PDBsum entry 6vba

Hydrolase

PDB id: 6vba

Contents

Protein chain

223 a.a.

Ligands

QU4

Waters ×259

PDB id:

6vba

Name:

Hydrolase

Title:

Structure of human uracil DNA glycosylase (udg) bound to aurintricarboxylic acid (ata)

Structure:

Uracil-DNA glycosylase. Chain: a. Synonym: udg. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: ung, dgu, ung1, ung15. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.80Å R-factor: 0.182 R-free: 0.234

Authors:

D.Moiani,A.S.Arvai,J.A.Tainer

Key ref:

M.T.Nguyen et al. (2021). An effective human uracil-DNA glycosylase inhibitor targets the open pre-catalytic active site conformation. Prog Biophys Mol Biol, 163, 143-159. PubMed id: 33675849 DOI: 10.1016/j.pbiomolbio.2021.02.004

Date:

18-Dec-19 Release date: 03-Mar-21

Protein chain

P13051  (UNG_HUMAN) -  Uracil-DNA glycosylase from Homo sapiens

Seq: 313 a.a.

Struc: 223 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.2.2.27 - uracil-DNA glycosylase.

DOI no: 10.1016/j.pbiomolbio.2021.02.004

Prog Biophys Mol Biol 163:143-159 (2021)

PubMed id: 33675849

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

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.

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 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.