An effective human uracil-DNA glycosylase inhibitor targets the open pre-catalytic active site conformation.

Autor: Nguyen MT; Case Western Reserve University, Department of Biochemistry, Cleveland, OH, 44106, USA., Moiani D; Departments of Cancer Biology and of Molecular & Cellular Oncology, University of Texas MD Anderson Cancer Center, 1515 Holcomb Blvd, Houston, TX, 77030, USA., Ahmed Z; Departments of Cancer Biology and of Molecular & Cellular Oncology, University of Texas MD Anderson Cancer Center, 1515 Holcomb Blvd, Houston, TX, 77030, USA., Arvai AS; Integrative Structural & Computational Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA., Namjoshi S; Departments of Cancer Biology and of Molecular & Cellular Oncology, University of Texas MD Anderson Cancer Center, 1515 Holcomb Blvd, Houston, TX, 77030, USA., Shin DS; Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA., Fedorov Y; Case Small-Molecule Screening Core, School of Medicine, Case Western Reserve University, Cleveland, OH, 44016, USA., Selvik EJ; Department of Pharmaceutical Sciences, The University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR, 72205, USA., Jones DE; Department of Pharmaceutical Sciences, The University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR, 72205, USA., Pink J; Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, 44106, USA., Yan Y; Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, 44106, USA., Laverty DJ; Department of Environmental Health, Harvard TH Chan School of Public Health, Boston, MA, 02115, USA., Nagel ZD; Department of Environmental Health, Harvard TH Chan School of Public Health, Boston, MA, 02115, USA., Tainer JA; Departments of Cancer Biology and of Molecular & Cellular Oncology, University of Texas MD Anderson Cancer Center, 1515 Holcomb Blvd, Houston, TX, 77030, USA; Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA. Electronic address: JTainer@mdanderson.org., Gerson SL; Case Western Reserve University, Department of Biochemistry, Cleveland, OH, 44106, USA; Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, 44106, USA. Electronic address: Slg5@case.edu.
Jazyk: angličtina
Zdroj: Progress in biophysics and molecular biology [Prog Biophys Mol Biol] 2021 Aug; Vol. 163, pp. 143-159. Date of Electronic Publication: 2021 Mar 03.
DOI: 10.1016/j.pbiomolbio.2021.02.004
Abstrakt: Human uracil DNA-glycosylase (UDG) is the prototypic and first identified DNA glycosylase with a vital role in removing deaminated cytosine and incorporated uracil and 5-fluorouracil (5-FU) from DNA. UDG depletion sensitizes cells to high APOBEC3B deaminase and to pemetrexed (PEM) and floxuridine (5-FdU), which are toxic to tumor cells through incorporation of uracil and 5-FU into DNA. To identify small-molecule UDG inhibitors for pre-clinical evaluation, we optimized biochemical screening of a selected diversity collection of >3,000 small-molecules. We found aurintricarboxylic acid (ATA) as an inhibitor of purified UDG at an initial calculated IC 50  < 100 nM. Subsequent enzymatic assays confirmed effective ATA inhibition but with an IC 50 of 700 nM and showed direct binding to the human UDG with a K D of <700 nM. ATA displays preferential, dose-dependent binding to purified human UDG compared to human 8-oxoguanine DNA glycosylase. ATA did not bind uracil-containing DNA at these concentrations. Yet, combined crystal structure and in silico docking results unveil ATA interactions with the DNA binding channel and uracil-binding pocket in an open, destabilized UDG conformation. Biologically relevant ATA inhibition of UDG was measured in cell lysates from human DLD1 colon cancer cells and in MCF-7 breast cancer cells using a host cell reactivation assay. Collective findings provide proof-of-principle for development of an ATA-based chemotype and "door stopper" strategy targeting inhibitor binding to a destabilized, open pre-catalytic glycosylase conformation that prevents active site closing for functional DNA binding and nucleotide flipping needed to excise altered bases in DNA.
(Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)
Databáze: MEDLINE
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