Acting on the glutamatergic synapse with nanobodies – nanoGluAct

In the central nervous system (CNS), the excitatory glutamatergic synapses are highly organized. They consist of post-synaptic ionotropic glutamate receptors and pre-synaptic glutamate localized inside pre-synaptic vesicles that mediate synaptic transmission responsible for learning and memory. Glutamate also induces the activation of G protein-coupled receptors (GPCRs), the metabotropic glutamate receptors (mGluRs) that modulate this synaptic transmission. mGluRs are involved in the pathophysiology of neuropsychiatric disorders, as well as neurodegenerative and neurodevelopmental diseases. Pharmacological targeting of mGluR can attenuate the development of symptoms and progression of many CNS disorders in animal models. No drug is on the market, but several small molecules targeting mGluRs are in clinical trials.
In human, there are eight different mGluRs (mGlu1 to mGlu8) that are expressed in different regions of the brain. There is a crucial lack of specific ligands to modulate the activity of subtype-specific mGluRs in vivo. Due to the conservation of the glutamate binding pocket, it is very difficult to develop ligand that bind specifically only one subtype of mGluRs. Therapeutics antibodies could present a number of advantages over small molecules.
The aim in the present program is to develop new pharmacological agents, camelids single domain antibodies (nanobodies) to modulate the activity of mGluRs both ex vivo and in animal models. We also aim to use these nanobodies as tools to investigate the localization and dynamics of the native mGluRs in neurons. The proof of concept will be done with two different mGluRs, mGluR2 and mGluR5, that represent the most studied mGluR drug targets. As preliminary results, we have characterized a high affinity nanobody that activates mGluR2 both in vitro and in vivo. We have shown that this nanobody is specific of the active conformation of mGluR2. In brain slices, this nanobody can inhibit the pre-synaptic increase in calcium in hippocampal mossy fiber terminals, as expected for the activation of mGluR2. We have further explored the effect of this nanobody in vivo, by injecting them alone, or in combination with DCG-IV (a mGluR2 agonist), and found that the nanobody potentiated the effect of a low and inactive concentration of DCG-IV in a fear conditioning test. It shows that nanobodies could be used as new drugs to modulate the activity of mGluRs in vivo.
Our project has three specific aims divided in five tasks: (i) to develop nanobodies specific of the states (inactive and active) of the mGluR2 and mGluR5 to use them as drugs in vivo (Task 2 and 3). The nanobodies will be tested in electrophysiology in hippocampal slices and in animals. Intravenous administration will be tested to see if they can reach the brain after crossing the brain-blood-barrier; (ii) to engineer the nanobodies to increase their affinities and their detection in native tissues (Task 4); (iii) to use them as tools to analyze the native mGluRs, their localization and dynamics (Task 5 and 6).
The program involves three research teams.
The group of P. Rondard (Partner 1, IGF, Montpellier), a world recognized expert in the GPCR field, with strong experience in the molecular pharmacology of the mGluRs. It will be responsible for the characterization in vitro and in vivo of the nanobodies generated by Partner 2.
The group of P. Chames and D. Baty (Partner 2, CRCM, Marseille) gained a strong recognition for his expertise in the development of llama nanobodies. It will be in charge of immunization, screening and engineering of the nanobodies against mGluRs.
Cisbio Bioassays (CBB, Partner 3) is an innovative biotech in the field of medical diagnostic in vitro, and in drug discovery to provide products and services for the discovery of therapeutic compounds acting on receptors such as GPCRs. Their products are based on the development of antibodies and own technologies of detection, the time-resolved FRET or HTRF®.