Selective inhibition of APOBEC3 enzymes by single-stranded DNAs containing 2′-deoxyzebularine

Barzak, Fareeda M; Harjes, Stefan; Kvach, Maksim V.; Kurup, Harikrishnan M.; Jameson, Geoffrey B.; Filichev, Vyacheslav V.; Harjes, Elena

doi:10.1039/c9ob01781j

Cited by 30 publications

(58 citation statements)

References 55 publications

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“…Our results also highlight the importance of protonation of the N3 atom in dZ for its inhibitory behaviour. Noteworthy differences in inhibition profiles among different A3 enzymes observed here point to possibilities of obtaining highly specific A3 inhibitors, thereby supporting our approach to development of oligonucleotide‐based A3 inhibitors with the aid of chemically modified nucleosides, the structures of which can stall enzymatic cytosine deamination . Future work will continue to focus on the chemical optimisation of our ssDNA‐based A3 inhibitors and their evaluation in vitro and in vivo.…”

Section: Resultssupporting

confidence: 60%

“…Noteworthy differences in inhibition profiles among different A3 enzymes observed here point to possibilities of obtaining highly specific A3 inhibitors, thereby supporting our approach to development of oligonucleotide-based A3 inhibitors with the aid of chemically modified nucleosides, the structures of which can stall enzymatic cytosine deamination. [36] Future work will continue to focus on the chemical optimisation of our ssDNA-based A3 inhibitors and their evaluation in vitro and in vivo. Nucleotides flanking the target dZ and 5FdZ motifs can be further modified to improve inhibitory potential and to enhance the lifetimes of oligonucleotides in biological media.…”

Section: Resultsmentioning

confidence: 99%

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Differential Inhibition of APOBEC3 DNA‐Mutator Isozymes by Fluoro‐ and Non‐Fluoro‐Substituted 2′‐Deoxyzebularine Embedded in Single‐Stranded DNA

et al. 2019

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The APOBEC3 (APOBEC3A‐H) enzyme family is part of the human innate immune system that restricts pathogens by scrambling pathogenic single‐stranded (ss) DNA by deamination of cytosines to produce uracil residues. However, APOBEC3‐mediated mutagenesis of viral and cancer DNA promotes its evolution, thus enabling disease progression and the development of drug resistance. Therefore, APOBEC3 inhibition offers a new strategy to complement existing antiviral and anticancer therapies by making such therapies effective for longer periods of time, thereby preventing the emergence of drug resistance. Here, we have synthesised 2′‐deoxynucleoside forms of several known inhibitors of cytidine deaminase (CDA), incorporated them into oligodeoxynucleotides (oligos) in place of 2′‐deoxycytidine in the preferred substrates of APOBEC3A, APOBEC3B, and APOBEC3G, and evaluated their inhibitory potential against these enzymes. An oligo containing a 5‐fluoro‐2′‐deoxyzebularine (5FdZ) motif exhibited an inhibition constant against APOBEC3B 3.5 times better than that of the comparable 2′‐deoxyzebularine‐containing (dZ‐containing) oligo. A similar inhibition trend was observed for wild‐type APOBEC3A. In contrast, use of the 5FdZ motif in an oligo designed for APOBEC3G inhibition resulted in an inhibitor that was less potent than the dZ‐containing oligo both in the case of APOBEC3GCTD and in that of full‐length wild‐type APOBEC3G.

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Section: Resultssupporting

confidence: 60%

Section: Resultsmentioning

confidence: 99%

Differential Inhibition of APOBEC3 DNA‐Mutator Isozymes by Fluoro‐ and Non‐Fluoro‐Substituted 2′‐Deoxyzebularine Embedded in Single‐Stranded DNA

et al. 2019

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“…Based on the position of the SEC elution profile, we conclude that peak 1 corresponds to homo-dimeric A3BCTD* (with a molecular weight of ~44 kDa, referred to as 'A3BCTD* dimer'). This contrasts with the other A3BCTD variants we reported in previous studies [59,61,80], A3BCTD-QM-ΔL3, A3BCTD-QM-ΔL3-E255A, and A3BCTD-DM, which eluted only as monomeric species. Previous reports have suggested that A3A exists as both a monomer and a dimer in vitro [52,53].…”

Section: Multimerization Of A3bctd* In Solutioncontrasting

confidence: 99%

“…The catalytically active A3BCTD* was expressed and purified as described previously [59,61,62]. This variant contained four substitution mutations (F200S, W228S, L230K, F308K), the truncation of loop 3, and the transplant of loop 1 from A3A.…”

