SWELL1-LRRC8 complex modulator

Abstract

The present invention is directed to various polycyclic compounds and methods of using these compounds for treating a variety of diseases, including metabolic diseases such as obesity, diabetes, non-alcoholic fatty liver disease, cardiovascular diseases such as hypertension and stroke, neurological diseases, male infertility, muscle disorders, and immune disorders.

Description

[Technical Field]

[0001] The present invention is directed to various polycyclic compounds and methods of using these compounds for treating various diseases associated with abnormal SWELL1 signaling, including metabolic diseases such as obesity, diabetes, non-alcoholic fatty liver disease, cardiovascular diseases such as hypertension and stroke, neurological diseases, male infertility, muscle disorders, and immune disorders. [Background technology]

[0002] Obesity-induced diabetes (type 2 diabetes, T2D) has reached epidemic proportions in the United States alone (2014, CDC), with more than one in three obese Americans (36%), >29 million people with diabetes, and approximately 86 million people with prediabetes. The economic impact of obesity and diabetes in the United States alone is close to $500 billion. Globally, this is an even more serious problem, with the incidence of type 2 diabetes estimated at 422 million in 2014 and a projected number of more than 700 million within the next decade. Nonalcoholic fatty liver disease (NAFLD) is highly associated with T2D and has a prevalence of 24% both in the United States and globally. NAFLD often progresses to advanced liver disease, cirrhosis, and hepatocellular carcinoma and is currently the second most common indication for liver transplantation in the United States, after hepatitis C.

[0003] While several medications are currently available to treat type 2 diabetes, physicians remain challenged in effectively treating this disease, as a significant proportion of patients continue to have poorly controlled blood glucose despite optimal medical therapy. The failure of medical therapies is related to several factors, including narrow mechanisms of action (insulin sensitizers vs. secretagogues), noncompliance (especially with drugs with frequent dosing regimens), and achieving euglycemia while avoiding life-threatening hypoglycemia. Furthermore, some current therapies, including TZDs also used to treat NAFLD, suffer from undesirable and dangerous side effects, such as congestive heart failure, weight gain, and edema.

[0004] Volume-regulated anion channels (VRACs) are thought to be cell swelling-induced anion channels. They regulate important functions in various organ systems and are implicated in pathologies associated with diabetes, obesity, nonalcoholic fatty liver disease, stroke, hypertension, and other conditions. Leucine-rich repeat-containing protein 8A (LRRC8A), also known as SWELL1, forms heteromeric VRACs with four other related homologs (LRRC8B-E).

[0005] SWELL1 (LRRC8a) is a necessary component of a volume-sensitive ion channel molecular complex that is activated in the setting of adipocyte hypertrophy and regulates adipocyte size, insulin signaling, and systemic blood glucose through a novel SWELL1-PI3K-AKT2-GLUT4 signaling axis. Adipocyte-specific SWELL1 ablation disrupts insulin-PI3K-AKT2 signaling and induces insulin resistance and glucose intolerance in vivo. Therefore, SWELL1 is identified as a positive regulator of adipocyte insulin signaling and glucose homeostasis, particularly in the setting of obesity.

[0006] In addition to impaired insulin sensitivity, type 2 diabetes is also characterized by a relative loss of insulin secretion from pancreatic β-cells. Regulation of β-cell excitability is the predominant mechanism controlling insulin secretion and systemic glycemia. Indeed, sulfonylurea receptor inhibitors (i.e., glibenclamide), the cornerstone of current diabetes pharmacotherapy, activate well-characterized, inhibitory, hyperpolarizing currents I to promote β-cell depolarization and activate voltage-gated calcium channels (VGCCs), thereby eliciting insulin secretion. K,ATP However, for such agents to be effective, excitatory currents must be present that allow membrane depolarization. SWELL1 is required for a pronounced swelling-activated chloride current in β-cells. SWELL1-mediated VRAC is activated by glucose-mediated β-cell swelling and provides the essential depolarizing current required for β-cell depolarization, glucose-stimulated Ca2+ signaling, and insulin secretion.

[0007] Normal SWELL1 function is necessary for the normal development of the human immune system. In one example, the expression of truncated SWELL1 protein caused by translocation of one allele of SWELL1 inhibits normal beta cell development and causes agammaglobulinemia 5 (AGM5) (Sawada, A., et al. Journal of Clinical Investigation 2003; Kubota, K. et al., FEBS Lett 2004). Because different types of immune system cells (e.g., B lymphocytes and T lymphocytes) use similar intracellular signaling pathways, the development and / or function of other immune system cells (e.g., T lymphocytes, macrophages, and / or NK cells) are likely to be affected by proper SWELL1 function.

[0008] Currently, the molecular causes of male infertility are only partially understood. Mice lacking SWELL1 in late spermatids fail to reduce cytoplasm during sperm development, leading to disorganized mitochondrial sheaths containing horn flagellates and reduced sperm motility. This indicates that SWELL1 is required for normal spermatid development and male infertility (Luck, JC, Journal of Biological Chemistry 2018).

[0009] SWELL1 and associated VRAC signaling are also associated with stroke-induced neurotoxicity and cardiovascular disease.

[0010] There is evidence that compounds that directly bind to SWELL1 can be used to inhibit or otherwise modulate SWELL1 to treat various conditions. One such compound is DCPIB (4[2[butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid) (referred to herein as Smod1), which has affinity for LRRC8A and is described in WO2018 / 027175. However, there is a need for compounds that have improved affinity and metabolic profiles and target a wider variety of LRRC8 homologs. Such compounds may be useful for improved therapy of diabetes, obesity, non-alcoholic fatty liver disease, stroke, hypertension, immunodeficiency, male infertility, and other conditions. Summary of the Invention

[0011] Various aspects of the present invention are directed to compounds of formula (I), and salts thereof:

[0012] [ka]

[0013] During the ceremony, R 1 and R 2 are each independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; R 3 is -YC(O)R 4 , -ZN(R 5 )(R 6 ), or -ZA, R 4 is hydrogen, substituted or unsubstituted alkyl, -OR 7 , or -N(R 8 )(R 9 ) and X 1 and X 2each independently represents a substituted or unsubstituted alkyl, halo, -OR 10 , or -N(R 11 )(R 12 ) and R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are each independently hydrogen or substituted or unsubstituted alkyl; Y and Z are each independently a substituted or unsubstituted carbon-containing moiety having at least two carbon atoms; A is at least one nitrogen heteroatom, boronic acid or

[0014] [ka]

[0015] and a substituted or unsubstituted 5- or 6-membered heterocycle having the formula: n is 1 or 2.

[0016] Further aspects are directed to various methods of using the compounds of Formula (I) to treat various conditions, including insulin sensitivity, obesity, diabetes, non-alcoholic fatty liver disease, metabolic disease, hypertension, stroke, vascular tone, systemic and / or pulmonary arterial pressure, blood flow, male infertility, muscle disorders, and / or immune deficiency, in a subject in need thereof. Generally, the methods include administering to the subject a therapeutically effective amount of a compound of Formula (I).

[0017] Other objects and features will be in part apparent and in part pointed out hereinafter. [Brief explanation of the drawings]

