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. 2015 Jun 17;7(292):292ra98.
doi: 10.1126/scitranslmed.aaa9134.

Phenotypic assays identify azoramide as a small-molecule modulator of the unfolded protein response with antidiabetic activity

Suneng Fu  1 , Abdullah Yalcin  2 , Grace Y Lee  2 , Ping Li  2 , Jason Fan  2 , Ana Paula Arruda  2 , Benedicte M Pers  2 , Mustafa Yilmaz  2 , Kosei Eguchi  2 , Gökhan S Hotamisligil  3
Affiliations

Phenotypic assays identify azoramide as a small-molecule modulator of the unfolded protein response with antidiabetic activity

Suneng Fu et al. Sci Transl Med. .

Abstract

The endoplasmic reticulum (ER) plays a critical role in protein, lipid, and glucose metabolism as well as cellular calcium signaling and homeostasis. Perturbation of ER function and chronic ER stress are associated with many pathologies ranging from diabetes and neurodegenerative diseases to cancer and inflammation. Although ER targeting shows therapeutic promise in preclinical models of obesity and other pathologies, the available chemical entities generally lack the specificity and other pharmacological properties required for effective clinical translation. To overcome these challenges and identify new potential therapeutic candidates, we first designed and chemically and genetically validated two high-throughput functional screening systems that independently measure the free chaperone content and protein-folding capacity of the ER. With these quantitative platforms, we characterized a small-molecule compound, azoramide, that improves ER protein-folding ability and activates ER chaperone capacity to protect cells against ER stress in multiple systems. This compound also exhibited potent antidiabetic efficacy in two independent mouse models of obesity by improving insulin sensitivity and pancreatic β cell function. Together, these results demonstrate the utility of this functional, phenotypic assay platform for ER-targeted drug discovery and provide proof of principle for the notion that specific ER modulators can be potential drug candidates for type 2 diabetes.