Section: Protein Expression and Purification Of A3bctd* Proteinmentioning

confidence: 99%

“…Therefore, to enhance the binding affinity and prevent deamination during experiments, we have studied the binding to active A3 enzymes of our recently characterized ssDNA-based A3 inhibitor containing 2'-deoxyzebularine (dZ, Scheme 1B). The sequence 5'-ATTT-dZ-ATTT (abbreviated as dZ-oligo) has a low (8-11) μM binding affinity for active A3 enzymes, including A3BCTD* and A3BCTD-DM [59,61]. The active A3BCTD*/dZ-oligo complex was analyzed using small-angle X-ray scattering (SAXS) to elucidate the changes in SAXS profiles upon ssDNA binding and subsequently to derive solution-state SAXS structural models to complement the solid-state X-ray structures.…”

Section: Introductionmentioning

confidence: 99%

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Small-Angle X-ray Scattering Models of APOBEC3B Catalytic Domain in a Complex with a Single-Stranded DNA Inhibitor

Barzak

Ryan

Kvach

et al. 2021

Viruses

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In normal cells APOBEC3 (A3A-A3H) enzymes as part of the innate immune system deaminate cytosine to uracil on single-stranded DNA (ssDNA) to scramble DNA in order to give protection against a range of exogenous retroviruses, DNA-based parasites, and endogenous retroelements. However, some viruses and cancer cells use these enzymes, especially A3A and A3B, to escape the adaptive immune response and thereby lead to the evolution of drug resistance. We have synthesized first-in-class inhibitors featuring modified ssDNA. We present models based on small-angle X-ray scattering (SAXS) data that (1) confirm that the mode of binding of inhibitor to an active A3B C-terminal domain construct in the solution state is the same as the mode of binding substrate to inactive mutants of A3A and A3B revealed in X-ray crystal structures and (2) give insight into the disulfide-linked inactive dimer formed under the oxidizing conditions of purification.

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Integrative analysis of genomic and transcriptomic data of normal, tumour, and co‐occurring leukoplakia tissue triads drawn from patients with gingivobuccal oral cancer identifies signatures of tumour initiation and progression

Ghosh

Das

Ghose

et al. 2022

The Journal of Pathology

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A thickened, white patch — leukoplakia — in the oral cavity is usually benign, but sometimes (in ~9% of individuals) it progresses to malignant tumour. Because the genomic basis of this progression is poorly understood, we undertook this study and collected samples of four tissues — leukoplakia, tumour, adjacent normal, and blood — from each of 28 patients suffering from gingivobuccal oral cancer. We performed multiomics analysis of the 112 collected tissues (four tissues per patient from 28 patients) and integrated information on progressive changes in the mutational and transcriptional profiles of each patient to create this genomic narrative. Additionally, we generated and analysed whole‐exome sequence data from leukoplakia tissues collected from 11 individuals not suffering from oral cancer. Nonsynonymous somatic mutations in the CASP8 gene were identified as the likely events to initiate malignant transformation, since these were frequently shared between tumour and co‐occurring leukoplakia. CASP8 alterations were also shown to enhance expressions of genes that favour lateral spread of mutant cells. During malignant transformation, additional pathogenic mutations are acquired in key genes (TP53, NOTCH1, HRAS) (41% of patients); chromosomal‐instability (arm‐level deletions of 19p and q, focal‐deletion of DNA‐repair pathway genes and NOTCH1, amplification of EGFR) (77%), and increased APOBEC‐activity (23%) are also observed. These additional alterations were present singly (18% of patients) or in combination (68%). Some of these alterations likely impact immune‐dynamics of the evolving transformed tissue; progression to malignancy is associated with immune suppression through infiltration of regulatory T‐cells (56%), depletion of cytotoxic T‐cells (68%), and antigen‐presenting dendritic cells (72%), with a concomitant increase in inflammation (92%). Patients can be grouped into three clusters by the estimated time to development of cancer from precancer by acquiring additional mutations (range: 4–10 years). Our findings provide deep molecular insights into the evolutionary processes and trajectories of oral cancer initiation and progression. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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Selective inhibition of APOBEC3 enzymes by single-stranded DNAs containing 2′-deoxyzebularine

Abstract: Selective inhibitors for APOBEC3B and APOBEC3A/G were obtained by substituting the preferred 2′-deoxycytidine by 2′-deoxyzebularine (Z) in a CCC DNA-motif.

Cited by 30 publications

References 55 publications

Differential Inhibition of APOBEC3 DNA‐Mutator Isozymes by Fluoro‐ and Non‐Fluoro‐Substituted 2′‐Deoxyzebularine Embedded in Single‐Stranded DNA

Differential Inhibition of APOBEC3 DNA‐Mutator Isozymes by Fluoro‐ and Non‐Fluoro‐Substituted 2′‐Deoxyzebularine Embedded in Single‐Stranded DNA

Small-Angle X-ray Scattering Models of APOBEC3B Catalytic Domain in a Complex with a Single-Stranded DNA Inhibitor

Integrative analysis of genomic and transcriptomic data of normal, tumour, and co‐occurring leukoplakia tissue triads drawn from patients with gingivobuccal oral cancer identifies signatures of tumour initiation and progression

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