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

[摘要] DNA-protein interactions regulate pivotal intracellular processes and thus provide an importanttarget for drug discovery efforts. Small molecules remain ineffective inhibitors of DNA bindingbecause of the lack of adequate high-throughput screening procedures, and the paucity of leadmolecules for drug design. High Mobility Group protein B1 (HMGB1) is a nuclear DNA-bindingprotein with no known sequence specificity or enzymatic activity, which plays an important rolein chemotherapy-induced apoptosis, tissue regeneration, and inflammation. The naturaltriterpene glycyrrhizin (GLA) has been reported to be a modest inhibitor of DNA-HMGB1binding interactions, though the mechanism of inhibition remained unclear. In the present work,we investigated inhibitory effects of GLA and its derivatives (carbenoxolone (CGA) andpotassium salt of glycyrrhetinate (GAK)) on DNA-binding activity of HMGB1. Using a newlydeveloped capillary electrophoresis mobility shift assay, we characterized binding of syntheticDNA duplexes with recombinant human HMGB1. Dynamic light scattering and fluorometricexperiments demonstrated that the inhibitory effects of these triterpenes on DNA bindingdepended on their ability to form supramolecular aggregates. GLA and GAK demonstratedinhibitory effect on DNA-HMGB1 binding at concentrations exceeding CMC (100-150mM). GLAinhibited DNA-HMGB1 binding with IC50 = 500 mM; 600 mM GAK showed 100% inhibition ofDNA-HMGB1 binding. In the presence of GLA, human lung carcinoma cells demonstrated morethan 300-fold increased viability when treated with chemotherapeutic drug cytarabine,consistent with the GLA inhibitory activity. The results of our study demonstrated that moleculescapable of forming stable aggregates are potential modulators of DNA-protein binding and openavenues to the development of novel classes of physiologically active compounds.

[学科分类] 药理学