by Jean-Luc Tardieu

Introduction

G-Protein Coupled Receptors (GPCRs) are currently the most important target class being investigated in the drug discovery process. Upon activation, GPCRs carry information within the cell via two major signaling pathways: one results in variations of the cAMP level, whereas the other results in a transient increase of intracellular Ca2+ triggered by inositol (1,4,5) tri-phosphate (IP3). The lifetime of IP3 is short. Therefore, there is great interest in following GPCR activation through the monitoring of IP3 degradation products, such as inositol (1) phosphate (IP1), which accumulates in the cell in the presence of LiCl (Figure 1).

     The IP-One Assay represents a novel and homogeneous screening platform that delivers second messenger measurement and represents a way of precisely investigating molecular events occurring after cell stimulation. Lithium chloride (LiCl) 50 mM in the stimulation buffer inhibits the phosphatase responsible for the degradation of IP1 in myo-inositol and allows IP1 accumulation in the cell.

Assay principle

The IP-One Assay was developed using Cisbio’s (Cedex, France; CIS US, Inc., Bedford, MA) HTRF (Homogeneous Time Resolved Fluorescence) technology. The assay is based on a monoclonal antibody specific to IP1, labeled with europium cryptate, competing with both native IP1 produced by cells and IP1 coupled with the dye d2 (a new HTRF acceptor). The specific signal (i.e. energy transfer) is inversely proportional to the concentration of IP1 in the calibrator or in the cell lysate.

     Within one hour after HTRF reagent distribution, the signal reaches equilibrium and remains stable even after several days of incubation at 4 C. Moreover, the signal is robust and not light sensitive, so the same plate can be read several times without any alteration of the signal level.

Optimizing an IP1 cell-based assay

The IP-One Assay validation must be carried out in the white cell cultured 384-well microplates (Greiner Bio-One, Monroe, NC). Figure 2 summarizes the three steps (A, B and C) to optimize an IP-One cell-based assay. All tests should include appropriate controls and in particular:

—Negative control (cryptate blank): Will allow the level of cryptate conjugate to be verified from the signal obtained at 620 nm. This control contains all components except the IP1-d2.

—Positive control: This control contains all components plus an IP1 standard solution without the ligand. HTRF signal proportionally decreases with IP1 concentration in the control.

—Non-stimulated cell: Contains all the components except the ligand. If the ligand is an agonist, the signal obtained with the ligand is maximum (HTRF basal signal) as the cells do not contain IP1, except for constitutively active GPCRs.

Step A — influence of the cell density on IP1 response

Cell density is the first parameter to be optimized in a cell-based assay. It depends on the expression level of the cell line tested.

Day 1: Plate 80 μl of the cell solution. The different densities tested are 10,000 — 20,000 — 40,000 and 60,000 cells per well. Cells are incubated overnight at 37°C under cell culture conditions (CO₂).

Day 2: Supernatant is discarded. The cells are stimulated for 90 minutes with the known agonist (vol. 10 μl). It is recommended to do a four-concentration agonist response around the IC50 expected for the ligand.

HTRF reagents are added and the reading is done after 60 minutes of incubation.

The optimal cell density is defined depending on the non-stimulated cell signal and the signal window.

This cell density is then used for the rest of the optimization.

Step B - influence of the stimulation buffer pre-incubation

The phosphatase responsible for IP1 degradation is inhibited by LiCl. Cell pre-incubation with the suggested stimulation buffer (LiCl 50 mM) could allow a better IP1 accumulation in the cell.

After an overnight incubation of the cells, the cell culture supernatant is discarded. The cells are either directly stimulated with 20 μl of a ligand solution in stimulation buffer, or pre-incubated for 15 minutes with 10 μl of stimulation buffer before addition of 10 μl of ligand.

Step C - kinetics study of the stimulation step

After GPCR activation, IP1 concentration increases in the cell, to quickly reach a maximum. Three stimulation times are tested: 30, 60 and 90 minutes. Plates are incubated at 37 C under cell culture conditions (CO₂).

An agonist dose response is performed. When the maximum IP1 production is reached, the working window of the assay is maximum and the IC50 for the agonist tested is at its lowest.

Improvement of screening conditions

When the IP-One cell-based assay is optimized with the GPCR target of interest, some additional parameters could be tested to improve the quality of the assay.

Evaluation of the stimulation buffer. The recommended stimulation buffer, supplied with the kit, satisfies most applications. Meanwhile, other stimulation buffers complemented with LiCl 50 mM can be tested. The use of buffers containing phosphate is prohibited. Phosphate strongly affects the sensitivity of the assay.

Miniaturization of the assay. HTRF allows very straightforward assay miniaturization to 96 half-well, 384 well or 1,536 well microplate formats. Proportional downsizing of all volumes will usually generate a comparable signal, providing of course that all the reagent concentrations are maintained at the same level.

Cell based assay with frozen cells. All the optimization steps were performed with cells plated the day before the assay. After an overnight incubation in cell culture conditions the cells became adherent. IP-One assays were also performed with frozen cells, thawed and plated a few hours before stimulation by the ligand.

     Cells were produced several days before the cell-based assay, and stored at 㪬 C. The cells were thawed and plated at the optimal cell density, as previously described, were stimulated a few hours later by the ligand, and the resulting IP1 production was measured.

GPCR targets already evaluated with IP-One

Table 1 represents agonist responses that have been obtained with IP-One, HTRF, and the radioactive method (Inositol cascade product’s isotopic detection, see Figure 1) on several GPCRs. EC50 were determined in a side-by-side comparison.

This table is not an exhaustive presentation of all targets already validated with IP-One as we only present the data obtained in our Research Center. Cisbio is committed to keeping all evaluations or validations involving our application scientists confidential.

About the author

Jean-Luc Tardieu is HTRF Product Manager at Cisbio international in Bagnols-sur-Cèze, France. He can be reached via email at jltardieu@cisbiointernational.fr. More information is available from:

CIS US, Inc.800-221-7554www.htrf-assays.com

Figure 1. The different ways to monitor Gq-coupled GPCR activation.

Table 1. Assessment of GPCR responses to different agonists as measured by IP-One.