Nucleotide-competing Reverse Transcriptase Inhibitors form a Stable Dead-end Complex with the HIV-1 Enzyme
M Ehteshami1, J Deval1, S Barry1, D Jochmans2, K Hertogs2, and Matthias Götte*1
1McGill Univ, Montreal, Canada and 2Tibotec, Mechelen, Belgium
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
(IC50ATP = 25 nM). The M184V mutation caused increases in IC50
measurements (IC50 = 650 nM; IC50ATP = 120 nM), while the
K65R mutant caused decreases (IC50 = 150 nM; IC50ATP = 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.