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.

Reduced incretin receptor trafficking upon activation enhances glycemic control and reverses obesity in diet-induced obese mice.

Activation of incretin receptors by their cognate agonist augments sustained cAMP generation both from the plasma membrane as well as from the endosome. To address the functional outcome of this spatiotemporal signaling, we developed a nonacylated glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptor dual agonist I-M-150847 that reduced receptor internalization following activation of the incretin receptors. The incretin receptor dual agonist I-M-150847 was developed by replacing the tryptophan cage of exendin-4 tyrosine substituted at the amino terminus with the C-terminal undecapeptide sequence of oxyntomodulin that placed lysine 30 of I-M-150847 in frame with the corresponding lysine residue of GIP. The peptide I-M-150847 is a partial agonist of GLP-1R and GIPR; however, the receptors, upon activation by I-M-150847, undergo reduced internalization that promotes agonist-mediated iterative cAMP signaling and augments glucose-stimulated insulin exocytosis in pancreatic ß cells. Chronic administration of I-M-150847 improved glycemic control, enhanced insulin sensitivity, and provided profound weight loss in diet-induced obese (DIO) mice. Our results demonstrated that despite being a partial agonist, I-M-150847, by reducing the receptor internalization upon activation, enhanced the incretin effect and reversed obesity.NEW & NOTEWORTHY Replacement of the tryptophan cage (Trp-cage) with the C-terminal oxyntomodulin undecapeptide along with the tyrosine substitution at the amino terminus converts the selective glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 to a novel GLP-1R and GIPR dual agonist I-M-150847. Reduced internalization of incretin receptors upon activation by the GLP-1R and GIPR dual agonist I-M-150847 promotes iterative receptor signaling that enhances the incretin effect and reverses obesity.

Class B1 GPCRs: insights into multireceptor pharmacology for the treatment of metabolic disease.

The secretin-like, class B1 subfamily of seven transmembrane-spanning G protein-coupled receptors (GPCRs) consists of 15 members that coordinate important physiological processes. These receptors bind peptide ligands and use a distinct mechanism of activation that is driven by evolutionarily conserved structural features. For the class B1 receptors, the C-terminus of the cognate ligand is initially recognized by the receptor via an N-terminal extracellular domain that forms a hydrophobic ligand-binding groove. This binding enables the N-terminus of the ligand to engage deep into a large volume, open transmembrane pocket of the receptor. Importantly, the phylogenetic basis of this ligand-receptor activation mechanism has provided opportunities to engineer analogs of several class B1 ligands for therapeutic use. Among the most accepted of these are drugs targeting the glucagon-like peptide-1 (GLP-1) receptor for the treatment of type 2 diabetes and obesity. Recently, multifunctional agonists possessing activity at the GLP-1 receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor, such as tirzepatide, and others that also contain glucagon receptor activity, have been developed. In this article, we review members of the class B1 GPCR family with focus on receptors for GLP-1, GIP, and glucagon, including their signal transduction and receptor trafficking characteristics. The metabolic importance of these receptors is also highlighted, along with the benefit of polypharmacologic ligands. Furthermore, key structural features and comparative analyses of high-resolution cryogenic electron microscopy structures for these receptors in active-state complexes with either native ligands or multifunctional agonists are provided, supporting the pharmacological basis of such therapeutic agents.

Customizable Click Biochemistry Strategy for the Design and Preparation of Glucagon-like Peptide-1 Conjugates and Coagonists.

The development of oligomeric glucagon-like peptide-1 (GLP-1) and GLP-1-containing coagonists holds promise for enhancing the therapeutic potential of the GLP-1-based drugs for treating type 2 diabetes mellitus (T2DM). Here, we report a facile, efficient, and customizable strategy based on genetically encoded SpyCatcher-SpyTag chemistry and an inducible, cleavable self-aggregating tag (icSAT) scheme. icSAT-tagged SpyTag-fused GLP-1 and the dimeric or trimeric SpyCatcher scaffold were designed for dimeric or trimeric GLP-1, while icSAT-tagged SpyCatcher-fused GLP-1 and the icSAT-tagged SpyTag-fused GIP were designed for dual GLP-1/GIP (glucose-dependent insulinotropic polypeptide) receptor agonist. These SpyCatcher- and SpyTag-fused protein pairs were spontaneously ligated directly from the cell lysates. The subsequent icSAT scheme, coupled with a two-step standard column purification, resulted in target proteins with authentic N-termini, with yields ranging from 35 to 65 mg/L and purities exceeding 99%. In vitro assays revealed 3.0- to 4.1-fold increased activities for dimeric and trimeric GLP-1 compared to mono-GLP-1. The dual GLP-1/GIP receptor agonist exhibited balanced activity toward the GLP-1 receptor or the GIP receptor. All the proteins exhibited 1.8- to 3.0-fold prolonged half-lives in human serum compared to mono-GLP-1 or GIP. This study provides a generally applicable click biochemistry strategy for developing oligomeric or dual peptide/protein-based drug candidates.

