Beta-lapachone, a specific competitive inhibitor of ligand binding to the glucocorticoid receptor.

Beta-Lapachone, a derivative of 1,2-naphthoquinone, inhibits the specific binding of [6,7-3H]triamcinolone acetonide (TA) to unbound hepatic and thymic glucocorticoid receptors in a dose-dependent manner with 50% of the maximal inhibition in thymus cytosol achieved at a final concentration of 5-10 microM. Preincubation of cytosol with 10 mM Na2MoO4, which stabilizes unbound receptors, potentiates the subsequent beta-lapachone-mediated inhibitory activity, while preincubation with 1 or 10 mM dithiothreitol blocks the subsequent inhibition of [6,7-3H]TA binding. A double reciprocal plot indicates that beta-lapachone is a competitive inhibitor of [6,7-3H]TA binding with an apparent Ki of approximately 6 microM. The ability of beta-lapachone to displace prebound [6,7-3H]TA and the ability of elevated concentrations of [6,7-3H]TA to reverse the beta-lapachone-mediated inhibition are totally consistent with this kinetic interpretation. The ability of beta-lapachone to interact directly with the ligand-binding site is confirmed by the fact that this compound can block the binding of [6,7-3H]TA to highly purified unactivated hepatic glucocorticoid receptors. Although beta-lapachone may interact specifically with receptor sulfhydryl groups, this compound is not a general oxidizing agent which inactivates the essential free sulfhydryl groups at the glucocorticoid-binding site. Beta-Lapachone does not affect activation of [6,7-3H]TA-receptor complexes nor does it itself act like a glucocorticoid and facilitate receptor activation (transformation). Interestingly, this compound does not affect the ligand-binding sites of estrogen, progesterone, androgen, or mineralocorticoid receptors or serum transcortin. Thus, beta-lapachone can be utilized as a specific probe for the ligand-binding site of the glucocorticoid receptor.