Paul Whiteaker - US grants

DESCRIPTION (provided by applicant): Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that play many important physiological roles. nAChRs naturally exist as a family of diverse subtypes. Each nAChR subtype is a pentamer and is defined by its composition of subunits, which in mammals are products of sixteen different genes. Each nAChR subtype has a distinctive distribution and set of characteristics. nAChR subtype diversity in the brain is physiologically important and provides opportunities for target-based drug treatment of neuropsychiatric conditions. Of particular interest in this project, native nAChR subtypes containing a6, b2, b3 a4 subunits (a6*-nAChR) are expressed in midbrain, dopaminergic, pleasure and reward centers. This means that drugs selectively targeting a6*-nAChR could be used to treat nicotine or other drug dependence, and/or Parkinson's disease. However, characterization of these a6*-nAChR subtypes in isolation has been elusive, in part because they are naturally expressed over a background of other nAChR subtypes, but also because of difficulties during heterologous expression in precisely controlling subunit composition, stoichiometry, and arrangement, perhaps due to the absence of chaperone-guided processes. We seek to overcome these obstacles toward a6*-nAChR characterization and heterologous expression using tethered pentameric subunit constructs. We will begin to test the hypothesis that nAChR construction from tethered subunits enforces correct subunit assembly in the absence of neuron-specific chaperone proteins, preserving properties of native nAChR subtypes. The specific aims are to heterologously express and characterize, first in Xenopus oocytes and then in a mammalian cell host, a6*-nAChR composed of defined, tethered subunits. These subunits will be created by systematically introducing a6 and b3 subunits into a construct proven to encode fully functional nAChR. Standard assays will be used to confirm construct expression from message to functional a6*-nAChR and to characterize functional a6*-nAChR. As befits an R21 project, successful heterologous expression of covalently-linked a6b2b3- and a6a4b2b3- nAChR constructs will provide proof of concept for expression of other, complex nAChR subtypes using the tethered pentamer approach. No other approach can ensure the subunit composition, stoichiometry and arrangement that define a given nAChR subtype. Pure, plentiful expression of a6*-nAChR as tethered pentamers would enable unambiguous determination of subunit contributions to subtype function, and tests of subtype selectivity of drugs at these targets. Of public health relevance, these advances could extend to improved treatment of diseases ranging from addictions to movement disorders. PUBLIC HEALTH RELEVANCE: Nicotinic acetylcholine receptors (nAChRs) containing the a6 subunit are implicated in diseases ranging from addictions to Parkinson's disease. These receptors have proved difficult to study using natural sources, due to a combination of scarcity and contamination with other nAChR subtypes. Artificial expression of a6-containing nAChRs would provide a valuable alternative, but obtaining appropriate expression of these complex receptors has been extremely challenging. We propose an approach using linked receptor subunits to make a6- containing nAChRs with properties matching those of their naturally-expressed counterparts, as an alternative to the usual association of individual subunits. This will enforce desired subunit interactions, facilitating expression in a variety of experimentally amenable systems. In turn, this will greatly simplify studies of these important receptors, enhancing knowledge of their properties and helping to guide development of nAChR- directed treatments for addictions and Parkinson's disease.

DESCRIPTION (provided by applicant): The overall objective is to develop and implement a High Throughput Screen suitable for identifying compounds with selectivity for nicotinic acetylcholine receptors which contain the alpha6 subunit (alpha6 nAChRs). To meet this objective, two Specific Aims are proposed: 1. An HTS-ready assay will be established and optimized for an existing, highly-functional, monoclonal cell line stably transfected with alpha6/Beta2Beta3 nAChRs. Both membrane-potential and intracellular Ca2+ dye approaches are suitable, and the approach which can be refined to provide the most robust and reliable results will be adopted for the second Aim. 2. The optimized assay will be configured for HTS conditions. An overall screening workflow is proposed. This workflow incorporates secondary orthogonal- potency-, and selectivity-counter-screening protocols to identify and discard false positives, and to characterize confirmed candidates, from the initial screen. Already-available assays suitable for orthogonal- and counter-screening, using different activities than the primary screen, are described. The second Aim also contains a proposal to generate proof-of-principle data from a pilot screen of ~2400 structurally-diverse test compounds (including a high proportion of established CNS-active drugs). The health relevance of this project arises from genetic association and neurochemical studies which link alpha6 nAChR subunit gene variants and alpa6 nAChR function to substance use and dependence (particularly to tobacco smoking), and to important features of Parkinson's Disease development and treatment. Each of these conditions is a major public-health issue. The underlying molecular mechanisms by which alpha6 nAChRs influence these phenomena are still not well understood. Compounds identified by the proposed screen could become valuable research tools to understand better the etiology of the major public health issues associated with alpha6 nAChRs. This would provide impact through new scientific insights and potentially by revealing novel therapeutic avenues / targets. Compounds identified by this screen could also be useful treatments for the same conditions. Notably, the impact on drug dependence therapies could be quite broad, given the established association of alpha6 nAChRs with abuse liability for diverse substances. Further, an alpha6 nAChR- selective radiotracer could be a valuable PET/SPECT ligand for Parkinson's Disease diagnosis and/or monitoring of treatment success. PUBLIC HEALTH RELEVANCE: This project is intended to develop and implement a technique for rapidly testing a large library of compounds, with the objective of identifying and characterizing compounds selective for nicotinic acetylcholine receptors which contain the 16 subunit. These receptors have been associated with the following major public- health issues: smoking, alcohol abuse, and Parkinson's Disease. Compounds identified by this project would be useful in understanding the underlying causes of these conditions, and may serve as leads for the development of compounds used in their treatment and / or diagnosis.

