ICE Bioscience Application Notes

Obesity, a complex and heterogeneous disease, has exhibited a nearly threefold global prevalence increase since 1975 according to World Health Organization (WHO) statistics. Projections indicate that over 4 billion individuals worldwide will be affected by obesity or be overweight by 2035, representing nearly 50% of the global population. Recognized by WHO as the fifth leading global mortality risk factor, obesity serves as a primary pathogenic driver and exacerbating factor for multiple disorders, including cardiovascular diseases, diabetes mellitus, and specific malignancies.

Amidst the persistent escalation of the diabetes and obesity pandemics, the pharmaceutical industry has intensified its focus on this high-potential market. Morgan Stanley forecasts predict the global obesity-related market will reach US$77 billion by 2030. Although emerging therapies such as glucagon-like peptide-1 (GLP-1) analogues (e.g., semaglutide) and dual glucose-dependent insulinotropic polypeptide (GIP)/GLP-1 receptor agonists (e.g., tirzepatide) have received clinical approval for obesity management, substantial demand persists for alternative or adjunctive therapies employing distinct mechanisms.

Besides GLP-1R, the amylin receptor is emerging as a promising next-generation therapeutic target, poised to potentially capture a multibillion-dollar market segment in glycemic control and weight management interventions. Amylin, a 37-amino acid peptide hormone known as islet amyloid polypeptide (IAPP), is a pancreatic beta-cell peptide hormone involved in satiation and controlling food intake. The metabolic regulatory functions of amylin are mediated by amylin receptors (AMYRs), which are a unique class of protein heterodimers formed by the combination of a calcitonin receptor (CTR) with three homologous receptor activity-modifying proteins (RAMP1, RAMP2, and RAMP3), resulting in three subtypes (AMY1R, AMY2R, and AMY3R).

Figure 1. Amylin receptor components comprise family B GPCR, the calcitonin receptor.

Amylin, an FDA-approved therapeutic agent for glycemic control, not only inhibits glucagon secretion and modulates postprandial blood glucose levels by augmenting leptin sensitivity but also exerts ancillary pharmacological effects. These include appetite suppression, induction of satiety, reduced caloric intake, suppression of gastric acid secretion and delayed gastric emptying.

Table 1. Key Advances in AMY3R/CTR-Targeted Drug Discovery

3.1 HTRF-cAMP Assay Development for CTR/AMY Receptors in COS7 Cell

To facilitate drug discovery targeting amylin/calcitonin receptors, the ICE team evaluated non-lipidated peptide hCT and human amylin and the lipidated peptide cagrilintide using the HTRF-cAMP assay across multiple host cell lines. The results showed that HEK293 and Flp-In-CHO cells produced strong responses to hCT and cagrilintide, whereas COS7 and UMUC-3 cells exhibited minimal background signals, making them ideal for genetic engineering.

Figure 2. hCT and hAmylin, Cagrilintide test in COS7, HEK293, Flp-In-CHO and UMUC-3 cells with HTRF cAMP assay.

Accordingly, COS7 cells were selected to generate isogenic cell lines overexpressing human CTR, AMY1R, AMY2R and AMY3R. This platform features low background noise and excellent signal amplification, supporting ultra-high-throughput screening and definitive pharmacological research. In addition, cell lines expressing CTR and AMY receptors from multiple species including rat, mouse, monkey and dog were constructed to meet the requirements for target selectivity and species-specific pharmacological assessment. Meanwhile, a full panel of overexpression cell lines based on UMUC-3 was also established.

Table 2. Establishment of Overexpressing Cell Lines and cAMP Assay Development

To assess the impact of albumin binding on peptides' cAMP activity, we tested lipidated/non-lipidated peptides with 0.1% casein, 0.1% bovine serum albumin (BSA) and 1% human serum albumin (HSA). As expected, 0.1% BSA and 1% HSA reduced the potency of the lipidated peptides (elorilintide and cagrilintide) at human AMY1R, AMY3R, and CTR, while the non-lipidated rat amylin and sCT showed no significant potency shifts.

Figure 3. sCT, Rat Amylin, Elorilintide, Cagrilintide and NN1213 were used as tool compounds to optimize the HTRF cAMP assay in COS7-CTR, AMY1R, AMY2R, AMY3R monoclonal cells.

Species selectivity evaluation is a key component of early-stage drug discovery, as it helps ensure that preclinical findings are translatable to human efficacy and safety. To enable such assessments, the ICE Bioscience team established stable COS7 cell lines overexpressing rat, mouse, dog, and monkey CTR and AMY3R, and developed a fully optimized HTRF-cAMP assay. This high-throughput platform offers robust cross-species selectivity profiling of compounds, thereby providing critical support for preclinical in vivo studies and high-throughput screening campaigns.

Figure 4. sCT, hCT, rat CT, and rat amylin were used as tool compounds to optimize the HTRF cAMP assay in COS7-rat CTR, COS7-rat AMY1R and COS7-rat AMY3R monoclonal cells.

3.2 HTRF-cAMP Assay Development for CTR/AMY Receptors in UMUC-3 Cell

UMUC-3 cells were selected as a host cell line for AMY and CTR drug screening due to their exceptionally low endogenous background activity toward amylin/calcitonin receptor signaling. UMUC-3 human CTR, AMY1R, AMY2R and AMY3R, as well as CGRP, AM1 and AM2 OE cells was established for cAMP assay validation.

Figure 5. Rat amylin, Elorilintide and Cagrilintide were used as tool compounds to optimize the HTRF cAMP assay in UMUC-3-hCTR AMY1R, AMY2R and AMY3R cell.

3.3 A NanoBiT Platform for β-Arrestin-2 Screening

In early drug discovery, assessing beta-arrestin recruitment helps determine whether a GPCR ligand induces receptor desensitization, internalization, or non-canonical signaling. This information is key for identifying pathway-biased agonists that avoid beta-arrestin-mediated side effects (e.g., tolerance), thereby supporting lead optimization and selection of safer, more efficacious candidates before preclinical studies.

Figure 6. NanoBiT platform for beta-arrestin recruitment assay.

A NanoBiT-based platform for beta-arrestin-2 recruitment has been established by the ICE Bioscience team. This sensitive, high-throughput, and quantitative assay is broadly applicable to agonist/antagonist screening, biased signaling investigation, and safety pharmacology assessment. The platform currently comprises 32 fully validated, screening-ready targets. Notably, several stable cell lines—including HEK293T-Arrestin2-hCTR, HEK293T-Arrestin2-hAMY1R, HEK293T-Arrestin2-hAMY2R, and HEK293T-Arrestin2-hAMY3R—have been generated and optimized using reference compounds such as sCT, human calcitonin, and rat amylin. This well-characterized system offers robust support for drug discovery efforts targeting the CTR and AMY family receptors.

Figure 7. NanoBiT assay in HEK293T-Arrestin2-hCTR, HEK293T-Arrestin2-hAMY3R stable cell.

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