WO2022217268A1

WO2022217268A1 - Low molecular weight upr inhibitors as therapeutics for treatment of diseases caused by unfolded protein and er stress

Info

Publication number: WO2022217268A1
Application number: PCT/US2022/071618
Authority: WO
Inventors: Arnab K. Chatterjee; Weijun Shen; Matt TREMBLAY; Nikki ALVAREZ; Manoj Kumar
Original assignee: The Scripps Research Institute
Priority date: 2021-04-08
Filing date: 2022-04-08
Publication date: 2022-10-13

Abstract

The present disclosure relates to the compound APC655 as an enhancer of the endoplasmic reticulum protein folding capacity; The compound promotes the expression of chaperones and activates the ATF6 branch of the unfolded protein response UPR and preserves P cells viability and function during stress conditions. The compound is useful in methods of treating protein misfolding diseases, such as metabolic disorders.

Description

LOW MOLECULAR WEIGHT UPR INHIBITORS AS THERAPEUTICS FOR TREATMENT OF DISEASES CAUSED BY UNFOLDED PROTEIN AND ER STRESS [0001] This application claims the benefit of priority to U.S. Provisional Patent Application No.63/172,164, filed on April 8, 2021, which application is incorporated in its entirety as if fully set forth herein. BACKGROUND [0002] The Endoplasmic Reticulum (ER) is an important organelle to regulate the folding capacity of the cell and maintain protein homeostasis. Perturbations of the ER due to accumulation of unfolded proteins, abnormal calcium regulation or chemical stress are associated with several diseases including metabolic and neurological syndromes¹. This perturbation of the ER homeostasis is known as ER stress, which leads to the activation of the Unfolded Protein Response (UPR)². Indeed, the ER can sense the unfolded proteins accumulating in the lumen and can regulate protein expression and degradation accordingly. Underpinning this process is the 78-kDa glucose-regulated protein (GRP78, also known as Bip)³. GRP78, an ER chaperone that is located in the ER lumen, activates UPR by binding to the three transmembrane UPR sensors: inositol requiring enzyme 1Į/ȕ (IRE1)⁴, PKR-like ER kinase (PERK)⁵, and activating transcription factor 6Į/ȕ (ATF6)⁶. During protein homeostasis conditions, GRP78 is bound to the three UPR sensors, keeping them in an inactive state. However, in the case of accumulated unfolded proteins in the lumen of the ER, GRP78 as a chaperone binds to the unfolded proteins and releases and activates the three UPR sensors on the membrane of the ER^{4, 7}. [0003] When activated, IRE1 undergoes dimerization and autophosphorylation through the kinase domain present in the cytosolic portion^{5, 8-10}. After phosphorylation, the endonuclease activity of IRE1 induces the splicing of the main downstream target, X-box-binding protein 1 (XBP1) mRNA, producing an active transcription factor that regulates the expression of genes necessary for improving the protein folding capacity, protein degradation and protein export from the cells^{11, 12}. [0004] Upon dimerization and phosphorylation, PERK’s main function is to block the translation of new proteins, by phosphorylating and inhibiting the eukaryotic translation initiation factor-2Į and activating the transcription of the Activating Transcription Factor 4 (ATF4), which, in turn, can direct an antioxidant response and induce the expression of DNA damage-inducible transcript 3, also known as C/EBP homologous protein (CHOP), a protein implicated in the UPR-induced apoptosis^{13, 14}. [0005] The last UPR sensor, ATF6, once released from the binding of GRP78, is free to translocate on the Golgi membrane where it is cleaved by 2 proteases, S1P and S2P, releasing a cytosolic portion that acts as transcription factor and induces the transcription of genes coding for chaperones, such as glucose-regulated protein 94 (GRP94), T), GRP78 and Catalase¹⁵. The induction of the ATF6 branch of the UPR is considered a protective response by increasing the ER folding capacity. Indeed, the activation of the ATF6 pathway is shown to be protective for several diseases including renal and cerebral ischemia/reperfusion¹⁵ and T2D^16-18. The pancreatic ȕ cells have a highly developed ER, required for the biosynthesis and folding of pre-insulin, with subsequent trafficking to the Golgi, packaging into granules, conversion to mature insulin and secretion in response to high glucose. For this reason, supporting protein homeostasis is particularly important for the function and survival of these cells. [0006] Accumulating evidence link ER stress with impaired ȕ cell function in type 1 and 2 diabetes, as well as peripheral insulin resistance associated with type 2 diabetes. Elevated levels of UPR markers like CHOP were observed in islets from individuals with type 1 diabetes (T1D)¹⁹. Elevated ER stress markers were observed in non-obese diabetic (NOD) mice and leptin-deficient (ob/ob) mice. In addition, administration of chemical chaperones rescues the harmful ER stress response and improves pathophysiological signs of diabetes in both diabetic models, validating UPR modulators as potential therapeutic agents for diabetes²⁰. In ob/ob mice, the overexpression of endogenous chaperones in the liver, such as GRP78, promotes the activation of a protective UPR and the expression of more chaperones, including GRP94 (also known as Heat shock protein 90kDa beta member 1, leading to a clearance of the lipids accumulated in the liver²¹. [0007] Overexpression of ATF6 in the liver has a similar beneficial effect of increasing fatty acid oxidation and protecting against hepatic steatosis with increased expression of endogenous chaperones like GRP78²². Similarly, overexpression of ATF6 in diet-induced obese mice has beneficial effects on insulin sensitivity.¹⁶UPR levels are elevated in the pre- diabetic stage of T1D. Previous studies showed that modulating the UPR at this stage, by enhancing the ATF6 pathway and promoting a pro-survival UPR, can prevent the disease onset in a diabetes mouse model^{18, 23}. In obese human, tauroursodeoxycholic acid, a bile acid derivative that acts as a chemical chaperone to enhance protein folding and ameliorate ER stress improved hepatic and muscle insulin sensitivity, although target cells and mechanisms require additional studies.^{20, 24, 25} [0008] Previously, Fu et al ²⁶ designed a high-throughput functional screening system to measure the protein folding capacity of the ER by tagging asialoglycoprotein receptor 1 with Cypridina Luciferase reporter (ASGR-CLuc)and identified Azoramide that promotes chaperones expression, protects hepatocytes against chemically induced ER stress, and ȕ cells survival and function and improves Glucose handling in a T2D mouse model. SUMMARY [0009] The present disclosure relates in various embodiments to the identification of drugs that target the folding capacity of the ER, and that are beneficial for treating several disorders, such as autoimmune and metabolic diseases. As disclosed in more detail herein, identification is based upon optimization of a system for ultra-high throughput screening (uHTS), and then screening a 1 million compound library. The resultant screen identified the compound APC655 that improves the ER folding capacity, activates the ATF6 pathway, induces chaperones expression and preserves ȕ cell viability and function during stress conditions [0010] Thus, in an embodiment, the present disclosure provides a method for treating a protein misfolding disease or disorder in a subject suffering therefrom. The method comprises administering to the subject an effective amount of the compound APC655:

