^{AJOENE IS AN INHIBITOR AND A "SUBVERSIVE" SUBSTRATE OF HUMAN GLUTATHIONE REDUCTASE AND T. CRUZI TRYPANOTHIONE REDUCTASE}

^{Susanne Bonse1,2, Helge Gallwitz1, Alfonso Martinez2, Ilme Schlichting2, and R. Luise Krauth-Siegel1}

¹Biochemie-Zentrum, Heidelberg University, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany,
²Abteilung Biophysik, Max-Planck-Institut für Medizinische Forschung, Jahnstrasse 29, D-69120 Heidelberg, Germany

^{Glutathione reductase (GR, EC 1.6.4.2; GSSG + NADPH + H+ --> 2 GSH + NADP+) and trypanothione reductase (TR, EC 1.6.4.8; TS}2 ^{+ NADPH + H+ --> T(SH)}2 ^{+ NADP+) are key enzymes in the cell's defense mechanisms against oxidative stress. The flavoproteins are responsible for maintaining the reducing milieu of their respective cell. Inhibitors of human GR are promising agents against parasitic diseases like malaria as well as in cancer therapy. TR has been found exclusively in trypanosomes, leishmania and Entamoeba histolytica. In these parasitic protozoa - to which belong the causative agents of severe tropical diseases - TR replaces GR. As trypanosomes and leishmania are very sensitive against oxidative stress, TR is a promising target molecule for the development of specific antiparasitic drugs.}

^{During catalysis these flavoenzymes change between two stable forms, E and EH}2^{. In GR, E is characterized by a disulfide bridge between the active site residues Cys58 and Cys63. Reduction of the enzyme leads to formation of the two-electron-reduced form EH}2^{, which contains Cys58 and Cys63 as a dithiol.}

^{Ajoene (E,Z-4,5,9-trithiadodeca-1,6,11-triene-9-oxide) is a garlic derived natural compound. It is well known for its antiplatelet, cytotoxic and antimicrobial activity. As shown here, ajoene is a covalent inhibitor of human GR and Trypanosoma cruzi TR. X-ray diffraction analysis of GR inhibited by E-ajoene in the presence of NADPH shows the S-CH=CH-CH}2 ^S(O)-CH2^-CH=CH2 ^{moiety of ajoene bound to Cys58 via a disulfide bridge. Both GR and TR which have been covalently modified by ajoene show a strongly increased oxidase activity when compared to the free enzyme.}

^{Ajoene acts also as a substrate of the flavoenzymes. 4,5,9-Trithiadodeca-1,6,11-triene (deoxyajoene) and 4,8,9,13-tetrathiahexadeca-1,5,8,10,15-tetraene have been identified as stable products of the GR catalyzed reaction. Besides these two-electron reduced products, single-electron reduced intermediates are generated which then react with molecular oxygen leading to the formation of superoxide anions.}

^{In conclusion, the interaction of ajoene with these key enzymes of the intracellular thiol metabolism may at least in part be responsible for the cytostatic, antimalarial and antitrypanosomal properties of this natural compound.}