Structural and mechanistic insights into dual activation of cagrilintide in amylin and calcitonin receptors.

Dual amylin and calcitonin receptor agonists (DACRAs) are novel candidates for treatment of T2D and obesity due to their beneficial effects on body weight, blood glucose and insulin sensitivity, effects that are superior to those of amylin. However, it is unknown how dual receptor activation by DACRAs compare to activation of either the amylin or the calcitonin receptor in vivo, and to what extent the calcitonin receptor is involved in the beneficial metabolic effects of DACRAs. In this study, we compared the effects of a highly potent DACRA, KBP, rat amylin (rAMY), rat calcitonin (rCT) and the combination of rAMY and rCT on long term efficacy on body weight, food intake and glucose tolerance. High fat fed Sprague Dawley rats were treated 4 weeks with KBP (5 µg/kg/day), rAMY (300 µg/kg/day), rCT, (300 µg/kg/day) and the combination of rAMY and rCT (300+300 µg/kg/day). To compensate for the different activity profiles, peptides were delivered by continuous subcutaneous infusion. Chronic treatment with KBP, rAMY and the combination of rAMY and rCT resulted in a 17%, 11% and 8% vehicle-corrected weight loss, respectively, while rCT alone had no effect. All treatments significantly reduced food intake in the initial phase of the study, while KBP and rAMY resulted in a prolonged reduced food intake. Interestingly, KBP was superior in terms of body weight loss (p<0.001) even though rAMY and KBP reduced the accumulated food intake equally. Moreover, KBP and rAMY improved oral glucose tolerance with significantly reduced insulin levels. Again, KBP was superior to the other treatments (p<0.001). In conclusion, dual activation of amylin and calcitonin receptors by KBP has beneficial effects on body weight and glucose tolerance superior to that of activating either receptor alone. This was despite correcting for the different activity of the peptides. Understanding of the dual or single receptor activation mechanisms is high pharmaceutical interest. Disclosure A.T. Larsen: None. N. Sonne: None. K.V. Andreassen: Employee; Self; Nordic Bioscience. M.A. Karsdal: Employee; Self; Nordic Bioscience. K. Henriksen: Employee; Self; Nordic Bioscience. Employee; Spouse/Partner; Novo Nordisk A/S. Stock/Shareholder; Self; Nordic Bioscience. Other Relationship; Self; Nordic Bioscience.

Amylin treatment improves body weight and glucose control, although it is limited by a short action and need for high doses. Dual amylin and calcitonin receptor agonists (DACRAs) are dual amylin and calcitonin receptor agonists with beneficial effects beyond those of amylin. However, to what extent the additional benefits reside in their higher potency or their targeting of the calcitonin receptor remains unclear. Here we deconstruct the receptors involved in the effects of a DACRA, KBP-088, by comparing it to rat amylin (rAMY), rat calcitonin (rCT), and their combination in obese high-fat diet (HFD) and diabetic Zucker diabetic fatty (ZDF) rats. HFD-fed Sprague-Dawley rats and ZDF rats were treated for 4 weeks with KBP-088 (5 µg/kg per day), rAMY (300 µg/kg per day), rCT (300 µg/kg per day), and the combination of rAMY and rCT (300+300 µg/kg per day) using infusion pumps. Body weight, food intake, fasting glycemia, glycated hemoglobin type A1c levels, and glucose tolerance were assessed. In obese HFD-fed rats, KBP-088, rAMY, and the combination of rAMY and rCT significantly reduced body weight and improved glucose tolerance, whereas rCT alone had no effect. In diabetic ZDF rats, rCT was efficient in lowering fasting glycemia similar to rAMY, whereas dual activation by KBP-088 and the combination of rAMY and rCT were superior to activating either receptor alone. In conclusion, calcitonin therapy regulates fasting blood glucose in a diabetic rat model, thereby underscoring the importance of calcitonin receptor activation as well as the known role of amylin receptor agonism in the potent metabolic benefits of this group of peptides. SIGNIFICANCE STATEMENT: We deconstruct the receptors activated by dual amylin and calcitonin receptor agonist (DACRA) therapy to elucidate through which receptor the beneficial metabolic effects of the DACRAs are mediated. We show that calcitonin receptor activation is important for blood glucose regulation in diabetes. This is in addition to the known metabolic beneficial role of amylin receptor activation. These data help in understanding the potent metabolic benefits of the DACRAs and underline the potential of DACRAs as treatment for diabetes and obesity.

