Quinone reductase II
Dotaz
Zobrazit nápovědu
Primaquine is a traditional antimalarial drug with low parasitic resistance and generally good acceptance at higher doses, which has been used for over 60 years in malaria treatment. However, several limitations related to its hematotoxicity have been reported. It is believed that this toxicity comes from the hydroxylation of the C-5 and C-6 positions of its 8-aminoquinoline ring before binding to the molecular target: the quinone reductase II (NQO2) human protein. In this study we propose primaquine derivatives, with substitution at position C-6 of the 8-aminoquinoline ring, planned to have better binding to NQO2, compared to primaquine, but with a reduced toxicity related to the C-5 position being possible to be oxidized. On this sense the proposed analogues were suggested in order to reduce or inhibit hydroxylation and further oxidation to hemotoxic metabolites. Five C-6 substituted primaquine analogues were selected by de novo design and further submitted to docking and molecular dynamics simulations. Our results suggest that all analogues bind better to NQO2 than primaquine and may become better antimalarials. However, the analogues 3 and 4 are predicted to have a better activity/toxicity balance.
- MeSH
- chinonreduktasy antagonisté a inhibitory chemie MeSH
- inhibitory enzymů chemie MeSH
- katalytická doména MeSH
- lidé MeSH
- primachin analogy a deriváty chemie MeSH
- sekundární struktura proteinů MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- termodynamika MeSH
- vazba proteinů MeSH
- vodíková vazba MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Quercetin and galangin can change the activity of glutathione reductase. Quercetin (a catechol structure in the B-ring) and galangin (any hydroxyl group in the B-ring) have different biological activities but, both possess high antioxidant abilities. Quercetin during the antioxidative action, is converted into an oxidized products (o-semiquinone and o-quinone), and subsequently glutathionyl adducts may be formed or SH-enzyme can be inhibited. We have tried to see whether inhibition of glutathione reductase (GR) can be influenced by preincubation of enzyme with NADPH (a creation of reduced form of enzyme, GRH(2)) and whether diaphorase activity of the enzyme is decreased by these flavonoids. The results confirmed that quercetin inhibits GRH(2) and inhibition is reduced by addition of EDTA or N-acetylcysteine. Both of flavonoids have no effect on diaphorase activity of glutathione reductase and this enzyme could increase the production of free radicals by catalysis of reduction of o-quinone during action of quercetin in vivo.
Mitochondria play a pivotal role in apoptosis: permeabilization of the outer mitochondrial membrane and the release of pro-apoptotic proteins from the intermembrane space of mitochondria are regarded as the key event in apoptosis induction. Here we demonstrate how non-toxic doses of the mitochondrial Complex II inhibitor thenoyltrifluoroacetone (TTFA), which specifically inhibits the ubiquinone-binding site of succinate dehydrogenase (SDH), synergistically stimulated cell death, induced by harmless doses of cisplatin in a panel of chemoresistant neuroblastoma cell lines. Apoptotic cell death was confirmed by cytochrome c release from the mitochondria, cleavage of poly ADP-ribose polymerase, processing of caspase-3, which is an important executive enzyme in apoptosis, and caspase-3-like activity. Methyl malonate, an inhibitor of the SDHA subunit partially reversed apoptosis stimulated by TTFA in SK-N-BE(2) neuroblastoma cells (NB), indicating that sensitization requires oxidation of succinate. In contrast, in IMR-32 NB cells, the same concentrations of TTFA markedly suppressed cisplatin-induced apoptosis. Comparison of oxygen consumption in cisplatin-resistant SK-N-BE(2) and cisplatin-sensitive IMR-32 cells clearly demonstrated impaired Complex II activity in IMR-32 cells. We also found that in SK-N-BE(2) cells co-treatment with cisplatin and TTFA markedly stimulated formation of reactive oxygen species (ROS), whereas in IMR cells, cisplatin-mediated ROS production was attenuated by TTFA, which explains apoptosis suppression in these cells. Thus, functionally active SDH is a prerequisite for the ROS-mediated sensitization to treatment by TTFA.
