This article highlights a system that combines acoustic dispensing, small-volume pipetting, and robust assay chemistries to identify GPCR modulators. This combination enables rapid screening of GPCRs in a 1,536-well format. Implementing this miniaturized, high-throughput GPCR assay allows cost-effective screening to identify lead modulators of GPCR signaling.
The Promega (www.promega.com) cAMP-Glo™ assay measures changes in cAMP levels, a hallmark of GPCR modulation. This homogeneous, bioluminescent assay is easily scalable to 1,536-well plate formats. The assay measures cAMP levels in cells by monitoring cAMP production in response to the effects of a test compound on GPCRs.
GPCRs that couple with adenylate cyclase will increase or decrease intracellular cAMP levels. The assay is based on the principle that cAMP stimulates protein kinase A (PKA) holoenzyme activity, decreasing available ATP and leading to decreased light production in a coupled luciferase reaction (Figure 1).
Binding of an extracellular ligand to its receptor alters the conformation of the associated heterotrimeric G protein (shown in orange and yellow in Figure 1), causing dissociation of the Ga and GbY subunits and initiating a cascade of cellular events. The alpha subunit, shown in yellow, is categorized into one of several groups: Gas, Gai/o, Gaq, and Ga12/13. Gas activates adenylate cyclase, while Gai/o inhibits adenylate cyclase activity.
As the concentration of cAMP increases, cAMP binds to PKA, supplied in the kit, and the regulatory subunits undergo a conformational change to release the catalytic subunits. The free catalytic subunits then catalyze the transfer of the terminal phosphate of ATP to a PKA substrate, consuming ATP in the process. The level of remaining ATP is determined using Kinase-Glo® (Promega), a luciferase-based-reagent. Luminescence is inversely proportional to cAMP levels. Thus, as cAMP concentration increases, luminescence decreases.
The cAMP-Glo assay can be performed in standard multiwell formats, including 96-, 384- or 1,536-well plates. Individual test compounds or compound libraries are transferred into the plates using an acoustic liquid handler (Echo® 555, Labcyte). Sound energy enables the dispensing of droplets of test compounds in 2.5-nL increments directly from source plates to the assay plates. No pipette tip or pin tool ever touches the compound samples during transfer. Additional advantages to using an acoustic liquid handler for dispensing compounds include precision (< 8% CV at 2.5 nL), minimal compound sample required (only 10 nL per assay well for the application described here), minimal dilutions required, reduced consumable usage, and reduced screening cost.
After the compounds are dispensed into the assay plates, cells of interest are added. Adherent, suspension, or frozen cell cultures can be used with the cAMP-Glo Assay. Cells are treated with the test compound or compound library in an induction buffer containing broad-range phosphodiesterase inhibitors to inhibit cAMP hydrolysis for an appropriate period of time to modulate cAMP levels.
Cells and subsequent assay reagents can be dispensed into assay plates using a non-contact, liquid dispenser (Equator™ HTS, Deerac Fluidics) for accurate pipetting. Advantages of using the Equator include speed and accuracy, a 50 nL to 50 µL volume-dispense range, low dead volumes, and ease of use. Independent channel control can be used for dispensing gradients when optimizing assay conditions. Interchangeable deck components, including small- or large-volume capacity reservoirs, stirring devices, and multiple wash stations, or other customizable configurations, make this a flexible platform for high-throughput screening (HTS).