Bridging the gap between GLP1-receptor agonists and cardiovascular outcomes: evidence for the role of tirzepatide.

Tirzepatide is a new drug targeting glucagon-like peptide 1(GLP1) and gastric inhibitory polypeptide (GIP) receptors. This drug has demonstrated great potential in improving the clinical outcomes of patients with type 2 diabetes. It can lead to weight loss, better glycemic control, and reduced cardiometabolic risk factors. GLP1 receptor agonists have been proven effective antidiabetic medications with possible cardiovascular benefits. Even though they have been proven to reduce the risk of major adverse cardiovascular events, their effectiveness in treating heart failure is unknown. Unlike traditional GLP1 receptor agonists, tirzepatide is more selective for the GIP receptor, resulting in a more balanced activation of these receptors. This review article discusses the possible mechanisms tirzepatide may use to improve cardiovascular health. That includes the anti-inflammatory effect, the ability to reduce cell death and promote autophagy, and also its indirect effects through blood pressure, obesity, and glucose/lipid metabolism. Additionally, tirzepatide may benefit atherosclerosis and lower the risk of major adverse cardiac events. Currently, clinical trials are underway to evaluate the safety and efficacy of tirzepatide in patients with heart failure. Overall, tirzepatide's dual agonism of GLP1 and GIP receptors appears to provide encouraging cardiovascular benefits beyond glycemic control, offering a potential new therapeutic option for treating cardiovascular diseases and heart failure.

Design of a novel long-acting dual GLP-1/GIP receptor agonist.

Tirzepatide, the first approved dual GLP-1/GIP receptor agonist (RA), has achieved better clinical outcomes than other GLP-1RAs. However, it is an imbalanced dual GIP/GLP-1 RA, and it remains unclear whether the degree of imbalance is optimal. Here, we present a novel long-acting dual GLP-1/GIP RA that exhibits better activity than tirzepatide toward GLP-1R. A candidate conjugate, D314, identified via peptide design, synthesis, conjugation, and experimentation, was evaluated using chronic studies in db/db and diet induced obese (DIO) mice. D314 achieved favorable blood glucose and body weight-lowering effects, equal to those of tirzepatide. Its half-life in dogs (T1/2: 78.3 ± 14.01 h) reveals its suitability for once-weekly administration in humans. This preclinical study suggests the potential role of D314 as an effective agent for treating T2DM and obesity.

Effect of Dual Glucagon-Like Peptide 1/Glucose-Dependent Insulinotropic Polypeptide Receptor Agonist (Tirzepatide) versus Bariatric Surgery on Weight Loss and Nonalcoholic Fatty Liver Disease.

OBJECTIVES: Bariatric surgery is a well-established treatment for obesity and type 2 diabetes. Tirzepatide, a dual GIP/GLP-1 receptor agonist, has emerged as a promising therapy for type 2 diabetes. This study aimed to compare the effects of bariatric surgery, semaglutide (a GLP-1 receptor agonist), and tirzepatide in Sprague-Dawley rats fed a high-fat diet. METHODS: Rats were divided into surgery, semaglutide, and tirzepatide treatment groups, along with a control group (sham). Weight, oral glucose tolerance, and levels of metabolic markers were assessed, along with adipose and liver tissue analysis. RESULTS: Surgery led to a 15.5% weight reduction, while rats treated with semaglutide exhibited a 10.7% reduction. Tirzepatide treatment at various concentrations (10, 50, and 100 nmol/kg) resulted in weight reductions of 5.0%, 14.9%, and 17.7%, respectively, compared to the sham group. Metabolic analyte levels decreased in intervention groups compared to the sham group, indicating improved metabolic health and glucose tolerance. Adipose tissue weight and hepatic liver fat droplets decreased in the intervention groups. CONCLUSION: Bariatric surgery and tirzepatide treatment significantly improved metabolic parameters in obese rats. Tirzepatide, particularly at higher concentrations, showed pronounced improvements compared to surgery and semaglutide. These findings suggest that high doses of tirzepatide could be explored as an alternative to bariatric surgery for the treatment of obesity.

