Main research directions. Synthesis of prooxidant xenobiotics (quinones, aromatic nitrocompounds, N-oxides, etc.), studies of interaction of these compounds with flavoenzymes and their impact on their cytotoxicity, mechanisms of flavoenzyme catalysis, prooxidant cytotoxicity of polyphenolic compounds. Since 1992, the Department collaborates with research centers in Lithuania, Belarus, Great Britain, Spain, Italy, USA, New Zealand, the Netherlands, France, Sweden, and Ukraine. We have participated in projects supported by the EC (2), NATO (2), the Royal Swedish Academy of Sciences (1), bilateral projects with France (2), Belarus (1), and Ukraine (1), COST actions (4), received a number of grants from LSSSF and RCL including the project funded by European Social Fund (ESF) under the Human Resources Development Action Programme, the Global Grant measure (2011-2015).
Synthesis of prooxidant compounds. We have synthesized or resynthesized over 300 compounds of different groups – nitroaromatic and nitroheterocyclic compounds, aliphatic nitrates, aromatic N-oxides, quinones, benzofuroxans, etc. (Fig. 1), examined their electrochemical properties and characterized their reduction energetics by quantum-mechanical methods.
Fig. 1. High-energy substances tetryl (1), keto-RDX (2), PETN (3), and other groups of compounds: nitrobenzimidazolonesi (4), aziridinyl-substituted quinones (5) and nitrobenzenes (6), nitrofurans (7), benzofuroxanes (8), di-N-oxides of quinoxaline (9) and 1,2,4-benzotriazine (10), and heterocyclic oximes (11).
The mechanisms of single- and two-electron reduction of quinones, nitroaromatic compounds, and other prooxidants by flavoenzymes. Flavoenzymes electrontransferases reduce quinones, aromatic nitrocompounds and N-oxides into their free radicals. The latter are reoxidized by oxygen with the formation of superoxide and other reactive oxygen species (ROS) causing the oxidative stress which significantly determines their cytotoxicity , antitumour and other therapeutic activity of the compounds. The enzymatic single-electron reduction reactions of the compounds have been examined applying mammalian cytochrome P-450 reductase, NO-synthase (in collaboration with J.-L. Boucher, Universite de Paris V), Anabaena sp. and P. falciparum ferredoxin:NADP+ reductase (in collaboration with C. Gomez-Moreno, Universidad de Zaragoza, and A. Aliverti, Universita de Milano), and mixed single- and two-electron reduction reactions by using mammalian mitochondrial complex I (in collaboration with A.D. Vinogradov, Moscow University), apoptosis inducting factor (in collaboration with I.F. Sevrioukova, California University, Irvine), lipoamide dehydrogenase, plant and bacterial thioredoxin reductases (in collaboration with J.-P. Jacquot and N. Rouhier, Universite de Nancy), and bacterial flavohemoglobin (in collaboration with L. Baciou and F. Lederer, Universite de Paris Sud). The single-electron reduction of quinones and nitroaromatic compounds by aforementioned enzymes is described by Marcus' model, and the rates are weakly influenced by the structure and VdWvol of the compounds. In several cases, the redox states of the enzymes responsible for the rate-limiting stage have been identified. Mixed single- and two-electron reduction is characterized by e-,H+,e- mechanism determined by the destabilization of neutral flavin semiquinone.
Two-electron reduction by mammalian DT-diaphorase or bacterial nitroreductases decreases the cytotoxicity of simple quinone compounds, but increases the cytotoxicity of aziridinyl-substituted quinones and nitroaromatic compounds due to formation of DNA-alkylating products (Fig. 2).
Fig. 2. Two-electron reduction of aziridinyl-substituted quinones with subsequent alkylation of DNA.
It has been examined the reduction of quinones and nitroaromatic compounds mediated by DT-diaphorase, E. cloacae nitroreductase B (in collaboration with R. Koder, Kentucky University), PETN reductase (in collaboration with N.S. Scrutton, Manchester University), and E. coli nitroreductase A (in collaboration with D.F. Ackerley, Welington University). These reactions have been found to be characterized by a strong substrate structure specificity because of significant conformational changes of the enzyme active sites and by negative ∆S≠. Depending on the stability of anionic flavin semiquinone radical, these reactions proceed through single- (H-) or three-step (e-,H+,e-) hydride transfer.
Mammalian cell culture cytotoxicity of quinones and nitroaromatic compounds. Applying several transformed and primary mammalian cell cultures (Center of Innovative Medicine), the cytotoxic action of quinones and nitroaromatics has been found to be mainly determined by the oxidative stress, and that the cytotoxicity of compounds increases with an increase in their single-electron reduction potential, with ∆log cL50/∆E17 of -8 – -10 V-1. Significantly increased cytotoxicity of aziridinyl-substituted quinones is attributed to their activation by DT-diaphorase. Aziridinyl-benzoquinone resistant cell sub-lines possess 10-fold decreased activity with DT-diaphorase and glutathione-S-transferase, whereas the activity of prooxidant and antioxidant enzymes varies in the limits of ±50%.
