Protein disulfide isomerase A1 regulates fenestration dynamics in primary mouse liver sinusoidal endothelial cells (LSECs).

Protein disulfide isomerases (PDIs) are involved in many intracellular and extracellular processes, including cell adhesion and cytoskeletal reorganisation, but their contribution to the regulation of fenestrations in liver sinusoidal endothelial cells (LSECs) remains unknown. Given that fenestrations are supported on a cytoskeleton scaffold, this study aimed to investigate whether endothelial PDIs regulate fenestration dynamics in primary mouse LSECs. PDIA3 and PDIA1 were found to be the most abundant among PDI isoforms in LSECs. Taking advantage of atomic force microscopy, the effects of PDIA1 or PDIA3 inhibition on the fenestrations in LSECs were investigated using a classic PDIA1 inhibitor (bepristat) and novel aromatic N-sulfonamides of aziridine-2-carboxylic acid derivatives as PDIA1 (C-3389) or PDIA3 (C-3399) inhibitors. The effect of PDIA1 inhibition on liver perfusion was studied in vivo using dynamic contrast-enhanced magnetic resonance imaging. Additionally, PDIA1 inhibitors were examined in vitro in LSECs for effects on adhesion, cytoskeleton organisation, bioenergetics, and viability. Inhibition of PDIA1 with bepristat or C-3389 significantly reduced the number of fenestrations in LSECs, while inhibition of PDIA3 with C-3399 had no effect. Moreover, the blocking of free thiols by the cell-penetrating N-ethylmaleimide, but not by the non-cell-penetrating 4-chloromercuribenzenesulfonate, resulted in LSEC defenestration. Inhibition of PDIA1 did not affect LSEC adhesion, viability, and bioenergetics, nor did it induce a clear-cut rearrangement of the cytoskeleton. However, PDIA1-dependent defenestration was reversed by cytochalasin B, a known fenestration stimulator, pointing to the preserved ability of LSECs to form new pores. Importantly, systemic inhibition of PDIA1 in vivo affected intra-parenchymal uptake of contrast agent in mice consistent with LSEC defenestration. These results revealed the role of intracellular PDIA1 in the regulation of fenestration dynamics in LSECs, and in maintaining hepatic sinusoid homeostasis.

Aromatic sulphonamides of aziridine-2-carboxylic acid derivatives as novel PDIA1 and PDIA3 inhibitors.

In this study, we report a series of newly synthesised sulphonamides of aziridine-2-carboxylic acid (Az-COOH) ester and amide analogues as potent protein disulphide isomerase (PDI, EC 5.3.4.1) inhibitors. The inhibitory activity on PDI was determined against recombinant human PDIA1 and PDIA3 proteins using an insulin reduction assay. These compounds in low micromolar to low nanomolar concentrations showed the effective in vitro inhibitory properties of PDIA1 with weaker effects on PDIA3. Complexes of 15N- and 15N,13C- uniformly labelled recombinant human PDIA1a with two PDIA1 inhibitors were produced and investigated by a protein nuclear magnetic resonance (NMR) spectroscopy. It was found that both C53 and C56 of the PDIA1 enzyme were involved in covalent binding. Finally, in a range of pharmacological studies, we demonstrated that investigated compounds displayed anti-cancer and anti-thrombotic activity. These findings demonstrate that sulphonamides of Az-COOH derivatives are promising candidates for the development of novel anti-cancer and anti-thrombotic agents.

The protein disulfide isomerase inhibitor 3-methyltoxoflavin inhibits Chikungunya virus.

