Nucleoside
analogs have proven effective for the inhibition of
viral polymerases and are the foundation of many antiviral therapies.
In this work, the antiretroviral potential of 6-azauracil analogs
was assessed using activity-based protein profiling techniques and
functional assays. Probes based on the 6-azauracil scaffold were examined
and found to bind to HCV polymerase and HIV-1 reverse transcriptase
through covalent modification of residues near the active site. The
modified sites on the HIV-1 RT were examined using a mass spectrometry
approach, and it was discovered that the azauracil moieties modified
the enzyme in proximity to its active site. However, these scaffolds
gave little or no inhibition of enzyme activity. Instead, a bifunctional
inhibitor was prepared using click chemistry to link the 6-azauracil
moiety to azidothymidine (AzT) and the corresponding triphosphate
(AzTTP). These bifunctional inhibitors were found to have potent inhibitory
function through a mode of action that includes both alkylation and
chain termination. An in vitro assay demonstrated that the bifunctional
inhibitor was 23-fold more effective in inhibiting HIV-1 RT activity
than the parent AzTTP. The bifunctional inhibitor was also tested
in HIV-1 permissive T cells where it decreased Gag expression similarly
to the front-line drug Efavirenz with no evidence of cytotoxicity.
This new bifunctional scaffold represents an interesting tool for
inhibiting HIV-1 by covalently anchoring a chain-terminating nucleoside
analog in the active site of the reverse transcriptase, preventing
its removal and abolishing enzymatic activity, and represents a novel
mode of action for inhibiting polymerases including reverse transcriptases.