Inhibiting APOBEC3 Activity with Single-Stranded DNA Containing 2′-Deoxyzebularine Analogues

Abstract

APOBEC3 enzymes form part of the innate immune system by deaminating cytosine to uracil in single-stranded DNA (ssDNA) and thereby preventing the spread of pathogenic genetic information. However, APOBEC mutagenesis is also exploited by viruses and cancer cells to increase rates of evolution, escape adaptive immune responses, and resist drugs. This raises the possibility of APOBEC3 inhibition as a strategy for augmenting existing antiviral and anticancer therapies. Here we show that, upon incorporation into short ssDNAs, the cytidine nucleoside analogue 2′-deoxyzebularine (dZ) becomes capable of inhibiting the catalytic activity of selected APOBEC variants derived from APOBEC3A, APOBEC3B, and APOBEC3G, supporting a mechanism in which ssDNA delivers dZ to the active site. Multiple experimental approaches, including isothermal titration calorimetry, fluorescence polarization, protein thermal shift, and nuclear magnetic resonance spectroscopy assays, demonstrate nanomolar dissociation constants and low micromolar inhibition constants. These dZ-containing ssDNAs constitute the first substrate-like APOBEC3 inhibitors and, together, comprise a platform for developing nucleic acid-based inhibitors with cellular activity.

Differential Inhibition of APOBEC3 DNA‐Mutator Isozymes by Fluoro‐ and Non‐Fluoro‐Substituted 2′‐Deoxyzebularine Embedded in Single‐Stranded DNA

“…On the other hand, inhibition of A3B CTD ‐AL1 by 5FdZ‐oligo was more powerful than that by dZ‐oligo under identical conditions. Previously, we had confirmed that dZ‐oligo is a competitive inhibitor of this enzyme . By monitoring the reaction in the presence of inhibitor at various concentrations, we obtained the inhibition constant ( K i ) for 5FdZ‐oligo [(2.1±0.8) μ m , Supporting Information]; this was 3.5 times lower than the K i of dZ‐oligo [(7.5±1.7) μ m ].…”

“…To assess the inhibition of A3 enzymes directly, we used a previously described NMR‐based activity assay in which the DNA substrate deamination is monitored by 1 H NMR spectroscopy in the presence of enzyme with and without inhibitors . The NMR‐based inhibition assay is a direct assay using just A3 enzymes; it does not require a secondary enzyme, such as uracil‐DNA glycosylase (UDG), as used in many indirect assays.…”

“…Next, having two active A3G enzymes—the C‐terminal domain (A3G CTD ) with the wild‐type sequence and full‐length A3G (flA3G)—we decided to test whether inhibition of A3G CTD is a good model for investigation of inhibition of two‐domain enzymes such as flA3G. Our studies were performed with two oligos: an A3G‐preferred CC5FdZ‐oligo in which the dC residue that is first deaminated by A3G was changed to 5FdZ, and the previously reported inhibitor CCdZ‐oligo . Our data show that inhibition of A3G deaminase activity by targeting only the catalytically active C‐terminal domain, A3G CTD , accurately translates to the overall inhibition of flA3G (Figure A).…”

“…To date, no selective small‐molecule inhibitors of A3A or A3B have been reported. We recently developed the first rationally designed competitive inhibitor of A3 enzymes by incorporating a known inhibitor of CDA—2′‐deoxyzebularine (dZ, Scheme B)—into ssDNA oligonucleotides . We demonstrated that dZ does not inhibit A3 enzymes when present as the free nucleoside, but becomes a low‐micromolar inhibitor if, and only if, it is incorporated into ssDNA.…”

“…Herein, we report the first syntheses of the 2′‐deoxy forms of 3‐deazauridine and 5‐fluorozebularine (3dadU and 5FdZ, respectively). We also report the incorporation of these nucleosides into ssDNA and their evaluation as A3 inhibitors with the aid of our previously described NMR‐based and fluorescence‐based enzymatic assays. 3‐Deaza‐2′‐deoxyzebularine (3dadZ, Scheme B) has a CH motif instead of the N3 atom in comparison with dZ and so can be used to evaluate the importance of protonation of the N3 atom in dZ in its inhibitory mechanism.…”