Obesity is a major and increasingly prevalent chronic metabolic disease with numerous comorbidities. While recent incretin-based therapies have provided pharmaceutical inroads into treatment of obesity, there remains an ongoing need for additional medicines with distinct modes of action as independent or complementary therapeutics. Among the most promising candidates, supported by phase 1 and 2 clinical trials, is cagrilintide, a long-acting amylin and calcitonin receptor agonist. As such, understanding how cagrilintide functionally engages target receptors is critical for future development of this target class. Here, we determine structures of cagrilintide bound to Gs-coupled, active, amylin receptors (AMY1R, AMY2R, AMY3R) and calcitonin receptor (CTR) and compare cagrilintide interactions and the dynamics of receptor complexes with previously reported structures of receptors bound to rat amylin, salmon calcitonin or recently developed amylin-based peptides. These data reveal that cagrilintide has an amylin-like binding mode but, compared to other peptides, induces distinct conformational dynamics at calcitonin-family receptors that could contribute to its clinical efficacy.

Introduction and Objective: Amylin analogues are being investigated as potential obesity treatments. Amylin receptors (AMYRs) belong to the calcitonin receptor (CTR) family and consist of heterodimers of the calcitonin receptor and receptor activity modifying proteins (RAMPs). CTR and AMYRs exhibit distinct physiological roles with CTR activation being important for calcium homeostasis, while AMYRs are involved in appetite regulation and body weight. It is however uncertain what the clinical role is of different receptor profiles. In this study we compare three amylin analogues in clinical development (cagrilintide, eloralintide and NN1213) on CTR and AMYRs activation as well as calcium lowering ability in rats to evaluate AMYR selectivity and furthermore address the efficacy on food intake and body weight reduction. Methods: Receptor potency was measured in vitro. Receptor selectivity was further investigated in rats, by measuring Ca2+ plasma levels after a single subcutaneous injection of vehicle, cagrilintide (10 nmol/kg), eloralintide (10 or 30 nmol/kg) or NN1213 (10 or 30 nmol/kg). In addition, the effect of NN1213 (10 nmol/kg, qd, sc.) on body weight and food intake was investigated in diet-induced obese rats during 17 days of dosing. Body weight and Ca2+ levels and food intake were analysed by a mixed effect model or 2-way repeated measure ANOVA, respectively, followed by Dunnett’s multiple comparison test. Results: In vitro, cagrilintide and eloralintide activated both rat and human AMYRs and CTR whereas NN1213 was more selective for AMYRs. In rats, cagrilintide and eloralintide induced a prolonged decrease in Ca2+ plasma levels in rats while NN1213 did not. Furthermore, NN1213 decreased body weight and food intake in diet-induced obese rats. Conclusion: Cagrilintide and eloralintide are both AMYR and CTR agonists whereas NN1213 is a selective AMYRs agonist that also decreases body weight in rats. Disclosure D. Ipsen: Employee; Novo Nordisk A/S. B. Ballarin-Gonzalez: Employee; Novo Nordisk. Stock/Shareholder; Novo Nordisk. E. Moo: Employee; Novo Nordisk. A. Secher: Employee; Novo Nordisk A/S. Stock/Shareholder; Novo Nordisk A/S. K. Raun: Employee; Novo Nordisk A/S. Stock/Shareholder; Novo Nordisk A/S.

Amylin receptors (AMYRs), heterodimers of the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins, are promising obesity targets. A hallmark of AMYR activation by Amy is the formation of a 'bypass' secondary structural motif (residues S19-P25). This study explored potential tuning of peptide selectivity through modification to residues 19-22, resulting in a selective AMYR agonist, San385, as well as nonselective dual amylin and calcitonin receptor agonists (DACRAs), with San45 being an exemplar. We determined the structure and dynamics of San385-bound AMY3R, and San45 bound to AMY3R or CTR. San45, via its conjugated lipid at position 21, was anchored at the edge of the receptor bundle, enabling a stable, alternative binding mode when bound to the CTR, in addition to the bypass mode of binding to AMY3R. Targeted lipid modification may provide a single intervention strategy for design of long-acting, nonselective, Amy-based DACRAs with potential anti-obesity effects.

