A small molecule UPR modulator for diabetes identified by high throughput screening

Abstract

Unfolded protein response (UPR) is a stress response that is specific to the endoplasmic reticulum (ER). UPR is activated upon accumulation of unfolded (or misfolded) proteins in the ER's lumen to restore protein folding capacity by increasing the synthesis of chaperones. In addition, UPR also enhances degradation of unfolded proteins and reduces global protein synthesis to alleviate additional accumulation of unfolded proteins in the ER. Herein, we describe a cell-based ultra-high throughput screening (uHTS) campaign that identifies a small molecule that can modulate UPR and ER stress in cellular and in vivo disease models. Using asialoglycoprotein receptor 1 (ASGR) fused with Cypridina luciferase (CLuc) as reporter assay for folding capacity, we have screened a million small molecule library and identified APC655 as a potent activator of protein folding, that appears to act by promoting chaperone expression. Furthermore, APC655 improved pancreatic β cell viability and insulin secretion under ER stress conditions induced by thapsigargin or cytokines. APC655 was also effective in preserving β cell function and decreasing lipid accumulation in the liver of the leptin-deficient (ob/ob) mouse model. These results demonstrate a successful uHTS campaign that identified a modulator of UPR, which can provide a novel candidate for potential therapeutic development for a host of metabolic diseases.

Keywords: ASGR, asialoglycoprotein receptor 1; ATF4, activating transcription factor 4; ATF6, activating transcription factor 6α/β; BID, twice a day; CLuc, Cypridina luciferase; Cell signaling; Chaperones; Diabetes; EGFP-VSVG, enhanced green fluorescence protein-vesicular stomatitis virus ts045 G protein; ER stress; ER, endoplasmic reticulum; ERP72, endoplasmic reticulum proteins 72; Endoplasmic reticulum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GLuc, Gaussia luciferase; GRP78, 78-kDa glucose-regulated protein; GRPRP94, glucose-regulated protein 94; GSIS, glucose stimulated insulin secretion; IKKβ, inhibitor of nuclear factor kappa-B kinase subunit beta; IL1β, interleukin 1β; INFγ, interferon gamma; IRE1, inositol requiring enzyme 1α/β; Liver; Metabolic diseases; NASH, nonalcoholic steatohepatitis; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; Nod, non-obese diabetic; OGTT, oral glucose tolerance test; PERK, PKR-like ER kinase; Pancreas; Protein folding; SP1/2, serine protease1/2; Small molecules; T1/2D, type1/2 diabetes; TG, thapsigargin; TNFα, tumor necrosis factor alpha; Tm, tunicamycin; UPR, unfolded protein response; Unfolded protein response; XBP1, X-box-binding protein 1; i.p., intraperitoneal; uHTS, ultra-high throughput screening; β cells.