Effect of Food Consumption on the Pharmacokinetics, Safety, and Tolerability of Once-Daily Orally Administered Orforglipron (LY3502970), a Non-peptide GLP-1 Receptor Agonist.

INTRODUCTION: We assessed the effect of the prandial state on the pharmacokinetics, safety, and tolerability of single and multiple doses of orforglipron (LY3502970), an oral, non-peptide glucagon-like peptide 1 receptor agonist (GLP-1 RA), in two studies (A and B). METHODS: Study A and study B were phase 1, randomized, crossover studies in healthy adults aged 18-65 years and 21-70 years, respectively. Participants received single (3 mg, study A) or multiple (16 mg, study B) oral doses of orforglipron under fasted and fed conditions. Blood samples were collected pre- and postdose to assess area under the concentration-time curve (AUC), maximum observed drug concentration (Cmax), time of Cmax (tmax), and half-life (t1/2) associated with terminal rate constant. AUC and Cmax were analyzed using a linear mixed-effects model. Treatment differences were presented as ratios of geometric least squares means (GLSM). Treatment-emergent adverse events (TEAEs), adverse events of special interest, and serious adverse events were assessed. RESULTS: Study A included 12 participants (mean age 45.0 years; male 66.7%); study B included 34 participants (mean age 42.8 years; male 88.2%). GLSM AUC and Cmax were lower by 23.7% and 23.2% in study A, and 17.6% and 20.9% in study B, in the fed versus fasted states, respectively. In both studies, t1/2 and median tmax were comparable between fed and fasted states. The majority of TEAEs in both studies were gastrointestinal tract-related conditions. No serious adverse events or deaths were reported in either study. CONCLUSION: The observed pharmacokinetic differences due to the prandial state are unlikely to contribute to clinically meaningful differences in the efficacy of orforglipron. The safety profile was consistent with the known profiles of other GLP-1 RAs. Given the absence of prandial restrictions, orforglipron may emerge as a convenient oral treatment option for patients with type 2 diabetes or obesity. TRIAL REGISTRATION: ClinicalTrials.gov identifiers, NCT03929744 and NCT05110794.

GIPR Agonism Enhances TZD-Induced Insulin Sensitivity in Obese IR Mice.

Recent studies have found that glucose-dependent insulinotropic polypeptide receptor (GIPR) agonism can enhance the metabolic efficacy of glucagon-like peptide-1 receptor agonist treatment by promoting both weight-dependent and -independent improvements on systemic insulin sensitivity. These findings have prompted new investigations aimed at better understanding the broad metabolic benefit of GIPR activation. Herein, we determined whether GIPR agonism favorably influenced the pharmacologic efficacy of the insulin-sensitizing thiazolidinedione (TZD) rosiglitazone in obese insulin-resistant (IR) mice. Genetic and pharmacological approaches were used to examine the role of GIPR signaling on rosiglitazone-induced weight gain, hyperphagia, and glycemic control. RNA sequencing was conducted to uncover potential mechanisms by which GIPR activation influences energy balance and insulin sensitivity. In line with previous findings, treatment with rosiglitazone induced the mRNA expression of the GIPR in white and brown fat. However, obese GIPR-null mice dosed with rosiglitazone had equivalent weight gain to that of wild-type (WT) animals. Strikingly, chronic treatment of obese IR WT animals with a long-acting GIPR agonist prevented rosiglitazone-induced weight-gain and hyperphagia, and it enhanced the insulin-sensitivity effect of this TZD. The systemic insulin sensitization was accompanied by increased glucose disposal in brown adipose tissue, which was underlined by the recruitment of metabolic and thermogenic genes. These findings suggest that GIPR agonism can counter the negative consequences of rosiglitazone treatment on body weight and adiposity, while improving its insulin-sensitizing efficacy at the same time.

Pancreatic islet α cell function and proliferation requires the arginine transporter SLC7A2.

