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Publication Abstract from PubMed

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 IC50 < 100 nM. Subsequent enzymatic assays confirmed effective ATA inhibition but with an IC50 of 700 nM and showed direct binding to the human UDG with a KD 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.

An effective human uracil-DNA glycosylase inhibitor targets the open pre-catalytic active site conformation.,Nguyen MT, Moiani D, Ahmed Z, Arvai AS, Namjoshi S, Shin DS, Fedorov Y, Selvik EJ, Jones DE, Pink J, Yan Y, Laverty DJ, Nagel ZD, Tainer JA, Gerson SL Prog Biophys Mol Biol. 2021 Mar 3. pii: S0079-6107(21)00009-2. doi:, 10.1016/j.pbiomolbio.2021.02.004. PMID:33675849^[3]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.