INTERACTION OF BETA-L-2',3'-DIDEOXY-2',3'-DIDEHYDRO-5-FLUORO-CTP WITHHUMAN 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, a-L-2',3'-dideoxy-2',3'-didehydro-5-fluoro cytidine (beta-L-Fd4CTP), with that of beta-L-2',3'-dideoxy-5-fluorocy tidine (beta-L-FddCTP) and 2',3'-dideoxycytidine (ddCTP), on DNA stran d 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 be ta-L-Fd4CTP that inhibited the yield of products by 50% were 0.20 mu M , 1.8 mu M, and 4.0 mu M for HIV RT, pol gamma, and pol beta, respecti vely. The beta-L-Fd4CTP at a concentration as high as 40 mu M had no i nhibitory effect on pol epsilon, but could inhibit pol alpha by 10-20% at 20 mu M. The K-m and relative V-max values of beta-L-Fd4CTP, beta- L-FddCTP, and ddCTP for incorporation into the standing start point of 5'-[P-32]-oligonucleotide primer annealed with M13mp19 phage DNA by H IV RT and human DNA polymerases were evaluated. The efficiency of inco rporation (V-max/K-m) 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 V-max/K-m ratio of beta-L-Fd4CTP for pol gamma was 7-fol d lower than that of ddCTP, but 4-fold higher than that of beta-L-FddC TP. Pol alpha could use beta-L-Fd4CTP as a substrate, but only at a hi gh concentration (>20 mu M). Incorporation of beta-L-Fd4CTP by pol eps ilon could not be detected. A hypothesis about the preferable recognit ion 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 dis cussed.