Background:  We previously reported that a series of 4′-C-ethynyl nucleoside analogs was active against a wide spectrum of HIV-1 isolates, including a variety of laboratory and drug-resistant HIV-1 strains in vitro. Among such 4′-C-ethynyl analogs, 2'-deoxy-4'-C-ethynyl-2-fluoroadenosine (E2FdA), fluorine-substituted at the 2-position of adenine, represents 1 of the most potent agents against HIV-1 with IC₅₀ values of ~1nM. To further evaluate the characteristics of E2FdA, we examined the intracellular metabolism of E2FdA and its effects on the activity of human polymerases.

Methods:  CEM cells were seeded at 10⁶ cells/mL, incubated with 200 nM ³H-E2FdA or ³H-3′-azido-2′,3′-dideoxythymidine (AZT) for 6 hours, and thoroughly washed to remove extracellular drugs. After further incubation, nucleosides/nucleotides within the cells were extracted with 60% methanol at various time points, and the amounts of metabolites were determined using HPLC and liquid scintillation counter. Persistence of anti-HIV activity of E2FdA was also determined as follows: MT4 or MAGI-CCR5 cells were exposed to 0.1 or 1 mM E2FdA (or AZT) for 24 hours, thoroughly washed to deplete extracellular drugs, cultured for various periods of time, then exposed to HIV-1, and further cultured for 48h. Anti-HIV activity was monitored using p24 production or in MAGI assay.

Results:  Amounts of intracellular E2FdA-monophosphates (MP), -diphosphates (DP), and -triphosphates (TP) increased proportionately with increased E2FdA concentrations. When exposed to AZT, only AZT-MP levels markedly increased compared with AZT-DP and AZT-TP. Intracellular T_1/2 of E2FdATP, an active form of E2FdA, was >12 hours in CEM cells, which was significantly greater than T_1/2 of AZT-TP (~3 hours). Similar results were obtained in MT4 and MAGI cells. E2FdA (0.1 mM) protected ~50% of MT4 and MAGI cells against the infection of HIV-1, added 24 hours after E2FdA removal from cultures. The IC₅₀ value of E2FdATP to inhibit dATP (0.3 mM) incorporation mediated by DNA polymerase-g was 10 mM, which was significantly higher than that of ddATP (IC₅₀ value: 0.2 mM)

Conclusions:  E2FdA possesses long intracellular T_1/2 of its TP form (>12 hours) and exerts minimal inhibition to DNA polymerase-g. The present data suggest that once daily dosing schedule of E2FdA could be possible with few side effects and warrant that E2FdA be further developed as a potential therapeutic for HIV-1 infection.