ELSEVIER Biochimica et Biophysica Acta 1293 (1996) 1-8 BB Biochi~ic~a et Biophysica A~ta Synthesis and interactions with thymidylate synthase of 2,4-dithio analogues of dUMP and 5-fluoro-dUMP Jolanta M. Dzik a, Mafia Bretner b Tadeusz Kulikowski b, Barbara Gotos a, Adam Jarmuta a Jarostaw Poznafiski b, Wojciech Rode a,*, David Shugar b Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., 02-093 Warszawa, Poland b Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5a Pawihskiego St., 02-106 Warszawa, Poland Received 10 July 1995; accepted 17 October 1995 Abstract The 2,4-dithio analogues of 2'-deoxyuridine and 2'-deoxy-5-fluorouridine have been synthesized by thiation of the previously described 2-thio analogues, ~md then phosphorylated enzymatically or chemically to yield 2,4-dithio-dUMP and 2,4-dithio-5-fluoro-dUMP. In striking contrast to the 2-thio and 4-thio analogues of dUMP, which are good substrates of thymidylate synthase, 2,4-dithio-dUMP is not a substrate. But, surprisingly, it is a competitive inhibitor, relative to dUMP, of the purified enzymes from both parental and FdUrd-resistant L1210 cell,;, with K i values of 32 /zM and 55 /xM, respectively. Although 2,4-dithio-5-fluoro-dUMP behaved as a typical slow-binding inhibitor of the enzyme, its K i value was 103-10a-fold higher than those for the corresponding 2-thio and 4-thio congeners. Similarly, 2,4-dithio-FdUrd was a much weaker inhibitor of tumour cell growth (IC5o = 10 -5 M) than FdUrd (IC5o = 10 -9 M), 2-thio-FdUrd (IC5o = 10 -7 M) or 4-thio-FdUrd (ICso = 5 • 10 -8 M), while with 2,4-dithio-dUrd no influence on cell growth could be observed. Theoretical coasiderations, based on calculated aromaticities of the uracil and thiouracil rings, suggest that lack of substrate activity of 2,4-dithio-dUMP may result from increased pyrimidine ring aromaticity of the latter, leading to resistance of C(6) to nucleophilic attack by the enzyme active center cysteine, Keywords: Thymidylate synthase; Inhibition; 2,4-dithio-dUMP; 2,4-dithio-5-fluoro-dUMP; Aromaticity 1. Introduction Thymidylate synthase catalyzes a key step in the de novo biosynthesis of d'[MP, required for DNA synthesis. The enzyme catalyzes the transfer of a hydroxymethyl group from methylenetetrahydrofolate (CH2HaPteGIu) to the C5 of dUMP and reduction of the transferred one carbon unit to a methyl group, utilizing a hydride equiva- lent provided by tetrahydrofolate. The products are dTMP and dihydrofolate [1]. Among thymidylate synthase inhibitors, 5-fluoro-dUMP Abbreviations: CH 2H4PteGlu, NS'l°-methylenetetrahydrofolate; FdUrd, 5-fluoro-2'-deoxyuridine; FdUMP, 5-fluom-2'-eoxyuridine-5'- monophosphate. * Corresponding author. Fax: +48 22 225342; e-mail: rode@nencki.gov.pl. 0167-4838/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0167-4838(95)00219-7 (FdUMP), a metabolite and active form of the anticancer drugs, 5-fluorouracil and 5-fluorodeoxyuridine (FdUrd), and of the antifungal drug, 5-fluorocytosine, plays a key role because of its particular features as a mechanism-based inactivator of the enzyme [2,3]. FdUMP forms a ternary complex with thymidylate synthase and CH2H4PteGlu, analogous to the catalytic intermediate. A number of alter- native substrates and mechanism-based inhibitors [re- viewed in Ref. [1,4-6]] have been studied, in order to learn more about the mechanism, and possible new modes of inhibition, of the enzyme. Since 2-thio and 4-thio derivatives of dUMP are good substrates [7,8], and their 5-fluoro congeners effective inhibitors [9,10], of the enzyme from various sources, it appeared of interest to synthesize the 2,4-dithio congeners of dUMP and FdUMP, and to investigate their interactions with thymidylate synthase.