or a pharmaceutically acceptable salt thereof. [0011] In another embodiment, the present disclosure provides an effective amount of the compound APC655 or a pharmaceutically acceptable salt thereof for use in the treatment of a protein misfolding disease or disorder in a subject suffering therefrom. BRIEF DESCRIPTION OF THE DRAWINGS [0012] Figures 1A – 1E. Ultra-high throughput screening assay and APC655 in ASGR-CLuc reporter assay. A. Dose-dependent response of the ASGR-CLuc assay to thapsigargin. B. Dose-dependent response of the ASGR-CLuc assay to tunicamycin. C. Primary screening output; Forskolin andௗazoramideௗwere used as positive controls in the screening.0% represents DMSO treated cells, the orange dots represent the positive controls, the grey dots are the HITS and in yellow are the confirmed HITS. The red circled dot is APC655.^D. chemical structure of APC655. E. Dose response of APC655-induced increase of ASGR- CLuc secretion with and without tunicamycin 1uM; the data are shown as percentage to DMSO. Error bars are represented as mean ± SD; n=3. [0013] Figures 2A – 2H. APC655 enhances ER protein folding capacity through the activation of the ATF6 pathway and chaperones expression, during stress. A. APC655 induces the ATF6 promoter; the SP1 inhibitor PF429242 was used as control in the assay. B. APC655 promotes the release of ATF6LD in a dose-dependent way, with or without treatment with TG 0.5uM. c. ATF6 mRNA levels are increased by TG treatment in INS1e cells and boosted by the co-treatment with APC6555uM. D. BIP mRNA level is increased by 6h TG treatment in INS1e cells and boosted by the co-treatment with APC6555uM. E. ERP72 (PDI) mRNA level is increased by APC6555uM in co-treatment with TG for 6h in INS1e cells. F. The chaperone GRP94 expression is increased by APC6555uM co-treatment with TG. G. P-EGFP-VSVG protein trafficking assay after 16h at 40 ûC with DMSO (a) or APC6553PM (c) and after 15 minutes at 32 ûC with DMSO (b) or APC6553PM (d). The arrow indicates the folded P-EGFP-VSVG protein H. Cellomics quantification of P-EGFP- VSVG protein trafficking assay, normalized on number of nuclei and expressed as % of total cells analyzed. Error bars are represented as mean ±SD; n=4-6. Significance was determined by one-way ANOVA test. *P<0.05 **P<0.01, ***P<0.001. [0014] Figures 3A – 3D. APC655 protects cells survival and function in INS1e cells. A. Cell viability assay performed with Cell Titer Glo kit in INS1 Akita cells treated in dose- response with APC655, with or without the proteasome inhibitor MG132. B. Cell Titer Glo assay performed in INS1e cells treated with APC6552uM, 6 uM and 20 uM with or without 25mM Glucose and 500 µM Palmitate; Control (ctr) is the cells without any treatment, DMSO are the cells only treated with the stressor. C. Caspase 3/7 activation performed in INS1e cells treated with APC6552uM, 6 uM and 20 uM with or without IFNȖ (500 ng/mL) and IL1ȕ (50ng/mL); ctr is the cells without any treatment, DMSO are the cells only treated with the stressor. D. Glucose Stimulated Insulin Secretion (GSIS) in INS1e cells treated with APC6550.2uM, 1 uM and 5 uM and stressed with 20nM thapsigargin. Error bars are represented as mean ± SD; n = 4 - 6. Significance was determined by one-way ANOVA test. *P<0.05 **P<0.01, ***P<0.001. [0015] Figures 4A – 4D. APC655 protects cells survival and function in primary rat islets. A. Cell viability assay in primary Rat Islets treated with APC655 for 24h at different doses (0.6, 1.5, 4 and 10 uM). B. Cell viability assay in primary Rat Islets treated with APC655 for 24h at different doses (0.6, 1.5, 4 and 10 uM), together with DMSO or IFNȖ 100 ng/mL and IL1ȕ 5ng/mL. C. Caspase 3/7 activation performed in INS1e cells treated with APC6552uM, 6 uM and 20 uM, together with DMSO or IFNȖ 100 ng/mL, IL1ȕ 5ng/mL and TNFĮ 50ng/mL. D. Glucose Stimulated Insulin Secretion (GSIS) in INS1e cells treated with APC6550.2uM, 1 uM and 5 uM and stressed with Cytokines (IFNȖ 25 ng/mL and IL1ȕ 1.25ng/mL). Error bars are represented as mean ± SD; n= 4-6. Significance was determined by one-way ANOVA test. *P<0.05 **P<0.01, ***P<0.001. [0016] Figures 5A – 5G. APC655 decreased body weight, lowered fasted glucose, showed trend improved blood glucose and decreases lipids accumulation in the liver of ob/ob mice. A. Body Weight in grams of naïve and ob/ob mice treated daily with the vehicle, APC65515mg/kg and APC65550mg/kg for 6 days. b. Fast glucose analysis in naïve and ob/ob mice treated daily with the vehicle, APC65515mg/kg and APC65550mg/kg for 6 days. c. OGTT in naïve and ob/ob mice treated daily with the vehicle, APC65515mg/kg and APC65550mg/kg for 6 days. d. Area under the curve (AUC) of OGTT. B. a. O red Oil staining in liver sections from naïve and ob/ob mice treated daily with the vehicle, APC655 15mg/kg and APC65550mg/kg for 6 days. b. liver weight in grams. c. Oil Red O staining quantification. Error bars are represented as mean ±SD; n=4-6. Significance was determined by one-way ANOVA test. *P<0.05 **P<0.01, ***P<0.001. [0017] Figures 6A – 6F. sAPC655 decreases NF-kB pathway activation in the liver from ob/ob mice. Western blot analysis (A) and quantification (B and C) of NF-kB pathway in the total proteins extracted from liver of naïve mice or ob/ob mice treated with the vehicle or APC5515 and 50 mg/kg. D. GRP94 protein levels analyzed by western blot in the total proteins extracted from liver of naïve mice or ob/ob mice treated with the vehicle or APC55 15 and 50 mg/kg and quantification (E). (F) GRP94 mRNA level in livers from ob/ob mice treated with the vehicle or APC5515 and 50 mg/kg, measured by qPCR. Error bars are represented as mean ±SD; n=4-6. Significance was determined by the one-way ANOVA test. *P<0.05 **P<0.01, ***P<0.001. DETAILED DESCRIPTION [0018] The Endoplasmic Reticulum (ER) is important for calcium storage, lipid biosynthesis, protein synthesis and trafficking. Defects in the ER function translate into accumulation of unfolded proteins in the lumen of the ER and result in the activation of the UPR pathways. Abnormal and unresolved UPR is associated with the development of type1 and type2 diabetes and is responsible in part for the loss and malfunction of pancreatic ȕ cells ^{23, 38}. As the main function of the UPR is to restore the ER homeostasis by promoting the expression of chaperones and blocking the translation of new proteins, an unresolved ER-stress can transition into the apoptotic cell death. Although a “protective” UPR could be beneficial for the cells for restoring protein homeostasis, it is not easy to reach the exact