Balancing Antiviral Potency and Host Toxicity: Identifying a Nucleotide Inhibitor with an Optimal Kinetic Phenotype for HIV-1 Reverse Transcriptase.

Two novel thymidine analogs, 3'-fluoro-3'-deoxythymidine (FLT) and 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine (Ed4T), have been investigated as nucleoside reverse transcriptase inhibitors (NRTIs) for treating HIV infection. Ed4T, in Phase II clinical trials, appears very promising, while toxicity halted FLT during this Phase. To understand these different molecular mechanisms of toxicity, pre-steady-state kinetics were used to examine the interactions of FLT and Ed4T with wild type human mitochondrial DNA polymerase γ (WT pol γ), which is often associated with NRTI toxicity, as well as the viral target protein, WT HIV-1 reverse transcriptase (RT). We report Ed4T-TP to be the first analog preferred over native nucleotides by RT but have negligible incorporation by WT pol γ, showing an ideal balance between high antiretroviral efficacy and minimal host toxicity. WT pol γ could discriminate Ed4T-TP from dTTP 12,000-fold better than RT, with only a 8.3-fold difference seen in discrimination for FLT-TP. A structurally related NRTI, d4T, is the only other analog favored by RT over native nucleotides, but it has only a 13-fold difference (versus 12,000 for Ed4T) in discrimination between the two enzymes. We propose the 4'-ethynyl group of Ed4T serves as an enzyme selectivity moiety, critical for discerning between RT and WT pol γ. We also show the pol γ mutation R964C, which predisposes HIV patients taking d4T to mitochondrial toxicity, showed some loss of discrimination for FLT-TP and Ed4T-TP. These molecular mechanisms of analog incorporation, which are critical for understanding pol γ-related toxicity, shed light on the unique toxicity profiles observed during clinical trials.