biostudies-literature00590Nguyen MTNIEHS NIH HHSNHLBI NIH HHSNCI NIH HHSNational Institutes of HealthUniversity of Texas MD Anderson Cancer Center143-159https://www.ebi.ac.uk/biostudies/studies/S-EPMC8722130biostudies-literatureUnknown163Human 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 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.Progress in biophysics and molecular biologyAn effective human uracil-DNA glycosylase inhibitor targets the open pre-catalytic active site conformation.PMC8722130U01 ES029520P30 ES000002RP1808135T32HL007118P30ES0000025P30 CA043703T32 HL007118U01ES029520P30 CA043703R35 CA220430P01 CA092584Shin DSAhmed ZFedorov YNguyen MTJones DEYan YLaverty DJArvai ASNagel ZDTainer JAGerson SLNamjoshi SSelvik EJMoiani DPink J59falseAn effective human uracil-DNA glycosylase inhibitor targets the open pre-catalytic active site conformation.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 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.2021-01-01T00:00:00Z2021 Aug2024-11-06T19:27:34.55Z2022-02-11T14:48:01.369ZS-EPMC87221303367584910.1016/j.pbiomolbio.2021.02.004