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

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.

Tobacco smoking is a global killer on a huge scale. There are 1.25 billion smokers worldwide. 50% will die from smoking-related disease and many want to quit. Effective smoking cessation aids would prevent millions of premature deaths and reduce the huge burden of smoking-related illness. There is enormous demand for effective, safe and cheap smoking cessation therapies. This project will explore promising new compounds for smoking cessation. We will work directly with industrial partners, who will provide raw materials, specialist advice and testing. This provides a direct route to developing and bringing to market new therapies. This market is vast, and growing rapidly: it was worth $1.9 billion in 2011 and will grow to $4.4 billion by 2023. The major potential impact of this project is therefore on public health through the provision of safer, more effective and potentially cheaper smoking cessation agents; helping people to stop smoking will improve health (e.g. cancer, respiratory and heart disease etc.) and reduce the burden on the NHS.

Our industry partner provides a direct route to impact in developing new smoking cessation agents. Our novel and potentially highly selective ligands may also be useful as biochemical tools. Better understanding of nicotinic receptors and structure-activity relationships will have wide impact given their importance as targets in many areas of biomedical science, e.g. pharmaceutical and agrochemical industries. Our ligands and binding models will contribute new leads and understanding of receptor subtypes, with opportunities for enhanced selectivity. The computational models we will develop and validate will also impact in pharma and biotech, where better computational tools may reduce the need for animal experiments.

We have established an industry partnership to evaluate and progress new smoking cessation agents. Cytisine, currently sold in Eastern Europe, could soon be FDA-approved as a smoking cessation agent, but alternatives with improved pharmacology, enhanced "user acceptability" and better commercialization potential are actively sought. Our aim to generate better ligands offers potentially direct commercial impact, which is recognised by our industrial partners by their significant project contributions. Such products would be patentable: cytisine is not. A more effective novel variant would benefit indirectly from cytisine's approval and be protected in major markets. Any new product is also subject to FDA approval, so this partnership is essential for candidate assessment, formulation, process development and regulatory approval.

We will develop relationships with other industrial partners in areas distinct from (and not in competition with) smoking cessation. We have had early discussions to assess nicotinic ligand development for both agrochemical and pharmaceutical application with two companies, and have an agreement in place with one to provide substituted ligands for assessment. This will allow us to identify opportunities for other applications.

Impact in pharmacology will be aided by our experimental collaborations in Bath, Oxford and Milan. We will make ligands available to these partners; we have confidentiality agreements agreed.

There is also potential impact of the computational tools from this project. Both BUDE and Sire are already being used by external academic and industrial partners. We will continue to work with these companies to extend the applications of these methods as they develop to assist commercial development of BUDE (but licensed at no cost to academics). Sire is freely available via GPL. We will run training workshops via CCP-BioSim and HECBioSim to train academic and industry users and demonstrate the methods. The tools will also be demonstrated at conferences, and their impact will be enhanced by their application with the RSC National Compound Collection, showcasing them to users e.g. in pharma.