Project 3 University of Colorado, Boulder

烟草使用仍然是一个主要的健康问题，尽管人们广泛了解其有害后果。虽然目前的戒烟疗法，包括尼古丁替代，安非他酮和伐尼克兰，已经取得了一些成功，但它们是不够的，因为只有一小部分（30-40%）尝试这些药物治疗的吸烟者放弃了烟草使用。因此，需要更有效的或额外的援助。 烟草烟雾中的尼古丁通常被认为是导致吸烟成瘾性的主要化合物。包括尼古丁在内的许多滥用药物的强化作用部分受到多巴胺能神经传递的调节（Koob和LeMoal，1997; Di Chiara，2000; Koob，2000; Dani和De Biasi，2001; Nestler，2002; Wise，2008）。胆碱能系统已显示通过激活烟碱乙酰胆碱受体（nAChR）来调节多巴胺（DA）释放（Dani和Di Biasi，2001; Kaneko等人，2000; Mameli-Engvall等，2006; McKay等，2007; Exley等，2008; Exley和Cragg，2008; Deng等，2007; Maskos，2008）。我们小组的研究（Cui等人，2003; Salminen等人，2004，2007; Grady等人，2007; Drenan等人，2008; Grady等人，2009 b）以及其他人（Kulak等，1997; Kaiser等，1998; Zhou等，2001; Champtiaux等，2003;佩里等，2007; Sidhpura等，2007; Exley等，2008; Perez等，2008，2009; Meyer等，2008）表明，a6-nAChR（a6 a4 B2 B3，a6 B2 B3，a6 B2）和a4-nAChR（a4 B2，a4 a5 B2）nAChR在调节尼古丁诱导的多巴胺释放增加中起重要作用。当在突触体DA释放实验中测量时，a6 B2 * 受体对吸烟浓度的尼古丁的激活高度敏感（Champtiaux等人，2003; Salminen等人，2004和2007），并主导烟碱对丘脑核中多巴胺神经传递的控制（Exley et al.，2008）这些结果，加上最近的发现，人类基因中的多态性编码的亚基，构成 在DA神经元中表达的nAChR，a4、a5、a6、B2、B3（CHRNA 4（Li et al.，2005，2008; Hutchison等人，2007）（Amos等，2008; Berrettini等，2008;贝鲁特等，2008; Hung等，2008; Schlaepfer等，2008; Saccone等，2009; Wang等，2009;韦斯等，2008; Thorgeirsson等人，2008年），CHRNA 6（Hoft等人，2009; Saccone等人，2009）（Ehringer等人，2007）和CHRNAB 3（Hoft等人，2009年; Saccone等人，2009年）影响烟草成瘾和/或肺癌的易感性，这表明，开发调节在调节DA释放中起重要作用的nAChRs的药物可能会提供新的，更有效的， 治疗烟草成瘾的方法。 α 6 B2 * 类受体的选择性激动剂或拮抗剂可能用作戒烟疗法。寻找这种化合物的进展是我们最初合作的目标。在过去的几年里，在这笔赠款的资助下，我们最初筛选了16种烟碱化合物，这些化合物由我们在Targacept的合作者提供。使用分级方法在天然表达感兴趣的nAChR的小鼠脑组织中系统地测定这些化合物，以使用膜结合测定法确定对四类nAChR（α 4 B2 *、α 7、α 3 B4 * 和α 6 B2 *）的亲和力、效力和功效， 突触体功能测定和来自表达α 7-nAChR的细胞的电流记录（最后一个测定在Targacept进行）。该筛选帮助我们的NCDDG确定尼古丁结构的修饰，以指导更具选择性的化合物的合成。这项工作正在出版中（已提交）。第二轮的6种化合物是由Targacept的化学家根据我们从最初一组化合物的测定中了解到的情况合成的。这些为我们提供了6对化合物（吡啶与嘧啶）来研究我们的假设，即嘧啶化合物可能对 a6B2*-nAChR。这项研究已经发表（Breining et al，2009），将有助于指导第三轮化合物的合成。我们计划以类似的方式分析这些，并希望选择一些（~3-4）用于行为和慢性治疗的研究。 此外，该补助金还支持对a6 B2 *-nAChR功能的研究。我们的合作者在加州理工学院的莱斯特实验室产生了一种功能获得性突变a6 L9'S。对这些突变小鼠的表征揭示，它们确实具有大大增加的α 6 β 2 *-nAChR活性，以及具有独特的表型，并且将有助于研究该亚型对行为的调节（Drenan等人，2008年）。我们打算继续表征新的和潜在的更具选择性的化合物，以及使用a6 L9小鼠研究a4 B2 * 和a6 B2 *-nAChR功能的方面。与天然激动剂ACh不同，尼古丁不能快速代谢;因此，长期暴露于尼古丁可能通过nAChR的激活和脱敏联合作用发挥作用（Rose，2007）。潜在的戒烟药物也将有可能促进一些项目3（博尔德。放大图片作者：Lester，亨利A.脱敏水平。NCDDG资助产生的数据表明，a6 B2 *-nAChR可能比a4 B2 * 脱敏。IBG提供的α 4和α 6无效突变小鼠及其野生型同窝仔将是评估选择性、脱敏和研究受体功能其他方面的有价值的工具。

Tobacco use remains a major health problem despite widespread knowledge of the damaging consequences. Although current smoking cessation therapies, including nicotine replacement, bupropion, and varenicline, have had some success, they are inadequate in that only a fraction (30-40%) of smokers who try these drug treatments abstain from tobacco use. Consequently, more effective or additional aids are needed. Nicotine in tobacco smoke is generally regarded as the major compound that is responsible for the addictive properties of smoking. The reinforcing effects of numerous drugs of abuse, including nicotine, are partially modulated by dopaminergic neurotransmission (Koob and LeMoal, 1997; Di Chiara, 2000; Koob, 2000; Dani and De Biasi, 2001; Nestler, 2002; Wise, 2008). Cholinergic systems have been shown to modulate dopamine (DA) release by activating nicotinic acetylcholine receptors (nAChRs) (Dani and Di Biasi, 2001; Kaneko et al., 2000; Mameli-Engvall et al, 2006; McKay et al, 2007; Exley et al, 2008; Exley and Cragg, 2008; Deng et al, 2007; Maskos, 2008). Studies from our group (Cui et al, 2003; Salminen et al, 2004, 2007, Grady et al, 2007; Drenan et al, 2008; Grady et al, 2009b) as well as others (Kulak et al, 1997; Kaiser et al, 1998; Zhou et al, 2001; Champtiaux et al, 2003; Perry et al, 2007; Sidhpura et al, 2007; Exley et al, 2008; Perez et al, 2008, 2009; Meyer et al, 2008) indicate that a6-nAChRs (a6a4B2B3, a6B2B3, a6B2) and a4-nAChRs (a4B2, a4a5B2) nAChRs play important roles in modulating nicotine-induced increases in dopamine release. The