balance between protein synthesis and degradation, preventing the ER-stress induced cell death and promoting proteostasis, thus preserving cell survival. The present disclosure resides, in part, in the discovery of a small molecule that can improve the ER folding capacity by stimulating the chaperones expression and, at the same time, preserving cell viability, as means for preventing and/or curing many metabolic diseases. Indeed, it has been previously showed that the treatment with chemical chaperones such as tauroursodeoxycholic acid or phenyl butyric acid reduces the diabetes in T1D mouse models when administered in pre-diabetic stage and this protection is lost in b-cell-specific ATF6a-deficient mice²⁵. [0019] As described in more detail herein, a high throughput phenotypic screening assay identified the compound APC655 as a small molecule UPR modulator that can rescue ER protein folding capacity. In ȕ cells, APC655 can repristinate the protein folding capacity of the ER during stress conditions by boosting the ATF6 pathway and enhancing the expression of chaperons. APC655 showed a protective effect on ȕ cell viability and function in both ȕ cell lines and primary islets. Activation of ATF6 and chaperones expression are known to be associated with a protection of hepatic steatosis in diet-induced insulin resistant mice. The expression of a dominant negative ATF6 in high-Fat-High-Sucrose diet-fed mice increases the susceptibility to develop hepatic steatosis and insulin resistance. It has been shown that ATF6 can stimulate fatty acid oxidation in hepatic cells through the activation of peroxisome proliferator-activated receptors (PPARa), thus maintaining metabolic homeostasis²². The treatment of ob/ob mice showed a protective effect of APC655 on glucose handling and on the lipid accumulation in the liver. In vivo data in the liver of ob/ob mice showed a strong downregulation of the NF-kB inflammatory pathway with the treatment of APC655, and the gene expression analysis of the UPR markers showed significant increase of GRP94 and XBP1, with a trend of increase in ATF6 and BIP and ATF4, indicating an increase in chaperone expression and potential folding capacity, resulting in the improvement of UPR and the ER stress. Interaction between UPR and inflammation is not well defined, but there are evidences that they can both regulate each other.²³ Nevertheless, both pathways are operative in a complex disease like hepatic steatosis and NASH. [0020] Abbreviations. UPR, Unfolded Protein Response; ER, Endoplasmic Reticulum; TG, thapsigargin; Tm, Tunicamycin; uHTS, ultra-high throughput screening; GRP78, 78-kDa glucose-regulated protein; IRE1, inositol requiring enzyme 1Į/ȕ; PERK, PKR-like ER kinase; ATF6, activating transcription factor 6Į/ȕ; XBP1, X-box-binding protein 1; ATF4, Activating Transcription Factor 4; GRPRP94, glucose-regulated protein 94 ; ERP72, endoplasmic reticulum proteins 72; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; IKKE, inhibitor of nuclear factor kappa-B kinase subunit beta; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; T1/2D, Type1/2 Diabetes; Nod, Non-Obese Diabetic; ASGR, asialoglycoprotein receptor 1; CLuc, Cypridina luciferase; GLuc, Gaussia Luciferase; EGFP-VSVG, Enhanced Green Fluorescence Protein- vesicular stomatitis virus ts045 G protein; SP1/2, serine protease1/2; INFJ; Interferon gamma; IL1b; Interleukin beta; TNFD; Tumor Necrosis Factor alpha; GSIS, Glucose Stimulated Insulin Secretion; OGTT, oral glucose tolerance test; BID, twice a day; IP, Intraperitoneal; NASH, Nonalcoholic steatohepatitis. [0021] Definitions [0022] As used herein, and unless otherwise specified to the contrary, the term “compound” is inclusive in that it encompasses a compound or a pharmaceutically acceptable salt, stereoisomer, and/or tautomer thereof. Thus, for instance, a compound of Formula IA or Formula IB includes a pharmaceutically acceptable salt of a tautomer of the compound. [0023] In this disclosure, a “pharmaceutically acceptable salt” is a pharmaceutically acceptable, organic or inorganic acid or base salt of a compound described herein. Representative pharmaceutically acceptable salts include, e.g., alkali metal salts, alkali earth salts, ammonium salts, water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1,1-methene-bis-2-hydroxy-3- naphthoate, einbonate), pantothenate, phosphate/diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts. A pharmaceutically acceptable salt can have more than one charged atom in its structure. In this instance the pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterions. [0024] The terms “treat”, “treating” and “treatment” refer to the amelioration or eradication of a disease or symptoms associated with a disease. In various embodiments, the terms refer to minimizing the spread or worsening of the disease resulting from the administration of one or more prophylactic or therapeutic compounds described herein to a patient with such a disease. [0025] The terms “prevent,” “preventing,” and “prevention” refer to the prevention of the onset, recurrence, or spread of the disease in a patient resulting from the administration of a compound described herein. [0026] The term “effective amount” refers to an amount of a compound as described herein or other active ingredient sufficient to provide a therapeutic or prophylactic benefit in the treatment or prevention of a disease or to delay or minimize symptoms associated with a disease. Further, a therapeutically effective amount with respect to a compound as described herein means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or prevention of a disease. Used in connection with a compound as described herein, the term can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease, or enhances the therapeutic efficacy of or is synergistic with another therapeutic agent. [0027] A “patient” or subject” includes an animal, such as a human, cow, horse, sheep, lamb, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig. In accordance with some embodiments, the animal is a mammal such as a non-primate and a primate (e.g., monkey and human). In one embodiment, a patient is a human, such as a human infant, child, adolescent or adult. In the present disclosure, the terms “patient” and “subject” are used interchangeably. [0028] METHODS OF USE [0029] As summarized above, the present disclosure provides a method for treating a protein misfolding disease or disorder in a subject suffering therefrom. The method comprises administering to the subject an effective amount of the compound APC655:

or a pharmaceutically acceptable salt thereof. [0030] In an embodiment, the present disclosure provides an effective amount of the compound APC655 or a pharmaceutically acceptable salt thereof for use in the treatment of a protein misfolding disease or disorder in a subject suffering therefrom. [0031] In various embodiments, the disease or disorder is selected from the group consisting of amyloid disease, a cardiovascular disease, a metabolic disorder, a lysosomal storage disease, an autoimmune disease or disorder, cystic fibrosis, antitrypsin-associated emphysema, a channelopathy, a collagenopathy, osteogenesis imperfect, pain, epilepsy, antitrypsin-associated liver cancer, and retinitis pigmentosa associated with mutant rhodopsin aggregation. [0032] In illustrative embodiments, the disease or disorder is an amyloid disease. Examples include Alzheimer’s disease, light chain amyloidosis, prion disease, a transthyretin amyloidosis, Creutzfeldt-Jakob disease, gelsolin amyloidosis, and lysozyme amyloidosis. [0033] The disease or disorder, in other embodiments, is a metabolic disorder. A metabolic disorder includes, for example, diabetes, nephrogenic diabetes insipidus, obesity, inflammatory bowel disease, Tay-Sachs disease, and hepatic steatosis. Specific examples of diabetes are Type 1 diabetes and Type 2 diabetes. [0034] In additional embodiments, the disease or disorder is a lysosomal storage disease. Examples of a lysosomal storage disease are Gaucher disease and Fabry’s disease. [0035] In still other embodiments, the disease or disorder is a cardiovascular disease. Illustrative embodiments include ischemic-reperfusion injury, cardiac hypertrophy, cardiac failure, and atherosclerosis. For example, the ischemic-reperfusion injury is selected from renal and cerebral ischemia-reperfusion. PHARMACEUTICAL COMPOSITION [0036] The disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of compound APC655, or a pharmaceutically acceptable salt, stereoisomer, and/or tautomer thereof in admixture with a pharmaceutically acceptable carrier. In some embodiments, the composition further contains, in accordance with accepted practices of pharmaceutical compounding, one or more additional therapeutic agents, pharmaceutically acceptable excipients, diluents, adjuvants, stabilizers, emulsifiers, preservatives, colorants, buffers, flavor imparting agents. [0037] The pharmaceutical composition of the present disclosure is formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular subject being treated, the clinical condition of the subject, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. [0038] The “therapeutically effective amount” of the compound or its pharmaceutically acceptable salt that is administered is governed by such considerations, and it is the minimum amount necessary to activate the UPR, bind to or activate IRE1, PERK, or ATF6, or any combination thereof. Such amount may be below the amount that is toxic to normal cells, or the subject as a whole. Generally, the initial therapeutically effective amount of the compound or its pharmaceutically acceptable salt of the present disclosure that is administered is in the range of about 0.01 to about 200 mg/kg or about 0.1 to about 20 mg/kg of patient body weight per day, with the typical initial range being about 0.3 to about 15 mg/kg/day. Oral unit dosage forms, such as tablets and capsules, may contain from about 0.1 mg to about 1000 mg of compound or its pharmaceutically acceptable salt of the present disclosure. In another embodiment, such dosage forms contain from about 50 mg to about 500 mg of the compound or its pharmaceutically acceptable salt of the present disclosure. In yet another embodiment, such dosage forms contain from about 25 mg to about 200 mg of the compound or its pharmaceutically acceptable salt of the present disclosure. In still another embodiment, such dosage forms contain from about 10 mg to about 100 mg of the compound or its pharmaceutically acceptable salt of the present disclosure. In a further embodiment, such dosage forms contain from about 5 mg to about 50 mg of the compound or its pharmaceutically acceptable salt of the present disclosure. In any of the foregoing embodiments the dosage form can be administered once a day or twice per day. [0039] The compositions of the present disclosure can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. [0040] Suitable oral compositions as described herein include without limitation tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, syrups or elixirs. [0041] In another aspect, also encompassed are pharmaceutical compositions suitable for single unit dosages that comprise the compound or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier. [0042] The compositions of the present disclosure that are suitable for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions. For instance, liquid formulations of the compounds of the present disclosure contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically palatable preparations of a compound of the present disclosure. [0043] For tablet compositions, a compound of the present disclosure in admixture with non- toxic pharmaceutically acceptable excipients is used for the manufacture of tablets. Examples of such excipients include without limitation inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known coating techniques to delay disintegration and absorption in the gastrointestinal tract and thereby to provide a sustained therapeutic action over a desired time period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. [0044] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. [0045] For aqueous suspensions, a compound of the present disclosure is admixed with excipients suitable for maintaining a stable suspension. Examples of such excipients include without limitation are sodium carboxymethylcellulose, methylcellulose, hydroxpropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia. [0046] Oral suspensions can also contain dispersing or wetting agents, such as naturally- occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin. [0047] Oily suspensions may be formulated by suspending a compound of the present disclosure in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. [0048] Sweetening agents such as those set forth above, and flavoring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid. [0049] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide a compound of the present disclosure in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. [0050] Pharmaceutical compositions of the present disclosure may also be in the form of oil- in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monoleate, and condensation reaction products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monoleate. The emulsions may also contain sweetening and flavoring agents. [0051] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable, an aqueous suspension or an oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. [0052] The compound or its pharmaceutically acceptable salt of the present disclosure may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols. [0053] Compositions for parenteral administrations are administered in a sterile medium. Depending on the vehicle used and concentration the concentration of the drug in the formulation, the parenteral formulation can either be a suspension or a solution containing dissolved drug. Adjuvants such as local anesthetics, preservatives and buffering agents can also be added to parenteral compositions. [0054] EXAMPLES [0055] General Materials and Methods [0056] In vitro cell-based Assays [0057] ASGR-CLuc and ATF6-CLuc Assay. ASGR-CLuc and AFT6-CLuc cells that were maintained in growth medium (Dulbecco’s modified Eagle’s medium containing antibiotics and 10% fetal bovine serum) were detached using trypsin. After removing excess trypsin by gentle centrifugation (600g for 5 min), the cells were resuspended in growth medium containing 2% FBS at a density of 625 cells/µL. Using an automated liquid dispenser, 4 µL of this solution was then dispensed to each well of 1536-well plates (Greiner) that were pre- spotted with 20 nL of various concentrations compound. The plates were incubated for 24 h in the incubator at 37°C with constant supply of 5% CO2, and 95% humidity. After the incubation period, plates were taken out and each well was supplemented with 1 µL of growth medium (10% FBS) or 1 µL of growth medium (10% FBS) containing 1 µM tunicamycin (200 nM final concentration) using an automated liquid dispenser. Plates were put back into the incubator for an additional 24h. Quantification of secreted ASGR-CLuc or ATF6-CLuc was done by addition of 2 µL of CLuc reagent (Pierce ™ Cypridina Luciferase Glow Assay Kit (# 16171) and the evolved luminescence signal was read using Envision (0.1 second/well, Perkin-Elmer). [0058] Cell culture and in vitro UPR assays. INS-1e ȕ cells that were maintained in growth medium were detached using trypsin. After removing excess trypsin by gentle centrifugation (600g for 5 min), the cells were resuspended in growth medium at a density of 250 cells/µL. 40 µL of this solution was then dispensed to each well of 384-well plates (Greiner) that were pre-spotted with 20 nL of various concentrations compound. The plates were incubated for 24 h in the incubator at 37°C with constant supply of 5% CO2, and 95% humidity. After the incubation period, plates were taken out and each well was supplemented with 10 µL of growth medium or 10 µL of growth medium containing 150 nM thapsigargin (TG, 30 nM final concentration). Plates were put back into the incubator for an additional 24h before further analysis [0059] Cell viability Assay. Cell viability of INS1e cells and primary Rat Islands was detected by adding 10 µL of CellTiter-Glo® Luminescent Cell Viability Assay (# G7473) to each well in 384 plate. Luminescence signal was read using Envision (0.1 second/well, Perkin-Elmer) to determine cell viabilities. [0060] Caspase 3/7 Activity. Caspase 3/7 activity of cells was detected by adding 10 µL of Caspase-Glo® 3/7y Assay (# G8092) to each well in 384 plate. Luminescence signal was read using Envision (0.1 second/well, Perkin-Elmer) to determine the luciferase signal. [0061] pEGFP VSVG trafficking assay.