Interrupting glucagon signaling decreases gluconeogenesis and the fractional extraction of amino acids by liver from blood resulting in lower glycemia. The resulting hyperaminoacidemia stimulates α cell proliferation and glucagon secretion via a liver-α cell axis. We hypothesized that α cells detect and respond to circulating amino acids levels via a unique amino acid transporter repertoire. We found that Slc7a2ISLC7A2 is the most highly expressed cationic amino acid transporter in α cells with its expression being three-fold greater in α than ß cells in both mouse and human. Employing cell culture, zebrafish, and knockout mouse models, we found that the cationic amino acid arginine and SLC7A2 are required for α cell proliferation in response to interrupted glucagon signaling. Ex vivo and in vivo assessment of islet function in Slc7a2-/- mice showed decreased arginine-stimulated glucagon and insulin secretion. We found that arginine activation of mTOR signaling and induction of the glutamine transporter SLC38A5 was dependent on SLC7A2, showing that both's role in α cell proliferation is dependent on arginine transport and SLC7A2. Finally, we identified single nucleotide polymorphisms in SLC7A2 associated with HbA1c. Together, these data indicate a central role for SLC7A2 in amino acid-stimulated α cell proliferation and islet hormone secretion.

Orforglipron (LY3502970), a novel, oral non-peptide glucagon-like peptide-1 receptor agonist: A Phase 1a, blinded, placebo-controlled, randomized, single- and multiple-ascending-dose study in healthy participants.

AIM: To evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of single and multiple doses of orforglipron (LY3502970), an oral, non-peptide glucagon-like peptide-1 receptor agonist (GLP-1RA) in healthy participants. MATERIALS AND METHODS: This was a double-blind, placebo-controlled, Phase 1 study. Overtly healthy adults aged 18 to 65 years with body mass index of 20 to 40 kg/m2 and glycated haemoglobin concentration of 47.5 mmol/mol (<6.5%) were eligible. In Part A, participants received single-dose orforglipron, with four cohorts receiving escalating doses (0.3-6 mg). In Part B, participants received 4 weeks of daily repeated oral orforglipron with doses escalating weekly to four different final target doses (2-24 mg). RESULTS: Ninety-two participants enrolled and received at least one study drug dose (32 in Part A [mean age 43.4 years] and 60 in Part B [mean age 42.5 years]). The most common adverse events were gastrointestinal tract-related. Pharmacokinetics were approximately dose proportional, and the mean t1/2 was 24.6 to 35.3 hours after a single dose (0.3-6 mg). On Day 28, the mean t1/2 was 48.1 to 67.5 hours across the dose range (2-24 mg). Substantial reductions in body weight of up to 5.4 kg were observed after 4 weeks in orforglipron-treated participants, compared to a reduction of 2.4 kg with placebo (P < 0.05). Orforglipron decreased fasting glucose levels across Days 1 to 28, and gastric emptying was delayed on Day 28. CONCLUSIONS: Orforglipron's long half-life (25-68 hours) allows once-daily oral dosing, without water and food restrictions. Orforglipron had a pharmacodynamic and safety profile similar to that of injectable GLP-1RAs, which supports continued clinical development.

Orforglipron (LY3502970), a novel, oral non-peptide glucagon-like peptide-1 receptor agonist: A Phase 1b, multicentre, blinded, placebo-controlled, randomized, multiple-ascending-dose study in people with type 2 diabetes.

