The enzyme protein disulfide isomerase (PDI), a member of the thioredoxin super-family, acts as both an isomerase and a reductase of disulfide bonds. It approaches mM concentrations in the endoplasmic reticulum and is found at lower levels in the mitochondrial outer membrane, the cytosol, and the cell surface. PDI has recently grown in medicinal and therapeutic importance as it holds key roles in thrombosis formation, cell adhesion and invasion. Here we describe a sensitive PDI reductase activity assay utilizing a commercially available self-quenched fluorescent disulfide reagent (BD-SS). PDI-mediated reduction can be followed using a plate-reader and can detect 5-10 nM isomerase in the presence of a range of small molecule and peptide thiol substrates. We have utilized BD-SS to address the difficulties in assessing the in vitro efficacy of proposed inhibitors of PDI that are themselves thiol-reactive. One assay involves a 15-minute preincubation of reduced PDI with the inhibitors and then follows the rate of fluorescence development after the addition of BD-SS. Alternatively, to approximate intracellular thiol concentrations, PDI is mixed with inhibitor in the presence of 5 mM reduced glutathione (GSH) and the assay is started by the addition of a small volume of BD-SS. Four mechanistically diverse inhibitors were tested: 3,4-methylenedioxy-β-nitrostyrene (MNS), 4-amino-phenylarsine oxide (APAO), 16F16, and quercetin-3-O-rutinoside. Reversible thiol-directed compounds MNS and APAO were found to inhibit PDI in the absence of competing thiols, but inhibition was completely lost when pre-incubated and assayed in the presence of 5 mM GSH. The irreversible chloroacetamide reagent, 16F16, showed inhibition of PDI that was only moderately attenuated with 5 mM GSH. Quercetin-3-O-rutinoside, a reversible non-thiol directed compound, showed similar inhibitory effects in both assay conditions. The results demonstrate the complexities of evaluating thiol-directed inhibitors in classical in vitro assays of PDI and provide a framework to assess the intricacies of inhibitor specificity in vivo.

Inhibition in the face of thiols: Complexities of protein disulfide isomerase inhibitor evaluation

254th National Meeting (2017)

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