Stefan Harjes

Nucleoside and polynucleotide cytidine deaminases (CDAs), such as CDA and APOBEC3, share a similar mechanism of cytosine to uracil conversion. In 1984, phosphapyrimidine riboside was characterised as the most potent inhibitor of human CDA, but the quick degradation in water limited the applicability as a potential therapeutic. To improve stability in water, we synthesised derivatives of phosphapyrimidine nucleoside having a CH₂ group instead of the N3 atom in the nucleobase. A charge-neutral phosphinamide and a negatively charged phosphinic acid derivative had excellent stability in water at pH 7.4, but only the charge-neutral compound inhibited human CDA, similar to previously described 2'-deoxyzebularine (K_i = 8.0 ± 1.9 and 10.7 ± 0.5 µM, respectively). However, under basic conditions, the charge-neutral phosphinamide was unstable, which prevented the incorporation into DNA using conventional DNA chemistry. In contrast, the negatively charged phosphinic acid derivative was incorporated into DNA instead of the target 2'-deoxycytidine using an automated DNA synthesiser, but no inhibition of APOBEC3A was observed for modified DNAs. Although this shows that the negative charge is poorly accommodated in the active site of CDA and APOBEC3, the synthetic route reported here provides opportunities for the synthesis of other derivatives of phosphapyrimidine riboside for potential development of more potent CDA and APOBEC3 inhibitors.

Nucleoside and polynucleotide cytidine deaminases (CDA and APOBEC3) share similar mechanism of cytosine to uracil conversion. In 1984 phosphapyrimidine riboside was characterised as the most potent inhibitor of human CDA but its quick degradation in water limited its applicability as a potential therapeutic. To improve stability in water, we synthesised a derivative dPC of phosphapyrimidine nucleoside having CH₂ group instead of the N3 atom in the nucleobase. A charge-neutral phosphinamide dPC-NH₂ and a negatively-charged phosphinic acid derivative dPC-OH had excellent stability in water at pH 7.4 but only the charge-neutral dPC-NH₂ inhibited human CDA similar to previously described 2’-deoxyzebularine (K_i = 8.0 ± 1.9 and 10.7 ± 0.5 µM, respectively). However, at basic conditions the charge-neutral phosphinamide dPC-NH₂ was unstable which prevented its incorporation into DNA using conventional DNA chemistry. In contrast, the negatively charged phosphinic acid derivative dPC-OH was incorporated into DNA instead of the target dC using an automated DNA synthesiser but no inhibition of APOBEC3A was observed for modified DNAs. Although this shows that negative charge is poorly accommodated in the active site of CDA and APOBEC3, the synthetic route reported here provides opportunities for the synthesis of other derivatives of phosphapyrimidine riboside for potential development of more potent CDA and APOBEC3 inhibitors.

A DNA hairpin possessing 1,3-diazepin-2-one 2′-deoxyriboside or 5-fluoro-2′-deoxyzebularine in its loop inhibits APOBEC3A in the nM range in vitro .

The normally antiviral enzyme APOBEC3A ^1-4 is an endogenous mutagen in many different human cancers ^5-7 , where it becomes hijacked to fuel tumor evolvability. APOBEC3A’s single-stranded DNA C-to-U editing activity ^{1, 8} results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations ^5-7 . Transgenic expression in mice demonstrates its tumorigenic potential ⁹ . APOBEC3A inhibitors may therefore comprise a novel class of anticancer 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.