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 k_inact/ K_I 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 inhibitor and demonstrate that KSC-34 has minimal sustained effects on the cellular unfolded protein response, indicating that a-site inhibition does not induce global protein folding-associated ER stress. KSC-34 treatment significantly decreases the rate of secretion of a destabilized, amyloidogenic antibody LIGHT chain, thereby minimizing pathogenic amyloidogenic extracellular proteins that rely on high PDIA1 activity for proper folding and secretion. Given the poor understanding of the contribution of each PDIA1 active site to the (patho)physiological functions of PDIA1, site selective inhibitors like KSC-34 provide useful tools for delineating the pathological role and therapeutic potential of PDIA1.