NATURAL-PRODUCT INHIBITORS OF HUMAN DNA-LIGASE-I

Enzymic activity mediated by recombinant human DNA ligase I (hLI), in conjunction with tannin removal procedures, has been applied to a natu ral-product screen involving similar to 1000 plant extracts and variou s pure compounds. The primary hLI activity assay involved the measurem ent of the amount of radiolabelled phosphate in a synthetic nucleic ac id hybrid that becomes resistant to alkaline phosphatase as a result o f ligation. A bioactivity-guided fractionation scheme resulted in the isolation of ursolic [IC50 = 100 mu g/ml (216 mu M)] and oleanolic [IC 50 = 100 mu g/ml (216 mu M)] acids from Tricalysia niamniamensis Hiern (Rubiaceae), which demonstrated similar DNA ligase inhibition profile s to other triterpenes such as aleuritolic acid. Protolichesterinic ac id [IC50 = 6 mu g/ml (20 mu M)], swertifrancheside [IC50 = 8 mu g/ml ( 11 mu M)] and fulvoplumierin [IC50 = 87 mu g/ml (357 mu M)] represent three additional natural-product structural classes that inhibit hLI. Fagaronine chloride [IC50 = 10 mu g/ml (27 mu M)] and certain flavonoi ds are also among the pure natural products that were found to disrupt the activity of the enzyme, consistent with their nucleic acid interc alative properties, Further analyses revealed that some of the hLI-inh ibitory compounds interfered with the initial adenylation step of the ligation reaction, indicating a direct interaction with the enzyme pro tein. However, in all cases, this enzyme-inhibitor interaction did not disrupt the DNA relaxation activity mediated by hLI. These results in dicate that, although the same enzyme active site may be involved in b oth enzyme adenylation and DNA relaxation, inhibitors may exert allost eric effects by inducing conformational changes that disrupt only one of these activities. Studies with inhibitors are important for the ass ignment of specific cellular functions to these enzymes, as well as fo r their development into clinically useful antitumour agents.