AIM: To report the results of a Phase 1b trial evaluating the safety, pharmacokinetics and pharmacodynamics of orforglipron (LY3502970), an oral, non-peptide glucagon-like peptide-1 receptor agonist (GLP-1RA), in patients with type 2 diabetes (T2D). MATERIALS AND METHODS: This was a double-blind, placebo-controlled Phase 1 study evaluating five different dosing regimens. The first group established that weekly dose escalation of the daily doses of orforglipron was generally well tolerated. This enabled a parallel-arm design for the four groups following. Participants were randomized 3:1 to daily doses of orforglipron or placebo for 12 weeks. Eligible participants with T2D were aged 18 to 70 years and had glycated haemoglobin (HbA1c) levels ≥53.0 mmol/mol (7.0%) and ≤91.3 mmol/mol (10.5%). RESULTS: A total of 51 participants received orforglipron and 17 received placebo. In the placebo and orforglipron groups, respectively, baseline HbA1c was 8.1% and 8.0%, and baseline body weight was 90.3 and 88.4 kg. The most common adverse events were gastrointestinal-related, and occurred early in treatment, similar to findings with other GLP-1RAs. At Week 12, mean t1/2 ranged from 29 to 49 hours. Mean HbA1c change ranged from -1.5% to -1.8% across orforglipron doses, versus -0.4% with placebo, and body weight change was -0.24 to -5.8 kg across orforglipron doses, versus 0.5 kg with placebo. CONCLUSIONS: Orforglipron treatment resulted in meaningful reductions in HbA1c and body weight, with an adverse event profile consistent with that of other GLP-1RAs. Orforglipron may provide a safe and effective once-daily oral treatment alternative to injectable GLP-1RAs or peptide oral formulations without water and food restrictions.

The incretin co-agonist tirzepatide requires GIPR for hormone secretion from human islets.

The incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) mediate insulin responses that are proportionate to nutrient intake to facilitate glucose tolerance1. The GLP-1 receptor (GLP-1R) is an established drug target for the treatment of diabetes and obesity2, whereas the therapeutic potential of the GIP receptor (GIPR) is a subject of debate. Tirzepatide is an agonist at both the GIPR and GLP-1R and is a highly effective treatment for type 2 diabetes and obesity3,4. However, although tirzepatide activates GIPR in cell lines and mouse models, it is not clear whether or how dual agonism contributes to its therapeutic benefit. Islet beta cells express both the GLP-1R and the GIPR, and insulin secretion is an established mechanism by which incretin agonists improve glycemic control5. Here, we show that in mouse islets, tirzepatide stimulates insulin secretion predominantly through the GLP-1R, owing to reduced potency at the mouse GIPR. However, in human islets, antagonizing GIPR activity consistently decreases the insulin response to tirzepatide. Moreover, tirzepatide enhances glucagon secretion and somatostatin secretion in human islets. These data demonstrate that tirzepatide stimulates islet hormone secretion from human islets through both incretin receptors.

LY3437943, a novel triple glucagon, GIP, and GLP-1 receptor agonist for glycemic control and weight loss: From discovery to clinical proof of concept.

With an increasing prevalence of obesity, there is a need for new therapies to improve body weight management and metabolic health. Multireceptor agonists in development may provide approaches to fulfill this unmet medical need. LY3437943 is a novel triple agonist peptide at the glucagon receptor (GCGR), glucose-dependent insulinotropic polypeptide receptor (GIPR), and glucagon-like peptide-1 receptor (GLP-1R). In vitro, LY3437943 shows balanced GCGR and GLP-1R activity but more GIPR activity. In obese mice, administration of LY3437943 decreased body weight and improved glycemic control. Body weight loss was augmented by the addition of GCGR-mediated increases in energy expenditure to GIPR- and GLP-1R-driven calorie intake reduction. In a phase 1 single ascending dose study, LY3437943 showed a safety and tolerability profile similar to other incretins. Its pharmacokinetic profile supported once-weekly dosing, and a reduction in body weight persisted up to day 43 after a single dose. These findings warrant further clinical assessment of LY3437943.

Tirzepatide induces a thermogenic-like amino acid signature in brown adipose tissue.

