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Published on: July 26, 2010
Design, Synthesis, and Evaluation of a Cross-Linked Oligonucleotide as the First Nanomolar Inhibitor of APOBEC3A
Harikrishnan M Kurup1,2, Maksim V Kvach1, Stefan Harjes1
1School of Natural Sciences, Massey University, Private Bag 11 222, Palmerston North 4442, New Zealand.
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 A3BCTD and wild-type A3A (Ki = 690 ± 140 and 360 ± 120 nM, respectively), providing a platform for further development of powerful A3 inhibitors.
Insights
APOBEC3 enzymes drive cancer drug resistance by mutating DNA. Researchers developed a novel DNA inhibitor targeting these enzymes, offering a new strategy to enhance cancer therapies and combat tumor evolution.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Drug resistance is a significant challenge in cancer treatment, with APOBEC3 (A3) enzymes identified as key contributors to resistance mechanisms in various cancers.
- A3 enzymes, specifically A3A and A3B, are cytidine deaminases that deaminate cytosine in single-stranded DNA (ssDNA), promoting cancer cell evolution and evasion of immune responses and therapies.
- Inhibition of A3A and A3B presents a promising therapeutic strategy to overcome cancer drug resistance and suppress tumor evolution, as these enzymes are non-essential for primary cell metabolism.
Purpose of the Study:
- To investigate if pre-shaped ssDNA, mimicking the U-shape in ssDNA-A3 complexes, can serve as a superior binder for A3 enzymes.
- To develop a novel inhibitor targeting A3 enzymes to augment existing anticancer therapies.
Main Methods:
- Utilized Cu(I)-catalyzed azide-alkyne cycloaddition to cross-link modified nucleobases in ssDNA, creating a U-shaped substrate.
- Engineered a substrate with cytosine at the apex of the loop for enhanced deamination by A3B C-terminal domain.
- Modified the cross-linked ssDNA substrate by replacing 2'-deoxycytidine with 2'-deoxyzebularine to create an A3 inhibitor.
Main Results:
- The U-shaped ssDNA substrate with cytosine at the loop apex demonstrated faster deamination by the engineered A3B C-terminal domain compared to linear substrates.
- The developed 2'-deoxyzebularine-containing ssDNA yielded the first nanomolar inhibitor for engineered A3B C-terminal domain (Kᵢ = 690 ± 140 nM) and wild-type A3A (Kᵢ = 360 ± 120 nM).
Conclusions:
- Pre-shaped ssDNA substrates can be effectively designed to enhance binding and inhibition of A3 enzymes.
- This study presents a novel platform for developing potent A3 inhibitors, offering a potential strategy to overcome drug resistance and suppress cancer evolution in combination therapies.
