Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors.

Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic ß-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

Growth differentiation factor 15 as a potential therapeutic for treating obesity.

BACKGROUND: Obesity is rapidly becoming one of the world's most critical health care concerns. Comorbidities accompanying excess weight include cardiovascular disease, diabetes, and certain cancers. These comorbidities result in greater hospitalization and other health care-related costs. Economic impacts are likely to be felt more acutely in developing countries, where obesity rates continue to rise and health care resources are already insufficient. Some of the more effective treatments are invasive and expensive surgeries, which some economies in the world cannot afford to offer to a broad population. Pharmacological therapies are needed to supplement treatment options for patients who cannot, or will not, undergo surgical treatment. However, the few drug therapies currently available have either limited efficacy or safety concerns. A possible exception has been glucagon-like peptide-1 analogs, although these have shown a number of adverse events. New drug therapies that are safe and produce robust weight loss are needed. SCOPE OF REVIEW: Herein, we review the role of growth differentiation factor 15 (GDF15) in feeding behavior and obesity, summarize some of the new and exciting biological discoveries around signaling pathways and tissue sites of action, and highlight initial efforts to develop GDF15-based therapies suitable for inducing weight loss in humans. MAJOR CONCLUSIONS: Within the last several years, great strides have been made in understanding the biology of GDF15. Recent developments include identification of an endogenous receptor, biological localization of the receptor system, impact on energy homeostasis, and identification of molecules suitable for administration to humans as anti-obesity treatments. New and exciting research on GDF15 suggests that it holds promise as a novel obesity treatment as new molecules progress toward clinical development.

GIPR antagonist antibodies conjugated to GLP-1 peptide are bispecific molecules that decrease weight in obese mice and monkeys.

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) regulate glucose and energy homeostasis. Targeting both pathways with GIP receptor (GIPR) antagonist antibody (GIPR-Ab) and GLP-1 receptor (GLP-1R) agonist, by generating GIPR-Ab/GLP-1 bispecific molecules, is an approach for treating obesity and its comorbidities. In mice and monkeys, these molecules reduce body weight (BW) and improve many metabolic parameters. BW loss is greater with GIPR-Ab/GLP-1 than with GIPR-Ab or a control antibody conjugate, suggesting synergistic effects. GIPR-Ab/GLP-1 also reduces the respiratory exchange ratio in DIO mice. Simultaneous receptor binding and rapid receptor internalization by GIPR-Ab/GLP-1 amplify endosomal cAMP production in recombinant cells expressing both receptors. This may explain the efficacy of the bispecific molecules. Overall, our GIPR-Ab/GLP-1 molecules promote BW loss, and they may be used for treating obesity.

The evolving systemic biomarker milieu in obese ZSF1 rat model of human cardiometabolic syndrome: Characterization of the model and cardioprotective effect of GDF15.

Cardiometabolic syndrome has become a global health issue. Heart failure is a common comorbidity of cardiometabolic syndrome. Successful drug development to prevent cardiometabolic syndrome and associated comorbidities requires preclinical models predictive of human conditions. To characterize the heart failure component of cardiometabolic syndrome, cardiometabolic, metabolic, and renal biomarkers were evaluated in lean and obese ZSF1 19- to 32-week-old male rats. Histopathological assessment of kidneys and hearts was performed. Cardiac function, exercise capacity, and left ventricular gene expression were also analyzed. Obese ZSF1 rats exhibited multiple features of human cardiometabolic syndrome by pathological changes in systemic renal, metabolic, and cardiovascular disease circulating biomarkers. Hemodynamic assessment, echocardiography, and decreased exercise capacity confirmed heart failure with preserved ejection fraction. RNA-seq results demonstrated changes in left ventricular gene expression associated with fatty acid and branched chain amino acid metabolism, cardiomyopathy, cardiac hypertrophy, and heart failure. Twelve weeks of growth differentiation factor 15 (GDF15) treatment significantly decreased body weight, food intake, blood glucose, and triglycerides and improved exercise capacity in obese ZSF1 males. Systemic cardiovascular injury markers were significantly lower in GDF15-treated obese ZSF1 rats. Obese ZSF1 male rats represent a preclinical model for human cardiometabolic syndrome with established heart failure with preserved ejection fraction. GDF15 treatment mediated dietary response and demonstrated a cardioprotective effect in obese ZSF1 rats.

