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    "count": 334,
    "next": "",
    "previous": null,
    "results": [
            "pap_id": "1",
            "title": "Acetaminophen-induced liver injury in rats and mice: comparison of protein adducts, mitochondrial dysfunction, and oxidative stress in the mechanism of toxicity.",
            "authors": "McGill, Mitchell R; Williams, C David; Xie, Yuchao; Ramachandran, Anup; Jaeschke, Hartmut",
            "chapter": "",
            "pages": "387-394",
            "journal": "Toxicology and Applied Pharmacology",
            "pub_date": "2012-11-01",
            "pub_year": 2012,
            "volume": "264",
            "issue": "3",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/j.taap.2012.08.015",
            "pmid": "22980195.0",
            "pmcid": "PMC3478469",
            "abstract": "Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the West. In mice, APAP hepatotoxicity can be rapidly induced with a single dose. Because it is both clinically relevant and experimentally convenient, APAP intoxication has become a popular model of liver injury. Early data demonstrated that rats are resistant to APAP toxicity. As a result, mice are the preferred species for mechanistic studies. Furthermore, recent work has shown that the mechanisms of APAP toxicity in humans are similar to mice. Nevertheless, some investigators still use rats. New mechanistic information from the last forty years invites a reevaluation of the differences between these species. Comparison may provide interesting insights and confirm or exclude the rat as an option for APAP studies. To this end, we treated rats and mice with APAP and measured parameters of liver injury, APAP metabolism, oxidative stress, and activation of the c-Jun N-terminal kinase (JNK). Consistent with earlier data, we found that rats were highly resistant to APAP toxicity. Although overall APAP metabolism was similar in both species, mitochondrial protein adducts were significantly lower in rats. Accordingly, rats also had less oxidative stress. Finally, while mice showed extensive activation and mitochondrial translocation of JNK, this could not be detected in rat livers. These data support the hypothesis that mitochondrial dysfunction is critical for the development of necrosis after APAP treatment. Because mitochondrial damage also occurs in humans, rats are not a clinically relevant species for studies of APAP hepatotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.",
            "references": [
            "pap_id": "10",
            "title": "Mefloquine selectively increases asynchronous acetylcholine release from motor nerve terminals.",
            "authors": "McArdle, Joseph J; Sellin, Lawrence C; Coakley, Kathleen M; Potian, Joseph G; Hognason, Kormakur",
            "chapter": "",
            "pages": "345-353",
            "journal": "Neuropharmacology",
            "pub_date": "2006-03-01",
            "pub_year": 2006,
            "volume": "50",
            "issue": "3",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/j.neuropharm.2005.09.011",
            "pmid": "16288931.0",
            "pmcid": "",
            "abstract": "Effectiveness against chloroquine-resistant Plasmodia makes mefloquine a widely used antimalarial drug. However, mefloquine's neurologic effects offset this therapeutic advantage. Cellular actions which might contribute to the neurologic effects of mefloquine are not understood. Structural similarity to tacrine suggested that mefloquine might alter cholinergic synaptic transmission. Therefore, we examined mefloquine's effects at a model cholinergic synapse. Triangularis sterni nerve-muscle preparations were isolated from adult mice and examined with sharp electrode current clamp technique. Within 30 min of exposure to 10 microM mefloquine, miniature endplate potentials (mepps) occurred in summating bursts and their mean frequency increased 10-fold. The threshold concentration for the increase of mean mepp frequency was 0.6 microM mefloquine. Mefloquine continued to increase mean mepp frequency for preparations bathed in extracellular solution lacking Ca2+. In contrast, mefloquine no longer increased mean mepp frequency for preparations pre-treated with the intracellular Ca2+ buffer BAPTA-AM. Although mefloquine disrupts a thapsigargin-sensitive neuronal Ca2+ store, pre-treatment with thapsigargin did not alter the mefloquine-induced alterations of mepps. Since mefloquine, like oligomycin, inhibits mitochondrial FOF1H+ ATP synthase we tested the interaction between these two chemicals. Like mefloquine, oligomycin induced bursts and increased mean frequency of mepps. Furthermore, pre-treatment with oligomycin precluded the mefloquine-induced alterations of asynchronous transmsitter release. These data suggest that mefloquine inhibits ATP production which increases the concentration of Ca2+ within the cytosol of nerve terminals. This elevation of Ca2+ concentration selectively increases asynchronous transmitter release since 10 microM mefloquine did not alter stimulus-evoked transmsitter release.",
            "references": []
            "pap_id": "100",
            "title": "Phytotoxic Compounds Cochlioquinones Are Inhibitors of Mitochondrial NADH-Ubiquinone Reductase",
            "authors": "Lim, Chi-Hwan; Ueno, Hideki; Miyoshi, Hideto; Miyagawa, Hisashi; Iwamura, Hajime; Ueno, Tamio",
            "chapter": "",
            "pages": "213-215",
            "journal": "Journal of pesticide science",
            "pub_date": "1905-06-18",
            "pub_year": 1905,
            "volume": "21",
            "issue": "2",
            "Issn": "1348-589X",
            "Isbn": "",
            "url": "",
            "doi": "10.1584/jpestics.21.213",
            "pmid": "",
            "pmcid": "",
            "abstract": "",
            "references": [
            "pap_id": "101",
            "title": "Pterulinic acid and pterulone, two novel inhibitors of NADH:ubiquinone oxidoreductase (complex I) produced by a Pterula species. II. Physico-chemical properties and structure elucidation.",
            "authors": "Engler, M; Anke, T; Sterner, O",
            "chapter": "",
            "pages": "330-333",
            "journal": "The Journal of Antibiotics",
            "pub_date": "1997-04-01",
            "pub_year": 1997,
            "volume": "50",
            "issue": "4",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.7164/antibiotics.50.330",
            "pmid": "9186559.0",
            "pmcid": "",
            "abstract": "The structures of two novel fungal antibiotics, isolated from a Pterula species, that interfere with the NADH:ubiquinone oxidoreductase and inhibit the respiration of eucaryotes, were determined by spectroscopic techniques. Both compounds, pterulinic acid (1a) and pterulone (2), contain a 1-benzoxepin ring system and are chlorinated. Pterulinic acid (1a), which was obtained as a 1:5 inseparable mixture of the two isomers (Z)-1a and (E)-1a, in addition contains a furan. Their structures were determined by mass spectrometry and NMR spectroscopy, and 2D heteronuclear correlation experiments permitted the assignment of all NMR signals.",
            "references": [
            "pap_id": "102",
            "title": "Capsaicin and its analogs inhibit the activity of NADH-coenzyme Q oxidoreductase of the mitochondrial respiratory chain.",
            "authors": "Shimomura, Y; Kawada, T; Suzuki, M",
            "chapter": "",
            "pages": "573-577",
            "journal": "Archives of Biochemistry and Biophysics",
            "pub_date": "1989-05-01",
            "pub_year": 1989,
            "volume": "270",
            "issue": "2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0003-9861(89)90539-0",
            "pmid": "2495767.0",
            "pmcid": "",
            "abstract": "Capsaicin and its analogs with different acyl moieties were found to inhibit the electron-transfer activity of NADH-coenzyme Q oxidoreductase isolated from beef heart mitochondria. The inhibitory potency of capsaicin was lower than those of dihydrocapsaicin and analogs with heptanoyl, capryl, undecanoyl, and lauroyl moieties, but was higher than those of analogs with palmitoyl and stearoyl moieties. The analog with the lauroyl moiety showed the strongest inhibition. These results suggest that hydrophobicity and the appropriate carbon chain length of the acyl moiety are important for the binding of compounds to the enzyme. On the other hand, capsaicin and its analogs did not interrupt rotenone-insensitive electron transfer from NADH to menadione. Furthermore, these compounds had almost no effect on the spectral properties and EPR signals arising from iron-sulfur clusters of the NADH-treated enzyme. Kinetic analyses with double-reciprocal plots showed that these compounds were competitive inhibitors with respect to coenzyme Q1, an electron acceptor. These results strongly suggest that capsaicin and its analogs bind to the coenzyme Q1 binding site of the enzyme.",
            "references": [
            "pap_id": "103",
            "title": "Comparison of the inhibitory action of synthetic capsaicin analogues with various NADH-ubiquinone oxidoreductases",
            "authors": "Satoh, Takashi; Miyoshi, Hideto; Sakamoto, Kimitoshi; Iwamura, Hajime",
            "chapter": "",
            "pages": "21-30",
            "journal": "Biochimica et Biophysica Acta (BBA) - Bioenergetics",
            "pub_date": "1996-01-01",
            "pub_year": 1996,
            "volume": "1273",
            "issue": "1",
            "Issn": "52728",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0005-2728(95)00131-X",
            "pmid": "",
            "pmcid": "",
            "abstract": "Capsaicin is a new naturally occurring inhibitor of proton-pumping NADH-ubiquinone oxidoreductase (NDH-1), that competitively acts against ubiquinone. A series of capsaicin analogues was synthesized to examine the structural factors required for the inhibitory action and to probe the structural property of the ubiquinone catalytic site of various NADH-ubiquinone reductases, including non-proton-pumping enzyme (NDH-2), from bovine heart mitochondria, potato tuber (Solanum tuberosum, L) mitochondria and Escherichia coli (GR 19N) plasma membranes. Some synthetic capsaicins were fairly potent inhibitors of each of the three NDH-1 compared with the potent rotenone and piericidin A. Synthetic capsaicin analogues inhibited all three NDH-1 activities in a competitive manner against an exogenous quinone. The modification both of the substitution pattern and of the number of methoxy groups on the benzene ring, which may be superimposable on the quinone ring of ubiquinone, did not drastically affect the inhibitory potency. In addition, alteration of the position of dipolar amide bond unit in the molecule and chemical modifications of this unit did not change the inhibitory potency, particularly with bovine heart and potato tuber NDH-1. These results might be explained assuming that the ubiquinone catalytic site of NDH-1 is spacious enough to accommodate a variety of structurally different capsaicin analogues in a dissimilar manner. Regarding the moiety corresponding to the alkyl side chain, a rigid diphenyl ether structure was more inhibitory than a flexible alkyl chain. Structure-activity studies and molecular orbital calculations suggested that a bent form is the active conformation of capsaicin analogues. On the other hand, poor correlations between the inhibitory potencies determined with the three NDH-1 suggested that the structural similarity of the ubiquinone catalytic sites of these enzymes is rather poor. The sensitivity to the inhibition by synthetic capsaicins remarkably differed between NDH-1 and NDH-2, supporting the notion that the sensitivity against capsaicin inhibition correlates well with the presence of an energy coupling site in the enzyme (Yagi, T. (1990) Arch. Biochem. Biophys. 281, 305–311). It is noteworthy that several synthetic capsaicins discriminated between NDH-1 and NDH-2 much better than natural capsaicin.",
            "references": [
            "pap_id": "104",
            "title": "Studies on the respiratory chain-linked nicotinamide adenine dinucleotide dehydrogenase. XXII. Rhein, a competitive inhibitor of the dehydrogenase.",
            "authors": "Kean, E A; Gutman, M; Singer, T P",
            "chapter": "",
            "pages": "2346-2353",
            "journal": "The Journal of Biological Chemistry",
            "pub_date": "1971-04-25",
            "pub_year": 1971,
            "volume": "246",
            "issue": "8",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "4324213.0",
            "pmcid": "",
            "abstract": "",
            "references": [
            "pap_id": "105",
            "title": "",
            "authors": "",
            "chapter": "",
            "pages": "",
            "journal": "",
            "pub_date": "",
            "pub_year": 1900,
            "volume": "",
            "issue": "",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "",
            "pmcid": "",
            "abstract": "",
            "references": []
            "pap_id": "106",
            "title": "The specificity of mitochondrial complex I for ubiquinones.",
            "authors": "Degli Esposti, M; Ngo, A; McMullen, G L; Ghelli, A; Sparla, F; Benelli, B; Ratta, M; Linnane, A W",
            "chapter": "",
            "pages": "327-334",
            "journal": "The Biochemical Journal",
            "pub_date": "1996-01-01",
            "pub_year": 1996,
            "volume": "313 ( Pt 1)",
            "issue": "",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1042/bj3130327",
            "pmid": "8546703.0",
            "pmcid": "PMC1216902",
            "abstract": "We report the first detailed study on the ubiquinone (coenzyme Q; abbreviated to Q) analogue specificity of mitochondrial complex I, NADH:Q reductase, in intact submitochondrial particles. The enzymic function of complex I has been investigated using a series of analogues of Q as electron acceptor substrates for both electron transport activity and the associated generation of membrane potential. Q analogues with a saturated substituent of one to three carbons at position 6 of the 2,3-dimethoxy-5-methyl-1,4-benzoquinone ring have the fastest rates of electron transport activity, and analogues with a substituent of seven to nine carbon atoms have the highest values of association constant derived from NADH:Q reductase activity. The rate of NADH:Q reductase activity is potently but incompletely inhibited by rotenone, and the residual rotenone-insensitive rate is stimulated by Q analogues in different ways depending on the hydrophobicity of their substituent. Membrane potential measurements have been undertaken to evaluate the energetic efficiency of complex I with various Q analogues. Only hydrophobic analogues such as nonyl-Q or undecyl-Q show an efficiency of membrane potential generation equivalent to that of endogenous Q. The less hydrophobic analogues as well as the isoprenoid analogue Q-2 are more efficient as substrates for the redox activity of complex I than for membrane potential generation. Thus the hydrophilic Q analogues act also as electron sinks and interact incompletely with the physiological Q site in complex I that pumps protons and generates membrane potential.",
            "references": [
            "pap_id": "107",
            "title": "Organic structural specificity and sites of coenzyme Q in succinoxidase and DPNH-oxidase systems",
            "authors": "Lenaz, Giorgio; Daves, G.Doyle; Folkers, Karl",
            "chapter": "",
            "pages": "539-550",
            "journal": "Archives of Biochemistry and Biophysics",
            "pub_date": "1968-03-01",
            "pub_year": 1968,
            "volume": "123",
            "issue": "3",
            "Issn": "39861",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0003-9861(68)90175-6",
            "pmid": "",
            "pmcid": "",
            "abstract": "In succinoxidase, CoQ1 had low activity, and CoQ2 through CoQ10 had comparable activities in both the acetone- and pentane-extracted systems. In the DPNH-oxidase system, CoQ1 had low activity; CoQ2, CoQ3, and CoQ4 had about one-fourth the activity; and CoQ5 and CoQ6 had about one-half the activity of CoQ10. CoQ7, CoQ8, and CoQ9 had activity comparable with that of CoQ10. Hexahydro- and octahydrocoenzyme Q4 had activities comparable with that of CoQ4 and CoQ10 for succinoxidase, but about 10–15% that of CoQ10 for DPNH-oxidase. The double bond in the isoprenoid unit adjacent to the nucleus, and possible cyclization, are not necessary for activity. 2,3-Dimethoxy-5-methyl-6-heptadecyl-l,4-benzoquinone shows succinoxidase and DPNH-oxidase activities comparable with that of hexahydrocoeuzyme Q4. The 5-methyl group is not essential for succinoxidase activity, but has some significance for DPNH-oxidase. A tertiary hydroxyl group in the isoprenoid unit adjacent to the nucleus abolishes activity for succinoxidase but not completely for DPNH-oxidase. Rhodoquinone had no activity in the succinoxidase system and 25% that of CoQ10 in the DPNH-oxidase system. Representatives of biosynthetic precursors, 2-methoxy-5-methyl-6-phytyl-1,4-benzoquinone and 2-methoxy-3-hydroxy-5-methyl-6-phytyl-1,4-benzoquinone, had no activity in either the succinoxidase or DPNH-oxidase systems. Four compounds having a mechanistic relationship between CoQ and vitamin E had no activity. These data support the concept of two sites for the electron transfer of CoQ. The site for CoQ in succinoxidase is not very specific in the structural and steric requirement for the isoprenoid side chain, and has an electron potentiality that is not met by rhodoquinone. The site for CoQ in DPNH-oxidase has a rather specific requirement for a given length of side chain, and an electron potentiality that is significantly met by rhodoquinone.",
            "references": [
            "pap_id": "108",
            "title": "Inhibitors of NADH-ubiquinone reductase: an overview.",
            "authors": "Degli Esposti, M",
            "chapter": "",
            "pages": "222-235",
            "journal": "Biochimica et Biophysica Acta",
            "pub_date": "1998-05-06",
            "pub_year": 1998,
            "volume": "1364",
            "issue": "2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/s0005-2728(98)00029-2",
            "pmid": "9593904.0",
            "pmcid": "",
            "abstract": "This article provides an updated overview of the plethora of complex I inhibitors. The inhibitors are presented within the broad categories of natural and commercial compounds and their potency is related to that of rotenone, the classical inhibitor of complex I. Among commercial products, particular attention is dedicated to inhibitors of pharmacological or toxicological relevance. The compounds that inhibit the NADH-ubiquinone reductase activity of complex I are classified according to three fundamental types of action on the basis of available evidence and recent insights: type A are antagonists of the ubiquinone substrate, type B displace the ubisemiquinone intermediate, and type C are antagonists of the ubiquinol product. Copyright 1998 Elsevier Science B.V.",
            "references": [
            "pap_id": "109",
            "title": "New inhibitors of complex I of the mitochondrial electron transport chain with activity as pesticides.",
            "authors": "Hollingworth, R M; Ahammadsahib, K I; Gadelhak, G; McLaughlin, J L",
            "chapter": "",
            "pages": "230-233",
            "journal": "Biochemical Society Transactions",
            "pub_date": "1994-02-01",
            "pub_year": 1994,
            "volume": "22",
            "issue": "1",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1042/bst0220230",
            "pmid": "8206238.0",
            "pmcid": "",
            "abstract": "",
            "references": [
            "pap_id": "11",
            "title": "Generation of free radicals during the reductive metabolism of nilutamide by lung microsomes: possible role in the development of lung lesions in patients treated with this anti-androgen.",
            "authors": "Berger, V; Berson, A; Wolf, C; Chachaty, C; Fau, D; Fromenty, B; Pessayre, D",
            "chapter": "",
            "pages": "654-657",
            "journal": "Biochemical Pharmacology",
            "pub_date": "1992-02-04",
            "pub_year": 1992,
            "volume": "43",
            "issue": "3",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0006-2952(92)90593-8",
            "pmid": "1311586.0",
            "pmcid": "",
            "abstract": "The pulmonary metabolism of nilutamide, a nitroaromatic anti-androgen drug leading to pulmonary lesions in a few recipients, has been investigated in rats. Incubation of nilutamide (1 mM) with rat lung microsomes and NADPH under anaerobic conditions led to the formation of the nitro anion free radical, as indicated by ESR spectroscopy. The steady state concentration of this radical was not decreased by CO or SKF 525-A (two inhibitors of cytochrome P450), but was decreased by NADP+ (10 mM) or p-chloromercuribenzoate (0.47 mM) (two inhibitors of NADPH-cytochrome P450 reductase activity). Anaerobic incubations of [3H]nilutamide (0.1 mM) with rat lung microsomes and a NADPH-generating system resulted in the in vivo covalent binding of [3H]nilutamide metabolites to microsomal proteins; covalent binding required NADPH; it was decreased in the presence of NADP+ (10 mM), or in the presence of the nucleophile glutathione (10 mM), but was unchanged in the presence of carbon monoxide. Under aerobic conditions, in contrast, the nitro anion free radical was reoxidized by oxygen, and its ESR signal was not detected. Covalent binding was essentially suppressed. Instead, there was consumption of NADPH and oxygen, and production of superoxide anion and hydogen peroxide. We conclude that nilutamide is reduced by rat lung microsomes NADPH-cytochrome P450 reductase into a nitro anion free radical. In anaerobiosis, the radical is reduced further to covalent binding species. In the presence of oxygen, in contrast, this nitro anion free radical undergoes redox cycling, with the generation of reactive oxygen species.",
            "references": [
            "pap_id": "110",
            "title": "The pyrethroids permethrin and cyhalothrin are potent inhibitors of the mitochondrial complex I.",
            "authors": "Gassner, B; Wüthrich, A; Scholtysik, G; Solioz, M",
            "chapter": "",
            "pages": "855-860",
            "journal": "The Journal of Pharmacology and Experimental Therapeutics",
            "pub_date": "1997-05-01",
            "pub_year": 1997,
            "volume": "281",
            "issue": "2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "9152394.0",
            "pmcid": "",