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Figures

Figure 1
Figure 1. Reporter systems monitor ER chaperone availability and activity
A. Two dual luciferase reporter constructs were generated, expressing the ATF6LD (yellow) or ASGR (black) fused to Cluc (red). Gluc (blue) was expressed from a separate promoter as an internal control. ER chaperones (green) mediate retention of ATF6LD-Cluc in the ER lumen by protein-protein interaction through a sensor domain in the ATFLD reporter. ASGR-Cluc secretion is mediated by the folding capacity of the ER. B. siRNA-mediated suppression of GRP78 increases ATF6LD-Cluc secretion compared to non treated and nonspecific controls (siCont). Efficiency of GRP78 suppression was validated by western blot analysis (lower panel). C. Thapsigargin (Tg) induced a dose-dependent increase in secretion of ATF6LD-Cluc compared to control (Gluc). D. siRNA-mediated suppression of GRP78 reduces ASGR-Cluc secretion compared to non treated and nonspecific controls. E. Tg induced a dose-dependent decrease in secretion of ASGR-Cluc compared to control (Gluc). For B and D, n=6 per group, graphs represent mean +/− SEM. For C and E, the mean of two technical replicates is shown. Experiments are representative of at least three independent experiments.
Figure 2
Figure 2. Azoramide regulates ER folding and secretion capacity without inducing ER stress
A. Chemical structure of azoramide (Azo). B. Expression profile of UPR genes in Huh7 cells following Tg and Azo treatment, average of two experimental duplicates is shown. CHOP induction in the 1µM Tg condition was 23.6 fold higher than control. C. Time course demonstrates differential regulation of GRP78 and CHOP expression and phosphorylation of eIF2a in Tg (upper panel) or Azo-treated Huh7 cells (lower panel). D. Dose response of Azo-induced increase of ASGR-Cluc secretion compared to control (Gluc). E. Dose response of Azo-induced decrease of ATF6LD-Cluc secretion compared to control (Gluc). F. ATF6LD-Cluc secretion in Azo-treated cells overexpressing GRP78 and ERDJ3. G. ATF6LD-Cluc secretion in Azo-treated cells following siRNA-mediated reduction of PERK, XBP-1 or ATF6. H. ATF6LD-Cluc secretion in Azo-treated cells following treatment with the PERK inhibitor GSK2606414 or the IRE1 inhibitor 4µ8C. For D–H, the mean of two technical replicates are shown. Experiments are representative of at least three independent experiments.
Figure 3
Figure 3. Azoramide protects against chemically-induced ER stress in vitro
A. Azoramide co- and pre-treatment counteracts tunicamycin (Tm)-induced ATF6LD-Cluc secretion and Tm-induced decrease of ASGR-Cluc secretion. The mean of two technical replicates for each condition is shown. Experiments are representative of at least two independent experiments. B. Azo pretreatment suppresses Tm-induced GRP78 and CHOP protein expression. C. Hypoxia induces ER stress, as measured by increased ATF6LD-Cluc secretion. Pretreatment with Azo abrogates this effect. Graph indicates mean +/− SD, n=8 per treatment. D. Expression of the P23H Rhodopsin mutant in HEK293A cells induces ER stress, as measured by CHOP expression. Treatment with Azo dose-dependently abrogates this effect. Graph indicates mean +/− SEM, n=4 per treatment. E. Azo treatment dose-dependently restores viability in RhodopsinP23H-expressing cells. Graph indicates mean+/− SEM, n=8 per treatment, **p<0.01 by t-test.
Figure 4
Figure 4. Azoramide treatment alters ER calcium homeostasis
A. Azoramide treatment increased the basal ER calcium concentration compared to vehicle in Hepa1-6 cells. Graph represents mean +/− SEM, n=91–184 per group. B,C. Azo pre-treatment increased ER calcium retention following Tg treatment. Graph represents mean +/− SEM, n=40–55 per group. D,E. Azo treatment delays the influx of calcium into the cytoplasm induced by Tg treatment. Graph represents mean +/− SEM, n=3–4 independent experiments (panel D), and n=63 (panel E). F. Azo treatment induces SERCA2 expression in Hepa 1–6 cells. *P<0.05, **P<0.01 by t-test.
Figure 5
Figure 5. Azoramide reduces ER stress and improves metabolism in ob/ob mice
A. Evaluation of ER stress markers in liver lysate from the ob/ob mice treated with oral Azo for one week. B. Body weights of the ob/ob mice during treatment with Azo or vehicle. C. Overnight fasting blood glucose levels, and, D. Glucose tolerance tests performed in the same groups of mice illustrate the impact of Azo treatment on glucose metabolism. In panels B–D, graphs represent mean +/− SEM, n=10 mice per group. *P<0.05, **P<0.01 by t-test (panel C), or repeated measures ANOVA (panel D).
Figure 6
Figure 6. Azoramide improves ER function, insulin secretion and survival in beta cells
A. Measurement of insulin and Pdx1 mRNA levels in islets isolated from ob/ob mice treated with vehicle or Azo for one week. Graph indicates mean +/− SEM, n=3. B. Glucose stimulated insulin secretion in vivo during glucose tolerance test of Azo- or vehicle-treated ob/ob mice, presented as change from fasting insulin level. Graph represents mean +/− SEM, n=9–10 mice/group. C. Glucose stimulated insulin secretion in Azo- or vehicle-treated Min6 cells. Graph indicates mean +/− SEM, n=4. D. Survival of vehicle and Azo-treated Ins-1 cells in the context of gluco-lipotoxicity (25mM Glucose and 500µM Palmitate). Graph indicates mean +/− SEM, n=8. *p<0.05 by t-test.
Figure 7
Figure 7. Azoramide induces weight loss, changes in energy expenditure and improved metabolic profile in mice with diet-induced obesity
A. Daily oral dosing of Azo for 1 week in HFD-induced obese mice decreased 6-hour fasting blood glucose. B. Body weight of HFD-fed mice before and after one week of Azo or vehicle treatment. C. Glucose infusion rate during hyperinsulinemic euglycemic clamp. D. Glucose disposal during clamp. E. Hepatic glucose production during clamp F. Glucose uptake to muscle (gastrocnemius) during clamp. G. Glucose uptake to white adipose (WAT) during clamp. H. Glycogen synthesis during clamp. Graphs represent mean +/− SEM, n=8–10/group, *P<0.05, **P<0.01 by t-test.
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