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.

A GIPR antagonist conjugated to GLP-1 analogues promotes weight loss with improved metabolic parameters in preclinical and phase 1 settings.

Obesity is a major public health crisis. Multi-specific peptides have emerged as promising therapeutic strategies for clinical weight loss. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are endogenous incretins that regulate weight through their receptors (R). AMG 133 (maridebart cafraglutide) is a bispecific molecule engineered by conjugating a fully human monoclonal anti-human GIPR antagonist antibody to two GLP-1 analogue agonist peptides using amino acid linkers. Here, we confirm the GIPR antagonist and GLP-1R agonist activities in cell-based systems and report the ability of AMG 133 to reduce body weight and improve metabolic markers in male obese mice and cynomolgus monkeys. In a phase 1, randomized, double-blind, placebo-controlled clinical study in participants with obesity ( NCT04478708 ), AMG 133 had an acceptable safety and tolerability profile along with pronounced dose-dependent weight loss. In the multiple ascending dose cohorts, weight loss was maintained for up to 150 days after the last dose. These findings support continued clinical evaluation of AMG 133.

An apoA-I mimetic peptibody generates HDL-like particles and increases alpha-1 HDL subfraction in mice.

The aim of this study is to investigate the capability of an apoA-I mimetic with multiple amphipathic helices to form HDL-like particles in vitro and in vivo. To generate multivalent helices and to track the peptide mimetic, we have constructed a peptibody by fusing two tandem repeats of 4F peptide to the C terminus of a murine IgG Fc fragment. The resultant peptidbody, mFc-2X4F, dose-dependently promoted cholesterol efflux in vitro, and the efflux potency was superior to monomeric 4F peptide. Like apoA-I, mFc-2X4F stabilized ABCA1 in J774A.1 and THP1 cells. The peptibody formed larger HDL particles when incubated with cultured cells compared with those by apoA-I. Interestingly, when administered to mice, mFc-2X4F increased both pre-ß and α-1 HDL subfractions. The lipid-bound mFc-2X4F was mostly in the α-1 migrating subfraction. Most importantly, mFc-2X4F and apoA-I were found to coexist in the same HDL particles formed in vivo. These data suggest that the apoA-I mimetic peptibody is capable of mimicking apoA-I to generate HDL particles. The peptibody and apoA-I may work cooperatively to generate larger HDL particles in vivo, either at the cholesterol efflux stage and/or via fusion of HDL particles that were generated by the peptibody and apoA-I individually.

Pharmacological targeting of glucagon and glucagon-like peptide 1 receptors has different effects on energy state and glucose homeostasis in diet-induced obese mice.

Pharmacologic contributions of directly agonizing glucagon-like peptide 1 (GLP-1) receptor or antagonizing glucagon receptor (GCGR) on energy state and glucose homeostasis were assessed in diet-induced obese (DIO) mice. Metabolic rate and respiratory quotient (RQ), hyperglycemic clamp, stable isotope-based dynamic metabolic profiling (SiDMAP) studies of (13)C-labeled glucose during glucose tolerance test (GTT) and gene expression were assessed in cohorts of DIO mice after a single administration of GLP-1 analog [GLP-1-(23)] or anti-GCGR antibody (Ab). GLP-1-(23) and GCGR Ab similarly improved GTT. GLP-1-(23) decreased food intake and body weight trended lower. GCGR Ab modestly decreased food intake without significant effect on body weight. GLP-1-(23) and GCGR Ab decreased RQ with GLP-1, causing a greater effect. In a hyperglycemic clamp, GLP-1-(23) reduced hepatic glucose production (HGP), increased glucose infusion rate (GIR), increased glucose uptake in brown adipose tissue, and increased whole-body glucose turnover, glycolysis, and rate of glycogen synthesis. GCGR Ab slightly decreased HGP, increased GIR, and increased glucose uptake in the heart. SiDMAP showed that GLP-1-(23) and GCGR Ab increased (13)C lactate labeling from glucose, indicating that liver, muscle, and other organs were involved in the rapid disposal of glucose from plasma. GCGR Ab and GLP-1-(23) caused different changes in mRNA expression levels of glucose- and lipid metabolism-associated genes. The effect of GLP-1-(23) on energy state and glucose homeostasis was greater than GCGR Ab. Although GCGR antagonism is associated with increased circulating levels of GLP-1, most GLP-1-(23)-associated pharmacologic effects are more pronounced than GCGR Ab.