[0018] [Figure 1]Chemical structures of Smod1 / DCPIB, Smod4, Smod2, Smod3, Smod5, Smod6 and Snot1 described herein. [Figure 2A] Patch clamp screening of Smod compounds for ICL,SWELL inhibitory activity. Time course of outward (black) and inward (blue) ICL,SWELL currents upon application of (A) Smod compounds lacking Snot1:ICL,SWELL inhibitory activity, (B) active Smod2, and (C) active Smod3. [Figure 2B] Patch clamp screening of Smod compounds for ICL,SWELL inhibitory activity. Time course of outward (black) and inward (blue) ICL,SWELL currents upon application of (A) Smod compounds lacking Snot1:ICL,SWELL inhibitory activity, (B) active Smod2, and (C) active Smod3. [Figure 2C] Patch clamp screening of Smod compounds for ICL,SWELL inhibitory activity. Time course of outward (black) and inward (blue) ICL,SWELL currents upon application of (A) Smod compounds lacking Snot1:ICL,SWELL inhibitory activity, (B) active Smod2, and (C) active Smod3. [Figure 3A] Patch clamp screening of Smod compounds for ICL,SWELL inhibitory activity. Time course of outward (black) and inward (blue) currents in ICL,SWELL upon application of (A) Smod compounds lacking Snot1:ICl,SWELL inhibitory activity, (B) Smod3 with maintained and enhanced activity, (C) Smod4 with maintained activity, and (D) Smod5 with maintained activity. [Figure 3B] Patch clamp screening of Smod compounds for ICL,SWELL inhibitory activity. Time course of outward (black) and inward (blue) currents in ICL,SWELL upon application of (A) Smod compounds lacking Snot1:ICl,SWELL inhibitory activity, (B) Smod3 with maintained and enhanced activity, (C) Smod4 with maintained activity, and (D) Smod5 with maintained activity. [Figure 3C] Patch clamp screening of Smod compounds for ICL,SWELL inhibitory activity. Time course of outward (black) and inward (blue) currents in ICL,SWELL upon application of (A) Smod compounds lacking Snot1:ICl,SWELL inhibitory activity, (B) Smod3 with maintained and enhanced activity, (C) Smod4 with maintained activity, and (D) Smod5 with maintained activity. [Figure 3D] Patch clamp screening of Smod compounds for ICL,SWELL inhibitory activity. Time course of outward (black) and inward (blue) currents in ICL,SWELL upon application of (A) Smod compounds lacking Snot1:ICl,SWELL inhibitory activity, (B) Smod3 with maintained and enhanced activity, (C) Smod4 with maintained activity, and (D) Smod5 with maintained activity. [Figure 4] Dose-response curves plotting the percentage of current (% of control) for increasing concentrations of Smod3, Smod1(+), and Smod1(-). The EC50 for Smod(+) is indicated by the red dashed line, and the EC50 for Smod3 is indicated by the blue dashed line. [Figure 5] Representative representation of the synthesis of Smod1 and the corresponding modifications to the synthesis of Smod compounds. Modifications to the synthetic scheme that can be made to synthesize various compounds described herein are indicated by double arrows. Methods: i) AlCl3, DCM, 5°C to room temperature; ii) 12N HCl; iii) 1) paraformaldehyde, dimethylamine, acetic acid, 85°C; iv) DMF, 85°C; v) H2SO4; vi) KOtBu, butyl iodide; vii) Pyridine-HCl, 195°C; viii) BrCH2CO2Et, K2CO3, DMF, 60°C; ix) 10N NaOH. [Figure 6] Induction of SWELL1 protein by Smod3 and Smod5, but not by vehicle or Snot1, in 3T3-F442A adipocytes. [Figure 7]Representative glucose tolerance test data, area under the curve (AUC), and fasting glucose from mice treated with vehicle, 5 mg / kg / day of Smod3, or Snot1 for 5 days. Smod3, but not Snot1, improves glucose tolerance (measured by area under the curve, AUC) and fasting glucose in HFD T2D mice. N = 5 mice per group. *p<0.05, **p<0.01, ***p<0.001. [Figure 8] Glucose loading in obese T2D mice (16 weeks HFD): before Smod6 (black circles), after Smod6 (5 mg / kg i.p. x 5 days, pink triangles), 4 weeks after i.p. vehicle injection (blue diamonds), and 4 weeks after Smod6 withdrawal (maroon squares). [Figure 9] Glucose loading in obese T2D mice (16 weeks HFD): 4 weeks after intraperitoneal vehicle injection (black circles), 4 weeks after Snot1 (5 mg / kg intraperitoneal x 5 days, blue squares), and 4 weeks after Smod6 (5 mg / kg intraperitoneal x 5 days, maroon triangles). [Figure 10] Cryo-EM structure of the SWELL1 homohexamer with Smod1 / DCPIB within the pore. The negatively charged carboxylate interacts electrostatically with the positively charged arginine (R103) at the pore constriction from SWELL1 / LRRC8a and / or LRRC8b. Figure adapted from Kern et al. eLife (2019). [Figure 11] Docking of Smod1 to SWELL1 using structure PDB ID: 6NZW. (A) Docking using the molecular manipulation environment (MOE) generated a docking pose consistent with the orientation of Smod1 observed in the cryo-EM structure (Figure 8). (B) Docking using the LeadIT software package and SeeSAR generated a higher-scoring binding pose than the pose from the cryo-EM structure, in which Smod1 is flipped 180 degrees. (C) Overlay of the highest-scoring docked poses of Smod1 and SWELL1 from MOE (red) and SeeSAR (yellow). [Figure 12]Patch clamp screening of UIPC-03-099 compound for ICL,SWELL inhibitory activity at 10 μM. [Figure 13] Patch clamp screening of UIPC-03-099 compound for ICL,SWELL inhibitory activity at 5 μM. [Figure 14] Patch clamp screening of UIPC-03-099 compound for ICL,SWELL inhibitory activity at 5 μM. [Figure 15] Patch clamp screening of UIPC-03-099 compound for ICL,SWELL inhibitory activity at 5 μM. [Figure 16] Patch clamp screening of UIPC-03-099 compound for ICL,SWELL inhibitory activity at 1 μM. [Figure 17] 1 shows the reaction scheme for producing compounds SN-401, SN-403, SN-406, SN-407 and SN071. [Figure 18] 1 shows a reaction scheme for producing SN072. [Figure 19] 1 shows a reaction scheme for producing the racemic compound of SN-401. [Figure 20A] Current-voltage plots of ICl,SWELL measured at baseline (Iso, black line) and hypotonic (210 mOsm) stimulation (Hypo, gray line) in non-T2D and T2D mice are shown. [Figure 20B] Current-voltage plots of ICl,SWELL measured at baseline (Iso, black line) and hypotonic (210 mOsm) stimulation (Hypo, gray line) in non-T2D and T2D human cells are shown. [Figure 20C] Shown are the mean inward and outward ICl,SWELL current densities at +100 and -100 mV from non-T2D (n=3 cells) and T2D (n=6 cells) mouse cells. [Figure 20D] Average inward and outward ICl,SWELL current densities at +100 and -100 mV from non-T2D (n=6 cells) and T2D (n=22 cells) human cells are shown. [Figure 20E] Average inward and outward ICl,SWELL current densities at +100 and -100 mV are shown from adipocytes isolated from the visceral fat of lean (n = 7 cells), obese non-T2D (n = 13 cells), and T2D patients (n = 5 cells). Data for lean and obese non-T2D adipocytes replotted from previously reported data in Zhang et al., 2017 for comparison. [Figure 20F] Western blot of SWELL1 protein expression in inguinal adipose tissue isolated from polygenic-T2D KKAY mice compared to the parental control strain KKAa (n=5 each). [Figure 20G] 1 shows a Western blot comparing SWELL1 protein expression in visceral adipose tissue isolated from lean, obese non-T2D, and obese T2D patients, respectively. [Figure 20H] Western blot of SWELL1 protein isolated from cadaveric islets of non-T2D and T2D donors (n=3 each) is shown. [Figure 21A] Western blots detecting SWELL1, pAKT2, AKT2, and -actin in wild-type (WT, black), SWELL1 knockout (KO, light gray), and KO (KO+SWELL1 O / E, dark gray) 3T3-F442A adipocytes (top) overexpressing SWELL1 with adenoviral stimulation for 15 minutes with 0 and 10 nM insulin are shown. The corresponding densitometry ratios of pAKT2 / -actin are shown below (n = 3 independent experiments for each condition). All densitometry measurements are normalized to the value of 0 nM insulin in WT 3T3-F442A preadipocytes, except for the bottom panel. Data are expressed as mean ± SEM. A two-tailed unpaired t-test was used, with *, **, and *** representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 21B]Average inward and outward current densities at +100 and -100 mV from WT (black, n = 5 cells), KO (light gray, n = 4 cells), and KO + SWELL1 O / E (dark gray, n = 4 cells) 3T3-F442A preadipocytes are shown. Data are expressed as mean ± SEM. Two-tailed unpaired t-test was used, with *, **, and *** representing p < 0.05, p < 0.01, and p < 0.001, respectively. [Figure 21C] Western blots comparing the levels of SWELL1, pAKT2, AKT2, and -actin (c) in wild-type (WT, black) and WT (WT+SWELL1 O / E, gray) 3T3-F442A adipocytes overexpressing SWELL1 with 0 and 10 nM insulin stimulation are shown (n = 6 independent experiments for each condition). The corresponding densitometric ratios of pAKT2 / -actin and total AKT2 are shown below. All densitometric measurements are normalized to the value of 0 nM insulin in WT 3T3-F442A preadipocytes, except for the bottom panel. Data are expressed as mean ± SEM. A two-tailed unpaired t-test was used, with *, **, and *** representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 21D] Western blots comparing pAS160, AS160, and -actin levels with 0 and 10 nM insulin stimulation in wild-type (WT, black) and WT (WT+SWELL1 O / E, gray) 3T3-F442A adipocytes overexpressing SWELL1 are shown (n = 6 independent experiments for each condition). Corresponding densitometric ratios of pAS160 / -actin (top right) and total AS160 (bottom right) are also shown. All densitometric measurements are normalized to the value of 0 nM insulin in WT 3T3-F442A preadipocytes, except for the bottom panel. Data are expressed as mean ± SEM. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively, by two-tailed unpaired t-test. [Figure 21E]A cartoon model of homomeric mouse LRRC8a / SWELL1 derived from the cryo-electron microscopy (EM) and X-ray crystallography structure (PDB ID: 6G90#) is shown, along with SN-401 / DCPIB binding in the pore region derived from the cryo-EM structure of DCPIB-bound SWELL1 (PDB ID: 6NZW$, shown as a dimer for illustration) and the chemical structure of SN-401 (top). [Figure 21F] Shown is the time course of Icl,swELL inward and outward currents in HEK-293 cells following hypotonic (210 mOsm) stimulation and inhibition by 10 μM SN-401. [Figure 21G] Western blots detecting SWELL1, pAKT2, and -actin in WT 3T3-F442A preadipocytes upon 0, 3, and 10 nM insulin stimulation (n = 2 independent experiments for each condition, top panel) and the corresponding densitometric ratios of SWELL1 / -actin and pAKT2 / -actin (bottom panel) are shown. All densitometric measurements are normalized to the value of 0 nM insulin in WT 3T3-F442A preadipocytes, except for the bottom panel. Data are expressed as mean ± SEM. Two-tailed unpaired t-tests were used, with *, **, and *** representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 21H] Western blots detecting SWELL1, pAKT2, AKT2, and -actin at 0 and 10 nM insulin in WT and KO 3T3-F442A adipocytes (n=6 independent experiments for each condition) are shown. [Figure 21I] The corresponding densitometry ratios of SWELL1 / -actin from Figure 21H are shown. All densitometry measurements are normalized to the value of 0 nM insulin in WT 3T3-F442A preadipocytes, except for the bottom panel. Data are presented as mean ± SEM. Two-tailed unpaired t-test was used, with *, **, and *** representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 21J]Corresponding densitometry ratios of pAKT / actin (top) and pAKT2 / AKT2 (bottom) from Figure 21H are shown. Densitometry in the top panel is normalized to 0 nM insulin for WT 3T3-F442A preadipocytes. pAKT2 / AKT2 normalization in the bottom panel was performed to 0 nM insulin for WT and 0 nM insulin for KO due to differential expression of total AKT2 in WT and KO. (Deneka et al. (2018) and Kern et al. (2019)). Data are presented as mean ± SEM. A two-tailed unpaired t-test was used, with *, **, and *** representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 21K] Western blots of pAS160, AS160, and -actin expression in WT 3T3-F442A adipocytes stimulated with 0 and 10 nM insulin (n = 3 independent experiments for each condition, left) and the corresponding densitometric ratios of pAS160 / AS160 (right) incubated in either vehicle or 10 μM SN-401 for 96 h are shown. All densitometric measurements are normalized to the value of 0 nM insulin in WT 3T3-F442A preadipocytes, except for the bottom panel. Data are expressed as mean ± SEM. Two-tailed unpaired t-tests were used, with *, **, and *** representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 22A] The chemical structures of SN-401, SN-403, SN-406, SN-407, SN071 and SN072 are shown. [Figure 22B] Time course of ICl,SWELL inward and outward currents upon inhibition by 7 μM SN-401 / SN-406 or 10 μM SN071 / SN072 after hypotonic (210 mOsm) stimulation in HEK-293 cells is shown. [Figure 22C]Figure 1 shows the mean percentage of maximal outward current blocked by SN-401 (n = 6), SN-403 (n = 3), SN-406 (n = 4), SN071 (n = 3), and SN072 (n = 3) at 10 μM (left) and by SN-403 (n = 3), SN-406 (n = 5), and SN-407 (n = 3) at 7 μM (right) in HEK-293 cells. Means shown ± SEM. Two-tailed unpaired t-test was used. *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively. [Figure 22D] Shown is a side view (i) without the protein surface occupying the pore and a top view (ii) with the protein surface of SN-401 (pink sticks) (pink sticks) resolved in a cryo-EM structure adapted from RCSB PDB:6NZZ, where the SN-401 carboxylate group interacts electrostatically with the guanidine group of the R103 residue (cyan sticks), and the SN-401 cyclopentyl and butyl groups do not interact with any channel residues. [Figure 22E] The pose generated for SN-401 by docking into PDB 6NZZ using the Molecular Operation Environment 2016 (MOE) software package is shown. SN-401 is shown as a yellow stick, with and without the molecular surface, and R103, D102, and L101 are shown as cyan sticks. Panel (i) shows a side view without the protein surface, while panel (ii) shows a top view with the protein surface of the upper binding pose of SN-401. The SN-401 carboxylate group interacts with the guanidine group of the R103 residue, and the SN-401 cyclopentyl group occupies a shallow hydrophobic cleft at the interface of the two monomers formed by SWELL1 D102 and L101. [Figure 22F]Poses generated for SN071 by docking into PDB 6NZZ using the Molecular Operation Environment 2016 (MOE) software package are shown. SN071, with and without the molecular surface, is shown as orange sticks, and R103, D102, and L101 are shown as cyan sticks. Panel (i) shows a top-down view of the first binding pose of SN071, which exhibits a potential electrostatic interaction with R103 (dotted circle) but cannot reach and occupy the hydrophobic cleft (black arrow). Panel (ii) shows a top-down view of the second pose of SN071, which has the cyclopentyl group occupying the hydrophobic cleft (dotted circle), but whose carboxylate group cannot reach and interact with R103 (black arrow). [Figure 22G] Poses generated for SN-406 by docking into PDB 6NZZ using the Molecular Operation Environment 2016 (MOE) software package are shown. SN-406 is shown as a yellow stick, with R103, D102, and L101 shown as cyan sticks, with and without the molecular surface. Panel (i) shows a top view of the best binding pose of SN-406, with the carboxylate group interacting with R103, the cyclopentyl group occupying the hydrophobic cleft, and the alkyl side chain of SN-406 interacting with the alkyl side chain of R103. Panel (ii) shows SN-406 shown as a yellow space-filling model. [Figure 23A] Western blots detecting SWELL1 and -actin in 3T3-F442A adipocytes treated with vehicle (n=8), SN-401 (n=10), SN-406 (n=6), or SN072 (n=6) (the SWELL1-inactive SN-401 homolog) at 10 μM for 96 hours, and the corresponding densitometric ratios of SWELL1 / -actin are shown. Data are presented as mean ± SEM. A two-tailed unpaired t-test (compared to vehicle) was used. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively. [Figure 23B]Western blots detecting SWELL1 and -actin in 3T3-F442A adipocytes treated with vehicle (n=6), SN-401 (n=6), SN-406 (n=3), SN071 (n=3) (an inactive SN-401 homolog), or SN072 (n=4) at 1 μM for 96 hours, and the corresponding densitometric ratios of SWELL1 / -actin are shown. Data are presented as mean ± SEM. A two-tailed unpaired t-test (compared to vehicle) was used. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively. [Figure 23C] Immunostaining images showing the localization of endogenous SWELL1 in 3T3-F442A preadipocytes treated with vehicle (n=19), SN-401 (n=21), SN-406 (n=13 at 1 and 10 μM), or SN071 (n=9 at 1 μM and n=13 at 10 μM) at 1 or 10 μM for 48 hours (scale bar - 20 μm), as well as corresponding quantification of SWELL1 membrane versus cytoplasmic localization fractions are shown. Data are presented as mean ± SEM. One-way ANOVA (compared to vehicle) was used. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively. [Figure 23D] Shown are ICl,SWELL inward and outward currents recorded over time from HEK-293 cells preincubated with vehicle, SN-401, SN-406, SN071, or SN072 at 1 μM and then stimulated with hypotonic solution. [Figure 23E] Figure 23D shows the mean outward Icl,swELL current density at +100 mV measured 7 minutes after hypotonic stimulation. Data are presented as mean ± SEM. One-way ANOVA (compared to vehicle) was used. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively. [Figure 23F] Shown are ICl,SWELL inward and outward currents recorded over time from HEK-293 cells preincubated with vehicle, SN-401, SN-406, SN071, or SN072 at 250 nM concentration and then stimulated with hypotonic solution. [Figure 23G] Figure 23F shows the mean outward Icl,swELL current density at +100 mV measured 7 minutes after hypotonic stimulation. Data are presented as mean ± SEM. One-way ANOVA (compared to vehicle) was used. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively. [Figure 23H] Western blots detecting pAKT2, AKT2, and -actin in 3T3-F442A adipocytes treated with vehicle (n=3 for 0 nM insulin, n=5 for 10 nM insulin) or 1 μM SN-401 (n=3 for 0 nM insulin, n=6 for 10 nM insulin) and the corresponding densitometry ratios of pAKT2 / -actin and pAKT2 / AKT2 are shown. Data are presented as mean ± SEM. A two-tailed unpaired t-test (compared to vehicle) was used. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively. [Figure 23I] Western blots detecting SWELL1 and -actin in 3T3-F442A adipocytes treated with vehicle, 1 mM palmitate + vehicle, 1 mM palmitate + 10 μM SN-401, 1 mM palmitate + 10 μM SN-406, or 1 mM palmitate + 10 μM SN072 (n=3 for each condition) are shown, along with the corresponding densitometric ratios of SWELL1 / -actin. Data are presented as mean ± SEM. A two-tailed unpaired t-test (compared to vehicle) was used. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively. [Figure 24A] Western blots detecting SWELL1 protein in visceral fat of C57BL / 6 mice fed a high-fat diet (HFD) and treated with either vehicle or SN-401 (5 mg / kg i.p.) for 21 weeks, and the corresponding densitometry ratios of SWELL1 / -actin (right) are shown (n=6 mice in each group). Means shown ± SEM. Two-tailed unpaired t-test. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively. [Figure 24B] FIG. 1 shows a Western blot comparing SWELL1 protein expression in inguinal adipose tissue of polygenic-T2D KKAY mice treated with SN-401 (5 mg / kg daily intraperitoneally x 14 days) compared to untreated control KKAA and wild-type C57BL / 6 mice. [Figure 24C] Glucose tolerance test (GTT) and insulin tolerance test (ITT) results of 8-week HFD-fed C57BL / 6 mice treated with either vehicle or SN-401 (5 mg / kg i.p.) for 10 days (n=7 mice in each group) are shown. Means shown ± SEM. Two-way ANOVA (p values ​​in the lower corners of the graphs) was used. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively. [Figure 24D] Fasting glucose levels of T2D KKAY mice (n=6) and their control strain KKAa mice (n=3) were compared before and after 4 days of SN-401 (5 mg / kg i.p.) treatment, respectively. Means shown ± SEM. Paired t-test. *, **, and *** indicate p<0.05, p<0.01, and p<0.001, respectively. [Figure 24E] Fasting glucose levels (d), GTT (e), and ITT (f) are shown for T2D KKAY mice (n=6) and their control strain KKAa (n=3) before and after 4 days of SN-401 (5 mg / kg i.p.) treatment, respectively. Means are shown ± SEM. Two-way ANOVA (p values ​​in the lower corners of the graphs) was used. *, **, and *** indicate p<0.05, p<0.01, and p<0.001, respectively. [Figure 24F] Fasting glucose levels (d), GTT (e), and ITT (f) are shown for T2D KKAY mice (n=6) and their control strain KKAa (n=3) before and after 4 days of SN-401 (5 mg / kg i.p.) treatment, respectively. Two-way ANOVA (p values ​​in the lower corners of the graphs) was used. *, **, and *** indicate p<0.05, p<0.01, and p<0.001, respectively. [Figure 24G]Fasting glucose levels (g) are shown for lean mice fed a normal chow diet (RC) treated with either vehicle or SN-401 (5 mg / kg i.p.) for 6 days (n=6 in each group). Means shown ± SEM. Two-tailed unpaired t-test. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively. [Figure 24H] GTT corresponding to fasting glucose levels in Figure 24G is shown for lean mice fed a normal chow diet (RC) treated with either vehicle or SN-401 (5 mg / kg i.p.) for 6 days (n=6 in each group). [Figure 24I] Figure 1 shows fasting glucose levels in HFD-T2D mice treated with either vehicle or SN-401 (5 mg / kg i.p.). Means shown ± SEM. Two-tailed unpaired t-test. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively. [Figure 24J] GTT (16 weeks of HFD, 4 days of treatment) and ITT (18 weeks of HFD, 4 days of treatment) of HFD-T2D mice treated with either vehicle or SN-401 (5 mg / kg i.p.) are shown. Means shown ± SEM. Two-way ANOVA (p values ​​in the lower corners of the graphs) was used. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively. [Figure 24K] Figure 1 shows relative insulin secretion in plasma of HFD-T2D mice (18 weeks of HFD, 4 days of treatment) after intraperitoneal glucose (0.75 g / kg BW) treated with either vehicle (n=3) or SN-401 (n=4, 5 mg / kg i.p.). [Figure 24L]Glucose-stimulated insulin secretion (GSIS) perfusion assays from pancreatic islets isolated from HFD-T2D mice (at 21 weeks) treated with either vehicle (n = 3 mice, and 3 experimental replicates) or SN-401 (n = 3 mice, and 2 experimental replicates, 5 mg / kg i.p.) and their corresponding area under the curve (AUC) comparisons (right) are shown. Means shown ± SEM. Two-tailed unpaired t-test. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively. [Figure 24M] Glucose-stimulated insulin secretion (GSIS) perfusion assays from pancreatic islets isolated from polygenic-T2D KKAY mice treated with either vehicle or SN-401 (5 mg / kg i.p. for 6 days, n=3 in each group, 3 experimental replicates) and their corresponding area under the curve (AUC) comparisons (right) are shown. Means shown ± SEM. Two-tailed unpaired t-test. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively. [Figure 25A] Figure 1 shows the mean glucose infusion rate during a euglycemic-hyperinsulinemic clamp in polygenic-T2D KKAY mice treated with vehicle (n=7) or SN-401 (n=8) for 4 days. Means shown ± SEM. Two-tailed unpaired t-test. Statistical significance is indicated by *, **, and ***, representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 25B] Figure 1 shows hepatic glucose production at baseline and during a euglycemic-hyperinsulinemic clamp in T2D KKAY mice treated with vehicle or SN-401 (n=9 in each group). Means shown ± SEM. Two-tailed unpaired t-test. Statistical significance is indicated by *, **, and ***, representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 25C]Glucose uptake determined from 2-deoxyglucose (2-DG) uptake in inguinal and gonadal white adipose tissue (iWAT) and heart during trace clamp in T2D KKAY mice treated with vehicle or SN-401 (n=9 in each group) is shown. Means shown ± SEM. Two-tailed unpaired t-test. Statistical significance is indicated by *, **, and ***, representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 25D] Glucose uptake into glycogen as determined from 2-DG uptake in liver (n=9 for vehicle, n=8 for SN-401), adipose (iWAT, n=7 for vehicle, n=6 for SN-401), and gastrocnemius muscle (n=7 for vehicle, n=6 for SN-401) during clamping in T2D KKAY mice is shown. Means shown ± SEM. Two-tailed unpaired t-test. Statistical significance is denoted by *, **, and ***, representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 25E] Schematic diagram of the treatment protocol for C57BL / 6 mice injected with either vehicle or SN-401 (n=6 in each group) while HFD-fed. [Figure 25F] Liver mass (left) and normalized ratio to body weight (right) of HFD-T2D mice after treatment with either vehicle or SN-401 (5 mg / kg i.p.) are shown. Means shown ± SEM. Two-tailed unpaired t-test. Statistical significance is indicated by *, **, and ***, representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 25G] Corresponding hematoxylin and eosin stained liver sections are shown. Scale bar - 100 μm. [Figure 25H] Liver triglycerides (6 mice in each group) are shown. Means shown ± SEM. Two-tailed unpaired t-test. Statistical significance is indicated by *, **, and ***, representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 25I]Histological scores for steatosis, lobular inflammation, hepatocellular injury (ballooning), and the NAFLD-activity score (NAS) are shown, which integrates the scores for steatosis, inflammation, and ballooning. Means are shown ± SEM. Two-tailed unpaired t-test. Statistical significance is indicated by *, **, and ***, representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 26A] Figure 1 shows fasting glucose levels, GTT, and their corresponding areas under the curve (AUC) of 8-week HFD-fed mice treated with either SWELL1-inactive SN-071 or SWELL1-active SN-403 (5 mg / kg i.p.) for 4 days (n=5 in each group). Data are presented as mean ± SEM. Two-way ANOVA for GTT. Two-tailed unpaired t-tests were used for FG, GTT AUC, GSIS AUC, and HOMA-IR. Statistical significance is indicated by *, **, and ***, representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 26B] Figure 1 shows fasting glucose levels, GTT, and corresponding AUC of mice fed a 12-week HFD before and after treatment with SN-406 (5 mg / kg i.p.) for 4 days (n=5 in each group). Two-way ANOVA for GTT. Paired t-test for GTT and GTT AUC. Statistical significance is indicated by *, **, and ***, representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 26C] Figure 1 shows the GTT and corresponding AUC of 12-week HFD-fed mice treated with either SWELL1-inactive SN-071 or SWELL1-active SN-406 (5 mg / kg i.p.) for 4 days (n=7 in each group). Data are expressed as mean ± SEM. Two-tailed unpaired t-tests were used for FG, GTT AUC, GSIS AUC, and HOMA-IR. Two-way ANOVA in a-c and f for GTT statistical significance is denoted by *, **, and ***, which represent p<0.05, p<0.01, and p<0.001, respectively. [Figure 26D]The corresponding HOMA-IR index for the data shown in Figure 26C is shown. Data are expressed as mean ± SEM. Two-tailed unpaired t-tests were used for FG, GTT AUC, GSIS AUC, and HOMA-IR. Statistical significance is indicated by *, **, and ***, representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 26E] Glucose-stimulated insulin secretion (GSIS) perfusion assay of pancreatic islets isolated from mice is shown in 26C. Data are expressed as mean ± SEM. Two-tailed unpaired t-tests were used for FG, GTT AUC, GSIS AUC, and HOMA-IR. Statistical significance is indicated by *, **, and ***, which represent p<0.05, p<0.01, and p<0.001, respectively. [Figure 26F] Figure 1 shows GTT and corresponding AUC in polygenic-T2D KKAY mice treated with either SWELL1-inactive SN-071 (n=5) or SWELL1-active SN-407 (n=6) (5 mg / kg i.p.) for 4 days. Data are expressed as mean ± SEM. Two-tailed unpaired t-tests were used for FG, GTT AUC, GSIS AUC, and HOMA-IR. Two-way ANOVA in a-c and f for GTT. Statistical significance is denoted by *, **, and ***, representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 26G] Glucose-stimulated insulin secretion (GSIS) perfusion assay of pancreatic islets isolated from mice is shown in 26F. Data are expressed as mean ± SEM. Two-tailed unpaired t-tests were used for FG, GTT AUC, GSIS AUC, and HOMA-IR. Statistical significance is indicated by *, **, and ***, representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 27A] Current-voltage plots of lc1,swELL measured in 3T3-F442A preadipocytes WT at baseline (Iso, black line) and hypotonic (Hypo, red line) stimulation, respectively, are shown. [Figure 27B]Current-voltage plots of lc1,swELL measured in 3T3-F442A preadipocyte KO at baseline (Iso, black line) and hypotonic (Hypo, red line) stimulation, respectively, are shown. [Figure 27C] Adenoviral overexpression of SWELL1 in KO (KO+SWELLL1 O / E) at baseline (Iso, black line) and hypotonic (Hypo, red line) stimulation, respectively, is shown. [Figure 27D] Immunostaining images showing the localization of endogenous or overexpressed SWELL1 with anti-Flag or anti-SWELL1 antibodies (scale bar -20 μm) are shown. [Figure 27E] Validation of SWELL1 antibody in WT 3T3-F442A compared to SWELL1 KO preadipocytes (scale bar - 20 μm) is shown, revealing a punctate pattern of endogenous SWELL1 localization (insert). [Figure 28] Figure 1 shows the relative mRNA expression of LRRC8 family members to GAPDH as assessed by qPCR (n=3 each) for 3T3 F-442A preadipocytes treated with vehicle or SN-401 at 10 μM for 96 hours. Data are presented as mean ± SEM. A two-tailed unpaired t-test was used, with *, **, and *** representing p<0.05, p<0.01, and p<0.001, respectively. [Figure 29A] Chemical structure of SN-401 / DCPIB (top) and time course of lc1,swELL inward and outward currents (bottom) upon inhibition by 7 μM SN-401 after hypotonic (210 mOsm) stimulation in HEK-293 cells. [Figure 29B] The chemical structure of SN-403 and the time course of lc1,swELL inward and outward currents (bottom) upon inhibition by 7 μM SN-403 after hypotonic (210 mOsm) stimulation in HEK-293 cells are shown. [Figure 29C] The chemical structure of SN-407 and the time course of lc1,swELL inward and outward currents (bottom) upon inhibition by 7 μM SN-407 after hypotonic (210 mOsm) stimulation in HEK-293 cells are shown. [Figure 29D]The binding pose for SN072 reveals that the carboxylate group can reach and interact electronically with R103, but in the absence of the butyl group, the cyclopentyl ring cannot be oriented to occupy the hydrophobic cleft without introducing excessive structural strain on the carbon connecting the core and the cyclopentyl ring. [Figure 29E] An alternative view of the best binding pose of SN-406 is shown, with the carboxylate group interacting with R103, the cyclopentyl group occupying the hydrophobic cleft, and the alkyl side chain of SN-406 interacting with the alkyl side chain of R103. [Figure 29F] Panel (i) shows a side view without the protein surface, and panel (ii) shows a top view with the protein surface in the upper binding pose of SN-403. The carboxylate group interacts with the guanidine group of residue R103 (solid circle), and the cyclopentyl group occupies a shallow hydrophobic cleft at the interface of the two monomers formed by D102 and L101 (dotted circle). [Figure 29G] (i) Side view without protein surface and (ii) top view with the protein surface in the top binding pose of SN-407 are shown, with the carboxylate group interacting with R103 (solid circle), the cyclopentyl group occupying the hydrophobic cleft (dotted circle), and the alkyl side chain of SN-407 interacting with the alkyl side chain of R103. [Figure 29H] Time course of ICl,SWELL inward and outward currents upon inhibition by 7 μM SN-406 after hypotonic stimulation in WT (left) and R103E mutant overexpressing (right) HEK-293 cells, respectively. [Figure 29I] Figure 1 shows the average percentage of maximal outward current blocked by SN-406 at 10 μM (left) and 7 μM (right) in WT (n = 4 at 10 μM and n = 5 at 7 μM) and R103E mutant (n = 5 at 10 μM and n = 6 at 7 μM) overexpressing HEK-293 cells. Data are presented as mean ± SEM. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively, using a two-tailed unpaired t-test. [Figure 30-1] Shown are immunostaining images showing the localization of endogenous SWELL1 in WT 3T3-F442A preadipocytes treated with vehicle or SN-401, SN-406, and SN071 at 1 or 10 μM for 48 hours (scale bar − 20 μm). [Figure 30-2] Shown are immunostaining images showing the localization of endogenous SWELL1 in WT 3T3-F442A preadipocytes treated with vehicle or SN-401, SN-406, and SN071 at 1 or 10 μM for 48 hours (scale bar − 20 μm). [Figure 31A] Figure 1 shows fasting glucose levels in C57BL / 6 lean mice fed a normal chow diet and treated with either vehicle or SN-401 (5 mg / kg i.p.) for 10 days (n=7 males in each group). Two-tailed unpaired t-tests were used for FG and AUC. [Figure 31B] Figure 1 shows GTT in C57BL / 6 lean mice fed a normal chow diet treated with either vehicle or SN-401 (5 mg / kg i.p.) for 10 days (n=7 males in each group). Data are presented as mean ± SEM. Two-way ANOVA was used for GTT and ITT. Statistical significance is represented by *, **, and ***, representing p<0.05, p<0.01, and p<0.001, respectively, and "ns" indicates that the difference was not significant. [Figure 31C] Figure 1 shows the ITT of C57BL / 6 lean mice fed a normal chow diet treated with either vehicle or SN-401 (5 mg / kg i.p.) for 10 days (n = 7 males in each group). Data are presented as mean ± SEM. Two-way ANOVA was used for b-d and i, GTT and ITT. Statistical significance is represented by *, **, and ***, representing p<0.05, p<0.01, and p<0.001, respectively, and "ns" indicates that the difference was not significant. [Figure 31D]Figure 1 shows GTT in HFD-T2D mice (8-week HFD) treated with either vehicle (n=5 males) or SN-401 (5 mg / kg i.p., n=4 males) for 8 weeks. Data are presented as mean ± SEM. Two-way ANOVA was used for GTT and ITT. Statistical significance is represented by *, **, and ***, which represent p<0.05, p<0.01, and p<0.001, respectively, and "ns" indicates that the difference was not significant. [Figure 31E] 1 shows the in vivo pharmacokinetics of SN-401 administered intraperitoneally (ip) at 5 mg / kg. [Figure 31F] 1 shows the in vivo pharmacokinetics of SN-406 administered intraperitoneally (ip) at 5 mg / kg. [Figure 31G] 1 shows the in vivo pharmacokinetics of SN-401 administered by oral gavage (po) at 5 mg / kg. [Figure 31H] 1 shows the in vivo pharmacokinetics of SN-406 administered by oral gavage (po) at 5 mg / kg. [Figure 31I] Fasting glucose levels, GTT, and AUC of HFD-T2D mice (10 weeks on HFD) treated with either vehicle (n = 6 males) or SN-401 (5 mg / kg orally, n = 7 males) for 5 days are shown. Data are expressed as mean ± SEM. Two-way ANOVA was used for b-d, and i, GTT and ITT. Two-tailed unpaired t-test was used for FG and AUC. Statistical significance is represented by *, **, and ***, representing p<0.05, p<0.01, and p<0.001, respectively, and "ns" indicates that the difference was not significant. [Figure 32A] Glucose uptake determined from 2-DG uptake in brown fat, extensor digitorum longus (EDL), soleus, and gastrocnemius muscles harvested under clamps in KKAY mice treated with vehicle or SN-401 (n=9 in each group, 5 mg / kg i.p.) for 4 days is shown. Data are presented as mean ± SEM. A two-tailed unpaired t-test was used for analysis. "ns" indicates that the difference was not significant. [Figure 32B-1]Images of hematoxylin and eosin stained liver tissue sections from HFD-T2D mice treated with either vehicle or SN-401 (5 mg / kg i.p.) are shown. Scale - (10X: 100 μm and 20X: 50 μm). [Figure 32B-2] Images of hematoxylin and eosin stained liver tissue sections from HFD-T2D mice treated with either vehicle or SN-401 (5 mg / kg i.p.) are shown. Scale - (10X: 100 μm and 20X: 50 μm). [Figure 32B-3] Images of hematoxylin and eosin stained liver tissue sections from HFD-T2D mice treated with either vehicle or SN-401 (5 mg / kg i.p.) are shown. Scale - (10X: 100 μm and 20X: 50 μm). [Figure 32B-4] Images of hematoxylin and eosin stained liver tissue sections from HFD-T2D mice treated with either vehicle or SN-401 (5 mg / kg i.p.) are shown. Scale - (10X: 100 μm and 20X: 50 μm). [Figure 33A] Western blots from WT and SWELL1 KO C2C12 (left) and primary myotubes (right) are shown. [Figure 33B] Current-voltage curves from WT and SWELL1 KO C2C12 myoblasts measured during voltage ramps of -100 to +100 mV + / - isotonic and hypotonic (210 mOsm) solutions are shown. [Figure 33C] Brightfield images merged with fluorescent images of differentiated WT and SWELL1 KO C2C12 myotubes (left, center) and primary skeletal muscle cells (right). DAPI stains cell nuclei blue (center). Red is the fluorescence of the mCherry reporter via adenoviral transduction. Scale bar: 100 μm. Average myotube surface area measured from WT (n = 21) and SWELL1 KO (n = 21) C2C12 myotubes (left) and WT (n = 22) and SWELL1 KO (n = 15) primary skeletal myotubes (right). Fusion index (% multinucleated cells) measured from WT (n = 5 fields) and SWELL1 KO (n = 5 fields) C2C12 (shown below representative images). [Figure 33D] A heatmap of the top 17 differentially expressed genes in WT vs. SWELL1 KO C2C12 myotubes derived from RNA sequencing is shown. [Figure 33E] Reads per million kilobases for selected myogenic differentiation genes (n=3 each) are shown. [Figure 33F] IPA canonical pathway analysis of significantly regulated genes in SWELL1 KO C2C12 myotubes compared to WT. n=3 for each group. For analysis using IPA, an FPKM cutoff of 1.5, a fold change of ≥1.5, and a false discovery rate of <0.05 were used for significantly differentially regulated genes. Statistical significance between values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent mean ± sem. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. n=3 independent experiments. [Figure 34A] Western blots of SWELL1, pAKT2, AKT2, pAS160, AS160, pAMPK, AMPK, pFoxO1, FoxO1, and β-actin in WT and SWELL1 KO C2C12 myotubes upon insulin stimulation (10 nM) are shown. [Figure 34B] Western blots of SWELL1, AKT2, pAKT2, pAS160, pAKT1, AKT1, and GAPDH in WT (Ad-CMV-mCherry) and SWELL1 KO (Ad-CMV-Cre-mCherry) primary skeletal myotubes after insulin stimulation (10 nM) are shown. [Figure 34C] Densitometric quantification of proteins shown on Western blots normalized to β-actin is shown. Statistical significance between the values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent mean ± sem. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. n=3 independent experiments. [Figure 34D]Densitometric quantification of proteins shown on Western blots normalized to GAPDH is shown. Statistical significance between values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent mean ± sem. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. n=3 independent experiments. [Figure 34E] Gene expression analysis of insulin signaling-related genes AKT2, FOXO3, FOXO4, FOXO6, and GLUT4 in WT and SWELL1 KO C2C12 myotubes. Statistical significance between the values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent mean ± s.e.m. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. n=3 independent experiments. [Figure 35A] Brightfield images of differentiated WT, SWELL1 KO, and SWELL1 KO+SWELL1 O / E C2C12 myotubes are shown. Scale bar: 100 μm. [Figure 35B] Quantification of mean myotube surface area in WT (n = 35), SWELL1 KO C2C12 (n = 26), and SWELL1 KO + SWELL1 O / E C2C12 (n = 45) cells is shown. Statistical significance between the indicated groups was calculated using one-way ANOVA with Tukey's multiple comparison test. Error bars represent mean ± s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. n = 3 independent experiments. [Figure 35C] Shown are SWELL1, AKT2, pAKT2, pAS160, pAKT1, AKT1, pP70S6K, P70S6K, pS6K, pERK1 / 2, ERK1 / 2, β-actin, and GAPDH Western blots from WT, SWELL1 KO, and SWELL1 KO+SWELL1 O / E C2C12 myotubes. [Figure 35D]Densitometric quantification of proteins shown on Western blots normalized to β-actin and GAPDH, respectively. Statistical significance between the indicated groups was calculated using one-way ANOVA with Tukey's multiple comparison test. Error bars represent mean ± sem. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. n=3 independent experiments. [Figure 36A] Western blots of SWELL1, AKT2, pAKT2, pAKT1, pAS160, pERK1 / 2, ERK1 / 2, and β-actin in WT and SWELL1 KO myotubes in response to 15 min of 0% and 5% static stretch are shown. [Figure 36B] Densitometric quantification of each signaling protein relative to β-actin is shown. Statistical significance between indicated groups was calculated using one-way ANOVA with Tukey's multiple comparison test. Error bars represent mean ± sem. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. n=3 independent experiments. [Figure 37A] SWELL1-3xFlag overexpressed in C2C12 cells followed by immunoprecipitation (IP) with Flag antibody. Western blot of Flag, SWELL1, GRB2, and GAPDH. IgG served as a negative control. [Figure 37B] Western blot of GRB2 is shown to verify GRB2 knockdown efficiency in SWELL1 KO / GRB2 knockdown (Ad-shGRB2-GFP) compared with WT C2C12 (Ad-shSCR-GFP) and SWELL1 KO (Ad-shSCR-GFP). Densitometry quantification of GRB2 knockdown relative to GAPDH (right). Statistical significance between the indicated groups was calculated using one-way ANOVA with Tukey's multiple comparison test. Error bars represent mean ± s.e.m. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. n=3 independent experiments. [Figure 37C]Fluorescence images of WT C2C12 / shSCR-GFP, SWELL1 KO / shSCR-GFP, and SWELL1 KO / shGRB2-GFP myotubes are shown. Scale bar: 100 μm. [Figure 37D] Quantification of mean myotube area for WT C2C12 / shSCR-GFP (n=25), SWELL1 KO / shSCR-GFP (n=28), and SWELL1 KO / shGRB2-GFP (n=24) is shown. Statistical significance between the indicated groups was calculated using one-way ANOVA with Tukey's multiple comparison test. Error bars represent mean ± s.e.m. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. n=3 independent experiments. [Figure 37E] Figure 1 shows the relative mRNA expression of selected myogenic differentiation genes in SWELL1 KO / shSCR and SWELL1 KO / shGRB2 compared to WT C2C12 / shSCR (n = 3 each), as well as the relative mRNA expression of SWELL1 KO / shGRB2 compared to SWELL1 KO / shSCR. Statistical significance between the indicated groups was calculated using one-way ANOVA with Tukey's multiple comparison test. Error bars represent the mean ± s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. n = 3 independent experiments. [Figure 37F] Shown is the fold change in mRNA in KO shGRB2 compared to KO cells with preserved GRB2 expression. [Figure 38A] Schematic diagram of Cre-mediated recombination of loxP sites flanking exon 3 using muscle-specific Myf5-Cre mice to generate skeletal muscle-targeted SWELL1 KO mice. [Figure 38B]Western blots of gastrocnemius muscle proteins isolated from WT and Myf5-Cre, SWELL1fl / fl (Myf5 KO) mice are shown. Liver samples from Myf5 KO and C2C12 cell lysates were used as positive controls for SWELL1. The Coomassie gel below serves as a loading control for skeletal muscle proteins. Densitometric quantification of SWELL1 deletion in skeletal muscle from Myf5 KO mice (n=3) compared to WT (n=3, SWELL1fl / fl) (right). Statistical significance between the values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent the mean ± sem. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 38C] NMR measurements of lean mass (%) and absolute fat mass are shown for WT (n = 11) and Myf5 KO (n = 7) mice. Statistical significance between the values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent the mean ± s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. [Figure 38D] Absolute muscle mass of freshly isolated muscle groups from WT (n = 3) and Myf5 KO (n = 4) mice is shown. Statistical significance between the values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent the mean ± s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. [Figure 38E]Hematoxylin and eosin staining of tibialis muscle from WT and Myf5 KO mice fed a normal chow diet for 28 weeks is shown (top). Scale bar: 100 μm. Bottom: ImageJ-converted image highlights the distinct surface boundaries of myotubes. Inset: Enlarged image shows smaller fiber size in Myf5 KO muscle tissue. Quantification of the average cross-sectional area of ​​muscle fibers from WT (n = 300) and Myf5 KO (n = 300) mice from 10–12 different fields of view (right). Statistical significance between the values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent the mean ± s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. [Figure 39A] Exercise treadmill tolerance testing of Myf5 KO mice (n = 14) compared to WT littermates (n = 15) is shown. Statistical significance between values ​​was calculated using a two-tailed Student's t-test. Error bars represent mean ± s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. [Figure 39B] Hang time in the reversal test for Myf5 KO (n = 8) and WT (n = 9) mice is shown. Statistical significance between the values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent the mean ± s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. [Figure 39C] Park values ​​of ex vivo isometric tetanic tension of soleus muscles isolated from Myf5 KO (n = 7) compared to WT (n = 7) mice are shown. Statistical significance between the values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent the mean ± s.e.m. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 39D]Figure 1 shows the ex vivo time to fatigue of soleus muscles isolated from Myf5 KO (n = 7) compared to WT (n = 7) mice. Statistical significance between the values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent the mean ± s.e.m. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 39E] Figure 1 shows ex vivo half-relaxation times of soleus muscles isolated from Myf5 KO (n = 7) mice compared to WT (n = 7) mice. Statistical significance between values ​​was calculated using a two-tailed Student's t-test. Error bars represent mean ± s.e.m. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 39F] Quantification of oxygen consumption rate (OCR) in WT and SWELL1 KO primary myotubes + / - insulin stimulation (10 nM) (n = 6 independent experiments), as well as basal OCR, OCR after oligomycin, OCR after FCCP, and OCR after antimycin A, is shown. Statistical significance between the indicated values ​​was calculated using a two-tailed Student's t-test. Error bars represent the mean ± s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. [Figure 39G] ATP-linked respiration is shown as baseline intracellular OCR minus post-oligomycin OCR. Statistical significance between values ​​was calculated using a two-tailed Student's t-test. Error bars represent mean ± s.e.m. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 39H] Quantification of extracellular acidification rate (ECAR), as well as basal OCR, OCR after oligomycin, OCR after FCCP, and OCR after antimycin A, in WT and SWELL1 KO primary myotubes + / - insulin stimulation (10 nM) (n = 6 independent experiments) is shown. Statistical significance between the indicated values ​​was calculated using a two-tailed Student's t-test. Error bars represent the mean ± s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. [Figure 40A] Glucose and insulin tolerance tests of WT (n=11) and Myf5 KO (n=10) mice fed a chow diet were performed using a two-way ANOVA (p values ​​in the bottom corners of the graphs). [Figure 40B] NMR measurements of percent fat mass and absolute fat mass are shown for WT (n = 11) and Myf5 KO (n = 7) mice. Statistical significance was calculated using a two-tailed Student's t-test. Error bars represent mean ± s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001. [Figure 40C] Body weights of WT (n = 11) and Myf5 KO (n = 7) mice fed a normal chow diet are shown. Statistical significance was calculated using a two-tailed Student's t-test. Error bars represent the mean ± s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001. [Figure 40D] Glucose tolerance test of WT (n = 8) and Myf5 KO (n = 7) mice fed a HFD for 16 weeks after 14 weeks of age is shown. Two-way ANOVA was used with p values ​​in the lower corners of the graphs. On the right, the corresponding area under the curve (AUC) for glucose tolerance in WT and Myf5 KO mice is shown. Statistical significance was calculated using a two-tailed Student's t-test. Error bars represent the mean ± sem. *, P < 0.05; **, P < 0.01; ***, P < 0.001. [Figure 40E] Figure 1 shows insulin tolerance tests of WT (n = 5) and Myf5 KO (n = 4) mice fed a HFD for 18 weeks after 14 weeks of age. Two-way ANOVA was used with p values ​​in the lower corners of the graphs. The right shows the corresponding area under the curve (AUC) for insulin resistance in WT and Myf5 KO mice. Statistical significance was calculated using a two-tailed Student's t-test. Error bars represent the mean ± sem. *, P < 0.05; **, P < 0.01; ***, P < 0.001. [Figure 41]Differentially expressed glucose and glycogen metabolism-related genes are shown after RNA-seq analysis of C2C12 WT and SWELL1 KO myotubes (n=3 each). Statistical significance between the values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent mean ± s.e.m. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 42A] NMR measurements of fat mass (%) and lean mass (%) are shown for WT (n=8) and Myf5 KO (n=7) mice fed a HFD (16 weeks) after 14 weeks of age. [Figure 42B] The body weights of WT (n=8) and Myf5 KO (n=7) mice are shown. [Figure 43A] A schematic diagram of Cre-mediated recombination of loxP sites flanking exon 3 using muscle-specific Myl1-Cre mice to generate skeletal muscle-targeted SWELL1 KO mice (Myl1-Cre, SWELL1fl / fl, Myl1 KO) is shown. [Figure 43B] PCR bands of SWELL1 recombination in Myl1 KO mice from isolated tissues are shown. [Figure 43C] Glucose tolerance test of WT (n = 6) and Myl1KO (n = 6) mice fed a chow diet for 14 weeks. Fasting glucose levels of WT and Myl1KO mice (right). Statistical significance between the values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent the mean ± s.e.m. *, P<0.05. [Figure 43D] Exercise treadmill tolerance test of Myl1KO (n=6) compared to WT (n=6) littermates is shown. Statistical significance between the values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent the mean ± s.e.m. *, P<0.05. [Figure 43E]Figure 1 shows epithelial (eWAT) and inguinal (iWAT) fat mass normalized to body weight (BM) isolated from Myl1 KO (n = 5) and WT (n = 4) mice. Statistical significance between the values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent the mean ± s.e.m. *, P < 0.05. [Figure 43F] Skeletal muscle mass normalized to body weight (BM) isolated from Myl1 KO (n = 5) and WT (n = 4) mice is shown. Statistical significance between the values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent the mean ± s.e.m. *, P < 0.05. [Figure 43G] The body weights of Myl1 KO (n = 5) and WT (n = 4) mice fed a normal chow diet are shown. Statistical significance between the values ​​shown was calculated using a two-tailed Student's t-test. Error bars represent the mean ± s.e.m. *, P<0.05. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention is directed to various polycyclic compounds and various methods of using these compounds for treating a variety of conditions in subjects in need thereof, including insulin sensitivity, obesity, diabetes, nonalcoholic fatty liver disease, metabolic disease, hypertension, stroke, vascular tone, systemic and / or pulmonary arterial blood pressure, and / or blood flow. Various neurological disorders, infertility problems, muscle disorders, and immune deficiencies can also be treated with these compounds.

