

in-1

x m

g-1

H2O

2pr

oduc

tion

, pm

olx m

in-1

x m

g-1 100

90

80

70

60

50

40

30

100

90

80

70

60

50

40

30

100

90

80

70

60

50

40

30 State 3

Sta

te 3

State 3

Sta

te 3

-Ketoglutarate…+ ADP-Ketoglutarate…+ ADP

Glutamate + malate…+ ADPGlutamate + malate…+ ADP

succinate

0

10

20

30

40

50

60

70

80

90

100

60 70 80 90 100

H2O

2ge

nera

tion

, %

in

Sta

te 3

V in State 3 H2O2

succinate

0

10

20

30

40

50

60

70

80

90

100

60 70 80 90 100

H2O

2ge

nera

tion

, %

in

Sta

te 3

V in State 3 H2O2V in State 3 H2O2

H2O

2 e

mis

sion

, %

of

max

H2O

2 e

mis

sion

, pm

ol/m

in/m

g