            "abstract": "The synthetic pyrethroid derivatives permethrin and cyhalothrin are widely used insecticides that are considered to be relatively nontoxic to higher animals. However, a variety of toxic effects on mammals have been reported. We investigated the effect of these drugs on energy coupling by mitochondria and on the activity of the individual respiratory complexes. Using isolated rat liver mitochondria, a concentration-dependent inhibition of glutamate and succinate sustained state 3 respiration was found for both compounds in the micromolar range. The effect of pyrethroids on the activities of the complexes I to V were assessed individually in submitochondrial particles (complex I) and in freeze-thawed mitochondria (complexes II-V). Complex I (EC was found to be the most sensitive link within the electron transport chain. Half-maximal inhibition was observed at 0.73 microM permethrin and 0.57 microM cyhalothrin, respectively, and exhibited sigmoidal inhibition kinetics. Complexes II, III, IV and V (EC,,, were not significantly inhibited by up to 50 microM of these drugs. Thus, our results reveal a model of action of synthetic pyrethroid insecticides not previously reported.",
            "references": [
            "pap_id": "111",
            "title": "The 1,2,3-benzothiadiazoles. A new type of compound acting on coupling site I, in rat liver mitochondria.",
            "authors": "Gil, D L; Ferreira, J; Reynafarje, B",
            "chapter": "",
            "pages": "7-15",
            "journal": "Xenobiotica; the Fate of Foreign Compounds in Biological Systems",
            "pub_date": "1980-01-01",
            "pub_year": 1980,
            "volume": "10",
            "issue": "1",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.3109/00498258009033725",
            "pmid": "7385916.0",
            "pmcid": "",
            "abstract": "1. In rat liver mitochondria, 6-chloro-1,2,3-benzothiadiazole inhibited ADP phosphorylation and Ca2+-transport when the energy required for these processes came from the oxidation of NAD-linked substrates. The inhibition was characterized by substantial reduction in oxygen consumption, H+-movement and disappearance of acceptor control ratio. 2. When the substrate oxidized was succinate, depending on the 6-chloro-1,2,3-benzothiadiazole concn., little or no effect was observed on ADP phosphorylation and Ca2+-transport. 3. The results suggest that 6-chloro-1,2,3-benzothiadiazole can block site I at low concn., but at higher concn. can affect site I and site II, although site I is always more affected.",
            "references": [
            "pap_id": "112",
            "title": "Thienylimidazo[2,1-b]thiazoles as inhibitors of mitochondrial NADH dehydrogenase.",
            "authors": "Andreani, A; Rambaldi, M; Leoni, A; Locatelli, A; Ghelli, A; Ratta, M; Benelli, B; Degli Esposti, M",
            "chapter": "",
            "pages": "1090-1097",
            "journal": "Journal of Medicinal Chemistry",
            "pub_date": "1995-03-31",
            "pub_year": 1995,
            "volume": "38",
            "issue": "7",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1021/jm00007a006",
            "pmid": "7707312.0",
            "pmcid": "",
            "abstract": "The synthesis of 6-substituted 5-(thienylvinyl)imidazo[2,1-b]thiazoles and 6-thienylimidazo[2,1-b]thiazoles is reported. These compounds were tested as specific inhibitors of the NADH: ubiquinone (UBQ) reductase activity of NADH dehydrogenase in mitochondrial membranes. The 6-thienylimidazo[2,1-b]thiazoles were more potent in mammalian than in nematode mitochondria and had an average titer of 0.11 mM for 2-methyl-6-(2-thienyl)imidazo[2,1-b]thiazole (10). This compound is noncompetitive with the ubiquinone substrate and interacts with a site which is mutually exclusive with that of rotenone but nonexclusive with that of piericidin and several other inhibitors of NADH dehydrogenase. In the series of 5-(thienylvinyl)imidazothiazoles, the hydrobromide of (E)-6-chloro-5-(2-thienylvinyl)imidazo[2,1-b]thiazole (E-5.HBr) was found to be more potent as an inhibitor of the NADH:UBQ activity (IC50 = 15-17 microM) than the 6-thienylimidazoles such as 10. The inhibitory action of E-5.HBr and its analogs is different from that of compound 10 as indicated by the mutual exclusivity with other inhibitors and the relative inhibition of the activity with various electron acceptors.",
            "references": [
            "pap_id": "113",
            "title": "Pethidine analogues, a novel class of potent inhibitors of mitochondrial NADH: ubiquinone reductase.",
            "authors": "Filser, M; Werner, S",
            "chapter": "",
            "pages": "2551-2558",
            "journal": "Biochemical Pharmacology",
            "pub_date": "1988-07-01",
            "pub_year": 1988,
            "volume": "37",
            "issue": "13",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0006-2952(88)90245-6",
            "pmid": "3134033.0",
            "pmcid": "",
            "abstract": "Analogues of the analgetic drug pethidine were synthesized. Two N-aralkylen derivatives displayed a superior inhibitory effect on the activity of NADH:ubiquinone reductase in beef heart mitochondrial membranes. Dose-response curves revealed that the potency of these compounds is very comparable to that of the standard probe rotenone. The inhibitors were characterized by (a) their action on the reductase activity in various (eukaryotic and prokaryotic) organisms, (b) their influence on the enzyme kinetics, (c) their effects on the NADH dependent reduction of different electron acceptors, (d) their interference with the activities of other mitochondrial oxido-reductases. With regard to many of these aspects a close analogy between pethidine derivatives and rotenone was observed. A computer simulation of the steric structures of these molecules indicates that both classes of the chemically rather unrelated inhibitors may imitate very similar conformations. The potential advantages of the pethidine derivatives for the investigation of structure - function relationships within complex I of the respiratory chain is discussed.",
            "references": []
            "pap_id": "114",
            "title": "Inhibition of NADH-linked oxidation in brain mitochondria by 1-methyl-4-phenyl-pyridine, a metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine.",
            "authors": "Nicklas, W J; Vyas, I; Heikkila, R E",
            "chapter": "",
            "pages": "2503-2508",
            "journal": "Life Sciences",
            "pub_date": "1985-07-01",
            "pub_year": 1985,
            "volume": "36",
            "issue": "26",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0024-3205(85)90146-8",
            "pmid": "2861548.0",
            "pmcid": "",
            "abstract": "1-methyl-4-phenylpyridine (MPP+), a major metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) inhibited the ADP-stimulated and uncoupled oxidation of NADH-linked substrates by brain mitochondrial preparations. MPTP itself was ineffective. The apparent Ki's for MPP+ inhibition of pyruvate or glutamate oxidation by purified rat brain mitochondria were approximately 300 and 400 microM, respectively; with mouse brain mitochondria the values were lower, 60 and 150 microM, respectively. Succinate oxidation was unaffected by either compound. Compromise of mitochondrial oxidative capacity by MPP+ could be an important factor in mechanisms underlying the toxicity of MPTP.",
            "references": [
            "pap_id": "115",
            "title": "Characterization of the inhibitory mechanism of 1-methyl-4-phenylpyridinium and 4-phenylpyridine analogs in inner membrane preparations.",
            "authors": "Gluck, M R; Krueger, M J; Ramsay, R R; Sablin, S O; Singer, T P; Nicklas, W J",
            "chapter": "",
            "pages": "3167-3174",
            "journal": "The Journal of Biological Chemistry",
            "pub_date": "1994-02-04",
            "pub_year": 1994,
            "volume": "269",
            "issue": "5",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "8106350.0",
            "pmcid": "",
            "abstract": "We have investigated the mechanism of the inhibition of membrane-bound NADH dehydrogenase by 1-methyl-4-phenylpyridinium (MPP+) and a series of its 4'-alkyl-substituted analogs of increasing hydrophobicity, as well as their neutral, desmethyl congeners. Comparison of hydrophobicity, as measured by partition coefficients, with the IC50 for the inhibition of NADH oxidase activity in mitochondrial inner membrane preparations shows a negative correlation, but the cationic inhibitors are more effective than the neutral analogs with similar hydrophobicity. The presence of 10 microM tetraphenylboron (TPB-) potentiates the inhibitory power of positively charged analogs up to 4'-pentyl-MPP+, while the neutral inhibitors are unaffected by TPB-. Without TPB-, the more hydrophilic analogs give incomplete inhibition, but the inclusion of TPB- permits the attainment of complete inhibition, accompanied by the appearance of sigmoidal titration curves. These data support the hypothesis that MPP+ analogs, like rotenone, are bound at two sites on the enzyme and occupancy of both is required for complete inhibition. TPB-, by forming ion pairs with the cationic analogs, facilitates their equilibration to both sites in membrane preparations. When present in molar excess over the MPP+ analog, TPB- partially reverses the inhibition by decreasing its concentration in the more hydrophilic binding site. The effect of temperature and of pH on the IC50 values for inhibition support the concept of dual binding sites, and the pH dependence of the inhibition reveals the participation of two ionized protein groups in the binding, one of which may be a thiol group.",
            "references": [
            "pap_id": "116",
            "title": "Probing the ubiquinone reduction site of mitochondrial complex I using novel cationic inhibitors.",
            "authors": "Miyoshi, H; Inoue, M; Okamoto, S; Ohshima, M; Sakamoto, K; Iwamura, H",
            "chapter": "",
            "pages": "16176-16183",
            "journal": "The Journal of Biological Chemistry",
            "pub_date": "1997-06-27",
            "pub_year": 1997,
            "volume": "272",
            "issue": "26",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1074/jbc.272.26.16176",
            "pmid": "9195916.0",
            "pmcid": "",
            "abstract": "A wide variety of N-methylpyridinium and quinolinium cationic inhibitors of mitochondrial complex I was synthesized to develop potent and specific inhibitors acting selectively at one of the two proposed ubiquinone binding sites of this enzyme (Gluck, M. R., Krueger, M. J., Ramsay, R. R., Sablin, S. O., Singer, T. P., and Nicklas, W. J. (1994) J. Biol. Chem. 269, 3167-3174). N-Methyl-2-n-dodecyl-3-methylquinolinium (MQ18) inhibited electron transfer of complex I at under microM order regardless of whether exogenous or endogenous ubiquinone was used as an electron acceptor. The presence of tetraphenylboron (TPB-) potentiated the inhibition by MQ18 in a different way depending upon the molar ratio of TPB- to MQ18. In the presence of a catalytic amount of TPB-, the inhibitory potency of MQ18 was remarkably enhanced, and the extent of inhibition was almost complete. The presence of equimolar TPB- partially reactivated the enzyme activity, and the inhibition was saturated at an incomplete level (approximately 50%). These results are explained by the proposed dual binding sites model for ubiquinone (cited above). The inhibition behavior of MQ18 for proton pumping activity was similar to that for electron transfer activity. The good correlation of the inhibition behavior for the two activities indicates that both ubiquinone binding sites contribute to redox-driven proton pumping. On the other hand, N-methyl-4-[2-methyl-3-(p-tert-butylphenyl)]propylpyridinium (MP6) without TPB- brought about approximately 50% inhibition at 5 microM, but the inhibition reached a plateau at this level over a wide range of concentrations. Almost complete inhibition was readily obtained at low concentrations of MP6 in the presence of TPB-. Thus MP6 appears to be a selective inhibitor of one of the two ubiquinone binding sites. With a combined use of MP6 and 2,3-diethoxy-5-methyl-6-geranyl-1,4-benzoquinone, we also provided kinetic evidence for the existence of two ubiquinone binding sites.",
            "references": [
            "pap_id": "117",
            "title": "Mitochondrial respiratory inhibition by N-methylated beta-carboline derivatives structurally resembling N-methyl-4-phenylpyridine.",
            "authors": "Albores, R; Neafsey, E J; Drucker, G; Fields, J Z; Collins, M A",
            "chapter": "",
            "pages": "9368-9372",
            "journal": "Proceedings of the National Academy of Sciences of the United States of America",
            "pub_date": "1990-12-01",
            "pub_year": 1990,
            "volume": "87",
            "issue": "23",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1073/pnas.87.23.9368",
            "pmid": "2251279.0",
            "pmcid": "PMC55166",
            "abstract": "Mitochondrial accumulation and respiratory inhibition are critical steps in the actions of N-methyl-4-phenylpyridinium ion (MPP+), the toxic metabolite of the parkinsonism-inducing agent, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. We examined the respiratory characteristics of 2-methylated beta-carbolines (2-Me beta Cs) and 2-methylated 3,4-dihydro-beta-carbolines (2-MeDH beta Cs), which encompass the MPP+ structure. As indoleamine derivatives, they could have endogenous roles in idiopathic parkinsonism. With rat liver mitochondria, the order for inhibition of NAD(+)-linked O2 consumption (6-min preincubations) was as follows: MPP+ = 2-methylharmine greater than 2-methylharmol = 2-methylharmaline much greater than 2-methylharmalol greater than 2-methylnorharman greater than 6-OH-2-methylharmalan much greater than 2-methylharman. Similar to MPP+, 2-MeDH beta C/2-Me beta C inhibition was potentiated by tetraphenylboron and reversed by dinitrophenol, consistent with the involvement of cationic forms. However, the participation of neutral forms was indicated by the 2-MeDH beta C/2-Me beta C inhibitory time courses, which were unlike MPP+. The neutral forms probably arise via indolic nitrogen deprotonation because the characteristics of a cationic beta-carboline that cannot N-deprotonate, 2,9-dimethylnorharman, mirrored MPP+ rather than 2-Me beta Cs. Succinate-supported respiration was also significantly blocked by 2-MeDH beta Cs/2-Me beta Cs, but results with tetraphenylboron and 2,9-dimethylnorharman indicated that cationic forms were less important than in the inhibition of NAD(+)-linked respiration. We suggest that the relatively potent inhibition by certain 2-MeDH beta Cs/2-Me beta Cs involves neutral forms for passive mitochondrial entry and cationic as well as neutral forms that act at several respiratory sites. Respiratory inhibition could reasonably underlie the reported neurotoxicity of 2-Me beta Cs.",
            "references": [
            "pap_id": "118",
            "title": "Aminoethylcysteine ketimine decarboxylated dimer inhibits mitochondrial respiration by impairing electron transport at complex I level.",
            "authors": "Pecci, L; Montefoschi, G; Fontana, M; Cavallini, D",
            "chapter": "",
            "pages": "755-760",
            "journal": "Biochemical and Biophysical Research Communications",
            "pub_date": "1994-03-15",
            "pub_year": 1994,
            "volume": "199",
            "issue": "2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1006/bbrc.1994.1293",
            "pmid": "8135820.0",
            "pmcid": "",
            "abstract": "The product of the spontaneous dimerization and decarboxylation of aminoethylcysteine ketimine (simply named the dimer in this note) has been investigated for a possible biochemical activity. It has been found that the dimer inhibits the ADP-dependent oxidation of NAD(+)-linked substrates in rat liver mitochondria and electron transport from NADH to O2 in bovine heart submitochondrial particles (SMP). Oxidation of succinate by SMP is not impaired by concentrations of the dimer inhibiting almost totally NADH oxidation. Furthermore, the dimer did not affect the rotenone-insensitive electron transfer from NADH to menadione. These results give a preliminary indication suggesting that the dimer inhibits electron flow from NADH dehydrogenase to ubiquinone at or near the rotenone binding site(s). The dimer inhibition falls in the same range exhibited by some neurotoxins which are known to interact with the rotenone binding site.",
            "references": [
            "pap_id": "119",
            "title": "The contribution of mitochondrial respiratory complexes to the production of reactive oxygen species.",
            "authors": "McLennan, H R; Degli Esposti, M",
            "chapter": "",
            "pages": "153-162",
            "journal": "Journal of Bioenergetics and Biomembranes",
            "pub_date": "2000-04-01",
            "pub_year": 2000,
            "volume": "32",
            "issue": "2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1023/a:1005507913372",
            "pmid": "11768748.0",
            "pmcid": "",
            "abstract": "This work was focused on distinguishing the contribution of mitochondrial redox complexes to the production of reactive oxygen species (ROS) during cellular respiration. We were able to accurately measure, for the first time, the basal production of ROS under uncoupled conditions by using a very sensitive method, based on the fluorescent probe dichlorodihydrofluorescein diacetate. The method also enabled the detection of the ROS generated by the oxidation of the endogenous substrates in the mitochondrial preparations and could be applied to both mitochondria and live cells. Contrary to the commonly accepted view that complex III (ubiquinol:cytochrome c reductase) is the major contributor to mitochondrial ROS production, we found that complex I (NADH-ubiquinone reductase) and complex II (succinate-ubiquinone reductase) are the predominant generators of ROS during prolonged respiration under uncoupled conditions. Complex II, in particular, appears to contribute to the basal production of ROS in cells.",
            "references": [
            "pap_id": "12",
            "title": "The uncoupling effect of the nonsteroidal anti-inflammatory drug nimesulide in liver mitochondria from adjuvant-induced arthritic rats.",
            "authors": "Caparroz-Assef, S M; Salgueiro-Pagadigorria, C L; Bersani-Amado, C A; Bracht, A; Kelmer-Bracht, A M; Ishii-Iwamoto, E L",
            "chapter": "",
            "pages": "117-124",
            "journal": "Cell Biochemistry and Function",
            "pub_date": "2001-06-01",
            "pub_year": 2001,
            "volume": "19",
            "issue": "2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1002/cbf.904",
            "pmid": "11335936.0",
            "pmcid": "",
            "abstract": "The aim of the present study was to evaluate the changes caused by adjuvant-induced arthritis in liver mitochondria and to investigate the effects of the nonsteroidal anti-inflammatory drug nimesulide. The main alterations observed in liver mitochondria from arthritic rats were: higher rates of state IV and state III respiration with beta-hydroxybutyrate as substrate; reduced respiratory control ratio and impaired capacity for swelling dependent on beta-hydroxybutyrate oxidation. No alterations were found in the activities of NADH oxidase and ATPase. Nimesulide produced: (1) stimulation of state IV respiration; (2) decrease in the ADP/O ratio and in the respiratory control ratio; (3) stimulation of ATPase activity of intact mitochondria; (4) inhibition of swelling driven by the oxidation of beta-hydroxybutyrate; (5) induction of passive swelling due to NH(3)/NH(4)+ redistribution. The activity of NADH oxidase was insensitive to nimesulide. Mitochondria from arthritic rats showed higher sensitivity to nimesulide regarding respiratory activity. The results of this work allow us to conclude that adjuvant-induced arthritis leads to quantitative changes in some mitochondrial functions and in the sensitivity to nimesulide. Direct evidence that nimesulide acts as an uncoupler was also presented. Since nimesulide was active in liver mitochondria at therapeutic levels, the impairment of energy metabolism could lead to disturbances in the liver responses to inflammation, a fact that should be considered in therapeutic intervention. Copyright 2001 John Wiley & Sons, Ltd.",
            "references": [
            "pap_id": "120",
            "title": "Inhibition of bovine heart mitochondrial and Paracoccus denitrificans NADH----ubiquinone reductase by dequalinium chloride and three structurally related quinolinium compounds.",
            "authors": "Anderson, W M; Patheja, H S; Delinck, D L; Baldwin, W W; Smiley, S T; Chen, L B",
            "chapter": "",
            "pages": "673-685",
            "journal": "Biochemistry international",
            "pub_date": "1989-10-01",
            "pub_year": 1989,
            "volume": "19",
            "issue": "4",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "2515858.0",
            "pmcid": "",
            "abstract": "1. Dequalinium chloride (DECA) and three related quinolinium compounds inhibit bovine heart mitochondrial and Paracoccus denitrificans electron transport activity, with inhibition localized between NADH and ubiquinone in both electron transport chains. 2. Structure-activity studies reveal that two quinolinium rings and a long bridging group are necessary for significant inhibition of reduction of artificial electron acceptors and coenzyme Q, whereas only one quinolinium ring and a long hydrocarbon side chain are required for significant inhibition of NADH oxidase activity. 3. Inhibition of coenzyme Q reduction by DECA is not reversed by dialysis. 4. Studies comparing DECA inhibition of rotenone-sensitive with rotenone-insensitive preparations indicate that DECA acts by a different inhibitory mechanism than rotenone on mammalian mitochondrial and P. denitrificans NADH----ubiquinone reductase.",
            "references": [
            "pap_id": "121",
            "title": "Flunarizine and cinnarizine inhibit mitochondrial complexes I and II: possible implication for parkinsonism.",
            "authors": "Veitch, K; Hue, L",
            "chapter": "",
            "pages": "158-163",
            "journal": "Molecular Pharmacology",
            "pub_date": "1994-01-01",
            "pub_year": 1994,
            "volume": "45",
            "issue": "1",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "8302275.0",
            "pmcid": "",
