Small molecule-induced oxidation of protein disulfide isomerase is neuroprotective

Abstract

Protein disulfide isomerase (PDI) is a chaperone protein in the endoplasmic reticulum that is up-regulated in mouse models of, and brains of patients with, neurodegenerative diseases involving protein misfolding. PDI's role in these diseases, however, is not fully understood. Here, we report the discovery of a reversible, neuroprotective lead optimized compound (LOC)14, that acts as a modulator of PDI. LOC14 was identified using a high-throughput screen of ∼10,000 lead-optimized compounds for potent rescue of viability of PC12 cells expressing mutant huntingtin protein, followed by an evaluation of compounds on PDI reductase activity in an in vitro screen. Isothermal titration calorimetry and fluorescence experiments revealed that binding to PDI was reversible with a K d of 62 nM, suggesting LOC14 to be the most potent PDI inhibitor reported to date. Using 2D heteronuclear single quantum correlation NMR experiments, we were able to map the binding site of LOC14 as being adjacent to the active site and to observe that binding of LOC14 forces PDI to adopt an oxidized conformation. Furthermore, we found that LOC14-induced oxidation of PDI has a neuroprotective effect not only in cell culture, but also in corticostriatal brain slice cultures. LOC14 exhibited high stability in mouse liver microsomes and blood plasma, low intrinsic microsome clearance, and low plasma-protein binding. These results suggest that LOC14 is a promising lead compound to evaluate the potential therapeutic effects of modulating PDI in animal models of disease.small molecule | protein disulfide isomerase | drug | inhibitor | neuroprotection

Human Milk Oligosaccharides Increase Mucin Expression in Experimental Necrotizing Enterocolitis

“…To understand the link between ER stress chaperones and HMO‐mediated barrier protection, we next assessed the impacts of PDI inhibitors 16F16 and rutin hydrate on barrier function in vitro. As shown in Figure E and F, HMO‐pretreated Caco‐2Bbe1 cells exhibited fewer EHEC‐induced AE lesions, but this effect was blocked when cells were inhibited with 16F16.…”

Novel Protein Disulfide Isomerase Inhibitor with Anticancer Activity in Multiple Myeloma

“…The central role of PDI for ER productivity and stress reduction suggests exocrine cancers and histologies with high levels of ER stress should be tested in future studies. Two leading PDI inhibitors have remained preclinical so far; PACMA 31 covalently binds to active site cysteines of PDI and has shown promise in ovarian cancer (22), whereas LOC14 was found to have antiapoptotic function on nerve cells in a model of Huntington disease and binds noncovalently at nanomolar concentration but requires high micromolar doses for PDI inhibition in the insulin aggregation assay (23), which measures its reducing potency (24). To date, no reliable method for measuring PDI activity in cells has been described and in vitro assays used in drug discovery poorly reproduce PDI physiology where cysteine oxidation, reduction, and isomerization of disulfide bonds has to be accomplished in short sequence and is influenced by additional enzymes, like Ero1-a (37) that restore oxidative potential of PDI required for immunoglobulin folding (5).…”

“…5C). To test whether CCF642 affects PDI function, we performed di-E-GSSG assays, which measure PDI's reducing capacity (17), and compared inhibition to known PDI inhibitors PACMA 31 (22) and LOC14 (23). CCF642 at 1 mmol/L inhibited reduction of di-E-GSSG about as much as 100 mmol/L of LOC14 or PACMA 31 (Fig.…”

Discovery and Mechanistic Elucidation of a Class of Protein Disulfide Isomerase Inhibitors for the Treatment of Glioblastoma

“…A reversible PDI modulator, LOC14 (EC 50 = 500 nM), has neuroprotective effects in cellular and rat models of Huntington’s disease. [10] Furthermore, PDI inhibitor CCF642 was demonstrated to be effective in a mouse xenograft model of multiple myeloma. [11] Mounting evidence highlights PDI as an important target against several diseases including cancer, emphasizing the need for potent, clinically relevant PDI inhibitors for cancer treatment.…”