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

Couturier, Cyril; Deprez, Benoı̂t

doi:10.3389/fendo.2012.00100

Cited by 43 publications

(29 citation statements)

References 126 publications

(168 reference statements)

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“…Our observation that LINGO‐1 forms cis ‐homodimers in living cells using a BRET‐based homogeneous assay allowed us to suggest that this simple and rapid method could be applied to the screening of LINGO‐1 ligands and/or SMPPIMs (Couturier and Deprez, ). Because BRET depends strictly on the molecular proximity and the orientation between donor and acceptor, it is theoretically applicable to study conformational changes as a consequence of ligand binding (De Vries et al ., ; Couturier and Deprez, ). Our present data demonstrate that our LINGO‐1 BRET‐based assay is of high quality and suitable for use in screening ( Z ′ score > 0.85) and the screening of around 1000 chemical compounds led us to identify one molecule, phenoxybenzamine, that specifically and dose‐dependently decreased the LINGO‐1 BRET signal.…”

Section: Discussionmentioning

confidence: 99%

Targeting the cis‐dimerization of LINGO‐1 with low MW compounds affects its downstream signalling

Cobret

Tauzia

Ferent

et al. 2014

British J Pharmacology

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BACKGROUND AND PURPOSEThe transmembrane protein LINGO-1 is a negative regulator in the nervous system mainly affecting axonal regeneration, neuronal survival, oligodendrocyte differentiation and myelination. However, the molecular mechanisms regulating its functions are poorly understood. In the present study, we investigated the formation and the role of LINGO-1 cis-dimers in the regulation of its biological activity. EXPERIMENTAL APPROACHLINGO-1 homodimers were identified in both HEK293 and SH-SY5Y cells using co-immunoprecipitation experiments and BRET saturation analysis. We performed a hypothesis-driven screen for identification of small-molecule protein-protein interaction modulators of LINGO-1 using a BRET-based assay, adapted for screening. The compound identified was further assessed for effects on LINGO-1 downstream signalling pathways using Western blotting analysis and AlphaScreen technology. KEY RESULTSLINGO-1 was present as homodimers in primary neuronal cultures. LINGO-1 interacted homotypically in cis-orientation and LINGO-1 cis-dimers were formed early during LINGO-1 biosynthesis. A BRET-based assay allowed us to identify phenoxybenzamine as the first conformational modulator of LINGO-1 dimers. In HEK-293 cells, phenoxybenzamine was a positive modulator of LINGO-1 function, increasing the LINGO-1-mediated inhibition of EGF receptor signalling and Erk phosphorylation. CONCLUSIONS AND IMPLICATIONSOur data suggest that LINGO-1 forms constitutive cis-dimers at the plasma membrane and that low MW compounds affecting the conformational state of these dimers can regulate LINGO-1 downstream signalling pathways. We propose that targeting the LINGO-1 dimerization interface opens a new pharmacological approach to the modulation of its function and provides a new strategy for drug discovery.

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Section: Discussionmentioning

confidence: 99%

Targeting the cis‐dimerization of LINGO‐1 with low MW compounds affects its downstream signalling

Cobret

Tauzia

Ferent

et al. 2014

British J Pharmacology

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“…The donor saturation assay described in Basic Protocol 1 is a mandatory step to determine the right donor/acceptor ratio to express within the cells in order to avoid the titration of inhibitors in a competition assay. Indeed, the acceptor molecule has to be expressed to a higher level compared to the donor in order to maximize the BRET signal (Couturier & Deprez, 2012). However, excess of acceptor but also of both acceptor/donor pair (excess of the monitored complex) might titer a potential active molecule (or macromolecule) leading to its inability to promote the expected decrease in signal.…”

Section: Bret2 Competition Assay With Anti-ras Macromoleculesmentioning

confidence: 99%

“…The major advantage of BRET2 over BRET1 method lies in the better spectral separation of the donor and acceptor emission peaks (115 nm). This implies less bleed through at the acceptor emission maximum and therefore a lower background (Bacart, Corbel, Jockers, Bach, & Couturier, 2008;Couturier & Deprez, 2012;Pfleger & Eidne, 2006).…”

Section: Background Informationmentioning

confidence: 99%

Bioluminescence Resonance Energy Transfer 2 (BRET2)‐Based RAS Biosensors to Characterize RAS Inhibitors