            "abstract": "Cinnarizine and flunarizine are piperazine derivatives with calcium antagonist and anticonvulsant properties and are used widely in the treatment of vertigo and circulatory disorders. They have been implicated recently in the aggravation, or even the induction, of parkinsonism in elderly patients. Because the aetiology of parkinsonism has been suggested as having a mitochondrial component, we have investigated the effects of both compounds on mitochondrial respiration and on the activities of the individual respiratory chain complexes. In intact mitochondria from rat liver, both drugs inhibited respiration rates, with substrates entering at Complex I (glutamate/malate) and Complex II (succinate). These effects could be explained by potent inhibitions (Ki 3-10 microM) of both complexes. Complex I is inhibited at a site near the ubiquinone-binding site, which is not competitive with respect to ubiquinone, whereas the inhibition of Complex II is apparently caused by competition with ubiquinone. Furthermore, the inhibition of NADH oxidation by flunarizine in submitochondrial particles caused an NADH-dependent generation of superoxide. These inhibitory properties of both compounds could be significant factors in the aggravation or induction of parkinsonism in elderly patients, in whom mitochondrial function already may be impaired.",
            "references": [
            "pap_id": "122",
            "title": "The antianginal agent ranolazine is a weak inhibitor of the respiratory complex I, but with greater potency in broken or uncoupled than in coupled mitochondria.",
            "authors": "Wyatt, K M; Skene, C; Veitch, K; Hue, L; McCormack, J G",
            "chapter": "",
            "pages": "1599-1606",
            "journal": "Biochemical Pharmacology",
            "pub_date": "1995-11-09",
            "pub_year": 1995,
            "volume": "50",
            "issue": "10",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0006-2952(95)02042-x",
            "pmid": "7503762.0",
            "pmcid": "",
            "abstract": "Ranolazine (RS-43285) has shown antianginal effects in clinical trials and cardiac anti-ischaemic activity in several in vivo and in vitro animal models, but without affecting haemodynamics. Its mechanism is thought to mainly involve a switch in substrate utilisation from fatty acids to glucose to, thus, improve efficiency of O2 use; however, its precise molecular target(s) are unknown. In studies to investigate its action further, using isolated rat heart mitochondria, ranolazine was found to weakly inhibit (pIC50 values > 300 microM) respiration by coupled mitochondria provided with NAD(+)-linked substrates but not with succinate. With broken mitochondrial membranes or submitochondrial particles, ranolazine inhibited NADH but not succinate oxidation and with pIC50 values in the lower range of 3-50 microM. Studies with different electron acceptors and respiratory inhibitors indicated that it inhibits respiratory Complex I at a site between ferricyanide and menadione and ubiquinone-1 reduction (i.e. at a similar locus to rotenone). However, unlike rotenone, ranolazine was an uncompetitive inhibitor with respect to ubiquinone-1. Ranolazine inhibition of Complex I was reversible and occurred also with mitochondria from pig, guinea pig, and human heart, and rat liver. Further studies using rat heart mitochondria in different energisation states (i.e. broken, uncoupled, or coupled) showed a 50-100-fold shift to greater potency of ranolazine in the broken compared to the coupled; with the uncoupled it was about 2-fold less potent than the broken. These shifts in potency were not found with rotenone or amytal. Studies with radiolabelled ranolazine showed that it bound to mitochondrial membranes with greater affinity in the broken compared to the coupled or uncoupled conditions. Rotenone displaced radiolabelled ranolazine from its binding site. This property of ranolazine may play some role in its anti-ischaemic activity.",
            "references": []
            "pap_id": "123",
            "title": "Comparison of the structures of the quinone-binding sites in beef heart mitochondria.",
            "authors": "Tan, A K; Ramsay, R R; Singer, T P; Miyoshi, H",
            "chapter": "",
            "pages": "19328-19333",
            "journal": "The Journal of Biological Chemistry",
            "pub_date": "1993-09-15",
            "pub_year": 1993,
            "volume": "268",
            "issue": "26",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "8396133.0",
            "pmcid": "",
            "abstract": "The ubiquinone pool in mitochondrial membranes serves as an electron carrier between both NADH-coenzyme Q oxidoreductase (Complex I) and succinate-coenzyme Q oxidoreductase (Complex II) and ubiquinol-cytochrome-c oxidoreductase (Complex III). It has been reported (Saitoh, I., Miyoshi, H., Shimizu, R., and Iwamura, H. (1992) Eur. J. Biochem. 209, 73-79) that 2-alkyl-4,6-dinitrophenols compete with exogenous coenzyme Q (Q) to inhibit electron transport through cytochromes b and c1 in mammalian mitochondria as well as in photosystem II. We have probed the similarities and differences in the reaction sites of exogenous Q in all three segments of the respiratory chain using selected 2-alkyl-4,6-dinitrophenols. The inhibition of Q analog reduction by the dinitrophenol derivatives was competitive for Complex I and noncompetitive for Complex II. The inhibition of Complex III was competitive with the pentyl analog, but was uncompetitive with the decyl analog, which may be due to different interactions of the two quinol analogs with Complex III. The degree of inhibition by several of these compounds was comparable for Complexes I and III, but Complex II was inhibited to a much smaller extent. The inhibitory potency of these compounds for Complexes I and III was increased by branching and by lengthening the carbon chain at the 2-position equivalent to the isoprenoid side chain of ubiquinone. Hydrophobic substituents increased the inhibition of Complex II. Replacement of the phenolic OH group by a chlorine atom decreased the maximum inhibition of Complex III, but increased that of Complex I. These data suggest that the structures of the exogenous Q-binding sites in Complexes I and III may be similar, but not identical, and that they are different from that in Complex II.",
            "references": []
            "pap_id": "124",
            "title": "The acridones, new inhibitors of mitochondrial NADH: ubiquinone oxidoreductase (complex I)",
            "authors": "Oettmeier, Walter; Masson, Klaus; Soll, Michael",
            "chapter": "",
            "pages": "262-266",
            "journal": "Biochimica et Biophysica Acta (BBA) - Bioenergetics",
            "pub_date": "1992-03-01",
            "pub_year": 1992,
            "volume": "1099",
            "issue": "3",
            "Issn": "52728",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0005-2728(92)90036-2",
            "pmid": "",
            "pmcid": "",
            "abstract": "Acridones (9-azaanthracen-10-ones) were found to be powerful inhibitors of mitochondrial NADH: ubiquinone oxidoreductase. Then inhibitory activity was best if an alkyl or alkyloxy substituent resided in the 4-position. Biological activity reached a maximum at a chain length of 9–10 Å. Halogen substitution in position 7, but not in positions 6 and 7, further enhanced activity. 2 Alkylacridones were much less active. Inhibitory activity in a Quantitative Structure-Activity Relationship (QSAR) could be correlated to Verloop. STERIMOL parameters L and L2 (Verloop, A., Hoogenstraten, W. and Tipker, J. (1976) in Drug Design (Anenz, E.I. ed), Vol. 7, pp. 165-207, Academic Press, New York). The QSAR could be further improved by inclusion of the lipophilicity parameter π.",
            "references": []
            "pap_id": "125",
            "title": "Quinolones and their N-oxides as inhibitors of mitochondrial complexes I and III",
            "authors": "Reil, Ellen; Höfle, Gerhard; Draber, Wilfried; Oettmeier, Walter",
            "chapter": "",
            "pages": "291-298",
            "journal": "Biochimica et Biophysica Acta (BBA) - Bioenergetics",
            "pub_date": "1997-01-01",
            "pub_year": 1997,
            "volume": "1318",
            "issue": "1-2",
            "Issn": "52728",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/S0005-2728(96)00150-8",
            "pmid": "",
            "pmcid": "",
            "abstract": "4(1H)-quinolones (2-alkyl- (1), 2-alkyl-3-methyl- (2), 2-methyl-3-alkyl- (3), 1-hydroxy-2-methyl-3-alkyl- (4) and 1-hydroxy-2-alkyl- (5)) with n-alkyl side chains varying from C5 to C17 have been synthesized and tested for biological activity in mitochondrial complexes. Whereas all quinolones were efficient inhibitors of electron transport in the cytochrome b/c1-complex from either beef heart or Rhodospirillum rubrum, in complex I from beef heart quinolones 1 and 2 only were highly active. In a Quantitative Structure-Activity Relationship (QSAR) inhibitory activity in the cytochrome b/c1-complexes could be correlated to the physicochemical parameters lipophilicity π and/or to STERIMOL L. Maximal inhibitory potency was achieved at a carbon chain length of 12–14 Å. Oxidant-induced reduction of cytochrome b established that some quinolones are inhibitors of the Qp rather than the Qn site.",
            "references": []
            "pap_id": "126",
            "title": "Evidence for three separate electron flow pathways through complex I: an inhibitor study",
            "authors": "Marshall Anderson, W.; Trgovcich-Zacok, Diane",
            "chapter": "",
            "pages": "186-193",
            "journal": "Biochimica et Biophysica Acta (BBA) - Bioenergetics",
            "pub_date": "1995-06-01",
            "pub_year": 1995,
            "volume": "1230",
            "issue": "3",
            "Issn": "52728",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0005-2728(95)94411-Q",
            "pmid": "",
            "pmcid": "",
            "abstract": "The mammalian mitochondrial electron transport chain catalyzes the oxidation of NADH at pH 8.0 and pH 6.5, and the oxidation of NADPH at pH 6.5. The pH-dependencies of the rate of steady-state oxidation of NADPH and NADH by Complex I as well as by its flavoprotein fraction have been extensively studied by the laboratory of Hatefi. One model to explain these pH-dependent oxidations was proposed by Bakker and Albracht (Biochim. Biophys. Acta 850 (1986) 413–422 and 423–428, modified by Van Belzen and Albracht (Biochim. Biophys Acta 974 (1989) 311–320), which predicts that Complex I is a heterodimer with protomer B, containing FMN and FeS clusters 1–4 in stiochiometric amounts, catalyzing NADH oxidation at pH 8, and Protomer A, containing FMN and FeS clusters 2, 4, catalyzing NAD(P)H oxidation at pH 6.5. A pH-dependent transfer of electrons from protomer A FeS clusters 2, 4 to protomer B FeS clusters 2, 4 is an obligate step in the oxidation of NAD(P)H at low pH. Strict interpretation of this model allows for only three types of inhibitor: one which inhibits all three oxidase activities (type 1); one which inhibits NADH oxidase, pH 8.0 (type 4) and a third which inhibits NAD(P)H oxidase, pH 6.5 (type 5). Another possibility is that there are three separate pathways of oxidation of NAD(P)H, which would allow for a total of seven different types of inhibitor, e.g., the three types above plus type 2 inhibiting NADH oxidase pH 8.0 and pH 6.5; type 3 inhibiting NADH oxidase pH 8.0, and NADPH oxidase pH 6.5; type 6 inhibiting NADH oxidase pH 6.5; and type 7 inhibiting NADPH oxidase pH 6.5. Using a series of thirteen inhibitors of Complex I activity and the chemical modification reagent ethoxyformic anhydride (EFA), four different inhibitor types were found: seven inhibitors of type 1, four inhibitors of type 2, one inhibitor of type 3 and one inhibitor of type 4. Treatment of submitochondrial particles (SMP) with EFA abolished NADH-dependent reduction of coenzyme Q at both pH 8.0 and 6.5, while inhibiting NADPH-dependent reduction of coenzyme Q at pH 6.5 by only 30%. These results do not support the heterodimer model of Complex I electron transport of Bakker and Albracht, but do support three separate electron flow pathways through complex 1 from reduced pyridine nucleotides to coenzyme Q. A new model of electron flow through Complex I based on these finding is proposed.",
            "references": [
            "pap_id": "127",
            "title": "Measurement of the membrane potential generated by complex I in submitochondrial particles",
            "authors": "Ghelli, A.; Benelli, B.; Degli Esposti, M.",
            "chapter": "",
            "pages": "746-755",
            "journal": "Journal of Biochemistry",
            "pub_date": "1997-04-01",
            "pub_year": 1997,
            "volume": "121",
            "issue": "4",
            "Issn": "0021-924X",
            "Isbn": "",
            "url": "",
            "doi": "10.1093/oxfordjournals.jbchem.a021649",
            "pmid": "",
            "pmcid": "",
            "abstract": "",
            "references": [
            "pap_id": "128",
            "title": "Specific labelling of a constituent polypeptide of bovine heart mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone reductase by the inhibitor diphenyleneiodonium.",
            "authors": "Ragan, C I; Bloxham, D P",
            "chapter": "",
            "pages": "605-615",
            "journal": "The Biochemical Journal",
            "pub_date": "1977-06-01",
            "pub_year": 1977,
            "volume": "163",
            "issue": "3",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1042/bj1630605",
            "pmid": "18140.0",
            "pmcid": "PMC1164741",
            "abstract": "1. NADH-ubiquinone-1 and NADH-menadione reductase activities of Complex I were inhibited by diphenyleneiodonium (apparent Ki 23 and 30 nmol/mg of protein respectively). Reduction of K3Fe(CN)6 and juglone was relatively unaffected. 2. Iodoniumdiphenyl and derivatives were much less effective inhibitors. Compounds with similar ring structures to diphenyleneiodonium, in particular dibenzofuran, were inhibitors of NADH-ubiquinone-1 oxidoreductase. 3. Diphenylene[125I]iodonium specifically labelled a polypeptide of mol.wt. 23500. Maximum incorporation was 1 mol/mol of Complex-I flavin or 1 mol/mol of the 23500-mol.wt. polypeptide. 4. The label associated with this polypeptide was of limited stability, especially at lower pH. 5. Complete inhibition of ubiquinone reduction was achieved when 1 mol of inhibitor was incorporated/mol of Complex-I flavin, but the relationship between inhibition and labelling was not linear. 6. No evidence for covalent interaction between diphenyleneiodonium and the phospholipids of Complex I was obtained. 7. Rotenone increased the apparent affinity of diphenyleneiodonium for the 23500-mol.wt. polypeptide without affecting the maximum incorporation. 8. The 23500-mol.wt. polypeptide was not solubilized by chaotropic agents. Prior treatment of Complex I with chaotropic agents or sodium dodecyl sulphate prevented incorporation of diphenyleneiodonium into this polypeptide.",
            "references": [
            "pap_id": "129",
            "title": "Inhibition of NADH-ubiquinone reductase activity by N,N'-dicyclohexylcarbodiimide and correlation of this inhibition with the occurrence of energy-coupling site 1 in various organisms.",
            "authors": "Yagi, T",
            "chapter": "",
            "pages": "2822-2828",
            "journal": "Biochemistry",
            "pub_date": "1987-05-19",
            "pub_year": 1987,
            "volume": "26",
            "issue": "10",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1021/bi00384a025",
            "pmid": "3111526.0",
            "pmcid": "",
            "abstract": "The NADH-ubiquinone reductase activity of the respiratory chains of several organisms was inhibited by the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD). This inhibition correlated with the presence of an energy-transducing site in this segment of the respiratory chain. Where the NADH-quinone reductase segment involved an energy-coupling site (e.g., in bovine heart and rat liver mitochondria, and in Paracoccus denitrificans, Escherichia coli, and Thermus thermophilus HB-8 membranes), DCCD acted as an inhibitor of ubiquinone reduction by NADH. By contrast, where energy-coupling site 1 was absent (e.g., in Saccharomyces cerevisiae mitochondria and Bacillus subtilis membranes), there was no inhibition of NADH-ubiquinone reductase activity by DCCD. In the bovine and P. denitrificans systems, DCCD inhibition was pseudo first order with respect to incubation time, and reaction order with respect to inhibitor concentration was close to unity, indicating that inhibition resulted from the binding of one inhibitor molecule per active unit of NADH-ubiquinone reductase. In the bovine NADH-ubiquinone reductase complex (complex I), [14C]DCCD was preferentially incorporated into two subunits of molecular weight 49,000 and 29,000. The time course of labeling of the 29,000 molecular weight subunit with [14C]DCCD paralleled the time course of inhibition of NADH-ubiquinone reductase activity.",
            "references": [
            "pap_id": "13",
            "title": "Nitrofurantoin inhibition of mouse liver mitochondrial respiration involving NAD-linked substrates",
            "authors": "Lim, Lori O.; Bortell, Rita; Neims, Allen H.",
            "chapter": "",
            "pages": "493-499",
            "journal": "Toxicology and Applied Pharmacology",
            "pub_date": "1986-07-01",
            "pub_year": 1986,
            "volume": "84",
            "issue": "3",
            "Issn": "0041008X",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0041-008X(86)90254-1",
            "pmid": "",
            "pmcid": "",
            "abstract": "In our study, nitrofurantoin (NF) and nitrofurazone (NZ) inhibited respiration of isolated mouse (C57B/6J, adult, male) liver mitochondria. Other aromatic nitro compounds, nitroimidazole, metronidazole, and p-nitrobenzoic acid, did not have any significant effect. The primary site of activity for NF was complex I NADH-ubiquinone oxidoreductase mediated respiration, since only complex I substrates, glutamate, β-hydroxybutyrate, and α-ketoglutarate-mediated respiration were decreased. Respiration supported by succinate, a complex II substrate, was not affected by any of the compounds. NF at a concentration of 50 μm decreased state 3 and dinitrophenol-uncoupled respiration to 28 ± 1 and 25 ± 5% of control, respectively, of mitochondria oxidizing glutamate. Studies with mitoplasts oxidizing glutamate showed that NF inhibited both state 3 and 4 respiration. The inhibition of state 3 was prevented by the simultaneous addition of superoxide dismutase (240 μg/ml) and catalase (200 μg/ml). These results suggest that the mitochondrion, in particular complex I of the electron transport system, is a target for NF toxicity. The effect on respiration may be mediated by NF redox cycling and the generation of reactive oxygen intermediates resulting in the interference of electron flow.",
            "references": [
            "pap_id": "130",
            "title": "Studies on the proton-translocating NADH:ubiquinone oxidoreductases of mitochondria and Escherichia coli using the inhibitor 1,10-phenanthroline.",
            "authors": "Finel, M; Majander, A",
            "chapter": "",
            "pages": "142-146",
            "journal": "FEBS Letters",
            "pub_date": "1994-02-14",
            "pub_year": 1994,
            "volume": "339",
            "issue": "1-2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0014-5793(94)80402-8",
            "pmid": "8313963.0",
            "pmcid": "",
            "abstract": "Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is uncompetitively inhibited by 1,10-phenanthroline (OP). EPR spectroscopy of submitochondrial particles indicates that OP, similarly to rotenone, inhibits electron transfer between the Fe-S clusters of complex I and the ubiquinone pool. The proton-translocating NADH dehydrogenase (NDH1) of E. coli is more sensitive to OP than is NDH1 of Paracoccus. EPR spectroscopy of membranous E. coli NDH1 shows that two slow- and one fast-relaxing Fe-S clusters become detectable upon reduction by NADH in the presence of OP. However, none of them resembles the mitochondrial cluster 2.",
            "references": [
            "pap_id": "131",
            "title": "Effects of inhibitors on mitochondrial adenosine triphosphatase of Crithidia fasciculata: an unusual pattern of specificities.",
            "authors": "Yarlett, N; Lloyd, D",
            "chapter": "",
            "pages": "13-17",
            "journal": "Molecular and Biochemical Parasitology",
            "pub_date": "1981-05-01",
            "pub_year": 1981,
            "volume": "3",
            "issue": "1",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0166-6851(81)90073-6",
            "pmid": "6454844.0",
            "pmcid": "",
            "abstract": "The mitochondrial adenosine triphosphatase of the kinetoplastid protozoon, Crithidia fasciculata, is inhibited by oligomycin, venturicidin, triethyltin sulphate, N,N'-dicyclohexylcarbodiimide, leucinostatin, Dio-9 and quercetin, but not spegazzinine or by compounds which interact with the beta-subunit of mitochondrial F1-ATPase (efrapeptin, aurovertin, citreoviridin or 4-chloro-7-nitrobenzofurazan). These results suggest that the F1 portion of the crithidial enzyme has an unusual type of beta-subunit. Further evidence for the atypical nature of this enzyme is provided by the observation that F1-inhibitor proteins from Acanthamoeba castellanii or bovine heart mitochondria do not inhibit the C. fasciculata enzyme activity.",
            "references": [
            "pap_id": "132",
            "title": "Inhibition of spinach chloroplasts photophosphorylation by the antibiotics leucinostatin and efrapeptin.",
            "authors": "Lucero, H A; Ravizzine, R A; Vallejos, R H",
            "chapter": "",
            "pages": "141-144",
            "journal": "FEBS Letters",
            "pub_date": "1976-09-15",
            "pub_year": 1976,
            "volume": "68",
            "issue": "1",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0014-5793(76)80423-1",
            "pmid": "9316.0",
            "pmcid": "",
            "abstract": "",
            "references": [
            "pap_id": "133",
            "title": "Uncoupling and specific inhibition of phosphoryl transfer reactions in mitochondria by antibiotic A20668.",
            "authors": "Reed, P W; Lardy, H A",
            "chapter": "",
            "pages": "3704-3708",
            "journal": "The Journal of Biological Chemistry",
            "pub_date": "1975-05-25",
            "pub_year": 1975,
            "volume": "250",
            "issue": "10",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "165181.0",
            "pmcid": "",