Chikungunya virus (CHIKV) is the etiological agent of chikungunya fever, a (re)emerging arbovirus infection, that causes severe and often persistent arthritis, as well as representing a serious health concern worldwide for which no antivirals are currently available. Despite efforts over the last decade to identify and optimize new inhibitors or to reposition existing drugs, no compound has progressed to clinical trials for CHIKV and current prophylaxis is based on vector control, which has shown limited success in containing the virus. Our efforts to rectify this situation were initiated by screening 36 compounds using a replicon system and ultimately identified the natural product derivative 3-methyltoxoflavin with activity against CHIKV using a cell-based assay (EC50 200 nM, SI = 17 in Huh-7 cells). We have additionally screened 3-methyltoxoflavin against a panel of 17 viruses and showed that it only additionally demonstrated inhibition of the yellow fever virus (EC50 370 nM, SI = 3.2 in Huh-7 cells). We have also showed that 3-methyltoxoflavin has excellent in vitro human and mouse microsomal metabolic stability, good solubility and high Caco-2 permeability and it is not likely to be a P-glycoprotein substrate. In summary, we demonstrate that 3-methyltoxoflavin has activity against CHIKV, good in vitro absorption, distribution, metabolism and excretion (ADME) properties as well as good calculated physicochemical properties and may represent a valuable starting point for future optimization to develop inhibitors for this and other related viruses.

Identifying Interaction Partners of Yeast Protein Disulfide Isomerases Using a Small Thiol-Reactive Cross-Linker: Implications for Secretory Pathway Proteostasis.

Protein disulfide isomerases (PDIs) function in forming the correct disulfide bonds in client proteins, thereby aiding the folding of proteins that enter the secretory pathway. Recently, several PDIs have been identified as targets of organic electrophiles, yet the client proteins of specific PDIs remain largely undefined. Here, we report that PDIs expressed in Saccharomyces cerevisiae are targets of divinyl sulfone (DVSF) and other thiol-reactive protein cross-linkers. Using DVSF, we identified the interaction partners that were cross-linked to Pdi1 and Eug1, finding that both proteins form cross-linked complexes with other PDIs, as well as vacuolar hydrolases, proteins involved in cell wall biosynthesis and maintenance, and many ER proteostasis factors involved ER stress signaling and ER-associated protein degradation (ERAD). The latter discovery prompted us to examine the effects of DVSF on ER quality control, where we found that DVSF inhibits the degradation of the ERAD substrate CPY*, in addition to covalently modifying Ire1 and blocking the activation of the unfolded protein response. Our results reveal that DVSF targets many proteins within the ER proteostasis network and suggest that these proteins may be suitable targets for covalent therapeutic development in the future.

PDIA3: Structure, functions and its potential role in viral infections.

The catalysis of disulphide (SS) bonds is the most important characteristic of protein disulphide isomerase (PDI) family. Catalysis occurs in the endoplasmic reticulum, which contains many proteins, most of which are secretory in nature and that have at least one s-s bond. Protein disulphide isomerase A3 (PDIA3) is a member of the PDI family that acts as a chaperone. PDIA3 is highly expressed in response to cellular stress, and also intercept the apoptotic cellular death related to endoplasmic reticulum (ER) stress, and protein misfolding. PDIA3 expression is elevated in almost 70% of cancers and its expression has been linked with overall low cell invasiveness, survival and metastasis. Viral diseases present a significant public health threat. The presence of PDIA3 on the cell surface helps different viruses to enter the cells and also helps in replication. Therefore, inhibitors of PDIA3 have great potential to interfere with viral infections. In this review, we summarize what is known about the basic structure, functions and role of PDIA3 in viral infections. The review will inspire studies of pathogenic mechanisms and drug targeting to counter viral diseases.

Elaboration of a benzofuran scaffold and evaluation of binding affinity and inhibition of Escherichia coli DsbA: A fragment-based drug design approach to novel antivirulence compounds.

Bacterial thiol-disulfide oxidoreductase DsbA is essential for bacterial virulence factor assembly and has been identified as a viable antivirulence target. Herein, we report a structure-based elaboration of a benzofuran hit that bound to the active site groove of Escherichia coli DsbA. Substituted phenyl groups were installed at the 5- and 6-position of the benzofuran using Suzuki-Miyaura coupling. HSQC NMR titration experiments showed dissociation constants of this series in the high µM to low mM range and X-ray crystallography produced three co-structures, showing binding in the hydrophobic groove, comparable with that of the previously reported benzofurans. The 6-(m-methoxy)phenyl analogue (2b), which showed a promising binding pose, was chosen for elaboration from the C-2 position. The 2,6-disubstituted analogues bound to the hydrophobic region of the binding groove and the C-2 groups extended into the more polar, previously un-probed, region of the binding groove. Biochemical analysis of the 2,6-disubsituted analogues showed they inhibited DsbA oxidation activity in vitro. The results indicate the potential to develop the elaborated benzofuran series into a novel class of antivirulence compounds.

