Interaction of beta-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.

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