            "abstract": "A20668 A, B, and C are polypeptide antibiotics that inhibit phosphorylation of ADP, Mg2t-ATPase, and the ATP-driven transhydrogenase of rat liver submitochondrial particles, but not the purified F1 ATPase. In intact mitochondria, 120668 inhibits uncoupler-induced ATPase, State 3 respiration, and phosphorylation; the A and B forms are approximately equipotent with rutamycin, whereas A20668 C is less effective. Concentrations of A20668 slightly greater than required for complete inhibition of phosphoryl transfer stimulate rapid, uncoupled respiration by mitochondria under State 3 of 4 conditions. A20668 A and B are more effective uncouplers than A20668 C. In the presence of venturicidin or ossamycin, concentrations of A20668, which alone do not uncouple, stimulate oxygen consumption of mitochondria incubated under either State 3 of 4 conditions. A20668 uncoupling is not potentiated by prior inhibition of phosphoryl transfer by venturicidin X, rutamycin, aurovertin, or efrapeptin. A20668 increases mitochondrial permeability to protons in passive swelling experiments where facilitation of proton conductance correlates well with potency to uncouple. A20668 apparently binds initially at a unique locus to inhibit mitochondrial phosphoryl transfer reactions. When this site is saturated, additional antibiotic may uncouple by increasing proton conductance of mitochondria. Binding of venturicidin or ossamycin appears to interfere with the binding of A20668 to its adjacent inhibitory site, thus effectively increasing the concentration of A20668 available to uncouple.",
            "references": [
            "pap_id": "134",
            "title": "The mode of inhibition of oxidative phosphorylation by efrapeptin (A23871). Evidence for an alternating site mechanism for ATP synthesis.",
            "authors": "Cross, R L; Kohlbrenner, W E",
            "chapter": "",
            "pages": "4865-4873",
            "journal": "The Journal of Biological Chemistry",
            "pub_date": "1978-07-25",
            "pub_year": 1978,
            "volume": "253",
            "issue": "14",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "149791.0",
            "pmcid": "",
            "abstract": "Results are presented that confirm and extend earlier findings that efrapeptin is a potent inhibitor of oxidative phosphorylation. Binding of efrapeptin is shown to be reversible, and a dissociation constant for the enzyme-inhibitor complex is estimated to be 10(-8) M under conditions for either ATP synthesis or hydrolysis. Fifty per cent inhibition of the ATP hydrolysis activity of submitochondrial particles is obtained at a ratio of 0.56 mol of inhibitor/mol of enzyme. Studies of efrapeptin binding under pseudo-first order conditions show that the onset of inhibition is first order with respect to efrapeptin. Combined with the inhibition titer, these results indicate that there is one inhibitor binding site per molecule of enzyme. Steady state velocity studies using a substrate regenerating system show that efrapeptin is competitive with both ADP and phosphate during ATP synthesis. However, during ATP hydrolysis, a distinctly different mode of inhibition is indicated with respect to ATP. Data are presented which suggest that ATP promotes the binding of efrapeptin to the enzyme. Indications that efrapeptin is a catalytic site inhibitor make these results difficult to reconcile with a simple mechanistic scheme involving a single independnet catalytic site for ATP synthesis and hydrolysis. Our results are discussed in terms of support for catalytic cooperativity between adjacent subunits as recently proposed by Kayalar et al. (Kayalar, C., Rosing, J., and Boyer, P. D. (1977) J. Biol. Chem. 252, 2486-2491).",
            "references": [
            "pap_id": "135",
            "title": "Mg2+-activated adenosine triphosphatase from Crithidia fasciculata: purification and inhibition by suramin and efrapeptin.",
            "authors": "Higa, A I; Cazzulo, J J",
            "chapter": "",
            "pages": "357-367",
            "journal": "Molecular and Biochemical Parasitology",
            "pub_date": "1981-10-01",
            "pub_year": 1981,
            "volume": "3",
            "issue": "6",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0166-6851(81)90036-0",
            "pmid": "6117795.0",
            "pmcid": "",
            "abstract": "The mitochondrial Mg2+-activated adenosine triphosphatase (ATPase; EC from the insect flagellate Crithidia fasciculata ATCC 11745 has been extracted from the membrane by chloroform treatment and purified to electrophoretic homogeneity by a method involving ammonium sulphate fractionation, gel filtration on Sephadex G-200 and DEAE-cellulose chromatography. The molecular weight of the native enzyme, determined by gel filtration, was about 350 000. Five subunits were detected by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, with molecular weights of 54 000, 45 000, 35 000, 20 000 and 10 000. The membrane-bound, but not the soluble (F1) ATPase was inhibited by oligomycin and leucinostatin. Both forms of the enzyme were strongly inhibited by the antibiotic efrapeptin and the trypanocidal drug suramin. The inhibition by efrapeptin was of the mixed type, with double-reciprocal plots intersecting below the abscissa, as in the case of the enzyme present in beef heart submitochondrial particles. Suramin, on the other hand, acted as a non-competitive inhibitor of the membrane-bound ATPase and as a strictly competitive inhibitor of the purified F1 ATPase.",
            "references": [
            "pap_id": "136",
            "title": "Possible role of cell surface H+ -ATP synthase in the extracellular ATP synthesis and proliferation of human umbilical vein endothelial cells.",
            "authors": "Arakaki, Naokatu; Nagao, Tomoko; Niki, Rie; Toyofuku, Ayako; Tanaka, Hiroaki; Kuramoto, Yoshinori; Emoto, Yuka; Shibata, Hirofumi; Magota, Koji; Higuti, Tomihiko",
            "chapter": "",
            "pages": "931-939",
            "journal": "Molecular Cancer Research",
            "pub_date": "2003-11-01",
            "pub_year": 2003,
            "volume": "1",
            "issue": "13",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "14638865.0",
            "pmcid": "",
            "abstract": "Extracellular ATP synthesis on human umbilical vein endothelial cells (HUVECs) was examined, and it was found that HUVECs possess high ATP synthesis activity on the cell surface. Extracellular ATP generation was detected within 5 s after addition of ADP and inorganic phosphate and reached a maximal level at 15 s. This type of ATP synthesis was almost completely inhibited by mitochondrial H(+)-ATP synthase inhibitors (e.g., efrapeptins, resveratrol, and piceatannol), which target the F(1) catalytic domain. Oligomycin and carbonyl cyanide m-chlorophenylhydrazone, but not potassium cyanide, also inhibited extracellular ATP synthesis on HUVECs, suggesting that cell surface ATP synthase employs the transmembrane electrochemical potential difference of protons to synthesize ATP as well as mitochondrial H(+)-ATP synthase. The F(1)-targeting H(+)-ATP synthase inhibitors markedly inhibited the proliferation of HUVECs, but intracellular ATP levels in HUVECs treated with these inhibitors were only slightly affected, as shown by comparison with the control cells. Interestingly, piceatannol inhibited only partially the activation of Syk (a nonreceptor tyrosine kinase), which has been shown to play a role in a number of endothelial cell functions, including cell growth and migration. These findings suggest that H(+)-ATP synthase-like molecules on the surface of HUVECs play an important role not only in extracellular ATP synthesis but also in the proliferation of HUVECs. The present results demonstrate that the use of small molecular H(+)-ATP synthase inhibitors targeting the F(1) catalytic domain may lead to significant advances in potential antiangiogenic cancer therapies.",
            "references": [
            "pap_id": "137",
            "title": "Inhibition of energy conservation reactions in chromatophores of Rhodospirillum rubrum by antibiotics.",
            "authors": "Lucero, H; Lescano, W I; Vallejos, R H",
            "chapter": "",
            "pages": "9-14",
            "journal": "Archives of Biochemistry and Biophysics",
            "pub_date": "1978-02-01",
            "pub_year": 1978,
            "volume": "186",
            "issue": "1",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0003-9861(78)90457-5",
            "pmid": "147053.0",
            "pmcid": "",
            "abstract": "",
            "references": [
            "pap_id": "138",
            "title": "Effects of inhibitors on mitochondrial adenosine triphosphatase of Tetrahymena pyriformis ST.",
            "authors": "Unitt, M D; Lloyd, D",
            "chapter": "",
            "pages": "261-266",
            "journal": "Journal of general microbiology",
            "pub_date": "1981-10-01",
            "pub_year": 1981,
            "volume": "126",
            "issue": "2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1099/00221287-126-2-261",
            "pmid": "6461727.0",
            "pmcid": "",
            "abstract": "Mitochondrial adenosine triphosphatase (ATPase) of the ciliate protozoon Tetrahymena pyriformis ST is completely inhibited by antiserum prepared against F1-ATPase purified from Schizosaccharomyces pombe, and by naturally occurring inhibitor proteins from this yeast and from bovine heart mitochondria. An ATPase inhibitor protein is also present in extracts of T. pyriformis. Mitochondrial ATPase of T. pyriformis is only partially inhibited by the F0-ATPase inhibitors N,N'-dicyclohexylcarbodiimide, oligomycin, leucinostatin, triethyltin sulphate and venturicidin, and (at high titres) by the F1-ATPase inhibitors Dio-9, efrapeptin, 4-chloro-7-nitrobenzofurazan and spegazzinine. Aurovertin, citreoviridin and quercetin were not inhibitory. Resistance to inhibitors distinguishes this mitochondrial ATPase from all those previously examined.",
            "references": [
            "pap_id": "139",
            "title": "Properties of F1-ATPase from the uncD412 mutant of Escherichia coli.",
            "authors": "Wise, J G; Duncan, T M; Latchney, L R; Cox, D N; Senior, A E",
            "chapter": "",
            "pages": "343-350",
            "journal": "The Biochemical Journal",
            "pub_date": "1983-11-01",
            "pub_year": 1983,
            "volume": "215",
            "issue": "2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1042/bj2150343",
            "pmid": "6228224.0",
            "pmcid": "PMC1152402",
            "abstract": "Properties of purified F1-ATPase from Escherichia coli mutant strain AN484 (uncD412) have been studied in an attempt to understand why the amino acid substitution in the beta-subunit of this enzyme causes a tenfold reduction from normal MgATP hydrolysis rate. In most properties that were studied, uncD412 F1-ATPase resembled normal E. coli F1-ATPase. Both enzymes were found to contain a total of six adenine-nucleotide-binding sites, of which three were found to be non-exchangeable and three were exchangeable (catalytic) sites. Binding of the non-hydrolysable substrate analogue adenosine 5'-[beta gamma-imido]triphosphate (p[NH]ppA) to the three exchangeable sites showed apparent negative co-operativity. The binding affinities for p[NH]ppA, and also ADP, at the exchangeable sites were similar in the two enzymes. Both enzymes were inhibited by efrapeptin, aurovertin and p[NH]ppA, and were inactivated by dicyclohexylcarbodi-imide, 4-chloro-7-nitrobenzofurazan and p-fluorosulphonyl-benzoyl-5'-adenosine. Km values for CaATP and MgATP were similar in the two enzymes. uncD412 F1-ATPase was abnormally unstable at high pH, and dissociated into subunits readily with consequent loss of activity. The reason for the impairment of catalysis in uncD412 F1-ATPase cannot be stated with certainty from these studies. However we discuss the possibility that the mutation interrupts subunit interaction, thereby causing a partial impairment in the site-site co-operativity which is required for 'promotion' of catalysis in this enzyme.",
            "references": [
            "pap_id": "14",
            "title": "Complex I is the major site of mitochondrial superoxide production by paraquat.",
            "authors": "Cochemé, Helena M; Murphy, Michael P",
            "chapter": "",
            "pages": "1786-1798",
            "journal": "The Journal of Biological Chemistry",
            "pub_date": "2008-01-25",
            "pub_year": 2008,
            "volume": "283",
            "issue": "4",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1074/jbc.M708597200",
            "pmid": "18039652.0",
            "pmcid": "",
            "abstract": "Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride) is widely used as a redox cycler to stimulate superoxide production in organisms, cells, and mitochondria. This superoxide production causes extensive mitochondrial oxidative damage, however, there is considerable uncertainty over the mitochondrial sites of paraquat reduction and superoxide formation. Here we show that in yeast and mammalian mitochondria, superoxide production by paraquat occurs in the mitochondrial matrix, as inferred from manganese superoxide dismutase-sensitive mitochondrial DNA damage, as well as from superoxide assays in isolated mitochondria, which were unaffected by exogenous superoxide dismutase. This paraquat-induced superoxide production in the mitochondrial matrix required a membrane potential that was essential for paraquat uptake into mitochondria. This uptake was of the paraquat dication, not the radical monocation, and was carrier-mediated. Experiments with disrupted mitochondria showed that once in the matrix paraquat was principally reduced by complex I (mammals) or by NADPH dehydrogenases (yeast) to form the paraquat radical cation that then reacted with oxygen to form superoxide. Together this membrane potential-dependent uptake across the mitochondrial inner membrane and the subsequent rapid reduction to the paraquat radical cation explain the toxicity of paraquat to mitochondria.",
            "references": [
            "pap_id": "140",
            "title": "Inhibition of mitochondrial proton F0F1-ATPase/ATP synthase by polyphenolic phytochemicals.",
            "authors": "Zheng, J; Ramirez, V D",
            "chapter": "",
            "pages": "1115-1123",
            "journal": "British Journal of Pharmacology",
            "pub_date": "2000-07-01",
            "pub_year": 2000,
            "volume": "130",
            "issue": "5",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1038/sj.bjp.0703397",
            "pmid": "10882397.0",
            "pmcid": "PMC1572158",
            "abstract": "Mitochondrial proton F0F1-ATPase/ATP synthase synthesizes ATP during oxidative phosphorylation. In this study, we examined the effects of several groups of polyphenolic phytochemicals on the activity of the enzyme. Resveratrol, a stilbene phytoalexin that is present in grapes and red wine, concentration-dependently inhibited the enzymatic activity of both rat brain and liver F0F1-ATPase/ATP synthase (IC(50) of 12 - 28 microM). Screening of other polyphenolic phytochemicals using rat brain F0F1-ATPase activity resulted in the following ranking potency (IC(50) in parenthesis): piceatannol (8 microM)>resveratrol (19 microM)=(-)epigallocatechin gallate (17 microM)>(-)epicatechin gallate, curcumin (45 microM)>genistein=biochanin A=quercetin=kaempferol=morin (55 - 65 microM)>phloretin=apigenin=daidzein (approx. 100 microM). Genistin, quercitrin, phloridzin, (+)catechin, (+)epicatechin, (-)epicatechin and (-)epigallocatechin had little effect at similar concentrations. Tannic acid, theaflavins (tea extract) and grape seed proanthocyanidin extract (GSPE) had IC(50) values of 5, 20 and 30 microg ml(-1), respectively. Several monophenolic antioxidants and non-phenolic compounds were ineffective at concentrations of 210 microM or higher. The inhibition of F0F1-ATPase by resveratrol and genistein was non-competitive in nature. The effects of polyphenolic phytochemicals were additive. Both resveratrol and genistein had little effect on the Na(+)/K(+)-ATPase activity of porcine cerebral cortex, whereas quercetin had similar inhibitory potency as for F0F1-ATPase. In conclusion, the ATP synthase is a target for dietary phytochemicals. This pharmacological property of these phytochemicals should be included in the examination of their health benefits as well as potential cytotoxicity.",
            "references": [
            "pap_id": "141",
            "title": "Piceatannol, a stilbene phytochemical, inhibits mitochondrial F0F1-ATPase activity by targeting the F1 complex.",
            "authors": "Zheng, J; Ramirez, V D",
            "chapter": "",
            "pages": "499-503",
            "journal": "Biochemical and Biophysical Research Communications",
            "pub_date": "1999-08-02",
            "pub_year": 1999,
            "volume": "261",
            "issue": "2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1006/bbrc.1999.1063",
            "pmid": "10425214.0",
            "pmcid": "",
            "abstract": "Piceatannol is a stilbene phytochemical from the seeds of Euphorbia lagascae, previously identified as an antileukemic principle. Piceatannol is considered an inhibitor of several tyrosine kinases. We recently reported that resveratrol, another stilbene phytoalexin from grape seeds, was an inhibitor of ATP synthase. Here, we demonstrated that piceatannol potently inhibited the rat brain mitochondrial F0F1-ATPase activity in both solubilized and submitochondrial preparations (IC50 of 8-9 microM), while having relatively small effect on the Na(+), K(+)-ATPase activity of porcine cerebral cortex (no effect up to 7 microM). Piceatannol inhibited the ATPase activity of the purified rat liver F1 with IC50 of about 4 microM, while resveratrol was slightly less active (IC50 of about 14 microM). Our results indicate that piceatannol and resveratrol inhibit the F-type ATPase by targeting the F1 sector, which is located to the inner membrane of mitochondria and plasma membrane of normal endothelial cells and several cancer cell lines. This mechanism could potentially contribute to the multiple effects of these chemopreventive phytochemicals. Copyright 1999 Academic Press.",
            "references": [
            "pap_id": "142",
            "title": "Inhibition sites in F1-ATPase from bovine heart mitochondria.",
            "authors": "Gledhill, Jonathan R; Walker, John E",
            "chapter": "",
            "pages": "591-598",
            "journal": "The Biochemical Journal",
            "pub_date": "2005-03-15",
            "pub_year": 2005,
            "volume": "386",
            "issue": "Pt 3",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1042/BJ20041513",
            "pmid": "15537385.0",
            "pmcid": "PMC1134879",
            "abstract": "High-resolution crystallographic studies of a number of inhibited forms of bovine F1-ATPase have identified four independent types of inhibitory site: the catalytic site, the aurovertin B-binding site, the efrapeptin-binding site and the site to which the natural inhibitor protein IF1 binds. Hitherto, the binding sites for other inhibitors, such as polyphenolic phytochemicals, non-peptidyl lipophilic cations and amphiphilic peptides, have remained undefined. By employing multiple inhibition analysis, we have identified the binding sites for these compounds. Several of them bind to the known inhibitory sites. The amphiphilic peptides melittin and synthetic analogues of the mitochondrial import pre-sequence of yeast cytochrome oxidase subunit IV appear to mimic the natural inhibitor protein, and the polyphenolic phytochemical inhibitors resveratrol and piceatannol compete for the aurovertin B-binding site (or sites). The non-peptidyl lipophilic cation rhodamine 6G acts at a separate unidentified site, indicating that there are at least five inhibitory sites in the F1-ATPase. Each of the above inhibitors has significantly different activity against the bacterial Bacillus PS3 alpha3beta3gamma subcomplex compared with that observed with bovine F1-ATPase. IF1 does not inhibit the bacterial enzyme, even in the absence of the epsilon-subunit. An understanding of these inhibitors may enable rational development of therapeutic agents to act as novel antibiotics against bacterial ATP synthases or for the treatment of several disorders linked to the regulation of the ATP synthase, including ischaemia-reperfusion injury and some cancers.",
            "references": [
            "pap_id": "143",
            "title": "Diethylstilbestrol. A novel F0-directed probe of the mitochondrial proton ATPase.",
            "authors": "McEnery, M W; Pedersen, P L",
            "chapter": "",
            "pages": "1745-1752",
            "journal": "The Journal of Biological Chemistry",
            "pub_date": "1986-02-05",
            "pub_year": 1986,
            "volume": "261",
            "issue": "4",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "2868005.0",
            "pmcid": "",
            "abstract": "At low concentrations, diethylstilbestrol (DES) is shown to be a potent F0-directed inhibitor of the F0F1-ATPase of rat liver mitochondria. In analogy to other F0-directed inhibitors, DES inhibits both the ATPase and ATP-dependent proton-translocation activities of the purified and membrane bound enzyme. When added at low concentrations with dicyclohexylcarbodiimide (DCCD), a covalent inhibitor, DES acts synergistically to inhibit ATPase activity of the complex. At higher concentrations, DES restores DCCD-inhibited ATPase activity. However, there is no restoration of ATP-dependent proton translocation. Under these conditions DCCD remains covalently bound to the F0F1-ATPase complex and F1 remains bound to Fo. Significantly, when the F0F1-ATPase is inhibited by the Fo-directed inhibitor venturicidin rather than DCCD, DES is also able to restore ATPase activity. In contrast, DES is unable to restore ATPase activity to F0F1 preparations inhibited by the Fo-directed inhibitors oligomycin or tricyclohexyltin. However, combinations of [DES + DCCD] or [DES + venturicidin] can restore ATPase activity to F0F1 preparations inhibited by either oligomycin or tricyclohexyltin. Results presented here indicate that the F0 moiety of the rat liver mitochondrial proton ATPase contains a distinct binding site for DES. In addition, they suggest that at saturating concentrations simultaneous occupancy of the DES binding site and sites for either DCCD or venturicidin promote \"uncoupled\" ATP hydrolysis.",
            "references": [
            "pap_id": "144",
            "title": "F0F1-ATPase of Vibrio parahaemolyticus: purification using new detergents and characterization.",
            "authors": "Ogawa, W; Izawa, S; Sakai-Tomita, Y; Moritani, C; Tsuda, M; Kinomura, K; Kitazawa, S; Tsuchiya, T",
            "chapter": "",
            "pages": "69-74",
            "journal": "Biochimica et Biophysica Acta",
            "pub_date": "1994-11-01",
            "pub_year": 1994,
            "volume": "1188",
