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

Authors: Nguyen, My T; Moiani, Davide; Ahmed, Zamal; Arvai, Andrew S; Namjoshi, Sarita; Shin, Dave S; Fedorov, Yuriy; Selvik, Edward J; Jones, Darin E; Pink, John; Yan, Yan; Laverty, Daniel J; Nagel, Zachary D; Tainer, John A; Gerson, Stanton L

Published In Prog Biophys Mol Biol, (2021 08)

Abstract: 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.

PubMed ID: 33675849

MeSH Terms: Catalytic Domain; Cytidine Deaminase; DNA Damage; DNA Repair*; Humans; Minor Histocompatibility Antigens; Uracil; Uracil-DNA Glycosidase*/genetics; Uracil-DNA Glycosidase*/metabolism