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.
Highlights
Enzymes of the human APOBEC3 (A3A-H) family normally combat retroviruses and other pathogenic elements by deaminating 2′-deoxycytidine to 2′-deoxyuridine in singlestranded DNA (Figure 1A)
The cytidine analogues zebularine [Z (Figure 1B)], 2′-deoxyzebularine, and tetrahydrouridine (THU) are known transition-state analogues (TSAs) of cytidine deaminase (CDA).[23−25] These competitive inhibitors bind tightly to the active site of CDA, as indicated by crystal structures.[23−28] Here we show that these TSAs as free nucleosides do not alter the activity of A3 enzymes (Figure S1), but micromolar-potent A3 inhibitors are obtained upon introduction of dZ in place of the target 2′-deoxycytidine in DNA substrates
We confirmed that pyrimidine-based TSA nucleosides (Figure 1B) do not inhibit A3 enzymes (Figure S1)
Summary
Detailed methods are provided in the Supporting Information. Synthesis of 2′-Deoxyzebularine (dZ), Its Phosphoramidite, and Oligonucleotides Containing dZ and dZMe. No binding of ssDNA to A3BCTD-QM-ΔL3 was seen by ITC under our standard conditions [50 mM citrate-phosphate buffer, 200 mM NaCl, and 2 mM β-mercaptoethanol (pH 5.5)], consistent with this mutant’s very low deaminase activity. For the binding of inactivated A3BCTD-QMΔL3-E255A to different DNA oligos, the binding of dZ oligo cannot be distinguished from binding of the substrate, illustrating the importance of the active-site glutamate for binding of transition-state analogues These results demonstrate that the choice of buffer, especially the ionic strength, affects strongly, by several orders of magnitude, the affinity of ssDNA for the A3 proteins. These data are consistent with 5-Me-cytidine being a much poorer substrate than cytidine for A3 proteins, with the exception of A3H.51−53 for further investigations, we focused on the evaluation of dZ-containing oligos For both A3BCTD-QM-ΔL3-AL1swap and A3BCTD-DM (Figure 3 and the Supporting Information), using preferred substrate 5′AT3CAT3 (Oligo-2), significant inhibition was observed with dZ-ssDNA [Oligo-9 (Figure 3C,D)].
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