ViewLux with modified kinetics of the aequorin reaction

Using the substrate coelenterazine i PerkinElmer has been able to adapt the flash luminescence aequorin assay to standard luminometers and potentially remove the requirement for integrated injectors. The aequorin technology has now been demonstrated on the ViewLux^® ultra HTS Imaging platform.

Coelenterazine i can be used as an alternative to coelenterazine h to reconstitute the active aequorin, enabling a delay in the cellular signal.

Compared to coelenterazine h, coelenterazine i has an iodine atom, which slows down the kinetics of the aequorin enzyme in biochemical assays.

Coelenterazine i slows kinetics

The use of coelenterazine i also slows down the kinetics of the cell-based assay. The response of CHO-H1 cells to histamine (H1 receptor agonist) and to ATP (agonist of the endogenous CHO cells’ P2Y receptor) is slower when derivative i was used (figure 2) than when derivative h was used (figure 1).

• Receptor Ligand Binding
• Receptor Membranes
• GPCR Cell Lines
• cAMP Quantitation
• Phosphorylation of ERK
• GTP Binding
• Reporter Gene Assays
• Intracellular Calcium

• Reagents and Assays
• Photoproteins
  
• Cell Lines
• Radioligands
• Detection Instruments
• Automated Liquid Handling
• High Content Screening
• Microplates
• Assay Development & Labeling

• MicroBeta²™ LumiJET™
• EnSpire™ Alpha Plate Reader

Figure 1.
 
Figure 2.
 

Comparable pharmacology can be obtained with the i derivative

Coelenterazine i was tested on 10 GPCR AequoScreen^® (aequorin) cell lines, and was found to generate a strong enough luminescent response for 7 of the 10 tested cell lines. The rank order of potency of the agonists and antagonists was conserved. In the agonist assay, pEC₅₀ values were on average greater by 0.85 (min +0.5, max +1.7) when using coelenterazine i compared to h, while in the antagonist assay pIC₅₀ values were on average smaller by 0.2 (min -0.9, max +0.4) when using coelenterazine i compared to h. Full kinetic data acquired on the LumiLux^® in the 384-well format was used to draw the dose-response curves in figures 3 and 4 and the data summarized in tables 1 and 2. See poster for the antagonist data, link below.

Agonist responses of the Vasopressin 1b receptor with coelenterazines h and i

Figure 3.

Figure 4.

Table 1. Agonist and antagonist assay on the V_1b receptor


Table 2. Agonist assay on the Adenosine A_2b receptor