            "issue": "1-2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0005-2728(94)90023-x",
            "pmid": "7947906.0",
            "pmcid": "",
            "abstract": "Previous attempts to isolate a stable F0F1-ATPase complex (H(+)-translocating ATPase) from Vibrio parahaemolyticus have been unsuccessful. Using new non-ionic detergents (alkyl thiomaltosides), a stable F0F1 complex with a high specific activity (15-25 units/mg protein) was purified and characterized. The purified F0F1-ATPase consists of eight subunits (alpha, beta, gamma, delta, epsilon, a, b and c). The new detergents, in combination with sucrose (or glycerol), lipid, dithiothreitol and phenylmethylsulfonyl fluoride, effectively stabilized the F0F1 complex. The ATPase activity of the F0F1 complex was greatly increased by anions, such as SO4(2-) and SO3(2-). Sodium ion increased the activity by about 2-fold. Dicyclohexylcarbodiimide, Zn2+, 4-acetamido-4'-isothiocyanostilben-2,2'disulfonate and tetrachlorosalicylanilide inhibited F0F1-ATPase activity. Ethanol, which stimulated F1-ATPase activity, inhibited F0F1-ATPase activity. Methanol, Na3VO4 and bafilomycin A1 did not have any significant effect on F0F1-ATPase activity, although methanol, like ethanol, stimulated F1-ATPase activity.",
            "references": [
            "pap_id": "145",
            "title": "Rapid purification and characterization of F1-ATPase of Vibrio parahaemolyticus.",
            "authors": "Sakai, Y; Kanazawa, H; Tsuda, M; Tsuchiya, T",
            "chapter": "",
            "pages": "18-22",
            "journal": "Biochimica et Biophysica Acta",
            "pub_date": "1990-07-17",
            "pub_year": 1990,
            "volume": "1018",
            "issue": "1",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0005-2728(90)90104-c",
            "pmid": "2142893.0",
            "pmcid": "",
            "abstract": "The F1 portion of H(+)-translocating ATPase as purified from membrane vesicles of Vibrio parahaemolyticus by a rapid procedure. The whole purification process (from culture of cells to purification of the enzyme) could be completed in 1 day. The F1-ATPase consists of five subunits (alpha, beta, gamma, delta and epsilon) like F1 of Escherichia coli and other microorganisms. The F1-ATPase of V. parahaemolyticus showed some interesting properties. Its activity was greatly stimulated by high concentrations (about 0.5 M) of SO4(2-), SO3(2-) and CH3COO-, their effects decreasing in this order. Among the anions tested, Cl- and NO3- were ineffective, or rather inhibitory, and cations had no significant effects. Ethanol (or methanol) stimulated the activity 2- to 3-fold. The activity was inhibited by 4-acetamido-4'-isothiocyanostilbene 2,2'-disulfonate (SITS) (an anion exchanger inhibitor), tetrachlorosalicylanilide (TCS) (an H+ conductor), azide and N-ethylmaleimide. Zinc inhibited the activity only slightly, although it strongly inhibited the ATPase activity in membrane vesicles.",
            "references": [
            "pap_id": "146",
            "title": "Inhibition of succinic dehydrogenase and F0F1-ATP synthase by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS).",
            "authors": "Bernardes, C F; Meyer-Fernandes, J R; Martins, O B; Vercesi, A E",
            "chapter": "",
            "pages": "799-806",
            "journal": "Zeitschrift fur Naturforschung. C, Journal of Biosciences",
            "pub_date": "1997-12-01",
            "pub_year": 1997,
            "volume": "52",
            "issue": "11-12",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1515/znc-1997-11-1212",
            "pmid": "9463937.0",
            "pmcid": "",
            "abstract": "This study shows that incubation of rat liver mitochondria in the presence of the thiol/ amino reagent 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) is followed by inhibition of both succinate supported respiration and oxidative phosphorylation. Half-maximal inhibition of succinic dehydrogenase activity and succinate oxidation by mitochondria was attained at 55.3 and 60.8 microM DIDS, respectively. DIDS did inhibit the net ATP synthesis and ATP< =>[32P]Pi exchange reaction catalyzed by submitochondrial particles in a dose-dependent manner (Ki = 31.7 microM and Ki = 32.7 microM), respectively. The hydrolytic activities of uncoupled heart submitochondrial particles and purified F1-ATPase were also inhibited 50% by 31.9 and 20.9 microM DIDS, respectively.",
            "references": [
            "pap_id": "147",
            "title": "Inhibitors of the ATP synthethase system.",
            "authors": "Linnett, P E; Beechey, R B",
            "chapter": "",
            "pages": "472-518",
            "journal": "Methods in Enzymology",
            "pub_date": "1905-06-01",
            "pub_year": 1905,
            "volume": "55",
            "issue": "",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0076-6879(79)55061-7",
            "pmid": "156854.0",
            "pmcid": "",
            "abstract": "",
            "references": [
            "pap_id": "148",
            "title": "Resistance of thermophilic ATPase (TF1) to specific F1-atpase inhibitors including local anesthetics.",
            "authors": "Saishu, T; Kagawa, Y; Shimizu, R",
            "chapter": "",
            "pages": "822-826",
            "journal": "Biochemical and Biophysical Research Communications",
            "pub_date": "1983-05-16",
            "pub_year": 1983,
            "volume": "112",
            "issue": "3",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0006-291x(83)91691-1",
            "pmid": "6221726.0",
            "pmcid": "",
            "abstract": "F1-ATPase obtained from mesophilic organisms is inhibited by specific inhibitors, such as aurovertin, efrapeptin, quercetin and several local anesthetics. This property has been explained by the common structure at the catalytic center of F1. However thermophilic F1 (TF1), which has the same primary structure at the center as other F1's, was shown to be resistant to these F1-specific inhibitors. Thus, the inhibitory mechanism may be explained not by the common structure at the catalytic site, but by some conformational changes of the flexible mesophilic F1 molecules or the absence of an inhibitor binding site in thermophilic F1.",
            "references": [
            "pap_id": "149",
            "title": "Pig heart mitochondrial ATPase : properties of purified and membrane-bound enzyme",
            "authors": "Di Pietro, Attilio; Godinot, Catherine; Bouillant, Marie-Louise; Gautheron, Danièle C.",
            "chapter": "",
            "pages": "959-967",
            "journal": "Biochimie",
            "pub_date": "1975-11-01",
            "pub_year": 1975,
            "volume": "57",
            "issue": "8",
            "Issn": "3009084",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/S0300-9084(75)80218-5",
            "pmid": "",
            "pmcid": "",
            "abstract": "Soluble ATPase (F1)has been purified from pig heart mitochondria. The purified enzyme had a high specific activity and was homogeneous as checked by ultracentrifugation and electrofocusing. It could be dissociated into subunits by cold-treatment or sodium dodecyl sulfate denaturation. The molecular weights of the two major and three minor subunits could be estimated by sodium dodecyl sulfate gel electrophoresis. The native enzyme had an isoelectric point of 5.2 while the cold-denatured enzyme showed three main bands focusing at pH 5.0, 5.2 and 5.4. Kinetic properties (Vm and Km (ATP)) have been compared for the soluble and membrane bound ATPase in presence of various anions. Inhibitory effects of Quercetin and other flavonoids have been tested in order to get an insight on the interaction between ATPase and its natural inhibitor.",
            "references": [
            "pap_id": "15",
            "title": "Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro.",
            "authors": "Dykens, James A; Jamieson, Joseph; Marroquin, Lisa; Nadanaciva, Sashi; Billis, Puja A; Will, Yvonne",
            "chapter": "",
            "pages": "203-210",
            "journal": "Toxicology and Applied Pharmacology",
            "pub_date": "2008-12-01",
            "pub_year": 2008,
            "volume": "233",
            "issue": "2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/j.taap.2008.08.013",
            "pmid": "18817800.0",
            "pmcid": "",
            "abstract": "As a class, the biguanides induce lactic acidosis, a hallmark of mitochondrial impairment. To assess potential mitochondrial impairment, we evaluated the effects of metformin, buformin and phenformin on: 1) viability of HepG2 cells grown in galactose, 2) respiration by isolated mitochondria, 3) metabolic poise of HepG2 and primary human hepatocytes, 4) activities of immunocaptured respiratory complexes, and 5) mitochondrial membrane potential and redox status in primary human hepatocytes. Phenformin was the most cytotoxic of the three with buformin showing moderate toxicity, and metformin toxicity only at mM concentrations. Importantly, HepG2 cells grown in galactose are markedly more susceptible to biguanide toxicity compared to cells grown in glucose, indicating mitochondrial toxicity as a primary mode of action. The same rank order of potency was observed for isolated mitochondrial respiration where preincubation (40 min) exacerbated respiratory impairment, and was required to reveal inhibition by metformin, suggesting intramitochondrial bio-accumulation. Metabolic profiling of intact cells corroborated respiratory inhibition, but also revealed compensatory increases in lactate production from accelerated glycolysis. High (mM) concentrations of the drugs were needed to inhibit immunocaptured respiratory complexes, supporting the contention that bioaccumulation is involved. The same rank order was found when monitoring mitochondrial membrane potential, ROS production, and glutathione levels in primary human hepatocytes. In toto, these data indicate that biguanide-induced lactic acidosis can be attributed to acceleration of glycolysis in response to mitochondrial impairment. Indeed, the desired clinical outcome, viz., decreased blood glucose, could be due to increased glucose uptake and glycolytic flux in response to drug-induced mitochondrial dysfunction.",
            "references": [
            "pap_id": "150",
            "title": "Purification and characterization of the F1-ATPase from Clostridium thermoaceticum.",
            "authors": "Ivey, D M; Ljungdahl, L G",
            "chapter": "",
            "pages": "252-257",
            "journal": "Journal of Bacteriology",
            "pub_date": "1986-01-01",
            "pub_year": 1986,
            "volume": "165",
            "issue": "1",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1128/jb.165.1.252-257.1986",
            "pmid": "2867087.0",
            "pmcid": "PMC214397",
            "abstract": "The F1 portion of the H+-ATPase from Clostridium thermoaceticum was purified to homogeneity by solubilization at low ionic strength, ion-exchange chromatography, and gel filtration. The last indicated the Mr to be 370,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the pure enzyme revealed four bands with Mr corresponding to 60,000, 55,000, 37,000, and 17,000 in an apparent molar ratio of 3:3:1:1. The purified enzyme would bind to stripped membranes to reconstitute dicyclohexylcarbodiimide-sensitive ATPase activity. Phosphohydrolase activity, measured at 58 degrees C, was optimal at pH 8.5. In the presence of a 1 mM excess of Mg2+ over the concentration of ATP, the Km for ATP was 0.4 mM, and the Vmax was 6.7 mumol min-1 mg-1. Unlike the membrane-bound F1F0 complex, the F1-ATPase was relatively insensitive to the inhibitors dicyclohexylcarbodiimide and tributyltin chloride. Both the complex and the F1-ATPase were inhibited by quercetin, azide, 7-chloro-4-nitro-benz-2-oxa-1,3-diazole, and free magnesium, and both were stimulated by primary alcohols and sulfite. In whole cells, the F1F0-ATPase catalyzed the synthesis of ATP in response to a pH gradient.",
            "references": [
            "pap_id": "151",
            "title": "Structural preferences for the binding of chromium nucleotides by beef heart mitochondrial ATPase.",
            "authors": "Bossard, M J; Schuster, S M",
            "chapter": "",
            "pages": "6617-6622",
            "journal": "The Journal of Biological Chemistry",
            "pub_date": "1981-07-10",
            "pub_year": 1981,
            "volume": "256",
            "issue": "13",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "6453868.0",
            "pmcid": "",
            "abstract": "The mono- and bidentate forms of adenosine 5'-diphosphate, chromium (III) salt (CrADP) were separated using Sephadex G-10 column chromatography. The isomeric purity of the two forms was monitored using high voltage electrophoresis and column chromatography. The same techniques were employed to assess the purity of the mono-, bi-, and tridentate forms of adenosine 5'-triphosphate, chromium (III) salt (CrATP). Distinct differences in the interaction of beef heart mitochondrial ATPase with the various isomers of chromium nucleotides were seen in kinetic studies. Monodentate CrADP was a competitive inhibitor of the ATP hydrolysis activity of both purified ATPase and submitochondrial particles. However, when ITPase activity was examined, noncompetitive inhibition was observed. The bidentate isomer of CrADP did not affect ATPase activity. Enzymatic synthesis of the transition state analog of ATP synthesis and hydrolysis, Pi-CrADP occurred exclusively with the monodentate isomer of CrADP. It was also found that only the mono- and tridentate forms of CrATP were potent inhibitors of ATP hydrolysis by beef heart mitochondrial ATPase. These results are discussed in terms of possible ATP synthesis and hydrolysis mechanisms.",
            "references": [
            "pap_id": "152",
            "title": "Inhibition of Escherichia coli H+-ATPase by venturicidin, oligomycin and ossamycin.",
            "authors": "Perlin, D S; Latchney, L R; Senior, A E",
            "chapter": "",
            "pages": "238-244",
            "journal": "Biochimica et Biophysica Acta",
            "pub_date": "1985-05-31",
            "pub_year": 1985,
            "volume": "807",
            "issue": "3",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0005-2728(85)90254-3",
            "pmid": "2859888.0",
            "pmcid": "",
            "abstract": "The antibiotics venturicidin, oligomycin and ossamycin were investigated as potential inhibitors of the Escherichia coli H+-ATPase. It was found that venturicidin strongly inhibited ATP-driven proton transport and ATP hydrolysis, while oligomycin weakly inhibited these functions. Inhibition of the H+-ATPase by venturicidin and oligomycin was correlated with inhibition of F0-mediate proton transport. Both inhibitors were found to interfere with the covalent reaction between dicyclohexyl[14C]carbodiimide and the F0 subunit c (uncE protein). Ossamycin had no direct inhibitory effect on E. coli F0 or F1; rather, it was found to uncouple ATP hydrolysis from proton transport.",
            "references": [
            "pap_id": "153",
            "title": "Studies on energy-linked reactions: modified mitochondrial ATPase of oligomycin-resistant mutants of Saccharomyces cerevisiae.",
            "authors": "Griffiths, D E; Houghton, R L",
            "chapter": "",
            "pages": "157-167",
            "journal": "European Journal of Biochemistry / FEBS",
            "pub_date": "1974-07-01",
            "pub_year": 1974,
            "volume": "46",
            "issue": "1",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1111/j.1432-1033.1974.tb03608.x",
            "pmid": "4277672.0",
            "pmcid": "",
            "abstract": "",
            "references": [
            "pap_id": "154",
            "title": "Understanding and exploiting the mechanistic basis for selectivity of polyketide inhibitors of F(0)F(1)-ATPase.",
            "authors": "Salomon, A R; Voehringer, D W; Herzenberg, L A; Khosla, C",
            "chapter": "",
            "pages": "14766-14771",
            "journal": "Proceedings of the National Academy of Sciences of the United States of America",
            "pub_date": "2000-12-19",
            "pub_year": 2000,
            "volume": "97",
            "issue": "26",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1073/pnas.97.26.14766",
            "pmid": "11121076.0",
            "pmcid": "PMC18993",
            "abstract": "Recently, a family of polyketide inhibitors of F(0)F(1)-ATPase, including apoptolidin, ossamycin, and oligomycin, were shown to be among the top 0.1% most cell line selective cytotoxic agents of 37, 000 molecules tested against the 60 human cancer cell lines of the National Cancer Institute. Many cancer cells maintain a high level of anaerobic carbon metabolism even in the presence of oxygen, a phenomenon that is historically known as the Warburg effect. A mechanism-based strategy to sensitize such cells to this class of potent small molecule cytotoxic agents is presented. These natural products inhibit oxidative phosphorylation by targeting the mitochondrial F(0)F(1) ATP synthase. Evaluation of gene expression profiles in a panel of leukemias revealed a strong correlation between the expression level of the gene encoding subunit 6 of the mitochondrial F(0)F(1) ATP synthase (known to be the binding site of members of this class of macrolides) and their sensitivity to these natural products. Within the same set of leukemia cell lines, comparably strong drug-gene correlations were also observed for the genes encoding two key enzymes involved in central carbon metabolism, pyruvate kinase, and aspartate aminotransferase. We propose a simple model in which the mitochondrial apoptotic pathway is activated in response to a shift in balance between aerobic and anaerobic ATP biosynthesis. Inhibitors of both lactate formation and carbon flux through the Embden-Meyerhof pathway significantly sensitized apoptolidin-resistant tumors to this drug. Nine different cell lines derived from human leukemias and melanomas, and colon, renal, central nervous system, and ovarian tumors are also sensitized to killing by apoptolidin.",
            "references": [
            "pap_id": "155",
            "title": "Nuclear mutations conferring oligomycin resistance in Neurospora crassa.",
            "authors": "Edwards, D L; Unger, B W",
            "chapter": "",
            "pages": "4254-4258",
            "journal": "The Journal of Biological Chemistry",
            "pub_date": "1978-06-25",
            "pub_year": 1978,
            "volume": "253",
            "issue": "12",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "149126.0",
            "pmcid": "",
            "abstract": "Mutants of Neurospora crassa have been isolated that are highly resistant to inhibition by oligomycin, an inhibitor of mitochondrial ATPase activity. Dixon plots (Dixon, M., and Webb, E.C. (1964) Enzymes, 2nd Ed, pp. 328-330, Academic Press, New York) of oligomycin inhibition curves of the parent strain and the resistant mutants are linear, indicating that oligomycin interacts at a single site within the ATPase complex. The Ki values obtained from the mutants vary from 150 to 900 times greater than the Ki obtained for the parent strain. The parent strain and the oligomycin-resistant mutants are also inhibited by bathophenanthroline, a lipophilic chelating agent that inhibits F1 ATPase activity. Dixon plots of bathophenanthroline inhibition curves are also linear and Ki values obtained are all approximately equal. Crosses of the oligomycin-resistant mutants to the oligomycin-sensitive parent strain show a mendelian segregation of the resistance characteristic. These data show that mutations leading to oligomycin resistance in Neurospora are due to alterations in nuclear genes.",
            "references": [
            "pap_id": "156",
            "title": "The relationship between the bovine heart mitochondrial adenosine triphosphatase, lipophilic compounds, and oligomycin.",
            "authors": "Cunningham, C C; George, D T",
            "chapter": "",
            "pages": "2036-2044",
            "journal": "The Journal of Biological Chemistry",
            "pub_date": "1975-03-25",
            "pub_year": 1975,
            "volume": "250",
            "issue": "6",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "123247.0",
            "pmcid": "",
            "abstract": "The lipid-free particulate preparations of the mitochondrial ATPase require phospholipid for activity and can be inhibited by oligomycin, as has been demonstrated previously. In this communication a steady state analysis of the activation of a particulate preparation of the ATPase by phospholipids and its subsequent inhibition by oligomycin has been carried out. The relative affinity of the ATPase for purified phospholipids has been determined by measuring the Km for activation (Ka) for several phospholipids. The Ka values varied from 30 to 100 mum. The Vmax in the presence of phosphatides varies from 0.29 to 1.11 mumol ATP hydrolyzed/min/mg of protein; no correlation is noted between the relative affinity of the enzyme for a phospholipid and the V max value. Higher V max values are noted with the more acidic phospholipids, however. Sodium dodecyl sulfate and monoolein also activate with Ka values of 25 and 800 mum, respectively. Diglycerides, however, do not activate. With all lipids the ATPase activity stimulated is oligomycin-sensitive. The Ki values for oligomycin range from 0.1 to 0.6 mum. Oligomycin is a competitive inhibitor with respect to all the phospholipids tested except phosphatidylethanolamine and phosphatidyglycerol. It is also competitive with respect to sodium dodecyl sulfate (k-i equals 0.94 mum). In reciprocal plots of activity versus ATP concentration, with and without oligomycin, an intercept consistent with either mixed or partial noncompetitive inhibition kinetics is noted. Comparable K-i values for oligomycin are obtained when calculated assuming either mixed or partial noncompetitive inhibition. The Km for ATP is the same in the unactivated and the lipid activated particulate ATPase; the value obtained is slightly lower than the Km for ATP in the solubilized, purified ATPase. Using a spectrophotometric assay the time required for activation with phospholipid and inhibition with oligomycin has also been determined. This investigation suggests the possibility that activation of the ATPase is due a position to interact with the water-soluble substrate. Consistent with the above suggestion is the supposition that the lipids do not necessarily confer inhibitor sensitivity to the ATPase, but rather allow an oligomycin-sensitive activity to be expressed.",
            "references": [
            "pap_id": "157",
            "title": "Comparison of the effects of oligomycin and dicyclohexylcarbodiimide on mitochondrial ATPase and related reactions.",