Béry

Rabbitts

2019

CP Cell Biology

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Protein-protein interactions (PPIs) are principle biological processes that control normal cell growth, differentiation, and homeostasis but are also crucial in diseases such as malignancy, neuropathy, and infection. Despite the importance of PPIs in biology, this target class has been very challenging to convert to therapeutics. In the last decade, much progress has been made in the inhibition of PPIs involved in diseases, but many remain difficult such as RAS-effector interactions in cancers. We describe here a protocol for using Bioluminescence Resonance Energy Transfer 2 (BRET2)-based RAS biosensors to detect and characterize RAS PPI inhibition by macromolecules and small molecules. This method could be extended to any other small GTPases or any other PPIs of interest. C 2019 by John Wiley & Sons, Inc.Keywords: biosensors r BRET r protein-protein interaction inhibition r RAS family GTPases of 22RAS associating (RA) domain of RALGDS. We also used as acceptor the full-length CRAF and PI3Kα effectors to study their respective downstream signaling pathways. Therefore, using this BRET-based RAS biosensors toolbox, we characterized the cellular properties of various RAS inhibitors comprising anti-RAS Design Ankyrin Repeat Proteins (DARPins) (Guillard et al., 2017) and anti-RAS intracellular domain antibody (iDAb RAS) (Bery et al., 2018) macromolecules and antibody derived anti-RAS compounds Quevedo et al., 2018).Here we describe a set of protocols that include step-by-step instructions to monitor the interaction between RAS and a partner (Basic Protocol 1), to identify inhibitors of RAS PPIs including macromolecules (Basic Protocol 2) and small molecules (Basic Protocol 3). We also describe a protocol to detect the effect of RAS inhibitors on the RAS downstream pathways by immunoblot (Basic Protocol 4) and finally a protocol explaining how to calculate the BRET ratio obtained in the previous protocols (Basic Protocol 5). Figure 1Use of the BRET-based RAS biosensors to assess a PPI. (A) A schematic representation of the BRET-based biosensors. A protein (P1) is fused to the donor moiety RLuc8 and a protein (P2 or P3) is tagged with the acceptor moiety GFP 2 . If P1 does not bind to P2, it only produces a background BRET signal. However, when P1 interacts with P2, it induces a BRET signal, if the RLuc8 and GFP 2 domains are within 10 nm. This BRET signal can be decreased by addition of a competitor (either by a macromolecule or a small molecule inhibitor). (B) A representative donor saturation assay between KRAS G12D , KRAS WT and KRAS S17N (donors) and CRAF RBD (acceptor) with total GFP 2 and RLuc8 controls. Where error bars are presented, these correspond to mean values ± SD of quadruplicates technical repeats. Day 2: Cell transfection2. Mix together a fixed amount of RLuc8 construct (typically 50 ng) and a varying amount of GFP 2 construct (see quantity in Table 2).The pEF-myc-cyto empty plasmid is used to equalize total amount of transfected DNA between wells. The total DNA amount transfected per well is 1.6 μg. ...

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“…These assays, usually carried out with human tumor cell lines, allow the study of the cytotoxic effect of small molecules, as well as their impact on p53–MDMs interactions. The RET assays, including fluorescence (FRET) and bioluminescence (BRET), have been highly used to study PPIs in a cellular environment . For FRET, a robust and affordable system for HTS is not available yet, whereas BRET has been widely applied in HTS, namely in the search for p53–MDM2 inhibitors .…”

Section: Screening Approaches To Search For Inhibitors Of the P53–mdmmentioning

confidence: 99%

Medicinal Chemistry Strategies to Disrupt the p53–MDM2/MDMX Interaction

Lemos

Leão

Soares

et al. 2016

Medicinal Research Reviews

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The growth inhibitory activity of p53 tumor suppressor is tightly regulated by interaction with two negative regulatory proteins, murine double minute 2 (MDM2) and X (MDMX), which are overexpressed in about half of all human tumors. The elucidation of crystallographic structures of MDM2/MDMX complexes with p53 has been pivotal for the identification of several classes of inhibitors of the p53-MDM2/MDMX interaction. The present review provides in silico strategies and screening approaches used in drug discovery as well as an overview of the most relevant classes of small-molecule inhibitors of the p53-MDM2/MDMX interaction, their progress in pipeline, and highlights particularities of each class of inhibitors. Most of the progress made with high-throughput screening has led to the development of inhibitors belonging to the cis-imidazoline, piperidinone, and spiro-oxindole series. However, novel potent and selective classes of inhibitors of the p53-MDM2 interaction with promising antitumor activity are emerging. Even with the discovery of the 3D structure of complex p53-MDMX, only two small molecules were reported as selective p53-MDMX antagonists, WK298 and SJ-172550. Dual inhibition of the p53-MDM2/MDMX interaction has shown to be an alternative approach since it results in full activation of the p53-dependent pathway. The knowledge of structural requirements crucial to the development of small-molecule inhibitors of the p53-MDMs interactions has enabled the identification of novel antitumor agents with improved in vivo efficacy.

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

Cited by 43 publications

References 126 publications

Targeting the cis‐dimerization of LINGO‐1 with low MW compounds affects its downstream signalling

Targeting the cis‐dimerization of LINGO‐1 with low MW compounds affects its downstream signalling

Bioluminescence Resonance Energy Transfer 2 (BRET2)‐Based RAS Biosensors to Characterize RAS Inhibitors

Medicinal Chemistry Strategies to Disrupt the p53–MDM2/MDMX Interaction

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