title: Mechanism of selective incorporation of the melanoma seeker 2-thiouracil into growing melanin.
abstract: The mechanism of selective incorporation of 2-thiouracil (TU), a highly specific melanoma seeker, into growing melanins was investigated both in vitro and in vivo. Methods used included direct analysis of the melanins, by evaluation of the absorption at 350 nm (A350) and chemical degradation coupled with HPLC quantitation of pigment makers, 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-dihydroxyindole-2-carboxylic acid (DHICA). Injection of radiolabeled TU into melanoma-bearing mice resulted in a rapid incorporation of the drug into the tumor pigment, with a substantial decrease in A350 and in PTCA 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 profoundly modified the normal course of melanogenesis, favoring formation of a complex mixture of addition products consisting mainly of 6-S-thiouracil-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, and the trimer 4. Similar reactions carried out on DHICA yielded the 4-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 to transient quinonoid intermediates in the melanin pathway, chiefly dopaquinone and 5,6-indolequinones, followed by entrainment of the resulting adducts into the growing pigment via oxidative copolymerization with DHICA and/or DHI.
authors.affiliation: Department of Organic and Biological Chemistry, University of Naples, Federico II, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Napolitano
authors.forename: A
authors.initials: A
authors.lastname: Palumbo
authors.forename: M
authors.initials: M
authors.lastname: d'Ischia
authors.forename: G
authors.initials: G
authors.lastname: Prota
mesh_terms: D000818:Animals; D001704:Biopolymers; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D005260:Female; D009682:Magnetic Resonance Spectroscopy; D008543:Melanins / Q000378:metabolism*; D008546:Melanoma, Experimental / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D014442:Monophenol Monooxygenase / Q000378:metabolism; D010084:Oxidation-Reduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013889:Thiouracil / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D001704:Biopolymers; D008543:Melanins; D014443:Tyrosine; D013889:Thiouracil; D014442:Monophenol Monooxygenase