            "authors": "Glaser, E; Norling, B; Kopecký, J; Ernster, L",
            "chapter": "",
            "pages": "525-531",
            "journal": "European Journal of Biochemistry / FEBS",
            "pub_date": "1982-01-01",
            "pub_year": 1982,
            "volume": "121",
            "issue": "3",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1111/j.1432-1033.1982.tb05818.x",
            "pmid": "6276175.0",
            "pmcid": "",
            "abstract": "",
            "references": [
            "pap_id": "158",
            "title": "Antibiotics as tools for metabolic studies. X. Inhibition of phosphoryl transfer reactions in mitochondria by peliomycin, ossamycin, and venturicidin.",
            "authors": "Walter, P; Lardy, H A; Johnson, D",
            "chapter": "",
            "pages": "5014-5018",
            "journal": "The Journal of Biological Chemistry",
            "pub_date": "1967-11-10",
            "pub_year": 1967,
            "volume": "242",
            "issue": "21",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "4228672.0",
            "pmcid": "",
            "abstract": "",
            "references": [
            "pap_id": "159",
            "title": "Studies on energy-linked reactions: isolation and properties of mitochondrial venturicidin-resistant mutants of Saccharomyces cerevisiae.",
            "authors": "Griffiths, D E; Houghton, R L; Lancashire, W E; Meadows, P A",
            "chapter": "",
            "pages": "393-402",
            "journal": "European Journal of Biochemistry / FEBS",
            "pub_date": "1975-02-21",
            "pub_year": 1975,
            "volume": "51",
            "issue": "2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1111/j.1432-1033.1975.tb03939.x",
            "pmid": "238835.0",
            "pmcid": "",
            "abstract": "Venturicidin is a specific inhibitor of aerobic growth of yeast and has no effect on fermentative growth, a result which is consistent with its known mode of action on mitochondrial oxidative phosphorylation. Venturicidin-resistant mutants of Saccharomyces cerevisiae have been isolated and form two general classes: class 1, nuclear mutants which are resistant to a variety of mitochondrial inhibitors and uncouplers, and class 2, mitochondrial mutants of phenotype VENR OLYR and VENR TETR in vivo. VENR OLYR mutants show a high degree of resistance to venturicidin and oligomycin at the whole cell and mitochondrial ATPase level but, in contrast, no resistance at the mitochondrial level is observed with VENR TETR mutants. Venturicidin resistance/sensitivity can be correlated with two binding sites on mitochondrial ATPase, one of which is common to the oligomycin binding site and the other is common to the triethyl tin binding site. Biochemical genetic studies indicate that two mitochondrial genes specify venturicidin resistance/sensitivity and that the mitochondrial gene products are components of the mitochondrial ATPase complex.",
            "references": [
            "pap_id": "16",
            "title": "Perhexiline.",
            "authors": "Ashrafian, Houman; Horowitz, John D; Frenneaux, Michael P",
            "chapter": "",
            "pages": "76-97",
            "journal": "Cardiovascular drug reviews",
            "pub_date": "1905-06-29",
            "pub_year": 1905,
            "volume": "25",
            "issue": "1",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1111/j.1527-3466.2007.00006.x",
            "pmid": "17445089.0",
            "pmcid": "",
            "abstract": "Perhexiline, 2-(2,2-dicyclohexylethyl)piperidine, was originally developed as an anti-anginal drug in the 1970s. Despite its success, its use diminished due to the occurrence of poorly understood side effects including neurotoxicity and hepatotoxicity in a small proportion of patients. Recently, perhexiline's mechanism of action and the molecular basis of its toxicity have been elucidated. Perhexiline reduces fatty acid metabolism through the inhibition of carnitine palmitoyltransferase, the enzyme responsible for mitochondrial uptake of long-chain fatty acids. The corresponding shift to greater carbohydrate utilization increases myocardial efficiency (work done per unit oxygen consumption) and this oxygen-sparing effect explains its antianginal efficacy. Perhexiline's side effects are attributable to high plasma concentrations occurring with standard doses in patients with impaired metabolism due to CYP2D6 mutations. Accordingly, dose modification in these poorly metabolizing patients identified through therapeutic plasma monitoring can eliminate any significant side effects. Herein we detail perhexiline's pharmacology with particular emphasis on its mechanism of action and its side effects. We discuss how therapeutic plasma monitoring has led to perhexiline's safe reintroduction into clinical practice and how recent clinical data attesting to its safety and remarkable efficacy led to a renaissance in its use in both refractory angina and chronic heart failure. Finally, we discuss the application of pharmacogenetics in combination with therapeutic plasma monitoring to potentially broaden perhexiline's use in heart failure, aortic stenosis, and other cardiac conditions.",
            "references": [
            "pap_id": "160",
            "title": "Inhibition of Thylakoid ATPase by Venturicidin as an Indicator of CF1-CF0 Interaction.",
            "authors": "Zhang, S.; Letham, D. D.; Jagendorf, A. T.",
            "chapter": "",
            "pages": "127-133",
            "journal": "Plant Physiology",
            "pub_date": "1993-01-01",
            "pub_year": 1993,
            "volume": "101",
            "issue": "1",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1104/pp.101.1.127",
            "pmid": "12231671.0",
            "pmcid": "PMC158656",
            "abstract": "Venturicidin inhibits the F0 portion of membrane-located, H+-pumping ATPases. We find it meets the criteria for an energy transfer inhibitor for spinach (Spinacia oleracea) thylakoids: complete inhibition of photophosphorylation and of photophosphorylation-stimulated and basal electron flow rates, but not of electron flow under uncoupled conditions. The extent of H+ uptake in the light is stimulated by venturicidin (vtcd), as expected for a compound blocking H+ efflux through CF0. Vtcd had no effect on the nonproton pumping, methanol-stimulated ATPase of thylakoids or on soluble CF1 ATPase. Under totally uncoupled conditions (saturating NH4Cl + gramicidin), vtcd can still inhibit sulfite-stimulated thylakoid ATPase completely. The concentration of vtcd needed for inhibition of ATPase was proportional to the concentration of thylakoids present in the assay, with an apparent stoichiometry of about 10 vtcd molecules per CF1/CF0 for 50% inhibition. Vtcd raised the Km for ATP somewhat, but had a stronger effect on the Vmax with respect to ATP. Inhibition by saturating vtcd ranged from 50 to 100%, depending on the condition of the thylakoids. Grinding leaves in buffer containing 0.2 M choline chloride (known to provide superior photophosphorylation rates) helped bring on maximum vtcd inhibition; trypsin treatment or aging of thylakoids brought on vtcd-resistant ATPase. We conclude that the extent of inhibition by vtcd can be used as an indicator of the tightness of coupling between CF1 and CF0.",
            "references": [
            "pap_id": "161",
            "title": "Structure-activity relationships within a family of selectively cytotoxic macrolide natural products.",
            "authors": "Salomon, A R; Zhang, Y; Seto, H; Khosla, C",
            "chapter": "",
            "pages": "57-59",
            "journal": "Organic Letters",
            "pub_date": "2001-01-11",
            "pub_year": 2001,
            "volume": "3",
            "issue": "1",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1021/ol006767d",
            "pmid": "11429871.0",
            "pmcid": "",
            "abstract": "[figure: see text] We describe a semi-synthetic deglycosylated derivative of apoptolidin that retains considerable activity against the mitochondrial ATPase but has greatly reduced cellular cytotoxicity. We also demonstrate that a related antifungal natural product, cytovaricin, inhibits the same molecular target. Structural comparison of these macrolides provides insights into their conserved features that are presumably important for biological activity and identifies promising avenues at the interface of organic synthesis and biosynthesis for the generation of new selective cytotoxic agents.",
            "references": [
            "pap_id": "162",
            "title": "Novel inhibitors of mitochondrial sn-glycerol 3-phosphate dehydrogenase.",
            "authors": "Orr, Adam L; Ashok, Deepthi; Sarantos, Melissa R; Ng, Ryan; Shi, Tong; Gerencser, Akos A; Hughes, Robert E; Brand, Martin D",
            "chapter": "",
            "pages": "e89938",
            "journal": "Plos One",
            "pub_date": "2014-02-24",
            "pub_year": 2014,
            "volume": "9",
            "issue": "2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1371/journal.pone.0089938",
            "pmid": "24587137.0",
            "pmcid": "PMC3933693",
            "abstract": "Mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a ubiquinone-linked enzyme in the mitochondrial inner membrane best characterized as part of the glycerol phosphate shuttle that transfers reducing equivalents from cytosolic NADH into the mitochondrial electron transport chain. Despite the widespread expression of mGPDH and the availability of mGPDH-null mice, the physiological role of this enzyme remains poorly defined in many tissues, likely because of compensatory pathways for cytosolic regeneration of NAD⁺ and mechanisms for glycerol phosphate metabolism. Here we describe a novel class of cell-permeant small-molecule inhibitors of mGPDH (iGP) discovered through small-molecule screening. Structure-activity analysis identified a core benzimidazole-phenyl-succinamide structure as being essential to inhibition of mGPDH while modifications to the benzimidazole ring system modulated both potency and off-target effects. Live-cell imaging provided evidence that iGPs penetrate cellular membranes. Two compounds (iGP-1 and iGP-5) were characterized further to determine potency and selectivity and found to be mixed inhibitors with IC₅₀ and K(i) values between ∼1-15 µM. These novel mGPDH inhibitors are unique tools to investigate the role of glycerol 3-phosphate metabolism in both isolated and intact systems.",
            "references": [
            "pap_id": "163",
            "title": "Comprehensive Analyses and Prioritization of Tox21 10K Chemicals Affecting Mitochondrial Function by in-Depth Mechanistic Studies.",
            "authors": "Xia, Menghang; Huang, Ruili; Shi, Qiang; Boyd, Windy A; Zhao, Jinghua; Sun, Nuo; Rice, Julie R; Dunlap, Paul E; Hackstadt, Amber J; Bridge, Matt F; Smith, Marjolein V; Dai, Sheng; Zheng, Wei; Chu, Pei-Hsuan; Gerhold, David; Witt, Kristine L; DeVito, Michael; Freedman, Jonathan H; Austin, Christopher P; Houck, Keith A; Thomas, Russell S; Paules, Richard S; Tice, Raymond R; Simeonov, Anton",
            "chapter": "",
            "pages": "2110-11-04",
            "journal": "Environmental Health Perspectives",
            "pub_date": "2018-07-26",
            "pub_year": 2018,
            "volume": "126",
            "issue": "7",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1289/EHP2589",
            "pmid": "30059008.0",
            "pmcid": "PMC6112376",
            "abstract": "BACKGROUND: A central challenge in toxicity testing is the large number of chemicals in commerce that lack toxicological assessment. In response, the Tox21 program is re-focusing toxicity testing from animal studies to less expensive and higher throughput in vitro methods using target/pathway-specific, mechanism-driven assays. OBJECTIVES: Our objective was to use an in-depth mechanistic study approach to prioritize and characterize the chemicals affecting mitochondrial function. METHODS: We used a tiered testing approach to prioritize for more extensive testing 622 compounds identified from a primary, quantitative high-throughput screen of 8,300 unique small molecules, including drugs and industrial chemicals, as potential mitochondrial toxicants by their ability to significantly decrease the mitochondrial membrane potential (MMP). Based on results from secondary MMP assays in HepG2 cells and rat hepatocytes, 34 compounds were selected for testing in tertiary assays that included formation of reactive oxygen species (ROS), upregulation of p53 and nuclear erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE), mitochondrial oxygen consumption, cellular Parkin translocation, and larval development and ATP status in the nematode Caenorhabditis elegans. RESULTS: A group of known mitochondrial complex inhibitors (e.g., rotenone) and uncouplers (e.g., chlorfenapyr), as well as potential novel complex inhibitors and uncouplers, were detected. From this study, we identified four not well-characterized potential mitochondrial toxicants (lasalocid, picoxystrobin, pinacyanol, and triclocarban) that merit additional in vivo characterization. CONCLUSIONS: The tier-based approach for identifying and mechanistically characterizing mitochondrial toxicants can potentially reduce animal use in toxicological testing.",
            "references": [
            "pap_id": "164",
            "title": "5-Fluorouracil induces apoptosis in human colon cancer cell lines with modulation of Bcl-2 family proteins.",
            "authors": "Nita, M E; Nagawa, H; Tominaga, O; Tsuno, N; Fujii, S; Sasaki, S; Fu, C G; Takenoue, T; Tsuruo, T; Muto, T",
            "chapter": "",
            "pages": "986-992",
            "journal": "British Journal of Cancer",
            "pub_date": "1998-10-01",
            "pub_year": 1998,
            "volume": "78",
            "issue": "8",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1038/bjc.1998.617",
            "pmid": "9792140.0",
            "pmcid": "PMC2063141",
            "abstract": "Recently, apoptosis has been implicated as one of the end points of cells exposed to chemotherapeutic agents. The p53 and Bcl-2 family of proteins are involved in chemotherapy-induced apoptosis, but in a cell type-dependent manner. We sought to determine the roles played by the p53 and Bcl-2 family of proteins in 5-fluorouracil (5-FU)-induced apoptosis of human colon cancer cell lines. We first studied the p53 genetic and functional status, and then 5-FU, at inhibitory concentration of 50% (IC50) doses, was used to induce apoptosis, which was confirmed by morphological analysis and enzyme-linked immunosorbent assay (ELISA). Bcl-2, Bcl-X(L), Bax, Bad, Bak and p53 protein expression was analysed by Western blotting. Using five human colon cancer cell lines, we found that equitoxic (IC50) doses of 5-FU induced apoptosis in both wild-type p53 and mutant p53 cells. Analysis of the steady-state levels of Bcl-2 family proteins showed high expression of Bcl-X(L) in all of the cell lines except Colo320. Bcl-2 was expressed in two of them. Bax presented with the lowest basal expression and Bad showed homogeneous expression. On the other hand, Bak expression varied more than fivefold among these cells. In cells containing wild-type p53 (e.g. LoVo), 5-FU-induced apoptosis was accompanied by increased expression of Bax and Bak without consistent modulation of other bcl-2 family proteins. In contrast in cells containing mutant p53 (e.g. DLD1), Bak expression was remarkably increased. There was a significant correlation between chemosensitivity and Bcl-X(L) to Bax ratio, rather than Bcl-2 to Bax. In conclusion, these results suggest that some members of the Bcl-2 family of proteins, in human colon cancer cell lines, are modulated by 5-FU and that the ratio of Bcl-X(L) to Bax may be related to chemosensitivity to 5-FU.",
            "references": [
            "pap_id": "165",
            "title": "",
            "authors": "",
            "chapter": "",
            "pages": "",
            "journal": "",
            "pub_date": "",
            "pub_year": 1900,
            "volume": "",
            "issue": "",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "",
            "pmcid": "",
            "abstract": "",
            "references": []
            "pap_id": "166",
            "title": "Structural basis for the quinone reduction in the bc1 complex: a comparative analysis of crystal structures of mitochondrial cytochrome bc1 with bound substrate and inhibitors at the Qi site.",
            "authors": "Gao, Xiugong; Wen, Xiaoling; Esser, Lothar; Quinn, Byron; Yu, Linda; Yu, Chang-An; Xia, Di",
            "chapter": "",
            "pages": "9067-9080",
            "journal": "Biochemistry",
            "pub_date": "2003-08-05",
            "pub_year": 2003,
            "volume": "42",
            "issue": "30",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1021/bi0341814",
            "pmid": "12885240.0",
            "pmcid": "",
            "abstract": "Cytochrome bc(1) is an integral membrane protein complex essential to cellular respiration and photosynthesis. The Q cycle reaction mechanism of bc(1) postulates a separated quinone reduction (Q(i)) and quinol oxidation (Q(o)) site. In a complete catalytic cycle, a quinone molecule at the Q(i) site receives two electrons from the b(H) heme and two protons from the negative side of the membrane; this process is specifically inhibited by antimycin A and NQNO. The structures of bovine mitochondrial bc(1) in the presence or absence of bound substrate ubiquinone and with either the bound antimycin A(1) or NQNO were determined and refined. A ubiquinone with its first two isoprenoid repeats and an antimycin A(1) were identified in the Q(i) pocket of the substrate and inhibitor bound structures, respectively; the NQNO, on the other hand, was identified in both Q(i) and Q(o) pockets in the inhibitor complex. The two inhibitors occupied different portions of the Q(i) pocket and competed with substrate for binding. In the Q(o) pocket, the NQNO behaves similarly to stigmatellin, inducing an iron-sulfur protein conformational arrest. Extensive binding interactions and conformational adjustments of residues lining the Q(i) pocket provide a structural basis for the high affinity binding of antimycin A and for phenotypes of inhibitor resistance. A two-water-mediated ubiquinone protonation mechanism is proposed involving three Q(i) site residues His(201), Lys(227), and Asp(228).",
            "references": [
            "pap_id": "167",
            "title": "Inhibitors of cytochrome c oxidase",
            "authors": "Erecińska, Maria; Wilson, David F.",
            "chapter": "",
            "pages": "1-20",
            "journal": "Pharmacology & Therapeutics",
            "pub_date": "1980-01-01",
            "pub_year": 1980,
            "volume": "8",
            "issue": "1",
            "Issn": "1637258",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/0163-7258(80)90057-1",
            "pmid": "",
            "pmcid": "",
            "abstract": "",
            "references": [
            "pap_id": "168",
            "title": "Identification of small molecule inhibitors of human cytochrome c oxidase that target chemoresistant glioma cells.",
            "authors": "Oliva, Claudia R; Markert, Tahireh; Ross, Larry J; White, E Lucile; Rasmussen, Lynn; Zhang, Wei; Everts, Maaike; Moellering, Douglas R; Bailey, Shannon M; Suto, Mark J; Griguer, Corinne E",
            "chapter": "",
            "pages": "24188-24199",
            "journal": "The Journal of Biological Chemistry",
            "pub_date": "2016-11-11",
            "pub_year": 2016,
            "volume": "291",
            "issue": "46",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1074/jbc.M116.749978",
            "pmid": "27679486.0",
            "pmcid": "PMC5104942",
            "abstract": "The enzyme cytochrome c oxidase (CcO) or complex IV (EC is a large transmembrane protein complex that serves as the last enzyme in the respiratory electron transport chain of eukaryotic mitochondria. CcO promotes the switch from glycolytic to oxidative phosphorylation (OXPHOS) metabolism and has been associated with increased self-renewal characteristics in gliomas. Increased CcO activity in tumors has been associated with tumor progression after chemotherapy failure, and patients with primary glioblastoma multiforme and high tumor CcO activity have worse clinical outcomes than those with low tumor CcO activity. Therefore, CcO is an attractive target for cancer therapy. We report here the characterization of a CcO inhibitor (ADDA 5) that was identified using a high throughput screening paradigm. ADDA 5 demonstrated specificity for CcO, with no inhibition of other mitochondrial complexes or other relevant enzymes, and biochemical characterization showed that this compound is a non-competitive inhibitor of cytochrome c When tested in cellular assays, ADDA 5 dose-dependently inhibited the proliferation of chemosensitive and chemoresistant glioma cells but did not display toxicity against non-cancer cells. Furthermore, treatment with ADDA 5 led to significant inhibition of tumor growth in flank xenograft mouse models. Importantly, ADDA 5 inhibited CcO activity and blocked cell proliferation and neurosphere formation in cultures of glioma stem cells, the cells implicated in tumor recurrence and resistance to therapy in patients with glioblastoma. In summary, we have identified ADDA 5 as a lead CcO inhibitor for further optimization as a novel approach for the treatment of glioblastoma and related cancers. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.",
            "references": [
            "pap_id": "169",
            "title": "Tetrathiomolybdate inhibits mitochondrial complex IV and mediates degradation of hypoxia-inducible factor-1α in cancer cells.",
            "authors": "Kim, Kyu Kwang; Abelman, Sarah; Yano, Naohiro; Ribeiro, Jennifer R; Singh, Rakesh K; Tipping, Marla; Moore, Richard G",
            "chapter": "",
            "pages": "1939-02-20",
            "journal": "Scientific Reports",
            "pub_date": "2015-10-15",
            "pub_year": 2015,
            "volume": "5",
            "issue": "",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1038/srep14296",
            "pmid": "26469226.0",
            "pmcid": "PMC4606568",