DESCRIPTION (provided by applicant): A pair of High Throughput Screening (HTS) assays suitable for identifying non-nicotine tobacco product compounds with activity at ¿3¿4 and ¿4¿2 nicotinic receptors is proposed. Two Specific Aims are described: 1. HTS-ready assays will be established and optimized for a pair of existing, highly-functional, monoclonal cell lines. One is stably transfected with ¿3¿4-, the other with ¿4¿2- nicotinic acetylcholine receptors (nAChRs). Both membrane-potential and Ca2+ dye fluorescence approaches are suitable and the approach which can be refined to provide the most robust and reliable results will be adopted for Aim 2. 2. The optimized assays will be configured for HTS conditions. An overall screening workflow is proposed, and will be applied to each of the optimized ¿3¿4 and ¿4¿2 HTS-ready assays. This workflow incorporates secondary orthogonal- potency-, and selectivity-counter-screening protocols to identify and discard false positives, and to characterize confirmed candidates, from the initial screens. Already-available assays suitable for orthogonal- and counter-screening, using different activities than the primary screen, are described. We will generate proof-of-principle data for each fully-configured assay using a pilot screen of ~2400 structurally-diverse test compounds. Relevance of this proposal to PAR-12-266, and the Family Smoking Prevention and Tobacco Control Act (FSPTCA): As detailed in the Specific Aims, Significance and Innovation sections, this proposal will provide a set of validated assays targeting nAChR subtypes tied to use of, and dependence on, tobacco products. The resulting screens will produce impact by accelerating progress on two stated goals of PAR-12-266: 1) Reducing Addiction - understanding ... other constituents and components beyond nicotine that affect addiction of combustible and non-combustible tobacco products. 2) Diversity of Tobacco Products - understanding the constituents, components, ingredients, additives, and design features; ... of conventional and new and emerging tobacco products. They will also address a specific research area of the FDA Center for Tobacco Products (http://www.fda.gov/downloads/TobaccoProducts/NewsEvents/UCM293998.pdf; #15; What high-throughput screens can be developed and/or used to evaluate compounds in tobacco products and smoke that may affect addiction (e.g., act on nicotinic or dopaminergic receptors...etc.)?). Availability of this suite of screens will also advance priorities of the FSPTCA. Tobacco product components identified by such screens will be valuable leads for follow-up studies to understand their relevance to tobacco use and dependence, and their precise mechanisms of action. These studies would provide impact through new scientific insights and potentially by revealing novel tobacco-cessation therapeutic avenues/targets. Compounds identified by these screens may also be candidates for regulation under the FSPTCA.