OBJECTIVES: Tirzepatide, a dual GIP and GLP-1 receptor agonist, delivered superior glycemic control and weight loss compared to selective GLP-1 receptor (GLP-1R) agonism in patients with type 2 diabetes (T2D). These results have fueled mechanistic studies focused on understanding how tirzepatide achieves its therapeutic efficacy. Recently, we found that treatment with tirzepatide improves insulin sensitivity in humans with T2D and obese mice in concert with a reduction in circulating levels of branched-chain amino (BCAAs) and keto (BCKAs) acids, metabolites associated with development of systemic insulin resistance (IR) and T2D. Importantly, these systemic effects were found to be coupled to increased expression of BCAA catabolic genes in thermogenic brown adipose tissue (BAT) in mice. These findings led us to hypothesize that tirzepatide may lower circulating BCAAs/BCKAs by promoting their catabolism in BAT. METHODS: To address this question, we utilized a murine model of diet-induced obesity and employed stable-isotope tracer studies in combination with metabolomic analyses in BAT and other tissues. RESULTS: Treatment with tirzepatide stimulated catabolism of BCAAs/BCKAs in BAT, as demonstrated by increased labeling of BCKA-derived metabolites, and increases in levels of byproducts of BCAA breakdown, including glutamate, alanine, and 3-hydroxyisobutyric acid (3-HIB). Further, chronic administration of tirzepatide increased levels of multiple amino acids in BAT that have previously been shown to be elevated in response to cold exposure. Finally, chronic treatment with tirzepatide led to a substantial increase in several TCA cycle intermediates (α-ketoglutarate, fumarate, and malate) in BAT. CONCLUSIONS: These findings suggest that tirzepatide induces a thermogenic-like amino acid profile in BAT, an effect that may account for reduced systemic levels of BCAAs in obese IR mice.

14-3-3-zeta mediates GLP-1 receptor agonist action to alter α cell proglucagon processing.

Recent studies demonstrate that α cells contribute to glucose-stimulated insulin secretion (GSIS). Glucagon-like peptide-1 receptor (GLP-1R) agonists potently potentiate GSIS, making these drugs useful for diabetes treatment. However, the role of α and ß cell paracrine interactions in the effects of GLP-1R agonists is undefined. We previously found that increased ß cell GLP-1R signaling activates α cell GLP-1 expression. Here, we characterized the bidirectional paracrine cross-talk by which α and ß cells communicate to mediate the effects of the GLP-1R agonist, liraglutide. We find that the effect of liraglutide to enhance GSIS is blunted by α cell ablation in male mice. Furthermore, the effect of ß cell GLP-1R signaling to activate α cell GLP-1 is mediated by a secreted protein factor that is regulated by the signaling protein, 14-3-3-zeta, in mouse and human islets. These data refine our understanding of GLP-1 pharmacology and identify 14-3-3-zeta as a potential target to enhance α cell GLP-1 production.

Is Glucagon Receptor Activation the Thermogenic Solution for Treating Obesity?

A major challenge of obesity therapy is to sustain clinically relevant weight loss over time. Achieving this goal likely requires both reducing daily caloric intake and increasing caloric expenditure. Over the past decade, advances in pharmaceutical engineering of ligands targeting G protein-coupled receptors have led to the development of highly effective anorectic agents. These include mono-agonists of the GLP-1R and dual GIPR/GLP-1R co-agonists that have demonstrated substantial weight loss in experimental models and in humans. By contrast, currently, there are no medicines available that effectively augment metabolic rate to promote weight loss. Here, we present evidence indicating that activation of the GCGR may provide a solution to this unmet therapeutic need. In adult humans, GCGR agonism increases energy expenditure to a magnitude sufficient for inducing a negative energy balance. In preclinical studies, the glucagon-GCGR system affects key metabolically relevant organs (including the liver and white and brown adipose tissue) to boost whole-body thermogenic capacity and protect from obesity. Further, activation of the GCGR has been shown to augment both the magnitude and duration of weight loss that is achieved by either selective GLP-1R or dual GIPR/GLP-1R agonism in rodents. Based on the accumulation of such findings, we propose that the thermogenic activity of GCGR agonism will also complement other anti-obesity agents that lower body weight by suppressing appetite.

GIPR Agonism Inhibits PYY-Induced Nausea-Like Behavior.