Endothelial microparticle-associated protein disulfide isomerase increases platelet activation in diabetic coronary heart disease.

BACKGROUND: Endothelial microparticles (EMPs) carrying the protein disulfide isomerase (PDI) might play a key role in promoting platelet activation in diabetes. This study aimed to examine the activation of platelets, the amounts of MPs, PMPs, and EMPs, and the concentration and activity of PDI in patients with diabetic coronary heart disease (CHD) and non-diabetic CHD. METHODS: Patients with CHD (n=223) were divided as non-diabetic CHD (n=121) and diabetic CHD (n=102). Platelet activation biomarkers, circulating microparticles (MPs), the concentration of protein disulfide isomerase (PDI), and MP-PDI activity were determined. The effect of EMPs on platelet activation was investigated in vitro. Allosteric GIIb/IIIa receptors that bind to PDI were detected by a proximity ligation assay (PLA). RESULTS: Platelet activation, platelet-leukocyte aggregates, circulating MPs, EMPs, PDI, and MP-PDI activity in the diabetic CHD group were significantly higher than in the non-diabetic CHD group (P<0.05). Diabetes (P=0.006) and heart rate <60 bpm (P=0.047) were associated with elevated EMPs. EMPs from diabetes increased CD62p on the surface of the platelets compared with the controls (P<0.01), which could be inhibited by the PDI inhibitor RL90 (P<0.05). PLA detected the allosteric GIIb/IIIa receptors caused by EMP-PDI, which was also inhibited by RL90. CONCLUSIONS: In diabetic patients with CHD, platelet activation was significantly high. Diabetes and heart rate <60 bpm were associated with elevated EMPs and simultaneously increased PDI activity on EMP, activating platelets through the allosteric GPIIb/IIIa receptors.

Protein disulphide isomerase inhibition as a potential cancer therapeutic strategy.

The protein disulphide isomerase (PDI) gene family is a large, diverse group of enzymes recognised for their roles in disulphide bond formation within the endoplasmic reticulum (ER). PDI therefore plays an important role in ER proteostasis, however, it also shows involvement in ER stress, a characteristic recognised in multiple disease states, including cancer. While the exact mechanisms by which PDI contributes to tumorigenesis are still not fully understood, PDI exhibits clear involvement in the unfolded protein response (UPR) pathway. The UPR acts to alleviate ER stress through the activation of ER chaperones, such as PDI, which act to refold misfolded proteins, promoting cell survival. PDI also acts as an upstream regulator of the UPR pathway, through redox regulation of UPR stress receptors. This demonstrates the pro-protective roles of PDI and highlights PDI as a potential therapeutic target for cancer treatment. Recent research has explored the use of PDI inhibitors with PACMA 31 in particular, demonstrating promising anti-cancer effects in ovarian cancer. This review discusses the properties and functions of PDI family members and focuses on their potential as a therapeutic target for cancer treatment.

A computer-aided approach to identify novel Leishmania major protein disulfide isomerase inhibitors for treatment of leishmaniasis.

Leishmaniasis is an infectious disease caused by parasites of the genus Leishmania and transmitted by the bite of a sand fly. To date, most available drugs for treatment are toxic and beyond the economic means of those affected by the disease. Protein disulfide isomerase (PDI) is a chaperone protein that plays a major role in the folding of newly synthesized proteins, specifically assisting in disulfide bond formation, breakage, or rearrangement in all non-native proteins. In previous work, we demonstrated that Leishmania major PDI (LmPDI) has an essential role in pathogen virulence. Furthermore, inhibition of LmPDI further blocked parasite infection in macrophages. In this study, we utilized a computer-aided approach to design a series of LmPDI inhibitors. Fragment-based virtual screening allowed for the understanding of the inhibitors' modes of action on LmPDI active sites. The generated compounds obtained after multiple rounds of virtual screening were synthesized and significantly inhibited target LmPDI reductase activity and were shown to decrease in vitro parasite growth in human monocyte-derived macrophages. This novel cheminformatics and synthetic approach led to the identification of a new series of compounds that might be optimized into novel drugs, likely more specific and less toxic for the treatment of leishmaniasis.

