Elena 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.

The normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its 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. APOBEC3A inhibitors may therefore comprise a unique class of anti-cancer agents that work by blocking mutagenesis, slowing tumor evolvability, and preventing 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 substrate motif that is part of a 3-nucleotide loop. In addition, the structural basis of APOBEC3A’s preference for YTCD motifs (Y = T, C; D = A, G, T) is explained. The nuclease-resistant phosphorothioated derivatives of these inhibitors have nanomolar potency in vitro and block APOBEC3A activity in human cells. These inhibitors may be useful probes for studying APOBEC3A activity in cellular systems and leading toward, potentially as conjuvants, next-generation, combinatorial anti-mutator and anti-cancer therapies.

The human antiviral enzyme APOBEC3A has mutagenic activity in many human cancers, where it is used to fuel tumour evolution. Its single-stranded DNA and RNA C-to-U editing activities contribute to undesirable mutagenic outcomes in cancers. Inhibitors of APOBEC3A may therefore block mutagenesis and prevent tumour evolution and thus reduce detrimental outcomes such as drug resistance and metastasis. Here we demonstrate that use of a carbazole pseudo-nucleoside as a part of a covalent cross-link between distant nucleotides in DNA leads to a faster deaminating substrate and upon changing of dC to 2'-deoxy-5-fluorozebularine to a more potent inhibitor of APOBEC3A (Ki = 29 ± 5 nM) in comparison with a DNA hairpin and previously described cross-linked DNAs. Truncation of the DNA sequence led to the first 4-mer cross-linked DNA inhibitor of APOBEC3A with nanomolar potency that is also stable against digestion by a phosphodiesterase. This provides a platform for a rational design of competitive inhibitors of APOBEC3 with properties of small-molecule drugs.

Glycocin F (GccF), a ribosomally synthesized, post-translationally modified peptide secreted by Lactobacillus plantarum KW30, rapidly inhibits the growth of susceptible bacteria at nanomolar concentrations. Previous studies have highlighted structural features important for its activity and have shown the absolute requirement for the Ser18 O-linked GlcNAc on the eight-residue loop linking the two short helices of the (C-X6-C)(2) structure. Here, we show that an ostensibly very small chemical modification to Ser18, the substitution of the C-alpha proton with a methyl group, reduces the antimicrobial activity of GccF 1000-fold (IC50 1.5 mu M cf. 1.5 nM). A comparison of the GccF(alpha-methylSer18) NMR structure (PDB 8DFZ) with that of the native protein (PDB 2KUY) showed a marked difference in the orientation and mobility of the loop, as well as a markedly different positioning of the GlcNAc, suggesting that loop conformation, dynamics, and glycan presentation play an important role in the interaction of GccF with as yet unknown but essential physiological target molecules.

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. 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.

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.

Drug resistance is a major problem associated with anticancer chemo- and immunotherapies. Recent advances in the understanding of resistance mechanisms have revealed that enzymes of the APOBEC3 (A3) family contribute to the development of drug resistance in multiple cancers. A3 enzymes are polynucleotide cytidine deaminases that convert cytosine to uracil (C→U) in single-stranded DNA (ssDNA) and in this way protect humans against viruses and mobile retroelements. On the other hand, cancer cells use A3s, especially A3A and A3B, to mutate human DNA, and thus by increasing rates of evolution, cancer cells escape adaptive immune responses and resist drugs. However, as A3A and A3B are non-essential for primary metabolism, their inhibition opens up a strategy to augment existing anticancer therapies and suppress cancer evolution. To test our hypothesis that pre-shaped ssDNA mimicking the U-shape observed in ssDNA-A3 complexes can provide a better binder to A3 enzymes, a Cu(I)-catalyzed azide-alkyne cycloaddition was used to cross-link two distant modified nucleobases in ssDNA. The resultant cytosine-containing substrate, where the cytosine sits at the apex of the loop, was deaminated faster by the engineered C-terminal domain of A3B than a standard, linear substrate. The cross-linked ssDNA was converted into an A3 inhibitor by replacing the 2'-deoxycytidine in the preferred TCA substrate motif by 2'-deoxyzebularine, a known inhibitor of single nucleoside cytidine deaminases. This strategy yielded the first nanomolar inhibitor of engineered A3B_CTD and wild-type A3A (K_i = 690 ± 140 and 360 ± 120 nM, respectively), providing a platform for further development of powerful A3 inhibitors.

APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2′-deoxycytidine embedded in 40-mer ssDNA was replaced by 2′-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G–ssDNA complex that gives insight into the observed “jumping” behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3–ssDNA complexes.