2-amino-1,3-thiazol-4(5H)-ones as potent and selective 11beta-hydroxysteroid dehydrogenase type 1 inhibitors: enzyme-ligand co-crystal structure and demonstration of pharmacodynamic effects in C57Bl/6 mice.

11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) has attracted considerable attention during the past few years as a potential target for the treatment of diseases associated with metabolic syndrome. In our ongoing work on 11beta-HSD1 inhibitors, a series of new 2-amino-1,3-thiazol-4(5 H)-ones were explored. By inserting various cycloalkylamines at the 2-position and alkyl groups or spirocycloalkyl groups at the 5-position of the thiazolone, several potent 11beta-HSD1 inhibitors were identified. An X-ray cocrystal structure of human 11beta-HSD1 with compound 6d (Ki=28 nM) revealed a large lipophilic pocket accessible by substitution off the 2-position of the thiazolone. To increase potency, analogues were prepared with larger lipophilic groups at this position. One of these compounds, the 3-noradamantyl analogue 8b, was a potent inhibitor of human 11beta-HSD1 (Ki=3 nM) and also inhibited 11beta-HSD1 activity in lean C57Bl/6 mice when evaluated in an ex vivo adipose and liver cortisone to cortisol conversion assay.

Development of 11beta-HSD1 inhibitors for the treatment of type 2 diabetes.

Glucocorticoid action is linked to the development of obesity and insulin resistance. Inhibition of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) has been proposed as a strategy to suppress glucocorticoid action in a tissue-specific manner. A large variety of 11beta-HSD1 inhibitor classes are under investigation by the pharmaceutical industry to treat type 2 diabetes and obesity.

FGF21 acts as a negative regulator of bile acid synthesis.

Fibroblast growth factor 21 (FGF21) is a potent regulator of glucose and lipid homeostasis in vivo; its most closely related subfamily member, FGF19, is known to be a critical negative regulator of bile acid synthesis. To delineate whether FGF21 also plays a functional role in bile acid metabolism, we evaluated the effects of short- and long-term exposure to native FGF21 and long-acting FGF21 analogs on hepatic signal transduction, gene expression and enterohepatic bile acid levels in primary hepatocytes and in rodent and monkey models. FGF21 acutely induced ERK phosphorylation and inhibited Cyp7A1 mRNA expression in primary hepatocytes and in different rodent models, although less potently than recombinant human FGF19. Long-term administration of FGF21 in mice fed a standard chow diet resulted in a 50-60% decrease in bile acid levels in the liver and small intestines and consequently a 60% reduction of bile acid pool size. In parallel, colonic and fecal bile acid was decreased, whereas fecal cholesterol and fatty acid excretions were elevated. The long-acting FGF21 analog showed superiority to recombinant human FGF21 and FGF19 in decreasing bile acid levels with long duration of effect action in mice. Long-term administration of the long-acting FGF21 analogs in obese cynomolgus monkeys suppressed plasma total bile acid and 7α-hydroxy-4-cholesten-3-one levels, a biomarker for bile acid synthesis. Collectively, these data reveal a previously unidentified role of FGF21 in bile acid metabolism as a negative regulator of bile acid synthesis.

Anti-obesity effects of GIPR antagonists alone and in combination with GLP-1R agonists in preclinical models.

Glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) has been identified in multiple genome-wide association studies (GWAS) as a contributor to obesity, and GIPR knockout mice are protected against diet-induced obesity (DIO). On the basis of this genetic evidence, we developed anti-GIPR antagonistic antibodies as a potential therapeutic strategy for the treatment of obesity and observed that a mouse anti-murine GIPR antibody (muGIPR-Ab) protected against body weight gain, improved multiple metabolic parameters, and was associated with reduced food intake and resting respiratory exchange ratio (RER) in DIO mice. We replicated these results in obese nonhuman primates (NHPs) using an anti-human GIPR antibody (hGIPR-Ab) and found that weight loss was more pronounced than in mice. In addition, we observed enhanced weight loss in DIO mice and NHPs when anti-GIPR antibodies were codosed with glucagon-like peptide-1 receptor (GLP-1R) agonists. Mechanistic and crystallographic studies demonstrated that hGIPR-Ab displaced GIP and bound to GIPR using the same conserved hydrophobic residues as GIP. Further, using a conditional knockout mouse model, we excluded the role of GIPR in pancreatic ß-cells in the regulation of body weight and response to GIPR antagonism. In conclusion, these data provide preclinical validation of a therapeutic approach to treat obesity with anti-GIPR antibodies.

