Characterization of an A-Site Selective Protein Disulfide Isomerase A1 Inhibitor

Protein disulfide isomerase A1 (PDIA1) is an endoplasmic reticulum (ER)-localized thiol-disulfide oxidoreductase that is an important folding catalyst for secretory pathway proteins. PDIA1 contains two active-site domains (a and a′), each containing a Cys-Gly-His-Cys (CGHC) active-site motif. The two active-site domains share 37% sequence identity and function independently to perform disulfide-bond reduction, oxidation, and isomerization. Numerous inhibitors for PDIA1 have been reported, yet the selectivity of these inhibitors toward the a and a′ sites is poorly characterized. Here, we identify a potent and selective PDIA1 inhibitor, KSC-34, with 30-fold selectivity for the a site over the a′ site. KSC-34 displays time-dependent inhibition of PDIA1 reductase activity in vitro with a kinact/KI of 9.66 × 103 M–1 s–1 and is selective for PDIA1 over other members of the PDI family, and other cellular cysteine-containing proteins. We provide the first cellular characterization of an a-site selective PDIA1 inh...