[0020] In various embodiments, compounds of the present invention include compounds of formula (I) and salts thereof:

[0021] [ka]

[0022] During the ceremony, R 1 and R 2are each independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; R 3 is -YC(O)R 4 , -ZN(R 5 )(R 6 ), or -ZA, R 4 is hydrogen, substituted or unsubstituted alkyl, -OR 7 , or -N(R 8 )(R 9 ) and X 1 and X 2 are each independently hydrogen, substituted or unsubstituted alkyl, halo, -OR 10 , or -N(R 11 )(R 12 ) and R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are each independently hydrogen or substituted or unsubstituted alkyl; Y and Z are each independently a substituted or unsubstituted carbon-containing moiety having at least two carbon atoms; A is at least one nitrogen heteroatom, boronic acid, or

[0023] [ka]

[0024] and a substituted or unsubstituted 5- or 6-membered heterocycle having the formula: n is 1 or 2.

[0025] In various embodiments, R 1 or R 2In a further embodiment, at least one of R is a substituted or unsubstituted straight or branched chain alkyl having at least two carbon atoms. 1 is hydrogen or C1-C6 alkyl. For example, in some embodiments, R 1 is butyl. In various embodiments, R 2 is cycloalkyl (e.g., cyclopentyl).

[0026] In various embodiments, R 1 and R 2 teeth,

[0027] [ka]

[0028] is selected from the group consisting of:

[0029] In various embodiments, R 3 is -YC(O)R 4 In some embodiments, R 3 is -ZN(R 5 )(R 6 In a further embodiment, R 3 is -ZA.

[0030] As described above, A can be a substituted or unsubstituted 5- or 6-membered heterocyclic ring having at least one nitrogen heteroatom. In some embodiments, A is a substituted or unsubstituted 5- or 6-membered heterocyclic ring having at least two, three, or four nitrogen heteroatoms. In some embodiments, A is a substituted or unsubstituted 5- or 6-membered heterocyclic ring having at least one nitrogen heteroatom and at least one other heteroatom selected from oxygen or sulfur. In various embodiments, A is a boronic acid or

[0031] [ka]

[0032] It could be.

[0033] In various embodiments, A is

[0034] [ka]

[0035] is.

[0036] In certain embodiments, A is

[0037] [ka]

[0038] is.

[0039] In certain embodiments, R 3 teeth,

[0040] [ka]

[0041] is selected from the group consisting of:

[0042] In various embodiments, R 4 -OR 7 or -N(R 8 )(R 9 )

[0043] In various embodiments, X 1 and X 2 are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or halo. 1 and X 2 are each independently C1-C6 alkyl, fluoro, chloro, bromo, or iodo. 1 and X 2are each independently methyl, fluoro, or chloro.

[0044] In various embodiments, R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are each independently hydrogen or alkyl. For example, in some embodiments, R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are each independently hydrogen or C1-C3 alkyl.

[0045] In various embodiments, Y and Z are each independently a substituted or unsubstituted alkylene having 2 to 10 carbons, a substituted or unsubstituted alkenylene having 2 to 10 carbons, or a substituted or unsubstituted arylene. In some embodiments, Y and Z are each independently an alkylene having 2 to 10 carbons, an alkenylene having 2 to 10 carbons, or a phenylene. Y and Z can each independently be a cycloalkylene having 4 to 10 carbons. In certain embodiments, Y is an alkylene or alkenylene having 3 to 8 carbons or 3 to 7 carbons. For example, Y can be an alkylene or any alkenylene having 4 carbons. In further embodiments, Z is an alkylene having 2 to 4 carbons. For example, Z can be an alkylene having 3 or 4 carbons.

[0046] In various embodiments, Y or Z is

[0047] [ka]

[0048] may be selected from the group consisting of:

[0049] In various embodiments, when Y is alkylene having 2 to 3 carbons, X 1 and X 2 and R are each fluoro or each substituted or unsubstituted alkyl (e.g., methyl or ethyl). In some embodiments, Y is not an alkylene having 3 carbons. In certain embodiments, R 7 is not hydrogen or C1-C6 alkyl. 1 and / or X 2 is not halo. In certain embodiments, X 1 and / or X 2 is not chloro. In some embodiments, R 1 and / or R 2 is not alkyl.

[0050] According to embodiments described herein, the compound of formula (I) is

[0051] [ka]

[0052] may be selected from the group consisting of:

[0053] Various compounds of formula (I) can advantageously regulate or inhibit SWELL1 channel. In certain embodiments, compounds of formula (I) have higher potency in regulating or inhibiting SWELL1 channel than equivalent amounts of DCPIB (4[2[butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid). Therefore, they can be used to treat conditions and diseases associated with impaired SWELL1 activity.

[0054] Various embodiments of the present invention include methods for increasing insulin sensitivity and / or treating obesity, diabetes (e.g., type 1 or type 2 diabetes), non-alcoholic fatty liver disease, metabolic disease, hypertension, stroke, vascular tone, and systemic and / or pulmonary arterial pressure and / or blood flow in a subject in need thereof. Various embodiments of the present invention also include methods for treating immune deficiency or infertility caused by insufficient or inappropriate SWELL1 activity in a subject in need thereof. In various embodiments, the immune deficiency can include agammaglobulinemia. In a further embodiment, the infertility can be, for example, male infertility caused by abnormal sperm development due to insufficient or inappropriate SWELL1 activity. Various embodiments of the present invention also include methods for treating or restoring athletic performance and / or improving muscle endurance. In a further embodiment, a method for treating a muscle disorder in a subject in need thereof is provided. The muscle disorder can include skeletal muscle atrophy. Because the SWELL1-LRRC8 complex also regulates myogenesis, methods are also provided for regulating myogenic differentiation in myotubes and insulin-P13K-AKT-AS160, ERK1 / 2 and mTOR signaling. Generally, these methods include administering a therapeutically effective amount of a compound of formula (I) to a subject.

[0055] In various methods described herein, administration of the compound is sufficient to upregulate the expression of SWELL1 or alter the expression of a SWELL1-associated protein. In some embodiments, administration of the compound is sufficient to stabilize the SWELL1-LRRC8 channel complex or a SWELL1-associated protein. In further embodiments, administration of the compound is sufficient to promote membrane trafficking and activity of the SWELL1-LRRC8 channel complex or a SWELL1-associated protein. In some embodiments, the SWELL1-associated protein is selected from the group consisting of LRRC8, GRB2, Cav1, IRS1, or IRS2. In various methods described herein, administration of the compound is sufficient to enhance SWELL1-mediated signal transduction.

[0056] According to various methods of the present invention, a pharmaceutical composition comprising a compound of Formula (I) is administered to a subject in need thereof. The pharmaceutical composition can be administered by a route including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracerebroventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. In various embodiments, administration is selected from the group consisting of oral, intranasal, intraperitoneal, intravenous, intramuscular, rectal, and transdermal.

[0057] Determining a therapeutically effective dose of any one or more of the compounds described herein is within the capabilities of those skilled in the art. A therapeutically effective amount refers to the amount of active ingredient that produces the desired result. The exact dosage will be determined by the practitioner in light of factors related to the subject requiring treatment. Dosage and administration will be adjusted to provide sufficient levels of the active ingredient or maintain the desired effect. Factors that may be considered include the severity of the disease state, the subject's general health, the subject's age, weight, and sex, diet, administration time and frequency, drug combination(s), reaction sensitivities, and tolerance / response to therapy. Long-acting pharmaceutical compositions may be administered every 3-4 days, every week, or once every two weeks, depending on the half-life and clearance rate of the particular formulation.

[0058] Typically, the usual dosage of a compound can vary from about 0.05 to about 100 mg per kg of body weight, depending on the route of administration. Guidance regarding specific dosages and delivery methods is provided in the literature and is generally available to practitioners in the art. Generally, a daily oral dose ranging from about 0.1 mg to about 75 mg, about 0.5 mg to about 50 mg, or about 1 mg to about 25 mg per kg of body weight is administered. The active ingredient can be administered in a single dose per day, or alternatively, in divided doses (e.g., twice daily, three times daily, four times daily, etc.). Generally, lower doses can be administered when parenteral routes are used. Thus, for example, for intravenous administration, doses ranging from about 0.05 mg to about 30 mg, about 0.1 mg to about 25 mg, or about 0.1 mg to about 20 mg per kg of body weight can be used.

[0059] Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers known in the art in dosages suitable for oral administration. Such carriers allow pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for ingestion by a subject. In certain embodiments, compositions are formulated for parenteral administration. Further details on formulation and administration techniques can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., Easton, Pa., which is incorporated herein by reference). After pharmaceutical compositions are prepared, they can be placed in appropriate containers and labeled for the treatment of the indicated condition. Such labeling will include the dosage, frequency of administration, and method of administration.

[0060] In addition to the active ingredient (e.g., a compound of Formula (I)), pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers, including excipients and adjuvants that facilitate the processing of the active compound into pharmaceutically usable preparations. As used herein, the term "pharmaceutically acceptable carrier" refers to any type of non-toxic, inert solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; and ethyl oleate. Examples of suitable additives include esters such as ethanol and ethyl laurate, agar, detergents such as Tween 80, buffers such as magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, artificial cerebrospinal fluid (CSF), and phosphate buffer, as well as other non-toxic, compatible lubricants such as sodium lauryl sulfate and magnesium stearate; coloring agents, release agents, coating agents, sweeteners, flavors and fragrances, preservatives, and antioxidants can also be included in the compositions, according to the judgment of the formulator based on the desired route of administration.

[0061] Unless otherwise specified, alkyl, alkenyl, and alkynyl groups described herein preferably contain 1 to 20 carbon atoms in their main chain. They can be straight or branched, or cyclic (e.g., cycloalkyl). Alkenyl groups can contain saturated or unsaturated carbon chains, so long as there is at least one carbon-carbon double bond. Alkynyl groups can contain saturated or unsaturated carbon chains, so long as there is at least one carbon-carbon triple bond. Unless otherwise specified, alkoxy groups described herein contain saturated or unsaturated, branched or unbranched carbon chains having 1 to 20 carbon atoms in their main chain.

[0062] Unless otherwise specified herein, the term "aryl" refers to a monocyclic, bicyclic, or tricyclic aromatic group containing 6 to 14 ring carbon atoms and including, for example, phenyl. The term "heteroaryl" refers to a monocyclic, bicyclic, or tricyclic aromatic group having 5 to 14 ring atoms and containing carbon atoms and at least 1, 2, or 3 oxygen, nitrogen, or sulfur heteroatoms.

[0063] Having described the invention in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention as defined in the appended claims. [Example]

[0064] The following non-limiting examples are provided to further illustrate the present invention.

[0065] Example 1: Synthesis and screening of compounds with improved affinity for SWELL1. A series of compounds (Smod compounds) were synthesized to evaluate the role of the butyrate side chain and aryl substituents on activity (see Figure 1 and Table 1 below). In preliminary patch clamp experiments to screen for compounds that preserve or enhance SWELL1 modulating activity, unique structural derivatives were synthesized using I Cl,SWELLInhibitory activity (Smod2-6, Figures 2, 3, and 12-16, and Table 1 below) was identified. Notably, the aminopropyl group conferred active Smod2. In vitro channel inhibitory activity was also maintained in Smod3-5 (Figure 3). Note that compounds lacking activity and therefore not SWELL1 modulators were also identified (i.e., Snot1, Figures 2A and 3A). Figure 4 summarizes the dose-response curves for the three isolated enantiomers of Smod1 (+ and -) compared to Smod3. Smod3 exhibited an EC 50 These compounds exhibit a strong shift in β-glucan, indicating higher potency. Figure 5 summarizes the synthetic scheme used to generate these compounds.

[0066] [Table 1] TIFF0007771497000013.tif232170TIFF0007771497000014.tif137170

[0067] Example 2: Effects of compounds on SWELL1 protein expression and glucose metabolism in vivo. We compared SWELL1 expression in vivo with channel-inactive Snot1 compared with channel-active Smod3 and Smod5. Both Smod3 and Smod5 induced SWELL1 protein in 3T3-F442A adipocytes compared with vehicle, whereas Snot1 had no effect (Figure 6). Furthermore, Smod3 (5 mg / kg i.p. x 4 days), but not Snot1, improved glucose tolerance (GTT, area under the curve) and fasting glucose (FG) in mice fed an HFD for 8 weeks in a preliminary study (Figure 7). Similarly, SWELL1 channel-active Smod6 maintained the improvement in glucose tolerance in HFD-fed T2D mice 4 weeks after treatment was discontinued and after 20 weeks of HFD, whereas SWELL1 channel-inactive Snot1 and vehicle did not (Figures 8 and 9).

[0068] Example 3: Structure-function studies on Smod compounds and their interaction with SWELL channels. To explain the activity profile of the Smod compounds described in Example 1, we generated a binding model using a new cryo-EM structure of Smod1 bound to the SWELL1 homohexamer. As shown in Figures 10 and 11A and 11C, the butyrate chain of Smod1 protrudes through the neck of the SWELL1 channel and interacts with the R103 residue(s). The remainder of the Smod1 structure occupies a hydrophobic binding space along the arginine side chain and immediately above the channel neck. This binding mode, and similar docking of Smods and Snots assessed in preliminary work, explains 1) the role of the butyrate chain and chain length for SWELL1 binding (i.e., Snot1 versus Smod1, 3, and 4), 2) the requirement for a carboxylate for activity (an amide in place of the Smod1 carboxylate group results in an inactive Smod), and 3) changing the aryl chlorin to an arylmethyl group (Smod5) did not significantly alter activity. This binding mode may seem paradoxical, as cationic Smod2 regulates SWELL1 activity because the tertiary amine likely does not interact with the R103 residue. However, one explanation for Smod2 activity is that the SWELL1-LRRC8 channel complex is not essentially a homohexamer of SWELL1 (Figure 10), but rather the pattern of F103, in which L103 replaces some R103 subunits (i.e., a SWELL1-LRRC8c / d / e heterohexamer), can create an environment for cation-Pi interactions. A second possible explanation for Smod2-bound SWELL1 was revealed through modeling studies, where in silico docking showed that Smod2 is flipped 180 degrees in the preferred docking pose (Figure 11B). In this alternative binding mode, hydrophobic binding interactions are maintained on the channel neck, and terminal cationic or anionic groups on the alkoxy chains interact with amino acid side chains or main-chain amides in the channel wall. Combined, these results indicate that different Smods may bind in different orientations within different SWELL1 channels.Therefore, differences in LRRC8 subunit composition in different tissues (differences at position 103 for different heterohexamers as well as amino acid mutations above the channel neck) may offer the possibility of identifying Smod compounds that exhibit tissue-selective inhibition of the SWELL1-LRRC8 channel complex. Indeed, given the widespread tissue expression of the SWELL1-LRRC8 channel complex, the ability to selectively modulate specific SWELL1-LRRC8 stoichiometry in different tissues or cell types may be of great importance.

[0069] Example 4: Materials and methods for Examples 6-12. patient Human pancreatic islets and adipocytes were obtained and cultured as previously described (Kang et al., 2018; Zhang et al., 2017). Patients involved in this study were anonymous, and information such as gender, age, HbA1c, glucose levels, and BMI was only available to the research team.

