Background:  Nucleotide-competing reverse transcriptase inhibitors (NcRTI) represent a novel class of compounds that has been shown to block the DNA polymerase activity of HIV-1 RT. Previous data suggested that the prototype compound NcRTI-1 can compete with natural dNTP substrates, despite significant structural differences, including the lack of a sugar-phosphate moiety. In addition, antiviral inhibition studies have shown that the M184V mutation is associated with decreased susceptibility to NcRTI-1, while the K65R mutation confers hypersusceptibility. Here we have studied the detailed biochemical mechanisms involved in both inhibition and resistance to NcRTI-1.

Methods:  We purified RT enzymes and employed kinetic assays, site-specific footprinting techniques, and band-shift experiments to study the binding properties and inhibitory effects of NcRTI-1 in cell-free assays.

Results:  Enzyme inhibition studies revealed that NcRTI-1 can efficiently block DNA synthesis (IC₅₀ = 250 nM). The potency of this compound is significantly increased in the presence of 3 mM ATP (IC_50ATP = 25 nM). The M184V mutation caused increases in IC₅₀ measurements (IC₅₀ = 650 nM; IC_50ATP = 120 nM), while the K65R mutant caused decreases (IC₅₀ = 150 nM; IC_50ATP = 15 nM). Like dNTP substrates, NcRTI-1 can form a stable ternary complex with RT bound to a DNA/DNA primer/template. The presence of 30 mM dNTP caused a band shift of 50% of the labeled DNA substrate, and concentrations as low as 6 µM of NcRTI-1 are sufficient to cause the same effect. Moreover, we measured sub-micromolar concentrations when this complex was simultaneously incubated with ATP, which suggests that the presence of ATP facilitates the formation of the inhibitory complex. In contrast, the M184V mutation diminishes the stability of a stable complex with RT-DNA and NcRTI-1. Site-specific footprinting studies revealed that NcRTI-1 can trap the RT-primer/template complex in its post-translocation configuration and that this effect is also diminished with the M184V mutant enzyme.

Conclusions:  The results provide strong evidence to suggest that NcRTI-1 can partially occupy the nucleotide binding site of HIV-1 RT. The compound forms a dead-end complex that prevents the incorporation of dNTP substrates. Our biochemical data are consistent with cell-based inhibition measurements showing that the M184V change is associated with decreased susceptibility to NcRTI-1, while K65R confers hypersusceptibility.