T-型钙通道天然活性分子挖掘及其在治疗疼痛中的应用

	T-型钙通道天然活性分子挖掘及其在治疗疼痛中的应用
	李松谕
导师	年寅
关键词	T-型钙通道，PPAP类化合物，黄酮类化合物 Key words:　T-type calcium channel, PPAP, isoprenylated flavonoids
摘要	摘　要离子通道介导的生物电和化学(钙)信号几乎参与所有重要的生命过程，是目前药物研发的第二大靶点。T-型钙通道（T-type Calcium Channel，TTCC，Cav3.1-Cav3.3）是一类特殊的电压门控离子通道，广泛分布于心血管，神经和内分泌等系统，其功能紊乱或基因水平的突变可导致多种严重疾病，如：失神癫痫、神经性疼痛、小脑性共济失调、帕金森氏震颤症及原发性醛固酮增多症等，是目前离子通道药物研发的热门靶点。遗憾的是现阶段暂无针对TTCC的上市药物，亟需进一步挖掘作用机制独特、选择性强的活性分子。与国际药企以合成分子为主的研发不同，本课题组从天然产物结构新颖、作用独特及先导性强出发，以TTCC为靶点对3000余个天然化合物进行活性筛选，发现活性分子167个，结构类型包括二萜、环烯醚萜、多环多异戊烯基取代酰化间苯三酚（PPAP）及生物碱等。在前期工作基础上，本研究继续对金丝桃属（Hypericum spp.）中的PPAP类和毛地黄鼠尾草（Salvia digitaloides.）中的异戊烯基取代黄酮类化合物利用膜片钳（Patch Clamp）等方法进行了活性筛选，同时也开展了对其选择性、作用机制及体内药效学等一系列研究，取得以下进展：（1）系统地完成了82个PPAP（1-82；63个新化合物）分子和4个黄酮类（3个新分子）化合物的活性筛选。共发现27个Cav3.1抑制剂和5个Cav3.2抑制剂，包括6个活性突出分子（IC50 ≤ 6.0 μM）。上述活性化合物中包含26个PPAP类分子（1-26），结构类型涉及A型BPAP、B型BPAP、seco-BPAP、nor-BPAP。PPAP类和黄酮类活性突出分子1、3、13、20、21、rxu-61对Cav3.1或Cav3.2通道抑制作用的IC50分别为3.8 μM、3.5 μM 、5.6 μM、3.5 μM 、6.0 μM和3.5 μM。（2）发现一系列PPAP类Cav3.1选择性抑制分子3、8、24。其中3对Cav3.1（IC50：3.5 μM）和Cav3.2选择性SI > 20，超过临床研究分子Z944（SI < 4）。（3）构效关系分析表明，Cav3.1选择性抑制剂3、8和24结构中O-2成环以及C-10位的苯甲酰基取代是其产生选择性的关键所。初步机理研究发现13与1、7、18、20、21、23相比结合位点更靠近通道的胞外区域。此外，通过与孔道阻断剂Ni2+的相互叠加实验发现活性突出分子的作用方式不是通过直接阻断孔道，而是与通道其他位点结合对电流产生抑制作用。基于分子对接的结果构建了Cav3.1和Cav3.2单突变和双突变共计36个，并在p.Ser407Ala Cav3.2突变体上对7、13和21活性进行了研究，发现该位点突变对上述分子活性无显著影响，不是潜在的结合位点。（4）活性分子HJ（1 mg/kg）对福尔马林诱导的Ⅰ相（1-5分钟）和Ⅱ相疼痛（15-60分钟）总体而言没有缓解作用。但将Ⅱ相疼痛分时段（10分钟）统计发现HJ可以显著缓解45-60分钟内小鼠的疼痛反应。上述结果提示HJ的吸收相对缓慢，需要进一步调整给药时间开展深入的药效学研究。综上，本研究进一步丰富了对TTCC天然活性分子结构类型、构效关系及作用机制的认识，为后续药物研发，特别是Cav3.1选择性抑制剂的挖掘与应用提供了结构模板等信息。; Ion channels mediate bioelectrical and chemical (calcium) signalings which involve in almost every essetial physiological process. They are currently the secondary biggest targets for drug development. T-type Calcium Channel (TTCC, Cav3.1-Cav3.3) is a special class of voltage-gated ion channels, which widely distributes in cardiovascular, neurological and endocrine systems. Dysfunctions or mutations of these channels can lead to many serious diseases, such as epilepsy, neuropathic pain, cerebellar ataxia, Parkinson's tremor and primary aldosteronism, etc. Thus, they are becoming promising targets for ion channel drug development. However, there are currently no drugs targeting TTCC be clinically used. Therefore, further exploration of active molecules with unique mechanisms of action and higher selectivity are desired. Unlike pharmaceutical companies who focus on synthetic molecules for TTCC drug development, our group have mainly paid attention to natural products and discovered 167 active molecules, including diterpenes, iridoid, polycyclic polyprenylated acylphloroglucinols (PPAPs) and alkaloids. In this study, we continuedly investigated the activity, selectivity, mechanism of action and in vivo pharmacodynamics of PPAPs from Hypericum spp. and the isoprenylated flavonoids isolated from Salvia digitaloides, by Patch Clamp and other methods. The following findings have been made: (1) The activities of eighty-two PPAPs (1-82; 63 new compounds) and four flavonoids (3 new compounds) were evaluated. Twenty-seven Cav3.1 inhibitors and five Cav3.2 inhibitors were identified, including 6 molecules, 1, 3, 13, 20, 21 and rxu-61, with noticeable activities (IC50 ≤ 6.0 μM). The IC50 values of molecules with noticeable activities, are 3.8 μM, 3.5 μM, 5.6 μM, 3.5 μM, 6.0 μM, and 3.5 μM, respectively. Among the active compounds include twenty-six PPAP molecules (1-26), with structural types as A-type BPAP, B-type BPAP, seco-BPAP, nor-BPAP. (2) A series of PPAPs, 3, 8 and 24, with selective Cav3.1 inhibition were identified. Compound 3 has selective index (SI) > 20 between Cav3.1 (IC50: 3.5 μM) and Cav3.2, which is much better than Z944 (SI < 4), a molecule under clinical study. (3) The structure-activity relationship analyses showed that the O-2 cyclization and the substitution of a benzoyl at the C-10 are the key structural features of selective Cav3.1 inhibitors of 3, 8 and 24. Preliminary mechanistic studies revealed that, other than 1, 7, 18, 20, 21, and 23, the binding sites of 13 are closer to the extracellular domain of the channel. In addition, interaction experiments with the TTCC pore blocker Ni2+ revealed that the modes of actions of the active molecules were not by directly blocking the pore but by binding with other sites of the channel. A total of thirty-six Cav3.1 and Cav3.2 mutations (single and double) were constructed based on the results of molecular docking. In addition, 7, 13 and 21 activities were investigated on the p.Ser407Ala Cav3.
语种	中文
	2022-05
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75127
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	李松谕. T-型钙通道天然活性分子挖掘及其在治疗疼痛中的应用[D]. 中国科学院大学,2022.

条目包含的文件
文件名称/大小	文献类型	版本类型	开放类型	使用许可
李松谕-李松谕-T-型钙通道天然活性分子（5173KB）	学位论文		限制开放	CC BY-NC-SA	请求全文