^^A temperature-sensitive variant (ts045) of vesicular stomatitis virus G protein tagged with GFP (VSVG-GFP) -based assay was used to follow the protein trafficking. ts045-VSVG protein reversibly misfolds and is retained in the ER at 40°C, but upon temperature shift to 32°C it correctly folds and is transported out of the ER into the secretory pathway²⁷. HEK293 cells were transfected with the pEGFP VSVG vector and cultured for 16h at 40 ûC and treated with DMSO or APC6553PM. After 16h the temperature was shifted to 32ûC for 15 minutes. A cellomics technique was used to quantify the P-EGFP-VSVG protein trafficking assay. [0062] ATF6 promoter reporter assay. For the ATF6 promoter reporter assay, HEK293 cells were transfected with the pGL4.39[luc2P/ATF6 RE/Hygro] Vector from Promega (# 9PIE366). Cells were resuspended in growth medium and plated at a density of 250 cells/µL in 384 wells pre-spotted with 20nL of various concentrations compound. The plates were incubated for 24 h in the incubator at 37°C with constant supply of 5% CO2, and 95% humidity. After the incubation period, plates were taken out and each well was supplemented with 10 µL of growth medium (10% FBS) or 10 µL of growth medium (10% FBS) containing thapsigargin for a final concentration of 500nM. Plates were put back into the incubator for an additional 8h. Quantification of ATF promoter activity was done by addition of 10 µL of Bright-Glo ™ Luciferase Assay System from Promega (#E2650) and the evolved luminescence signal was read using Envision (0.1 second/well, Perkin-Elmer). [0063] Glucose Stimulated Insulin Secretion. Rat primary islets (5k in one 10cm dish) were dispersed following a dispersal protocol; cells were counted and diluted to a final concentration of 5000 cells/100ul in 96-well “V” bottom non-treated Nunc plates (Nunc#249935) with 5000 cells/well (100ul/well). Plates were spin at 1000 RPM (RT6000B Sorvall tabletop = 208 RCF) for 5 min room temp, then placed in 37oC 5%CO2 incubator overnight. INS1 cells are plated at the density of 100K/100 ul/well in 96 well plate in 11 mM glucose medium overnight. The day after, the cells are treated with APC655 for 16h. After 16h, the cells are switched in 5.5mM RPMI medium and co-treated with APC655 and stressor (palmitate acid 0.2 mM or Thapsigargin 25nM) for 24 hours. After 24h, the cells are incubated in Krebs-Ringer bicarbonate HEPES buffer /2.8mM glucose for 2hrs for serum deprivation. After serum deprivation, the cells are stimulated with different glucose levels (2.8mM/20mM) in Krebs-Ringer bicarbonate HEPES buffer /fatty-acid free 0.1%BSA buffer for 2hr. At this point the medium is collected to determine the insulin level using HTRF Insulin Assay Kit (Cisbio Assay, 62INSPEC). Briefly, 10 µL antibodies solution containing two monoclonal antibodies that recognize the insulin was added for each 10 µL of the medium. These antibodies are labeled with fluorophores that are Fluorescence Resonance Energy Transfer pair in proximity. Fluorescence Resonance Energy Transfer signal is measured using Envision plate reader with excitation at 320 nm and emission at 665 nm and 615 nm. The cell layer is lysate with RIPA buffer and used to determine protein quantification using the BCA assay. Protein quantification is used to normalize the Insulin level.^ [0064] Western blot. For total protein extract preparation from cell preparation, the cell pellet was lysate with RIPA buffer, sonicated and incubate on the shaker at 4 C for 30 minutes and centrifuged at 12,000g for 15 min at 4 °C. The pellet was discarded. An aliquot of the supernatants was used for protein determination using the BCA protein assay reagent, according to the manufacturer's instruction, while the remainder was used for Western blot analysis. An equal amount of protein from each sample was loaded onto 4-12% SDS- polyacrylamide gel and transferred to a PVDF membrane. The membranes were incubated with the appropriate primary and secondary antibodies, and the signal was detected using the Odissey Li-Cor platform and the densitometric analysis was performed using the Image J software (NIH, Bethesda, MA, USA). For total protein extract from livers, a fragment of the liver from each mouse was homogenized in RIPA buffer using the Precellys Lysing Kit and the precellys 24 homogenizer. Then sonicated and processed the same way of the cell lysate. [0065] Gene expression analysis. For total RNA extract from cell preparation, the total RNA extraction was performed using the RNeasy ®Mini Kit (Quiagen), following the manufacture’s protocol. Total RNA from liver tissues was extracted using the TRIZOL. Total RNA was converted into cDNA using the High-Capacity cDNA-RT kit from Applied Biosystem, according to supplier's instructions. PCR amplification was performed using the PowerUp™ SYBR™ Green Master Mix from Applied Biosystem, following the manufacturer's protocol. For data analysis, the 7500 Software v2.0.5, provided by Applied Biosystem, was used. All PCR reactions were carried out in triplicate (duplicate for the standard curves). All qPCR results are expressed as relative ratio of the target cDNA transcripts to GGAPDH and normalized to that of the reference condition. [0066] Animal model and in vivo treatment [0067] Animal Studies. All animal care and experimental procedures were approved by the Intarcia Testing Facility Institutional Animal Care and Use Committee or the Institutional Animal Care and Use Committee of the California Institute for Biomedical Research (Calibr) and strictly followed the National Institutes of Health guidelines for humane treatment of animals. [0068] Animal. C57BL/6J (JAX stock# 000664) and ob/ob (JAX stock #000632) male mice 12/13 weeks of age were transferred to Jackson Labs in vivo research laboratory in Bar Harbor, ME (Protocol # AUS19001). The mice were ear notched for identification and housed in individually and positively ventilated polysulfonate cages with HEPA filtered air at a density of 3-4 mice per cage. The animal room was lit entirely with artificial fluorescent lighting, with a controlled 12 h light/dark cycle (6 am to 6 pm light). The normal temperature and relative humidity ranges in the animal rooms were 22 ± 4°C and 50 ± 15%, respectively. The animal rooms were set up to have 15 air exchanges per hour. Filtered tap water, acidified to a pH of 2.5 to 3.0, and normal rodent chow were provided ad libitum. [0069] PK Plasma Collection. Approximately 75^L whole blood were collected at 0, 1, 3, 5 and 24 hours post dose on study day 7. Whole blood will be collected into lithium heparin plasma separator tubes. Plasma will be extracted after centrifugation (14000rpm; 4°C). Plasma was stored at -80°C for future analyses. [0070] Dosing and Non-fasted blood glucose. All animals enrolled on study were dosed twice a day (BID) by intraperitoneal Injections at volume of 5 mL/kg. Body weights were recorded each day of dosing in the morning. Non-fasted blood glucose values were measured three times weekly and were assessed at the same time each day pre dose (e.g. between 8- 10am). [0071] Intraperitoneal glucose and insulin tolerance tests and measurement of insulin release. Oral glucose tolerance (OGTT) tests were conducted on day 7 after an overnight fast. For OGTT, initial blood glucose values were determined prior to administration (1.5g/kg BW) of a 30% glucose solution (300mg/ml) D-glucose in sterile PBS) by oral gavage. Blood glucose was measured using the AlphaTrak2 at 0,15, 30, 60- 90 and 120-minutes post glucose administration. Any values exceeding the limitation of the glucometer (>750mg/dL) were diluted 1:1 with sterile PBS and re-measured. Sera insulin was determined using the Mercodia Mouse Ultrasensitive Insulin ELISA kit (Mercodia Cat. No.10-1249-01) [0072] Histological analysis and staining quantification. The livers were collected from the mice, fixed in formalin for 24h and subsequentially frozen. Thereafter, cryosections were prepared for histological and immunohistochemical analyses. Sections stained with Oil Red O (from Abcam) were photographed and the images were analyzed using Image J. [0073] Reagents [0074] Thapsigargin (#T9033) and PF429242 (SML0667) were purchased from Millipore Sigma; Tunicamycin was purchased from Tocris (#5316).

