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    Design, synthesis, and evaluation of cross-linked single-stranded DNAs as inhibitors of APOBEC3 enzymes : a thesis presented in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Chemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2021) Kurup, Harikrishnan Mohana
    Drug resistance is a major problem associated with anti-cancer chemo- and immunotherapies. Recent advances in the understanding of resistance mechanisms revealed that APOBEC3 (A3) family enzymes 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 retro-elements. 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 a strategy to augment existing anticancer therapies and suppress cancer evolution. It is known that ssDNA bound to both A3A and to a chimeric A3B (A3BCTD) is not linear but adopts a distinctive U-shape, projecting the target cytosine into the active site. We hypothesized that locking DNA sequences into the observed U-shape may provide not only better substrates but also, appropriately modified, better inhibitors of A3 enzymes. 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 create a cross-link between two modified nucleobases in ssDNA. The resultant cytosine-containing substrate, where the cytosine sits at the apex of the loop, was deaminated faster by A3B than a standard, linear substrate. The cross-linked ssDNA was converted into an A3B inhibitor by replacing the 2'-deoxycytidine in the preferred TCA substrate motif by 2'-deoxyzebularine (dZ) or 5-fluoro-2'-deoxyzebularine (5-FdZ), a known inhibitor of single nucleoside cytidine deaminases. This strategy yielded the first nanomolar A3B inhibitor (Kᵢ = 100 ± 16 nM) and provides a platform for further development of modified ssDNAs as powerful A3 inhibitors. We also synthesized three seven-membered ring-containing nucleosides as transition-state analogues of cytosine deamination and incorporated them in a ssDNA sequence to test the inhibition of A3 enzymes. In this work we successfully synthesized a nucleoside with a seven-membered ring-containing double bond (ddiazep) and two nucleosides having a seven-membered ring with hydroxyl groups (R and S isomers) as a nucleobase. However, the inhibition of A3 by these compounds was not as good as by a ssDNA containing dZ. Interestingly, there was a difference in the inhibition produced by seven-membered ring-containing R and S isomers. The inhibition data showed that the relative S stereochemistry was essential in the inhibition of A3.
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    Biophysical and biochemical characterisation of DNA-based inhibitors of the cytosine-mutating APOBEC3 enzymes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2020) Barzak, Fareeda Maged Yahya Mohammad
    With the rise of antiviral and anticancer drug resistance, a new approach must be taken to overcome this burden. The APOBEC3 (A3) family of cytosine deaminases hypermutate cytosines to uracils in single-stranded DNA (ssDNA). These enzymes act as double-edged swords: on one side they protect humans against a range of retroviruses and other pathogens, but several A3s are exploited by viruses and cancer cells to increase their rate of evolution using the enzyme’s mutagenic actions. This latter mode permits escape of cancer cells from the adaptive immune response and leads to the development of drug resistance. In particular, APOBEC3B (A3B) is considered to be a main driving source of genomic mutations in cancer cells. Inhibition of A3B, while retaining the beneficial actions of the other A3 in the immune system, may be used to augment existing anticancer therapies. In this study, we showed for the first time that short ssDNAs containing cytosine analogue nucleosides, 2'-deoxyzebularine (dZ) or 5-fluoro-2'-deoxyzebularine (5FdZ) in place of the substrate 2'-deoxycytidine (dC) in the preferred 5'-TC motif, inhibit the catalytic activity of A3B. However, as most A3 enzymes (except A3G) prefer to deaminate ssDNA with a 5'-TC motif, selective A3B inhibition was uncertain. We noted that nucleotides adjacent to the 5'-CCC motif influence the dC deamination preference of A3A, A3B, and A3G’s. Replacement of the A3B’s preferred dC in the 5'-CCC motif with dZ (5'-dZCC) led to the first selective inhibitor of A3B, in preference to A3A and A3G. Furthermore, using small-angle X-ray scattering (SAXS) we obtained the first model of a full-length two-domain A3 in complex with a dZ-ssDNA inhibitor. Our model showed that the ssDNA was largely bound to the C-terminal domain (CTD) with limited contact to the N-terminal domain in solution, due to the high affinity of the dZ for the CTD active-site. Our work provides a new platform for use of ssDNA-based inhibitors in targeting the mutagenic action of the A3B. Further developments using more potent inhibitors will help to achieve inhibition in cellular studies, with the ultimate goal to complement anti-cancer and antiviral treatments.