ESI-FTMS Assay of HIV-1 Reverse Transcriptase Activity and Possible Uncompetitive Enzyme Inhibitors

摘要

The rapid emergence of drug resistant strains constitutes a powerful motivation for investigating alternative strategies for the long-term treatment of human immunodeficiency virus type 1 (HIV-1). An important component of standard highly active-antiretroviral therapies (HAART) consists of reverse transcriptase (RT) inhibitors that are particularly affected by resistance-inducing mutations. This multifunctional enzyme catalyzes the conversion of single-stranded viral RNA into double-stranded DNA that can be incorporated into the host's genome to initiate viral replication. For this reason, RT performs both RNA- and DNA-dependent DNA polymerase, as well as RNase H activities, which are the targets of current antiviral drugs. Alternative therapeutic strategies could be aimed at disrupting selected enzyme-substrate interactions, such as those between RT and the polypurine tract (PPT, Fig. 1). This RNA-DNA hybrid structure is not cleaved by the RNase H function and plays a critical role in initiating the (+)DNA synthesis. The very specific interactions that mediate the recognition of PPT by RT suggest unique structural features that could constitute very favorable targets for novel inhibitors. With the goal of enabling the rapid screening of potential drug candidates, we are pursuing MS-based methods to discriminate among the three distinctive aspects of RT activity: substrate binding, DNA polymerization, and RNA hydrolysis. Protein-nucleic acids interactions can be effectively studied by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry by taking advantage of the intrinsically gentle character of electrospray ionization (ESI), which allows for the direct observation of relatively weak non-covalent complexes in the gas-phase. ESI-FTICR can be also employed to follow the conversion of nucleic acid substrates into final products to assess enzyme activity. In this direction, ad hoc substrates were designed to include a primer consisting of a 28mer DNA oligonucleotide and a template consisting of a 25mer RNA. A RT-mutant devoid of RNase activity was tested for its ability to extend the DNA primer through the addition of bases complementary to the RNA overhang (Fig. 1). In preliminary studies, the highly-homologous E. Coli RNase H was used to test separately the hydrolytic activity of RT using the same RNA-DNA hybrid construct.