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.

Glucagon receptor antagonist-mediated improvements in glycemic control are dependent on functional pancreatic GLP-1 receptor.

Antagonism of the glucagon receptor (GCGR) is associated with increased circulating levels of glucagon-like peptide-1 (GLP-1). To investigate the contribution of GLP-1 to the antidiabetic actions of GCGR antagonism, we administered an anti-GCGR monoclonal antibody (mAb B) to wild-type mice and GLP-1 receptor knockout (GLP-1R KO) mice. Treatment of wild-type mice with mAb B lowered fasting blood glucose, improved glucose tolerance, and enhanced glucose-stimulated insulin secretion during an intraperitoneal glucose tolerance test (ipGTT). In contrast, treatment of GLP-1R KO mice with mAb B had little efficacy during an ipGTT. Furthermore, pretreatment with the GLP-1R antagonist exendin-(9-39) diminished the antihyperglycemic effects of mAb B in wild-type mice. To determine the mechanism whereby mAb B improves glucose tolerance, we generated a monoclonal antibody that specifically antagonizes the human GLP-1R. Using a human islet transplanted mouse model, we demonstrated that pancreatic islet GLP-1R signaling is required for the full efficacy of the GCGR antagonist. To identify the source of the elevated GLP-1 observed in GCGR mAb-treated mice, we measured active GLP-1 content in pancreas and intestine from db/db mice treated with anti-GCGR mAb for 8 wk. Elevated GLP-1 in GCGR mAb-treated mice was predominantly derived from increased pancreatic GLP-1 synthesis and processing. All together, these data show that pancreatic GLP-1 is a significant contributor to the glucose-lowering effects observed in response to GCGR antagonist treatment.

Long-term inhibition of the glucagon receptor with a monoclonal antibody in mice causes sustained improvement in glycemic control, with reversible alpha-cell hyperplasia and hyperglucagonemia.

Uncontrolled hepatic glucose output (HGO) contributes significantly to the pathological hyperglycemic state of patients with type 2 diabetes. Glucagon, through action on its receptor, stimulates HGO, thereby leading to increased glycemia. Antagonizing the glucagon signaling pathway represents an attractive therapeutic approach for the treatment of type 2 diabetes. We previously reported the generation and characterization of several high-affinity monoclonal antibodies (mAbs) targeting the glucagon receptor (GCGR). In the present study, we demonstrate that a 5-week treatment of diet-induced obese mice with mAb effectively normalized nonfasting blood glucose. Similar treatment also reduced fasting blood glucose without inducing hypoglycemia or other undesirable metabolic perturbations. In addition, no hypoglycemia was found in db/db mice that were treated with a combination of insulin and mAb. Long-term treatment with the mAb caused dose-dependent hyperglucagonemia and minimal to mild alpha-cell hyperplasia in lean mice. There was no evidence of pancreatic alpha-cell neoplastic transformation in mice treated with mAb for as long as 18 weeks. Treatment-induced hyperglucagonemia and alpha-cell hyperplasia were reversible after treatment withdrawal for periods of 4 and 10 weeks, respectively. It is noteworthy that pancreatic beta-cell function was preserved, as demonstrated by improved glucose tolerance throughout the 18-week treatment period. Our studies further support the concept that long-term inhibition of GCGR signaling by a mAb could be an effective approach for controlling diabetic hyperglycemia.

Fully human monoclonal antibodies antagonizing the glucagon receptor improve glucose homeostasis in mice and monkeys.

Antagonizing the glucagon signaling pathway represents an attractive therapeutic approach for reducing excess hepatic glucose production in patients with type 2 diabetes. Despite extensive efforts, there is currently no human therapeutic that directly inhibits the glucagon/glucagon receptor pathway. We undertook a novel approach by generating high-affinity human monoclonal antibodies (mAbs) to the human glucagon receptor (GCGR) that display potent antagonistic activity in vitro and in vivo. A single injection of a lead antibody, mAb B, at 3 mg/kg, normalized blood glucose levels in ob/ob mice for 8 days. In addition, a single injection of mAb B dose-dependently lowered fasting blood glucose levels without inducing hypoglycemia and improved glucose tolerance in normal C57BL/6 mice. In normal cynomolgus monkeys, a single injection improved glucose tolerance while increasing glucagon and active glucagon-like peptide-1 levels. Thus, the anti-GCGR mAb could represent an effective new therapeutic for the treatment of type 2 diabetes.

