Vol. 53, Issue 5, 801-807, May 1998

Interaction of -L-2',3'-dideoxy-2',3'-didehydro-5-fluoro-CTP with Human Immunodeficiency Virus-1 Reverse Transcriptase and Human DNA Polymerases: Implications for Human Immunodeficiency Virus Drug Design

Marina Kukhanova, Xiuyan Li, Shu-Hui Chen, Ivan King, Terrence Doyle, William Prusoff, and Yung-Chi Cheng

Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510 (M.K., W.P., Y.-C. C.), Vion Pharmaceuticals, Inc., New Haven, Connecticut 06511 (X.L., S.-H. C., I.K., T.D.)

The work reported in this article has evaluated the relative molecular activity of the 5'-triphosphate of a novel -L-nucleoside with an unsaturated ribose residue, -L-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine (-L-Fd4CTP), with that of -L-2',3'-dideoxy-5-fluorocytidine (-L-FddCTP) and 2',3'-dideoxycytidine (ddCTP), on DNA strand elongation by human immunodeficiency virus-1 reverse transcriptase (HIV RT) and human DNA polymerases  (pol ),  (pol ),  (pol ), and  (pol ). The concentrations of -L-Fd4CTP that inhibited the yield of products by 50% were 0.20 µM, 1.8 µM, and 4.0 µM for HIV RT, pol , and pol , respectively. The -L-Fd4CTP at a concentration as high as 40 µM had no inhibitory effect on pol , but could inhibit pol  by 10-20% at 20 µM. The K_m and relative V_max values of -L-Fd4CTP, -L-FddCTP, and ddCTP for incorporation into the standing start point of 5'-[³²P]-oligonucleotide primer annealed with M13mp19 phage DNA by HIV RT and human DNA polymerases were evaluated. The efficiency of incorporation (V_max/K_m) of -L-Fd4CTP by HIV RT was about 4-fold and 12-fold higher than that of ddCTP and -L-FddCTP, respectively. In contrast, the V_max/K_m ratio of -L-Fd4CTP for pol  was 7-fold lower than that of ddCTP, but 4-fold higher than that of -L-FddCTP. Pol  could use -L-Fd4CTP as a substrate, but only at a high concentration (>20 µM). Incorporation of -L-Fd4CTP by pol  could not be detected. A hypothesis about the preferable recognition of the 2',3'-dideoxy-2',3'-didehydro- structure of -L-Fd4CTP to that of the 2',3'-dideoxy-structure of -L-FddCTP by HIV RT is discussed.