The induction of nausea and emesis is a major barrier to maximizing the weight loss profile of obesity medications, and therefore, identifying mechanisms that improve tolerability could result in added therapeutic benefit. The development of peptide YY (PYY)-based approaches to treat obesity are no exception, as PYY receptor agonism is often accompanied by nausea and vomiting. Here, we sought to determine whether glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) agonism reduces PYY-induced nausea-like behavior in mice. We found that central and peripheral administration of a GIPR agonist reduced conditioned taste avoidance (CTA) without affecting hypophagia mediated by a PYY analog. The receptors for GIP and PYY (Gipr and Npy2r) were found to be expressed by the same neurons in the area postrema (AP), a brainstem nucleus involved in detecting aversive stimuli. Peripheral administration of a GIPR agonist induced neuronal activation (cFos) in the AP. Further, whole-brain cFos analyses indicated that PYY-induced CTA was associated with augmented neuronal activity in the parabrachial nucleus (PBN), a brainstem nucleus that relays aversive/emetic signals to brain regions that control feeding behavior. Importantly, GIPR agonism reduced PYY-mediated neuronal activity in the PBN, providing a potential mechanistic explanation for how GIPR agonist treatment reduces PYY-induced nausea-like behavior. Together, the results of our study indicate a novel mechanism by which GIP-based therapeutics may have benefit in improving the tolerability of weight loss agents.

Structural determinants of dual incretin receptor agonism by tirzepatide.

SignificanceTirzepatide is a dual agonist of the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon-like peptide-1 receptor (GLP-1R), which are incretin receptors that regulate carbohydrate metabolism. This investigational agent has proven superior to selective GLP-1R agonists in clinical trials in subjects with type 2 diabetes mellitus. Intriguingly, although tirzepatide closely resembles native GIP in how it activates the GIPR, it differs markedly from GLP-1 in its activation of the GLP-1R, resulting in less agonist-induced receptor desensitization. We report how cryogenic electron microscopy and molecular dynamics simulations inform the structural basis for the unique pharmacology of tirzepatide. These studies reveal the extent to which fatty acid modification, combined with amino acid sequence, determines the mode of action of a multireceptor agonist.

GIPR Function in the Central Nervous System: Implications and Novel Perspectives for GIP-Based Therapies in Treating Metabolic Disorders.

During the past decade, pharmaceutical engineering of unimolecular agents has revealed the therapeutic potential of glucose-dependent insulinotropic polypeptide receptor (GIPR) agonism. From this work, one of the most intriguing findings is that engagement of GIPR enhances the weight loss profile of glucagon-like peptide 1 (GLP-1)-based therapeutics. Consequently, this pharmacological approach, in combination with novel Gipr mouse models, has provided evidence indicating that activation of GIPR in certain areas of the brain that regulate energy balance is required for the synergistic weight loss of dual GIPR and GLP-1 receptor (GLP-1R) agonism. This has led to significant interest in understanding how GIPR activity in the brain functions to reduce caloric intake, induce negative energy balance, and drive weight loss. Herein, we review key findings in this field and provide a novel perspective explaining how GIP may act in the brain to affect energy balance both alone and in concert with GLP-1R agonism.

GIPR agonism mediates weight-independent insulin sensitization by tirzepatide in obese mice.

Tirzepatide (LY3298176), a dual GIP and GLP-1 receptor (GLP-1R) agonist, delivered superior glycemic control and weight loss compared with GLP-1R agonism in patients with type 2 diabetes. However, the mechanism by which tirzepatide improves efficacy and how GIP receptor (GIPR) agonism contributes is not fully understood. Here, we show that tirzepatide is an effective insulin sensitizer, improving insulin sensitivity in obese mice to a greater extent than GLP-1R agonism. To determine whether GIPR agonism contributes, we compared the effect of tirzepatide in obese WT and Glp-1r-null mice. In the absence of GLP-1R-induced weight loss, tirzepatide improved insulin sensitivity by enhancing glucose disposal in white adipose tissue (WAT). In support of this, a long-acting GIPR agonist (LAGIPRA) was found to enhance insulin sensitivity by augmenting glucose disposal in WAT. Interestingly, the effect of tirzepatide and LAGIPRA on insulin sensitivity was associated with reduced branched-chain amino acids (BCAAs) and ketoacids in the circulation. Insulin sensitization was associated with upregulation of genes associated with the catabolism of glucose, lipid, and BCAAs in brown adipose tissue. Together, our studies show that tirzepatide improved insulin sensitivity in a weight-dependent and -independent manner. These results highlight how GIPR agonism contributes to the therapeutic profile of dual-receptor agonism, offering mechanistic insights into the clinical efficacy of tirzepatide.