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.

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.

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.

Enhancement of therapeutic protein in vivo activities through glycoengineering.

Delivery of protein therapeutics often requires frequent injections because of low activity or rapid clearance, thereby placing a burden on patients and caregivers. Using glycoengineering, we have increased and prolonged the activity of proteins, thus allowing reduced frequency of administration. Glycosylation analogs with new N-linked glycosylation consensus sequences introduced into the protein were screened for the presence of additional N-linked carbohydrates and retention of in vitro activity. Suitable consensus sequences were combined in one molecule, resulting in glycosylation analogs of rHuEPO, leptin, and Mpl ligand. All three molecules had substantially increased in vivo activity and prolonged duration of action. Because these proteins were of three different classes (rHuEPO is an N-linked glycoprotein, Mpl ligand an O-linked glycoprotein, and leptin contains no carbohydrate), glycoengineering may be generally applicable as a strategy for increasing the in vivo activity and duration of action of proteins. This strategy has been validated clinically for glycoengineered rHuEPO (darbopoetin alfa).

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.

5-Alkyl-2-urea-Substituted Pyridines: Identification of Efficacious Glucokinase Activators with Improved Properties.

Two 1-(4-aryl-5-alkyl-pyridin-2-yl)-3-methylurea glucokinase activators were identified with robust in vivo efficacy. These two compounds possessed higher solubilities than the previously identified triaryl compounds (i.e., AM-2394). Structure-activity relationship studies are presented along with relevant pharmacokinetic and in vivo data.

Novel Series of Potent Glucokinase Activators Leading to the Discovery of AM-2394.

Glucokinase (GK) catalyzes the phosphorylation of glucose to glucose-6-phosphate. We present the structure-activity relationships leading to the discovery of AM-2394, a structurally distinct GKA. AM-2394 activates GK with an EC50 of 60 nM, increases the affinity of GK for glucose by approximately 10-fold, exhibits moderate clearance and good oral bioavailability in multiple animal models, and lowers glucose excursion following an oral glucose tolerance test in an ob/ob mouse model of diabetes.

Pharmacologic Effects of FGF21 Are Independent of the "Browning" of White Adipose Tissue.

"Browning," the appearance and activation of brown-in-white (brite) adipose cells within inguinal white adipose tissue (iWAT), and induction of uncoupling protein 1 (UCP1) correlate with fibroblast growth factor-21 (FGF21)-induced weight loss and glucose homeostasis improvements. Therefore, antiobesity therapies targeting browning and brite adipocyte activation are currently being sought. To test the dependence of weight loss on browning, we examined whether this event was responsible for FGF21-Fc's beneficial effects. Lean and diet-induced obese mice housed at 21°C or 30°C that received FGF21-Fc exhibited similar degrees of body weight reduction and glucose homeostasis improvement. Substantial browning of iWAT occurred only in FGF21-Fc-treated lean mice housed at 21°C. Further, FGF21-Fc-treated Ucp1(-/-) mice showed robust improvements in body weight, glucose homeostasis, and plasma lipids, associated with increased energy expenditure and FGF21-Fc-induced Ppargc1 expression in iWAT. We conclude that FGF21 requires neither UCP1 nor brite adipocytes to elicit weight loss and improve glucose homeostasis.

FGF21 can be mimicked in vitro and in vivo by a novel anti-FGFR1c/ß-Klotho bispecific protein.

The endocrine hormone FGF21 has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. As an alternative to the native cytokine, we generated bispecific Avimer polypeptides that bind with high affinity and specificity to one of the receptor and coreceptor pairs used by FGF21, FGFR1c and ß-Klotho. These Avimers exhibit FGF21-like activity in in vitro assays with potency greater than FGF21. In a study conducted in obese male cynomolgus monkeys, animals treated with an FGFR1c/ß-Klotho bispecific Avimer showed improved metabolic parameters and reduced body weight comparable to the effects seen with FGF21. These results not only demonstrate the essential roles of FGFR1c and ß-Klotho in mediating the metabolic effects of FGF21, they also describe a first bispecific activator of this unique receptor complex and provide validation for a novel therapeutic approach to target this potentially important pathway for treating diabetes and obesity.