[0075] Example 1: Phenotypic screening to measure the protein capacity of the ER identified APC655 as a UPR modulator [0076] We utilized a reporter system in which ASGR1, a transmembrane receptor whose folding is affected by chemically induced ER stress, is tagged with Cypridina luciferase reporter (ASGR-CLuc fusion protein) and outfitted with a secretion signal to monitor and quantify the folding capacity of the ER²⁶. As an internal control, this cell line also constitutively expressed a Gaussia Luciferase, which is not modulated by ER stress. The reporter assay was optimized and miniaturized to 1536 well format suitable for uHTS. Two molecules were used as positive controls: Azoramide, which is an UPR modulator with beneficial effects in obese mice²⁶ and Forskolin, a known activator of the enzyme adenylyl cyclase which was identified as a strong activator of the reporter system from a small pilot screening ²⁸. The treatment of HEK293 cells expressing ASGR-CLuc with thapsigargin and tunicamycin inhibits the folding of ASGR1, thus decreasing the luciferase signal in a dose responsive manner (Fig 1A and 1B). [0077] We screened compounds at 4 µM concentration in the presence of 100 nM tunicamycin. Compounds that inhibited 60% of the reduction of ASGR-CLuc signal were identified as hits for follow-up studies. The hit compounds were subsequentially confirmed in triplicate at 4 µM (Fig.1C) and counter-screened for luciferase stabilizers using a non-UPR related CLuc control construct and for cytotoxicity using the cell titer-glo assay in Ins1e cells (< 30% inhibition). Some of the known small molecules were identified from this screening, validating our screening assay. Among the confirmed hits (Fig.1C), APC655 was identified as a potent hit in preserving protein folding capacity in dose-responsive manner, both when treated simultaneously or following pre-treatment with Tunicamycin (Figs.1D and 1E). [0078] Example 2: APC655 activates ATF6 pathway and chaperones expression. [0079] To investigate whether the increased protein folding capacity of the ER induced by APC655 is due to the modulation of the UPR, we analyzed different signaling markers of the three UPR branches. Western blot analysis of proteins involved in the IRE1 and PERK branches of the UPR pathways, such as BIP, IRE1, XBP1 and ATF4, showed that APC655 does not affect these signaling components in the presence of stress. To investigate the involvement of the ATF6 branch, we looked at ATF6LD-CLuc reporter assay to monitor ATF6 release by GRP78²⁶. APC655 induced the release of ATF6LD in the medium, in a dose-dependent manner and enhanced the effect of 0.5 µM thapsigargin on ATF6 activation (Fig.2B). [0080] To further delineate whether APC655 affects the expression level of ATF6, we generated an ATF6 reporter assay to study the promoter activity of ATF6. For the promoter reporter assay we used PF429242, an inhibitor of S1P, as control in the experiment²⁹, which provides a contrast to the activating effect of thapsigargin on the ATF6 promoter. While APC655 has the capacity to induce the ATF6 reporter, the S1P inhibitor did not (Fig 2B). ATF6 is also upregulated by APC655 in the presence of stress at the mRNA level in INS1e cells pretreated overnight with APC6555uM and co-treated with thapsigargin 100nM for 6 hours (Fig 2C). [0081] To confirm the activation of ATF6 by APC655 in vitro, we overexpressed HEK293 cells with EGFP-ATF6 construct and observed higher EGFP signal in cells treated with thapsigargin (100nM) and APC655 (5 PM) compared to control. These data suggested that APC655 enhances the upregulation of the ATF6 branch of UPR during stress conditions. As a measure of ATF6 activation, we also analyzed the ATF6 downstream targets by qPCR in INS1e cells treated with thapsigargin, showing the upregulation of the chaperones GRP78, GRP94 and ERP72 (ER protein 72; also known as Protein disulfide-isomerase A4), all known targets of ATF6 (Fig.2D, 2E, 2F). Interestingly, chaperones expression was also associated with the reestablishment of the protein trafficking in the cells as shown in Fig 2G and 2H by the pEGFP-VSVG trafficking assay. Taken together these data show that APC655 is a modulator of the ATF6 pathway and induces the expression of endogenous chaperones, thus incrementing the protein folding capacity of the ER in INS1e during stress. [0082] Example 3: APC655 protect ȕ cells from chemically induced ER-stress dependent cell death. [0083] APC655 promotes the expression of chaperones and increases the protein folding capacity of the ER. The purpose of this example is to show that this activity translates into the protection or improved ȕ cell survival in the various ER stress-induced cell death. We used ȕ cells carrying the Akita mutation (C96Y) in the insulin gene, which leads to incorrect folding of the insulin protein^{30, 31}. Blocking protein degradation with the proteasome inhibitor MG132 in these cells leads to the accumulation of the unfolded insulin in the ER and the subsequent UPR activation and cell death. As shown in fig.3A, Akita cells treated with MG132 showed 90% reduction of cell viability, and cotreatment with APC655 protected the Akita cells from UPR-induced cell death. In the glucolipotoxicity settings, treatment of INS1e cells with 25mM Glucose and 500µM Palmitate for 48 hours leads to significant reduction of cell viability and APC655 protected cell survival in dose-response (Fig 3B). [0084] Cytokines have been directly implicated in the pathogenesis of type 1 diabetes (T1D) as the major drivers of inflammation and play crucial roles in controlling ongoing ȕ cell destruction³². The in vitro treatment of INS1e cells with interferon J^^IFNJ^^(500ng/ml) and interleukin-1^E (IL1E^ (50 ng/ml) leads to the activation of caspase 3/7, and co-treatment with APC655 reduced the caspase 3/7 activation in dose response (Fig.3C). Because APC655 protects pancreatic ȕ cells from the UPR-induced apoptosis, next we tested whether this improved ȕ cell survival is associated with the protection of ȕ cells function. ȕ cells release insulin in response to high glucose (Glucose Stimulated Insulin Secretion, GSIS) in INS1e cells is greatly reduced by the treatment with the ER stressor thapsigargin. The results show that APC655 treatment totally recovered the attenuated ȕ cell function. At 5 µM concentration, the total insulin secreted at 20 mM glucose stimulation surpassed the normal GSIS response without thapsigargin stressor, indicating an enhancement of ȕ cell viability and function. [0085] Example 4: APC655 improves ȕ cell viability and function from cytokine induced ER-stress in primary islets. [0086] The purpose of this example is to show that improved survival in INS1e cells can be translated into primary ȕ cells from freshly isolated rat islets. APC655 treatment alone does not impact the ȕ cell viability in rat islets (Fig.4A). We have developed the cytokine stress assay in primary rat islets. ȕ cell viability was greatly reduced with the treatment of a cytokine cocktail composed of IFNJ^(100ng/ml), IL1E^(5 ng/ml) and TNF^E^(50ng/ml) and APC655 treatment reduced this loss of viability in dose response (Fig.4B). Similarly, the caspase activation induced by the cytokine cocktail was greatly inhibited by APC655 treatment (Fig.4C). In a similar way to INS1e cells, the islet function is totally abolished by the treatment of cytokine cocktail, and APC655 partially recovered its GSIS function, although the effect is mild, potentially due to the harsh assay conditions (Fig.4D). All these results indicated that APC655 has beneficial effects in diabetes and can protect ȕ cells survival during stress in diabetes models. [0087] Example 5: APC655 decreased body weight, lowered fast blood glucose and improved liver steatosis in the ob/ob mice. [0088] Leptin deficient ob/ob mice are characterized by insulin resistance and liver lipid accumulation. Previous work demonstrated that the ER stress level in this mouse model is high in both pancreatic ȕ cells and in hepatocytes³³. Encouraged by the in vitro protection of ȕ cell viability and function in INS1e and primary islets, we tested this compound in the ob/ob mouse model. Intraperitoneal (IP)administration of APC655 at 15 and 50 mg/kg twice a day (BID) for 7 days reduced body weight (Fig.5A) and fasting glucose (Fig.5B) in ob/ob mice. In addition, APC655 also shows a trend in improving glucose handling as demonstrated in the OGTT (Fig.5C and 5D). [0089] A significant reduction of liver size was observed with APC655 treatment (50 mg/kg) in the ob/ob mice during takedown. Indeed, the liver weight normalized by the body weight showed a significant reduction in the mice treated with APC655 at 50mg/kg and a trend of reduction in the ob/ob mice at 15 mg/kg treatment (Fig.5F). We also analyzed the lipids accumulation in the livers