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.