D-penicillamine causes free radical-dependent inactivation of activator protein-1 DNA binding

D-Penicillamine (beta, beta-dimethyl cysteine) is an antirheumatic thiol drug with a poorly understood mechanism of action. On the basis that gold(I) thiolates and D-penicillamine are both capable of forming stable bonds with endogenous thiols, we sought a common target of action. Cysteine residues in the basic DNA binding domains of Jun and Fos, members of the activator protein-1 (AP-1) transcription factor family, have been identified as likely targets for the therapeutic action of antirheumatic gold(I) thiolates. The current study demonstrates that AP-1 DNA binding is inhibited by D-penicillamine in the presence of Fenton reagents (Fe2+/EDTA and H2O2) but not with either agent alone. The effect is biphasic, with maximum inhibition in the concentration range of approximately 100-250 microM. Cysteine has qualitatively similar properties, although the effect is less pronounced. In contrast, glutathione and thiomalate do not inhibit AP-1 DNA binding, even in the presence of Fenton reagents. Mutant proteins were used to identify the cysteine residues within the DNA binding domains of Jun and Fos that are essential for the inhibitory action of D-penicillamine. The results suggest that D-penicillamine is distinguished from other thiols by its formation of sulfur-containing radicals capable of inhibiting AP-1 DNA binding by a mechanism involving the cysteine residues of Jun and Fos.