a6B2* receptors are highly sensitive to activation by smoking concentrations of nicotine when measured in synaptosomal DA release experiments (Champtiaux et al, 2003; Salminen et al., 2004 and 2007) and dominate nicotine control of dopamine neurotransmission in the nucleus accumbens (Exley et al., 2008) These results, coupled with recent findings that polymorphisms in the human genes that code for the subunits that make up nAChRs expressed in DA neurons, a4, a5, a6, B2, B3 (CHRNA4 (Li et al., 2005, 2008; Hutchison et al, 2007) CHRNAS (Amos et al, 2008; Berrettini et al, 2008; Beirut et al. 2008; Hung et al, 2008; Schlaepfer et al, 2008; Saccone et al, 2009; Wang et al, 2009; Weiss et al, 2008; Thorgeirsson et al, 2008), CHRNA6 (Hoft et al, 2009; Saccone et al, 2009) CHRNB2 (Ehringer et al, 2007) and CHRNAB3 (Hoft et al, 2009; Saccone et al, 2009) influence vulnerability to tobacco addiction and/or lung cancer, suggest that developing drugs that modulate the nAChRs that play vital roles in modulating DA release may provide new, more effective approaches to treat tobacco addiction. It is possible that a selective agonist or antagonist for the a6B2* class of receptors could be of use as a therapy for smoking cessation. Progress toward finding such a compound was a goal of our initial collaboration. In the past few years with funding from this grant, we initially screened 16 nicotinic compounds, supplied by our collaborators at Targacept. These compounds were assayed systematically in mouse brain tissues that naturally express the nAChR of interest using a hierarchical approach to determine affinity, potency, and efficacy at four classes of nAChRs, a4B2*, a7, a3B4* and a6B2* using membrane binding assays, synaptosomal function assays, and current recordings from cells expressing a7-nAChR (this last assay was carried out at Targacept). This screen has helped our NCDDG to define modifications to the structure of nicotine to guide the synthesis of more selective compounds. Publication of this work is in progress (submitted). A second round of 6 compounds was synthesized by chemists at Targacept based on what we learned from our assays of the initial set of compounds. These provided us with 6 pairs of compounds (pyridine vs pyrimidine) to investigate our hypothesis that the pyrimidine compounds may be more selective for a6B2*-nAChR. This study has been published (Breining et al, 2009), and will help guide synthesis of the third round of compounds. We plan to assay these in a similar manner, and hope to choose a few (~3-4) for studies of behavior and chronic treatment. In addition this grant has supported research into the function of the a6B2*-nAChR. A gain-of-function mutation, a6L9'S, has been generated by our collaborators in the Lester lab at Caltech. Characterization of these mutant mice revealed that they do indeed have greatly increased activity of the a6B2*-nAChR, as well as having a distinct phenotype, and will facilitate study of modulation of behaviors by this subtype (Drenan et al., 2008). We intend to continue characterizing new and potentially more selective compounds, as well as study aspects of a4B2* and a6B2*-nAChR function using the a6L9'S mice. Unlike the natural agonist, ACh, nicotine is not metabolized quickly; therefore, chronic exposure to nicotine may act by a combination of activation and desensitization of nAChRs (Rose, 2007). Potential smoking cessation drugs will also be likely to promote some Project 3 (Boulder. Marks, PI) Lester, Henry A. level of desensitization. Data generated with the NCDDG grant suggests that a6B2*-nAChRs may desensitize less than a4B2*. The a4 and a6 null mutant mice and their wild-type littermates available at IBG will be valuable tools to assess selectivity, desensitization and to study other aspects of receptor function.