Section9:IP-3/IP-1 Assays

From Assay Guidance Wiki

Agonist stimulation of G protein coupled receptors that are coupled to Gαq (or to Gβγ subunits) leads to activation of phospholipase C β (PLC-β) followed by production of D-myo-inositol 1,4,5-triphosphate (IP3). IP3 initiates the release of Ca2+ from intracellular stores before it is rapidly degraded to IP2 then IP1. Activation of this pathway is usually measured by detection of intracellular calcium using fluorescent calcium indicator dyes and fluorescence plate readers (FLIPR). Although calcium assays are robust and easily amenable to HTS, there are some important limitations: calcium flux is very rapid and transient, and does not allow detection of constitutive activity (or inverse agonism); interference by fluorescent and nuisance compounds is a problem; and sensitivity is often insufficient to allow the use of primary cells. Alternatively, it is possible to measure IP3 production directly or indirectly as a read-out of PLC-β activation.

Reprinted from Cisbio with permission

Overview of Technology

Traditional assays for total inositol phosphate accumulation used radioactivity and were complicated and not amenable to HTS. In addition, IP3 production is very rapid and transient before it is metabolized to IP2 and IP1. There are a few alternative technologies available for measurement of IP-3 including AlphaScreen (Perkin Elmer) and HitHunter™ Fluorescence Polarization (DiscoveRx). Recently a homogeneous time resolved fluorescence assay for IP1 (IP-One HTRF®), has been developed by Cisbio (see below). This assay format takes advantage of the fact that lithium chloride (LiCl) inhibits the degradation of IP1, the final step in the inositol phosphate cascade, allowing it to accumulate in the cell, where it can be measured as a substitute for IP3. Data from Cisbio show that the assay can be used with endogenously or heterologously expressed receptors in either adherent or suspension cells, to quantitate the activity of agonists, antagonists, and inverse agonists. Agonist EC50’s and antagonist IC50’s using the IP-One HTRF® assay correlate very well with those from calcium assays and traditional IP3 detection assays.

IP-One HTRF® (Cisbio)

General Background

The IP-One HTRF® assay kit allows direct quantification of myo-Inositol 1 phosphate (IP1) in cultured cells. The assay is a competitive immunoassay. IP1 produced by cells (in the presence of LiCl) after receptor activation competes with an IP1 analog coupled to a d2 fluorophore (acceptor) for binding to an anti-IP1 monoclonal antibody labeled with Eu Cryptate (donor). The resulting signal is inversely proportional to the concentration of IP1 in the sample. A standard curve is constructed to convert raw data to IP1 concentration.

Reprinted from Cisbio with permission

See the following link for more information: http://www.htrf.com/products/gpcr/ipone/

Sample Protocol

Assay may be conducted in 96-well, 384-well or 1536-well formats. Only white plates should be used for IP-One HTRF. Suggested plate types:

96 half-well plate Costar cat # 3688 (white, opaque flat bottom, TC-treated). Total working volume = 100 ul.
96-well Costar cat # 3917 (white, opaque flat bottom, TC-treated). Total working volume = 200 ul.
See the following link for additional plate recommendations: http://www.htrf.com/technology/assaytips/microplate/

Adherent cells may be seeded into tissue culture treated, white microplates 24 hours before assay. Just before the assay, media is removed from adherent cells and replaced with Stimulation Buffer (included in the kit). Note: buffers containing phosphate can not be used.
Alternatively, cells may be prepared in suspension using the Stimulation Buffer provided in the kit, and plated immediately before the assay.
Serial dilutions of the IP1standard included in the kit are made using Stimulation Buffer, and pipetted into the assay plate for the standard curve.
Cells are pre-treated with antagonist compounds prepared in Stimulation Buffer for 15-30 minutes at 37oC, 5% CO2.
Agonist prepared in Stimulation Buffer is added and plates are incubated at 37oC, 5% CO2 for required stimulation time (to be optimized).
Diluted IP1 d2 conjugate is added to wells.
Diluted anti-IP1 Eu Cryptate is added to wells.
Plates are incubated for 1 hour at room temperature.
Plates are read on an HTRF® compatible reader (eg: Envision, Tecan GENios, BMG Rubystar). See the following link for other readers: http://www.htrf.com/technology/htrfmeasurement/compatible_readers/
Excitation is at 320 nm. CisBio recommends using a ratiometric measurement for HTRF® emissions at both 620 nm and 665 nM. Emissions at 620 nm are used as an internal reference and emissions at 665 nM reflect the biological response. The ratio of 665/620 allows normalization for well-to- well variability and interference due to assay components.
Signal Stability: plates may be read repeatedly for determination of kinetics, and signal is stable for at least 24 hours at RT.

