MECHANISM OF SELECTIVE INCORPORATION OF THE MELANOMA SEEKER 2-THIOURACIL INTO GROWING MELANIN

The mechanism of selective incorporation of 2-thiouracil (TU), a highl y specific melanoma seeker, into growing melanins was investigated bot h in vitro and in vivo. Methods used included direct analysis of the m elanins, by evaluation of the absorption at 350 nm (A(350)) and chemic al degradation coupled with HPLC quantitation of pigment markers, i.e. , pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA), as well as biosynthetic experiments involving tyrosinase -catalyzed oxidation of DOPA, 5,6-dihydroxyindole (DHI), and 5,6-dihyd roxyindole-2-carboxylic acid (DHICA). Injection of radiolabeled TU int o melanoma-bearing mice resulted in a rapid incorporation of the drug into the tumor pigment, with a substantial decrease in A(350) and in P TCA yields. Similar changes in the absorption properties were observed in biosynthetic melanins prepared in the presence of TU, whereas the yields of PTCA and PDCA varied depending on the pigment precursor used . When incubated with DOPA in the presence of tyrosinase, TU profoundl y modified the normal course of melanogenesis, favoring formation of a complex mixture of addition products consisting mainly of 6-S-thioura cil-DOPA as well as DHI-TU adducts. The latter were obtained in larger amounts by enzymatic oxidation of DHI in the presence of TU and were identified as the 3- and 2-substituted adducts 1 and 2, the dimer 3, a nd the trimer 4. Similar reactions carried out on DHICA yielded the C- substituted adduct 5, the dimer 6, and the trimer 7. A new mechanistic scheme for the incorporation of TU into growing melanin is proposed, which envisages nucleophilic attack of the thioureylene moiety of TU t o transient quinonoid intermediates in the melanin pathway, chiefly do paquinone and 5,6-indolequinones, followed by entrainment of the resul ting adducts into the growing pigment via oxidative copolymerization w ith DHICA and/or DHI.