Glucagon-like peptide-1/glucose-dependent insulinotropic polypeptide receptor co-agonists for cardioprotection, type 2 diabetes and obesity: a review of mechanisms and clinical data.

PURPOSE OF REVIEW: Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are approved for the management of type 2 diabetes (T2D) and obesity, and some are recommended for cardiorenal risk reduction in T2D. To enhance the benefits with GLP-RA mono-agonist therapy, GLP-1/glucose-dependent insulinotropic polypeptide (GIP) receptor co-agonists are in development to capitalize on the synergism of GLP-1 and GIP agonism. We review the mechanisms of action and clinical data for GLP-1/GIP receptor co-agonists in T2D and obesity and their potential role in cardiovascular protection. RECENT FINDINGS: Tirzepatide, a first-in-class unimolecular GLP-1/GIP receptor co-agonist, is approved for T2D and is awaiting approval for obesity management. Phase 3 trials in T2D cohorts revealed significant reductions in glycemia and body weight and superiority compared with GLP-1R mono-agonism with semaglutide. Tirzepatide has demonstrated significant body weight reductions in individuals with obesity but not diabetes. It enhances lipid metabolism, reduces blood pressure, and lowers liver fat content. Pooled phase 2/3 data showed cardiovascular safety in T2D while a post hoc analysis suggested tirzepatide slows the decline of kidney function in T2D. SUMMARY: GLP-1/GIP receptor co-agonists are a novel addition to the diabetes and obesity armamentarium. The cardiorenal-metabolic benefits position them as promising multiprong tools for metabolically complex individuals with chronic vascular complications.

GIP and GLP-2 together improve bone turnover in humans supporting GIPR-GLP-2R co-agonists as future osteoporosis treatment.

The intestinal hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2) are key regulators of postprandial bone turnover in humans. We hypothesized that GIP and GLP-2 co-administration would provide stronger effect on bone turnover than administration of the hormones separately, and tested this using subcutaneous injections of GIP and GLP-2 alone or in combination in humans. Guided by these findings, we designed series of GIPR-GLP-2R co-agonists as template for new osteoporosis treatment. The clinical experiment was a randomized cross-over design including 10 healthy men administered subcutaneous injections of GIP and GLP-2 alone or in combination. The GIPR-GLP-2R co-agonists were characterized in terms of binding and activation profiles on human and rodent GIP and GLP-2 receptors, and their pharmacokinetic (PK) profiles were improved by dipeptidyl peptidase-4 protection and site-directed lipidation. Co-administration of GIP and GLP-2 in humans resulted in an additive reduction in bone resorption superior to each hormone individually. The GIPR-GLP-2R co-agonists, designed by combining regions of importance for cognate receptor activation, obtained similar efficacies as the two native hormones and nanomolar potencies on both human receptors. The PK-improved co-agonists maintained receptor activity along with their prolonged half-lives. Finally, we found that the GIPR-GLP-2R co-agonists optimized toward the human receptors for bone remodeling are not feasible for use in rodent models. The successful development of potent and efficacious GIPR-GLP-2R co-agonists, combined with the improved effect on bone metabolism in humans by co-administration, support these co-agonists as a future osteoporosis treatment.

The dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist tirzepatide: a novel cardiometabolic therapeutic prospect.

Incretin hormones are peptides released in the intestine in response to the presence of nutrients in its lumen. The main incretins are glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). GLP-1 stimulates insulin secretion, inhibits glucagon secretion at pancreatic α cells and has also extrapancreatic influences as slowing of gastric emptying which increases the feeling of satiety. GIP is the main incretin hormone in healthy people, causative of most the incretin effects, but the insulin response after GIP secretion in type 2 diabetes mellitus (T2DM) is strongly reduced. Therefore, in the past GIP has been considered an unappealing therapeutic target for T2DM. This conception has been changing during recent years, since it has been reported that resistance to GIP can be reversed and its effectiveness restored by improving glycemic control. This fact paved the way for the development of a GIP receptor agonist-based therapy for T2DM, looking also for the possibility of finding a combined GLP-1/GIP receptor agonist. In this framework, the novel dual GIP and GLP-1 receptor agonist tirzepatide seems to be not just a new antidiabetic medication. Administered as a subcutaneous weekly injection, it is a manifold single pharmacological agent that has the ability to significantly lower glucose levels, as well as improve insulin sensitivity, reduce weight and amend dyslipidemia favorably modifying the lipid profile. Tirzepatide and additional dual GLP-1/GIP receptor agonists that could eventually be developed in the future seem to be a promising furthest advance for the management of several cardiometabolic settings. Obviously, it is too early to be overly hopeful since it is still necessary to determine the long-term effects of these compounds and properly verify the potential cardiovascular benefits. Anyway, we are currently facing a novel and very appealing therapeutic option.