The role of tyrosine 177 in human 11beta-hydroxysteroid dehydrogenase type 1 in substrate and inhibitor binding: an unlikely hydrogen bond donor for the substrate.

The catalytic motif (YSASK) at the active site of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is conserved across different species. The crystal structures of the human, guinea pig and mouse enzymes have been resolved to help identify the non-conserved residues at the active site. A tyrosine residue (Y177) upstream of the catalytic motif in human 11beta-HSD1 represents the largest difference at the active sites between the human and the rodent enzyme where the corresponding residue is glutamine. Although Y177 was postulated as a potential hydrogen bond donor in substrate binding in crystal structure-based modeling, no experimental evidence is available to support this notion. Here, we report that Y177 is not a hydrogen bond donor in substrate binding because removal of the hydroxyl group from its side chain by mutagenesis (Y177F) did not significantly change the Km value for cortisone. However, removal of the hydrophobic side chain by changing tyrosine to alanine (Y177A) or substitution with a hydrophilic side chain by changing tyrosine to glutamine (Y177Q) increased Km values for cortisone. These data suggest that Y177 is involved in substrate binding through its hydrophobic side chain but not by hydrogen bonding. In addition, the three mutations had little effect on the binding of the rodent substrate 11-dehydrocorticosterone, suggesting that Y177 does not confer substrate specificity. However, the same mutations reduced the affinity of the licorice derived 11beta-HSD1 inhibitor glycyrrhetinic acid by about 6- to 10-fold. Interestingly, the affinity of carbenoxolone, the hemisuccinate ester of glycyrrhetinic acid with a similar potency against the wildtype enzyme, was not drastically affected by the same mutations at Y177. These data suggest that Y177 has a unique role in inhibitor binding. Molecular modeling with glycyrrhetinic acid led to findings consistent with the experimental data and provided potential interaction mechanisms. Our data suggest that Y177 plays an important role in both substrate and inhibitor binding but it is unlikely a hydrogen bond donor for the substrate.

The selectivity of tyrosine 280 of human 11beta-hydroxysteroid dehydrogenase type 1 in inhibitor binding.

11beta-Hydroxysteroid dehydrogenase type 1 is a homodimer where the carboxyl terminus of one subunit covers the active site of the dimer partner. Based on the crystal structure with CHAPS, the carboxyl terminal tyrosine 280 (Y280) has been postulated to interact with the substrate/inhibitor at the binding pocket of the dimer partner. However, the co-crystal structure with carbenoxolone argues against this role. To clarify and reconcile these findings, here we report our mutagenesis data and demonstrate that Y280 is not involved in substrate binding but rather plays a selective role in inhibitor binding. The involvement of Y280 in inhibitor binding depends on the inhibitor chemical structure. While Y280 is not involved in the binding of carbenoxolone, it is critical for the binding of glycyrrhetinic acid.

2-(S)-phenethylaminothiazolones as potent, orally efficacious inhibitors of 11beta-hydroxysteriod dehydrogenase type 1.

11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is the enzyme that converts cortisone to cortisol. A growing body of evidence suggests that selective inhibition of 11beta-HSD1 could potentially treat metabolic syndrome as well as type 2 diabetes. Through modification of our initial lead 1, we have discovered trifluoromethyl thiazolone 17. This compound had a Ki of 22 nM, possessed low in vivo clearance, and showed a 91% inhibition of adipose 11beta-HSD1 enzymatic activity in a mouse ex vivo pharmacodynamic model.

High capacity homogeneous non-radioactive cortisol detection assays for human 11beta-hydroxysteroid dehydrogenase type 1.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion of inert glucocorticoid (cortisone) to the active glucocorticoid (cortisol) and is enriched in liver and fat tissues. Increasing evidence suggests that selective inhibition of 11beta-HSD1 may reduce the excess glucocorticoid levels that underlie the etiology of many common disorders that constitute the metabolic syndrome. Measurement of 11beta-HSD1 activity has historically involved the detection of cortisol by methods unfavorable for large-scale screening, such as high performance liquid chromatography or thin layer chromatography. Here we describe the development and validation of novel homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) and electrochemiluminescence assays for the measurement of cortisol. These non-radioactive assays were easy to perform and produced robust results with reference compound values comparable to those obtained by conventional methods. The TR-FRET assay was easily automated and was successfully employed for the high-throughput screening of a large compound library for inhibitors of purified human recombinant 11beta-HSD1.