- MeSH
- apoptóza účinky léků MeSH
- chemorezistence MeSH
- cílená molekulární terapie * MeSH
- cisplatina farmakologie MeSH
- kyselina jantarová metabolismus MeSH
- lidé MeSH
- mitochondrie účinky léků enzymologie MeSH
- nádorové buněčné linie MeSH
- nádorové proteiny antagonisté a inhibitory MeSH
- neuroblastom patologie MeSH
- oxidace-redukce MeSH
- protinádorové látky farmakologie MeSH
- reaktivní formy kyslíku metabolismus MeSH
- respirační komplex II antagonisté a inhibitory MeSH
- screeningové testy protinádorových léčiv MeSH
- spotřeba kyslíku účinky léků MeSH
- superoxidy metabolismus MeSH
- synergismus léků MeSH
- thenoyltrifluoraceton farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Mitochondrial respiratory chain is organised into supramolecular structures that can be preserved in mild detergent solubilisates and resolved by native electrophoretic systems. Supercomplexes of respiratory complexes I, III and IV as well as multimeric forms of ATP synthase are well established. However, the involvement of complex II, linking respiratory chain with tricarboxylic acid cycle, in mitochondrial supercomplexes is questionable. Here we show that digitonin-solubilised complex II quantitatively forms high molecular weight structures (CIIhmw) that can be resolved by clear native electrophoresis. CIIhmw structures are enzymatically active and differ in electrophoretic mobility between tissues (500 - over 1000 kDa) and cultured cells (400-670 kDa). While their formation is unaffected by isolated defects in other respiratory chain complexes, they are destabilised in mtDNA-depleted, rho0 cells. Molecular interactions responsible for the assembly of CIIhmw are rather weak with the complexes being more stable in tissues than in cultured cells. While electrophoretic studies and immunoprecipitation experiments of CIIhmw do not indicate specific interactions with the respiratory chain complexes I, III or IV or enzymes of the tricarboxylic acid cycle, they point out to a specific interaction between CII and ATP synthase.
- MeSH
- buněčné linie MeSH
- elektronový transportní řetězec chemie metabolismus MeSH
- lidé MeSH
- metabolické sítě a dráhy MeSH
- mitochondrie genetika metabolismus MeSH
- molekulová hmotnost MeSH
- orgánová specificita MeSH
- oxidativní fosforylace MeSH
- respirační komplex II chemie metabolismus MeSH
- transport elektronů MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Increasing evidence points to the respiratory Complex II (CII) as a source and modulator of reactive oxygen species (ROS). Both functional loss of CII as well as its pharmacological inhibition can lead to ROS generation in cells, with a relevant impact on the development of pathophysiological conditions, i.e. cancer and neurodegenerative diseases. While the basic framework of CII involvement in ROS production has been defined, the fine details still await clarification. It is important to resolve these aspects to fully understand the role of CII in pathology and to explore its therapeutic potential in cancer and other diseases.
- MeSH
- cílená molekulární terapie * MeSH
- lidé MeSH
- mitochondriální nemoci farmakoterapie metabolismus patologie MeSH
- mitochondrie metabolismus patologie MeSH
- reaktivní formy kyslíku metabolismus MeSH
- respirační komplex II metabolismus MeSH
- transport elektronů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Respiratory complex II (CII, succinate dehydrogenase, SDH) inhibition can induce cell death, but the mechanistic details need clarification. To elucidate the role of reactive oxygen species (ROS) formation upon the ubiquinone-binding (Qp) site blockade, we substituted CII subunit C (SDHC) residues lining the Qp site by site-directed mutagenesis. Cell lines carrying these mutations were characterized on the bases of CII activity and exposed to Qp site inhibitors MitoVES, thenoyltrifluoroacetone (TTFA) and Atpenin A5. We found that I56F and S68A SDHC variants, which support succinate-mediated respiration and maintain low intracellular succinate, were less efficiently inhibited by MitoVES than the wild-type (WT) variant. Importantly, associated ROS generation and cell death induction was also impaired, and cell death in the WT cells was malonate and catalase sensitive. In contrast, the S68A variant was much more susceptible to TTFA inhibition than the I56F variant or the WT CII, which was again reflected by enhanced ROS formation and increased malonate- and catalase-sensitive cell death induction. The R72C variant that accumulates intracellular succinate due to compromised CII activity was resistant to MitoVES and TTFA treatment and did not increase ROS, even though TTFA efficiently generated ROS at low succinate in mitochondria isolated from R72C cells. Similarly, the high-affinity Qp site inhibitor Atpenin A5 rapidly increased intracellular succinate in WT cells but did not induce ROS or cell death, unlike MitoVES and TTFA that upregulated succinate only moderately. These results demonstrate that cell death initiation upon CII inhibition depends on ROS and that the extent of cell death correlates with the potency of inhibition at the Qp site unless intracellular succinate is high. In addition, this validates the Qp site of CII as a target for cell death induction with relevance to cancer therapy.
