APOBEC enzymes are single-stranded DNA (ssDNA) cytosine to uracil (C to U) deaminases that belong to a larger family of DNA and RNA editing enzymes with innate immune functions. APOBEC-mediated mutagenesis in viruses and cancer cells results in an increased rate of genomic evolution, and ultimate drug resistance,whichadvocates for the development of APOBEC inhibitors. To discover small molecule inhibitors for APOBEC enzymes, we developed a single-timepoint ssDNA assay that reports on APOBEC-catalyzed cytosine deamination using a fluorescence readout. Although very useful as a high-throughput screening assay, we now desire to evaluate the time-dependent enzyme inhibition kinetics of covalent APOBEC inhibitors for which a real-time deamination assay would be ideal. To achieve this goal, we modified 5-furyl-2'-deoxycytidine developed by Tor and coworkers to 6-furyl-2'-fluorocytidine, synthesized both nucleoside phosphoramidites, and prepared ssDNA substrates of APOBEC enzymes containing the fluorescent cytidine probes. This presentation will showcase the development of this new assay and its utility for screening small molecule APOBEC inhibitors.

Development of fluorescent nucleosides for real-time measurements of single-stranded DNA cytidine deamination by APOBEC enzymes

2019 Fall National Meeting

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