A high-throughput cell-based assay pipeline for the preclinical development of bacterial DsbA inhibitors as antivirulence therapeutics.

Antibiotics are failing fast, and the development pipeline remains alarmingly dry. New drug research and development is being urged by world health officials, with new antibacterials against multidrug-resistant Gram-negative pathogens as the highest priority. Antivirulence drugs, which inhibit bacterial pathogenicity factors, are a class of promising antibacterials, however, their development is stifled by lack of standardised preclinical testing akin to what guides antibiotic development. The lack of established target-specific microbiological assays amenable to high-throughput, often means that cell-based testing of virulence inhibitors is absent from the discovery (hit-to-lead) phase, only to be employed at later-stages of lead optimization. Here, we address this by establishing a pipeline of bacterial cell-based assays developed for the identification and early preclinical evaluation of DsbA inhibitors, previously identified by biophysical and biochemical assays. Inhibitors of DsbA block oxidative protein folding required for virulence factor folding in pathogens. Here we use existing Escherichia coli DsbA inhibitors and uropathogenic E. coli (UPEC) as a model pathogen, to demonstrate that the combination of a cell-based sulfotransferase assay and a motility assay (both DsbA reporter assays), modified for a higher throughput format, can provide a robust and target-specific platform for the identification and evaluation of DsbA inhibitors.

Juglone prevents human platelet aggregation through inhibiting Akt and protein disulfide isomerase.

BACKGROUND/PURPOSE: Juglone, a natural compound widely found in Juglandaceae plants, has been suggested as a potential drug candidate for treating cancer, inflammation, and diabetic vascular complications. In the present study, the antiplatelet effect and underlying mechanisms of juglone were investigated for the first time. STUDY DESIGN/METHODS: Human platelet aggregation and activation were measured by turbidimetric aggregometry, flow cytometry, and Western blotting. In vitro antithrombotic activity of juglone was assessed using collagen-coated flow chambers under whole-blood flow conditions. The effect of juglone on protein disulfide isomerase (PDI) activity was determined by the dieosin glutathione disulfide assay. RESULTS: Juglone (1 - 5 µM) inhibited platelet aggregation and glycoprotein (GP) IIb/IIIa activation caused by various agonists. In a whole blood flow chamber system, juglone reduced thrombus formation on collagen-coated surfaces under arterial shear rates. Juglone abolished intracellular Ca2+ elevation and protein kinase C activation caused by collagen, but had no significant effect on that induced by G protein-coupled receptor agonists. In contrast, Akt activation caused by various agonists were inhibited in juglone-treated platelets. Additionally, juglone showed inhibitory effects on both recombinant human PDI and platelet surface PDI at concentrations similar to those needed to prevent platelet aggregation. CONCLUSION: Juglone exhibits potent in vitro antiplatelet and antithrombotic effects that are associated with inhibition of Akt activation and platelet surface PDI activity.

Crosstalk between endoplasmic reticulum stress and oxidative stress: Focus on protein disulfide isomerase and endoplasmic reticulum oxidase 1.

