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

摘要 慢性疼痛是公共卫生领域的一个主要问题，预计今年的财务成本将超过6000亿美元 明年患有慢性疼痛的患者忍受着极端的情感、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 管理，如NSAID，阿片类药物，神经元稳定剂和抗抑郁药，通常不提供 足够的救济，以恢复全面的生活质量，在许多情况下，这些治疗本身限制 患者，如阿片类药物治疗，防止患者合法驾驶。最近的临床前研究 确定神经元腺苷酸环化酶1型（AC1）作为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， AC1基因敲除小鼠提供了将AC1与慢性炎性疼痛疾病联系起来的直接证据。 此外，AC1抑制剂将缺乏与用于治疗的其他药剂（例如阿片类药物）相关的副作用。 治疗慢性炎性疼痛。AC1抑制剂的开发是一个独特的挑战， 通过先前的临床前AC1抑制剂NB 001证实。NB001具有显著的缺点，包括 相对于其他腺苷酸环化酶同工型的适度选择性，可能是由于其腺嘌呤样结构。化合物 这种类型的抑制剂称为P位点抑制剂，通过与AC的活性位点结合而起作用，AC的活性位点在所有 同种型。对于含腺嘌呤的分子如NB001的其他关注包括对其他细胞的影响。 例如DNA合成。我们假设，开发一种AC1的小分子抑制剂， 使我们能够模拟AC1敲除表型，并为治疗慢性 炎性疼痛。我们设计我们的研究目标不是保守的P位点或毛喉素结合位点， 相反，这是一种新的方法，靶向AC1和钙调素（CaM）的独特蛋白质-蛋白质相互作用。AC1 和AC8都被CaM激活，然而，CaM结合结构域在结构上是独特的， 这一位置为实现AC1选择性提供了前所未有的机会。因此，本提案的目标 目的是：1）开发一种新的AC1/CaM生物化学筛选测定法，2）在高水平上实施这种新的测定法， 通量筛选，以询问AC1/CaM蛋白抑制剂的100，000种化合物的文库- 蛋白质相互作用，以及3）使用细胞测定法验证和化学优化先导分子， 选择性和效力，以指导药物化学工作。到目前为止，我们已完成初步研究， 开发新的筛选试验，建立必要的试验子集，并巩固 爱荷华州和普渡大学之间的合作，为我们的成功完成 目标。我们期待最终得到改进并应用于临床的选择性AC1抑制剂的鉴定。 慢性炎性疼痛的模型。

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.