Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A

Abstract

The normally antiviral enzyme APOBEC3A is an endogenous mutagen in many different human cancers, where it becomes hijacked to fuel tumor evolvability. APOBEC3A's single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations. Transgenic expression in mice demonstrates its tumorigenic potential. APOBEC3A inhibitors may therefore comprise a novel class of anti-cancer agents that work by blocking mutagenesis, preventing tumor evolvability, and lessening detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2'-deoxy-5-fluorozebularine in place of the cytidine in the TC recognition motif that is part of a three-nucleotide loop. The nuclease-resistant phosphorothioated derivatives of these inhibitors maintain nanomolar in vitro potency against APOBEC3A, localize to the cell nucleus, and block APOBEC3A activity in human cells. These results combine to suggest roles for these inhibitors to study A3A activity in living cells, potentially as conjuvants, leading toward next-generation, combinatorial anti-mutator and anti-cancer therapies.

Seven-membered ring nucleobases as inhibitors of human cytidine deaminase and APOBEC3A

“…60,61 During our study two articles have been published on drug discovery efforts to obtain small molecule inhibitors of A3. 62,63 None approaches the nM potency exhibited by our FdZ 43 and dDiAzep -containing hairpins. This highlights the interest and need for powerful A3 inhibitors.…”

“…41 DNA hairpins with short loops have also been shown to provide selective inhibitors of A3A when the target dC in the loop was changed to dZ-derivatives. [42][43][44] We also demonstrated that dZ and THU as free nucleosides did not inhibit A3 enzymes, which indicates that ssDNA delivers dZ into the active site of A3. 17 In the past, the diazepinone riboside was described as a more powerful inhibitor of CDA than dZ (K i = 25 nM for 1,3diazepin-2-one [31][32][33][34][35] and 2.9 µM for dZ).…”

“…41 DNA hairpins with short loops have also been shown to provide selective inhibitors of A3A when the target dC in the loop was changed to dZ-derivatives. 42–44 We also demonstrated that dZ and THU as free nucleosides did not inhibit A3 enzymes, which indicates that ssDNA delivers dZ into the active site of A3. 17…”

“…58 As a control for the evaluation of dDiAzep in the structure of a DNA hairpin, we synthesised and tested an FdZ-hairpin that showed powerful inhibition of wild-type A3A with a K i of 117 nM. 43 Full characterisation of this compound and its crystal structure with wild-type A3A will be published elsewhere. 43 Unfortunately, due to chemical difficulties we had to abandon the synthesis of the 2′-deoxy form of dDiAzep-OH (Fig.…”

“…43 Full characterisation of this compound and its crystal structure with wild-type A3A will be published elsewhere. 43 Unfortunately, due to chemical difficulties we had to abandon the synthesis of the 2′-deoxy form of dDiAzep-OH (Fig. 1B).…”