Interaction of β-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 Kukhanova1,
- Xiuyan Li2,
- Shu-Hui Chen2,
- Ivan King2,
- Terrence Doyle2,
- William Prusoff1 and
- Yung-Chi Cheng1
- 1Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510 (M.K., W.P., Y.-C. C.), 2Vion Pharmaceuticals, Inc., New Haven, Connecticut 06511 (X.L., S.-H. C., I.K., T.D.)
Abstract
The work reported in this article has evaluated the relative molecular activity of the 5′-triphosphate of a novel β-l-nucleoside with an unsaturated ribose residue, β-l-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (β-l-Fd4CTP), with that of β-l-2′,3′-dideoxy-5-fluorocytidine (β-l-FddCTP) and 2′,3′-dideoxycytidine (ddCTP), on DNA strand elongation by human immunodeficiency virus-1 reverse transcriptase (HIV RT) and human DNA polymerases α (pol α), β (pol β), γ (pol γ), and ε (pol ε). The concentrations of β-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 γ, and pol β, respectively. The β-l-Fd4CTP at a concentration as high as 40 μm had no inhibitory effect on pol ε, but could inhibit pol α by 10–20% at 20 μm. TheKm and relativeVmax values of β-l-Fd4CTP, β-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 β-l-Fd4CTP by HIV RT was about 4-fold and 12-fold higher than that of ddCTP and β-l-FddCTP, respectively. In contrast, theVmax/Kmratio of β-l-Fd4CTP for pol γ was 7-fold lower than that of ddCTP, but 4-fold higher than that of β-l-FddCTP. Pol α could use β-l-Fd4CTP as a substrate, but only at a high concentration (>20 μm). Incorporation of β-l-Fd4CTP by pol ε could not be detected. A hypothesis about the preferable recognition of the 2′,3′-dideoxy-2′,3′-didehydro- structure of β-l-Fd4CTP to that of the 2′,3′-dideoxy-structure of β-l-FddCTP by HIV RT is discussed.
Footnotes
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Send reprint requests to: Dr. Yung-Chi Cheng, Pharmacology Department, Yale University School of Med., 333 Cedar Street., New Haven, CT 06510.
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This work was supported by National Institutes of Health Grant AI38204.
- Abbreviations:
- HIV
- human immunodeficiency virus type 1
- HBV
- human hepatitis B virus
- pol
- polymerase
- RT
- reverse transcriptase
- dNTP
- 2′-deoxynucleoside 5′-triphosphate
- dCTP
- 2′-deoxycytidine 5′-triphosphate
- ddNTP
- 2′,3′-dideoxynucleoside 5′-triposhate
- ddC
- 2′,3′-dideoxycytidine
- ddCTP
- 5′-triphosphate of ddC
- FddC
- 5′-fluoro-analog of ddC
- FddCTP
- 5′-triposphate of FddC
- Fd4C
- 2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine
- Fd4CTP
- 5′-triphosphate of FddC
- 3TC
- β-l-2′,3′-dideoxy-3′-thiocytidine
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- Received December 2, 1997.
- Accepted January 26, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
