Challenges in the evaluation of thiol-reactive inhibitors of human protein disulfide Isomerase

Abstract

This paper addresses how to evaluate the efficacy of the growing inventory of thiol-reactive inhibitors of mammalian protein disulfide isomerase (PDI) enzymes under realistic concentrations of potentially competing thiol-containing peptides and proteins. For this purpose, we introduce a variant of the widely-used reductase assay by using a commercially-available cysteine derivative (BODIPY FL L-Cystine; BD-SS) that yields a 55-fold increase in fluorescence (excitation/emission; 490/513 nm) on scission of the disulfide bond. This plate reader-compatible method detects human PDI down to 5–10 nM, can utilize a range of thiol substrates (including 5 μM dithiothreitol, 10 μM reduced RNase thiols, and 5 mM glutathione; GSH), and can operate from pH 6–9.5 in a variety of buffers. PDI assays often employ low micromolar levels of substrates leading to ambiguities when thiol-directed inhibitors are evaluated. The present work utilizes 5 mM GSH for both pre-incubation and assay phases to more realistically reflect the high concentration of thiols that an inhibitor would encounter intracellularly. Extracellular PDI faces a much lower concentration of potentially competing thiols; to assess reductase activity under these conditions, the pre-reduced PDI is treated with inhibitor and then fluorescence increase upon reduction of BD-SS is followed in the absence of additional competing thiols. Both assay modes were tested with four mechanistically diverse PDI inhibitors. Two reversible reagents, 3,4-methylenedioxy-β-nitrostyrene (MNS) and the arsenical APAO, were found to be strong inhibitors of PDI in the absence of competing thiols, but were ineffective in the presence of 5 mM GSH. A further examination of the nitrostyrene showed that MNS not only forms facile Michael adducts with GSH, but also with the thiols of unfolded proteins (Kd values of 7 and <0.1 μM, respectively) suggesting the existence of multiple potential intracellular targets for this membrane-permeant reagent. The inhibition of PDI by the irreversible alkylating agent, the chloroacetamide 16F16, was found to be only modestly attenuated by 5 mM GSH. Finally, the thiol-independent flavonoid inhibitor quercetin-3-O-rutinoside was found to show equal efficacy in reoxidation and turnover assay types. This work provides a framework to evaluate inhibitors that may target the CxxC motifs of PDI and addresses some of the complexities in the interpretation of the behavior of thiol-directed reagents in vivo.

Gaussia princeps luciferase: a bioluminescent substrate for oxidative protein folding

“…). Here, the arsenical is sequestered effectively in vitro by m M levels of reduced glutathione leaving oxidative protein folding unimpaired as observed for other proteins …”

“…With sGLuc 100 n M rPDI leads to a 1600‐fold enhancement in the bioluminescent activity over a 2 h incubation. This unscrambling assay sGLuc should provide a convenient bioluminescent screening assay for the isomerase activity of rPDI to complement the more widely employed reductase‐based assays …”

“…sGLuc now provides a sensitive alternative protein substrate for the unscrambling activity of rPDI. This new method may provide a convenient complement to the widely employed assays based on the ability of the isomerase to promote reduction of disulfide‐containing terminal oxidants …”

“…Human PDIA1 was expressed, purified and handled as described earlier with minor modifications . The previous protocol for the expression of HsQSOX in E. coli was modified as summarized below.…”

“…This unscrambling assay sGLuc should provide a convenient bioluminescent screening assay for the isomerase activity of rPDI to complement the more widely employed reductasebased assays. [29][30][31][32][33]…”