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.
The APOBEC3 (APOBEC3A‐H) enzyme family is part of the human innate immune system that restricts pathogens by scrambling pathogenic single‐stranded (ss) DNA by deamination of cytosines to produce uracil residues. However, APOBEC3‐mediated mutagenesis of viral and cancer DNA promotes its evolution, thus enabling disease progression and the development of drug resistance. Therefore, APOBEC3 inhibition offers a new strategy to complement existing antiviral and anticancer therapies by making such therapies effective for longer periods of time, thereby preventing the emergence of drug resistance. Here, we have synthesised 2′‐deoxynucleoside forms of several known inhibitors of cytidine deaminase (CDA), incorporated them into oligodeoxynucleotides (oligos) in place of 2′‐deoxycytidine in the preferred substrates of APOBEC3A, APOBEC3B, and APOBEC3G, and evaluated their inhibitory potential against these enzymes. An oligo containing a 5‐fluoro‐2′‐deoxyzebularine (5FdZ) motif exhibited an inhibition constant against APOBEC3B 3.5 times better than that of the comparable 2′‐deoxyzebularine‐containing (dZ‐containing) oligo. A similar inhibition trend was observed for wild‐type APOBEC3A. In contrast, use of the 5FdZ motif in an oligo designed for APOBEC3G inhibition resulted in an inhibitor that was less potent than the dZ‐containing oligo both in the case of APOBEC3GCTD and in that of full‐length wild‐type APOBEC3G.
Selective inhibitors for APOBEC3B and APOBEC3A/G were obtained by substituting the preferred 2′-deoxycytidine by 2′-deoxyzebularine (Z) in a CCC DNA-motif.
In normal cells APOBEC3 (A3A-A3H) enzymes as part of the innate immune system deaminate cytosine to uracil on single-stranded DNA (ssDNA) to scramble DNA in order to give protection against a range of exogenous retroviruses, DNA-based parasites, and endogenous retroelements. However, some viruses and cancer cells use these enzymes, especially A3A and A3B, to escape the adaptive immune response and thereby lead to the evolution of drug resistance. We have synthesized first-in-class inhibitors featuring modified ssDNA. We present models based on small-angle X-ray scattering (SAXS) data that (1) confirm that the mode of binding of inhibitor to an active A3B C-terminal domain construct in the solution state is the same as the mode of binding substrate to inactive mutants of A3A and A3B revealed in X-ray crystal structures and (2) give insight into the disulfide-linked inactive dimer formed under the oxidizing conditions of purification.
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