            "abstract": "Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that triggers adaptive responses upon low oxygen conditions and plays a crucial role in cancer metabolism and therapy resistance. Tetrathiomolybdate (TM), a therapy option for copper overload disorder, has also been shown to be capable of limiting tumor angiogenesis, although its underlying mechanism remains unclear. Using ovarian and endometrial cancer cell lines, we observed that TM downregulates HIF-1α protein levels and HIF-transcriptional targets involved in tumor angiogenesis and glycolysis, but did not affect HIF-1α protein synthesis. TM-mediated HIF-1α downregulation was suppressed when HIF-prolyl hydroxylase activity was pharmacologically inhibited using deferoxamine or dimethyloxaloylglycine, and also when the oxygen-dependent degradation domains of HIF-1α, which are responsible for the interaction with HIF-prolyl hydroxylase, were deleted. These findings suggest that TM causes HIF-1α downregulation in a HIF-prolyl hydroxylase-dependent manner. Our studies showed that TM inhibits the activity of the copper-dependent mitochondrial complex IV and reduces mitochondrial respiration, thereby possibly increasing oxygen availability, which is crucial for HIF-prolyl hydroxylase activity. Pimonidazole staining also showed that TM elevates oxygen tension in hypoxic cells. Our studies provide mechanistic evidence for TM-mediated HIF-1α regulation and suggest its therapeutic potential as a method of blocking angiogenesis in ovarian and endometrial tumors.",
            "references": [
            "pap_id": "17",
            "title": "Metabolic manipulation in ischaemic heart disease, a novel approach to treatment.",
            "authors": "Lee, Leong; Horowitz, John; Frenneaux, Michael",
            "chapter": "",
            "pages": "634-641",
            "journal": "European Heart Journal",
            "pub_date": "2004-04-01",
            "pub_year": 2004,
            "volume": "25",
            "issue": "8",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/j.ehj.2004.02.018",
            "pmid": "15084367.0",
            "pmcid": "",
            "abstract": "Antianginal drugs that exert their anti-ischaemic effects primarily by altering myocardial metabolism have recently attracted attention. They have the potential to relieve symptoms in patients with refractory angina who are already on \"optimal\" medical therapy and have disease that is not amenable to revascularisation, making these drugs an attractive addition to therapy, particularly for the elderly population. In some cases, they may even be used as first-line treatment. These drugs increase glucose metabolism at the expense of free-fatty-acid metabolism, enhancing oxygen efficiency during myocardial ischaemia. Whilst they have been demonstrated to reduce ischaemia in several clinical trials, their use remains limited. This review aims to draw attention to these \"metabolic\" antianginal drugs while surveying the evidence supporting their use and mode of action. Four metabolic antianginal drugs are reviewed: perhexiline, trimetazidine, ranolazine, and etomoxir. We also discuss the metabolic actions of glucose-insulin-potassium and beta-blockers and describe myocardial metabolism during normal and ischaemic conditions. The potential of these metabolic agents may extend beyond the treatment of ischaemia secondary to coronary artery disease. They offer significant promise for the treatment of symptoms occurring due to inoperable aortic stenosis, hypertrophic cardiomyopathy, and chronic heart failure.",
            "references": [
            "pap_id": "170",
            "title": "ATP synthase and the actions of inhibitors utilized to study its roles in human health, disease, and other scientific areas.",
            "authors": "Hong, Sangjin; Pedersen, Peter L",
            "chapter": "",
            "pages": "590-641",
            "journal": "Microbiology and Molecular Biology Reviews",
            "pub_date": "2008-12-01",
            "pub_year": 2008,
            "volume": "72",
            "issue": "4",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1128/MMBR.00016-08",
            "pmid": "19052322.0",
            "pmcid": "PMC2593570",
            "abstract": "ATP synthase, a double-motor enzyme, plays various roles in the cell, participating not only in ATP synthesis but in ATP hydrolysis-dependent processes and in the regulation of a proton gradient across some membrane-dependent systems. Recent studies of ATP synthase as a potential molecular target for the treatment of some human diseases have displayed promising results, and this enzyme is now emerging as an attractive molecular target for the development of new therapies for a variety of diseases. Significantly, ATP synthase, because of its complex structure, is inhibited by a number of different inhibitors and provides diverse possibilities in the development of new ATP synthase-directed agents. In this review, we classify over 250 natural and synthetic inhibitors of ATP synthase reported to date and present their inhibitory sites and their known or proposed modes of action. The rich source of ATP synthase inhibitors and their known or purported sites of action presented in this review should provide valuable insights into their applications as potential scaffolds for new therapeutics for human and animal diseases as well as for the discovery of new pesticides and herbicides to help protect the world's food supply. Finally, as ATP synthase is now known to consist of two unique nanomotors involved in making ATP from ADP and P(i), the information provided in this review may greatly assist those investigators entering the emerging field of nanotechnology.",
            "references": [
            "pap_id": "171",
            "title": "The oligomycin-sensitivity conferring protein of mitochondrial ATP synthase: emerging new roles in mitochondrial pathophysiology.",
            "authors": "Antoniel, Manuela; Giorgio, Valentina; Fogolari, Federico; Glick, Gary D; Bernardi, Paolo; Lippe, Giovanna",
            "chapter": "",
            "pages": "7513-7536",
            "journal": "International Journal of Molecular Sciences",
            "pub_date": "2014-04-30",
            "pub_year": 2014,
            "volume": "15",
            "issue": "5",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.3390/ijms15057513",
            "pmid": "24786291.0",
            "pmcid": "PMC4057687",
            "abstract": "The oligomycin-sensitivity conferring protein (OSCP) of the mitochondrial F(O)F1 ATP synthase has long been recognized to be essential for the coupling of proton transport to ATP synthesis. Located on top of the catalytic F1 sector, it makes stable contacts with both F1 and the peripheral stalk, ensuring the structural and functional coupling between F(O) and F1, which is disrupted by the antibiotic, oligomycin. Recent data have established that OSCP is the binding target of cyclophilin (CyP) D, a well-characterized inducer of the mitochondrial permeability transition pore (PTP), whose opening can precipitate cell death. CyPD binding affects ATP synthase activity, and most importantly, it decreases the threshold matrix Ca²⁺ required for PTP opening, in striking analogy with benzodiazepine 423, an apoptosis-inducing agent that also binds OSCP. These findings are consistent with the demonstration that dimers of ATP synthase generate Ca²⁺-dependent currents with features indistinguishable from those of the PTP and suggest that ATP synthase is directly involved in PTP formation, although the underlying mechanism remains to be established. In this scenario, OSCP appears to play a fundamental role, sensing the signal(s) that switches the enzyme of life in a channel able to precipitate cell death.",
            "references": [
            "pap_id": "172",
            "title": "Metformin directly acts on mitochondria to alter cellular bioenergetics.",
            "authors": "Andrzejewski, Sylvia; Gravel, Simon-Pierre; Pollak, Michael; St-Pierre, Julie",
            "chapter": "",
            "pages": "1900-01-11",
            "journal": "Cancer & metabolism",
            "pub_date": "2014-08-28",
            "pub_year": 2014,
            "volume": "2",
            "issue": "",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1186/2049-3002-2-12",
            "pmid": "25184038.0",
            "pmcid": "PMC4147388",
            "abstract": "BACKGROUND: Metformin is widely used in the treatment of diabetes, and there is interest in 'repurposing' the drug for cancer prevention or treatment. However, the mechanism underlying the metabolic effects of metformin remains poorly understood. METHODS: We performed respirometry and stable isotope tracer analyses on cells and isolated mitochondria to investigate the impact of metformin on mitochondrial functions. RESULTS: We show that metformin decreases mitochondrial respiration, causing an increase in the fraction of mitochondrial respiration devoted to uncoupling reactions. Thus, cells treated with metformin become energetically inefficient, and display increased aerobic glycolysis and reduced glucose metabolism through the citric acid cycle. Conflicting prior studies proposed mitochondrial complex I or various cytosolic targets for metformin action, but we show that the compound limits respiration and citric acid cycle activity in isolated mitochondria, indicating that at least for these effects, the mitochondrion is the primary target. Finally, we demonstrate that cancer cells exposed to metformin display a greater compensatory increase in aerobic glycolysis than nontransformed cells, highlighting their metabolic vulnerability. Prevention of this compensatory metabolic event in cancer cells significantly impairs survival. CONCLUSIONS: Together, these results demonstrate that metformin directly acts on mitochondria to limit respiration and that the sensitivity of cells to metformin is dependent on their ability to cope with energetic stress.",
            "references": [
            "pap_id": "173",
            "title": "Sodium fluoroacetate poisoning.",
            "authors": "Proudfoot, Alex T; Bradberry, Sally M; Vale, J Allister",
            "chapter": "",
            "pages": "213-219",
            "journal": "Toxicological Reviews",
            "pub_date": "1905-06-28",
            "pub_year": 1905,
            "volume": "25",
            "issue": "4",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.2165/00139709-200625040-00002",
            "pmid": "17288493.0",
            "pmcid": "",
            "abstract": "Sodium fluoroacetate was introduced as a rodenticide in the US in 1946. However, its considerable efficacy against target species is offset by comparable toxicity to other mammals and, to a lesser extent, birds and its use as a general rodenticide was therefore severely curtailed by 1990. Currently, sodium fluoroacetate is licensed in the US for use against coyotes, which prey on sheep and goats, and in Australia and New Zealand to kill unwanted introduced species. The extreme toxicity of fluoroacetate to mammals and insects stems from its similarity to acetate, which has a pivotal role in cellular metabolism. Fluoroacetate combines with coenzyme A (CoA-SH) to form fluoroacetyl CoA, which can substitute for acetyl CoA in the tricarboxylic acid cycle and reacts with citrate synthase to produce fluorocitrate, a metabolite of which then binds very tightly to aconitase, thereby halting the cycle. Many of the features of fluoroacetate poisoning are, therefore, largely direct and indirect consequences of impaired oxidative metabolism. Energy production is reduced and intermediates of the tricarboxylic acid cycle subsequent to citrate are depleted. Among these is oxoglutarate, a precursor of glutamate, which is not only an excitatory neurotransmitter in the CNS but is also required for efficient removal of ammonia via the urea cycle. Increased ammonia concentrations may contribute to the incidence of seizures. Glutamate is also required for glutamine synthesis and glutamine depletion has been observed in the brain of fluoroacetate-poisoned rodents. Reduced cellular oxidative metabolism contributes to a lactic acidosis. Inability to oxidise fatty acids via the tricarboxylic acid cycle leads to ketone body accumulation and worsening acidosis. Adenosine triphosphate (ATP) depletion results in inhibition of high energy-consuming reactions such as gluconeogenesis. Fluoroacetate poisoning is associated with citrate accumulation in several tissues, including the brain. Fluoride liberated from fluoroacetate, citrate and fluorocitrate are calcium chelators and there are both animal and clinical data to support hypocalcaemia as a mechanism of fluoroacetate toxicity. However, the available evidence suggests the fluoride component does not contribute. Acute poisoning with sodium fluoroacetate is uncommon. Ingestion is the major route by which poisoning occurs. Nausea, vomiting and abdominal pain are common within 1 hour of ingestion. Sweating, apprehension, confusion and agitation follow. Both supraventricular and ventricular arrhythmias have been reported and nonspecific ST- and T-wave changes are common, the QTc may be prolonged and hypotension may develop. Seizures are the main neurological feature. Coma may persist for several days. Although several possible antidotes have been investigated, they are of unproven value in humans. The immediate, and probably only, management of fluoroacetate poisoning is therefore supportive, including the correction of hypocalcaemia.",
            "references": [
            "pap_id": "174",
            "title": "In vitro assessment of mitochondrial dysfunction and cytotoxicity of nefazodone, trazodone, and buspirone.",
            "authors": "Dykens, James A; Jamieson, Joseph D; Marroquin, Lisa D; Nadanaciva, Sashi; Xu, Jinghai J; Dunn, Margaret C; Smith, Arthur R; Will, Yvonne",
            "chapter": "",
            "pages": "335-345",
            "journal": "Toxicological Sciences",
            "pub_date": "2008-06-08",
            "pub_year": 2008,
            "volume": "103",
            "issue": "2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1093/toxsci/kfn056",
            "pmid": "18344530.0",
            "pmcid": "",
            "abstract": "Mitochondrial toxicity is increasingly implicated in a host of drug-induced organ toxicities, including hepatotoxicity. Nefazodone was withdrawn from the U.S. market in 2004 due to hepatotoxicity. Accordingly, we evaluated nefazodone, another triazolopyridine trazodone, plus the azaspirodecanedione buspirone, for cytotoxicity and effects on mitochondrial function. In accord with its clinical disposition, nefazodone was the most toxic compound of the three, trazodone had relatively modest effects, whereas buspirone showed the least toxicity. Nefazodone profoundly inhibited mitochondrial respiration in isolated rat liver mitochondria and in intact HepG2 cells where this was accompanied by simultaneous acceleration of glycolysis. Using immunocaptured oxidative phosphorylation (OXPHOS) complexes, we identified Complex 1, and to a lesser amount Complex IV, as the targets of nefazodone toxicity. No inhibition was found for trazodone, and buspirone showed 3.4-fold less inhibition of OXPHOS Complex 1 than nefazodone. In human hepatocytes that express cytochrome P450, isoform 3A4, after 24 h exposure, nefazodone and trazodone collapsed mitochondrial membrane potential, and imposed oxidative stress, as detected via glutathione depletion, leading to cell death. Our results suggest that the mitochondrial impairment imposed by nefazodone is profound and likely contributes to its hepatotoxicity, especially in patients cotreated with other drugs with mitochondrial liabilities.",
            "references": []
            "pap_id": "175",
            "title": "Strategies to reduce late-stage drug attrition due to mitochondrial toxicity.",
            "authors": "Dykens, James A; Marroquin, Lisa D; Will, Yvonne",
            "chapter": "",
            "pages": "161-175",
            "journal": "Expert Review of Molecular Diagnostics",
            "pub_date": "2014-03-07",
            "pub_year": 2014,
            "volume": "7",
            "issue": "2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1586/14737159.7.2.161",
            "pmid": "17331064.0",
            "pmcid": "",
            "abstract": "Mitochondrial dysfunction is increasingly implicated in the etiology of drug-induced toxicities and negative side-effect profiles. Early identification of mitochondrial liabilities for new chemical entities is therefore crucial for avoiding late-stage attrition during drug development. Limitations of traditional methods for assessing mitochondrial dysfunction have discouraged routine evaluation of mitochondrial liabilities. To circumvent this bottleneck, a high-throughput screen has been developed that measures oxygen consumption; one of the most informative parameters for the assessment of mitochondrial status. This technique has revealed that some, but not all, members of many major drug classes have mitochondrial liabilities. This dichotomy encourages optimism that efficacy can be disassociated from mitochondrial toxicity, resulting in safer drugs in the future.",
            "references": []
            "pap_id": "176",
            "title": "Drug-Induced Mitochondrial Dysfunction",
            "authors": "",
            "chapter": "",
            "pages": "",
            "journal": "",
            "pub_date": "2008-09-14",
            "pub_year": 2008,
            "volume": "",
            "issue": "",
            "Issn": "",
            "Isbn": "9.78E+12",
            "url": "",
            "doi": "10.1002/9780470372531",
            "pmid": "",
            "pmcid": "",
            "abstract": "",
            "references": []
            "pap_id": "177",
            "title": "Current concepts in drug-induced mitochondrial toxicity.",
            "authors": "Nadanaciva, Sashi; Will, Yvonne",
            "chapter": "",
            "pages": "2.15.1-2.15.9",
            "journal": "Current protocols in toxicology / editorial board, Mahin D. Maines (editor-in-chief) ... [et al.]",
            "pub_date": "2009-05-09",
            "pub_year": 2009,
            "volume": "2",
            "issue": "",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1002/0471140856.tx0215s40",
            "pmid": "20941696.0",
            "pmcid": "",
            "abstract": "Mitochondria generate most of the cell's ATP and play key roles in fatty acid oxidation, steroid synthesis, heme synthesis, thermogenesis, calcium homeostasis, and apoptosis. With the development of new methods to study mitochondrial function, it is becoming clear that drug-induced mitochondrial dysfunction is one of the causes of drug toxicity. Mitochondria can be impaired by drugs in a variety of ways. These include inhibition of oxidative phosphorylation, uncoupling of electron transport from ATP synthesis, irreversible opening of the mitochondrial permeability transition pore, inhibition of transporters within the mitochondrial inner membrane, increased oxidative stress, inhibition of the citric acid cycle, inhibition of fatty acid oxidation, and impairment of either mtDNA replication or mtDNA-encoded protein synthesis. This unit provides an overview on the physiological roles of mitochondria and the mechanisms by which they can be adversely affected by drugs.",
            "references": []
            "pap_id": "178",
            "title": "The toxicity of fluoroacetate and the tricarboxylic acid cycle. 1949.",
            "authors": "Li?becq, C; Peters, R A",
            "chapter": "",
            "pages": "254-269",
            "journal": "Biochimica et Biophysica Acta",
            "pub_date": "1905-06-11",
            "pub_year": 1905,
            "volume": "1000",
            "issue": "",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "2673373.0",
            "pmcid": "",
            "abstract": "",
            "references": []
            "pap_id": "179",
            "title": "Rosiglitazone Induces Mitochondrial Biogenesis in Differentiated Murine 3T3-L1 and C3H/10T1/2 Adipocytes.",
            "authors": "Rong, James X; Klein, Jean-Louis D; Qiu, Yang; Xie, Mi; Johnson, Jennifer H; Waters, K Michelle; Zhang, Vivian; Kashatus, Jennifer A; Remlinger, Katja S; Bing, Nan; Crosby, Renae M; Jackson, Tymissha K; Witherspoon, Sam M; Moore, John T; Ryan, Terence E; Neill, Sue D; Strum, Jay C",
            "chapter": "",
            "pages": "179454",
            "journal": "PPAR Research",
            "pub_date": "1905-07-03",
            "pub_year": 1905,
            "volume": "2011",
            "issue": "",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1155/2011/179454",
            "pmid": "22013433.0",
            "pmcid": "PMC3195302",
            "abstract": "Growing evidence indicates that PPAR? agonists, including rosiglitazone (RSG), induce adipose mitochondrial biogenesis. By systematically analyzing mitochondrial gene expression in two common murine adipocyte models, the current study aimed to further establish the direct role of RSG and capture temporal changes in gene transcription. Microarray profiling revealed that in fully differentiated 3T3-L1 and C3H/10T1/2 adipocytes treated with RSG or DMSO vehicle for 1, 2, 4, 7, 24, and 48 hrs, RSG overwhelmingly increased mitochondrial gene transcripts time dependently. The timing of the increases was consistent with the cascade of organelle biogenesis, that is, initiated by induction of transcription factor(s), followed by increases in the biosynthesis machinery, and then by increases in functional components. The transcriptional increases were further validated by increased mitochondrial staining, citrate synthase activity, and O(2) consumption, and were found to be associated with increased adiponectin secretion. The work provided further insight on the mechanism of PPAR?-induced mitochondrial biogenesis in differentiated adipocytes.",
            "references": [
            "pap_id": "18",
            "title": "Comparison of negative inotropic potency, reversibility, and effects on calcium influx of six calcium channel antagonists in cultured myocardial cells.",
            "authors": "Barry, W H; Horowitz, J D; Smith, T W",
            "chapter": "",
            "pages": "51-59",
            "journal": "British Journal of Pharmacology",
            "pub_date": "1985-05-01",
            "pub_year": 1985,
            "volume": "85",
            "issue": "1",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1111/j.1476-5381.1985.tb08830.x",
            "pmid": "4027472.0",
            "pmcid": "PMC1916780",
            "abstract": "The negative inotropic effects of calcium channel antagonists on the myocardium were used as a standard for the definition and determination of potency of this group of drugs. The effects of six calcium channel antagonists (verapamil, methoxyverapamil (D600), nifedipine, lidoflazine, perhexiline and diltiazem) were compared on cultured chick embryo ventricular cells. Drug concentrations producing 50% inhibition of contractile amplitude, derived from linearized concentration-response curves, varied from 2.8 X 10(-8)M for nifedipine to 8.3 X 10(-7)M for perhexiline. Equipotent negative inotropic concentrations of verapamil, D600, perhexiline, diltiazem and lidoflazine produced a similar inhibitory effect on 45Ca uptake into cultured cells. Nifedipine produced no significant inhibition of 45Ca uptake. The time required for recovery of contractility after cessation of drug superfusion varied in the order lidoflazine greater than perhexiline greater than D600 greater than verapamil greater than nifedipine greater than diltiazem. This relative order accords closely with the reported in vivo half-lives of these drugs. It is concluded that while some inhibition of 45Ca2+ uptake into cardiac cells can be demonstrated with five of the six calcium channel blockers studied, the relationship between the degree of inhibition of calcium influx and negative inotropic effects may not be uniform for all calcium channel antagonists.",
