Nicotinic Ligand Development to Target Smoking Cessation and Gain a Molecular Level Understanding of Partial Agonism

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This proposal addresses the mechanism of action of a new class of "drug" used for smoking cessation based on agents which bind to and partially activate the high affinity nicotine receptors in the brain.The "first to market" drug (varenicline) is a nicotinic partial agonist but is subject to FDA restrictions that reflect an inadequate level of receptor subtypes selectivity; varenicline targets the high affinity nicotinic binding site (alpha4beta2) but is also a full agonist at the alpha7 receptor. If poor subtype selectivity is responsible for varenicline's side effects, a better insight into how ligand structure links to the receptor (and subtype) function is key, but to do this we will need to broaden our appreciation the modes of ligand binding. Cytisine, which is our focus, is a naturally-occurring partial nicotinic agonist that has been used for smoking cessation, and so itself is also of commercial potential. We have recently developed robust and efficient chemistry that allows us to modify cytisine directly, specifically, and at an otherwise almost unexplored site on the molecule. This chemistry uses cytisine as a very accessible starting point to explore a range of new structural variants to probe novel binding modes and so potentially new ways to achieve nicotinic subtype specificity. Recent speculation as to those molecular features of cytisine (and varenicline) that mediate ligand binding to nicotinic receptors will be investigated by (i) making specific molecules whose structural features will attenuate ligand binding and by (ii) expanding the range of ligand-protein contacts made in a very directed manner. We will build on preliminary structural and computational studies and create new ligands associated with C3 and C4 positions of cytisine. Both classes, but especially C4 variants, offer an opportunity to engage existing (but currently "spectator") protein residues in ligand binding that are within that region of the binding site directly associated with differentiation between the key receptor subtypes. Modifying existing binding by accessing these additional modes will allow us to rationally "tune" subtype selectivity. Success will dramatically improve our view of structure-relationships within nicotinic ligands and offer insights to medicinal chemists as to the range and spatial distribution of an enhanced "active site" template.We will study the specific detail of these new interactions using crystallography to pinpoint the ligand within a model protein and so identify newly "engaged" residues. We will then link this to the full human receptor protein, which still cannot be crystallised, in order to understand how ligand binding, as well as any new modes of binding, translates into how the full protein receptor functions. We will do this with new and powerful computational methods to look at the detail of the consequences of ligand binding in order to correlate ligand structure to whole receptor function.We have a series of collaborators in place in what is already a multidisciplinary programme to enable us to explore the biological, structural properties and commercial potential of these new ligands, and to correlate that to our predictive and analytical computational methods. Success in this project will critically depend on a close interplay between our new computational techniques and the synthetic chemistry. We will shed light on molecular level features of ligand structure which determine subtype selectivity and this will have longer term implications for safety/user acceptability within smoking cessation agents. By understanding the detail of how ligand structure links to overall receptor function, and this is an especially challenging computational task, we hope to separate subtype selectivity issues from more generic "downstream" effects (associated with nicotinic activation) which has the potential to guide further development of nicotinic-based therapeutic agents.