Browsing by Author "Barzak FM"

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    Design, Synthesis, and Evaluation of a Cross-Linked Oligonucleotide as the First Nanomolar Inhibitor of APOBEC3A
    (American Chemical Society, 2022-11-15) Kurup HM; Kvach MV; Harjes S; Barzak FM; Jameson GB; Harjes E; Filichev VV
    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.
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    Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity
    (MDPI (Basel, Switzerland), 2022-09-06) Barzak FM; Ryan TM; Mohammadzadeh N; Harjes S; Kvach MV; Kurup HM; Krause KL; Chelico L; Filichev VV; Harjes E; Jameson GB; De la Torre JC; Andrei G
    APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2'-deoxycytidine embedded in 40-mer ssDNA was replaced by 2'-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G-ssDNA complex that gives insight into the observed "jumping" behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3-ssDNA complexes.
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    Small-Angle X-ray Scattering Models of APOBEC3B Catalytic Domain in a Complex with a Single-Stranded DNA Inhibitor
    (MDPI (Basel, Switzerland), 2021-02-12) Barzak FM; Ryan TM; Kvach MV; Kurup HM; Aihara H; Harris RS; Filichev VV; Harjes E; Jameson GB; Chelico L
    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.