Inhibition of glucocorticoid receptor transformation, subunit dissociation, and temperature-dependent inactivation by various N-substituted maleimides

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Abstract
A series of N-substituted maleimides were synthesized, and their effect on the activation to the DNA binding state of the rat liver glucocorticoid receptor was studied. Unactivated (preincubated at 0.degree. C) cytosolic [3H]triamcinolone acetonide-receptor complexes were pretreated with various N-alkylmaleimides at 0.degree. C and then heated at 25.degree. C and assayed for DNA-cellulose binding. No inhibition of the DNA binding activity was observed with either N-ethylmaleimide or N-substituted maleimides bearing an ionizable substituent, like N-(.omega.-carboxyalkyl)maleimides and N-[2-(trimethylammonio)ethyl]maleimide. On the contary, treatment with long-chain alkylmaleimides like N-heptylmaleimide resulted in significant inhibition. The highest inhibition was obtained with N-benzylmaleimide and, to a lesser extent, N-(ethylphenyl)-maleimide, whereas N-benzylsuccinimide was ineffective. Treatment of cytosol containing unactivated glucocorticoid complexes at 3.degree. C with N-benzylmaleimide also prevents the temperature-mediated conversion of 8S receptor to 4S. Moreover, N-benzylmaleimide was able to inhibit the inactivation of the receptor steroid-binding activity caused by heat. N-Benzylmaleimide shares with molybdate ions the ability to inhibit glucocorticoid receptor activation, dissociation, and inactivation. However, their respective mechanisms of action are probably distinct, since their effects on receptor inactivation appear additive. It is suggested from the comparison of the various maleimides tested that the sulfhydryl groups essential for receptor activation and dissociation lie in a rather nonpolar encironment including aromatic amino acid(s).