RPIB - Recherches Partenariales et Innovation Biomédicale

Development of innovative assays for the discovery of GPCRs biased signaling ligands – RSignal

Our assays will be based on the use of various types of binders, interacting with the GPCR effectors: some as general binders, while other able to intarct selectively with the active form of the effector. By adding fluorophores compatible with time resolved FRET measurement on each binders, the active form of the effectors will be tected by HTRF(R)

3 types of binders have been identified and validated that specifically recognize the active form of one effector. Interactors have also been identified for a second effector. All these tools have been validated using purified effectors.

Our next goal is to establish the conditions to use these tools in cell membranes. In parallel, we will be developping other tools to target a third GPCR effector.

If the program is aimed at developing tools to tackle the issue of biased ligands in the real life of GPCRs, the tools developped will also be the bases for assay kits that will be commercialized. Accordingly, patents will be filled, then preventing us from any communication so far.

Submission summary

The main objective in this program is to develop new technologies and products to measure multiple signalling pathways at the level of the G-proteins (G-protein conformational change) and of the ß-arrestin (ß-arrestin recruitment to the receptor) for high-throughput screening.
G protein-coupled receptors (GPCRs) play critical roles in cell-cell communication. Being encoded by the largest gene family, GPCRs still represent the most promising targets for the development of innovative therapies, especially considering that each cell type expresses a specific subset of these receptors. Despite intense research, only very few new drugs have been put on the market within the last few years. One of the difficulties resides not only in the identification of specific molecules, but also to make sure they have the correct action in native cells. Indeed, it has been demonstrated that GPCRs do not signal similarly depending on the molecule (either small molecules or antibodies) used to activate it. This concept of "functional selectivity" opens great promise for the development of novel therapeutic agents, not only targeting the receptor of interest, but also selectively acting on the pathway responsible for the therapeutic effect. Accordingly, it becomes essential to very early examine the action of novel therapeutic agents on the signaling properties of the targeted receptor in its native environment.
The aim in the present program is to develop simple, throughput compatible assays to selectively examine the coupling properties of any GPCRs in native cell preparations.
The program will concentrate on two first pathways for which no simple assays compatible with native signaling measures are available yet: Gi protein activation and beta-arrestin recruitment. The aim will be to develop two types of antibodies: antibodies recognizing the G-alpha proteins or ß-arrestins in any state, and antibodies selectively interacting with the active form of these proteins. To increase the chance of success, we will develop two types of antibodies targeting these proteins: nanobodies from llamas and classical monoclonal antibodies from mice. Nanobodies correspond to the variable domain of antibody fragments derived from a special class of camelid antibodies devoid of light chain. Because of their small size (13 kDa) they have a tendency to bind epitopes normally inaccessible to conventional antibodies such as grooves and cavities. Nanobody isolation will be based on the immunization of llamas with inactive and active forms of these proteins, generation of phage libraries and their selection using purified proteins in the indicated states. Nanobodies will be labeled with optimized HTRF compatible fluorophores, and used to selectively detect the active form of these proteins in native cell extracts after action of the potential therapeutic agent. After validation of the assays, kits will be commercialized, increasing the portfolio of CisBio Bioassays.
The program involves three research teams.
The group of JP Pin (IGF, Montpellier), a world recognized expert in the GPCR field, with strong experience in the purification of receptors, G proteins and arrestins. This group also has been very active in developing innovative assays within the last 15 years. It will be responsible for the production of the proteins used for the immunization, and in the development of the assays.
The group of D Baty (CRCM, Marseille) gained a strong recognition for his expertise in the development of llama nanobodies. It will be in charge of preparing the phage display libraries and their selection against the proteins provided by JP Pin's team.
CisBio Bioassays is a world leader in GPCR assays with the development of many kits to detect signaling responses, and many of them are based on the use of selective antibodies and HTRF. It will be in charge of developing the HTRF compatible nanobodies and monoclonal antibodies, and finalizing the assay kit development for commercialization.

Project coordination

Jean-Philippe PIN (Institut de Génomique Fonctionnelle)

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.


IGF Institut de Génomique Fonctionnelle
CBB Cisbio Bioassays
CRCM Centre de Recherche en Cancérologie de Marseille

Help of the ANR 791,010 euros
Beginning and duration of the scientific project: March 2014 - 48 Months

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