[0070] animal All C57BL / 6 mice involved in the study were purchased from Charles River Laboratories. Both KK.Cg-Ay / J (KKN) and KK.Cg-Aa / J (KKAa) mice were obtained from the Jackson Laboratory (strain number: 002468) and were sex- and age-matched and bred for the experiment. Mice were fed either a regular chow diet (RC) or a high-fat diet (Research Diets, Inc., 60 kcal% fat) with free access to water ad libitum and housed in a light-, temperature-, and humidity-controlled room. For high-fat diet (HFD) studies, only male mice were used, and the HFD regimen was initiated at 6–9 weeks of age. For all experiments involving KKN and KKAa mice, both males and females were used in an approximately 50 / 50 ratio. In all experiments involving mice, researchers remained blind both during the experiment and in subsequent analyses.

[0071] 3T3-F442A cell line 3T3-F442A (Sigma-Aldrich) cells were maintained in 90% DMEM (25 mM D-glucose and 4 mM L-glutamine) containing 10% fetal bovine serum (FBS), 100 IU penicillin, and 100 μg / ml streptomycin.

[0072] HEK-293 cell line HEK-293 (ATCC® CRL-1573™) cells were maintained in 90% DMEM (25 mM D-glucose and 4 mM L-glutamine) containing 10% fetal bovine serum (FBS), 100 IU penicillin, and 100 μg / ml streptomycin. Overexpression of plasmid DNA in HEK-293 cells was performed using Lipofectamine 2000 (Invitrogen) reagent.

[0073] Small molecule treatment All compounds were dissolved in Kolliphor® EL (Sigma, #C5135). Either vehicle (Kolliphor® EL), SN-401 (DCPIB, 5 mg / kg body weight / day, Tocris, D1540), SN-403, SN-406, SN-407, or SN071 was administered intraperitoneally as indicated using a 1 cc syringe / 26G × 1 / 2 inch needle once daily for 4–10 days; in one experiment, SN-401 was administered once daily for 8 weeks. SN-401 formulated as above was also administered by oral gavage at 5 mg / kg / day for 5 days using a 20G × 1.5 inch reusable metal oral gavage needle.

[0074] Adenovirus Ad5-RIP2-GFP (4.1 × 10 10 PFU / ml) and Ad5-CAG-LoxP-stop-LoxP-3XFlag-SWELL1 (1 × 10 10 Adenovirus type 5 containing 8 × 10 PFU / ml was obtained from Vector Biolabs. 10Adenovirus type 5 with 100 PFU / ml was obtained from the Iowa Viral Vector Core.

[0075] cell culture Wild-type (WT) and SWELL1 knockout (KO) 3T3-F442A (Sigma-Aldrich) cells were cultured and differentiated as previously described (Zhang et al., 2017). Preadipocytes were maintained on collagen-coated (rat tail type I collagen, Corning) plates in 90% DMEM (25 mM D-glucose and 4 mM L-glutamine) containing 10% fetal bovine serum (FBS), 100 IU penicillin, and 100 μg / ml streptomycin. Upon reaching confluency, cells were differentiated in the above medium supplemented with 5 μg / ml insulin (Cell Applications) and replenished with differentiation medium every other day. For insulin signaling studies on WT and KO adipocytes with or without SWELL1 overexpression (O / E), cells were differentiated for 10 days and transduced with Ad5-CAG-LoxP-stop-LoxP-SWELL1-3XFlag virus (MOI 12) on day 11 in 2% FBS-containing differentiation medium. To induce overexpression, Ad5-CMV-Cre-wt-lRES-eGFP (MOI 12) was added on day 13 in 2% FBS-containing differentiation medium. Then, on days 15–17, cells were switched to 10% FBS-containing differentiation medium. On day 18, cells were starved in serum-free medium for 6 hours and stimulated with 0 and 10 nM insulin for 5 or 15 minutes. Either Ad5-CAG-LoxP-stop-LoxP-SWELL1-3XFlag or Ad5-CMV-Cre-wt-lRES-eGFP virus-transduced cells alone were used as controls. Based on GFP fluorescence, viral transduction efficiency was approximately 90%.

[0076] For SN-401 treatment and insulin signaling studies in 3T3-F442A preadipocytes, cells were incubated with either vehicle (DMSO) or 10 μM SN-401 for 96 hours. Cells were starved for 6 hours (+DMSO or SN-401), washed three times with PBS, and stimulated with 0, 3, or 10 nM insulin-containing medium for 15 minutes before lysate collection. For 3T3-F442A preadipocytes, WT and KO cells were treated with either vehicle (DMSO), 1, or 10 μM SN-40X for 96 hours after 7–11 days of differentiation. For SWELL1 detection, cells were then stimulated with 0 and 10 nM insulin / serum-containing medium (+DMSO or SN-40X) for 15–30 minutes. For AKT and AS160 signaling, serum-starved cells in the presence of compounds were washed twice in hypotonic buffer (240 mOsm) and then incubated in hypotonic buffer for 10 min at 37°C, followed by stimulation with insulin / serum-containing medium. To simulate glucolipotoxicity, sodium palmitate was dissolved in 18.4% fatty acid-free BSA in DMEM medium with 25 mM glucose at 37°C, resulting in a palmitate:BSA conjugation ratio of 1:3 (Busch et al., 2002). 3T3-F442A adipocytes were incubated with vehicle or SN-401, SN-406, or SN072 at 10 μM for 96 h, then treated with 1 mM palmitate for an additional 16 h, and lysates were collected and further processed as described above.

[0077] Molecular docking SN-401 and its analogs were docked into the expanded-state structure of the LRRCBA-SN-401 homo-hexamer in the MSP1E3D1 nanodisc (PDB ID: 6NZZ) using the Molecular Operating Environment (MOE) 2016.08 software package [Chemical Computing Group, Montreal, Canada]. The 3D structure obtained from the PDB (PDB ID: 6NZZ) was prepared for docking by first generating missing loops using the loop generation function in the Yasara software package, followed by sequential hydrogen addition, adjusting the 3D protonation state, and energy minimization using the Amber10 force field in MOE. The docked ligand structure was prepared by adjusting partial charges, followed by energy minimization using the Amber10 force field. The docking site was defined by selecting protein residues within 5 Å of the co-crystallized ligand (SN-401). The docking parameters were set as follows: Configuration: Triangle matcher, Scoring function: London dG, Retention pose: 30, Refinement: Rigid receptor, Rescoring function: GBVI / WSA dG, Retention pose: 5. The binding poses of the compounds were predicted using the validated docking algorithms described above.

[0078] electrophysiology Patch clamp recordings of β cells and mature adipocytes were performed as previously described (Kang et al., 2018, Zhang et al., 2017). 3T3-F442A WT and KO preadipocytes were prepared as described in the cell culture section above. For SWELL1 overexpression recordings, preadipocytes were first transduced with Ad5-CAG-LoxP-stop-LoxP-3XFlag-SWELL1 (MOI 12) for 2 days in 2% FBS medium. Then, overexpression was induced by adding Ad5-CMV-Cre-wt-lRES-eGFP (MOI 10-12) for 2 days in 2% FBS medium, changed to 10% FBS-containing medium, and selected based on GFP expression (approximately 2-3 days). For cell recordings, pancreatic islets were transduced with Ad-RIP2-GFP and dispersed 48-72 hours later for patch clamp experiments. GFP+ cells marked β cells, which were selected for patch clamp recordings. Cl,SWELL I by SN-401 analogs after activation of Cl,SWELL To measure inhibition, HEK-293 cells were perfused with a hypotonic solution (hypo, 210 mOsm) as described below, and then SN-401 analogs + hypo were applied at 10 and 7 μM to measure I Cl,SWELL The % inhibition of I upon application of SN-401 analogs to closed SWELL1-LRRC8 channels was assessed. Cl,SWELLTo assess inhibition, HEK-293 cells were preincubated with vehicle (or SN-401, SN-406, SN071, and SN072) for 30 min before hypotonic stimulation and then stimulated with hypotonic solution plus SN-401 analogs. Recordings were recorded using an Axopatch 2008 amplifier coupled with a Digidata 1550 digitizer and pClamp 10.4 software. The extracellular buffer composition for hypotonic stimulation contained 90 mM NaCl, 2 mM CsCl, 1 mM MgCl, 1 mM CaCb, 10 mM HEPES, 10 mM mannitol, pH 7.4 with NaOH (210 mOsm / kg). The extracellular isotonic buffer composition was the same as above, except for the mannitol concentration of 110 mM (300 mOsm / kg). The composition of the intracellular buffer was 120 mM L-aspartic acid, 20 mM CsCl, 1 mM MgCl, 5 mM EGTA, 10 mM HEPES, 5 mM MgATP, 120 mM CsOH, 0.1 mM GTP, and pH 7.2 with CsOH. All recordings were performed at room temperature (RT) using HEK-293 cells, β cells, and 3T3-F442A cells performed in the perforated patch configuration, as previously described (Kang et al., 2018; Zhang et al., 2017).

[0079] Western blot Cells were washed twice in ice-cold phosphate-buffered saline and lysed in RIPA buffer (150 mM NaCl, 20 mM HEPES, 1% NP-40, 5 mM EDTA, pH 7.4) with protease / phosphatase inhibitors (Roche). The cell lysate was further sonicated 2–3 times with 10-second intervals and centrifuged at 14,000 rpm for 20 min at 4°C. The supernatant was collected and further estimated for protein concentration using a DC protein assay kit (Bio-Rad). Adipose tissue was homogenized and suspended in RIPA buffer with inhibitors as described above. Protein samples were further prepared by boiling in 4x Laemmli buffer. Approximately 10–20 μg of total protein was loaded onto a 4–15% gradient gel (Bio-Rad) and separated, followed by protein transfer onto a PVDF membrane (Bio-Rad). The membranes were blocked with 5% BSA (5% milk for SWELL1) in TBST buffer (0.2 M Tris, 1.37 M NaCl, 0.2% Tween-20, pH 7.4) for 1 hour and then incubated with the appropriate primary antibody (5% BSA or milk) overnight at 4 °C. After further washing in TBST buffer, secondary antibodies (Bio-Rad, goat anti-rabbit, #170-6515) in 1% BSA in TBST buffer (or 1% milk for SWELL1) were added for 1 hour at room temperature. Signals were developed by chemiluminescence (Pierce) and visualized using a Chemidoc imaging system (Biorad). Images were further analyzed for band intensity using ImageJ software. The following primary antibodies were used: anti-phospho-AKT2 (#8599s), anti-AKT2 (#3063s), anti-phospho-AS160 (#4288s), anti-AS160 (#2670s) anti-GAPDH (#D16H11) and anti-β-actin (#8457s) from Cell Signaling, and a rabbit polyclonal anti-SWELL1 antibody was raised against the epitope QRTKSRIEQGIVDRSE (SEQ ID NO: 13) (Pacific Antibodies).

[0080] Immunofluorescence 3T3-F442A preadipocytes (WT, KO) and adipocytes differentiated without or with SWELL1 overexpression (WT + SWELL1 O / E, KO + SWELL1 O / E) were prepared as described in the cell culture section on collagen-coated coverslips. For SWELL1 membrane transport, 3T3-F442A preadipocytes were incubated with either 1 or 10 μM of vehicle (or SN-401, SN-406, or SN071) for 48 hours and further treated. Cells were fixed in ice-cold acetone at -20°C for 15 minutes, washed four times with 1x PBS, permeabilized with 0.1% Triton X-100 in 1x PBS for 5 minutes at room temperature, and then blocked with 5% normal goat serum for 1 hour at room temperature. Either anti-SWELL1 (1:400) or anti-Flag (1:1500, Sigma #F3165) antibody was added to the cells and incubated overnight at 4°C. Cells were then washed three times (1x PBS) before and after the addition of 1:1000 Alexa Flour 488 / 568 secondary antibody (anti-rabbit, #A11034 or anti-mouse, #A11004) at room temperature for 1 hour. Cells were counterstained with nuclear TO-PRO-3 (Life Technologies, #T3605) or DAPI (Invitrogen, #D1306) stain (1 μM) for 20 minutes, followed by three washes with 1x PBS. Coverslips were then mounted onto slides using Prolong Diamond antifade medium. All images were acquired using a Zeiss LSM700 / LSM510 confocal microscope with a 63x objective (NA 1.4). SWELL1 membrane localization was quantified by stacking all z-images and converting it into a binary image in which the cytoplasmic intensity per unit area was subtracted from the total intensity of the cell per unit area using lmageJ software.

[0081] metabolic phenotype Before the glucose tolerance test (GTT), mice were fasted for 6 hours. Baseline glucose levels (fasting glucose, FG) at 0 min were measured from blood samples collected from the tail using a glucometer (Bayer Healthcare LLC). Lean or HFD mice were injected (intraperitoneally) with either 1 g or 0.75 g of D-glucose / kg body weight, respectively, and glucose levels were measured at 7, 15, 30, 60, 90, and 120 min after injection. For the insulin tolerance test (ITT), mice were fasted for 4 hours. Similar to the GTT, baseline blood glucose levels were measured at 0 min and 15, 30, 60, 90, and 120 min after injection (intraperitoneally) of insulin (Humulin R, 1 U / kg body weight for lean mice or 1.25 U / kg body weight for HFD mice). GTT or ITT in vehicle (or SN-401, SN-403, SN-406, SN-407, and SN071)-treated groups was performed approximately 24 hours after the last injection. For insulin secretion assays, vehicle- (or SN-401, SN-406, and SN071)-treated HFD mice were fasted for 6 hours and injected (intraperitoneally) with 0.75 g of D-glucose / kg body weight. Blood samples were collected at 0, 7, 15, and 30 minutes in microvette capillary tubes (SARSTEDT, #16.444) and centrifuged at 2000 × g for 20 minutes at 4 °C. The collected plasma was then measured for insulin content using an ultrasensitive mouse insulin ELISA kit (Crystal Chem, #90080). All mice and treatment groups were evaluated blindly throughout the experiment.

[0082] Mouse islet isolation and perfusion assay For patch clamp studies involving primary mouse cells, mice were anesthetized by injection of avertin (0.0125 g / ml in HO) followed by cervical dislocation. HFD or polygenic KKAy mice treated with either vehicle (or SN-401, SN-406, SN-407, and SN071) were anesthetized with 1–4% isoflurane followed by cervical dislocation. Pancreatic islets were further isolated as previously described (Kang et al., 2018). Islets were perfused using Biorep Perfusion experiments were performed using a PER14-02 system from BioRad Technologies. For each experiment, approximately 50 freshly isolated islets (all from the same isolation batch) were manually selected to match the size of the islets across the sample and loaded by the same experienced operator into a polycarbonate perfusion chamber between two layers of polyacrylamide-microbead slurry (Bio-Gel-P-4, BioRad). The perfusion buffer contained (in mM): 120 NaCl, 24 NaHCO3, 4.8 KCl, 2.5 CaCl2, 1.2 MgSO4, 10 HEPES, 2.8 glucose, 27.2 mannitol, 0.25% w / v bovine serum albumin, pH 7.4 with NaOH (300 mOsm / kg). The perfusion buffer, maintained at 37°C, was circulated at 120 μL / min. After washing with 2.8 mM glucose solution for 48 min for stabilization, the islets were stimulated with the following sequence: 16.7 mM glucose for 16 min, 2.8 mM glucose for 40 min, 30 mM KCl for 10 min, and 2.8 mM glucose for 12 min. The osmolarity was matched by adjusting the mannitol concentration when preparing a solution containing 16.7 mM glucose. Serial samples were collected every 1 or 2 min into 96-well plates stored at 4 °C. Insulin concentrations were further determined using a commercially available ELISA kit (Mercodia). The area under the curve (AUC) of high glucose-induced insulin release was calculated for the 50-, 74-, and 84-minute time points. Upon completion of the experiment, the islets were further lysed by adding RIPA buffer, and the amount of insulin was detected by ELISA.

[0083] Drug pharmacokinetics Pharmacokinetic studies for the SN-401 / SN-406 study were conducted at Charles River Laboratories as outlined below. Male C57 / BL6 mice were used in the study and evaluated for a single dose (5 mg / kg). The compounds were prepared at a final concentration of 1 mg / ml in Cremaphor for the intraperitoneal and oral gavage routes, and in a 5% ethanol, 10% Tween-20, and water mixture for the intravenous route. Terminal blood samples were collected via cardiac venipuncture under anesthesia at 0.08, 0.5, 2, and 8 hours post-dose in the intravenous group, and 0.25, 2, 8, and 24 hours post-dose in the intraperitoneal and oral gavage groups, respectively, with a sample size of three mice per time point. Blood samples were collected in tubes with K2 EDTA anticoagulant and further processed to recover plasma by centrifugation at 3500 rpm for 10 minutes at 5°C. The samples were further processed by LC / MS to determine the compound concentration. Non-compartmental analysis was performed to obtain PK parameters using the PKPlus software package (Simulation Plus). The area under the plasma concentration-time curve (AUCint) was calculated from time 0 to infinity, where Cmax is the maximum concentration achieved in plasma and t112 is the terminal elimination half-life. Oral bioavailability was calculated using AUC0railleurAUC1v * It was calculated as 100.

[0084] In vitro and in silico ADMET In vitro ADMET studies were performed at Charles River Laboratories as outlined below. For the Caco-2 permeability assay, cells were cultured for 21 days (DMEM, 10% FBS, 1% L-Glutamax, and 1% PenStrep). HBSS was used as the transport buffer, and TEER measurements were performed before the start of the assay. Compounds were added to the apical side to determine apical-to-basolateral transport (AB) and to the basolateral side to determine basolateral-to-apical transport (BA). Samples (10 μL) were collected at time 0 and 2 h and diluted (5 times) with transport buffer. After the quench reaction, samples were further diluted in MilliQ water for bioanalysis. TEER measurements were performed at the end of the assay. Wells with a significant decrease in TEER values ​​after the assay were not included in the data, and the measurements were repeated. Analyte levels (peak area ratios) were measured at the apical (A) and basolateral (B) sides at T0, and T2h-A-band BA flux was calculated by averaging three individual measurements. Apparent permeability (Papp, cm / sec) was calculated as dQ(flux) / (dt * area * The efflux ratio was calculated as Papp(BA) / Papp(BA). For the microsomal metabolic stability assay, microsomes were diluted in potassium phosphate buffer to maintain a final concentration of 0.5 mg / ml throughout the assay procedure. Compounds were diluted 10-fold in acetonitrile and incubated with microsomes at 37°C with gentle shaking. Samples were collected at different time points and quenched. The samples were mixed by vortexing for 10 minutes and centrifuged at 3100 rpm for 10 minutes at 4°C. The supernatant was diluted in water and further analyzed by LC / MS autosampler. The half-life (T) was calculated by the equation 0.692 / slope, where the slope is ln(% remaining relative to T zero vs. time). 1 / 2) was calculated. Intrinsic clearance was calculated using (CLn1) = T112 * 1 / initial concentration * mg preparation / g liver * g liver / kg body weight. For cytochrome P450 inhibition assays, cofactors and substrates were mixed in potassium phosphate buffer. A stock concentration of 10 mM compound (in DMSO) was diluted 5-fold in acetonitrile and mixed with the cofactor / substrate mixture (2x). Human liver microsomes were diluted to a final concentration of 0.2 mg / ml in potassium phosphate buffer (2x), and the reaction was initiated by mixing the microsomes with the compound / cofactor / substrate mixture at 37°C with gentle shaking. Samples were collected at T0 and T 30 The samples were collected at the 20 min time point and quenched. The samples were then centrifuged at 3100 rpm for 5 min at 5-10 °C, and the supernatant was diluted in water and further analyzed by LC / MS autosampler. % inhibition was calculated (using peak area ratios) for zero inhibition (full activity) and no activity (complete inhibition). Drug properties and drug-likeness predictions for SN-401 and SN-406 were performed using the FAF-Drugs4 and preADMET software packages.

[0085] Hyperinsulinemic-euglycemic clamp A sterile silicone catheter (Dow-Corning) was placed into the jugular vein of mice under isoflurane anesthesia. The catheter was flushed with 200 U / ml heparin in saline, and the free end of the catheter was directed subcutaneously through a blunt 14-gauge sterile needle and connected to a small tubing device exiting through the animal's back. Mice were allowed to recover from surgery for 3 days and then received intraperitoneal injections of vehicle or SN-401 (5 mg / kg) for 4 days. A hyperinsulinemic-euglycemic clamp was performed on day 8 after surgery in unrestrained, conscious mice as described elsewhere (Ayala et al., 2011; Kim et al., 2000), with some modifications. Mice were fasted for 6 hours, at which point insulin and glucose infusions were initiated (time 0). Basal sampling was performed 80 min before time 0, followed by a 1-min priming 5 μCi bolus followed by a 0.05 μCi / min infusion of D-[3-3H]-glucose (Perkin Elmer) to trace whole-body glucose flux. After the basal period, a continuous infusion of D-[3-3H]-glucose was initiated at time 0 at a rate of 0.2 μCi / min, and insulin (Humulin, Eli Lilly) was infused at a bolus of 80 mU / kg / min, followed by a continuous infusion of insulin at a dose of 8 mU / kg / min throughout the assay. Fifty percent dextrose (Hospira) was infused at a variable rate (GIR) starting simultaneously with the start of insulin infusion to maintain euglycemia at a target level of 150 mg / dL (8.1 mM). Blood glucose (BG) measurements were performed every 10 min via tail vein sampling using a Contour glucometer (Bayer). After mice reached stable BG and GIR (typically 75 min after starting the insulin infusion; some mice required longer times to achieve steady state), a single bolus of 12 μCi of [1-14C]-2-deoxy-D-glucose (Perkin Elmer) in 96 μL of saline was administered.Plasma samples (collected from centrifuged blood) for measuring tracer enrichment, glucose levels, and insulin concentrations were collected at -80, -20, -10, and 0 min, and every 10 min from 80 min post-insulin (5 min after administration of the [1-14C]-2-deoxy-D-glucose bolus) until the end of the assay at 140 min. Tissue samples were then collected from the organs of interest (e.g., liver, heart, kidney, white adipose tissue, brown adipose tissue, gastrocnemius muscle, soleus muscle, etc.) under isoflurane anesthesia to determine [1-14C]-2-deoxy-D-glucose tracer uptake. Plasma and tissue samples were processed as previously described (Ayala et al., 2011). Briefly, plasma samples were deproteinized with Ba(OH)2 and ZnSO4 and dried to remove tritiated water. Glucose turnover rate (mg / kg-min) was calculated as the tracer infusion rate (dpm / min) per kg of mouse body weight divided by the corrected plasma glucose specific activity (dpm / mg). Fluctuations from steady state were accounted for by the use of Steele's model. Plasma glucose was measured using an Analox GMD9 system (Analox Technologies).

[0086] Tissue samples (approximately 30 mg each) were homogenized in 750 μL of 0.5% perchloric acid, neutralized with 10 M KOH, and centrifuged. The supernatant was then used to initially measure the abundance of total [1-14C] signal (from both 1-14C-2-deoxy-D-glucose and 1-14C-2-deoxy-D-glucose hexaphosphate) and, following a precipitation step with 0.3 N Ba(OH)3 and 0.3 N ZnSO4, to measure non-phosphorylated 1-14C-2-deoxy-D-glucose. Glycogen was isolated by ethanol precipitation from 30% KOH tissue lysates as described (Shiota, 2012). T0 and T 140 Plasma insulin levels were measured using a Stellux ELISA rodent insulin kit (Alpco).

[0087] Quantitative RT-PCR 3T3-F442A preadipocytes treated with either vehicle (DMSO) or 10 μM SN-401 for 96 h were solubilized in TRlzol, and total RNA was isolated using the Purelink RNA kit (Life Technologies). cDNA synthesis, qRT-PCR reaction, and quantification were performed as previously described ( Zhang et al., 2017 ).

[0088] Liver isolation, triglycerides and histology HFD mice treated with either vehicle or SN-401 were anesthetized with 1-4% isoflurane followed by cervical dislocation. Total liver weight was measured, and identical sections from the right medial lobe of the liver were dissected for further examination. Total triglyceride content was determined by homogenizing 10-50 mg of tissue in 1.5 mL of chloroform:methanol (2:1 v / v) and centrifuging at 12,000 rpm for 10 min at 4 °C. A 20 μL aliquot was evaporated in a 1.5 mL microcentrifuge tube for 30 min. Triglyceride content was determined by adding 100 μL of Infinite Triglyceride Reagent (Fisher Scientific) to the dried sample, followed by a 30-min incubation at room temperature. Samples were then transferred to a 96-well plate along with standards (0-2,000 mg / di), and the final concentration was determined by measuring absorbance at 540 nm and normalizing to tissue weight. For histological examination, liver sections were fixed in 10% formalin-zinc and paraffin-embedded. Hematoxylin and eosin (H&E)-stained sections were then evaluated for steatosis grade, lobular inflammation, and hepatocyte ballooning for nonalcoholic fatty liver disease (NAFLD) scoring (Kleiner et al., 2005, Liang et al., 2014, Rauckhorst et al., 2017).

