Phosphorylation of 4'-thio--D-Arabinofuranosylcytosine and Its Analogs by Human Deoxycytidine Kinase HITOSHI SOMEYA, SUE C. SHADDIX, KAMAL N. TIWARI, JOHN A. SECRIST III, and WILLIAM B. PARKER Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama (H.S.); and Southern Research Institute, Birmingham, Alabama (S.C.S., K.N.T., J.A.S., W.B.P.) Received October 9, 2002; accepted December 6, 2002 ABSTRACT 4'-thio--D-Arabinofuranosylcytosine (T-araC) exhibits excel- lent in vivo antitumor activity against a variety of solid tumors despite its structural similarity to -D-arabinofuranosylcytosine (araC), an agent which is poorly active against solid tumors in vivo. It is of great interest to elucidate why these compounds show a profound difference in antitumor activity. Because de- oxycytidine kinase (dCK) is the critical enzyme in the activation of both compounds, here we report the differences in the substrate characteristics with human dCK between these com- pounds. The catalytic efficiency (V max /K m ) of araC was 100-fold higher than that of T-araC using either ATP or UTP as the phosphate donor. However, V max values of araC and T-araC were similar when UTP was the phosphate donor. Since UTP is believed to be the true phosphate donor for dCK in intact cells, these data indicated that the rates of phosphorylation of these two compounds at high pharmacologically relevant concentra- tions would be similar. This prediction was confirmed in intact cell experiments, which supported the hypothesis that UTP is the physiological phosphate donor for dCK phosphorylation in cells. The relative lack of importance of phosphate donor to the phosphorylation of T-araC by dCK revealed important insights into the activation of this compound in human cells at pharma- cological doses. These studies indicated that replacement of the 4'-oxygen with sulfur significantly reduced the substrate activity of nucleoside analogs with dCK and that the superior activity of T-araC with respect to araC against solid tumors was not due to superior activity with dCK. T-araC (Fig. 1) is the most promising antitumor agent among the 4'-thionucleoside analogs that have been synthe- sized in our drug development program (Tiwari et al., 2000a). It exhibits excellent in vivo antitumor activity against a variety of human solid tumor xenografts, such as CAK-1 (renal), NCI-H23 (non-small cell lung), HCT-116 (colon), LOX (melanomas), PANC-1 (pancreas), and DU-145 (prostate) (Waud et al., 1999; Tomkinson et al., 2002). Because of these results, it is being evaluated for effectiveness in clinical trials by OSI Pharmaceuticals Inc. (Melville, NY) as OSI-7836. T-araC is a structural analog of araC (Fig. 1), which is clin- ically used in the treatment for acute myelogenous leukemia and other hematological malignancies (Peters et al., 1987; Stryckmans et al., 1987; Mastrianni et al., 1992) but is poorly active against solid tumors in vivo (Davis et al., 1974; Cheng and Capizzi, 1982). It is of great interest to elucidate how the minor structural difference between araC and T-araC (the 4'-oxygen atom in the arabinofuranosyl ring is replaced by a sulfur atom; Fig. 1) results in the profound difference in antitumor activity that is observed with these two agents. Our previous studies indicated that the basic mechanisms of action of these two agents were similar. They were phos- phorylated to their respective triphosphates, which inhibited DNA replication. (Parker et al., 2000). Blajeski et al. (2002) have shown that araCTP and T-araCTP similarly inhibit DNA synthesis (i.e., they are both alternative substrates for DNA polymerase  and , which results in chain termina- tion). However, there were many quantitative differences in the metabolism and activity between araC and T-araC: 1) T-araC was phosphorylated to active metabolites at 1% the This work was supported by a grant from the National Cancer Institute, P01 CA 34200. A preliminary report of this work was presented at the 2002 annual meeting of the American Association for Cancer Research (Someya et al., 2002). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.045435. ABBREVIATIONS: T-araC, 4'-thio--D-arabinofuranosylcytosine; araC, -D-arabinofuranosylcytosine; araCMP, 5'-monophosphate of araC; araCTP, 5'-triphosphate of araC; Br-dCyd, 5-bromo-2'-deoxycytidine; Br-T-araC, 5-bromo-4'-thio--D-arabinofuranosylcytosine; CH 3 -dCyd, 5-methyl-2'-deoxycytidine; CH 3 -T-araC, 5-methyl-4'-thio--D-arabinofuranosylcytosine; Cl-dCyd, 5-chloro-2'-deoxycytidine; Cl-T-araC, 5-chloro-4'-thio--D-arabinofuranosylcytosine; dCK, deoxycytidine kinase; dCyd, 2'-deoxycytidine; dThd, thymidine; F-araC, 5-fluoro--D-ar- abinofuranosylcytosine; F-dCyd, 5-fluoro-2'-deoxycytidine; F-T-araC, 5-fluoro-4'-thio--D-arabinofuranosylcytosine; HPLC, high pressure liquid chromatography; MS, mass spectrometry; SAX, strong anion exchange; T-araCMP, 5'-monophosphate of T-araC; T-araCTP, 5'-triphosphate of T-araC; T-dCyd, 4'-thio-2'-deoxycytidine. 0022-3565/03/3043-1314 –1322$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 304, No. 3 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 45435/1048181 JPET 304:1314–1322, 2003 Printed in U.S.A. 1314 at ASPET Journals on August 16, 2018 jpet.aspetjournals.org Downloaded from