Non-Opioids for Inflammatory Pain: Adenylyl Cyclase 1 as a Novel Target

摘要 慢性疼痛是公共卫生的主要问题，预计每年的财务成本将超过6000亿美元 明年。患有慢性疼痛的患者承受着极端的情感、身体和社会负担， 导致生活质量严重下降。不幸的是，目前用于治疗慢性疼痛的药物 治疗，如非类固醇抗炎药、阿片类药物、神经稳定剂和抗抑郁药，通常不会提供 足够的缓解来恢复完全的生活质量，在许多情况下，这些治疗本身就限制了 患者，如阿片类药物治疗，阻止患者合法驾驶。最近的临床前研究表明 发现神经元腺苷酸环化酶1(AC1)是治疗慢性疼痛的新靶点。AC1高度 在与疼痛处理和神经元可塑性相关的神经元组织中表达，并利用 AC1基因敲除小鼠提供了将AC1与慢性炎症性疼痛条件联系起来的直接证据。 此外，AC1抑制剂没有其他药物(如阿片类药物)的副作用。 治疗慢性炎症性疼痛。AC1抑制剂的开发是一个独特的挑战，因为 先前的临床前AC1抑制剂NB001证明了这一点。NB001有很大的缺点，包括 与其他腺酰环化酶异构体相比有一定的选择性，这可能是由于其腺嘌呤样结构所致。化合物 这种类型的被称为P-位点抑制物，通过与AC的活性部位结合而起作用，该活性部位在所有 异构体。对NB001等含腺嘌呤分子的其他担忧还包括对其他细胞的影响 DNA合成等过程。我们假设开发一种AC1的小分子抑制剂将 使我们能够模拟AC1基因敲除表型，为慢性粒细胞白血病的治疗提供了一条新的途径 炎症性疼痛。我们设计我们的研究不是针对保守的P-位点或forsklin结合位点，而是 相反，这是一种新的方法，针对AC1和钙调蛋白(CaM)独特的蛋白质-蛋白质相互作用。AC1 和AC8都被CaM激活，然而，CaM结合域在结构上是独特的， 地理位置为实现AC1选择性提供了前所未有的机会。因此，这项提案的目标是 1)建立一种新的AC1/CaM生化筛选方法；2)将该方法在较高的 对AC1/CaM蛋白抑制剂的100,000个化合物文库进行吞吐量筛查- 蛋白质相互作用，以及3)使用细胞分析验证和化学优化铅分子 用于指导药物化学工作的选择性和有效性。到目前为止，我们已完成初步研究，以 开发新的筛选试验，建立必要的分析子集，并巩固 爱荷华大学和普渡大学合作，成功完成我们的 目标。我们期待最终改进和应用的选择性AC1抑制剂的鉴定。 慢性炎症性疼痛模型。

ABSTRACT Chronic pain is a major concern in public health with financial costs projected to surmount $600 billon in the next year. Patients afflicted with chronic pain endure extreme emotional, physical, and social burdens, resulting in severely diminished quality of life. Unfortunately, drugs currently used for chronic pain management, such as NSAIDs, opioids, neuronal stabilizers, and antidepressants, do not typically provide sufficient relief to restore full quality of life, and in many instances these treatments themselves limit patients, such as opioid treatment preventing a patient from legally driving. Recent preclinical studies have identified neuronal adenylyl cyclase type 1 (AC1) as a novel target for treating chronic pain. AC1 is highly expressed in neuronal tissues associated with pain processing and neuronal plasticity, and studies using AC1 knockout mice provide direct evidence linking AC1 to chronic inflammatory pain conditions. Furthermore, AC1 inhibitors would lack the side effects associated with other agents (e.g. opioids) used to treat chronic inflammatory pain. The development of AC1 inhibitors represents a unique challenge, as demonstrated by a prior preclinical AC1 inhibitor, NB001. NB001 has significant shortcomings, including modest selectivity over other adenylyl cyclase isoforms, likely due to its adenine-like structure. Compounds of this type are called P-site inhibitors and act by binding to the active site of AC that is conserved among all isoforms. Additional concerns for adenine-containing molecules like NB001 include effects on other cellular processes such as DNA synthesis. We hypothesize that developing a small molecule inhibitor of AC1 will allow us to mimic the AC1 knockout phenotype and provide a new avenue for the treatment of chronic inflammatory pain. We designed our studies to target NOT the conserved P-site or forskolin-binding site, but rather a novel approach, targeting the unique protein-protein interaction of AC1 and calmodulin (CaM). AC1 and AC8 are both activated by CaM, however, the CaM binding domains are unique in structure and location providing an unprecedented opportunity to achieve AC1 selectivity. Thus, the goals of this proposal are to: 1) develop a novel AC1/CaM biochemical screening assay, 2) implement this novel assay in a high throughput screen to interrogate a library of 100,000 compounds for inhibitors of the AC1/CaM protein- protein interaction, and 3) validate and chemically optimize lead molecules using cellular assays focused on selectivity and potency to guide medicinal chemistry efforts. To date, we have completed initial studies to develop the novel screening assay, established a subset of the necessary assays, and cemented the collaboration between the University of Iowa and Purdue University for the successful completion of our aims. We anticipate the identification of selective AC1 inhibitors that ultimately be improved and applied in models of chronic inflammatory pain.