Background:  NRTI are among the most potent therapeutics, and are often considered for first-line therapy. All approved NRTI act as chain terminators because they lack a 3'OH, and it has long been considered that the absence of the 3'OH is essential for antiviral activity. However, this feature can also impart detrimental properties to the inhibitor, such as reduced affinity for RT compared to the analogous dNTP substrate, as well as reduced intracellular conversion to the active nucleoside triphosphate. We have found that certain nucleosides that retain the 3'OH group and have substitutions at the 4' and 2 positions of the deoxyribose sugar and base respectively, have exceptional antiviral properties and are highly potent inhibitors of HIV RT. One of these compounds, 4'-ethynyl,2-fluorodeoxyadenosine (4'E-2FdA) is the most potent RT inhibitor described to date.

Methods:  Using enzymology techniques, we characterized the mechanisms of action and resistance of this novel inhibitor of the reverse transcriptase.

Results: 4'E-2FdA can inhibit RT by multiple mechanisms. At physiological concentrations of dNTP it acts as a chain terminator despite the presence of an accessible 3'OH. We report here that this apparent chain termination arises from difficulty of the primer 3'-terminus to translocate following incorporation of the compound. Therefore, we propose that 4'E-2FdA is a translocation-deficient reverse transcriptase inhibitor (TDRTI) that acts by a novel mechanism. We show that the M184V mutation in HIV RT confers low-level resistance to 4'-ethynyl modified nucleosides. In a primer extension assay, we observed a ~4-fold increase in the IC₅₀ of 4'E‑2FdA-TP, reflecting the viral resistance observed in cell culture assays. Steady-state kinetic experiments demonstrated that resistance is primarily due to a decrease in the affinity of M184V RT for 4'E-2FdA triphosphate. Molecular modeling analysis suggests that the decrease in binding affinity is the result of steric hindrance between the Val184 of the mutant RT and the 4' ethynyl group of the inhibitor.

Conclusions:  In conclusion, 4'E-2FdA is a highly potent antiviral that acts by a novel mechanism, inhibiting the translocation function of the reverse transcriptase.