[0089] Quantification and statistical analysis Standard unpaired or paired two-tailed Student's t-tests were performed when comparing two groups. One-way analysis of variance was used for multiple group comparisons. For GTT and ITT, two-way analysis of variance (Anova) was used. A p-value of less than 0.05 was considered statistically significant. *, **, and *** represent p-values ​​less than 0.05, 0.01, and 0.001, respectively. All data are expressed as mean ± SEM. All statistical details and analyses are presented in the figure briefs.

[0090] Example 5: Synthesis General information: All commercially available reagents and solvents were used directly without further purification unless otherwise noted. Reactions were monitored either by thin-layer chromatography (silica plates, silica gel 60 F2s4, performed by Merck) and visualized under UV light. Flash chromatography was performed using silica gel 60 as the stationary phase under positive air pressure. Unless otherwise noted, 1H NMR spectra were recorded on a CDCb on a Bruker Avance spectrometer operating at 300 MHz at ambient temperature. All peaks are reported in ppm, scaled downfield from TMS, using the residual solvent peak in CDCb (H 5 = 7.26) or TMS (5 = 0.0) as the internal standard. H NMR data are reported as follows: chemical shift (ppm, scale), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet and / or multiplet resonance, dd = double doublet, dt = double triplet, br = broad), overall constant (Hz), and integral. All high-resolution mass spectrometry (HRMS) was performed on a Waters Q-Tof Premier mass spectrometer using electrospray ionization (ESI) time-of-flight (TOF).

[0091] 2-Cyclopentyl-1-(2,3-dichloro-4-methoxyphenyl)ethan-1-one (3) was prepared according to Scheme 1 (Figure 17).

[0092] [ka]

[0093] To a stirred solution of aluminum chloride (13.64 g, 102 mmol, 1.1 equiv.) in dichloromethane (250 mL) at 0 °C, cyclopentylacetyl chloride (15 g, 102 mmol, 1.1 equiv.) was added, and the resulting solution was stirred at 0 °C for 10 min under a nitrogen atmosphere. To this, a solution of 2,3-dichloroanisole (16.46 g, 92.9 mmol, 1 equiv.) in dichloromethane (50 mL) was added at 0 °C, and the resulting solution was warmed to room temperature and stirred for 16 h. Upon completion, the reaction was added to cold concentrated hydrochloric acid (100 mL), followed by extraction with dichloromethane (150 mL × 3). The organic fractions were pooled, concentrated, and purified by silica gel chromatography using 0–15% ethyl acetate in hexane as the eluent to give compound 3 (22.41 g, 84%) as a white solid. 1 H NMR(300MHz,CDCl3)δ 7.39(d,J=8.7Hz,1H),6.89(d,J=8.7Hz,1H),3.96(s,3H),2.96(d,J=7.2Hz,2H ),2.38-2.21(m,1H),1.92-1.75(m,2H),1.69-1.46(m,4H),1.28-1.05(m,2H). HRMS(ESI), C 14 H 17 Cl2O2[M+H] + The calculated m / z value for this compound was 287.0605, and the observed value was 287.0603.

[0094] 6,7-Dichloro-2-cyclopentyl-5-methoxy-2,3-dihydro-1H-inden-1-one (4) was prepared according to Scheme 1 (Figure 17).

[0095] [ka]

[0096] To 2-cyclopentyl-1-(2,3-dichloro-4-methoxyphenyl)ethan-1-one (3) (21.5 g, 74.8 mmol, 1 equiv.) in a round-bottom flask was added paraformaldehyde (6.74 g, 224.5 mmol, 3 equiv.), dimethylamine hydrochloride (30.52 g, 374 mmol, 5 equiv.), and acetic acid (2.15 ml), and the resulting mixture was stirred at 85 °C for 16 h. Dimethylformamide (92 ml) was then added to the reaction, and the resulting solution was stirred at 85 °C for 4 h. Upon completion, the reaction was diluted with ethyl acetate and then washed with 1 N hydrochloric acid. The organic fraction was collected, concentrated under vacuum, and used in the next step without purification. To the concentrated product in the round-bottom flask at 0 °C, cold concentrated sulfuric acid (120 ml) was added, and the resulting solution was stirred at room temperature for 18 h. Upon completion, the reaction was diluted with cold water and extracted three times with ethyl acetate (100 ml). The organic fractions were pooled, concentrated, and purified by silica gel chromatography using 0-15% ethyl acetate in hexane as the eluent to give compound 4 (18.36 g, 82%) as a beige solid. 1 H NMR(300MHz,CDCl3)δ 6.88(s,1H),4.00(s,3H),3.16(dd,J=18.1,8.7Hz,1H),2.80(d,J=14.4Hz,2H),2.43 -2.22(m,1H),1.96(s,1H),1.73-1.48(m,5H),1.46-1.33(m,1H),1.17-1.00(m,1H). LRMS(ESI), C 15 H 17 Cl2O2[M+H] + The calculated m / z value for this compound was 299.0605, and the observed value was 299.0614.

[0097] 2-Butyl-6,7-dichloro-2-cyclopentyl-5-methoxy-2,3-dihydro-1H-inden-1-one (5) was prepared according to Scheme 1 (Figure 17).

[0098] [ka]

[0099] A stirred suspension of 4 (23 gm, 76.8 mmol, 1 equiv.) in anhydrous tert-butanol (220 ml) was refluxed at 95°C for 30 minutes. To the resulting solution was added potassium tert-butanol (1 M in tert-butanol) (84 ml, 84.5 mmol, 1.1 equiv.), and the resulting solution was refluxed for 30 minutes. The reaction was then cooled to room temperature, followed by the addition of iodobutane (44.2 ml, 384 mmol, 5 equiv.), and the reaction was then refluxed for an additional 60 minutes. The reaction was cooled, concentrated, and purified by silica gel chromatography using 0-10% ethyl acetate in hexane as the eluent to give compound 5 (17.75 g, 65%) as a clear oil. 1 H NMR(300MHz,CDCl3)δ 6.89(s,1H),4.09-3.90(m,3H),2.98-2.70(m,2H),2.36-2.18(m,1H),1.89-1.71( m,2H),1.58-1.42(m,5H),1.33-1.09(m,4H),1.09-0.94(m,2H),0.93-0.73(m,4H). HRMS(ESI), C 19 H 25 Cl2O2[M+H] + Calculated m / z value for 355.1231, observed value 355.1231.

[0100] 2-Butyl-6,7-dichloro-2-cyclopentyl-5-hydroxy-2,3-dihydro-1H-inden-1-one (6) was prepared according to Scheme 1 (Figure 17).

[0101] [ka]

[0102] To 5 (3.14 g, 8.87 mmol, 1 equiv.) was added aluminum chloride (2.36 g, 17 mmol, 2 equiv.) and sodium iodide (2.7 g, 17 mmol, 2 equiv.), and the resulting solid mixture was triturated and stirred at 70 °C for 60 min. Upon completion, the reaction was diluted with dichloromethane and washed with saturated aqueous sodium thiosulfate. The organic fractions were collected and concentrated to give a beige solid, which was then washed multiple times with hexane to give compound 6 (2.87 g, 95%) as a white solid. 1 H NMR(300MHz,CDCl3)δ 7.03(s,1H),6.32(s,1H),2.97-2.73(m,2H),2.36-2.17(m,1H),1.88-1.68(m,2H),1.62-1 .39(m,6H),1.31-1.11(m,3H),1.08-0.97(m,2H),0.97-0.87(m,1H),0.83(t,J=7.3Hz,3H). HRMS(ESI), C 18 H 23 Cl2O2[M+H] + The calculated m / z value for this compound was 341.1075, and the observed value was 341.1089.

[0103] 2-((2-Butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy)acetic acid (7) (SN071) was prepared according to Scheme 1 (Figure 17).

[0104] [ka]

[0105] To a stirred solution of 5 (170 mg, 0.50 mmol, 1 equiv) in anhydrous dimethylformamide (1 ml), potassium carbonate (76 mg, 0.56 mmol, 1.1 equiv) and ethyl 2-bromoacetate (61 μl, 0.56 mmol, 1.1 equiv) were added and the reaction was stirred at 60 °C for 2 h. Upon completion, 4 N NaOH (1 ml) was added to the reaction and the reaction was stirred at 100 °C for 60 min. Upon completion, the reaction was concentrated and purified by column chromatography using 0-10% methanol in dichloromethane as the eluent to give SN071 as a clear solid (173 mg, 87%). 1 H NMR(300MHz,CDCl3)δ 6.80(s,1H),5.88(s,1H),4.88(s,2H),2.87(q,J=17.9Hz,2H),2.34-2.20(m,1H),1.91-1.69(m,2H) ,1.66-1.39(m,6H),1.32-1.13(m,3H),1.10-0.95(m,2H),0.94-0.86(m,1H),0.83(t,J=7.3Hz,3H). HRMS(ESI), C 20 H 25 Cl2O4[M+H] + The calculated m / z value for this compound was 399.1130, and the observed value was 399.1132.

[0106] 4-((2-Butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy)butanoic acid (8) (SN-401) was prepared according to Scheme 1 (Figure 17).

[0107] [ka]

[0108] To a stirred solution of 5 (100 mg, 0.29 mmol, 1 equiv) in anhydrous dimethylformamide (1 ml) was added potassium carbonate (45 mg, 0.32 mmol, 1.1 equiv) and ethyl 4-bromobutyrate (46 μl, 0.32 mmol, 1.1 equiv) and the reaction was stirred at 60 °C for 2 h. Upon completion, 4 N NaOH (1 ml) was added to the reaction and the reaction was stirred at 100 °C for 60 min. Upon completion, the reaction was concentrated and purified by column chromatography using 0-10% methanol in dichloromethane as the eluent to afford SN-401 as a clear solid (111 mg, 89%). 1 H NMR(300MHz,CDCl3)δ 10.77(s,1H),6.86(s,1H),4.21(t,J=5.9Hz,2H),2.88(t,J=14.4Hz,2H),2.69(t,J=7.0Hz,2H),2.26(dd,J=12.6,6.1Hz ,3H),1.87-1.73(m,2H),1.64-1.44(m,6H),1.35-1.10(m,4H),1.08-0.95(m,J=15.0,7.7Hz,2H),0.82(t,J=7.3Hz,3H). HRMS(ESI), C 22 H 29 Cl2O4[M+H] + The calculated m / z value for this compound was 427.1443, and the observed value was 427.1446.

[0109] 5-((2-Butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy)pentanoic acid (9) (SN-403) was prepared according to Scheme 1 (Figure 17).

[0110] [ka]

[0111] To a stirred solution of 5 (100 mg, 0.29 mmol, 1 equiv.) in anhydrous dimethylformamide (1 ml), potassium carbonate (45 mg, 0.32 mmol, 1.1 equiv.) and ethyl 6-bromovalerate (51 μl, 0.32 mmol, 1.1 equiv.) were added and the reaction was stirred at 60 °C for 2 h. Upon completion, 4 N NaOH (1 ml) was added to the reaction and the reaction was stirred at 100 °C for 60 min. Upon completion, the reaction was concentrated and purified by column chromatography using 0-10% methanol in dichloromethane as the eluent to afford SN-403 as a clear solid (114 mg, 88%). 1 H NMR(300MHz,CDCl3)δ 10.95(s,1H),6.85(brs,1H),4.16(t,J=5.7Hz,2H),2.96-2.75(m,2H),2.61-2.44(m,2H),2.35-2.17(m,1H),2.10-1.87( m,4H),1.86-1.70(m,2H),1.66-1.38(m,6H),1.32-1.13(m,3H),1.08-0.96(m,2H),0.94-0.86(m,1H),0.86-0.73(m,3H). HRMS(ESI), C 23 H 31 Cl2O4[M+H] + The calculated m / z value for this compound was 441.1599, and the observed value was 441.1601.

[0112] 6-((2-Butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy)hexanoic acid (10) (SN-406) was prepared according to Scheme 1 (Figure 17).

[0113] [ka]

[0114] To a stirred solution of 5 (100 mg, 0.29 mmol, 1 equiv) in anhydrous dimethylformamide (1 ml) was added potassium carbonate (45 mg, 0.32 mmol, 1.1 equiv) and ethyl 6-bromohexanoate (58 μl, 0.32 mmol, 1.1 equiv) and the reaction was stirred at 60 °C for 2 h. Upon completion, 4 N NaOH (1 ml) was added to the reaction and the reaction was stirred at 100 °C for 60 min. Upon completion, the reaction was concentrated and purified by column chromatography using 0-10% methanol in dichloromethane as the eluent to afford SN-406 as a clear solid (115 mg, 86%). 1H NMR(300MHz,CDCl3)δ 11.70(s,1H),6.85(s,1H),4.13(t,J=6.2Hz,2H),2.93-2.74(m,2H),2.43(t,J=7.3Hz,2H),2.32-2.17(m,1H),1 .98-1.87(m,2H),1.85-1.68(m,4H),1.66-1.40(m,8H),1.28-1.12(m,3H),1.07-0.93(m,2H),0.91-0.70(m,4H). HRMS(ESI), m / z calculated value for C24H33Cl2O4[M+H]+ 455.1756, measured value 455.1756.

[0115] 7-((2-Butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy)heptanoic acid (11) (SN-407) was prepared according to Scheme 1 (Figure 17).

[0116] [ka]

[0117] To a stirred solution of 5 (100 mg, 0.29 mmol, 1 equiv) in anhydrous dimethylformamide (1 ml) was added potassium carbonate (45 mg, 0.32 mmol, 1.1 equiv) and ethyl 7-bromoheptanoate (63 μl, 0.32 mmol, 1.1 equiv) and the reaction was stirred at 60 °C for 2 h. Upon completion, 4 N NaOH (1 ml) was added to the reaction and the reaction was stirred at 100 °C for 60 min. Upon completion, the reaction was concentrated and purified by column chromatography using 0-10% methanol in dichloromethane as the eluent to afford SN-407 as a clear solid (122 mg, 89%). 1 H NMR(300MHz,CDCl3)δ 11.52(s,1H),6.85(s,1H),4.12(t,J=6.3Hz,2H),2.84(q,J=18.2Hz,2H),2.47-2.32(m,2H),2.32-2.18(m,1H),1.96-1.84(m, 2H),1.83-1.64(m,4H),1.62-1.39(m,10H),1.28-1.14(m,3H),1.08-0.94(m,2H),0.91(d,J=8.5Hz,1H),0.81(t,J=7.3Hz,3H). HRMS(ESI), C 25 H 35 Cl2O4[M+H] + The calculated m / z value for this compound was 469.1912, and the observed value was 469.1896.

[0118] 4-((6,7-Dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy)butanoic acid (12) (SN072) was synthesized according to Scheme 2 (Figure 18).

[0119] [ka]

[0120] To 4 (100 mg, 0.36 mmol, 1 equiv.) were added aluminum chloride (89 mg, 0.67 mmol, 2 equiv.) and sodium iodide (101 mg, 0.67 mmol, 2 equiv.), and the resulting solid mixture was triturated and stirred at 70 °C for 60 min. Upon completion, the reaction was diluted with dichloromethane and washed with saturated aqueous sodium thiosulfate. The organic fractions were collected and concentrated to give a beige solid, which was then washed multiple times with hexane to give compound 6 as a white solid, which was used in the next step. To a stirred solution of the product from the first step in anhydrous dimethylformamide (1 ml), potassium carbonate (53 mg, 0.39 mmol, 1.1 equiv.) and ethyl 4-bromobutyrate (55 μl, 0.39 mmol, 1.1 equiv.) were added, and the reaction was stirred at 60 °C for 2 h. Upon completion, 4 N NaOH (1 ml) was added to the reaction, and the reaction was stirred at 100 °C for 60 min. Upon completion, the reaction was concentrated and purified by column chromatography using 0-10% methanol in dichloromethane as the eluent to give SN072 as a clear solid (107 mg, 86%). 1 H NMR(300MHz,CDCl3)δ 6.87(s,1H),4.21(t,J=5.9Hz,2H),3.26-3.02(m,1H),2.94-2.56(m,4H),2.40-2.19 (m,3H),2.03-1.90(m,1H),1.74-1.50(m,5H),1.47-1.32(m,1H),1.19-1.00(m,1H). HRMS(ESI), C 18 H 21 Cl2O4[M+H] + The calculated m / z value for this compound was 371.0817, and the observed value was 371.0808.

[0121] Enantiomerically enriched SN-401 isomers were synthesized according to a literature procedure (Cragoe et al., 1982), as shown in Scheme 3, Figure 19. Briefly, racemic compound 7 (1 equiv.) was dissolved with cinchonine (1 equiv.) in a minimal amount of hot DMF and allowed to cool. The precipitated salt was separated (the filtrate used to obtain the opposite enantiomer) and recrystallized five more times from DMF. The salt was then acidified with aqueous HCl and extracted into ether. The ether was evaporated under vacuum to afford enantiomerically enriched (+)-7A in 23% yield, [α]25D + 16.8 °C (c 5, EtOH). Here, the DMF filtrate from the first step, enriched with (-)-7B, was concentrated, acidified with aqueous HCl, extracted into ether, and concentrated to a solid. The resulting solid (1 equiv.) was dissolved in a minimum amount of hot ethanol along with cinchonidine (1 equiv.) and allowed to cool. The precipitated salt was separated and recrystallized five more times from DMF. The salt was then acidified with aqueous HCl and extracted into ether. The ether was evaporated under vacuum to give enantiomerically enriched (-)-7A in 19% yield, [α]25D -15.6 °C (c 5, EtOH). Enantiomerically enriched 7A and 7B were then transformed into the respective phenols (+)-6A and (-)-6B, followed by the desired enantiomerically enriched oxybutyric acid (+)-8A [α]25D. 25D +15.9℃ (c 5, EtOH) and (-)-8B[α] 25D The enantiomerically enriched product was subjected to the same two-step reaction sequence with conversion to -14.5 °C (c 5, EtOH). The H NMR and HRMS of the enantiomerically enriched product are not reported as they are identical to the racemate.

[0122] Example 6: In T2D beta cells and adipocytes, I Cl,SWELL and SWELL1 protein is reduced. SWELL1 / LRRC8a ablation impairs insulin signaling in target tissues and insulin secretion from pancreatic β3 cells, inducing a prediabetic state of glucose intolerance. These recent findings indicate that reduction of SWELL1 may contribute to type 2 diabetes (T2D). To determine whether SWELL1-mediated currents are altered in T2D, we analyzed I currents in freshly isolated pancreatic β3 cells from T2D mice fed a HFD for 5–7 months (Figure 20A) and T2D patients (Figure 20B, Tables 2 and 3 below) compared with non-T2D controls. Cl,SWELL In both mouse and human T2D β cells, the maximum I upon stimulation with hypotonic swelling was measured. Cl,SWELL Current density (measured at +100 mV) is significantly reduced compared to non-T2D controls (83% in mice and 63% in humans, Figures 20C and 20D), similar to the reduction observed in SWELL1 knockout (KO) and knockdown (KO) mice and human β-cells, respectively (Kang et al., 2018). β-cell I in the setting of T2D Cl,SWELL These reductions in VRAC / I in the murine KKN T2D model Cl,SWELL Consistent with previous measurements of I in adipocytes isolated from T2D KKN mice compared with non-T2D controls, these results suggest that I Cl,SWELL Similarly, SWELL1-mediated I was reduced by >50% compared to 100%. Similarly, SWELL1-mediated I was measured in isolated human adipocytes from obese T2D patients (BMI = 52.3, HgbA1c = 6.9%, fasting glucose = 148-151 mg / di). Cl,SWELL showed a trend towards a 50% reduction in I in adipocytes from lean patients compared with previously reported obese non-T2D patients. Cl,SWELL There was no difference between the I and I in both adipose tissues (Figure 20E, Table 4 below). Cl,SWELL IV RAC is an important component of I in the setting of T2D. Cl,SWELLWe investigated whether these reductions in SWELL1 protein expression were associated with reduced SWELL1 protein expression. Indeed, SWELL1 protein is reduced in adipose tissue from T2D KKN mice compared with parental control KKAa mice (Figure 20F). Similarly, SWELL1 protein is lower in adipose tissue from obese T2D patients (BMI = 53.7, HgbA1c = 8.0%, fasting glucose = 183-273 mg / di) compared with adipose tissue from normoglycemic obese patients (BMI = 50.2, HgbA1c = 5.0%, fasting glucose = 84-97 mg / di; Figure 20G, Table 5 below). Furthermore, total SWELL1 protein in diabetic human cadaveric islets shows a trend toward a 50% reduction compared with islets from non-diabetic individuals (Figure 20H, Table 6 below). Taken together, these findings indicate that reduced SWELL1 activity in adipocytes and beta cells (and possibly other tissues) may underlie the insulin resistance and impaired insulin secretion associated with T2D. Furthermore, SWELL1 protein expression is increased in both adipose tissue and liver in the setting of early-stage, hyperglycemic obesity, and shRNA-mediated suppression of this SWELL1 induction exacerbates insulin tolerance and glucose intolerance. Therefore, we speculate that maintaining or inducing SWELL1 expression / signaling in peripheral tissues may support insulin sensitivity and secretion to preserve systemic blood glucose in the setting of T2D.

[0123] [Table 2]

[0124] [Table 3]

[0125] [Table 4]

[0126] [Table 5]

[0127] [Table 6]

[0128] Example 7: SWELL1 protein expression regulates insulin-stimulated Pl3K-AKT2-AS160 signaling. To test whether SWELL1 regulates insulin signaling, we overexpressed Flag-tagged SWELL1 (SWELL1 O / E) in both WT and SWELL1 KO 3T3-F442A adipocytes and measured insulin-stimulated phosphorylated AKT2 (pAKT2) as a readout of insulin sensitivity (Figure 21A). SWELL1 KO 3T3-F442A adipocytes exhibited significantly blunted insulin-mediated pAKT2 signaling compared to WT adipocytes, as previously described (Zhang et al., 2017). This was fully restored by re-expression of SWELL1 in SWELL1 KO adipocytes (KO+SWELL1 O / E, Figure 21A), demonstrating a significant reduction in SWELL1-mediated I in response to hypotonic stimulation. Cl,SWELLThis, along with the restoration of the SWELL1-LRRC8a signaling complex at the plasma membrane (Figure 21B and Figures 27A-C), is consistent with the restoration of the SWELL1-LRRC8a signaling complex at the plasma membrane. Notably, the reduction in total AKT2 protein expression observed in SWELL1 KO adipocytes was not restored by SWELL1 re-expression, indicating that transient changes in SWELL1 protein expression, as opposed to AKT2 protein expression, preferentially regulate insulin-pAKT2 signaling. SWELL1 overexpression in WT adipocytes also increases both basal and insulin-stimulated pAKT2, as well as the downstream phosphorylation of AS160 signaling (pAS160) in WT adipocytes (Figures 21C and 21D). Using anti-FLAG and SWELL1 KO-validated custom-made anti-SWELL1 antibodies, respectively, we confirmed that FLAG-tagged SWELL1 was successfully transported to the plasma membrane when expressed in both WT and SWELL1 KO adipocytes, visualized by immunofluorescence (IF). Overexpression of FLAG-tagged SWELL1 in WT and SWELL1 KO adipocytes resulted in a punctate pattern at the cell periphery, similar to endogenous SWELL1 in WT adipocytes (Figures 27D and 27E). Overall, these data indicate that SWELL1 expression levels regulate insulin-Pl3K-AKT2-AS160 signaling in adipocytes. Furthermore, these data suggest that pharmacological SWELL1 induction in peripheral tissues may enhance insulin signaling and improve whole-body insulin sensitivity and glucose homeostasis in the setting of T2D.