Cellular stress and inflammation, establishing as disease pathology, have reached great heights in the last few decades. Stress conditions such as hyperglycemia, hyperlipidemia and lipoproteins are known to disturb proteostasis resulting in the accumulation of unfolded or misfolded proteins, alteration in calcium homeostasis culminating in unfolded protein response. Protein disulfide isomerase and endoplasmic reticulum oxidase-1 are the key players in protein folding. The protein folding process assisted by endoplasmic reticulum oxidase-1 results in the production of reactive oxygen species in the lumen of the endoplasmic reticulum. Production of reactive oxygen species beyond the quenching capacity of the antioxidant systems perturbs ER homeostasis. Endoplasmic reticulum stress also induces the production of cytokines leading to inflammatory responses. This has been proven to be the major causative factor for various pathophysiological states compared to other cellular triggers in diseases, which further manifests to increased oxidative stress, mitochondrial dysfunction, and altered inflammatory responses, deleterious to cellular physiology and homeostasis. Numerous studies have drawn correlations between the progression of several diseases in association with endoplasmic reticulum stress, redox protein folding, oxidative stress and inflammatory responses. This review aims to provide an insight into the role of protein disulfide isomerase and endoplasmic reticulum oxidase-1 in endoplasmic reticulum stress, unfolded protein response, mitochondrial dysfunction, and inflammatory responses, which exacerbate the progression of various diseases.

Protein Disulfide Isomerase 4 Drives Docetaxel Resistance in Prostate Cancer.

BACKGROUND: Protein disulfide isomerase 4 (PDIA4) has been reported to be closely associated with chemoresistance in several types of malignancies. But the pathogenic mechanisms of PDIA4 involved in docetaxel (DTX) resistance in prostate cancer (PCa) are still unknown. Hence, this study was conducted to evaluate the potential effect of PDIA4 on chemoresistance to DTX in PCa cells and to investigate the underlying mechanisms. METHODS: Two types of DTX-resistant PCa cells, that is, DTX-resistant PC-3 cells (PC-3/DTXR) and C4-2B cells (C4-2B/DTXR) were developed, as well as the parental PC-3 and C4-2B cells were obtained to investigate these issues. Short hairpin RNAs targeting human PDIA4 to knockdown the expression of PDIA4 or PDIA4-expressing adenoviral vectors to overexpress the PDIA4 were transfected into PCa cells to study the underlying mechanisms of PDIA4 involving in PCa DTX resistance. RESULTS: Results showed that PDIA4 exhibited a dramatic overexpression in PC-3/DTXR and C4-2B/DTXR cells. Down-regulation of PDIA4 by infecting PC-3/DTXR and C4-2B/DTXR cells with shPDIA4 lentivirus stimulated cell death by prompting apoptosis. Up-regulation of PDIA4 by infecting PC-3 and C4-2B cells with PDIA4-expressing adenovirus showed severer resistance to DTX. In addition, PDIA4 up-regulation induced phosphorylated protein kinase B (Akt) expression, while PDIA4 knockdown significantly inhibited the expression in PCa cells. CONCLUSIONS: Our study indicates that PDIA4 is a negative regulator of PCa cell apoptosis and plays a critical role in PCa DTX resistance by activating the Akt-signaling pathway. Thereby, it implies that targeting PDIA4 could be a potential adjuvant therapeutic approach against DTX resistance in PCa.

Molecular docking-assisted screening reveals tannic acid as a natural protein disulphide isomerase inhibitor with antiplatelet and antithrombotic activities.

Protein disulphide isomerase (PDI) promotes platelet activation and constitutes a novel antithrombotic target. In this study, we reported that a PDI-binding plant polyphenol, tannic acid (TA), inhibits PDI activity, platelet activation and thrombus formation. Molecular docking using plant polyphenols from dietary sources with cardiovascular benefits revealed TA as the most potent binding molecule with PDI active centre. Surface plasmon resonance demonstrated that TA bound PDI with high affinity. Using Di-eosin-glutathione disulphide fluorescence assay and PDI assay kit, we showed that TA inhibited PDI activity. In isolated platelets, TA inhibited platelet aggregation stimulated by either GPVI or ITAM pathway agonists. Flow cytometry showed that TA inhibited thrombin- or CRP-stimulated platelet activation, as reflected by reduced granule secretion and integrin activation. TA also reduced platelet spreading on immobilized fibrinogen and platelet adhesion under flow conditions. In a laser-induced vascular injury mouse model, intraperitoneal injection of TA significantly decreased the size of cremaster arteriole thrombi. No prolongation of mouse jugular vein and tail-bleeding time was observed after TA administration. Therefore, we identified TA from natural polyphenols as a novel inhibitor of PDI function. TA inhibits platelet activation and thrombus formation, suggesting it as a potential antithrombotic agent.

