Identification of small molecule synthetic inhibitors of DNA polymerase beta by NMR chemical shift mapping.
DNA polymerase beta (beta-pol) plays a central role in repair of damaged DNA bases by base excision repair (BER) pathways. A predominant phenotype of beta-pol null mouse fibroblasts is hypersensitivity to the DNA-methylating agent methyl methanesulfonate. Residues in the 8-kDa domain of beta-pol that seem to interact with a known natural product beta-pol inhibitor, koetjapic acid, were identified by NMR chemical shift mapping. The data implicate the binding pocket as the hydrophobic cleft between helix-2 and helix-4, which provides the DNA binding and deoxyribose phosphate lyase activities of the enzyme. Nine structurally related synthetic compounds, containing aromatic or other hydrophobic groups in combination with two carboxylate groups, were then tested. They were found to bind to the same or a very similar region on the surface of the enzyme. The ability of these compounds to potentiate methyl methanesulfonate cytotoxicity, an indicator of cellular BER capacity, in wild-type and beta-pol null mouse fibroblasts, was next ascertained. The most active and beta-pol-specific of these agents, pamoic acid, was further characterized and found to be an inhibitor of the deoxyribose phosphate lyase and DNA polymerase activities of purified beta-pol on a BER substrate. Our results illustrate that NMR-based mapping techniques can be used in the design of small molecule enzyme inhibitors including those with potential use in a clinical setting.
Hu
Hong-Yu
HY
Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032, USA.
Horton
Julie K
JK
Gryk
Michael R
MR
Prasad
Rajendra
R
Naron
Jana M
JM
Sun
Di-An
DA
Hecht
Sidney M
SM
Wilson
Samuel H
SH
Mullen
Gregory P
GP
D000818:Animals; D001557:Benzenesulfonates / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D002229:Carbenoxolone / Q000737:chemistry / Q000494:pharmacology; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D019951:DNA Polymerase beta / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D019695:Glycyrrhizic Acid / Q000737:chemistry* / Q000494:pharmacology; D051379:Mice; D008817:Mice, Mutant Strains; D009284:Naphthols / Q000737:chemistry / Q000494:pharmacology; D019906:Nuclear Magnetic Resonance, Biomolecular; D019761:Phosphorus-Oxygen Lyases / Q000037:antagonists & inhibitors / Q000378:metabolism; D017434:Protein Structure, Tertiary
D001557:Benzenesulfonates; D004791:Enzyme Inhibitors; D009284:Naphthols; C010332:solochrome cyanine R; D019695:Glycyrrhizic Acid; C004368:pamoic acid; D004247:DNA; C423906:5'-deoxyribose phosphate lyase; D019951:DNA Polymerase beta; D019761:Phosphorus-Oxygen Lyases; D002229:Carbenoxolone