Quinones and nitroaromatic compounds as inhibitors and subversive substrates of antioxidant flavoenzymes. Quinones and nitroaromatic compounds inhibit the antioxidant mammalian and parasite flavoenzymes glutathione reductase (GR) and trypanothione reductase (TR), and mammalian thioredoxin reductase (TrxR), thus inhibiting the regeneration of –SH groups in the cell. In parallel, they may be reduced by the aforementioned enzymes in mixed single- and two-electron way, thus subverting their antioxidant functions. The above mechanisms may be employed in the design of new antiparasitic and anticancer agents. The interaction of the above compounds has been examined with erythrocyte and P. falciparum GR (in collaboration with E. Davioud-Charvet, Universite de Strasbourg), T. congolense TR (in collaboration with J. Blanchard, Albert Einstein College of Medicine, NY), and mammalian TrxR (in collaboration with E. Arner, Karolinska Institutet). A number of efficient inhibitors of the above enzymes have been discovered. It has been established that apart from the reduced flavin, the above compounds may be reduced by the catalytic SeH-SH-moiety of TrxR. In collaboration with P. Grellier (MNHN, Paris), the relation between the efficiency of inhibition of P. falciparum GR by quinones and nitroaromatic compounds and their in vitro antiplasmodial activity has been established.
Prooxidant cytotoxicity of polyphenols. The antioxidant activity of polyphenols (flavonoids, polihydroxybenzenes, etc.) relies on neutralization of ROS. Therefore they are considered as useful food components; their antitumour and antiparasitic activity have been examined. However, polyphenols possess prooxidant cytotoxicity, because during their (auto)oxidation, H2O2 and quinone/quinomethide oxidation products are formed which alkylate –SH groups in the cell. The cytotoxicity of polyphenols in several cell lines increases with a decrease in their single-electron oxidation potential (∆log cL50/∆E27 = 1 – 2 V-1 ), and with an increase in their lipophilicity. Hydroxylation and oxidative demethylation of polyphenols by cytochromes P-450 increases their cytotoxicity, whereas the methylation by catechol-O-methyltransferase decreases it. During the collaboration with P. Venskutonis (Kaunas Technological University), prooxidant cytotoxicity of a number of plant and herb polyphenolic extracts has been characterized.
Key grants 2012-2017
National Research Projects funded by Research Council of Lithuania
- Heterocyclic N-oxides: synthesis, cytotoxicity, and interaction with target enzymes (MIP-080/2011). Dr. J. Šarlauskas. 2011-2012.
- European Social Fund (ESF) under the Human Resources Development Action Programme, the Global Grant measure, project No. VP1-3.1-ŠMM-07-K-01-103. Molecular mechanisms of toxicity and antitumor activity of quinones and polyphenols: enzymatic redox reactions, cytotoxicity, signal transduction and proteomics. Habil. Dr. N. Čėnas. 2011–2015.
- New generation N-heterocyclic quinones: rational synthesis and elucidation of anticancer activity (MIP-032/2014). Dr. Ž. Anusevičius. 2014-2016.
International Research Projects
- COST Action CM0801. New drugs for neglected diseases. Dr. J. Šarlauskas. Habil. Dr. N. Čėnas. 2009-2012.
- COST Action CM1307. Targeted chemotherapy towards diseases by endoparasites. Dr. J. Šarlauskas. Habil. Dr. N. Čėnas. 2014-2017.
- Bilateral Lithuanian-French programme Gilibert. Quinones and nitroaromatic compounds as the subversive substrates of flavohemoglobins: mechanisms and biomedicinal implications (No. TAP LZ 07/2013). Habil. Dr. N. Čėnas 2013-2014.
- Bilateral Lithuania-Belarus programme. The characterization of the inter-protein interaction and the modulators of the redox equivalent transfer in steroid hydroxylation systems (No. TAP LB 02/2013). Dr. Ž. Anusevičius. 2013-2014.
- 5. Bilateral Lithuania-Ukraine programme. Investigation of L- and D-lactate: cytochrome с oxidoreductases isolated from the recombinant yeast Hansenula polymorpha and their usage for construction of amperometric biosensors (No. TAP LU 03/2014). Dr. K. Krikštopaitis. 2014-2015.
Valiauga, B., Misevičienė, L., Rich, M., Ackerley, D., Šarlauskas, J. and Čėnas, N., 2018. Mechanism of Two-/Four-Electron Reduction of Nitroaromatics by Oxygen-Insensitive Nitroreductases: The Role of a Non-Enzymatic Reduction Step. Molecules, 23(7), p.1672.
