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

这项建议阐述了一类新的戒烟药物的作用机制，该药物基于与大脑中高亲和力尼古丁受体结合并部分激活的药物。首次上市的药物(Varenicline)是尼古丁部分激动剂，但受到FDA的限制，反映了受体亚型选择性的不足；varenicline针对高亲和力尼古丁结合部位(α4beta2)，但也是α7受体的完全激动剂。如果较差的亚型选择性是varenicline副作用的原因，那么更好地了解配体结构与受体(和亚型)功能的联系是关键，但要做到这一点，我们需要扩大我们对配体结合模式的认识。我们的重点是胞苷，它是一种自然产生的部分尼古丁激动剂，已被用于戒烟，因此本身也具有商业潜力。我们最近开发了强大而高效的化学，使我们能够直接，具体地，在分子上几乎没有探索过的位置上修饰胞苷。该化学以胞苷为起点，探索一系列新的结构变体，探索新的结合模式，从而有可能找到实现尼古丁亚型特异性的新方法。最近关于胞苷(和varenicline)介导配体与尼古丁受体结合的分子特征的推测将通过(I)制造其结构特征将减弱配体结合的特定分子以及(Ii)扩大以非常定向的方式形成的配体-蛋白质接触的范围来研究。我们将建立在初步的结构和计算研究的基础上，并创建与胱氨酸的C3和C4位置相关的新配体。这两类，尤其是C4变异体，都提供了一个机会，使现有的(但目前是“旁观者”)蛋白质残基与配体结合，这些结合部位位于与关键受体亚型之间的分化直接相关的结合部位。通过访问这些附加模式来修改现有绑定将允许我们合理地“调整”子类型选择性。成功将极大地改善我们对尼古丁配体内部结构关系的看法，并为药物化学家提供关于增强的“活性部位”模板的范围和空间分布的见解。我们将使用结晶学来研究这些新相互作用的具体细节，以精确定位模型蛋白质中的配体，从而识别新的“参与”残基。然后，我们将把它与仍不能结晶的完整的人类受体蛋白联系起来，以了解配体结合以及任何新的结合模式如何转化为完整的蛋白质受体如何发挥作用。我们将用新的和强大的计算方法来研究配体结合的结果的细节，以便将配体结构与整个受体功能相关联。我们已经在一个已经是多学科的计划中建立了一系列的合作者，使我们能够探索这些新配体的生物学、结构特性和商业潜力，并将其与我们的预测和分析计算方法相关联。这个项目的成功将关键取决于我们的新计算技术和合成化学之间的密切相互作用。我们将阐明决定亚型选择性的配体结构的分子水平特征，这将对戒烟剂的安全性/使用者可接受性产生较长期的影响。通过了解配体结构与整体受体功能之间的联系的细节，这是一项特别具有挑战性的计算任务，我们希望将亚型选择性问题与更普遍的(与尼古丁激活相关的)“下游”效应分开，这有可能指导尼古丁类治疗剂的进一步发展。

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.