from the vehicle and APC655 treated ob/ob mice, using the oil red O staining (Fig.5E). As shown in figure 5E and quantified in figure 5G, lipids staining is strongly reduced in ob/ob mice treated with APC65550mg/kg, indicating a beneficial effect of APC655 in liver steatosis and NASH. [0090] Example 6: Systemic Action of APC655. The link between obesity, UPR and inflammation is not completely understood but there is much evidence that UPR and inflammation are connected, regulated by each other, and involved in obesity^34-36. We analyzed inflammatory response and UPR chaperones in the liver from the ob/ob mice treated with the vehicle or APC655 and compared them with the naïve control mice. The activation of NF-kB by UPR has been reported as a common signaling cascade in the 3 UPR branches³⁷. Western blot analysis of the total proteins isolated from livers of ob/ob mice showed that the treatment with APC655 modulated NF-kB signaling pathway, as demonstrated by the reduced NF-kB (p65) phosphorylation and increased protein level of the NF-kB inhibitor IKKȕ (Fig.6A-6C). Also, protein levels and mRNA levels of GRP94 were upregulated in the livers from ob/ob mice treated with APC655, supporting our in vitro observations (Fig.6D-E and Fig.6F). [0091] Next, we analyzed the expression level of genes involved in the UPR pathways. APC655 treatment highlighted a trend but not significant increase of chaperone expression in the livers of ob/ob mice treated with APC655. Taken together, these data suggest that the upregulation of chaperones might be consequential to the reduced inflammation and suggests a more systemic mechanism of action of APC655 in vivo. [0092] Numbered references cited in the present disclosure are as follows: 1. Lin, J. H.; Walter, P.; Yen, T. S., Endoplasmic reticulum stress in disease pathogenesis. Annu Rev Pathol 2008, 3, 399-425. 2. Karagoz, G. E.; Acosta-Alvear, D.; Walter, P., The Unfolded Protein Response: Detecting and Responding to Fluctuations in the Protein-Folding Capacity of the Endoplasmic Reticulum. Cold Spring Harb Perspect Biol 2019, 11,a033886. 3. Gardner, B. M.; Pincus, D.; Gotthardt, K.; Gallagher, C. M.; Walter, P., Endoplasmic reticulum stress sensing in the unfolded protein response. Cold Spring Harb Perspect Biol 2013, 5, a013169. 4. Mori, K.; Ma, W.; Gething, M. J.; Sambrook, J., A transmembrane protein with a cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus. Cell 1993, 74, 743-56. 5. Harding, H. P.; Zhang, Y.; Ron, D., Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 1999, 397, 271-4. 6. Haze, K.; Yoshida, H.; Yanagi, H.; Yura, T.; Mori, K., Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol Biol Cell 1999, 10, 3787-99. 7. Cox, J. S.; Shamu, C. E.; Walter, P., Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell 1993, 73, 1197-206. 8. Prischi, F.; Nowak, P. R.; Carrara, M.; Ali, M. M., Phosphoregulation of Ire1 RNase splicing activity. Nat Commun 2014, 5, 3554. 9. Shamu, C. E.; Walter, P., Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus. EMBO J 1996, 15 (12), 3028-39. 10. Tirasophon, W.; Welihinda, A. A.; Kaufman, R. J., A stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells. Genes Dev 1998, 12, 1812-24. 11. He, Y.; Sun, S.; Sha, H.; Liu, Z.; Yang, L.; Xue, Z. et al Emerging roles for XBP1, a sUPeR transcription factor. Gene Expr 2010, 15, 13-25. 12. Lerner, A. G.; Upton, J. P.; Praveen, P. V.; Ghosh, R.; Nakagawa, Y.; Igbaria, A. et al., IRE1alpha induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress. Cell Metab 2012, 16, 250-64. 13. DuRose, J. B.; Scheuner, D.; Kaufman, R. J.; Rothblum, L. I.; Niwa, M., Phosphorylation of eukaryotic translation initiation factor 2alpha coordinates rRNA transcription and translation inhibition during endoplasmic reticulum stress. Mol Cell Biol 2009, 29, 4295-307. 14. Fribley, A.; Zhang, K.; Kaufman, R. J., Regulation of apoptosis by the unfolded protein response. Methods Mol Biol 2009, 559, 191-204. 15. Blackwood, E. A.; Azizi, K.; Thuerauf, D. J.; Paxman, R. J.; Plate, L.; Kelly, J. W. et al, Pharmacologic ATF6 activation confers global protection in widespread disease models by reprograming cellular proteostasis. Nat Commun 2019, 10, 187. 16. Ozcan, L.; Ghorpade, D. S.; Zheng, Z.; de Souza, J. C.; Chen, K.; Bessler, M. et al., Hepatocyte DACH1 Is Increased in Obesity via Nuclear Exclusion of HDAC4 and Promotes Hepatic Insulin Resistance. Cell Rep 2016, 15, 2214-25. 17. Li, Y.; Wang, X.; Yang, B.; Wang, H.; Ma, Z.; Lu, Z. et al., 3beta-Hydroxysteroid- Delta24 Reductase (DHCR24) Protects Pancreatic beta Cells from Endoplasmic Reticulum Stress-Induced Apoptosis by Scavenging Excessive Intracellular Reactive Oxygen Species. J Diabetes Res 2020, 2020, 3426902. 18. Engin, F.; Yermalovich, A.; Nguyen, T.; Hummasti, S.; Fu, W.; Eizirik, D. L. et al., Restoration of the unfolded protein response in pancreatic beta cells protects mice against type 1 diabetes. Sci Transl Med 2013, 5, 211ra156. 19. Marhfour, I.; Lopez, X. M.; Lefkaditis, D.; Salmon, I.; Allagnat, F.; Richardson, S. J. et al., Expression of endoplasmic reticulum stress markers in the islets of patients with type 1 diabetes. Diabetologia 2012, 55, 2417-20. 20. Ozcan, U.; Yilmaz, E.; Ozcan, L.; Furuhashi, M.; Vaillancourt, E.; Smith, R. O. et al., Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science 2006, 313, 1137-40. 21. Ye, R.; Jung, D. Y.; Jun, J. Y.; Li, J.; Luo, S.; Ko, H. J. et al., GRP78 heterozygosity promotes adaptive unfolded protein response and attenuates diet- induced obesity and insulin resistance. Diabetes 2010, 59, 6-16. 22. Chen, X.; Zhang, F.; Gong, Q.; Cui, A.; Zhuo, S.; Hu, Z. et al., Hepatic ATF6 Increases Fatty Acid Oxidation to Attenuate Hepatic Steatosis in Mice Through Peroxisome Proliferator-Activated Receptor alpha. Diabetes 2016, 65, 1904-15. 23. Engin, F., ER stress and development of type 1 diabetes. J Investig Med 2016, 64, 2-6. 24. Kars, M.; Yang, L.; Gregor, M. F.; Mohammed, B. S.; Pietka, T. A.; Finck, B. N. et al., Tauroursodeoxycholic Acid may improve liver and muscle but not adipose tissue insulin sensitivity in obese men and women. Diabetes 2010, 59, 1899-905. 25. Bronczek, G. A.; Vettorazzi, J. F.; Soares, G. M.; Kurauti, M. A.; Santos, C.; Bonfim, M. F. et al., The Bile Acid TUDCA Improves Beta-Cell Mass and Reduces Insulin Degradation in Mice With Early-Stage of Type-1 Diabetes. Front Physiol 2019, 10, 561. 26. Fu, S.; Yalcin, A.; Lee, G. Y.; Li, P.; Fan, J.; Arruda, A. P. et al., Phenotypic assays identify azoramide as a small-molecule modulator of the unfolded protein response with antidiabetic activity. Sci Transl Med 2015, 7, 292ra98. 27. Presley, J. F.; Cole, N. B.; Schroer, T. A.; Hirschberg, K.; Zaal, K. J.; Lippincott- Schwartz, J., ER-to-Golgi transport visualized in living cells. Nature 1997, 389, 81-5. 28. Cunha, D. A.; Ladriere, L.; Ortis, F.; Igoillo-Esteve, M.; Gurzov, E. N.; Lupi, R. et al., Glucagon-like peptide-1 agonists protect pancreatic beta-cells from lipotoxic endoplasmic reticulum stress through upregulation of BiP and JunB. Diabetes 2009, 58, 2851-62. 29. Lebeau, P.; Byun, J. H.; Yousof, T.; Austin, R. C., Pharmacologic inhibition of S1P attenuates ATF6 expression, causes ER stress and contributes to apoptotic cell death. Toxicol Appl Pharmacol 2018, 349, 1-7. 30. Liu, M.; Haataja, L.; Wright, J.; Wickramasinghe, N. P.; Hua, Q. X.; Phillips, N. F. et al., Mutant INS-gene induced diabetes of youth: proinsulin cysteine residues impose dominant-negative inhibition on wild-type proinsulin transport. PLoS One 2010, 5, e13333. 31. Weiss, M. A., Diabetes mellitus due to the toxic misfolding of proinsulin variants. FEBS Lett 2013, 587, 1942-50. 32. Lu, J.; Liu, J.; Li, L.; Lan, Y.; Liang, Y., Cytokines in type 1 diabetes: mechanisms of action and immunotherapeutic targets. Clin Transl Immunology 2020, 9, e1122. 33. Yang, L.; Li, P.; Fu, S.; Calay, E. S.; Hotamisligil, G. S., Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance. Cell Metab 2010, 11, 467-78. 34. Amen, O. M.; Sarker, S. D.; Ghildyal, R.; Arya, A., Endoplasmic Reticulum Stress Activates Unfolded Protein Response Signaling and Mediates Inflammation, Obesity, and Cardiac Dysfunction: Therapeutic and Molecular Approach. Front Pharmacol 2019, 10, 977. 35. Hotamisligil, G. S., Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell 2010, 140, 900-17. 36. Hummasti, S.; Hotamisligil, G. S., Endoplasmic reticulum stress and inflammation in obesity and diabetes. Circ Res 2010, 107, 579-91. 37. Garg, A. D.; Kaczmarek, A.; Krysko, O.; Vandenabeele, P.; Krysko, D. V.; Agostinis, P., ER stress-induced inflammation: does it aid or impede disease progression? Trends Mol Med 2012, 18, 589-98. 38. Engin, F.; Nguyen, T.; Yermalovich, A.; Hotamisligil, G. S., Aberrant islet unfolded protein response in type 2 diabetes. Sci Rep 2014, 4, 4054.

