6-Carboxyfluorescein and structurally similar molecules inhibit DNA binding and repair by O6-alkylguanine DNA alkyltransferase

Human O-6-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O-6-alkylguanine and O-4-alkylthymine adducts in single-stranded and duplex DNAs. These activities protect normal cells and tumor cells against drugs that alkylate DNA; drugs that inactivate ACT are under test as chemotherapeutic enhancers. In studies using 6-carboxyfluorescein (FAM)-labeled DNAs, ACT reduced the fluorescence intensity by similar to 40% at binding saturation, whether the FAM was located at the 5' or the 3' end of the DNA. ACT protected residual fluorescence from quenching, indicating a solute-inaccessible binding site for FAM. Sedimentation equilibrium analyses showed that saturating AGT-stoichiometries were higher with FAM-labeled DNAs than with unlabeled DNAs, suggesting that the FAM provides a protein binding site that is not present in unlabeled DNAs. Additional fluorescence and sedimentation measurements showed that ACT forms a 1:1 complex with free FAM. Active site benzylation experiments and docking calculations support models in which the primary binding site is located in or near the active site of the enzyme. Electrophoretic analyses show that FAM inhibits DNA binding (IC50 similar to 76 mu M) and repair of DNA containing an O-6-methylguanine residue (IC50 similar to 63 mu M). Similar results were obtained with other polycyclic aromatic compounds. These observations demonstrate the existence of a new class of non-covalent ACT-inhibitors. After optimization for binding-affinity, members of this class might be useful in cancer chemotherapy. (C) 2011 Elsevier B.V. All rights reserved.