Setting Up a Bioluminescence Resonance Energy Transfer High throughput Screening Assay to Search for Protein/Protein Interaction Inhibitors in Mammalian Cells

Abstract

Each step of the cell life and its response or adaptation to its environment are mediated by a network of protein/protein interactions termed "interactome." Our knowledge of this network keeps growing due to the development of sensitive techniques devoted to study these interactions. The bioluminescence resonance energy transfer (BRET) technique was primarily developed to allow the dynamic monitoring of protein/protein interactions (PPI) in living cells, and has widely been used to study receptor activation by intra- or extra-molecular conformational changes within receptors and activated complexes in mammal cells. Some interactions are described as crucial in human pathological processes, and a new class of drugs targeting them has recently emerged. The BRET method is well suited to identify inhibitors of PPI and here is described why and how to set up and optimize a high throughput screening assay based on BRET to search for such inhibitory compounds. The different parameters to take into account when developing such BRET assays in mammal cells are reviewed to give general guidelines: considerations on the targeted interaction, choice of BRET version, inducibility of the interaction, kinetic of the monitored interaction, and of the BRET reading, influence of substrate concentration, number of cells and medium composition used on the Z' factor, and expected interferences from colored or fluorescent compounds.

Keywords: BRET; HTS; P2I2; PPI; RET; inhibitor compound; modulator compound; screening assay.

Figure 1

Resonance energy transfer methods. (A) Basic properties of donor/acceptor compatible couple in order to gain RET. Principles of (B): the FRET method, (C): The BRET method, and (D): the HTRF method. D, Donor; A, Acceptor; S, Substrate.

Figure 2

Bioluminescence resonance energy transfer donor saturation assay. (A) Basic donor saturation assay expressed as milliBRET unit (mBU); (B) Donor Saturation Analysis for dimeric complexes formation: in red, theoretical curve if 100% donor and acceptor interact with each other at a 1/1 molar ratio. In blue, the common DSA curves obtained showing lower percentage of donor/acceptor complexes in cells. (C) Donor saturation assay for analysis to set up a P2I2 BRET-based screening assay. In hatched black, the dynamic windows of BRET monitoring. In hatched red, the two areas of the DSA curve to avoid. In green, the ideal window to be chosen when setting up a P2I2 BRET-based screening assay.

Figure 3

Setting up and optimize a P2I2 BRET-based screening assay. (A) Basic protocol of P2I2 BRET-based screening assay; (B–H) parameters analysis of the BRET signal monitored using OBRc/OBRGRP (●) and CD4/PLSCR1 (○) BRET-based screening assays, engineered using CHO-Trex cell lines to allow doxycyclin induction of donor. n = 3; (B) kinetic of the BRET induction: BRET signal monitored as a function of time after inducer addition (doxycyclin 0.1 μg/ml). (C) Kinetic of the BRET reading after coelenterazine h addition and (D) effect on the Z′ factor calculated from 8 points. n = 3. (E) Effect of substrate concentration or cell number used (F) on the Z′ factor (from 8 points). n = 3; (G) dose dependent effect of red phenol or Fluorescein (H) interfering compound in the medium when reading BRET. In gray when medium was removed before reading: OBRc/OBRGRP () and CD4/PLSCR1 ().

Figure 4

Expected results from a P2I2 BRET-based screening assay. (A) Different expected BRET change upon inhibitor or modulator compound action compared to basal BRET signal. X and Y: Protein X and Y; D: energy donor; A: energy Acceptor; S: BRET Substrate. (B) Results of a 320 compounds miniscreen using OBRc/OBRGRP (●) and CD4/PLSCR1 (○) BRET-based screening assays, expressed as% of basal BRET in absence of compound in each plate. (C) Fluorescence measured from the same plates as in (B) expressed as % of fluorescence value in absence of compound (represented by ).