CE44 - Biochimie du Vivant

Overcoming the limitations of existing ion channel high-throughput screening and analysis methods by using Bioluminescence Resonance Energy Transfer – CANALBRET

B Overcoming the limitations of existing ion channel high-throughput screening and analysis methods by using Bioluminescence Resonance Energy Transfer

Our consortium hypothesizes that the advent of well-designed intra- and intermolecular Bioluminescence Resonance Energy Transfer (BRET) probes targeting ion channels will provoke a paradigm shift in the field of ion channel biology.

General objectives of the project

To develop intermolecular BRET assays monitoring the dynamics of protein-protein interactions (PPIs) engaged by ion channels following their activation, using known protein partners and newly discovered ones through an innovative BRET-based PPIs screening assay. 2/ To exemplify the use of BRET intramolecular probes, successfully applied by our consortium on the TRPV1 channel (1) on a set of a dozen of voltage-gated, stretch-activated, ligand-gated and polymodal ion channels, and to build and characterize a second-generation intramolecular BRET probes that will measure the ionic flux in the microenvironment of the channel pore of several calcium-permeable cation channels, one sodium channel, two potassium channels and one chloride channel. 3/ To compare efficiency and robustness of the results obtained in real high throughput screening conditions using conventional methods (fluorescent probes & planar patch-clamp) and the most efficient intra- and intermolecular BRET assays developed and characterized in the framework of the CANALBRET project, in view of the transfer of the technology to Domain Therapeutics, the industrial partner of our consortium, and 4/ To assess, using a remote BRET measurement setup, whether or not efficacy and potency of chemical inhibitors targeting polymodal ion channel are affected by physical forces (temperature, pressure and stretching) and hypoxia, mimicking physiopathological conditions.

For the last fifteen years, resonance energy-transfer (RET) based approaches have offered new opportunities for probing the activity of an ever-growing list of proteins in living cells, in real-time (6). These techniques are based on the nonradiative transfer of energy between an energy donor and a compatible fluorescent energy acceptor. This is a system of choice for monitoring both constitutive and regulated inter- and intra-molecular interactions, in view of the strict dependence on molecular proximity (~100 Å) and orientation between donor and acceptor molecules for energy transfer. Among the various RET techniques, Bioluminescence Resonance Energy Transfer (BRET) is a popular, broadly-applicable method, increasingly widely-used for studying protein activity in living systems. Eliminating the need for an external light source for donor excitation gives BRET some advantages over related techniques like Fluorescence Resonance Energy Transfer (FRET). Thanks to these advantages, BRET assays have been widely implemented for GPCR and kinases drug screening.
We recently demonstrated that intra- and inter-molecular BRET probes could be useful for probing the activation of TRPV ion channels in real-time on live-cell under either temperature or chemical activation. Such innovative BRET probes monitor molecular events related to ion channel activation (conformational changes and dynamic of protein-protein interactions) that are intractable using either the conventional fluorescent-based probes or the patch-clamp technique. They therefore opened up new prospects for developing simple cell-based assays that may improve the effectiveness of ion channel drug screening.

Halfway through, we validated new concepts of intramolecular BRET probes targeting ion channels and carried out a first screening of a drug bank using the TRPV1 channel.

The CANALBRET project enjoys excellent momentum, and is driven by a very fruitful interaction between academic partners as well as with our private partner. The experiments, although slightly behind schedule due to adaptation to the COVID crisis, do not encounter major technical problems. Results are therefore coming out on a regular basis. Several publications can be considered for the coming years.
The immediate prospects concern the realization of a second screen, the deepening of new concepts in molecular pharmacology of ionic channels updated thanks to the different generations of probes that we have developed, and the exploration of the effects of physical and physico-chemical constraints on ion channel activity.

1. Chappe et al. (2021). Molecular Pharmacology. En révision
2. Brevet n°EP 20163892.1. Déposé le 18 Mars 2020. Titre : Novel ion conducting channel fusion subunits and methods of use thereof.

Ion channels are pore-forming membrane proteins that allow ions to pass through the channel pore. Widely regarded as attractive drug targets for many therapeutic applications, ion channels represent the third largest class of targets in drug discovery after G-protein coupled receptors and kinases, and account for worldwide sales of US$ 12 billion highlighting their 'tractable' nature. Nonetheless, in spite of this ranking, ion channels continue to be under exploited as drug targets and only 20% of ion channels are currently commercially exploited (among more than 300 known members). Historically high throughput screening of drug candidates on ion channels has proven to be technically difficult and remains very expensive. For the last fifteen years, resonance energy-transfer (RET) based approaches have offered new opportunities for probing the activity of an ever-growing list of proteins in living cells, in real-time. These techniques are based on the nonradiative transfer of energy between an energy donor and a compatible fluorescent energy acceptor. This is a system of choice for monitoring both constitutive and regulated inter- and intra-molecular interactions, in view of the strict dependence on molecular proximity (around 100 Å) and orientation between donor and acceptor molecules for energy transfer. Among the various RET techniques, Bioluminescence Resonance Energy Transfer (BRET) is a popular, broadly-applicable method, increasingly widely-used for studying protein activity in living systems. Moreover the BRET assay has been widely implemented for G Protein-Coupled Receptors (GPCR) and kinases drug screening. Our consortium recently demonstrated that intra- and intermolecular BRET probes targeting P2X2 or TRPV1/3/4 ion channels could be useful for probing the activation of these ion channels in real-time on live-cell under chemical activation. Such innovative BRET probes monitor molecular events related to ion channel activation (conformational changes and dynamic of protein-protein interactions) that are intractable using either the conventional fluorescent-based probe or the patch-clamp technique. The CANALBRET project now opens the avenue of developing similar high added value platforms for drug discovery in the ion channel field as well as innovative analysis methods for academic research. Strengthened by the interaction of three experienced academic research groups and one private company, the CANALBRET project will (i) extend the proof of concept of the ion channel BRET probes to other ion channels than those belonging to the P2X and TRP family alone, (ii) demonstrate the power of the CANALBRET technology in comparison to the conventional techniques used for ion channel drug screening and (iii) further the basic knowledge of polymodal activation of ion channel and the dynamic of their protein-protein interactions.

Project coordination

Yann Percherancier (LABORATOIRE D'INTEGRATION DU MATERIAU AU SYSTEME)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IMS LABORATOIRE D'INTEGRATION DU MATERIAU AU SYSTEME
CRCTB CENTRE DE RECHERCHE CARDIO-THORACIQUE DE BORDEAUX
IGF Institut de génomique fonctionnelle
DT DOMAIN THERAPEUTICS
University College Dublin (UCD) / UCD Cell Screening Laboratory

Help of the ANR 523,955 euros
Beginning and duration of the scientific project: October 2019 - 42 Months

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