DNA base analogs, 2,4,5,6-substituted pyrimidines and 2,6-substituted purines were tested as potential inhibitors of E. coli Fpg protein (formamidopyrimidine -DNA glycosylase). Three of the seventeen compounds tested revealed inhibitory properties. 2-Thioxanthine was the most efficient, inhibiting 50% of 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeG) excision activity at 17.1 mM concentration. The measured K i was 4.44 ± 0.15 mM. Inhibition was observed only when the Fpg protein was first challenged to its substrate followed by the addition of the base analog, suggesting uncompetitive (catalytic) inhibition. For two other com-Vol. 52 No. 1/2005 167-178 QUARTERLY . ; [ 3 H]MNU-DNA, DNA alkylated with [ 3 H]methylnitrosourea; 8-oxoG, 8-oxoguanine; Trp-P-1, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole. pounds, 2-thio-or 2-oxo-4,5,6-substituted pyrimidines, IC 50 was only 343.3 ± 58.6 and 350 ± 24.4 mM, respectively. No change of the Fpg glycosylase activity was detected in the presence of Fapy-7MeG, up to 5 mM. We also investigated the effect of DNA structure modified by tryptophan pyrolysate (Trp-P-1) on the activity of base excision repair enzymes: Escherichia coli and human DNA glycosylases of oxidized (Fpg, Nth) and alkylated bases (TagA, AlkA, and ANPG), and for bacterial AP endonuclease (Xth protein). Trp-P-1, which changes the secondary DNA structure into non-B, non-Z most efficiently inhibited excision of alkylated bases by the AlkA glycosylase (IC 50 = 1 mM). The ANPG, TagA, and Fpg proteins were also inhibited although to a lesser extent (IC 50 = 76.5 mM, 96 mM, and 187.5 mM, respectively). Trp-P-1 also inhibited incision of DNA at abasic sites by the b-lyase activity of the Fpg and Nth proteins, and to a lesser extent by the Xth AP endonuclease. Thus, DNA conformation is critical for excision of damaged bases and incision of abasic sites by DNA repair enzymes.
