Using IP-One HTRF® Assay to Identify Low Affinity Compounds

Introduction

The HTRF technology invented by French company Cisbio has been developed further to provide kits, reagents and services for drug discovery research. In life science, the company’s homogeneous assays can be used to research kinases & signaling, biomarkers, epigenetics and G-protein coupled receptors (GPCRs).

The company offers different HTRF-certified, multidetection microplate readers such as the FLUOstar Omega, POLARstar Omega, CLARIOstar and PHERAstar. Microplate readers are only certified if they meet the Cisbio’s standards in terms of dynamic range, sensitivity, CV%, DF% and S/N.

This article describes the IP-One HTRF® assay from Cisbio carried out using two different HTS microplate readers with different detection technologies.

G-Protein Coupled Receptors

GPCRs are transmembrane proteins involved in the signal transduction of extracellular stimuli. They are associated with a complex arrangement of intracellular proteins controlling a wide range of downstream effectors. One such second messenger that is produced in response to the activation of Gq-coupled receptors, is inositol 1,4,5-triphosphate (IP3). However, owing to a very short half-life, IP3 is too difficult to assess in drug screening assays and instead, the release of calcium, triggered by IP3, has been widely used as a downstream readout of this signaling pathway. Another way to monitor IP3 is to measure the accumulation of a downstream metabolite of IP3 called inositol monophosphate (IP1).

Homogeneous time-resolved fluorescence assay (HTRF®) can be optimized by raising monoclonal antibodies against IP1, taking advantage of the stability of IP1 and its accumulation in cells. Comparison of HTRF® IP-One assay with existing methods has shown that it produces similar compound potency data. In addition, the end point accumulation of IP1 enables discrimination of slow acting compounds that cannot be observed by calcium sensing. Through the quantification of constitutively active GPCRs, the HTRF® IP-One assay also enables the characterization of inverse agonists which cannot be achieved by measuring calcium release. Furthermore, HTRF® IP-One detection confers superior assay robustness and significantly lower false positive rates when compared to calcium detection. The IP-One assay from Cisbio Bioassays is based on the proprietary HTRF® technology (Figure 1).

Figure 1. Biosynthesis of IP1.

Principle of IP-One HTRF® Assay

The assay uses an IP1-specific monoclonal antibody and is based on a format where the fluorescence resonance energy transfer (FRET) signal between the HTRF® acceptor and donor is inhibited by the intracellular accumulation of IP1. An IP1 calibration curve can estimate the concentration of IP1 accumulated in cells as a function of the compound concentration.

Figure 2. IP-One HTRF® Assay Principle

Materials and Methods

The following materials are used in the IP-One HTRF® assay experiment:

  • HTS CCD-based microplate imager from a different vendor
  • Next generation PMT based HTS microplate reader, PHERAstar FS

Black Greiner 1536-well microplates with an assay volume of 5μL were used. The last four columns of a microplate were filled with positive (POS, n=64) and negative (NEG, n=64) controls, and 1408 different compounds were pipetted into the first 44 columns, as shown in Figure 3.  

Figure 3. 1536-well microplate layout

The PHERAstar FS provides a dedicated simultaneous dual emission (SDE) direct photon counting time-resolved fluorescence mode and a high power pulsed nitrogen laser emitting at 337nm. The laser is superior to a broadband xenon flash lamp when exciting the terbium (Tb) donor molecule. The energy emission of the laser takes advantage of the higher molecular extinction coefficient of the terbium cryptate peaking at around 337nm, compared to europium (Eu).

Results and Discussion

Results were assessed using the HTRF® ratio of the two emission wavelengths (Em 665nm/ Em 620nm). A surface graph representing the entire 1536-well microplate shows the hits as peaks, as illustrated below in Figure 4 and Figure 5.

Figure 4. HTRF® ratios obtained for the IP-One assay with the PHERAstar FS

Figure 5. HTRF® ratios obtained for the IP-One assay with a CCD-based HTS Device

Table 1. Speed and assay quality are compared between the PHERAstar FS and the CCD camera based HTS reader.

 

PHERAstar FS

CCD based HTS reader

Read Times (1536)

53 sec

2:12 min

Assay Window

6:1

2:1

Delta F%

490

76

Z’ Value

0.70

0.24

Conclusion

Cisbio’s IP-One HTRF® assay was carried out on two different HTS microplate readers using different detection technologies. The PMT based PHERAstar FS can readily resolve low affinity compounds that cannot be detected using a leading HTS CCD camera-based imaging microplate reader.

The PHERAstar FS from BMG LABTECH is a next-generation HTS reader that serves as an ideal solution for HTS screening assays.

Figure 6. BMG LABTECH’s multidetection microplate reader PHERAstar FS

Acknowledgement

Produced from articles authored by EJ Deli, BMG LABTECH GmbH; JL Tardieu and F Degorce, Cisbio Bioassays.

About BMG Labtech

BMG LABTECH is a leading developer and global manufacturer of microplate reader instrumentation with a wide range of measurement methods. Microplate readers are used in the pharmaceutical and biotech industries, as well as in academic research establishments, for both basic research analysis and High Throughput Screening. BMG LABTECH focuses solely on microplate readers and offers the most diverse selection of optical detection systems in conjunction with integrated liquid handling equipment.


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Last updated: Jul 14, 2018 at 6:44 PM

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