Salidroside can target both P4HB-mediated inflammation and melanogenesis of the skin.

Rationale: Many external factors can induce the melanogenesis and inflammation of the skin. Salidroside (SAL) is the main active ingredient of Rhodiola, which is a perennial grass plant of the Family Crassulaceae. This study evaluated the effect and molecular mechanism of SAL on skin inflammation and melanin production. It then explored the molecular mechanism of melanin production under ultraviolet (UV) and inflammatory stimulation. Methods: VISIA skin analysis imaging system and DermaLab instruments were used to detect the melanin reduction and skin brightness improvement rate of the volunteers. UV-treated Kunming mice were used to detect the effect of SAL on skin inflammation and melanin production. Molecular docking and Biacore were used to verify the target of SAL. Immunofluorescence, luciferase reporter assay, CO-IP, pull-down, Western blot, proximity ligation assay (PLA), and qPCR were used to investigate the molecular mechanism by which SAL regulates skin inflammation and melanin production. Results: SAL can inhibit the inflammation and melanin production of the volunteers. SAL also exerted a protective effect on the UV-treated Kunming mice. SAL can inhibit the tyrosinase (TYR) activity and TYR mRNA expression in A375 cells. SAL can also regulate the ubiquitination degradation of interferon regulatory factor 1 (IRF1) by targeting prolyl 4-hydroxylase beta polypeptide (P4HB) to mediate inflammation and melanin production. This study also revealed that IRF1 and upstream stimulatory factor 1 (USF1) can form a transcription complex to regulate TYR mRNA expression. IRF1 also mediated inflammatory reaction and TYR expression under UV- and lipopolysaccharide-induced conditions. Moreover, SAL derivative SAL-plus (1-(3,5-dihydroxyphenyl) ethyl-ß-d-glucoside) showed better effect on inflammation and melanin production than SAL. Conclusion: SAL can inhibit the inflammation and melanogenesis of the skin by targeting P4HB and regulating the formation of the IRF1/USF1 transcription complex. In addition, SAL-plus may be a new melanin production and inflammatory inhibitor.

Impairment of cell adhesion and migration by inhibition of protein disulphide isomerases in three breast cancer cell lines.

Protein disulphide isomerase A3 (PDIA3) is an endoplasmic reticulum (ER)-resident disulphide isomerase and oxidoreductase with known substrates that include some extracellular matrix (ECM) proteins. PDIA3 is up-regulated in invasive breast cancers and correlates in a mouse orthotopic xenograft model with breast cancer metastasis to bone. However, the underlying cellular mechanisms remain unclear. Here we investigated the function of protein disulphide isomerases in attachment, spreading and migration of three human breast cancer lines representative of luminal (MCF-7) or basal (MDA-MB-231 and HCC1937) tumour phenotypes. Pharmacological inhibition by 16F16 decreased initial cell spreading more effectively than inhibition by PACMA-31. Cells displayed diminished cortical F-actin projections, stress fibres and focal adhesions. Cell migration was reduced in a quantified 'scratch wound' assay. To examine whether these effects might result from alterations to secreted proteins in the absence of functional PDIA3, adhesion and migration were quantified in the above cells exposed to media conditioned by wildtype (WT) or Pdia3-/- mouse embryonic fibroblasts (MEFs). The conditioned medium (CM) of Pdia3-/- MEFs was less effective in promoting cell spreading and F-actin organisation or supporting 'scratch wound' closure. Similarly, ECM prepared from HCC1937 cells after 16F16 inhibition was less effective than control ECM to support spreading of untreated HCC1937 cells. Overall, these results advance the concept that protein disulphide isomerases including PDIA3 drive the production of secreted proteins that promote a microenvironment favourable to breast cancer cell adhesion and motility, characteristics that are integral to tumour invasion and metastasis. Inhibition of PDIA3 or related isomerases may have potential for anti-metastatic therapies.