[0129] The small molecule 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (DCPIB, Figure 21E), one of a series of structurally diverse (acylaryloxy)acetic acid derivatives, was synthesized in the late 1970s, studied for its diuretic properties, and evaluated as a potential treatment for cerebral edema in the 1980s. DCPIB is derived from the FDA-approved diuretic, ethacrylic acid, but possesses minimal diuretic activity and instead selectively inhibits VRAC / I. Cl,SWELLIt was used as an inhibitor (Figure 21F) and binds at the constriction point within the SWELL1-LRRC8 hexamer (Figure 21E), with an IC of approximately 5 μM. 50 After demonstrating that SWELL1 is required for normal insulin signaling in adipocytes, we investigated the VRAC / I activation by DCPIB (renamed SN-401 herein). Cl,SWELL We predicted that pharmacological inhibition of SWELL1 would reduce insulin signaling. Unexpectedly, when applied for 96 hours, SN-401 increased SWELL1 protein expression in 3T3-F442A preadipocytes (3-fold control expression, Figure 21G) and adipocytes (1.5-fold control expression, Figure 21I), which was associated with enhanced insulin-stimulated pAKT2 levels (Figures 21H and 21J) and enhanced insulin-stimulated pAS160 levels (Figure 21K). These SN-401-mediated effects on insulin-AKT2-AS160 signaling were absent in SWELL1 KO 3T3-F442A adipocytes, consistent with an on-target SWELL1-mediated mechanism of action of SN-401 (Figures 21H and 21J). SN-401-mediated increase in SWELL1 protein expression was not associated with an increase in SWELL1, LRRC8b, LRRC8c, LRRC8d, or LRRC8e mRNA expression, suggesting a posttranscriptional mechanism for the increased expression of these proteins (Figure 28).

[0130] Example 8: Structure-activity relationship and molecular docking simulations reveal specific SN-401-SWELL1 interactions required for on-target activity. To confirm that the SN-401-induced increase in SWELL1 protein was mediated by direct binding to the SWELL1-LRRC8 channel complex, as opposed to off-target effects, we performed a SN-401-induced increase in SWELL1 protein expression. Cl,SWELLWe designed and synthesized novel SN-401 congeners with subtle structural changes that either maintain or enhance (SN-403, SN-406, SN-407, Figure 22A) or completely eliminate (SN071, SN072, Figure 22A) SN-401 on-target inhibition (Figures 22B and 22C, Figures 29A-29C). During the course of this work, Kern DM and colleagues published a cryo-EM structure of SN-401 / DCPIB bound to a SWELL1 homomer (Kern et al., 2019). This structure revealed that SN-401 binds at the pore constriction of the SWELL1 / LRRC8a homohexamer, where the electronegative SN-401 carboxylate group electrostatically interacts with the R103 residue in one or more of the SWELL1 monomers (Figure 22D). Furthermore, SN-401 appears to stabilize SWELL1 hexamers and was required to obtain resolvable cryo-EM images in lipid-nanodiscs (Kern et al., 2019).

[0131] To characterize the structural features of SN-401 involved in binding to SWELL1-LRRC8, we performed molecular docking simulations of SN-401 and its analogs onto the SWELL1 homohexamer (PDB: 6NZZ) and identified two molecular determinants predicted to be important for SN-401-SWELL1-LRRC8 binding: (1) the length of the carbon chain connecting the anionic carboxylate group, which is predicted to electrostatically interact with one or more R103 guanidine groups (found in SWELL1 / LRRC8a and LRRC8b), and (2) the proper orientation of the hydrophobic cyclopentyl group (conserved at all LRRC8 subunit interfaces) that slides into the hydrophobic cleft at the interface of the LRRC8 monomer (Figure ​(Figure22E). Docking simulations predicted that shortening the carbon chain leading to the carboxylate by two carbons would yield a molecule, SN071, that could either interact with R103 via the carboxylate group (Figure 22F(,)) or the cyclopentyl ring could occupy the hydrophobic cleft (Figure 22F(it)), but could not simultaneously participate in both interactions (Figure 22F, black arrow). Similarly, SN072, an SN-401 analog lacking the butyl group, is predicted to be unable to orient the cyclopentyl group in a position favorable for interaction with the hydrophobic cleft without introducing structural strain into the molecule (Figure 29D, black arrow). Both of these structural modifications, predicted to abolish either the carboxylate-R103 electrostatic bond or the cyclopentyl-hydrophobic pocket bond, were shown to be ineffective in vitro. Cl,SWELL This was sufficient to eliminate inhibitory activity (Figures 22B and 22C). Conversely, extending the carbon chain attached to the carboxylate group by one to three carbons yields compounds predicted to strengthen the R103 electrostatic interaction (Figures 22G, 29E-29G, solid black circles), better orienting the cyclopentyl group to bind within the hydrophobic cleft (Figures 22G, 29E, and 29F, dashed black circles).

[0132] Additional binding interactions of the homologs SN-406 and SN-407 are also predicted along the channel due to the long carbon chain, which provides additional hydrophobic interactions with the side chain carbon of the R103 residue (Figure 22G, Figure 29E, gray dashed lines). This is due to the SN-406 / SN-407 I Cl,SWELL This would be expected to increase inhibitory activity, and indeed this was observed (Figures 22B and 22C, Figures 29A-29C). To further test this drug-channel binding model, the binding model was developed to show that decreasing the electropositivity of the pore constriction by replacing the electropositive R103 with an electronegative glutamic acid residue (E103) increased the inhibitory activity of SN-406 I. Cl,SWELL We overexpressed the R103E mutant SWELL1 construct in a WT background because it was predicted to have reduced inhibitory activity. Consistent with the predictions of this binding model, R103E-expressing HEK cells 、 SN-406 Mediation I CLl,SWELL A reduction in inhibition is shown (Figures 29H and 29I).

[0133] Collectively, these functional and molecular docking experiments suggest that SN-401 and SWELL1-active homologs (SN-403 / 406 / 407) bind to the SWELL1-LRRC8 hexamer both at R103 (via the carboxylate terminus) and at the interface between LRRC8 monomers (via the hydrophobic terminus), stabilizing the closed state of the channel, thereby activating I Cl,SWELL We demonstrate that these compounds inhibit SWELL1-LRRC8 activity. Guided by docking studies and binding models revealing that the SN-401 carboxylate group interacts with the R103 residues of multiple LRRC8 monomers within the hexameric channel and the SN-401 cyclopentyl group binds within the hydrophobic cleft between adjacent monomers, we hypothesize that these SN-40X compounds function as molecular tethers to stabilize the assembly of the SWELL1-LRRC8 hexameric channel. This reduces disassembly and subsequent proteasomal degradation of the SWELL1-LRRC8 complex, thereby enhancing its translocation from the endoplasmic reticulum to the plasma membrane signaling domain and functioning as a pharmacological chaperone.

[0134] Experiment 9: SN-401 and the SWELL1-active homolog SN-406 function as pharmacological chaperones at submicron concentrations. To test this hypothesis, we applied the SWELL1-active compounds SN-401 and SN-406 to 3T3-F442A adipocytes differentiated for 4 days under basal culture conditions, and then measured SWELL1 protein levels after 6 hours of serum starvation. At both 1 and 10 μM, SN-401 and SN-406 significantly enhanced SWELL1 protein levels to 1.5- to 2.3-fold levels compared to vehicle-treated controls, whereas the inactive homologs SN071 and SN072 did not significantly increase SWELL1 protein levels (Figures 23A and 23B). SN-401 and SN-406 also enhanced the plasma membrane (PM) localization of endogenous SWELL1 in preadipocytes compared to vehicle or SN071 (Figures 23C and 23D), consistent with increased endoplasmic reticulum (ER) trafficking and pharmacological chaperone activity of SWELL1. Notably, SN-401 and SN-406 can enhance both SWELL1 protein and trafficking at concentrations as low as 1 μM, demonstrating the EC of SN-401 and SWELL1-active homologs that bind to SWELL1-LRRC8 in the closed or resting state. 50 These data indicate that SN-401 or SN-406 were <1 μM, or an order of magnitude lower than the approximately 10 μM concentration required to inhibit activated SWELL1-LRRC8 (upon hypotonic stimulation). Indeed, applying SN-401 or SN-406 to HEK cells 30 min prior to hypotonic activation at both 1 μM (Figures 23D and 23E) and 250 nM (Figures 23F and 23G) significantly inhibited and delayed subsequent hypotonic SWELL1-LRRC8 activation, in contrast to either vehicle or the inactive SN071 and SN072 compounds (Figures 23D and 23E). These data suggest that the SN-40X compounds bind with higher affinity to the closed state of SWELL1-LRRC8 channels than to the open state, resulting in a greater I Cl,SWELLThis supports the idea that SN-401 putatively stabilizes the closed conformation of the channel to inhibit SWELL1-LRRC8 signaling. Furthermore, these data indicate that SN-401 and its SWELL1-active homolog, SN-40X, function as pharmacological chaperones at concentrations less than one-tenth the concentration required to inhibit activated SWELL1-LRRC8 channels. Indeed, treatment of 3T3-F442A adipocytes with 1 μM SN-401 for 96 h followed by washout also robustly increases insulin-pAKT2 signaling compared to vehicle (Figure 23H).

[0135] Second, glucolipotoxin-associated endoplasmic reticulum (ER) stress in metabolic syndrome impairs SWELL1-LRRC8 assembly and trafficking and promotes SWELL1 protein degradation, thereby contributing to the I in T2D. Cl,SWELL We investigated whether pharmacological chaperones (SN-401-406) might support SWELL1-LRRC8 assembly and rescue SWELL1-LRRC8 from degradation (Figures 20A-20F). In this context, we hypothesized that pharmacological chaperones (SN-401-406) might support SWELL1-LRRC8 assembly and rescue SWELL1-LRRC8 from degradation. To test this concept in vitro, we first treated 3T3-F442A adipocytes with either vehicle, SN-401, SN-406, or SN072, and then subjected these cells to 1 mM palmitate + 25 mM glucose to induce glucolipotoxic stress (Figure 23I). Upon palmitate / glucose treatment, we found that SWELL1 protein was reduced by 50%, consistent with ER stress-mediated SWELL1 degradation, and this reduction was completely prevented by both SWELL1-active SN-401 and SN-406, but not by SWELL1-inactive SN072 (Figure 23I). These data are consistent with the idea that SN-401 and SWELL1-active homologs function as pharmacological chaperones to stabilize SWELL1-LRRC8 assembly and signaling under glucolipotoxic conditions associated with T2D and metabolic syndrome.

[0136] Example 10: SN-401 improves systemic glucose homeostasis in a mouse T2D model by increasing SWELL1 and enhancing insulin sensitivity and secretion. To determine whether SN-401 improves insulin signaling and glucose homeostasis in vivo, we treated two T2D mouse models: obese HFD-fed mice and the polygenic T2D KKN mouse model with SN-401 (5 mg / kg i.p. for 4–10 days). In vivo, SN-401 enhances SWELL1 expression 2.3-fold in the adipose tissue of HFD-fed T2D mice (Figure 24A). Similarly, SN-401 increases SWELL1 expression in the adipose tissue of T2D KKN mice to levels comparable to both non-T2D C57 / B6 mice and the parental KKAa strain (Figure 24B). This restoration of SWELL1 expression is associated with normalized fasting blood glucose (FG), glucose tolerance (GTT), and significantly improved insulin tolerance (ITT) in both HFD-induced T2D mice (Figure 24C) and the polygenic T2D KKAy model (Figures 24D-24F). Notably, treating the control KKAa parental strain with SN-401 at the same treatment dose (5 mg / kg x 4-10 days) does not cause hypoglycemia or alter glucose and insulin tolerance (Figures 24D-24F). Similarly, lean, non-T2D, glucose-challenged mice treated with SN-401 have similar FG, GTT, and ITT compared to vehicle-treated mice (Figures 24G and 24H, and Figures 31A-31C). However, when made insulin-resistant and diabetic after 16 weeks of HFD feeding, these same mice treated with SN-401 (from Figures 24G and 24H) showed significant improvements in FG (Figure 24I), GTT, and ITT (Figure 24J) compared to vehicle. These data indicate that SN-401 restores glucose homeostasis in the setting of T2D but has little effect on glucose homeostasis in non-T2D mice. Importantly, this portends a low risk of inducing hypoglycemia. Despite its significant effect on glucose tolerance, SN-401 was well tolerated during the chronic intraperitoneal infusion protocol, with daily intraperitoneal infusions showing no overt signs of toxicity for up to 8 weeks (Figure 31D).Indeed, the in vivo pharmacokinetics (PK) of SN-401 and SN-406 in mice after intraperitoneal or oral administration of 5 mg / kg of SN-401 or SN-406 showed that plasma concentrations transiently increased to I. Cl,SWELL Inhibitory concentrations were approached (Figures 31E and 31F, intraperitoneal dosing) or I Cl,SWELL Although significantly lower than inhibitory concentrations (Figures 31G and 31H, oral dosing), we demonstrate that SWELL1 maintains concentrations sufficient for pharmacological chaperone activity (>≈100 nM) for over 8-12 h.

[0137] SN-401 has robust, in vitro, and in vivo characteristics that indicate it may be an effective oral therapy for T2D. First, several algorithms designed to identify candidate compounds with oral drug-like physicochemical properties (Lipinski (Lipinski et al., 2001), Veber (Veber et al., 2002), Egan (Egan et al., 2000), MDDR (Oprea, 2000)) indicate that SN-401 has oral drug-like properties compared to currently approved oral T2D drugs (Table 7 below).

[0138] [Table 7]

[0139] Second, in vitro studies show that SN-401 has good Caco-2 cell monolayer permeability and minimal cytochrome p450 isozyme inhibition (Table 8 below). Third, SN-401 has no effect on hERG, human Kv, and delayed rectifier channels. 、 I in guinea pig tuft cells at channel-inhibitory concentrations (approximately 5-10 μM) Cl,SWELLThis is consistent with silica ADMET predictions (Table 7), indicating a low potential for cardiac QT prolongation and arrhythmias. Fourth, in vivo PK studies in mice demonstrated that SN-401 has high oral bioavailability (AUC oral / AUC intravenous = 79%, Figures 31G and 31H, and Table 9 below), and that SN-401 administered via oral gavage to HFD-fed T2D C57 mice at 5 mg / kg / day fully retains therapeutic efficacy in vivo (Figure 31I).

[0140] [Table 8]

[0141] [Table 9]

[0142] To investigate the possible contribution of SN-401-mediated enhancement of insulin secretion from pancreatic β cells, we next measured glucose-stimulated insulin secretion (GSIS) in SN-401-treated mice subjected to a 21-week HFD. We found that the impaired GSIS classically observed in long-term HFD (21-week HFD) mice was significantly improved in SN-401-treated HFD mice based on serum insulin measurements (Figure 24K) and perfused GSIS from isolated islets (Figure 24L), consistent with the predicted effects of SWELL1 induction in pancreatic β cells. Similar results were obtained in perfusion assays performed with SN-401 compared to vehicle-treated T2D KKN mice (Figure 24M). Collectively, these data indicate that SN-401-mediated improvement in systemic glycemia in T2D occurs via enhancement of both peripheral insulin sensitivity and β-cell insulin secretion via SN-401 pharmacological chaperone-mediated SWELL1-LRRC8 gain-of-function, a phenotype opposite to in vivo loss-of-function studies ( Kang et al., 2018 and Zhang et al., 2017 ).

[0143] Example 11: SN-401 improves whole-body insulin sensitivity, tissue glucose uptake, and non-alcoholic fatty liver disease in a mouse model of T2D. To more rigorously evaluate the effects of SN-401 on insulin sensitization and glucose metabolism in T2D mice, we compared euglycemic hyperinsulinemic clamps traced with 3H-glucose and 14C-deoxyglucose in T2D KKN mice treated with SN-401 or vehicle. Consistent with enhanced systemic insulin sensitivity, SN-401-treated T2D KKN mice required higher glucose infusion rates (GIR) to maintain euglycemia compared with vehicle (Figure 25A). Hepatic glucose production from gluconeogenesis and / or glycogenolysis (Ra, glucose appearance rate) was reduced by 40% in SN-401-treated T2D KKN mice at baseline (basal, Figure 25B) and further suppressed by 75% during glucose / insulin infusion (clamp, Figure 25B). These data indicate that SN-401 increases hepatic insulin sensitivity.

[0144] Because SN-401-mediated increases in SWELL1 are expected to enhance insulin-pAKT2-pAS160 signaling, GLUT4 plasma membrane translocation, and tissue glucose uptake, we next measured the effects of SN-401 on glucose uptake in adipose tissue, cardiac muscle, and skeletal muscle using 2-deoxyglucose (2-DG). SN-401 enhanced insulin-stimulated 2-DG uptake in inguinal white adipose tissue (iWAT), gonadal white adipose tissue (gWAT), and cardiac muscle (Figure 25C), but not in brown fat or skeletal muscle (Figure 32A). Because adipocyte SWELL1 ablation significantly reduces insulin-pAKT2-pGSK3-regulated intracellular glycogen content, we next examined whether SN-401-mediated increases in SWELL1 increase glucose incorporation into tissue glycogen in the setting of T2D. Indeed, liver, adipose, and skeletal muscle glucose incorporation into glycogen is significantly increased in SN-401-treated mice (FIG. 25D), consistent with SWELL1-mediated insulin-pAKT2-pGSK3-glycogen synthase gain of function.

[0145] Nonalcoholic fatty liver disease (NAFLD), like T2D, is associated with insulin resistance. NASH is a progressive form of nonalcoholic liver disease defined by three histological features: hepatic steatosis, hepatic lobule inflammation, and hepatocellular injury (ballooning), with or without fibrosis. NAFLD and T2D likely share at least some pathophysiological mechanisms, as more than one-third (37%) of patients with T2D also have NASH, and nearly one-half (44%) of patients with NASH also have T2D. To evaluate the effect of SN-401 on the development of NAFLD, mice were maintained on a HFD for 16 weeks, followed by intermittent administration of SN-401 for 5 weeks (Figure 25E). SN-401-treated mice had reduced absolute and body weight-normalized liver masses and overall smaller livers (Figure 25F), and lower hepatic triglyceride concentrations (Figure 25H) compared with vehicle-treated mice. Histological evaluation showed that SN-401-treated mice had significantly reduced hepatic steatosis and hepatocellular injury compared with vehicle-treated mice (Figures 25F and 25J). SN-401-treated mice had a NAFLD activity score (NAS), which integrates histological scores of hepatic steatosis, lobular inflammation, and hepatocyte ballooning (Kleiner et al., 2014). (2005) (Figure 25I) also improved by >2 points in SN-401-treated mice compared with vehicle-treated mice. Taken together, these data demonstrate that SN-401 enhances SWELL1 protein and SWELL1-mediated signaling to simultaneously enhance both systemic insulin sensitivity and pancreatic β-cell insulin secretion, thereby normalizing systemic glycemia in a T2D mouse model. This improved metabolic state can reduce ectopic lipid deposition and NAFLD associated with obesity and T2D.

[0146] Example 12: SWELL1 active SN-401 analogs improve systemic glucose homeostasis in murine T2D. To determine whether the observed in vivo effects of SN-401 in T2D mice were due to SWELL1-LRRC8 binding, as opposed to off-target effects, we next measured fasting blood glucose and glucose tolerance in HFD T2D mice treated with either SWELL1-active SN-403 or SN-406 compared with SWELL1-inactive SN071 (all at 5 mg / kg / day for 4 days). In mice treated with an HFD for 8 weeks, SN-403 significantly reduced fasting blood glucose and improved glucose tolerance compared with SN071 (Figure 26A). In a cohort of mice fed an HFD for 12 to 18 weeks with more severe obesity-induced T2D, SN-406 also significantly reduced fasting blood glucose and improved glucose tolerance (Figure 26B). Similarly, in a separate experiment, SN-406 significantly improved glucose tolerance in HFD T2D mice compared with SWELL1-inactive SN071 (Figure 26C), which correlated with improved insulin sensitivity based on the homeostasis model assessment of insulin resistance (HOMA-IR) (Matthews et al., 1985) (Figure 26D) and a trend toward significantly enhanced insulin secretion in perfused GSIS (Figure 26E). Finally, based on GTT AUC, SN-407 also improved glucose tolerance and increased GSIS in T2D KKN mice compared with SN071 (Figure 26F) (Figure 26G). These data demonstrate the in vivo antihyperglycemic effect of SN-401, whose bioactive analog requires SWELL1-LRRC8 binding, thus supporting the notion of SWELL1 on-target activity in vivo.

[0147] Example 13: Discussion of Examples 6-12. The current working model is that the transition from compensated obesity (prediabetes, normoglycemia) to decompensated obesity (T2D, hyperglycemia) reflects a relative reduction in SWELL1 protein expression and signaling, particularly in peripheral insulin-sensitive tissues and pancreatic β cells, a metabolically phenotypic SWELL1 loss-of-function model. This contributes to the concomitant development of insulin resistance and impaired insulin secretion associated with poorly controlled T2D and hyperglycemia. SWELL1 forms a macromolecular signaling complex containing heterohexamers of SWELL1 and LRRC8b-e, with a stoichiometry that may vary depending on the tissue. The SWELL1-LRRC8 signaling complex is inherently unstable, and therefore, we propose that a certain proportion of the complex succumbs to disassembly and degradation. Glucolipotoxin and subsequent ER stress associated with the T2D state provide an unfavorable environment for SWELL1-LRRC8 complex assembly, potentially contributing to the SWELL1 protein and SWELL1-mediated I signaling observed in T2D. Cl,SWELL This contributes to SWELL1 degradation and reduction of SWELL1 activity. The small molecules SN-401 and SN-401 homologs, which have conserved SWELL1-binding activity, act as pharmacological chaperones to stabilize the formation of the SWELL1-LRRC8 complex. This reduces SWELL1 degradation and promotes the passage of SWELL1-LRRC8 isomers through the ER and Golgi apparatus to the plasma membrane, thereby correcting SWELL1 deficiency in multiple metabolically important tissues in the setting of T2D and metabolic syndrome, improving overall systemic glycemia through both insulin sensitization and secretory mechanisms. Indeed, the concept of small molecule inhibitors functioning as therapeutic molecular chaperones supporting the folding, assembly, and trafficking of proteins (including ion channels) has been demonstrated for Niemann-Pick disease type C and congenital hyperinsulinism (SUR1-KATP channel mutant). Moreover, this therapeutic mechanism is similar to the small molecule corrector for another chloride channel, CFTR (VX-659 / VX-445, Vertex Pharmaceuticals), which has proven to be a groundbreaking therapeutic approach for cystic fibrosis.

[0148] Through structure-activity relationship (SAR) and silica molecular docking studies, we identified hotspots on opposite ends of the SN-401 molecule that interact with distinct regions of the SWELL1-LRRC8 complex: a carboxylate group bearing R103 within multiple LRRC8 subunits at the constriction within the pore, and a cyclopentyl group within the hydrophobic cleft formed by adjacent LRRC8 monomers. These hotspots function like molecular staples or tethers to bind and stabilize loosely associated SWELL1 homomers into a more rigid hexameric structure, particularly in the setting of T2D. Indeed, cryo-EM structures obtained with lipid nanodiscs required DCPIB / SN-401 binding to obtain images with sufficient spatial resolution (Kern et al., 2019), supporting the notion that SN-401 stabilizes SWELL1 homomers. Another benefit provided by SAR studies was the identification and synthesis of SN-401 congeners with ablated (SN071 / SN072) or enhanced (SN-403 / 406 / 407) SWELL1 binding, providing a powerful tool for directly querying SWELL1 on-target activity in vitro and in vivo and validating proof of concept for developing novel SN-401 congeners with enhanced efficacy.

[0149] The SWELL1-LRRC8 complex is widely expressed in multiple tissues and consists of unknown combinations of SWELL1, LRRC8b, LRRC8c, LRRC8d, and LRRC8e, indicating that the SWELL1 complex is highly heterogeneous. However, SWELL1-LRRC8 stabilizers such as SN-401 can be designed to target many (if not all) possible channel complexes because they all contain elements required for SN-401 binding: at least one R103 (from the carboxyl group binding site, required for SWELL1 monomers) and a hydrophobic cleavage (cyclopentyl binding site) conserved among all LRRC8 monomers. Indeed, traced glucose clamps revealed insulin-sensitizing effects in multiple tissues, including adipose tissue, skeletal muscle, liver, and heart. Increased glucose uptake in the heart is particularly intriguing because it may have beneficial effects on cardiac energy agents, such as those observed with SGLT2 inhibitors, which favorably affect both systolic (HFrEF) and diastolic (HFpEF) function in diabetic cardiomyopathy, thereby potentially improving cardiac outcomes in T2D.

[0150] The current study provides initial proof-of-concept for pharmacological induction of SWELL1 signaling using a SWELL1 modulator (SN-40X congener) to treat metabolic diseases in multiple homeostatic lymph nodes, including adipose tissue, liver, and pancreatic β cells. SN-401 may therefore represent a tool compound from which novel drug classes can be derived to treat T2D, NASH, and other metabolic diseases.

[0151] Example 14: Materials and methods for Examples 15-22. Animals. All mice were housed in a temperature-, humidity-, and light-controlled room and allowed free access to water and food. Male and female SWELL1fl / fl (WT), Myl1Cre;SWELL1 fl / fl (Myl1 KO), Myf5Cre;SWELL1 fl / flBoth Myl1Cre (JAX#24713) and Myf5Cre (JAX#007893) mice (skeletal muscle-targeted SWELL1 KO) were generated and used in these studies. Myl1Cre (JAX#24713) and Myf5Cre (JAX#007893) mice were purchased from the Jackson Laboratory. For high-fat diet (HFD) studies, a Research Diets Inc. (catalog number D12492) (60 kcal% fat) regimen was used, starting at 14 weeks of age.

