Measure Real-Time cAMP Signaling in the Living Cell

Abstract

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that plays a key role in regulating glucose homeostasis via its activity at the GLP-1 receptor (GLP-1R). To date, GLP-1R has become an attractive therapeutic target for type 2 diabetes (T2D). Several GLP-1R agonists have been developed or are in clinical trials such as: Exenatide, Liraglutide, Lixisentide, Dulagutide, Semiglutide, Efpeglenatide, and Albenatide. These drugs stimulate G-protein-coupled receptors (GPCR) resulting in adenylated cyclase activation and consequently cyclic adenosine mono-phosphate (cAMP) production. This makes the cAMP assay platform is useful for identifying GLP-1 functional activities. However, current cAMP assays have two major limitations: 1) low accuracy with infeasibility of real time measurements of cAMP levels and 2) low precision among separate sample preparation from live cells. In order to overcome those restrictions and design a rapid and homogeneous protocol, a living cell based real-time potency assay has been developed for measuring bioactivity of GLP-1r agonists such as Albenatide. This assay, which combines the stimulating cell, lysis, and cAMP measurement in-situ, provides fast, accurate, reproducible, and highly sensitive results. The assay linearity has been tested with the proportionality of concentration/amount of analyte. The r2 of the linear curve in measuring relative potency (RP) vs. nominal RP was >0.99 and the recovery of the expected relative potency in the range of 50∼200% were 102.6∼112.4%. Compared with the traditional ELISA-based assay, this assay requires less sample volume (20ul vs. 100ul), low matrix effect with wide assay window (5 fold higher), high sensitivity (pM vs. nM), and shorter procedure time (hours vs. days). Thus, applications of this novel cell-based cAMP assay can be extended to high-throughput screening for the potency of GLP-1r agonists, and more specifically characterizing the anti-drug antibody (ADA) capabilities. Disclosure X. Liu: None. S. Hong: None. S. Wu: None. Z. Yao: None. Q.X. Yao: None.