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
Loading...
Date
2021
DOI
Open Access Location
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Massey University
Rights
The Author
Abstract
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.
Description
Keywords
Enzyme inhibitors, DNA, Drug resistance in cancer cells, Treatment