Elucidating the structural dynamics of A PENTAMERIC LIGAND-GATED ION CHANNELS for rational DRUG design – PENTA_CONTROL

Pentameric ligand-gated ion channels (pLGICs) are transmembrane protein assemblies that mediate neuronal signaling by opening an ion pore in response to the binding of a chemical messenger. Understanding their function at an atomic level of detail is key for the development of drug therapies against a range of neurological disorders, including Alzheimer’s, Parkinson’s, schizophrenia, and depression. Among the pLGICs, the glycine receptor (GlyR) is arguably the best-characterized channel with existing structures at high resolution in complex with agonist, antagonist, and modulators and has been recently identified as drug target for pain modulation. However, its structural dynamics underlying function, i.e. channel opening, closing, and desensitization, as well as its allosteric regulation remain to be understood. Here, we propose to characterize the functional dynamics of the human GlyR a1 by a synergistic approach combining all-atom Molecular Dynamics with voltage-clamp fluorometry (VCF) in living cells. To this aim, the structural dynamics of the known states of GlyR, including the highly dynamic and structurally heterogeneous resting state, will be characterized by all-atom Molecular Dynamics in native lipid membranes. The functional isomerization(s) of the channel, i.e. both gating and desensitization, will be probed by VCF measurement at the cell membrane, whose time and spatial resolution are suited to characterize short-lived intermediates, and explored with atomic resolution by “instructed” Molecular Dynamics incorporating the quenching results as biased collective variables. This original framework will bridge the gap between structural and functional data in pLGICs, thus opening to the structural characterization of the so far elusive gating intermediates involved in pre-activation and fast desensitization. The time-resolved atomistic description of the signal transduction pathways in GlyR with atomic resolution will be combined with conformation-dependent pharmacological studies. To this aim, the pharmacology of the structurally characterized states, including the pre-active intermediate supposed to be at the origin of partial agonism, will be explored by docking studies with free energy rescoring. High-throughput screening campaigns followed by experimental testing of the prioritized compounds by classical electrophysiology in oocytes will be used to search for modulatory ligands of the human GlyR a1. A fundamental aspect of the project is providing a proof of principle that the optimization of the differential binding affinity for one conformational state over the others, here named state-based pharmacology, is key for the rational design of positive and negative allosteric modulators in pLGICs.