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PDBsum entry 6vba
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References listed in PDB file
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Key reference
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Title
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An effective human uracil-Dna glycosylase inhibitor targets the open pre-Catalytic active site conformation.
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Authors
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M.T.Nguyen,
D.Moiani,
Z.Ahmed,
A.S.Arvai,
S.Namjoshi,
D.S.Shin,
Y.Fedorov,
E.J.Selvik,
D.E.Jones,
J.Pink,
Y.Yan,
D.J.Laverty,
Z.D.Nagel,
J.A.Tainer,
S.L.Gerson.
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Ref.
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Prog Biophys Mol Biol, 2021,
163,
143-159.
[DOI no: ]
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PubMed id
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Abstract
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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.
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