Results and Data Analysis

The ratio of absorbance 665/ absorbance 620 nm emissions is calculated. A standard curve is plotted of Ratio 665/620 vs IP-1 concentration using non-linear least squares fit (sigmoidal dose response variable slope, 4PL). Unknowns are read from the standard curve as nM concentration of IP1. Ratio 665/620 of unknowns should fall on the linear portion of the standard curve. Increased accumulation of IP-1 will result in a decrease in signal.

Example: IP-1 Standard Curve:

Assay Formats

Agonist Mode:

Cells are stimulated with agonist for optimum time and increase in IP1 produced by receptor activation is quantified.
Max response is maximum IP1 produced by full agonist stimulation.
Min response is IP1 produced in the presence of stimulation buffer and the absence of agonist.
Relative EC50 and Relative Efficacy (% maximum activity of a test compound relative to the reference agonist) may be obtained from concentration response curve.

Example: Agonist concentration response curve

Antagonist Mode:

Cells are treated with test antagonist compound for approximately 15 minutes.
Agonist is then added at approximately EC80 concentration and incubated for optimum time.
Inhibition of the agonist response is quantified.
Max response is IP1 produced by EC80 concentration of agonist in the absence of compound.
Min response is IP1 produced in the presence of stimulation buffer and the absence of agonist or test compound.
Relative IC50 may be obtained from concentration response curve and used to calculate antagonist Kb.

Example: Antagonist concentration response curve

Inverse Agonist Mode:

Cells expressing a constitutively active receptor are treated with test compound for optimum time (in the absence of agonist). Inhibition of the basal response (IP1 produced in the presence of stimulation buffer alone) by test compound is quantified.
Max response is basal level of IP1 produced in cells expressing the constitutively active receptor during the incubation time.
Min response is basal level of IP1 produced in cells without the receptor.
Relative EC50 Inverse and Relative Efficacy Inverse (% maximum response of reference inverse agonist) may be obtained from concentration response curve.

Assay Optimization

The following parameters should be optimized to ensure that the level of IP-1 produced in the wells falls within the linear range of the standard curve, signal window is maximized and variability is acceptable:

cell number
preincubation of cells with stimulation buffer
agonist stimulation time
incubation time after addition of conjugates

See the following link for Cisbio recommendations for assay optimization: http://www.htrf.com/files/resources/ip-one%20nature.pdf

References

Eglen RM. (2005) Functional G protein-coupled receptor assays for primary and secondary screening. Combinatorial Chmistry & High Throughput Screening 8 (4), 311-318.
Inglese, J., Johnson, R.L., Simeonov, A., Xia, M., Zheng, W., Austin, C.P., Auld, D.S. (2007) High-throughput screening assays for the identification of chemical probes. Nature Chemical Biology 3 (8), 466-479.
McLoughlin, D.J., Bertelli, F., Williams, C. (2007) The A, B, Cs of G-protein-coupled receptor pharmacology in assay development for HTS. Expert Opinion on Drug Discovery 2 (5), 603-619.
Trinquet E. et al. (2006) D-myo-Inositol 1-phosphate as a surrogate of d-myo-inositol 1,4,5-tris phosphate to monitor G protein-coupled receptor activation. Analytical Biochemistry 358 (1), 126-135.

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