Claims

WE CLAIM: 1. A method for treating a protein misfolding disease or disorder in a subject suffering therefrom, comprising administering to the subject an effective amount of APC655:

or a pharmaceutically acceptable salt thereof.

2. The method according to claim 1, wherein the disease or disorder is selected from the group consisting of amyloid disease, a cardiovascular disease, a metabolic disorder, a lysosomal storage disease, an autoimmune disease or disorder, cystic fibrosis, antitrypsin- associated emphysema, a channelopathy, a collagenopathy, osteogenesis imperfect, pain, epilepsy, antitrypsin-associated liver cancer, and retinitis pigmentosa associated with mutant rhodopsin aggregation.

3. The method according to claim 1 or 2, wherein the disease or disorder is an amyloid disease.

4. The method according to any one of claims 1 to 3, wherein the amyloid disease is selected from the group consisting of Alzheimer’s disease, light chain amyloidosis, prion disease, a transthyretin amyloidosis, Creutzfeldt-Jakob disease, gelsolin amyloidosis, and lysozyme amyloidosis.

5. The method according to claim 1 or 2, wherein the disease or disorder is a metabolic disorder.

6. The method according to any one of claims 1, 2, or 5, wherein the metabolic disorder is selected from the group consisting of diabetes, nephrogenic diabetes insipidus, obesity, inflammatory bowel disease, Tay-Sachs disease, and hepatic steatosis.

7. The method according to any one of claims 1, 2, 5, or 6, wherein the disease or disorder is selected from Type 1 diabetes and Type 2 diabetes.

8. The method according to claim 1 or 2, wherein the disease or disorder is a lysosomal storage disease.

9. The method according to any one of claims 1, 2, or 8, wherein the lysosomal storage disease is selected from Gaucher disease and Fabry’s disease.

10. The method according to claim 1 or 2, wherein the disease or disorder is a cardiovascular disease.

11. The method according to any one of claims 1, 2, or 10, wherein the cardiovascular disease is selected from the group consisting of ischemic-reperfusion injury, cardiac hypertrophy, cardiac failure, and atherosclerosis.

12. The method according to any one of claims 1, 2, 10, or 11, wherein the ischemic- reperfusion injury is selected from renal and cerebral ischemia-reperfusion.