Characterization of Aminobenzylphenols as Protein Disulfide Isomerase Inhibitors in Glioblastoma Cell Lines.

Disulfide bond formation is a critical post-translational modification of newly synthesized polypeptides in the oxidizing environment of the endoplasmic reticulum and is mediated by protein disulfide isomerase (PDIA1). In this study, we report a series of α-aminobenzylphenol analogues as potent PDI inhibitors. The lead compound, AS15, is a covalent nanomolar inhibitor of PDI, and the combination of AS15 analogues with glutathione synthesis inhibitor buthionine sulfoximine (BSO) leads to synergistic cell growth inhibition. Using nascent RNA sequencing, we show that an AS15 analogue triggers the unfolded protein response in glioblastoma cells. A BODIPY-labeled analogue binds proteins including PDIA1, suggesting that the compounds are cell-permeable and reach the intended target. Taken together, these findings demonstrate an extensive biochemical characterization of a novel series of highly potent reactive small molecules that covalently bind to PDI.

Treatment of Neuroblastoma Cells with Inhibitors of Protein Disulfide Isomerase Upregulates NQO1 Activity.

Hallmarks of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease include oxidative stress, accumulation of unfolded proteins, and neuronal cell death. One key player in maintaining redox homeostasis and oxidative protein folding is the protein disulfide isomerase (PDI). PDI has been the focus of drug discovery studies in neurodegenerative diseases, which have reported, paradoxically, that PDI inhibition is neuroprotective in cellular disease models. This study investigated the molecular implications of PDI inhibition by examining the effect of the PDI inhibitors securinine and 16F16 on the gene expression profile of SH-SY5Y neuroblastoma cells. Microarray analysis identified 36 genes that were differentially expressed in both inhibitor treatments. Computational approaches revealed that these differentially expressed genes are involved in apoptosis and cell death and that they are part of a protein-protein interaction network. Among the 36 identified genes, NAD(P)H quinone dehydrogenase 1 (NQO1) displayed the highest average expression change. As a central player in the cellular oxidative stress response, NQO1 was the focus of further investigation. Immunoblotting confirmed the increased expression level of NQO1, and activity assays demonstrated substantial increases in NQO1 activity in SH-SY5Y cells after treatment with PDI inhibitors. In summary, this study suggests a novel link between PDI inhibition and NQO1 activity, providing insights into the dynamic interplay between protein folding, oxidative stress, and cell death in neurodegenerative diseases, which can be exploited for drug development in the future.

ATF3 Coordinates Antitumor Synergy between Epigenetic Drugs and Protein Disulfide Isomerase Inhibitors.

Histone deacetylase inhibitors (HDACi) are largely ineffective in the treatment of solid tumors. In this study, we describe a new class of protein disulfide isomerase (PDI) inhibitors that significantly and synergistically enhance the antitumor activity of HDACi in glioblastoma and pancreatic cancer preclinical models. RNA-sequencing screening coupled with gene silencing studies identified ATF3 as the driver of this antitumor synergy. ATF3 was highly induced by combined PDI and HDACi treatment as a result of increased acetylation of key histone lysine residues (acetylated histone 3 lysine 27 and histone 3 lysine 18) flanking the ATF3 promoter region. These chromatin marks were associated with increased RNA polymerase II recruitment to the ATF3 promoter, a synergistic upregulation of ATF3, and a subsequent apoptotic response in cancer cells. The HSP40/HSP70 family genes DNAJB1 and HSPA6 were found to be critical ATF3-dependent genes that elicited the antitumor response after PDI and HDAC inhibition. In summary, this study presents a synergistic antitumor combination of PDI and HDAC inhibitors and demonstrates a mechanistic and tumor suppressive role of ATF3. Combined treatment with PDI and HDACi offers a dual therapeutic strategy in solid tumors and the opportunity to achieve previously unrealized activity of HDACi in oncology. SIGNIFICANCE: This study uses a first-in-class PDI inhibitor entering clinical development to enhance the effects of epigenetic drugs in some of the deadliest forms of cancer.