Design, Synthesis, and Evaluation of a Cross-Linked Oligonucleotide as the First Nanomolar Inhibitor of APOBEC3A

Abstract

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.

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

“…Prior studies have shown that linear ssDNA substrates with 2-deoxyzebularine (dZ) and 5-fluoro-dZ (FdZ) in place of the target cytosine are weak inhibitors of A3A (and A3B) [30][31][32] . Additional work by our group and others has shown greater inhibition of A3A in vitro with pre-formed U-shaped, hairpin dZ and FdZ transition-state trapping oligonucleotides [33][34] . Here, we use X-ray crystallography to reveal the first high-resolution structures of wildtype A3A-inhibited complexes and demonstrate the underlying mechanism of inhibition.…”

“…The general strategy for the synthesis of dZ and FdZ and their incorporation into DNA oligomers has been described by us elsewhere 33 . 3-[(Dimethylaminomethylidene)amino]-3 H -1,2,4-dithiazole-3-thione (DDTT, Sulfurizing Reagent II from GlenResearch, USA) was used for sulfurization of oligos.…”

“…number: 008a-500, Minsk, Belorussia). dZ and FdZ phosphoramidites were synthesized as previously described 31-33 .…”

“…23 A3A-E72A was used for structural studies and ITC experiments with the substrate. Wildtype A3A, which was recombinantly expressed with the His 6 tag at the C-terminal end in E. coli and purified as described 33 , was used for kinetic and structural studies with the inhibitor. The yield from 1-10 L expression was usually not enough to justify size-exclusion chromatography purification.…”

The cytidine deaminase APOBEC3A is required for large ribosomal subunit biogenesis

“…Our work on APOBEC3A highlights another example that links the integrity of the genome to ribosome biogenesis. Currently, cytidine deaminase inhibitors are being developed [reviewed in ( 26 )], including against APOBEC3A ( 90, 91 ), with a focus on their application in cancer therapy ( 92 ). APOBEC3A’s newfound function in ribosome biogenesis emphasizes it as a top candidate cytidine deaminase target for cancer therapeutics.…”