Hydroxynaphthoic Acids Identified in a High Throughput Screening Potently Ameliorate Endoplasmic Reticulum Stress as Novel Chemical Chaperones

Abstract

Folding of newly synthesized protein occurs in endoplasmic reticulum (ER) and is assisted by chaperone molecules. In ER stress conditions, misfolded proteins are enriched in a lumen of ER perturbing its normal function, which triggers cellular self-defense mechanism, the unfolded protein response (UPR). It was reported that tunicamycin-induced ER stress can be modulated with high concentration of chemicals such as 4-phenylbutyric acid and salicylate. In search of assay systems to identify such compounds, we have developed a cell-based reporter assay where renilla luciferase activity is driven by glucose-regulated protein 78 (GRP78) promoter. Using our reporter assay, we have screened chemical libraries and found that hydroxynaphthoic acids, especially 1-, 3-, and 6-hydroxy-2-naphthoic acids, potently decrease the ER stress signal, showing an order of magnitude better activity than salicylate. UPR markers such as GRP78, C/EBP homology protein (CHOP) and phosphorylated protein kinase RNA-activated (PKR)-like ER kinase (PERK) were significantly down-regulated with hydroxynaphthoic acids in western blot. Among the analogues, 1-hydroxy-2-naphthoic acid was the most potent in down-regulating those UPR markers. Further, both phosphorylated inositol-requiring enzyme 1α (IRE1α) and spliced form of X-box binding protein 1 (XBP1) were decreased in the protein and the mRNA level, implying both PERK and IRE1α branches in UPR mechanism are controlled with hydroxynaphthoic acids. Taken together, it was suggested that hydroxynaphthoic acids exert their ER stress-reducing activity prior to the UPR activation as chemical chaperones do. In summary, we report a cell-based assay system for the screening of ER stress-reducing compounds and hydroxynaphthoic acids as novel series of chemical chaperones.

Aspirin metabolite sodium salicylate selectively inhibits transcriptional activity of ATF6α and downstream target genes

“…NaSal can alleviate ER stress through the reduction of the expression of ATF6, CHOP and BiP on primary human adipocytes exposed to a number of distinct treatment conditions 33 . In hepatic cells, the simultaneous treatment with NaSal and tunicamycin decreased the phosphorylation of PERK and the expression of BiP and CHOP induced by tunicamycin alone 34 .…”

“…Previous studies had demonstrated reduction of the expression of ATF6α, CHOP and BiP on primary human adipocytes exposed to NaSal and ER-stress inducing stimuli for 24 hours 33 . In hepatic cells, the simultaneous treatment with 10 mM NaSal and tunicamycin decreased the phosphorylation of PERK and the expression of BiP and CHOP induced by tunicamycin alone 34 . We found that expressions of BiP , Pdia4 , Herpud-1 , Hrd-1 and p58 ipk were significantly but not completely inhibited in cells pre-treated with NaSal (Fig.…”

Bi-aryl Analogues of Salicylic Acids: Design, Synthesis and SAR Study to Ameliorate Endoplasmic Reticulum Stress

“…In search of novel chemical chaperones, we have reported that hydroxynaphthoic acids (HNAs) showed about an order of magnitude lower EC 50 than known chemical chaperones such as salicylic acid 14 and t-UDCA. 15 HNAs were identified through a cell-based assay we have developed and decreased ER stress markers including GRP78 and phosphorylated PERK.…”

“…Representative pharmacological chaperones include iminosugars to bind to β-glucocerebrosidase as potential treatment for Gaucher disease 11,12 and STF-083010 to treat atherosclerosis as an IRE1 inhibitor. 13 In search of novel chemical chaperones, we have reported that hydroxynaphthoic acids (HNAs) showed about an order of magnitude lower EC 50 than known chemical chaperones such as salicylic acid 14 and t--UDCA. 15 HNAs were identified through a cell-based assay we have developed and decreased ER stress markers including GRP78 and phosphorylated PERK.…”

“…15 HNAs were identified through a cell-based assay we have developed and decreased ER stress markers including GRP78 and phosphorylated PERK. 14,16 Especially, 3-Hydroxy-2-naththoic acid (3-HNA) showed excellent anti-diabetic effects including recovery of insulin sensitivity and glucose-lowering activity in ob/ob mouse when administered orally through ER stress-reducing activity. 16 Structurally, 3-HNA has an extended phenyl group of salicylic acid.…”