SUMMARY / ABSTRACT ?-Conotoxin MII (?-CtxMII) selectively antagonizes ?3?2*- and ?6?2*-nAChR. We have previously developed ?6?2*-nAChR-selective ?-Ctxs to define mesolimbic ?6?2*-nAChR contributions to nicotine and other drug abuse phenotypes. A lack of selective compounds, and lethality in ?3 nAChR null mutant mice means virtually no ?3?2*-nAChR studies have been performed. Nor have extra-limbic ?6?2*-nAChR contributions to addiction- relevant behaviors been investigated extensively. Therefore, we will develop ?-Ctx ligands to discriminate between, characterize, and define the roles of ?3?2*- and ?6?2*-nAChR. The medial habenula (MH) and interpeduncular nucleus (IPN) contain the densest CNS ?3?2*-nAChR populations (which outnumber MH and IPN ?6?2*-nAChR). ?3?4*- and ?5*-nAChR in MH and IPN support nicotine dependence but ganglionic expression of ?3?4*-nAChR and lack of ?5*-nAChR in orthosteric binding sites may make these problematic smoking cessation targets. To establish and differentiate MH and IPN ?3?2*- and ?6?2*-nAChR contributions to nicotine abuse and addiction phenotype, three Specific Aims are proposed: 1) To discover further lead ?-Ctx ligands with ?3?2*-nAChR selectivity by screening an existing panel of >400 novel peptides, and develop them for enhanced selectivity. We have already identified 2 ?-Ctx leads with >10-fold ?3?2*-nAChR selectivity over other subtypes. The most-selective leads will be developed using a novel and streamlined approach to improve ?-Ctx selectivity. 2) To elucidate MH and IPN ?3?2*-nAChR subunit composition. We will make radio- and fluorescence-labeled derivatives of the highly-selective peptides developed in Aim 1. Using these labeled peptides, detailed ?3?2*-nAChR composition will be confirmed for the first time using nAChR subunit-null mutant mice. 3) To define the importance of MH and IPN ?3?2*- and ?6?2*-nAChR in nicotine reinforcement and withdrawal. We will test in rats if local infusion of the selective antagonists ?-CtxMII (?3?2* & ?6?2*), ?- CtxPIA (?6?2*-only), or a novel ?3?2*-only ?-Ctx into MH, IPN or 2 more control regions a) affects motivation to work for nicotine (under a progressive ratio schedule) and b) affects spontaneous somatic and behavioral withdrawal symptoms. For Aims 2 & 3, we describe how to proceed using existing compounds in the extremely unlikely case that Aim 1 does not yield suitably ?3?2*-nAChR-selective ?-Ctxs. This proposal's new screening and peptide-development features will radically advance future utilization of the invaluable ?-Ctx resource. The resources developed in this proposal will be vital to enable future studies probing nAChR function within the addiction-related network to which MH and IPN are extensively connected. Using our novel and potent method for engineering ?-Ctx selectivity in combination with refined behavioral testing across male and female subjects greatly enhances translational impact. This proposal promises to identify and characterize ?3?2*-nAChR as novel, druggable, smoking therapeutic targets, and confirm ?6?2*-nAChR as a viable tobacco cessation target. These advances may produce a major impact on public health by promoting smoking cessation.

SUMMARY / ABSTRACT Tobacco use, and most-prominently cigarette smoking, is the leading cause of preventable death in the USA and across the world. Smoking behavior is driven by addiction to nicotine, which exerts its effects through nicotinic acetylcholine receptors (nAChR). Better understanding how nAChR function and expression is regulated is thus crucial. Prototoxins are an extensive family of proteins which serve as physiologically important regulators of multiple nAChR subtypes. However, the basis of prototoxins' nAChR subtype selectivities, their interaction sites, the mechanisms by which they alter nAChR function, and their roles within nicotine dependence pathways are largely undetermined. This application addresses these critical gaps in our knowledge. Our Preliminary Data indicate that the prototoxin lynx1 allosterically regulates multiple isoforms of ?3?4*-, ?4?2*- and ?5*-nAChR, which have repeatedly been linked to human smoking behavior. We have also observed differential macroscopic and single-channel functional effects of lynx1 across nAChR isoforms, which provide ideal readouts for use in defining the sites at which lynx1/?3?4*-nAChR interactions occur. These findings led us to our underlying hypothesis: that allosteric prototoxin effects arise from (generally well- conserved) interactions with non-agonist-binding nAChR ?(-)- subunit interfaces, and that differential outcomes arise from the details of interactions at each prototoxin/nAChR interface. New Preliminary data also indicate that rostral-IPN (IPR) GABA neurons, with a well-defined role in somatic nicotine withdrawal, coexpress ?3?4*- and ?4?2*-nAChR, together with high levels of both ?5 subunit and lynx1 mRNA. They therefore represent an excellent, dependence-related, native system for studying with which nAChR population(s) ?5 subunits associate, and how lynx1 modulates these same nAChR populations in the IPN. We therefore are ideally placed to compare functional outcomes of lynx1 modulation across the same defined nAChR populations in native neurons and in vitro models, enhancing validation and interpretation of findings across these systems. We will pursue this opportunity by combining precise experimental data from a multidisciplinary experimental approach with sophisticated molecular dynamics modeling. This closely integrated research plan will allow us to establish for the first time a generalized framework to understand how prototoxin modulators produce functional outcomes across multiple nAChR subtypes and isoforms, in both native neurons and in vitro expression systems. It will also ensure maintenance of scientific rigor, rapidly refine our experimental designs, and produce key biological and mechanistic insights. In addition, regionally-restricted prototoxin expression may permit modulation of nAChR function to be restricted to particular brain regions or cell types. Prototoxin/nAChR interactions may therefore represent promising new drug targets. By probing the nature and sites of prototoxin/nAChR interactions, this study promises to remove critical barriers to progress in scientific and clinical work related to nicotine addiction, as well as other conditions affected by nicotinic receptor function.