Inhibition of sequence independent DNA-binding activity of human recombinant HMGB1 protein with natural triterpenes

By Sivakumar Annadurai, Natalia F. Krynetskaia, Manali S. Phadke, Evgeny Krynetskiy,Daniel J. Canney



Abstract

DNA-protein interactions regulate pivotal intracellular processes and thus provide an important target for drug discovery efforts.  Small molecules remain ineffective inhibitors of DNA binding because of the lack of adequate high-throughput screening procedures, and the paucity of lead molecules for drug design. High Mobility Group protein B1 (HMGB1) is a nuclear DNA-binding protein with no known sequence specificity or enzymatic activity, which plays an important role in chemotherapy-induced apoptosis, tissue regeneration, and inflammation.  The natural triterpene glycyrrhizin (GLA) has been reported to be a modest inhibitor of DNA-HMGB1 binding interactions, though the mechanism of inhibition remained unclear. In the present work, we investigated inhibitory effects of GLA and its derivatives (carbenoxolone (CGA) and potassium salt of glycyrrhetinate (GAK)) on DNA-binding activity of HMGB1. Using a newly developed capillary electrophoresis mobility shift assay, we characterized binding of synthetic DNA duplexes with recombinant human HMGB1. Dynamic light scattering and fluorometric experiments demonstrated that the inhibitory effects of these triterpenes on DNA binding depended on their ability to form supramolecular aggregates. GLA and GAK demonstrated inhibitory effect on DNA-HMGB1 binding at concentrations exceeding CMC (100-150?M). GLA inhibited DNA-HMGB1 binding with IC50 = 500 ?M; 600 ?M GAK showed 100% inhibition of DNA-HMGB1 binding. In the presence of GLA, human lung carcinoma cells demonstrated more than 300-fold increased viability when treated with chemotherapeutic drug cytarabine, consistent with the GLA inhibitory activity. The results of our study demonstrated that molecules capable of forming stable aggregates are potential modulators of DNA-protein binding and open avenues to the development of novel classes of physiologically active compounds.

 

Key Words : DNA-binding proteins, HMGB1, Glycyrrhizin, inhibitors, supramolecular aggregates.

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