Moussaoui, M., Misevičienė, L., Anusevičius, Ž., Marozienė, A., Lederer, F., Baciou, L. and Čėnas, N., 2018. Quinones and nitroaromatic compounds as subversive substrates of Staphylococcus aureus flavohemoglobin. Free Radical Biology and Medicine, 123, pp.107-115.
Valiauga, B., Williams, E.M., Ackerley, D.F. and Čėnas, N., 2017. Reduction of quinones and nitroaromatic compounds by Escherichia coli nitroreductase A (NfsA): characterization of kinetics and substrate specificity. Archives of biochemistry and biophysics, 614, pp.14-22.
Smutok, O., Karkovska, M., Serkiz, R., Vus, В., Čenas, N. and Gonchar, M., 2017. A novel mediatorless biosensor based on flavocytochrome b 2 immobilized onto gold nanoclusters for non-invasive L-lactate analysis of human liquids. Sensors and Actuators B: Chemical, 250, pp.469-475.
Peciukaityte-Alksne, M., Šarlauskas, J., Miseviciene, L., Maroziene, A., Cenas, N., Krikštopaitis, K., Staniulyte, Z. and Anusevicius, Ž., 2017. Flavoenzyme-mediated reduction reactions and antitumor activity of nitrogen-containing tetracyclic ortho-quinone compounds and their nitrated derivatives. EXCLI journal, 16, p.663.
Šarlauskas, J., Tamulienė, J. and Čėnas, N., 2017. Aziridinyl-substituted benzo-1, 4-quinones: A preliminary investigation on the theoretical and experimental studies of their structure and spectroscopic properties. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 178, pp.136-141.
Jarasiene-Burinskaja, R., Alksne, M., Bartuskiene, V., Voisniene, V., Burinskij, J., Cenas, N. and Bukelskiene, V., 2017. Study of the cytotoxic effects of 2, 5-diaziridinyl-3, 6-dimethyl-1, 4-benzoquinone (MeDZQ) in mouse hepatoma cells. EXCLI journal, 16, p.151.
Šarlauskas, J., Pečiukaitytė-Alksnė, M., Misevičienė, L., Marozienė, A., Polmickaitė, E., Staniulytė, Z., Čėnas, N. and Anusevičius, Ž., 2016. Naphtho [1′, 2′: 4, 5] imidazo [1, 2-a] pyridine-5, 6-diones: Synthesis, enzymatic reduction and cytotoxic activity. Bioorganic & medicinal chemistry letters, 26(2), pp.512-517.
Ger, M., Kaupinis, A., Nemeikaite-Ceniene, A., Sarlauskas, J., Cicenas, J., Cenas, N. and Valius, M., 2016. Quantitative proteomic analysis of anticancer drug RH1 resistance in liver carcinoma. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1864(2), pp.219-232.
Stulpinas, A., Imbrasaitė, A., Krestnikova, N., Šarlauskas, J., Čėnas, N. and Kalvelytė, A.V., 2015. Study of bioreductive anticancer agent RH-1-induced signals leading the wild-type p53-bearing lung cancer A549 cells to apoptosis. Chemical research in toxicology, 29(1), pp.26-39.
Valiauga, B., Rouhier, N., Jacquot, J.P. and Čėnas, N., 2015. Quinone-and nitroreductase reactions of Thermotoga maritima thioredoxin reductase. Acta Biochimica Polonica, 62(2), pp.303-309.
Kosychova, L., Karalius, A., Staniulytė, Z., Sirutkaitis, R.A., Palaima, A., Laurynėnas, A. and Anusevičius, Ž., 2015. New 1-(3-Nitrophenyl)-5, 6-dihydro-4H-[1, 2, 4] triazolo [4, 3-a][1, 5] benzodiazepines: Synthesis and Computational Study. Molecules, 20(4), pp.5392-5408.
Miliukienė, V., Nivinskas, H. and Čėnas, N., 2014. Cytotoxicity of anticancer aziridinyl-substituted benzoquinones in primary mice splenocytes. Acta Biochimica Polonica, 61(4), pp.833-836.
Šarlauskas, J., Misevičienė, L., Marozienė, A., Karvelis, L., Stankevičiūtė, J., Krikštopaitis, K., Čėnas, N., Yantsevich, A., Laurynėnas, A. and Anusevičius, Ž., 2014. The study of NADPH-dependent flavoenzyme-catalyzed reduction of benzo [1, 2-c] 1, 2, 5-oxadiazole N-oxides (Benzofuroxans). International journal of molecular sciences, 15(12), pp.23307-23331.
Anusevičius, Ž., Nivinskas, H., Šarlauskas, J., Sari, M.A., Boucher, J.L. and Čėnas, N., 2013. Single-electron reduction of quinone and nitroaromatic xenobiotics by recombinant rat neuronal nitric oxide synthase. Acta Biochimica Polonica, 60(2).
Head of department