- MeSH
- buněčná smrt fyziologie MeSH
- konformace proteinů MeSH
- lidé MeSH
- mitochondrie metabolismus fyziologie MeSH
- molekulární sekvence - údaje MeSH
- mutageneze cílená MeSH
- respirační komplex II chemie genetika metabolismus fyziologie MeSH
- sekvence aminokyselin MeSH
- ubichinon chemie genetika metabolismus MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
With the arrival of the third millennium, in spite of unprecedented progress in molecular medicine, cancer remains as untamed as ever. The complexity of tumours, dictating the potential response of cancer cells to anti-cancer agents, has been recently highlighted in a landmark paper by Weinberg and Hanahan on hallmarks of cancer [1]. Together with the recently published papers on the complexity of tumours in patients and even within the same tumour (see below), the cure for this pathology seems to be an elusive goal. Indisputably, the strategy ought to be changed, searching for targets that are generally invariant across the landscape of neoplastic diseases. One such target appears to be the mitochondrial complex II (CII) of the electron transfer chain, a recent focus of research. We document and highlight this particularly intriguing target in this review paper and give examples of drugs that use CII as their molecular target. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease.
- MeSH
- apoptóza účinky léků MeSH
- biologické modely MeSH
- lidé MeSH
- mitochondrie účinky léků metabolismus MeSH
- molekulární struktura MeSH
- nádory farmakoterapie metabolismus patologie MeSH
- protinádorové látky chemie klasifikace terapeutické užití MeSH
- respirační komplex II antagonisté a inhibitory metabolismus MeSH
- transport elektronů účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
BACKGROUND: Renal cell carcinoma (RCC) is a disease typified by anomalies in cell metabolism. The function of mitochondria, including subunits of mitochondrial respiratory complex II (CII), in particular SDHB, are often affected. Here we investigated the state and function of CII in RCC patients. METHODS: We evaluated tumour tissue as well as the adjacent healthy kidney tissue of 78 patients with RCC of different histotypes, focusing on their mitochondrial function. As clear cell RCC (ccRCC) is by far the most frequent histotype of RCC, we focused on these patients, which were grouped based on the pathological WHO/ISUP grading system to low- and high-grade patients, indicative of prognosis. We also evaluated mitochondrial function in organoids derived from tumour tissue of 7 patients. RESULTS: ccRCC tumours were characterized by mutated von Hippel-Lindau gene and high expression of carbonic anhydrase IX. We found low levels of mitochondrial DNA, protein and function, together with CII function in ccRCC tumour tissue, but not in other RCC types and non-tumour tissues. Mitochondrial content increased in high-grade tumours, while the function of CII remained low. Tumour organoids from ccRCC patients recapitulated molecular characteristics of RCC tissue. CONCLUSIONS: Our findings suggest that the state of CII, epitomized by its assembly and SDHB levels, deteriorates with the progressive severity of ccRCC. These observations hold the potential for stratification of patients with worse prognosis and may guide the exploration of targeted therapeutic interventions.