Discovery of an Orally Efficacious Positive Allosteric Modulator of the Glucagon-like Peptide-1 Receptor.

The identification of LSN3318839, a positive allosteric modulator of the glucagon-like peptide-1 receptor (GLP-1R), is described. LSN3318839 increases the potency and efficacy of the weak metabolite GLP-1(9-36)NH2 to become a full agonist at the GLP-1R and modestly potentiates the activity of the highly potent full-length ligand, GLP-1(7-36)NH2. LSN3318839 preferentially enhances G protein-coupled signaling by the GLP-1R over ß-arrestin recruitment. Ex vivo experiments show that the combination of GLP-1(9-36)NH2 and LSN3318839 produces glucose-dependent insulin secretion similar to that of GLP-1(7-36)NH2. Under nutrient-stimulated conditions that release GLP-1, LSN3318839 demonstrates robust glucose lowering in animal models alone or in treatment combination with sitagliptin. From a therapeutic perspective, the biological properties of LSN3318839 support the concept that GLP-1R potentiation is sufficient for reducing hyperglycemia.

Glucagon blockade restores functional ß-cell mass in type 1 diabetic mice and enhances function of human islets.

We evaluated the potential for a monoclonal antibody antagonist of the glucagon receptor (Ab-4) to maintain glucose homeostasis in type 1 diabetic rodents. We noted durable and sustained improvements in glycemia which persist long after treatment withdrawal. Ab-4 promoted ß-cell survival and enhanced the recovery of insulin+ islet mass with concomitant increases in circulating insulin and C peptide. In PANIC-ATTAC mice, an inducible model of ß-cell apoptosis which allows for robust assessment of ß-cell regeneration following caspase-8-induced diabetes, Ab-4 drove a 6.7-fold increase in ß-cell mass. Lineage tracing suggests that this restoration of functional insulin-producing cells was at least partially driven by α-cell-to-ß-cell conversion. Following hyperglycemic onset in nonobese diabetic (NOD) mice, Ab-4 treatment promoted improvements in C-peptide levels and insulin+ islet mass was dramatically increased. Lastly, diabetic mice receiving human islet xenografts showed stable improvements in glycemic control and increased human insulin secretion.

GLP-1 receptor signaling increases PCSK1 and ß cell features in human α cells.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that potentiates glucose-stimulated insulin secretion. GLP-1 is classically produced by gut L cells; however, under certain circumstances α cells can express the prohormone convertase required for proglucagon processing to GLP-1, prohormone convertase 1/3 (PC1/3), and can produce GLP-1. However, the mechanisms through which this occurs are poorly defined. Understanding the mechanisms by which α cell PC1/3 expression can be activated may reveal new targets for diabetes treatment. Here, we demonstrate that the GLP-1 receptor (GLP-1R) agonist, liraglutide, increased α cell GLP-1 expression in a ß cell GLP-1R-dependent manner. We demonstrate that this effect of liraglutide was translationally relevant in human islets through application of a new scRNA-seq technology, DART-Seq. We found that the effect of liraglutide to increase α cell PC1/3 mRNA expression occurred in a subcluster of α cells and was associated with increased expression of other ß cell-like genes, which we confirmed by IHC. Finally, we found that the effect of liraglutide to increase bihormonal insulin+ glucagon+ cells was mediated by the ß cell GLP-1R in mice. Together, our data validate a high-sensitivity method for scRNA-seq in human islets and identify a potentially novel GLP-1-mediated pathway regulating human α cell function.

