Multivalency in the inhibition of oxidative protein folding by arsenic(III) species

The renewed use of arsenicals as chemotherapeutics has rekindled interest in the biochemistry of As(III) species. In this work, simple bis- and tris-arsenical derivatives were synthesized with the aim of exploiting the chelate effect in the inhibition of thiol-disulfide oxidoreductases (here, Quiescin sulfhydryl oxidase, QSOX, and protein disulfide isomerase, PDI) that utilize two or more CxxC motifs in the catalysis of oxidative protein folding. Coupling 4-aminophenylarsenoxide (APAO) to acid chloride or anhydride derivatives yielded two bis-arsenical prototypes, BA-1 and BA-2, and a tris-arsenical, TA-1. Unlike the monoarsenical, APAO, these new reagents proved to be strong inhibitors of oxidative protein folding in the presence of a realistic intracellular concentration of competing monothiol (here, 5 mM reduced glutathione, GSH). However, this inhibition does not reflect direct inactivation of QSOX or PDI, but avid binding of MVAs to the reduced unfolded protein substrates themselves. Titrations of reduced riboflavin-binding protein with MVAs show that all 18 protein -SH groups can be captured by these arsenicals. With reduced RNase, addition of substoichiometric levels of MVAs is accompanied by the formation of Congo Red- and Thioflavin T-positive fibrillar aggregates. Even with Kd values of ∼50 nM, MVAs are ineffective inhibitors of PDI in the presence of millimolar levels of competing GSH. These results underscore the difficulties of designing effective and specific arsenical inhibitors for folded enzymes and proteins. Some of the cellular effects of arsenicals likely reflect their propensity to associate very tightly and nonspecifically to conformationally mobile cysteine-rich regions of proteins, thereby interfering with folding and/or function.