            "references": []
            "pap_id": "180",
            "title": "Statin-Induced Myopathy Is Associated with Mitochondrial Complex III Inhibition.",
            "authors": "Schirris, Tom J J; Renkema, G Herma; Ritschel, Tina; Voermans, Nicol C; Bilos, Albert; van Engelen, Baziel G M; Brandt, Ulrich; Koopman, Werner J H; Beyrath, Julien D; Rodenburg, Richard J; Willems, Peter H G M; Smeitink, Jan A M; Russel, Frans G M",
            "chapter": "",
            "pages": "399-407",
            "journal": "Cell Metabolism",
            "pub_date": "2015-09-01",
            "pub_year": 2015,
            "volume": "22",
            "issue": "3",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/j.cmet.2015.08.002",
            "pmid": "26331605.0",
            "pmcid": "",
            "abstract": "Cholesterol-lowering statins effectively reduce the risk of major cardiovascular events. Myopathy is the most important adverse effect, but its underlying mechanism remains enigmatic. In C2C12 myoblasts, several statin lactones reduced respiratory capacity and appeared to be strong inhibitors of mitochondrial complex III (CIII) activity, up to 84% inhibition. The lactones were in general three times more potent inducers of cytotoxicity than their corresponding acid forms. The Qo binding site of CIII was identified as off-target of the statin lactones. These findings could be confirmed in muscle tissue of patients suffering from statin-induced myopathies, in which CIII enzyme activity was reduced by 18%. Respiratory inhibition in C2C12 myoblasts could be attenuated by convergent electron flow into CIII, restoring respiration up to 89% of control. In conclusion, CIII inhibition was identified as a potential off-target mechanism associated with statin-induced myopathies. Copyright ? 2015 Elsevier Inc. All rights reserved.",
            "references": [
            "pap_id": "181",
            "title": "High-throughput assessment of oxidative respiration in fish embryos: Advancing adverse outcome pathways for mitochondrial dysfunction.",
            "authors": "Souders, Christopher L; Liang, Xuefang; Wang, Xiaohong; Ector, Naomi; Zhao, Yuan H; Martyniuk, Christopher J",
            "chapter": "",
            "pages": "162-173",
            "journal": "Aquatic Toxicology",
            "pub_date": "2018-06-18",
            "pub_year": 2018,
            "volume": "199",
            "issue": "",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/j.aquatox.2018.03.031",
            "pmid": "29631217.0",
            "pmcid": "",
            "abstract": "Mitochondrial dysfunction is a prevalent molecular event that can result in multiple adverse outcomes. Recently, a novel high throughput method to assess metabolic capacity in fish embryos following exposure to chemicals has been adapted for environmental toxicology. Assessments of oxygen consumption rates using the Seahorse XF(e) 24/96 Extracellular Flux Analyzer (Agilent Technologies) can be used to garner insight into toxicant effects at early stages of development. Here we synthesize the current state of the science using high throughput metabolic profiling in zebrafish embryos, and present considerations for those wishing to adopt high throughput methods for mitochondrial bioenergetics into their research. Chemicals that have been investigated in zebrafish using this metabolic platform include herbicides (e.g. paraquat, diquat), industrial compounds (e.g. benzo-[a]-pyrene, tributyltin), natural products (e.g. quercetin), and anti-bacterial chemicals (i.e. triclosan). Some of these chemicals inhibit mitochondrial endpoints in the ?M-mM range, and reduce basal respiration, maximum respiration, and spare capacity. We present a theoretical framework for how one can use mitochondrial performance data in zebrafish to categorize chemicals of concern and prioritize mitochondrial toxicants. Noteworthy is that our studies demonstrate that there can be considerable variation in basal respiration of untreated zebrafish embryos due to clutch-specific effects as well as individual variability, and basal oxygen consumption rates (OCR) can vary on average between 100 and 300/ pmol/min/embryo. We also compare OCR between chorionated and dechorionated embryos, as both models are employed to test chemicals. After 24/ h, dechorionated embryos remain responsive to mitochondrial toxicants, although they show a blunted response to the uncoupling agent carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP); dechorionated embryos are therefore a viable option for investigations into mitochondrial bioenergetics. We present an adverse outcome pathway framework that incorporates endpoints related to mitochondrial bioenergetics. High throughput bioenergetics assays conducted using whole embryos are expected to support adverse outcome pathways for mitochondrial dysfunction. Copyright ? 2018 Elsevier B.V. All rights reserved.",
            "references": []
            "pap_id": "182",
            "title": "Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex.",
            "authors": "Esser, Lothar; Quinn, Byron; Li, Yong-Fu; Zhang, Minquan; Elberry, Maria; Yu, Linda; Yu, Chang-An; Xia, Di",
            "chapter": "",
            "pages": "281-302",
            "journal": "Journal of Molecular Biology",
            "pub_date": "1905-06-26",
            "pub_year": 1905,
            "volume": "341",
            "issue": "1",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/j.jmb.2004.05.065",
            "pmid": "15312779.0",
            "pmcid": "",
            "abstract": "Cytochrome bc(1) is an integral membrane protein complex essential for cellular respiration and photosynthesis; it couples electron transfer from quinol to cytochrome c to proton translocation across the membrane. Specific bc(1) inhibitors have not only played crucial roles in elucidating the mechanism of bc(1) function but have also provided leads for the development of novel antibiotics. Crystal structures of bovine bc(1) in complex with the specific Q(o) site inhibitors azoxystrobin, MOAS, myxothiazol, stigmatellin and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole were determined. Interactions, conformational changes and possible mechanisms of resistance, specific to each inhibitor, were defined. Residues and secondary structure elements that are capable of discriminating different classes of Q(o) site inhibitors were identified for the cytochrome b subunit. Directions in the displacement of the cd1 helix of cytochrome b subunit in response to various Q(o) site inhibitors were correlated to the binary conformational switch of the extrinsic domain of the iron-sulfur protein subunit. The new structural information, together with structures previously determined, provide a basis that, combined with biophysical and mutational data, suggest a modification to the existing classification of bc(1) inhibitors. bc(1) inhibitors are grouped into three classes: class P inhibitors bind to the Q(o) site, class N inhibitors bind to the Q(i) site and the class PN inhibitors target both sites. Class P contains two subgroups, Pm and Pf, that are distinct by their ability to induce mobile or fixed conformation of iron-sulfur protein.",
            "references": []
            "pap_id": "183",
            "title": "Inhibitors of NADH-ubiquinone reductase: an overview.",
            "authors": "Degli Esposti, M",
            "chapter": "",
            "pages": "222-235",
            "journal": "Biochimica et Biophysica Acta",
            "pub_date": "1998-05-06",
            "pub_year": 1998,
            "volume": "1364",
            "issue": "2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1016/s0005-2728(98)00029-2",
            "pmid": "9593904.0",
            "pmcid": "",
            "abstract": "This article provides an updated overview of the plethora of complex I inhibitors. The inhibitors are presented within the broad categories of natural and commercial compounds and their potency is related to that of rotenone, the classical inhibitor of complex I. Among commercial products, particular attention is dedicated to inhibitors of pharmacological or toxicological relevance. The compounds that inhibit the NADH-ubiquinone reductase activity of complex I are classified according to three fundamental types of action on the basis of available evidence and recent insights: type A are antagonists of the ubiquinone substrate, type B displace the ubisemiquinone intermediate, and type C are antagonists of the ubiquinol product. Copyright 1998 Elsevier Science B.V.",
            "references": []
            "pap_id": "184",
            "title": "Understanding and exploiting the mechanistic basis for selectivity of polyketide inhibitors of F(0)F(1)-ATPase.",
            "authors": "Salomon, A R; Voehringer, D W; Herzenberg, L A; Khosla, C",
            "chapter": "",
            "pages": "14766-14771",
            "journal": "Proceedings of the National Academy of Sciences of the United States of America",
            "pub_date": "1905-06-22",
            "pub_year": 1905,
            "volume": "97",
            "issue": "26",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1073/pnas.97.26.14766",
            "pmid": "11121076.0",
            "pmcid": "PMC18993",
            "abstract": "Recently, a family of polyketide inhibitors of F(0)F(1)-ATPase, including apoptolidin, ossamycin, and oligomycin, were shown to be among the top 0.1% most cell line selective cytotoxic agents of 37, 000 molecules tested against the 60 human cancer cell lines of the National Cancer Institute. Many cancer cells maintain a high level of anaerobic carbon metabolism even in the presence of oxygen, a phenomenon that is historically known as the Warburg effect. A mechanism-based strategy to sensitize such cells to this class of potent small molecule cytotoxic agents is presented. These natural products inhibit oxidative phosphorylation by targeting the mitochondrial F(0)F(1) ATP synthase. Evaluation of gene expression profiles in a panel of leukemias revealed a strong correlation between the expression level of the gene encoding subunit 6 of the mitochondrial F(0)F(1) ATP synthase (known to be the binding site of members of this class of macrolides) and their sensitivity to these natural products. Within the same set of leukemia cell lines, comparably strong drug-gene correlations were also observed for the genes encoding two key enzymes involved in central carbon metabolism, pyruvate kinase, and aspartate aminotransferase. We propose a simple model in which the mitochondrial apoptotic pathway is activated in response to a shift in balance between aerobic and anaerobic ATP biosynthesis. Inhibitors of both lactate formation and carbon flux through the Embden-Meyerhof pathway significantly sensitized apoptolidin-resistant tumors to this drug. Nine different cell lines derived from human leukemias and melanomas, and colon, renal, central nervous system, and ovarian tumors are also sensitized to killing by apoptolidin.",
            "references": [
            "pap_id": "185",
            "title": "Drug-induced mitochondrial toxicity.",
            "authors": "Chan, Katie; Truong, Don; Shangari, Nandita; O'Brien, Peter J",
            "chapter": "",
            "pages": "655-669",
            "journal": "Expert Opinion on Drug Metabolism & Toxicology",
            "pub_date": "2005-12-05",
            "pub_year": 2005,
            "volume": "1",
            "issue": "4",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1517/17425255.1.4.655",
            "pmid": "16863431.0",
            "pmcid": "",
            "abstract": "Mitochondria play a critical role in generating most of the cell's energy as ATP. They are also involved in other metabolic processes such as urea generation, haem synthesis and fatty acid beta-oxidation. Disruption of mitochondrial function by drugs can result in cell death by necrosis or can signal cell death by apoptosis (e.g., following cytochrome c release). Drugs that injure mitochondria usually do so by inhibiting respiratory complexes of the electron chain; inhibiting or uncoupling oxidative phosphorylation; inducing mitochondrial oxidative stress; or inhibiting DNA replication, transcription or translation. It is important to test for mitochondrial toxicity early in drug development as impairment of mitochondrial function can induce various pathological conditions that are life threatening or can increase the progression of existing mitochondrial diseases.",
            "references": [
            "pap_id": "186",
            "title": "Differential mitochondrial toxicity screening and multi-parametric data analysis.",
            "authors": "Tsiper, Maria V; Sturgis, Jennifer; Avramova, Larisa V; Parakh, Shilpa; Fatig, Raymond; Juan-Garc?a, Ana; Li, Nianyu; Rajwa, Bartek; Narayanan, Padma; Qualls, C W; Robinson, J Paul; Davisson, V Jo",
            "chapter": "",
            "pages": "",
            "journal": "Plos One",
            "pub_date": "1905-07-04",
            "pub_year": 1905,
            "volume": "7",
            "issue": "10",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1371/journal.pone.0045226",
            "pmid": "23077490.0",
            "pmcid": "PMC3471932",
            "abstract": "Early evaluation of new drug entities for their potential to cause mitochondrial dysfunction is becoming an important task for drug development. Multi-parametric high-content screening (mp-HCS) of mitochondrial toxicity holds promise as a lead in-vitro strategy for drug testing and safety evaluations. In this study, we have developed a mp-HCS and multi-parametric data analysis scheme for assessing cell responses to induced mitochondrial perturbation. The mp-HCS measurements are shown to be robust enough to allow for quantitative comparison of biological systems with different metabolic pathways simulated by alteration of growth media. Substitution of medium glucose for galactose sensitized cells to drug action and revealed novel response parameters. Each compound was quantitatively characterized according to induced phenotypic changes of cell morphology and functionality measured by fluorescent biomarkers for mitochondrial activity, plasma membrane permeability, and nuclear morphology. Descriptors of drug effects were established by generation of a SCRIT (Specialized-Cell-Response-to-Induced-Toxicity) vector, consisting of normalized statistical measures of each parameter at each dose and growth condition. The dimensionality of SCRIT vectors depends on the number of parameters chosen, which in turn depends on the hypothesis being tested. Specifically, incorporation of three parameters of response into SCRIT vectors enabled clustering of 84 training compounds with known pharmacological and toxicological activities according to the degree of toxicity and mitochondrial involvement. Inclusion of 6 parameters enabled the resolution of more subtle differences between compounds within a common therapeutic class; scoring enabled a ranking of statins in direct agreement with clinical outcomes. Comparison of drug-induced changes required variations in glucose for separation of mitochondrial dysfunction from other types of cytotoxicity. These results also demonstrate that the number of drugs in a training set, the choice of parameters used in analysis, and statistical measures are fundamental for specific hypothesis testing and assessment of quantitative phenotypic differences.",
            "references": []
            "pap_id": "187",
            "title": "High content of mitochondrial glycerol-3-phosphate dehydrogenase in pancreatic islets and its inhibition by diazoxide.",
            "authors": "MacDonald, M J",
            "chapter": "",
            "pages": "8287-8290",
            "journal": "The Journal of Biological Chemistry",
            "pub_date": "1981-08-25",
            "pub_year": 1981,
            "volume": "256",
            "issue": "16",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "",
            "pmid": "6790537.0",
            "pmcid": "",
            "abstract": "Homogenates of isolated pancreatic islets contain 40-70 times as much flavin-linked glycerol-3-phosphate dehydrogenase (EC as homogenates of whole pancreas, liver, heart, or skeletal muscle when the activity is assayed with either iodonitrotetrazolium or with dichloroindophenol as an electron acceptor. Intact mitochondria from islets release 3HOH from [2-3H]glycerol phosphate 7 times faster than do skeletal muscle mitochondria. The activity of the cytosolic, NAD-linked, glycerol phosphate dehydrogenase (EC in pancreatic islets is comparable to that of the mitochondrial dehydrogenase so a glycerol phosphate shuttle is possible in pancreatic islets. Diazoxide, an inhibitor of insulin release in vivo and in vitro, inhibits the islet mitochondrial glycerol phosphate dehydrogenase in all three of the assays mentioned above at concentrations that inhibit insulin release and CO2 formation from glucose by isolated pancreatic islets. Diazoxide does not inhibit the dehydrogenase in mitochondria from skeletal muscle, liver, and heart. A slight inhibition in mitochondria from whole pancreas can be accounted for as inhibition of the islet dehydrogenase because no inhibition is observed in mitochondria from pancreas of rats treated with alloxan, an agent that causes diabetes by destroying pancreatic beta cells. The results of this study are compatible with the hypothesis that the mitochondrial glycerol phosphate dehydrogenase has a key role in stimulus-secretion coupling in the pancreatic beta cell during glucose-induced insulin release.",
            "references": [
            "pap_id": "188",
            "title": "Mitochondrial ATP-sensitive potassium channels inhibit apoptosis induced by oxidative stress in cardiac cells.",
            "authors": "Akao, M; Ohler, A; O'Rourke, B; Marb?n, E",
            "chapter": "",
            "pages": "1267-1275",
            "journal": "Circulation Research",
            "pub_date": "2001-06-22",
            "pub_year": 2001,
            "volume": "88",
            "issue": "12",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1161/hh1201.092094",
            "pmid": "11420303.0",
            "pmcid": "",
            "abstract": "Mitochondria can either enhance or suppress cell death. Cytochrome c release from mitochondria and depolarization of the mitochondrial membrane potential (DeltaPsi) are crucial events in triggering apoptosis. In contrast, activation of mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels prevents lethal ischemic injury in vivo, implicating these channels as key players in the process of ischemic preconditioning. We probed the relationship between mitoK(ATP) channels and apoptosis in cultured neonatal rat cardiac ventricular myocytes. Incubation with 200 micromol/L hydrogen peroxide induced TUNEL positivity, cytochrome c translocation, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and dissipation of DeltaPsi. Pharmacological opening of mitoK(ATP) channels by diazoxide (100 micromol/L) preserved mitochondrial integrity and suppressed all markers of apoptosis. Diazoxide prevented DeltaPsi depolarization in a concentration-dependent manner (EC(50) approximately 40 micromol/L, with saturation by 100 micromol/L), as shown by both flow cytometry and quantitative image analysis of cells stained with fluorescent DeltaPsi indicators. These cytoprotective effects of diazoxide were reproduced by pinacidil, another mitoK(ATP) agonist, and blocked by the mitoK(ATP) channel antagonist 5-hydroxydecanoate (500 micromol/L). Our findings identify a novel mitochondrial pathway that is protective against apoptosis. The results also pinpoint mitoK(ATP) channels as logical therapeutic targets in diseases of enhanced apoptosis and oxidative stress.",
            "references": [
            "pap_id": "189",
            "title": "5-Hydroxydecanoate is metabolised in mitochondria and creates a rate-limiting bottleneck for beta-oxidation of fatty acids.",
            "authors": "Hanley, Peter J; Dr?se, Stefan; Brandt, Ulrich; Lareau, Rachel A; Banerjee, Abir L; Srivastava, D K; Banaszak, Leonard J; Barycki, Joseph J; Van Veldhoven, Paul P; Daut, J?rgen",
            "chapter": "",
            "pages": "307-318",
            "journal": "The Journal of Physiology",
            "pub_date": "2005-01-15",
            "pub_year": 2005,
            "volume": "562",
            "issue": "Pt 2",
            "Issn": "",
            "Isbn": "",
            "url": "",
            "doi": "10.1113/jphysiol.2004.073932",
            "pmid": "15513944.0",
            "pmcid": "PMC1665522",
            "abstract": "5-Hydroxydecanoate (5-HD) blocks pharmacological and ischaemic preconditioning, and has been postulated to be a specific inhibitor of mitochondrial ATP-sensitive K(+) (K(ATP)) channels. However, recent work has shown that 5-HD is activated to 5-hydroxydecanoyl-CoA (5-HD-CoA), which is a substrate for the first step of beta-oxidation. We have now analysed the complete beta-oxidation of 5-HD-CoA using specially synthesised (and purified) substrates and enzymes, as well as isolated rat liver and heart mitochondria, and compared it with the metabolism of the physiological substrate decanoyl-CoA. At the second step of beta-oxidation, catalysed by enoyl-CoA hydratase, enzyme kinetics were similar using either decenoyl-CoA or 5-hydroxydecenoyl-CoA as substrate. The last two steps were investigated using l-3-hydroxyacyl-CoA dehydrogenase (HAD) coupled to 3-ketoacyl-CoA thiolase. V(max) for the metabolite of 5-HD (3,5-dihydroxydecanoyl-CoA) was fivefold slower than for the corresponding metabolite of decanoate (l-3-hydroxydecanoyl-CoA). The slower kinetics were not due to accumulation of d-3-hydroxyoctanoyl-CoA since this enantiomer did not inhibit HAD. Molecular modelling of HAD complexed with 3,5-dihydroxydecanoyl-CoA suggested that the 5-hydroxyl group could decrease HAD turnover rate by interacting with critical side chains. Consistent with the kinetic data, 5-hydroxydecanoyl-CoA alone acted as a weak substrate in isolated mitochondria, whereas addition of 100 mum 5-HD-CoA inhibited the metabolism of decanoyl-CoA or lauryl-carnitine. In conclusion, 5-HD is activated, transported into mitochondria and metabolised via beta-oxidation, albeit with rate-limiting kinetics at the penultimate step. This creates a bottleneck for beta-oxidation of fatty acids. The complex metabolic effects of 5-HD invalidate the use of 5-HD as a blocker of mitochondrial K(ATP) channels in studies of preconditioning.",
            "references": [