QSAR features for inhibitors of mitochondrial bioenergetics. Anatoly A. Starkov.
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Transcript of QSAR features for inhibitors of mitochondrial bioenergetics. Anatoly A. Starkov.
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QSAR features for inhibitors of mitochondrial QSAR features for inhibitors of mitochondrial
bioenergetics.bioenergetics.
Anatoly A. StarkovAnatoly A. Starkov
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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
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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.
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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.
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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:.
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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.
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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.
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Terada H. Uncouplers of oxidative phosphorylation. Environ Health Perspect. 1990 Jul;87:213-8.
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[uncoupler], M
Sta
te 4
, nm
ol O
2/m
in/m
gState 3 respiration rate
[Uncoupler] max
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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.
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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.
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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)
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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.
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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:
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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
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McLaughlin SG, Dilger JP. Transport of protons across membranes by weak acids. Physiol Rev. 1980 Jul;60(3):825-63.
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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
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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.
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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.
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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:
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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
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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.
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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
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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)
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Antimycin A
Myxothiazol
Stigmatellin
Classical inhibitors of CoQ:Cytochrome c reductase (RC Complex III)
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N3FMN
N1bN4N5N7
N2
N6aN6b
N1a
Complexity of mammalian NADH:CoQ reductase (RC Complex I)
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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
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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.
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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.
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[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
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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)
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<
>
=
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)
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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.
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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
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