Long-acting MIC-1/GDF15 molecules to treat obesity: Evidence from mice to monkeys.

In search of metabolically regulated secreted proteins, we conducted a microarray study comparing gene expression in major metabolic tissues of fed and fasted ob/ob mice and C57BL/6 mice. The array used in this study included probes for ~4000 genes annotated as potential secreted proteins. Circulating macrophage inhibitory cytokine 1 (MIC-1)/growth differentiation factor 15 (GDF15) concentrations were increased in obese mice, rats, and humans in comparison to age-matched lean controls. Adeno-associated virus-mediated overexpression of GDF15 and recombinant GDF15 treatments reduced food intake and body weight and improved metabolic profiles in various metabolic disease models in mice, rats, and obese cynomolgus monkeys. Analysis of the GDF15 crystal structure suggested that the protein is not suitable for conventional Fc fusion at the carboxyl terminus of the protein. Thus, we used a structure-guided approach to design and successfully generate several Fc fusion molecules with extended half-life and potent efficacy. Furthermore, we discovered that GDF15 delayed gastric emptying, changed food preference, and activated area postrema neurons, confirming a role for GDF15 in the gut-brain axis responsible for the regulation of body energy intake. Our work provides evidence that GDF15 Fc fusion proteins could be potential therapeutic agents for the treatment of obesity and related comorbidities.

A high-throughput microfluidic assay for SH2 domain-containing inositol 5-phosphatase 2.

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) is a potential drug target for the treatment of type 2 diabetes. This enzyme serves as a negative regulator of insulin-mediated signal transduction by catalyzing the dephosphorylation of the second messenger lipid molecule phosphatidylinositol 3,4,5-triphosphate. Traditionally, assays for phosphoinositide phosphatases such as SHIP2 have relied on radiolabeled phosphatidylinositol-containing lipid membranes and chromatographic separation of labeled phospholipid substrate from product by thin-layer chromatography. We have expressed and purified catalytically active phosphatase domain constructs of SHIP2 from Escherichia coli and developed a sensitive and antibody- or binding protein-independent assay for SHIP2 amenable to high-throughput screening of phosphoinositide phosphatases or phosphoinositide kinases. This microfluidic assay, with Z' values approximately 0.8, is based upon the difference in mobility within an electric field between a fluorophore-labeled phosphatidylinositol 3,4,5-triphosphate substrate and the corresponding 3,4-bisphosphate product. High-throughput screening of a 91,060-member compound library in 384-well format resulted in the identification of SHIP2 inhibitors.

Molecular targeting of the GK-GKRP pathway in diabetes.

INTRODUCTION: Type 2 diabetes mellitus is a major healthcare concern. Significant efforts are being devoted toward developing new, safe, and more effective treatments. One approach involves activating glucokinase (GK). Earlier GK activator (GKA) approaches have focused on direct activation of GK through allosteric activators. AREAS COVERED: This review summarizes the roles of GK and its key partner glucokinase regulatory protein in glucose metabolism and describes approaches that may alleviate hypoglycemic risk observed with GKAs. EXPERT OPINION: The current GKA therapeutic approaches are associated with disappointing success rates. In rodent animal models, efficacy was observed with GKA. However, in all human studies, GKAs effectively lowered blood glucose, but at the expense of an increased risk of hypoglycemia. Other liabilities like loss of efficacy with time and increase in blood pressure or triglyceride levels have been reported with different molecules. To avoid hypoglycemic risk, alternative approaches to regulate GK activity have been initiated. Data from clinical trials using these agents are either not yet available to the public or the compounds are too early in development for humans. GK is a promising target for antidiabetic therapy. Despite encouraging biology, more research is required to fully understand GK as a drug target.

Small Molecule Disruptors of the Glucokinase-Glucokinase Regulatory Protein Interaction: 5. A Novel Aryl Sulfone Series, Optimization Through Conformational Analysis.

The glucokinase-glucokinase regulatory protein (GK-GKRP) complex plays an important role in controlling glucose homeostasis in the liver. We have recently disclosed a series of arylpiperazines as in vitro and in vivo disruptors of the GK-GKRP complex with efficacy in rodent models of type 2 diabetes mellitus (T2DM). Herein, we describe a new class of aryl sulfones as disruptors of the GK-GKRP complex, where the central piperazine scaffold has been replaced by an aromatic group. Conformational analysis and exploration of the structure-activity relationships of this new class of compounds led to the identification of potent GK-GKRP disruptors. Further optimization of this novel series delivered thiazole sulfone 93, which was able to disrupt the GK-GKRP interaction in vitro and in vivo and, by doing so, increases cytoplasmic levels of unbound GK.