- MeSH
- antigeny nádorové MeSH
- dospělí MeSH
- karboanhydrasa IX metabolismus genetika MeSH
- karcinom z renálních buněk * patologie metabolismus genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- mitochondriální DNA genetika metabolismus MeSH
- mitochondrie * metabolismus patologie genetika MeSH
- mutace MeSH
- nádorový supresorový protein VHL genetika metabolismus MeSH
- nádory ledvin * patologie metabolismus genetika MeSH
- respirační komplex II * metabolismus genetika MeSH
- senioři MeSH
- sukcinátdehydrogenasa genetika metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVES: Ingestion of aristolochic acid (AA) is associated with development of urothelial tumors linked with aristolochic acid nephropathy, and is implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. Aristolochic acid I (AAI), the major toxic component of AA, is more toxic than its demethoxylated derivate AAII. A different enzymatic conversion of both carcinogens might be one of the reasons explaining this feature. Therefore, the present study has been designed to compare efficiency of human NAD(P)H:quinone oxidoreductase (NQO1) and phase II enzymes such as sulfotransferases (SULTs) and N,O-acetyltransferases (NATs) to activate AAI and AAII in vitro. In addition, to investigate the molecular mechanisms of AAI and AAII reduction by human NQO1, molecular modeling was used to compare interactions of AAI and AAII with the active site of this enzyme. METHODS: DNA adduct formation by AAI and AAII was investigated by the nuclease P1 version of the 32P-postlabeling method. In silico docking, employing soft-soft (flexible) docking procedure, was used to study the interactions of AAI and AAII with the active site of human NQO1. RESULTS: Human NQO1 activated AAI and AAII, generating DNA adduct patterns reproducing those found in several species including human exposed to these compounds. These results demonstrate that NQO1 is capable of reducing both AAs to reactive species binding to DNA. However, concentrations required for half-maximum DNA binding mediated by NQO1 were higher for AAII (158 µM) than for AAI (17 µM). One of the reasons causing this phenomenon is a lower efficiency of NQO1 to reduce AAII than AAI we found in this work; although both AAI and AAII are bound with similar binding affinities to the NQO1 active site, the binding orientation of AAII in the active site of NQO1 does not favor the effective reduction of its nitro group. Because reduced nitro-aromatics are often further activated by SULTs or NATs, their roles in AAI and AAII activation were investigated. Our results indicate that phase II reactions do not stimulate the bioactivation of AAs; neither enzymes present in human hepatic cytosols nor human SULT1A1, 1A2, 1A3, 1E, or 2A nor NAT1 or NAT2 further enhanced DNA adduct formation by AAs. In contrast, human SULT1A1, 1A2 and 1A3 as well as NAT1 and NAT2 enzymes even inhibited NQO1-mediated bioactivation of AAII. Therefore, under the in vitro conditions used, DNA adducts arise by enzymatic reduction of AAs through the formation of N-hydroxyaristolactams that are spontaneously decomposed to the reactive species forming DNA adducts. CONCLUSION: The results found in this study emphasize the importance of NQO1 in the metabolic activation of AAI and AAII and provide the evidence that initial nitroreduction is the rate limiting step in their activation. This enzyme is more effective in activation of AAI relative to AAII, which might contribute to its lower binding to DNA found both in vitro and in vivo, Moreover, inhibition effects of conjugation reactions on AAII activation might further contribute to its decreased capability of forming DNA adducts and its lower toxicity comparing with AAI.
- MeSH
- acetyltransferasy chemie metabolismus fyziologie MeSH
- adukty DNA metabolismus MeSH
- aktivace enzymů MeSH
- biotransformace fyziologie MeSH
- katalytická doména MeSH
- kultivované buňky MeSH
- kyseliny aristolochové chemie farmakokinetika MeSH
- laktamy metabolismus farmakokinetika MeSH
- lidé MeSH
- molekulární konformace MeSH
- molekulární modely MeSH
- NAD(P)H dehydrogenasa (chinon) chemie metabolismus fyziologie MeSH
- sulfotransferasy chemie metabolismus fyziologie MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Mitochondrial complex II or succinate dehydrogenase (SDH) is at the crossroads of oxidative phosphorylation and the tricarboxylic acid cycle. It has been shown that Sdh5 (SDHAF2/SDH5 in mammals) is required for flavination of the subunit Sdh1 (SDHA in human cells) in yeast. Here we demonstrate that in human breast cancer cells, SDHAF2/SDH5 is dispensable for SDHA flavination. In contrast to yeast, CRISPR-Cas9 nickase-mediated SDHAF2 KO breast cancer cells feature flavinated SDHA and retain fully assembled and functional complex II, as well as normal mitochondrial respiration. Our data show that SDHA flavination is independent of SDHAF2 in breast cancer cells, employing an alternative mechanism.
- MeSH
- flaviny MeSH
- genový knockdown MeSH
- lidé MeSH
- mitochondriální proteiny genetika metabolismus MeSH
- mitochondrie genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- nádorové proteiny genetika metabolismus MeSH
- nádory prsu genetika metabolismus MeSH
- posttranslační úpravy proteinů * MeSH
- respirační komplex II genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH