Discovery of allosteric modulators specifically targeting alpha5-containing nAChRs. – NICOFIVE

The nicotinic acetylcholine receptors (nAChRs) are pentameric complexes belonging to the superfamily of pentameric ligand-gated ion channels (pLGICs), mediating excitatory cholinergic communication between cells in the nervous system. In the brain, the major nAChR is made by the association of the a4 and ß2 subunits, and a minor population also contains the a5 subunit (a4ß2a5 nAChRs). Recent genomic and epidemiologic data have demonstrated a link between a highly prevalent (single-nucleotide) polymorphism (SNP) of the a5 subunit and both smoking addiction and lung cancer. Since this a5SNP generates a loss of function of a4ß2a5 nAChRs, it is expected that activating specifically a4ß2a5 nAChRs by positive allosteric modulator targeting the a5 subunit (PAMa5) would be a treatment of smoking dependence. However, a5 is an « orphan » subunit for which no ligand has been found at present.
To discover de novo a PAMa5, we propose to target a unique binding site at the interface between the a5 and a4 subunits. This a5/a4 site is homologous to the ACh binding site located at the a4/ß2 interface, but shows significant differences in amino-acid composition and does not bind ACh and nicotine. We will develop a rational approach using engineered nAChRs. We already generated a protein surrogate of the a5/a4 site, namely AChBP-5/4. The AChBP protein is homologous to the extracellular domain of nAChRs, and we performed on it a set of 25 mutations reconstituting the entire a5/a4 site. AChBP-5/4 allows performing binding of ligands using surface plasmon resonance (SPR), as well as X-ray crystallography. We already identified a few alkaloids binding on AChBP-5/4 with micromolar affinity. In parallel, we developed pentameric concatemers of the full-length a4ß2a5 nAChR, strictly constraining their pentameric assembly and allowing precise electrophysiological experiments.
Based on these extensive preliminary data, we will implement a fragment-based drug discovery approach, deconstructing into small fragments a homemade library of selected alkaloids. Fragments will be screened on AChBP-5/4 by in silico docking, SPR-binding and X-ray crystallography assays. They will be further characterized on a4ß2a5 nAChRs, for their modulatory effect by electrophysiology and on in silico models of a4ß2a5 nAChR that simulate their transition pathway of activation. Molecular reconstruction of ligands will be performed both using standard fragment growing, linking or merging strategies, and using fragment recombination by kinetic target-guided synthesis (in situ click chemistry on AChBP-5/4).
We thus propose an integrated approach, combining biochemical, electrophysiological and structural experiments with in silico modeling and synthetic organic chemistry, to achieve the challenge of rationally developing a novel class of PAMa5. The project is based on extensive preliminary data, and we will combine in parallel several strategies to increase the chances to discover potent allosteric modulators. The discovered PAMa5 and NAMa5 will be precious to investigate the fundamental processes mediated by the a5 subunit at the molecular and allosteric level. They will also allow investigation of native tissues such as brain slices and living animal, to study the contribution of a5-containing nAChRs to physiopathological processes. Many pharmaceutical companies are developing nicotinic ligands, and the lead compounds identified will be patented before contacting industries, with a well-established indication for smoking cessation. Beside these expected impacts of the project, our AChBP/fragment-based strategy may be generalized to any interface of the multiple nAChRs present in the brain and at the periphery, opening the way to develop new classes of subtype-specific compounds.