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

Abstract

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.

Figure 1

Domain organization of PDIA1 and chemical structures of PDIA1 inhibitors. (A) PDIA1 comprises two active site a-type domains that contain a CGHC active site motif, together with two b-domains implicated in substrate recognition and binding. (B) Chemical structure of RB-11-ca, a previously reported a-site inhibitor of PDIA1. RB-11-ca contains a central triazine scaffold, a chloroacetamide reactive group for covalent cysteine modification, an alkyne bioorthogonal handle for CuAAC, and an octylamine diversity element. (C) Predicted binding pose of RB-11-ca in a domain active site of PDIA1 by the covalent docking algorithm from Schrodinger, Inc. (D) Structures of second-generation PDIA1 inhibitors obtained by varying the diversity element of RB-11-ca.

Figure 2

Characterization of PDIA1 second-generation library members (A) Library members (5 μM) were incubated with purified recombinant PDIA1 (50 μg/mL) in PBS, and protein labeling by each compound was evaluated after CuAAC-mediated incorporation of a TAMRA fluorophore, SDS-PAGE, and in-gel fluorescence (complete gel images provided in Figure S1). Library members with equal or greater potency than RB-11-ca progressed to the next step of the assay. (B) In-gel fluorescence studies of library members (5 μM) incubated with MCF-7 cell lysate (2 mg/mL) to assess potency and selectivity for endogenous PDIA1 in a complex proteome (complete gel images provided in Figure S3). (C) To assess cell permeability as well as selectivity of the library, probes (5 μM) were incubated with MCF-7 cells for 3 hours at 37 °C. Following cell lysis and in-gel fluorescence, KSC-34 was found to be ~8-fold more potent than the lead compound, RB-11-ca, in whole cells against PDIA1 (left panel). Coomassie gel (right panel is provided to show normalized protein abundance). Fluorescence intensity quantified by Image J (NIH, Bethesda, MD). All data are normalized to RB-11-ca and error bars represent standard deviation (SD) for n=3 experiments.

Figure 3

Evaluation of potency and selectivity of KSC-34. (A) Concentration and time-dependent inhibition of PDIA1 by KSC-34, RB-11-ca, and 16F16. PDIA1 reductase activity was measured using an insulin reduction assay in a 100 μL reaction volume containing 0.5 μM PDIA1, 0.16 mM insulin, and 1 mM DTT in assay buffer. Turbidity of the insulin solution was measured over time at various concentrations and pre-incubation times with KSC-34. Error bars represent SD from n=2 experiments. (B) Labeling of WT, C397A and C53A by KSC-34. Each recombinant PDIA1 protein (50 μg/ml) was incubated with KSC-34 (5 μM) for 1 hour, and subjected to CuAAC with TAMRA-azide prior to in-gel fluorescence. (C) Active-site selectivity for KSC-34, RB-11-ca, and 16F16. KSC-34 was found to have a 30-fold selectivity for the a domain over the a′ domain, compared to RB-11-ca which exhibited a 21-fold selectivity and 16F16 which exhibited 2-fold selectivity. Error is calculated by PRISM as +/− SEM from n=3 experiments. (D) PDI isoform selectivity. KSC-34 was found to only covalently modify PDIA1, over PDIA3 and PDIA4, two closely related family members. Fluorescent bands in cell lysate lanes for PDIA3 and PDIA4 samples represent endogenous PDIA1 protein.

Figure 4

Evaluation of proteome-wide selectivity of KSC-34. Mass spectrometry analysis of proteins enriched in KSC-34 (5 μM) and DMSO treated MCF-7 cells. The spectral count difference between KSC-34-treated and DMSO-treated samples are plotted for all proteins identified (Table S2). Error bars indicate SD for n=2 experiments.

Figure 5

Effects of KSC-34 on cell viability and the unfolded protein response. (A) qPCR analysis of UPR target genes following concentration-dependent treatment of MCF-7 cells with KSC-34 for 3 hours at 37 °C. qPCR data are reported as the mean fold change relative to the corresponding DMSO-treated cells +/− SEM from n=3 biological replicates. (B) qPCR analysis of UPR target genes following co-treatment of MCF-7 cells with KSC-34 (20 μM) and IRE1α inhibitor, 4u8c. qPCR data are reported relative to the corresponding DMSO-treated cells +/− SEM from n=3 biological replicates.

Figure 6

KSC-34 reduces secretion of destabilized ALLC from mammalian cells. (A) Immunoblot of anti-FLAG IPs of lysates prepared from HEK293^DAX cells transiently transfected with ^FTALLC and pre-treated for 1 h with vehicle or KSC-34 (40 μM). Cells were crosslinked for 30 min with DSP (500 μM) prior to lysis. Mock transfected cells are included as a control. (B) Graph showing secreted ^FTALLC (grey) and viability (blue) of HEK293^Trex cells stably expressing ^FTALLC pretreated for 4 h with KSC-34 (40 μM). Media was conditioned for 2 h in the presence or absence of KSC-34 (40 μM) prior to quantification of secreted ^FTALLC by ELISA. Viability was measured following media conditioning by Cell Titre Glo. All data are normalized to vehicle-treated cells. Error bars show SEM for n=3 experiments. ***p<0.005. (C) Representative autoradiogram and quantification of the fraction [³⁵S]-labeled ^FTALLC secreted from HEK293^DAX cells using the experimental paradigm shown. Experiments were performed in the absence or presence of KSC-34 (40 μM) added 1 h prior to labeling and then again throughout the experiment. Fraction secreted was calculated as described in the Supporting Information . Error bars show SEM for n=4. ***p<0.005. (D) Graph showing secreted ^FTALLC (grey) and viability (blue) of HEK293^DAX cells transiently expressing ^FTALLC pretreated for 4 h with KSC-34 (40 μM). HEK293^DAX cells stably expressing PDIA1 shRNA are indicated. Media was conditioned for 2 h in the presence or absence of KSC-34 (40 μM) prior to quantification of secreted ^FTALLC by ELISA. Viability was measured following media conditioning by Cell Titre Glo. All data are normalized to vehicle-treated controls. Error bars show SEM for n=3 experiments. *p<0.05. (E) Graph showing the fraction [³⁵S]-labeled ^FTALLC secreted at t=4 h from HEK293^DAX cells transiently transfected with ^FTALLC quantified using the same experimental paradigm shown in Fig. 6C. HEK293^DAX cells stably expressing PDIA1 shRNA are indicated. Experiments were performed in the absence or presence of KSC-34 (40 μM) added 1 h prior to labeling and then again throughout the experiment. Error bars show SEM for n=4 experiments. *p<0.05, ***p<0.005.