QSAR features for inhibitors of mitochondrial bioenergetics. Anatoly A. Starkov
H+H+H+H+ H+H+H+H+ Oxygen C-III SDH C C-IV C-I FMN IM NADH NAD + Succinate Fumarate Water e e e e e e e CoQ Fuel Supply System Electron transfer in the respiratory chain NADH Oxygen, CoQH 2 e H+H+H+H+ p.m.f. = + pH
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 routine mitochondrial functions, thus inducing a metabolically futile wasting of energy. Wallace KB, Starkov AA. Mitochondrial targets of drug toxicity. Annu Rev Pharmacol Toxicol. 2000;40: UNCOUPLING:.
Respiratory chain H+H+ H+H+ AH A-A- A-A- H+H+ H+H+ IM Matrix A-A- AH Respiratory chain H+H+ H+H+ AH A-A- H+H+ H+H+ IM Matrix pH HA 2 - AH A-A- Proton shuttling by lipophilic weak acids. substituted phenols trifluoromethylbenzimidazoles salicylanilides carbonylcyanide 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 Jun;26(3):
Terada H. Uncouplers of oxidative phosphorylation. Environ Health Perspect Jul;87:213-8.
[uncoupler], M State 4, nmol O 2 /min/mg State 3 respiration rate [Uncoupler] max
Respiratory chain H+H+ H+H+ RN + RN H+H+ H+H+ IM Matrix - + pH A-A- Respiratory chain H+H+ H+H+ RNA - H + RN H+H+ H+H+ IM Matrix pH A-A- RN - + Proton shuttling by lipophilic weak bases and ion pairs. amine local anesthetics 3. 4.
Respiratory chain H+H+ H+H+ AH A-A- A-A- H+H+ 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 Respiratory chain H+H+ H+H+ 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
Respiratory chain H+H+ H+H+ 2H + IM Matrix E U pH - + Ca 2+ U: Ca2+ uniporter. E: Ca2+ ionophores. 7. Ion cycling. (Variant : U=valinomycin, Ca 2+ =K +, E=nigericine)
RC H+H+ H+H+ Ca 2+ 2H + IM Matrix pH Ca 2+ 2H + U E precipitate + CypD + + Fuel Supply System PTP Cytosol Ca 2+ signal ER storage + 1. Normal Ca 2+ signaling: Uncoupling due to the permeability transition pore (mPTP). 8.
RC H+H+ H+H+ Ca 2+ 2H + IM Matrix pH Ca 2+ 2H + U E precipitate + CypD + + Fuel Supply System PTP Cytosol Ca 2+ flooding ER storage + 2. Pathological Ca 2+ flooding opens mPTP:
McLaughlin SG, Dilger JP. Transport of protons across membranes by weak acids. Physiol Rev Jul;60(3): Classical efficient uncoupler: 4<pKa<7.2, 3<logP<8
McLaughlin SG, Dilger JP. Transport of protons across membranes by weak acids. Physiol Rev Jul;60(3):
Steps:Mechanism typedescriptors Acquire H+1-5, 7pKa Adsorb to the membrane1-5, 7, (6)D(water-membrane) (partition coefficient) Partition into the membrane1-7, (6)K,K’(surface-core) (species partition coefficient) Cross the membrane1-5, 7k,k’(species) (translocation rate constant) Release H+ inside matrix1-5, 7pKa Cross the membrane1-5, 7k’(species) (translocation rate constant) Acquire H+1-5, 7pKa’ 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 Apr;21(4):
McLaughlin SG, Dilger JP. Transport of protons across membranes by weak acids. Physiol Rev Jul;60(3): 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 Jun;18(6): 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-IV IM NADH NAD + Water e e e e e CoQ ROSROS ROS ROSROS Succinate Fumarate e e Fuel Supply System
Q i site Q o site b low b high e e e e e e ISP Cyt.c1 Cyt.c Myxothiazol Antimycin Stigmatellin QiQi QoQo QH 2 Q Matrix side - + IM A QH 2 CoQ:Cytochrome c reductase (RC Complex III)
Antimycin A Myxothiazol Stigmatellin Classical inhibitors of CoQ:Cytochrome c reductase (RC Complex III)
N3 FMN N1b N4 N5 N7 N2 N6a N6b 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 Oct;57(10): 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 May 6;1364(2):
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.
[O 2 ]=0 Coupled respiration ADP Mito O 2 consumed 50 nmol O 2 1 min State 4 State 4’ State 3 ADP [O 2 ]=0 100 nmol ATP 1 min ADP [ATP] (~20 mV) V state 3 V state 4,4’ ADP:O
50 nmol O 2 1 min ADP [O 2 ]=0 2,4-DNP Mito O 2 consumed Uncoupled respiration ADP [O 2 ]=0 100 nmol ATP 1 min ADP [ATP] (~20 mV) 2,4-DNP V(u) state 3 V(u) state 4,4’ ADP:O(u)
< > = UncoupledCoupled Uncoupling: less ATP for the same O 2 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 Jun;18(6):
ROS production is regulated by H 2 O 2 emission, % of max H 2 O 2 emission, pmol/min/mg