[0152] Generation of CRISPR / Cas9-mediated SWELL1 flox (SWELL1 fl / fl) mice. SWELL1 fl / fl mice were generated as previously described (Zhang et al., 2017). Briefly, SWELL1 intron sequences were obtained from Ensembl Transcript ID ENSMUST00000139454. All CRISPR / Cas9 sites were identified using ZiFit Target version 4.2. Oligonucleotide pairs corresponding to selected CRISPR-Cas9 target sites were designed, synthesized, annealed, and cloned into the pX330-U6-Chimeric_BB-CBh-hSpCas9 construct (Addgene plasmid #42230) according to the protocol detailed in Cong et al., 2013. CRISPR-Cas9 reagents and ssODNs were injected into the pronuclei of F1 mixed C57 / 129 mouse strain embryos at injection concentrations of 5 ng / μl and 75–100 ng / μl, respectively. Correctly targeted mice were screened by PCR across the predicted loxP insertion site on either side of exon 3. These mice were then backcrossed for >8 generations onto a C57BL / 6 background.

[0153] Antibodies: Rabbit polyclonal anti-SWELL1 antibodies were raised against the epitope QRTKSRIEQGIVDRSE (SEQ ID NO: 13) (Pacific Antibodies). All other primary antibodies were purchased from Cells Signaling: anti-β-actin (#8457s), p-AKT1 (#9018s), Akt1 (#2938s), pAKT2 (#8599s), Akt2 (#3063s), p-AS160 (#4288s), AS160 (#2670s), AMPKα (#5831s), pAMPKα (#2535s), FoxO1 (#2880s) and pFoxO1 (#9464s), p70 S6 kinase (#9202s), p-p70 S6 kinase (#9205s), pS6 ribosomal (#5364s), GAPDH (#5174s), pErk1 / 2 (#9101s), Total Erk1 / 2 (#9102s). Purified mouse anti-Grb2 was purchased from BD (610111s). Purified anti-Flag mouse antibody was purchased from Sigma. Rabbit IgG Santa Cruz (sc-2027). All primary antibodies were used at a 1:1000 dilution, except for anti-Flag, which was at a 1:2000 dilution. All secondary antibodies (anti-rabbit-HRP and anti-mouse-HRP) were used at a 1:10000 dilution.

[0154] Adenovirus: Ad5-CMV-mCherry (1 × 10 10 PFU / ml), Ad5-CMV-Cre-mCherry (3×10 10 Adenovirus type 5 carrying 1 × 10 PFU / ml was obtained from the Iowa viral vector core facility. Ad5-CAG-LoxP-stop-LoxP-3XFlag-SWELL1 (1 × 10 10 Ad5-U6-shGRB2-GFP (1 × 10 PFU / ml) was obtained from Vector Biolabs. 9 PFU / ml) and Ad5-U6-shSCR-GFP (1 × 10 10 PFU / ml) were obtained from Vector Biolabs.

[0155] Electrophysiology. All recordings were performed in the whole-cell configuration at room temperature, as previously described (Zhang et al., 2017 and Kang et al., 2018). Briefly, currents were measured with either an Axopatch 200B amplifier or a MultiClamp 700B amplifier (Molecular Devices) paired to a Digidata 1550 digitizer using pClamp 10.4 software. The intracellular solution contained (in mM): 120 L-aspartate, 20 CsCl, 1 MgCl2, 5 EGTA, 10 HEPES, 5 MgATP, 120 CsOH, 0.1 GTP, pH 7.2 with CsOH. The extracellular solution for hypotonic stimulation contained (in mM): 90% NaCl, 2% CsCl, 1% MgCl2, 1% CaCl2, 10% HEPES, 5% glucose, 5% mannitol, and pH 7.4 with NaOH (210 mOsm / kg). The isotonic extracellular solution contained the same composition as above, except for the mannitol concentration of 105 (300 mOsm / kg). Osmolality was checked using a vapor pressure osmometer 5500 (Wescor). Currents were filtered at 10 kHz and sampled at 100 μs intervals. Patch pipettes were pulled from borosilicate glass capillary tubing (WPI) using a P-87 micropipette puller (Sutter Instruments). Pipette resistance was approximately 4-6 MΩ when the patch pipette was filled with the intracellular solution. The holding potential was 0 mV. Voltage ramps from -100 to +100 mV (0.4 mV / ms) were applied every 4 seconds.

[0156] Primary muscle satellite cell isolation: Satellite cell isolation and differentiation were performed as previously described with minor modifications (Hindi et al., 2017). Briefly, gastrocnemius and quadriceps muscles were cultured using SWELL1. flflMuscle tissue was excised from mice (8-10 weeks old) and washed twice with 1x PBS supplemented with 1% penicillin-streptomycin and fungizone (300 μl / 100 ml). The tissue was incubated in DMEM-F12 medium supplemented with collagenase II (2 mg / ml), 1% penicillin-streptomycin, and fungizone (300 μl / 100 ml) on a shaker for 90 minutes at 37°C. The tissue was washed with 1x PBS and again incubated in DMEM-F12 medium supplemented with collagenase II (1 mg / ml), dispase (0.5 mg / ml), 1% penicillin-streptomycin, and fungizone (300 μl / 100 ml) on a shaker for 30 minutes at 37°C. The tissue was then triturated, passed through a cell strainer (70 μm), and centrifuged. Satellite cells were then placed on BD Matrigel-coated dishes. Cells were stimulated to differentiate into myoblasts in DMEM-F12, 20% fetal bovine serum (FBS), 40 ng / ml basic fibroblast growth factor (bfgf, R&D Systems, 233-FB / CF), 1x non-essential amino acids, 0.14 mM β-mercaptoethanol, 1x penicillin / streptomycin, and fungizone. Myoblasts were maintained in 10 ng / ml bfgf and then differentiated in DMEM-F12, 2% FBS, 1x insulin-transferrin-selenium upon reaching 80% confluence.

[0157] Cell culture: WT C2C12 and SWELL1 KO C2C12 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, Atlanta Bioselect) and the antibiotic 1% penicillin-streptomycin (Gibco, USA) at 37°C and 5% CO2. Cells were grown to 80% confluence and then transferred to differentiation medium DMEM supplemented with antibiotics and 2% horse serum (HS, GIBCO) to induce differentiation. The differentiation medium was changed every two days. Cells were differentiated into myotubes for up to 6 days. Myotube images were then taken for quantification of myotube surface area and fusion index.

[0158] Quantification of myotube morphology, surface area, and fusion index: After differentiation (day 7), cells were imaged using an Olympus IX73 microscope (10x objective, Olympus, Japan). Five to six bright-field images were randomly acquired from each 6-well plate for each experimental condition. Myotube surface area was manually quantified using ImageJ software. Morphometric quantification was performed by an independent observer blinded to the experimental conditions. For the fusion index, differentiated myotubes growing on coverslips were washed with 1x PBS and fixed with 2% PFA. After washing three times with 1x PBS, cells were permeabilized with 0.1% TritonX100 for 5 minutes at room temperature, followed by blocking with 5% goat serum for 30 minutes. Cells were stained with DAPI (1 μM) for 15 minutes. After washing with 1x PBS, coverslips were mounted on slides using ProLong Diamond antifade agent. Cells were imaged under a bright-field and DAPI filter with an Olympus IX73 microscope (10x objective, Olympus, Japan). The fusion index (number of nuclei integrated into myotubes / total number of nuclei present in that field) was analyzed using ImageJ.

[0159] RNA sequencing: RNA quality was assessed using an Agilent BioAnalyzer 2100 by the University of Iowa Institute of Human Genetics, Genomics Division. RNA integrity scores greater than 8 were accepted for RNAseq library preparation. RNA libraries of 150 bp PolyA-enriched RNA were generated and sequenced on a HiSeq 4000 genome sequencing platform (Illumina). Sequencing results were uploaded and analyzed using BaseSpace (Illumina). Sequences were trimmed to 125 bp using the FASTQ Toolkit (version 2.2.0) and aligned to the Mus musculus mmp10 genome using RNA-Seq Alignment (version 1.1.0). Transcripts were assembled, and differential gene expression was determined using Cufflinks Assembly and DE (version 2.1.0). Significantly regulated genes were analyzed using Ingenuity Pathway Analysis (QIAGEN), filtering for cutoffs of >1.5 fragments per million reads, >1.5-fold change in gene expression, and a false discovery rate of <0.05. Heat maps were generated to visualize significantly regulated genes.

[0160] Myotube signaling studies: For insulin stimulation, differentiated C2C12 myotubes were incubated in serum-free medium for 6 hours and stimulated with 0 and 10 nM insulin for 15 minutes. Meanwhile, differentiated primary myotubes were incubated in serum-free medium for 4 hours and stimulated with 0 and 10 nM insulin for 2 hours. To examine intracellular signaling during SWELL1 overexpression (SWELL1 O / E), C2C12 myotubes were overexpressed by transducing Ad5-CAG-LoxP-stop-LoxP-SWELL1-3xFlag (MOI 50-60) and Ad5-CMV-Cre-mCherry (MOI 50-60) in DMEM (2% FBS and 1% penicillin-streptomycin) and polybrene (4 μg / ml) for 36 hours. Ad5-CMV-Cre-mCherry alone (MOI 50-60) with polybrene (4 μg / ml) was transduced into WT C2C12 or SWELL1 KO C2C12 as a control. Viral transduction efficiency (60-70%) was confirmed by mCherry fluorescence. Cells were further differentiated in differentiation medium for up to 6 days. Myotube images were taken before lysate collection for further signaling studies. GRB2 knockdown was achieved by transducing myotubes with Ad5-U6-shSCR-GFP (control, MOI 50-60) or Ad5-U6-shSWELL1-GFP (GRB2 knockdown, MOI 50-60) in DMEM (2% FBS and 1% penicillin-streptomycin) supplemented with polybrene (4 μg / ml) for 24 hours. Cells were further differentiated in differentiation medium for up to 6 days. Before harvesting the cells for RNA isolation, images of differentiated myotubes were taken for myotube surface area quantification.

[0161] Stretch stimulation: C2C12 myotubes were placed in each well of a 6-well BioFlex culture plate. Cells were differentiated in differentiation medium for up to 6 days and then placed in a Flexcell Jr. Tension System (FX-6000T) and incubated at 37°C and 5% CO2. C2C12 myotubes on the flexible membrane were subjected to either no tension or 5% static stretch for 15 minutes. Cells were lysed, and proteins were isolated for subsequent Western blot analysis.

[0162] Western blot: Cells were washed with ice-cold 1x PBS and lysed in ice-cold lysis buffer (150 mM NaCl, 20 mM HEPES, 1% NP-40, 5 mM EDTA, pH 7.5) supplemented with protease / phosphatase inhibitors (Roche). The cell lysate was further sonicated (20% pulse frequency, 20 seconds) and centrifuged at 14,000 rpm for 20 minutes at 4°C. The supernatant was collected and the protein concentration was estimated using a DC protein assay kit (Bio-Rad). For immunoblotting, an appropriate volume of 4x Laemmli (Bio-Rad) sample loading buffer was added to the sample (10–20 μg protein), which was then heated at 90°C for 5 minutes before loading onto a 4–20% gel (Bio-Rad). Proteins were separated using running buffer (Bio-Rad) at 110 V for 2 hours. Proteins were transferred to PVDF membranes (Bio-Rad) and blocked in 5% (w / v) BSA or 5% (w / v) milk in TBST buffer (0.2 M Tris, 1.37 M NaCl, 0.2% Tween-20, pH 7.4) for 1 hour at room temperature. Blots were incubated with primary antibodies overnight at 4°C, followed by secondary antibodies (Bio-Rad, goat anti-mouse #170-5047, goat anti-rabbit #170-6515, all used at 1:10,000) for 1 hour at room temperature. Membranes were washed three times and imaged by chemiluminescence (Pierce) using a Chemidoc imaging system (Bio-Rad). Images were further analyzed for band intensity using ImageJ software. β-actin or GAPDH levels were quantified for equal protein loading.

[0163] Immunoprecipitation: C2C12 myotubes were plated on 10 cm dishes in complete medium and grown to 80% confluence. For SWELL1-3xFlag overexpression, Ad5-CAG-LoxP-stop-LoxP-3XFlag-SWELL1 (MOI 50-60) and Ad5-CMV-Cre-mCherry (MOI 50-60) were added to cells in DMEM medium (2% FBS and 1% penicillin-streptomycin) with polybrene (4 μg / ml) and grown for 36 hours. Cells were then switched to differentiation medium for up to 6 days. Myotubes were then harvested in ice-cold lysis buffer (150 mM NaCl, 20 mM HEPES, 1% NP-40, 5 mM EDTA, pH 7.5) supplemented with protease / phosphatase inhibitors (Roche) and kept on ice for 15 minutes with gentle agitation to allow complete lysis. Lysates were incubated with anti-Flag antibody (Sigma no. F3165) or control rabbit IgG (Santa Cruz sc-2027) spin terminal overnight at 4°C. Protein G Sepharose beads (GE) were added for 4 h, and then samples were centrifuged at 10,000 g for 3 min, washed three times with RIPA buffer, resuspended in Laemmli buffer (Bio-Rad), boiled for 5 min, and separated by SDS-PAGE gel followed by Western blot protocol.

[0164] RNA isolation and quantitative RT-PCR: Differentiated cells were solubilized in TRIzol, and total RNA was isolated using the PureLink RNA kit (Life Technologies) and column DNase digestion kit (Life Technologies). cDNA synthesis, qRT-PCR reactions, and quantification were performed as previously described (Zhang et al., 2017). All experiments were performed in triplicate, and data were normalized using GAPDH as an internal control. All primers used for qRT-PCR are listed in Table 10 below.

[0165] [Table 10]

[0166] Muscle tissue homogenization: Mice were euthanized, and the gastrocnemius muscles were excised and washed with 1x PBS. The muscle tissue was pulverized with a surgical blade and placed in 8 volumes of ice-cold homogenization buffer (20 mM Tris, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1% Triton X-100, 10% (w / v) glycerol, 1 mM EDTA, 1 mM dithiothreitol, pH 7.8) supplemented with protease / phosphatase inhibitors (Roche). The tissue was homogenized on ice using a Dounce homogenizer (40-50 passes) and incubated overnight at 4°C with continuous rotation. The tissue lysate was further sonicated 2-3 times with 20-second cycle intervals and centrifuged at 14,000 rpm for 20 minutes at 4°C. The supernatant was collected for protein concentration estimation using a DC protein assay kit (Bio-Rad). Due to the high content of contractile proteins in this preparation, Coomassie gel staining was performed to demonstrate equal protein loading and normalize Western blot quantification.

[0167] Histology: Mice were anesthetized with isoflurane followed by cervical dislocation. The tibialis anterior (TA) muscle was carefully excised and gently immersed in Tissue-tek OCT medium placed on a wooden cork. The tissue orientation was maintained while embedding in the medium. The wooden cork with the tissue was then gently immersed in a bath of isopentane pre-cooled with liquid N2 for 10–14 seconds and stored at -80°C. Tissue sectioning (10 μm) was performed on a Leica cryostat, and all sections were collected onto positively charged microscope slides for H&E staining as previously described (Bonetto et al., 2015). Briefly, TA section slides were stained with hematoxylin for 2 minutes and eosin for 1 minute, then dehydrated in ethanol and xylene. Slides were then mounted with a coverslip, and images were captured with an EVOS cell imaging microscope (10x objective). For quantification of fiber cross-sectional area, images were processed using ImageJ software to enhance contrast and smooth / sharpen cell boundaries to clearly demarcate muscle fiber cross-sectional area. All measurements were performed by an independent observer blinded to the identity of the slides.

[0168] Exercise stress test and inversion test: The mouse treadmill exercise protocol was adapted from Dougherty et al., 2016. Briefly, mice were first acclimated to a motorized treadmill (Columbus Instruments Exer3 / 6 Treadmill (Columbus, OH)) for 3 days by running continuously at 7 m / min for 10–15 min (3-min intervals) with an electric shock grid (frequency 1 Hz) installed in each lane. During the treadmill test, mice ran while gradually increasing the speed (5.5 m / min–22 m / min) and incline (0°–15°), each at 3-min time intervals. The total running distance of each mouse was recorded at the end of the experiment. The predefined criteria for removing mice from the treadmill and recording their distance traveled were the delivery of 5-s consecutive shocks or 5–6 shocks within a 15-s time interval. These mice were then promptly removed from the treadmill. The mouse was then released, and the total duration and distance were recorded for further analysis. The mouse reversal test was performed using a wire grid screen apparatus elevated to 50 cm. The mouse was stabilized on a 60-degree inclined screen with its head facing the base of the screen. The screen was slowly rotated to 0 degrees (horizontal) so that the mouse was completely inverted and hanging upside down from the screen. Soft bedding was placed under the screen to protect the mouse from injury if it fell. The reversal test for each mouse was repeated twice with a 45-minute interval (rest period). The hanging time for each mouse was repeated three times with a 5-minute interval. The maximum hanging time limit for each mouse was set to 3 minutes.

[0169] Isolated muscle contractility assessment: The soleus muscle was carefully dissected and transferred to a specialized muscle stimulation system (1500A, Aurora Scientific, Aurora, ON, Canada), where physiological testing was performed in a blinded fashion. The muscle was immersed in Ringer's solution (in mM) (NaCl 137, KCl 5, CaCl 22, NaH 2 PO 4 1, NaHCO 3 24, MgSO 4 1, glucose 11, and curare 0.015) maintained at 37°C. The distal tendon was secured to the arm of a dual-mode ergometer (300C-LR, Aurora Scientific, Aurora, ON, Canada) with silk sutures, and the proximal tendon was secured to a fixed post. The muscle was stimulated with an electrical stimulator (701C, Aurora Scientific, Aurora, ON, Canada) using parallel platinum plate electrodes extending along the muscle. The relaxed muscle length was established by increasing the muscle length until a passive force above the transducer noise was detected, and fiber length was measured through a micrometer reticle in the eyepiece of a dissecting microscope. The optimal muscle length was then determined by increasing the muscle length by 10% of the slack fiber length until the isometric tetanic force plateaued. At this optimal length, force was recorded during twitch and isometric tetanic contractions (300 ms trains of 0.3 ms pulses at 225 Hz). The muscle was then fatigued with repeated tetanic contractions every 10 s until the force fell below 50% of its peak. At this point, the muscle was cut from the suture and weighed. This weight determined the peak fiber length and muscle density (1.056 g / cm). 3 ) were used to calculate the physiological cross-sectional area (PCSA) and convert it to specific force (tension). Experimental data were analyzed and quantified using Matlab (Mathworks) and presented as peak tetanic tension (Tenant Tension) - the peak force recorded during tetanus normalized to PCSA, time to fatigue (TTF) - the time for tetanic tension to fall below 50% of its peak value during the fatigue test, and half-relaxation time (HRT) - half the time from peak force to return to baseline during the twitch contraction.

[0170] XF-24 Seahorse assay: Cellular respiration was quantified in primary myotubes using the XF24 Extracellular Flux (XF) Bioanalyzer (Agilent Technologies / Seahorse Bioscience, North Billerica, MA, USA). flfl Primary skeletal muscle cells isolated from mice were cultured at 20 x 10 cells per well. 3 Cells were plated onto BD Matrigel-coated plates at a density of 1000 μg / ml. After 24 hours, cells were incubated in Ad5-CMV-mCherry or Ad5-CMV-Cre-mCherry (MOI 90-100) in DMEM-F12 medium (2% FBS and 1% penicillin-streptomycin) for 24 hours. Cells were then switched to differentiation medium for an additional 3 days. For insulin stimulation, cells were incubated in serum-free medium for 4 hours and stimulated with 0 and 10 nM insulin for 2 hours. The medium was then changed to XF-DMEM and kept in a non-CO2 incubator for 60 minutes. Basal oxygen consumption rate (OCR) was measured in XF-DMEM. Oxygen consumption was then measured after the addition of each of the following compounds: oligomycin (1 μg / ml) (ATP-linked OCR), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 1 μM) (maximum capacity OCR), and antimycin A (10 μM, reserve capacity OCR). For glycolysis stress tests, cells were switched to glucose-free XF-DMEM and kept in a non-CO2 incubator for 60 min before the experiment. Extracellular acidification rates (ECARs) were determined in XF-DMEM and subsequently in these additional conditions: glucose (10 mM), oligomycin (1 μM), and 2-DG (100 mM). Data from Seahorse experiments (normalized to protein) reflect the results of one Seahorse run / condition with six replicates.

[0171] Metabolic phenotype: Body composition (fat and lean mass) of mice was measured by nuclear magnetic resonance (NMR) using an Echo-MRI 3-in-1 analyzer (EchoMRI, LLC). For the glucose tolerance test (GTT), mice were fasted for 6 hours and then intraperitoneally injected with glucose (1 g / kg body weight for lean mice and 0.75 g / kg body weight for HFD mice). Glucose levels were monitored at the indicated times from blood at the tip of the tail using a glucometer (Bayer Healthcare LLC). For the insulin tolerance test (ITT), mice were fasted for 4 hours and then intraperitoneally injected with insulin (Humulin R, 1 U / kg for lean mice and 1.25 U / kg for HFD mice), followed by glucometer measurement at the indicated times.

[0172] Statistics. Data are expressed as mean ± sem. For comparisons between two groups, a two-tailed paired or unpaired Student's t-test was used. For three or more groups, data were analyzed by one-way analysis of variance and Tukey's post-hoc test. For GTT and ITT, a two-way analysis of variance (Anova) was used. A p-value of <0.05 was considered statistically significant. *, **, and *** represent p-values ​​less than 0.05, 0.01, and 0.001, respectively.

[0173] Example 15: SWELL1 is expressed and functional in skeletal muscle and is required for myotube formation. SWELL1 (LRRC8a) is a Cl,SWELLSWELL1-LRRC8 is an essential component of the hexameric ion channel signaling complex that encodes the volume-regulated anion current (VRAC). The SWELL1-LRRC8 complex has been shown to regulate cell volume in response to the application of non-physiological hypotonic extracellular solutions, but the physiological function(s) of this ubiquitously expressed ion channel signaling complex remain unknown. To determine the function of the SWELL1-LRRC8 channel complex in skeletal muscle, we isolated SWELL1 from C2C12 mouse myoblasts using CRISPR / cas9-mediated gene editing as previously described (Zhang et al., 2017 and Kim et al., 2000) and transduced with adenoviral Cre-mCherry (KO) or mCherry alone (WT control) (Zhang et al., 2017). flfl SWELL1 was genetically deleted from primary skeletal muscle cells isolated from mice. SWELL1 protein Western blot confirmed robust SWELL1 ablation in both SWELL1 KO C2C212 myotubes and SWELL1 KO primary skeletal myotubes (Figure 33A). Next, whole-cell patch clamp revealed that the hypotonicity-activated (210 mOsm) outward rectifying current present in WT C2C12 myoblasts was abolished in SWELL1 KO C2C12 myoblasts (Figure 33B), demonstrating the I in skeletal myoblasts. Cl,SWELL We confirmed that SWELL1 is also required for VRAC. Notably, SWELL1 ablation is associated with impaired myotube formation in both C2C12 myoblasts and primary skeletal satellite cells (Figure 33C), with myotube area in C2C12 and skeletal myotubes being reduced by 58% and 45%, respectively, compared to WT. As an alternative measure, myoblast fusion, as assessed by the myotube fusion index (number of nuclei in myotubes / total number of nuclei, Figure 33C), is also significantly reduced by 80% in SWELL1 KO C2C12 compared to WT.

