Selective action of 4'-azidothymidine triphosphate on reverse transcriptase of human immunodeficiency virus type 1 and human DNA polymerases .alpha. and .beta.

Abstract

4'-Azidothymidine (ADRT) is a novel nucleoside analogue that exhibits potent inhibitory activity against the replication of human immunodeficiency virus (HIV) in lymphocytes. The mechanisms by which ADRT inhibits HIV reverse transcriptase (HIV-RT) as ADRT 5'-triphosphate (ADRT-TP), the active intracellular metabolite of ADRT, and as the ADRT-MP molecule incorporated into DNA were examined and compared to their effects on human DNA polymerases alpha and beta. Inhibition of HIV-RT by ADRT-TP is competitive against TTP and is more potent against RNA to DNA synthesis (Ki = 0.009 microM versus Km = 3.3 microM for TTP) than it is against DNA to DNA synthesis (Ki = 0.95 microM versus Km = 16.3 microM for TTP). ADRT-TP is also a more potent inhibitor for primer elongation on RNA template than on DNA template. ADRT-TP is a poor inhibitor of human DNA polymerases alpha (Ki = 62.5 microM) and beta (Ki = 150 microM) (Chen et al., 1992). The consequences of ADRT incorporation into DNA are strikingly different for the HIV-RT and for human DNA polymerases alpha and beta. DNA polymerases alpha and beta incorporate a single ADRT-MP molecule into nascent DNA at a very slow rate and continue to elongate. They are unable to incorporate a second consecutive ADRT-MP. However, HIV-RT is able to efficiently incorporate two consecutive ADRT molecules. Incorporation of two consecutive ADRT-MP molecules by HIV-RT prevents further DNA chain elongation. Incorporation of two ADRT-MP molecules separated by one deoxyribonucleoside monophosphate (dAMP, dCMP, or dGMP) also abolishes DNA chain elongation by HIV-RT.

Mechanism of Inhibition of HIV-1 Reverse Transcriptase by 4′-Ethynyl-2-fluoro-2′-deoxyadenosine Triphosphate, a Translocation-defective Reverse Transcriptase Inhibitor

“…Additionally, nucleoside analogs substituted at the 4Ј-position have also been described previously (43)(44)(45)(46). For example, 4Ј-azidothymidine and 4Ј-azidoadenosine both inhibit HIV-1 replication (46) although with potencies 200 -2000-fold less than that of EFdA.…”

“…Both 4Ј-azido nucleosides also have poor in vitro selectivity indices because of significant cytotoxicity. Azidothymidine-TP was shown to inhibit RT-catalyzed DNA synthesis by a type of delayed chain termination; incorporation of two sequential azidothymidine-MP molecules blocked DNA synthesis (44,45). 4Ј-Methyl thymidine and 4Ј-ethyl thymidine both seem to cause pauses and stops in DNA synthesis at the point of incorporation (39).…”

Activity against Human Immunodeficiency Virus Type 1, Intracellular Metabolism, and Effects on Human DNA Polymerases of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine

“…In terms of the mechanism of antiviral activity of EFdA, previous reports of 4Ј-substituted-2Ј-deoxynucleosides, such as 4Ј-AZT, have shown that following intracellular anabolism to the 5Ј triphosphate, HIV-1 reverse transcriptase (RT) efficiently incorporated the nucleotide, which prevented further chain elongation of the viral DNA (4,5). Although the rate of incorporation for the 5Ј-triphosphate of 4Ј-AZT was quite low, HIV-1 RT was able to incorporate two consecutive molecules efficiently.…”

“…For elution, the following program was used: 5 min of buffer A, followed by 10 min of a highly convex gradient to 75% buffer A-25% buffer B, followed by 15 min of a slightly convex gradient to 100% buffer B, and finally followed by a 10-min isocratic elution with buffer B. One-minute elution fractions were collected, and the radioactivity of each fraction was measured using a liquid scintillation counter to determine the amount of metabolites. (2,4,8,12, and 24 h). The amounts of intracellular metabolites were determined by HPLC analysis as described above.…”

A New Paradigm for Developing Antiviral Drugs Exemplified by the Development of Supremely High Anti-HIV Active EFdA

“…While we were working on our project, the anti-HIV activity of several 4'SdNs was reported by the Syntex group [16][17][18][19][20][21][22] and others [23,24]. Therefore, the anti-HIV activities of 4'SdNs that we studied together with those reported by other groups are listed in Table 2.…”