The Family B G protein-coupled calcitonin receptor is an important drug target. The aim of this work was to elucidate the molecular mechanism of action of small-molecule agonist ligands acting at this receptor, comparing it with the action mechanism of the receptor's natural peptide ligand. cAMP responses to four non-peptidyl ligands and calcitonin were studied in COS-1 cells expressing wild-type and chimeric calcitonin-secretin receptors. All compounds were full agonists at the calcitonin receptor with no activity at the secretin receptor. Only chimeric constructs including the calcitonin receptor amino terminus exhibited responses to any of these ligands. We progressively truncated this domain and tested constructs for cAMP responses. Although calcitonin was able to activate the calcitonin receptor fully with the first 58 residues absent, its potency was 3 orders of magnitude lower than that at the wild-type receptor. After truncation of 114 residues, there was no response to calcitonin. In contrast, small-molecule ligands were fully active at receptors having up to 149 amino-terminal residues absent. Those compounds finally became inactive after truncation of 153 residues. Deletion and/or alanine replacement of the region of the calcitonin receptor between residues 150 and 153 resulted in marked reduction in cAMP responses to these compounds, with some compound-specific differences observed, supporting a critical role for this region. Binding studies further supported distinct sites of action of small molecules relative to that of calcitonin. These findings focus attention on the potential importance of the juxtamembranous region of the amino terminus of the Family B calcitonin receptor for agonist drug action.

Amylin receptors (AMYRs) are heterodimers of the calcitonin (CT) receptor (CTR) and one of three receptor activity-modifying proteins (RAMPs), AMY1R, AMY2R, and AMY3R. Selective AMYR agonists and dual AMYR/CTR agonists are being developed as obesity treatments; however, the molecular basis for peptide binding and selectivity is unknown. We determined the structure and dynamics of active AMYRs with amylin, AMY1R with salmon CT (sCT), AMY2R with sCT or human CT (hCT), and CTR with amylin, sCT, or hCT. The conformation of amylin-bound complexes was similar for all AMYRs, constrained by the RAMP, and an ordered midpeptide motif that we call the bypass motif. The CT-bound AMYR complexes were distinct, overlapping the CT-bound CTR complexes. Our findings indicate that activation of AMYRs by CT-based peptides is distinct from their activation by amylin-based peptides. This has important implications for the development of AMYR therapeutics.

Pharmacological discrimination of calcitonin receptor: receptor activity-modifying protein complexes.

Development of a Novel Assay for Direct Assessment of Selective Amylin Receptor Activation Reveals Novel Differences in Behavior of Selective and Nonselective Peptide Agonists.

Actions of β-Amyloid Protein on Human Neurons Are Expressed through the Amylin Receptor

Modulation of amylin and calcitonin receptor activation by hybrid peptides

Development of the novel amylin and calcitonin receptor activators by peptide mutagenesis

Parallel activation of the calcitonin receptor (CTR) and amylin receptor (AMYR) is considered a more effective weight-loss strategy. Although the novel dual amylin and calcitonin receptor agonist (DACRA), petrelintide, is currently undergoing phase II clinical trials, its agonistic activity remains insufficient compared with natural agonists. Further optimization of the agonistic capabilities of petrelintide is an attractive strategy for developing DACRAs. Due to the lack of structure-activity relationship (SAR) and target binding information, a step-by-step process involving three rounds of modifications was performed guided by structure-based drug design and molecular dynamics (MD) simulations. Two successful methylation strategies led to the identification of the more efficient novel DACRA, BGM1812, with excellent performance in terms of half-life, stability, and solubility. In both in vivo and in vitro studies, BGM1812 showed significantly enhanced efficacy. This finding provides valuable insights into the SAR of petrelintide and highlights the potential of BGM1812 as a promising obesity drug candidate.