[0174] Example 16: Global transcriptome analysis reveals that SWELL1 ablation blocks myogenic differentiation and dysregulates multiple myogenic signaling pathways. To further characterize the observed SWELL1-dependent impairment in myotube formation in C2C12 and primary muscle cells, we performed genome-wide RNA sequencing (RNA-seq) of SWELL1 KO C2C12 compared to control WT C2C12 myotubes. These transcriptome data revealed clear differences in the overall transcriptional profiles between WT and SWELL1 KO C2C12 myotubes (Figure 33D), accompanied by a significant suppression of numerous skeletal muscle differentiation genes, including Mef2a (0.2-fold), Myl2 (0.008-fold), Myl3 (0.01-fold), Myl4 (0.008-fold), Actc1 (0.005-fold), Tnnc2 (0.005-fold), and Igf2 (0.01-fold) (Figure 33E). Curiously, this suppression of myogenic differentiation is associated with a significant induction of ppargc1α (PGC1α, 14-fold) and PPARγ (3.7-fold). PGC1α and PPARγ are positive regulators of skeletal muscle differentiation, indicating that SWELL1-dependent defects in skeletal muscle differentiation are downstream of PGC1α and PPARγ. Next, to further define the putative pathway dysregulation underlying SWELL1-mediated disruption in myogenesis, we performed pathway analysis on the transcriptome data. We found that numerous signaling pathways essential for myogenic differentiation were disrupted, including insulin (2 × 10-3), MAP kinase (5 × 10-4), PI3K-AKT (1 × 10-4), AMPK (6 × 10-5), integrin (3 × 10-6), mTOR (2 × 10-6), integrin-linked kinase (4 × 10-7), and IL-8 (1 × 10-7) signaling pathways (Figure 33F).

[0175] Example 17: SWELL1 regulates multiple insulin-dependent signaling pathways in skeletal myotubes. Guided by the results of pathway analysis and the fact that skeletal myogenesis and maturation are regulated by insulin, PI3K, AKT, mTOR, and MAPK, we directly examined several insulin-stimulated pathways in WT and SWELL1 KO C2C12 myotubes, including insulin-stimulated AKT2-AS160, FOXO1, and AMPK signaling. Indeed, insulin-stimulated pAKT2, pAS160, pFOXO1, and pAMPK are abrogated in SWELL1 KO myotubes compared to WT C2C12 myotubes (Figures 34A and 34C). Importantly, insulin-AKT-AS160 signaling is also reduced in SWELL1 KO primary skeletal myotubes compared to WT primary myotubes (Figures 34B and 34D), consistent with the observed differentiation block (Figure 33C). This confirms that SWELL1-dependent insulin-AKT and downstream signaling are not unique to immortalized C2C12 myotubes but are conserved in primary skeletal myotubes. Furthermore, a reduction in total AKT2 protein was associated with SWELL1 ablation in both C2C12 and primary skeletal muscle cells, consistent with the 3-fold reduction in AKT2 mRNA expression observed in the RNA sequencing data (Figure 34E). Furthermore, transcription of several key insulin signaling and glucose homeostasis genes, including GLUT4 (SLC2A4, 51-fold), FOXO3 (2-fold), FOXO4 (2.8-fold), and FOXO6 (18-fold), was suppressed by SWELL1 ablation (Figure 34E). Indeed, FOXO signaling is thought to integrate insulin signaling and glucose metabolism in several insulin-sensitive tissues. Collectively, these data indicate that impaired SWELL1-dependent insulin-AKT-AS160-FOXO signaling is associated with the observed defects in myogenic differentiation upon SWELL1 ablation in cultured skeletal myotubes and also predicts putative impairments in skeletal muscle glucose metabolism and oxidative metabolism.

[0176] Example 18: SWELL1 overexpression in SWELL1-depleted C2C12 is sufficient to rescue myogenic differentiation and enhance intracellular signaling above baseline levels. To further verify the SWELL1-mediated effects on muscle differentiation and signal transduction, we re-expressed SWELL1 in SWELL1 KO C2C12 myoblasts (SWELL1 O / E) and then compared WT and SWELL1 KO C2C12 myotubes to examine myotube differentiation and basal activity of multiple intracellular signaling pathways by Western blot, including pAKT1, pAKT2, pAS160, p-p70S6K, pS6K, and pERK1 / 2. Increasing SWELL1 O / E to 2.12-fold WT levels completely rescued myotube development in SWELL1 KO myotubes (Figure 35A) and quantified by restoring SWELL1 KO myotube area to levels above WT (Figure 35B). This rescue of SWELL1 KO myotube development during SWELL1 O / E (Figures 35A and 35B) is associated with either restored (pAS160, AKT2, pAKT1, AKT1, p70S6K) or ultranormal (pAKT2, p-p70S6K, pS6K, pERK1 / 2) signaling compared to WT C2C12 myotubes (Figures 35C and 35D). These data demonstrate that SWELL1 protein expression levels strongly regulate skeletal muscle insulin signaling and myogenic differentiation.

[0177] Example 19: SWELL1-LRRC8 mediates stretch-dependent PI3K-pAKT2-pAS160-MAPK signaling in C2C12 myotubes. Numerous reports have demonstrated that VRAC and its functionally encoded SWELL1-LRRC8 complex are mechanoresponsive in cellular contexts. It is well established that mechanical stretch is a key regulator of skeletal muscle proliferation, differentiation, and hypertrophy, and can be mediated by PI3K-AKT-MAPK signaling and integrin signaling pathways. To determine whether SWELL1 is also required for stretch-mediated AKT and MAP kinase signaling in skeletal myotubes, we subjected WT and SWELL1 KO C2C12 myotubes to 0% or 5% equiaxial stretch using the FlexCell stretch system. Mechanical stretch (5%) is sufficient to stimulate PI3K-AKT2 / AKT1-pAS160-MAPK (ERK1 / 2) signaling in WT C2C12 myotubes in a SWELL1-dependent manner (Figures 36A and 36B). These data position SWELL1-LRRC8 as a coregulator of both insulin- and stretch-mediated PI3K-AKT-pAS160-MAPK signaling.

[0178] Example 20: SWELL1 interacts with GRB2 in C2C12 myotubes and regulates myogenic differentiation. The SWELL1-LRRC8 complex has previously been reported to interact with growth factor receptor-bound 2 (GRB2) and regulate PI3K-AKT signaling in both lymphocytes and adipocytes, whereby GRB2 binds to IRS1 / 2 and negatively regulates insulin signaling. Indeed, GRB2 knockdown enhances insulin-PI3K-MAPK signaling and induces myogenesis and myogenic differentiation genes. To determine whether SWELL1 and GRB2 interact in C2C12 myotubes, we overexpressed C-terminal 3xFlag-tagged SWELL1 in C2C12 cells, followed by immunoprecipitation (IP) with Flag antibody. Consistent with the GRB2-SWELL1 interaction, we observed significant GRB2 enrichment upon Flag IP from lysates of SWELL1-3xFlag expressing C2C12 myotubes (Figure 37A). Based on the idea that SWELL1 titrates GRB2-mediated suppression of AKT / MAPK signaling and that SWELL1 ablation results in unrestricted GRB2-mediated AKT / MAPK inhibition, we next tested whether GRB2 knockdown (KD) could rescue myogenic differentiation in SWELL1 KO C2C12 myotubes. ShRNA-mediated GRB2 KD in SWELL1 KO C2C12 myoblasts (SWELL1 KO / shGRB2, Figure 37B) stimulated myotube formation (Figure 37C) and increased myotube area to levels comparable to those of WT / shSCR (Figures 37C and 37D). Similarly, GRB2 KD in SWELL1 KO C2C12 myotubes induced myogenic differentiation markers IGF1, MyoHCl, MyoHCl1a, and MyoHCIIb compared to both SWELL1 KO / shSCR and WT / shSCR (Figures 37E and 37F). These data are consistent with GRB2 suppression rescuing myotube differentiation in SWELL1 KO C2C12 and support a model in which SWELL1 regulates myogenic differentiation by titrating GRB2-mediated signaling.

[0179] Example 21: Skeletal muscle-targeted SWELL1 knockout mice have reduced skeletal muscle cell size, muscle endurance, and force production ex vivo. To investigate the physiological consequences of SWELL1 ablation in vivo, we used Myf5-Cre mice and SWELL1 fl / fl We generated skeletal muscle-specific SWELL1 KO mice using Cre-LoxP technology by crossbreeding Myf5 KO mice (Myf5 KO, Figure 38A) and confirmed robust skeletal muscle SWELL1 depletion in Myf5 KO gastrocnemius muscle, 12.3-fold lower than WT controls (Figure 38B). Notably, in contrast to the severe impairment in skeletal muscle formation observed in both SWELL1 KO C2C12 and primary skeletal myotubes in vitro (Figures 33, 35, and 37), Myf5 KO mice developed skeletal muscle mass comparable to WT littermates based on Echo / MRI body composition (Figure 38C) and total muscle weight (Figure 38D) and were born at a normal Mendelian ratio (Table 11 below). However, histological examination revealed a 27% reduction in skeletal muscle cell cross-sectional area in Myf5 KO mice compared to WT mice (Figure 38E), indicating a requirement for SWELL1 in skeletal muscle cell size regulation in vivo. This may be due to reduced myotube fusion, as observed in C2C12 and primary skeletal muscle cells in vitro (Figure 33), but occurs to a lesser extent in vivo. These data indicate that the severe impairment in myogenesis observed in vitro may reflect a very early requirement for SWELL1 signaling in skeletal muscle development (before SWELL1 protein removal by Myf5-Cre-mediated SWELL1 recombination) or other fundamental differences in the myogenic differentiation process in vitro and in vivo.

[0180] [Table 11]

[0181] Because insulin signaling is a key regulator of skeletal muscle oxidative capacity and endurance, we next investigated the role of SWELL1 in Myf5 KO mice compared with Myf5 KO mice. fl / flWe examined exercise tolerance in a treadmill test in Myf5 KO mice (WT). Myf5 KO mice exhibited a 14% reduction in exercise capacity compared to age- and sex-matched WT controls (Figure 39A). Hang time in the reversal test was also reduced by 29% in Myf5 KO mice compared to controls, further supporting the reduction in skeletal muscle endurance upon SWELL1 depletion in vivo (Figure 39B). To determine whether these reductions in muscle function in vivo were due to muscle-specific dysfunction, we next performed ex vivo experiments in which soleus muscles were isolated from mice and subjected to twitch / train tests. Peak tetanic tension was observed to be reduced by 15% in Myf5 KO soleus muscles compared to WT controls (Figure 39C), indicating a skeletal muscle autonomic mechanism, with no differences in time to fatigue (TTF, Figure 39D) or time to 50% decay (Figure 39E).

[0182] To determine whether these SWELL1-dependent differences in muscle endurance and force are due to impaired oxidative capacity, we next measured the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in WT and SWELL1 KO primary skeletal muscle cells under basal and insulin-stimulated conditions (Figure 39F). Oxygen consumption in SWELL1 KO primary myotubes was 26% lower than in WT and, in contrast to WT cells, unresponsive to insulin stimulation (Figure 39F), consistent with abrogation of insulin-AKT / ERK1 / 2 signaling upon SWELL1 depletion in skeletal muscle. These relative changes persisted in the presence of complex V and III inhibitors, oligomycin and antimycin A (Figures 39F and 39G), indicating that the insulin-stimulated glycolytic pathway is primarily dysregulated upon SWELL1 depletion. In contrast, FCCP, which maximally uncouples mitochondria, abolished the difference in oxygen consumption between WT and SWELL1 KO primary muscle cells, suggesting that there may be no difference in functional mitochondrial content in SWELL1 KO muscle. To measure glycolysis more directly, we measured the extracellular acidification rate (ECAR) in WT and SWELL1 KO primary myotubes. The insulin-stimulated ECAR increase was abolished in SWELL1 KO compared to WT cells, and these differences persisted regardless of electron transport chain regulators (Figure 39H). These data indicate that SWELL1 regulation of skeletal muscle cell oxygen consumption occurs at the level of glucose metabolism, likely via SWELL1-dependent insulin-PI3K-AKT-AS160-GLUT4 signaling, glucose uptake, and utilization. These findings in primary skeletal muscle cells are supported by the significant transcriptional repression of numerous glycolytic genes, including Aldoa, Eno3, GAPDH, Pfkm, and Pgam2, as well as glucose and glycogen metabolism genes, including Phka1, Phka2, Ppp1r3c, and Gys1, upon SWELL1 ablation in C2C12 myotubes (Figure 41).

[0183] Example 22: Skeletal muscle-targeted SWELL1 ablation impairs systemic glucose metabolism and increases adiposity. Guided by the evidence of impaired insulin-PI3K-AKT-AS160-GLUT4 signaling observed in SWELL1 KO C2C12 and primary myotubes, we next examined systemic glucose homeostasis and insulin sensitivity in WT and Myf5 KO mice by measuring glucose and insulin tolerance. On a normal chow diet, there are no differences in either glucose or insulin tolerance between WT and Myf5 KO mice (Figure 40A). However, over 16–24 weeks, chow-fed Myf5 KO mice developed a 29% increased fat percentage based on body composition measurements compared to WT mice (Figure 40B), with no significant differences in lean mass (Figure 40C) or total body weight (Figure 40C). When Myf5 KO mice are fed a high-fat diet (HFD) for 16 weeks, there is no difference in adiposity observed compared to WT mice (Figure 42), but glucose tolerance is impaired (Figure 40D) and there is mild insulin resistance in HFD Myf5 KO mice compared to WT (Figure 40E).

[0184] Because Myf5 is also expressed in brown fat, these metabolic phenotypes may result from SWELL1-mediated effects in brown fat and associated changes in whole-body metabolism. To exclude this possibility, we performed a dual-injection assay using Myl1-Cre and SWELL1, as Myl1-Cre is restricted to mature skeletal muscle (Figure 43B) and excludes brown fat. fl / fl Mouse (Myl1-Cre, SWELL1 fl / flWe repeated a subset of the above experiments in skeletal muscle-targeted KO mice generated by crossing Myl1 KO mice with Myf5 KO mice (Figure 43A). Similar to Myf5 KO mice, Myl1 KO mice, fed a regular chow diet, have normal glucose tolerance (Figure 43C), but exhibit a 24% reduction in exercise capacity in a treadmill test compared to WT mice (Figure 43D). Myl1 KO mice also exhibit increased visceral fat percentage over time on a regular chow diet, based on a 24% increase in epididymal fat mass normalized to body weight (Figure 43E), with no differences in inguinal adipose tissue, muscle mass (Figure 43F), or total body weight (Figure 43G). These data indicate that impaired skeletal muscle glucose uptake in Myl1 KO and Myf5 KO mice is compensated for by increased adipose glucose uptake and de novo adipogenesis, contributing to a preserved glucose tolerance, at the expense of increased adipose tissue in skeletal muscle-targeted SWELL1 KO mice fed a regular chow diet. However, excess nutrition-induced obesity, and associated impairments in adipose and hepatic glucose disposal, may manifest glucose intolerance and insulin resistance in skeletal muscle-targeted SWELL1 KO mice.

[0185] Example 23: Discussion of Examples 15-22. Our data reveal that the SWELL1-LRRC8 channel complex regulates insulin / stretch-mediated AKT-AS160-GLUT4, MAP kinase, and mTOR signaling in differentiated myoblast cultures, thereby affecting myogenic differentiation, insulin-stimulated glucose metabolism, and oxygen consumption. In vivo, skeletal muscle-targeted SWELL1 KO mice exhibit smaller skeletal muscle cells, impaired muscle endurance and force production, and are prone to adiposity, glucose intolerance, and insulin resistance. Insulin / stretch-mediated PI3K-AKT and mTOR signaling are well-known key regulators of myogenic differentiation, metabolism, and muscle function, and impaired SWELL1-AKT-mTOR signaling may contribute to defects in myogenic differentiation. Indeed, consistent with previous findings and a proposed model in adipocytes in which SWELL1 mediates the interaction between GRB2 and IRS1 to regulate insulin-AKT signaling, SWELL1 also associates with GRB2 in skeletal myotubes, and GRB2 knockdown rescues the impaired myogenic differentiation in SWELL1-KO adipocyte myocytes. Thus, our working model for SWELL1-mediated regulation of insulin-PI3K-AKT and downstream signaling in adipocytes appears to be conserved in skeletal myotubes. The in vitro phenotypes observed in CRISPR / cas9-mediated SWELL1-KO C2C12 myotubes and SWELL1-KO primary myotubes are consistent with the observations of Chen et al., 2019, who used siRNA-mediated SWELL1 knockdown to demonstrate that the SWELL1-LRRC8 channel complex is required for myogenic differentiation. However, the ability of both GRB2 KD and SWELL1 O / E to rescue myogenic differentiation and insulin-AKT, MAP kinase, and mTOR signaling in SWELL1 KO myotubes implies an atypical, non-conductive signaling mechanism. Based on our and previous studies, SWELL1 O / E is a key player in the I Cl,SWELLAlthough SWELL1 does not increase SWELL1 / VRAC to supranormal levels, pAKT, pERK1 / 2, and mTOR levels are enhanced to two to three times the endogenous levels upon 2x SWELL1 O / E in C2C12 myotubes. These data indicate that alternative / atypical signaling mechanisms, as opposed to typical / conductive signaling mechanisms, underlie SWELL1-LRRC8 signaling.

[0186] It is also noteworthy that the profound myogenic differentiation block observed upon SWELL1 ablation in both C2C12 and primary myotubes is significantly milder in vivo, with only a 30% reduction in skeletal muscle cell cross-sectional area observed in Myf5 KO mice, without any changes in total muscle mass or lean content. This phenotypic discrepancy may reflect fundamental differences in the biology of skeletal muscle differentiation in in vitro and in vivo environments.

[0187] While overall muscle development is largely intact in both Myl1 KO and Myf5 KO mice, there is a consistent reduction in exercise capacity, muscle endurance, and force production, as well as a trend toward increased adiposity over time compared to age- and sex-matched controls. The observed impairment in exercise capacity in skeletal muscle SWELL1 KO mice is consistent with a level of insulin resistance, as in db / db mice and humans, and may be due to impaired skeletal muscle glycolysis and oxygen consumption in SWELL1-depleted skeletal muscle. Furthermore, the increased gonadal adiposity observed in Myl1 KO and Myf5 KO mice while maintaining glucose and insulin tolerance mimics both skeletal muscle-specific insulin receptor KO mice (MIRKO) and transgenic mice expressing a skeletal muscle dominant-negative insulin receptor mutant, suggesting that skeletal muscle-specific insulin resistance drives glucose redistribution from skeletal muscle to adipose tissue to promote adiposity. In the case of Myf5 KO mice, this underlying mild insulin resistance and glucose intolerance are not obscured by overnutrition and HFD feeding. Recent findings from skeletal muscle-specific AKT1 / AKT2 double KO mice indicate that these effects are not solely due to muscle AKT signaling but may potentially involve other insulin-sensitive signaling pathways.

[0188] In summary, we show that SWELL1-LRRC8 regulates myogenic differentiation in myotubes and insulin-PI3K-AKT-AS160, ERK1 / 2, and mTOR signaling via GRB2-mediated signaling. In vivo, SWELL1 is required for maintaining normal exercise capacity, muscle endurance, basal adiposity, and systemic glycemia in the setting of overnutrition. These findings further contribute to our understanding of the SWELL1-LRRC8 channel complex in regulating whole-body metabolism.

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[0190] When introducing elements of the invention or preferred embodiment(s) thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0191] In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.

[0192] Because various changes can be made in the compositions and methods described above without departing from the scope of the invention, it is intended that all subject matter contained in the above description and shown in the accompanying drawings be interpreted as illustrative and not in a limiting sense.

Claims

1. A compound of formula I, or a salt thereof, 【Chemistry 1】 During the ceremony, R １ and R ２ However, each independently, 【Chemistry 2】 selected from the group consisting of R ３ But -Y-C(O)R ４ , -Z-N(R ５ ) (R ６ ), or -Z-A, R ４ is hydrogen, substituted or unsubstituted alkyl, —OR ７ , or -N(R ８ ) (R ９ ) and X １ and X ２ are each independently hydrogen, substituted or unsubstituted alkyl, halo, -OR １０ , or -N(R １１ ) (R １２ ) and R ５ , R ６ , R ７ , R ８ , R ９ , R １０ , R １１ , and R １２ are each independently hydrogen or substituted or unsubstituted alkyl; Y is an unsubstituted carbon-containing moiety having at least two carbon atoms; X １ and X ２ is halo, then Y is not alkylene having 3 carbon atoms; Z is a substituted or unsubstituted carbon-containing moiety having at least two carbon atoms; A is, 【Transformation 3】 n is 1 or 2; compound, or a salt thereof.

2. R １ is butyl, or R ２ The compound of claim 1 , wherein is cyclopentyl.

3. R １ The compound of claim 1 , wherein is butyl.

4. R ２ The compound of claim 1 , wherein is cyclopentyl.

5. R ３ but, 【Chemistry 4】 The compound according to any one of claims 1 to 4, selected from the group consisting of:

6. R ４ But, -OR ７ or -N(R ８ ) (R ９ 6. The compound according to claim 1, wherein

7. A is, 【Transformation 5】 The compound according to any one of claims 1 to 6, selected from the group consisting of:

8. Y and Z are each independently an alkylene having 2 to 10 carbon atoms, an alkenylene having 2 to 10 carbon atoms, or an arylene; X １ and X ２ is halo, then Y is not alkylene having 3 carbon atoms, or Y and Z are each independently an alkylene having 2 to 10 carbon atoms, an alkenylene having 2 to 10 carbon atoms, or a phenylene; X １ and X ２ is halo, then Y is not alkylene having 3 carbon atoms, or Y and Z are each independently a cycloalkylene having 4 to 10 carbons; or Y and Z are each independently 【Transformation 6】 and X is selected from the group consisting of １ and X ２ is halo, then Y is not alkylene having 3 carbon atoms, or Y is an alkylene or alkenylene having 3 to 8 carbons or 3 to 7 carbons, and X １ and X ２ is halo, then Y is not alkylene having 3 carbon atoms, or Y is an alkylene or alkenylene having 4 carbons; or When Y is alkylene having 2 to 3 carbons, X １ and X ２ are each substituted or unsubstituted alkyl, or Z is an alkylene having 2 to 4 carbons, or The compound of any one of claims 1 to 7, wherein Z is an alkylene having 3 or 4 carbons.

9. X １ and X ２ are each independently substituted or unsubstituted C1-C6 alkyl or halo; X １ and X ２ is halo, then Y is not alkylene having 3 carbon atoms, or X １ and X ２ are each independently C1-C6 alkyl, fluoro, chloro, bromo, or iodo; X １ and X ２ is halo, then Y is not alkylene having 3 carbon atoms, or X １ and X ２ are each independently methyl, fluoro, or chloro; １ and X ２ The compound of any one of claims 1 to 8, wherein when is halo, Y is not alkylene having 3 carbon atoms.

10. R ５ , R ６ , R ７ , R ８ , R ９ , R １０ , R １１ , and R １２ is each independently hydrogen or C1-C3 alkyl. 【Request Item 11】 【Chemistry 7】 The compound according to any one of claims 1 to 10, selected from the group consisting of:

12. the compound modulates or inhibits the SWELL1 channel; 12. The compound of any one of claims 1 to 11, wherein the compound may have greater potency in modulating or inhibiting SWELL1 channels than an equivalent amount of DCPIB (4[2[butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid).

13. A pharmaceutical composition for use in a subject in need of increasing insulin sensitivity and / or treating obesity, type 1 diabetes, type 2 diabetes, non-alcoholic fatty liver disease, metabolic disease, hypertension, stroke, vascular tone, and systemic and / or pulmonary arterial pressure and / or blood flow, said pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 12; or A pharmaceutical composition for use in a subject in need of treatment for an immune deficiency caused by insufficient or inappropriate SWELL1 activity, said pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 12, The immune deficiency may include agammaglobulinemia, or A pharmaceutical composition for use in a subject in need of treatment for infertility caused by insufficient or inappropriate SWELL1 activity, said pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 12, The infertility may be male infertility caused by abnormal sperm development due to the insufficient or inappropriate SWELL1 activity, or A pharmaceutical composition for use in a subject in need of treating or restoring athletic performance and / or improving muscle endurance, said pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 12, or A pharmaceutical composition for use in a subject in need of modulating myogenic differentiation in myotubes and insulin-P13K-AKT-AS160, ERK1 / 2 and mTOR signaling, said pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 12; or A pharmaceutical composition for use in a subject in need of treatment for a muscle disorder, said pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 12, The pharmaceutical composition, wherein the muscle disorder may include skeletal muscle atrophy.

14. administration of said pharmaceutical composition is sufficient to upregulate the expression of SWELL1 or alter the expression of a SWELL1-associated protein; or administration of the pharmaceutical composition is sufficient to stabilize the SWELL1-LRRC8 channel complex or a SWELL1-associated protein; or administration of the pharmaceutical composition is sufficient to promote membrane trafficking and activity of the SWELL1-LRRC8 channel complex or a SWELL1-associated protein; or The pharmaceutical composition of claim 13, wherein administration of the pharmaceutical composition is sufficient to enhance SWELL1-mediated signaling.

15. 15. The pharmaceutical composition of claim 14, wherein the SWELL1-associated protein is selected from the group consisting of LRRC8, GRB2, Cav1, IRS1, and IRS2.

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