Structural basis for GLP-1 receptor activation by LY3502970, an orally active nonpeptide agonist.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are efficacious antidiabetic medications that work by enhancing glucose-dependent insulin secretion and improving energy balance. Currently approved GLP-1R agonists are peptide based, and it has proven difficult to obtain small-molecule activators possessing optimal pharmaceutical properties. We report the discovery and mechanism of action of LY3502970 (OWL833), a nonpeptide GLP-1R agonist. LY3502970 is a partial agonist, biased toward G protein activation over ß-arrestin recruitment at the GLP-1R. The molecule is highly potent and selective against other class B G protein-coupled receptors (GPCRs) with a pharmacokinetic profile favorable for oral administration. A high-resolution structure of LY3502970 in complex with active-state GLP-1R revealed a unique binding pocket in the upper helical bundle where the compound is bound by the extracellular domain (ECD), extracellular loop 2, and transmembrane helices 1, 2, 3, and 7. This mechanism creates a distinct receptor conformation that may explain the partial agonism and biased signaling of the compound. Further, interaction between LY3502970 and the primate-specific Trp33 of the ECD informs species selective activity for the molecule. In efficacy studies, oral administration of LY3502970 resulted in glucose lowering in humanized GLP-1R transgenic mice and insulinotropic and hypophagic effects in nonhuman primates, demonstrating an effect size in both models comparable to injectable exenatide. Together, this work determined the molecular basis for the activity of an oral agent being developed for the treatment of type 2 diabetes mellitus, offering insights into the activation of class B GPCRs by nonpeptide ligands.

Synthetic protease-activated class B GPCRs.

G-protein coupled receptors (GPCRs) are the ligand detection machinery of a majority of extracellular signaling systems in metazoans. Novel chemical and biological tools to probe the structure-function relationships of GPCRs have impacted both basic and applied GPCR research. To better understand the structure-function of class B GPCRs, we generated receptor-ligand fusion chimeric proteins that can be activated by exogenous enzyme application. As a prototype, fusion proteins of the glucagon-like peptide-1 receptor (GLP-1R) with GLP-1(7-36) and exendin-4(1-39) peptides incorporating enterokinase-cleavable N-termini were generated. These receptors are predicted to generate fusion protein neo-epitopes upon proteolysis with enterokinase that are identical to the N-termini of GLP-1 agonists. This system was validated by measuring enterokinase-dependent GLP-1R mediated cAMP accumulation, and a structure-activity relationship for both linker length and peptide sequence was observed. Moreover, our results show this approach can be used in physiologically relevant cell systems, as GLP-1R-ligand chimeras were shown to induce glucose-dependent insulin secretion in insulinoma cells upon exposure to enterokinase. This approach suggests new strategies for understanding the structure-function of peptide-binding GPCRs.

Tirzepatide is an imbalanced and biased dual GIP and GLP-1 receptor agonist.

Tirzepatide (LY3298176) is a dual GIP and GLP-1 receptor agonist under development for the treatment of type 2 diabetes mellitus (T2DM), obesity, and nonalcoholic steatohepatitis. Early phase trials in T2DM indicate that tirzepatide improves clinical outcomes beyond those achieved by a selective GLP-1 receptor agonist. Therefore, we hypothesized that the integrated potency and signaling properties of tirzepatide provide a unique pharmacological profile tailored for improving broad metabolic control. Here, we establish methodology for calculating occupancy of each receptor for clinically efficacious doses of the drug. This analysis reveals a greater degree of engagement of tirzepatide for the GIP receptor than the GLP-1 receptor, corroborating an imbalanced mechanism of action. Pharmacologically, signaling studies demonstrate that tirzepatide mimics the actions of native GIP at the GIP receptor but shows bias at the GLP-1 receptor to favor cAMP generation over ß-arrestin recruitment, coincident with a weaker ability to drive GLP-1 receptor internalization compared with GLP-1. Experiments in primary islets reveal ß-arrestin1 limits the insulin response to GLP-1, but not GIP or tirzepatide, suggesting that the biased agonism of tirzepatide enhances insulin secretion. Imbalance toward GIP receptor, combined with distinct signaling properties at the GLP-1 receptor, together may account for the promising efficacy of this investigational agent.