Piperazine oxadiazole inhibitors of acetyl-CoA carboxylase.

Acetyl-CoA carboxylase (ACC) is a target of interest for the treatment of metabolic syndrome. Starting from a biphenyloxadiazole screening hit, a series of piperazine oxadiazole ACC inhibitors was developed. Initial pharmacokinetic liabilities of the piperazine oxadiazoles were overcome by blocking predicted sites of metabolism, resulting in compounds with suitable properties for further in vivo studies. Compound 26 was shown to inhibit malonyl-CoA production in an in vivo pharmacodynamic assay and was advanced to a long-term efficacy study. Prolonged dosing with compound 26 resulted in impaired glucose tolerance in diet-induced obese (DIO) C57BL6 mice, an unexpected finding.

Long-acting FGF21 has enhanced efficacy in diet-induced obese mice and in obese rhesus monkeys.

Fibroblast growth factor 21 (FGF21), a hormone with short half-life, has consistently shown strong pharmacological efficacy. We first assessed the efficacy of murine recombinant FGF21 in C57BL6 lean mice for 5 wk. We then generated a long-acting FGF21 molecule by fusing a Fc to a variant of human recombinant FGF21 (hrFGF21) that contained two engineered mutations [L98R, P171G; Fc-FGF21(RG)] and tested it in C57BL6 diet-induced obese mice and obese rhesus monkeys. We compared its metabolic properties with those of the hrFGF21. Groups of diet-induced obese mice were treated for 36 d with different doses of hrFGF21 (01, 0.3, and 1 mg/kg twice daily) and with Fc-FGF21(RG) (2.3 mg/kg, every 5 d). Body weight, glucose, insulin, cholesterol, and triglyceride levels were decreased after treatment with either compound. A glucose tolerance test (GTT) was also improved. Obese rhesus monkeys were treated with hrFGF21 (once a day) and Fc-FGF21(RG) (once a week) in a dose-escalation fashion. Doses started at 0.1 and 0.3 mg/kg and ended at 3 and 5 mg/kg for hrFGF21 and Fc-FGF21(RG), respectively. Doses were escalated every 2 wk, and animals were followed up for a washout period of 3 wk. Body weight, glucose, insulin, cholesterol, and triglyceride levels and the GTT profile were decreased to a greater extent with Fc-FGF21(RG) than with hrFGF21. The PK-PD relationship of Fc-FGF21(RG) exposure and triglyceride reduction was also conducted with a maximum response model. In conclusion, in more than one species, Fc-FGF21(RG) chronically administered once a week showed similar or greater efficacy than hrFGF21 administered daily.

Treating diabetes and obesity with an FGF21-mimetic antibody activating the ßKlotho/FGFR1c receptor complex.

Fibroblast growth factor 21 (FGF21) is a distinctive member of the FGF family with potent beneficial effects on lipid, body weight, and glucose metabolism and has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. As an alternative to native FGF21, we have developed a monoclonal antibody, mimAb1, that binds to ßKlotho with high affinity and specifically activates signaling from the ßKlotho/FGFR1c (FGF receptor 1c) receptor complex. In obese cynomolgus monkeys, injection of mimAb1 led to FGF21-like metabolic effects, including decreases in body weight, plasma insulin, triglycerides, and glucose during tolerance testing. Mice with adipose-selective FGFR1 knockout were refractory to FGF21-induced improvements in glucose metabolism and body weight. These results in obese monkeys (with mimAb1) and in FGFR1 knockout mice (with FGF21) demonstrated the essential role of FGFR1c in FGF21 function and suggest fat as a critical target tissue for the cytokine and antibody. Because mimAb1 depends on ßKlotho to activate FGFR1c, it is not expected to induce side effects caused by activating FGFR1c alone. The unexpected finding of an antibody that can activate FGF21-like signaling through cell surface receptors provided preclinical validation for an innovative therapeutic approach to diabetes and obesity.