A New Class of Uracil–DNA Glycosylase Inhibitors Active against Human and Vaccinia Virus Enzyme

Abstract

Uracil–DNA glycosylases are enzymes that excise uracil bases appearing in DNA as a result of cytosine deamination or accidental dUMP incorporation from the dUTP pool. The activity of Family 1 uracil–DNA glycosylase (UNG) activity limits the efficiency of antimetabolite drugs and is essential for virulence in some bacterial and viral infections. Thus, UNG is regarded as a promising target for antitumor, antiviral, antibacterial, and antiprotozoal drugs. Most UNG inhibitors presently developed are based on the uracil base linked to various substituents, yet new pharmacophores are wanted to target a wide range of UNGs. We have conducted virtual screening of a 1,027,767-ligand library and biochemically screened the best hits for the inhibitory activity against human and vaccinia virus UNG enzymes. Although even the best inhibitors had IC50 ≥ 100 μM, they were highly enriched in a common fragment, tetrahydro-2,4,6-trioxopyrimidinylidene (PyO3). In silico, PyO3 preferably docked into the enzyme’s active site, and in kinetic experiments, the inhibition was better consistent with the competitive mechanism. The toxicity of two best inhibitors for human cells was independent of the presence of methotrexate, which is consistent with the hypothesis that dUMP in genomic DNA is less toxic for the cell than strand breaks arising from the massive removal of uracil. We conclude that PyO3 may be a novel pharmacophore with the potential for development into UNG-targeting agents.

Probing the Conformational Restraints of DNA Damage Recognition with β-L-Nucleotides

“…The following enzymes were overexpressed and purified essentially as described earlier: E. coli Fpg [ 78 ], MutY (the catalytic p25 fragment) [ 79 ], Nei [ 80 ], Nth [ 81 ], Nfo [ 82 ], human OGG1 [ 83 ], MBD4 (the catalytic fragment) [ 62 ], NEIL1 [ 84 ], NEIL2 [ 85 ], vaccinia virus D4 protein [ 86 ], exonuclease-deficient KF [ 87 ], exonuclease-deficient RBpol [ 88 ], human Pol β [ 89 ], the catalytic fragment (residues 242–575) of human Pol λ [ 90 ] and the catalytic fragment (residues 1–560) of human Pol κ [ 91 ]. Human MPG, NEIL3 and SMUG1 proteins were kindly provided by Dr. Murat Saparbaev (Institut Gustave Roussy, France).…”

Back-Up Base Excision DNA Repair in Human Cells Deficient in the Major AP Endonuclease, APE1

“…Nb.Bpu10I and Nt.Bpu10I nickases, human APE1, and E. coli Ung and Nfo were from New England Biolabs (Ipswich, MA, USA), phage T4 DNA ligase and polynucleotide kinase, from Thermo Fisher Scientific (Waltham, MA, USA). Human UNG was purified as described [ 102 ]. HEK293FT APEX1 KO cells 1C4 and 2A9 had been generated and described earlier [ 39 ] and were from the laboratory stocks.…”

Recognition of a Clickable Abasic Site Analog by DNA Polymerases and DNA Repair Enzymes

“…Human APEX1 [ 79 ], NEIL1 [ 80 ], NEIL2 [ 81 ], MBD4 [ 82 ], OGG1 [ 83 ], UNG [ 84 ], POLκ (catalytic core, residues 1–560) [ 85 ], POLλ (catalytic core, residues 242–575) [ 86 ], E. coli Fpg [ 87 ], KF exo − [ 88 ], MutY [ 89 ], Nei [ 90 ], Nfo [ 91 ], Nth [ 92 ], phage RB69 DNA polymerase exo − [ 93 ] and vaccinia virus UNG [ 84 ] were purified essentially as described ( Supplementary Figures S1 and S2 ). Human MPG, NEIL3 and SMUG1 proteins were a kind gift from Dr. Murat Saparbaev (Institut Gustave Roussy, France).…”