Project 3 University of Colorado, Boulder

尽管烟草的破坏性后果广为人知，但它仍然是一个主要的健康问题。尽管目前的戒烟疗法，包括尼古丁替代疗法、安非他酮和瓦伦尼克林，已经取得了一些成功，但它们还不够充分，因为只有一小部分(30%-40%)尝试这些药物治疗的吸烟者戒烟。因此，需要更有效或更多的援助。烟草烟雾中的尼古丁通常被认为是导致吸烟上瘾的主要化合物。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)。胆碱能系统已被证明通过激活烟碱型乙酰胆碱受体(NAChRs)来调节多巴胺(DA)的释放(Dani和Di Biasi，2001；Kaneko等人，2000；Mameli-Engvall等人，2006；McKay等人，2007；Exley等人，2008；Exley和Cragg，2008；邓等人，2007；Maskos，2008)。来自我们小组的研究(Cui等人，2003；Salminen等人，2004,2007；Grady等人，2007；Drenan等人，2008；Grady等人，2009b)以及其他人(Kulak等人，1997；Kaiser等人，1998；周等人，2001；Champtiaux等人，2003；Perry等人，2007；Sidhpua等人，2007；Exley等人，2008；Perez等人，2008；Perez等人，2009；Meyer等人，2008年)指出，a6-nAChRs(a6a4B2B3，a6B2B3，a6B2)和a4-nAChRs(a4B2，a4a5B2)nAChRs在调节尼古丁诱导的多巴胺释放增加中发挥重要作用。在突触体DA释放实验中，a6B2*受体对吸烟浓度的尼古丁激活高度敏感(Champtiaux等人，2003；Salminen等人，2004和2007)，并主导尼古丁对伏隔核多巴胺神经传递的控制(Exley等人，2008)。这些结果，加上最近的发现，人类基因中编码组成亚单位的基因的多态 在DA神经元中表达的nAChRs，A4，a5，a6，B2，B3(CHRNA4(Li等人，2005,2008；Hutchison等人，2007)CHRNAS(Amos等人，2008；Berrettini等人，2008；Beirut等人，2008；Beirut et al.2008年；Hung等人，2008年；Schlaepfer等人，2008年；Saccon等人，2009年；Wang等人，2009年；Weiss等人，2008年；Thorgeirsson等人，2008年)，CHRNA6(Hoft等人，2009；Saccon等人，2009)，CHRNB2(Ehringer等人，2007)和CHRNAB3(Hoft等人，2009；Saccon等人，2009)影响烟草成瘾和/或肺癌的易感性，表明开发调节nAChRs的药物在调节DA释放中发挥关键作用可能提供新的、更有效的药物 治疗烟草成瘾的方法。 A6B2*类受体的选择性激动剂或拮抗剂可能用于戒烟治疗。寻找这样一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，我们初步筛选了16种尼古丁化合物，这些化合物是由我们在Targacept的合作者提供的。在自然表达感兴趣的nAChR的小鼠脑组织中，使用分层方法系统地分析这些化合物，以使用膜结合分析来确定四类nAChR，a4B2*，A7，a3B4*和a6B2*的亲和力、效力和有效性， 突触体功能分析，以及表达A7-nAChR的细胞的电流记录(最后一项分析在Targacept进行)。这一筛选帮助我们的NCDDG定义了尼古丁结构的修饰，以指导更具选择性的化合物的合成。这部作品的出版正在进行中(已提交)。第二轮6个化合物是由Targacept的化学家根据我们从最初的一组化合物的分析中获得的结果合成的。这为我们提供了6对化合物(吡啶和嘧啶)来验证我们的假设，即嘧啶化合物可能对 A6B2*-nAChR。这项研究已经发表(Brening等人，2009年)，并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对这些进行测试，并希望选择几个(~3-4)用于行为和慢性治疗的研究。 此外，这笔赠款还支持对a6B2*-nAChR功能的研究。我们在加州理工大学莱斯特实验室的合作者已经产生了一个功能获得突变，A6L9‘S。对这些突变小鼠的特征表明，它们确实极大地提高了a6B2*-nAChR的活性，并且具有独特的表型，将有助于研究该亚型对行为的调节(Drenan等人，2008年)。我们打算继续鉴定新的和可能更具选择性的化合物，并使用a6L9‘S小鼠研究a4B2*和a6B2*-nAChR功能。与天然激动剂ACh不同，尼古丁代谢不快；因此，长期接触尼古丁可能通过激活和脱敏nAChRs发挥作用(Rose，2007)。潜在的戒烟药物也可能促进一些项目3(博尔德。脱敏水平。NCDDG赠款产生的数据表明，a6B2*-nAChRs可能会比a4B2*脱敏。IBG提供的a4和a6缺失突变小鼠和它们的野生型窝种将是评估选择性、脱敏和研究受体功能的其他方面的宝贵工具。

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.