Discovery and Structure-Guided Optimization of Diarylmethanesulfonamide Disrupters of Glucokinase-Glucokinase Regulatory Protein (GK-GKRP) Binding: Strategic Use of a N → S (nN → σ*S-X) Interaction for Conformational Constraint.

The HTS-based discovery and structure-guided optimization of a novel series of GKRP-selective GK-GKRP disrupters are revealed. Diarylmethanesulfonamide hit 6 (hGK-hGKRP IC50 = 1.2 µM) was optimized to lead compound 32 (AMG-0696; hGK-hGKRP IC50 = 0.0038 µM). A stabilizing interaction between a nitrogen atom lone pair and an aromatic sulfur system (nN → σ*S-X) in 32 was exploited to conformationally constrain a biaryl linkage and allow contact with key residues in GKRP. Lead compound 32 was shown to induce GK translocation from the nucleus to the cytoplasm in rats (IHC score = 0; 10 mg/kg po, 6 h) and blood glucose reduction in mice (POC = -45%; 100 mg/kg po, 3 h). X-ray analyses of 32 and several precursors bound to GKRP were also obtained. This novel disrupter of GK-GKRP binding enables further exploration of GKRP as a potential therapeutic target for type II diabetes and highlights the value of exploiting unconventional nonbonded interactions in drug design.

Structure-activity relationship of (1-aryl-2-piperazinylethyl)piperazines: antagonists for the AGRP/melanocortin receptor binding.

Agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin action.(1) In the hypothalamus, melanocortin peptide agonists act as satiety-inducing factors that mediate their action through the melanocortin-4 receptor (MC4R) whereas AGRP is an opposing orexigenic agent. Novel inhibitors of the AGRP/MC4 binding based on (piperazinylethyl)piperazines were prepared, and their structure-activity relationship was established.

Discovery of Small-Molecule Glucokinase Regulatory Protein Modulators That Restore Glucokinase Activity.

In the nuclei of hepatocytes, glucokinase regulatory protein (GKRP) modulates the activity of glucokinase (GK), a key regulator of glucose homeostasis. Currently, direct activators of GK (GKAs) are in development for the treatment of type 2 diabetes. However, this approach is generally associated with a risk of hypoglycemia. To mitigate such risk, we target the GKRP regulation, which indirectly restores GK activity. Here we describe a screening strategy to look specifically for GKRP modulators, in addition to traditional GKAs. Two high-throughput screening campaigns were performed with our compound libraries using a luminescence assay format, one with GK alone and the other with a GK/GKRP complex in the presence of sorbitol-6-phosphate (S6P). By a subtraction method in the hit triage process of these campaigns, we discovered two close analogs that bind GKRP specifically with sub-µM potency to a site distinct from where fructose-1-phosphate binds. These small molecules are first-in-class allosteric modulators of the GK/GKRP interaction and are fully active even in the presence of S6P. Activation of GK by this particular mechanism, without altering the enzymatic profile, represents a novel pharmacologic modality of intervention in the GK/GKRP pathway.

Small molecule disruptors of the glucokinase-glucokinase regulatory protein interaction: 4. Exploration of a novel binding pocket.

Structure-activity relationship investigations conducted at the 5-position of the N-pyridine ring of a series of N-arylsulfonyl-N'-2-pyridinyl-piperazines led to the identification of a novel bis-pyridinyl piperazine sulfonamide (51) that was a potent disruptor of the glucokinase-glucokinase regulatory protein (GK-GKRP) interaction. Analysis of the X-ray cocrystal of compound 51 bound to hGKRP revealed that the 3-pyridine ring moiety occupied a previously unexplored binding pocket within the protein. Key features of this new binding mode included forming favorable contacts with the top face of the Ala27-Val28-Pro29 ("shelf region") as well as an edge-to-face interaction with the Tyr24 side chain. Compound 51 was potent in both biochemical and cellular assays (IC50=0.005 µM and EC50=0.205 µM, respectively) and exhibited acceptable pharmacokinetic properties for in vivo evaluation. When administered to db/db mice (100 mg/kg, po), compound 51 demonstrated a robust pharmacodynamic effect and significantly reduced blood glucose levels up to 6 h postdose.