Long-acting dual amylin and calcitonin receptor agonists (DACRAs) are novel candidates for treatment of type 2 diabetes (T2D) and obesity due to their beneficial effects on both body weight, glucose control and insulin action. Cagrilintide, which is currently in clinical trials, has shown promising effects on weight loss. In this study we compared a new long-acting DACRA (KBP) to cagrilintide in pre-clinical models of obesity and T2D. In vitro potencies were assessed using receptor assays. In vivo efficacies were investigated head-to-head in high fat diet (HFD) fed obese and T2D (ZDF) rat models. In vitro data showed that both peptides active both the amylin and the calcitonin receptor, with KBP being more potent on both receptors. This was further confirmed in vivo by assessment of acute effects on food intake and CTX suppression. KBP (1.5, 4.5 and 13.5 nmol/kg) and cagrilintide (10, 30 and 100 nmol/kg) induced a potent and dose-dependent weight loss in HFD rats, with the highest dose of KBP being superior to cagrilintide. In diabetic ZDF rats, DACRA treatment improved glucose control and preserved plasma insulin compared to vehicle. Interestingly, despite similar levels of plasma insulin, KBP treatment was superior to cagrilintide in improving glucose control. This was further reflected in the HbA1c levels at study end, where KBP treatment resulted in significantly lower levels compared to cagrilintide. In summary, both DACRAs induced weight loss and improved glucose tolerance, insulin action as well as glucose control. However, KBP treatment results in superior efficacy on both weight loss and glucose control. These findings highlight KBP as a promising once-weekly agent for treatment of obesity and T2D. Disclosure A.T.Larsen: Employee; Nordic Bioscience. N.Sonne: None. K.Mohamed: None. E.Bredtoft: None. F.Andersen: None. M.A.Karsdal: Employee; Nordic Bioscience A/S, Nordic Bioscience A/S, Nordic Bioscience A/S. K.Henriksen: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S.

Introduction and Objective: Amylin, a peptide hormone co-secreted with insulin by pancreatic beta cells in response to food intake, suppresses appetite and delays gastric emptying, making it an attractive target for obesity therapy. Cagrilintide, a long-acting dual amylin and calcitonin receptor agonist (DACRA) peptide, has demonstrated promising efficacy and safety profile in clinical trials, but an oral small molecule DACRA with good bioavailability and stability could provide a more convenient and accessible alternative for obesity treatment. This study evaluates the in vitro and in vivo pharmacological properties of ACCG-2671 for obesity treatment. Methods: The in vitro binding affinity and functional potency of ACCG-2671 were assessed for the calcitonin receptor (CTR) and amylin receptor (AMY3R). Chronic weight-loss effects were evaluated in diet-induced obese (DIO) rats by oral daily dosing of ACCG-2671 as single treatments or in combination treatments with semaglutide using two treatment regimens: add-on and concurrent combination. Results: ACCG-2671 demonstrated high binding affinity and balanced potency in human CTR and AMY3R functional assays. In DIO rats, oral administration of ACCG-2671 resulted in significant, dose-dependent body weight reductions. Combination treatments (add-on and concurrent) resulted in superior weight loss compared to single treatments. Conclusion: ACCG-2671 is a novel oral small molecule DACRA that significantly reduces body weight in obese rats. Its pharmacological profile and preclinical efficacy support its advancement as a development candidate, alone and in combination for obesity treatment, potentially offering a novel, convenient, and accessible therapeutic option. Disclosure X. Fang: Employee; Structure Therapeutics, Inc. Z. Zhang: Employee; Structure Therapeutics, Inc. H. Zhang: Employee; Structure Therapeutics, Inc. C. Lu: None. D. Xue: Employee; Structure Therapeutics, Inc. Z. Wang: Employee; Structure Therapeutics, Inc. S. Zhan: Employee; Structure Therapeutics, Inc. X. Wang: Employee; Structure Therapeutics, Inc. W. Liang: Employee; Structure Therapeutics, Inc. T. Ma: Employee; Structure Therapeutics, Inc. N. Hu: Employee; Structure Therapeutics, Inc. J.J. Zhang: None. L. Chen: None. X. Lai: None. D. He: None. C. Li: Employee; Structure Therapeutics, Inc. Z. Gao: Employee; Structure Therapeutics, Inc. X. Lin: None. X. Jiang: Employee; Structure Therapeutics, Inc. H. Lei: Employee; Structure Therapeutics, Inc. F. Zhang: Stock/Shareholder; Eli Lilly and Company, Novo Nordisk A/S. Employee; Structure Therapeutics, Inc.