Discovery of a novel GLP-1/GIP dual receptor agonist CY-5 as long-acting hypoglycemic, anti-obesity agent.

Glucagon-like peptide-1 (GLP-1) receptor agonists as an effective approach for type 2 diabetes mellitus (T2DM) has been explored extensively, multi agonists based on GLP-1 may have better clinical benefits on obesity, Nonalcoholic steatohepatitis (NASH) and other metabolic diseases. To get multi agonists based on GLP-1, 15 conjugates were designed, synthesized, and tested for biological activity. GLP-1/glucagon dual receptor agonist E1 showed moderate long-acting hypoglycemic effect, CY-5 and CY-16 with GLP-1/GIP dual receptor agonistic activity exhibited longer duration of continuous blood glucose stabilization. The long-acting hypoglycemic effect was equal to that of semaglutide. Although they have lost the agonistic activity on glucagon receptor, chronic in vivo studies on T2DM mice and diet-induced obesity (DIO) mice showed that CY-5 can effectively reduce food intake, inhibit body weight gain, repair islets damage and improve the glucose tolerance. One month treatment on NASH mice showed that CY-5 can significantly lower the TG, TC, AST, ALT and LDL-C and increase the HDL-C. CY-5 can also improve the liver vacuolation, reduce fat accumulation and delay the process of the fibrosis. The liver protection effect is better than that of semaglutide. In summary, CY-5 is a promising candidate for the treatment of metabolic diseases and worthy for further development.

The novel GLP-1/GIP dual agonist DA3-CH is more effective than liraglutide in reducing endoplasmic reticulum stress in diabetic rats with cerebral ischemia-reperfusion injury.

BACKGROUND AND AIMS: Diabetes is one of the most important risk factors and comorbidities of ischemic stroke. Endoplasmic reticulum stress (ERS) is considered to be the major injury mechanism of ischemic stroke with diabetes. Studies have found that incretin can inhibit ERS in ischemia-reperfusion injury of the liver and heart. We aimed to explore the effects of GLP-1/GIP double agonist DA3-CH and GLP-1 single agonist liraglutide on ERS and apoptosis in diabetic rats with cerebral ischemia-reperfusion injury. METHODS AND RESULTS: 72 Sprague-Dawley (SD) male rats were randomly divided into 4 groups: ① blank group (Sham group, n = 18); model group (Saline group, n = 18); DA3 treatment group (DA3 group, n = 18); liraglutide treatment group (Lir group, n = 18). The Sham group was not given any treatment and was only raised in the same environment as the other groups. The remaining 3 groups used STZ-induced diabetes models. After the successful membrane formation of diabetes, DA3-CH and liraglutide (10 mmol/kg, once-daily for 14 days) were injected intraperitoneally. Thereafter, rats were subjected to middle cerebral artery occlusion followed by 24-h reperfusion. Animals were evaluated for neurologic deficit score, infarct volume, and biomarker analyses of the brain after ischemia. The DA3-CH-treated and liraglutide-treated groups showed significantly reduced scores of neurological dysfunction and cerebral infarction size, and reduced the expression of ERS markers GRP78, CHOP and Caspase-12, and the expression of apoptosis marker bax. Anti-apoptotic markers bcl-2 and neuronal numbers increased significantly. CONCLUSIONS: DA3-CH and liraglutide have obvious neuroprotective effects in a rat model of cerebral ischemia-reperfusion injury with diabetes, which can reduce the infarct size and the neurological deficit score. Their exert neuroprotective effects in a rat model of cerebral ischemia-reperfusion injury with diabetes by inhibiting endoplasmic reticulum stress and thereby reducing apoptosis. DA3 is better than liraglutide.

The Design of a GLP-1/PYY Dual Acting Agonist.

The two gut hormones GLP-1 and PYY3-36 , which are both secreted from the L-cells upon food stimuli, have a stronger inhibitory effect on food intake when they are combined, compared to their individual effects as single agonists. Although they are not homologous and share no sequence similarity, we show that a GLP-1 analogue can be designed to exhibit potent activity on both the Y2 and GLP-1 receptors. Dual acting hybrid analogues were realized by designing truncated and potent Y2 receptor PYY analogues, followed by integrating the critical residues into GLP-1. In this study, we show that one of these dual acting agonists acutely reduces food intake significantly more than the respective mono-agonist counterparts.