GPR119调节剂与糖皮质激素受体拮抗剂的筛选与药理学研究(硕士)(论文38000字)
中文摘要
本论文包括三部分工作：第一部分围绕G蛋白偶联受体119（G protein-coupled receptor 119,GPR119）建立细胞水平的激动剂及正向变构调节剂高通量筛选模型，通过大规模筛选发现一个小分子激动剂，并进行了细胞水平的功能验证，最后应用糖尿病db/db小鼠初步研究了体内药效。第二部分涉及一个糖皮质激素受体（Glucocorticoid receptor, GR）拮抗剂的分子与细胞药理学研究，以及通过构效关系分析和结构优化改造发现的一个活性更好的衍生物。第三部分介绍了应用结核杆菌二氢叶酸还原酶（DHFR）的分子水平筛选模型和高通量筛选发现的DHFR特异性小分子抑制剂。
糖尿病是一组由遗传和环境因素共同作用引起的临床综合征，主要包括1型（Type 1 diabetes mellitus, T1DM）和2型糖尿病（Type 2 diabetes mellitus, T2DM）这两种类型，其中以T2DM最为普遍，症状主要表现为胰岛素抵抗及胰岛β细胞功能障碍。GPR119是一个通过基因组测序、序列比对分析发现的一个孤儿受体。该受体被激活后，可以发挥治疗糖尿病的作用。由于GPR119主要与Gs蛋白发生偶联，我们建立了基于萤光素酶报告基因的细胞水平筛选系统，旨在寻找GPR119的小分子激动剂或正向变构调节剂。通过对320000个化合物的筛选，找到了一个结构新颖的小分子激动剂MW1219。MW1219可以剂量依赖性的刺激cAMP产生以及下游萤光素酶的表达。在功能细胞株Min6和GluTag上，MW1219可以特异性地通过GPR119途径刺激胰岛素和胰高血糖素样肽-1的分泌。当GPR119的表达水平被siRNA下调后，MW1219的促分泌功能也相应减弱。给予雄性db/db小鼠MW1219（100 mg/kg、每日一次灌胃给药）连续6周治疗后，其空腹血糖和糖化血红蛋白水平较之未治疗组明显降低，外周血胰岛素含量显著升高，糖耐量也有所改善。糖皮质激素受体是核受体超家族的成员之一，为配体激活的转录因子。糖皮质激素主要通过与受体结合发挥代谢调节作用。库欣氏综合症和一些长期接受糖皮质激素治疗的患者体内糖皮质激素的水平异常升高，严重时则出现糖尿病、血脂异常和肥胖等代谢紊乱症状。NC3327是前期经过高通量筛选获得的一个糖皮质激素受体小分子抑制剂。研究表明，NC3327可以与地塞米松竞争性结合糖皮质激素受体，抑制前者诱导的基因表达，但对糖皮质激素受体本身没有激动活性。NC3327也不能诱导糖皮质激素受体从细胞质向细胞核的转移。然而，NC3327可以下调肝细胞HepG2内糖异生的关键性限速酶PEPCK和G6pase的表达，降低TAT酶的活性，而对NFκβ无抑制活性。对NC3327开展的一系列结构优化改造产生出一个活性明显改善的衍生物YZ105。该化合物不仅可以阻断肝糖异生，而且能够拮抗地塞米松诱导的3T3-L1前脂肪细胞分化作用。综上所述，NC3327和YZ105都是 糖皮质激素受体的“竞争性”拮抗剂，对于库欣氏综合症引发的代谢紊乱也许具有潜在的治疗效果。二氢叶酸还原酶是结核分枝杆菌生命活动中不可或缺的蛋白酶，主要参与核苷酸和蛋白质生物合成等重要的生命活动。因此，针对此酶开发抑制剂有望找到治疗结核病的新药。通过将二氢叶酸还原酶反应与一个产生荧光产物的反应相偶联，建立了高效稳定的筛选模型。经过大规模筛选，发现了三个可抑制二氢叶酸还原酶活性的小分子化合物，其中一个化合物的抑制活性对二氢叶酸还原酶呈现高特异性。
关键词：2型糖尿病，G蛋白偶联受体119，激动剂，正向变构调节剂，高通量筛选，糖皮质激素受体，拮抗剂，结核分枝杆菌，二氢叶酸还原酶
Screening and pharmacological studies of a GPR119 agonist and a glucocorticoid receptor antagonist
Abstract
This thesis contains there parts: (1) application of a cell-based high-through screening (HTS) assay for G protein-coupled receptor 119 (GPR119) at to identify agonists and positive allosteric modulators through large-scale compound library screening and subsequent functional assessments both in vitro and in vivo in diabetic db/db mice; (2) molecular and cellular pharmacological studies on a glucocorticoid receptor antagonist and discovery of an analogue with a better bioactivity profile through structural modification and optimization; (3) improvement of a HTS assay for dihydrofolate reductase (DHFR) derived from Mycobacterium tuberculosis and subsequent identification a specific DHFR inhibitors.Diabetes mellitus is a syndrome induced by genetic and environmental factors, which is divided into type 1 (T1DM) and type 2 diabetes mellitus (T2DM). T2DM is the most common form of diabetes with insulin resistance and pancreatic β cell dysfunction. GPR119 is an orphan receptor discovered by genome sequencing and sequence alignment analysis. It shows therapeutic effects once activated. Since it is coupled to Gs protein, we established a reporter gene (luciferase) assay to screening agonists and positive allosteric modulators to GPR119. Following a HTS campaign involving 320,000 compounds, one hit with novel structure, MW1219, was identified which could specifically stimulate the secretion of both insulin and glucagon-like peptide-1 through GPR119 signaling pathway in MIN6 and GluTag cells, respectively. Male diabetic db/db mice showed lowered fasting glucose and HbA1c levels follow 6-week daily oral administration of MW1219 (100 mg/kg)compared to the vehicle treatment group. Both insulin response and glucose tolerance were improved after the therapy.
Glucocorticoid receptor (GR) is a ligand-activated transcription factor which belongs to the nuclear receptor superfamily. Glucocorticoid regulates metabolism through binding to this receptor. Excessive glucocorticoid seen in Cushing’s syndrome or longtime administration of glucocorticoids can lead to high circulating levels of the steroid that may induce dyslipidemia, obesity and diabetes. NC3327 is a GR antagonist discovered via a HTS campaign. Previous studies have shown that it can compete with dexamethasone in binding to GR. It also inhibits gene expression induced by dexamethasone while display no agonist activity. NC3327 fails to initiate the translocation of GR from cytoplasm to nucleus but is capable of suppress the expression of key rate-limiting enzymes, including PEPCK, G6Pase and TAT, during gluconeogenesis in HepG2 cells, while exhibiting no negative impact on NFκβ. Follow a series of structural modification and optimization, YZ105，a analog to NC3327, demonstrated a better bioactivity profile that the parent compound. It not only inhibits gluconeogenesis but also prevents differentiation of preadipocyte 3T3L-1 induced by dexamethasone. Taken together, this class of compounds possesses therapeutic potential for metabolic disorders.
Dihydrofolate reductase (DHFR) is a critical enzyme for the growth of Mycobacterium tuberculosis. It is involved in multiple important pathways, such as the synthesis of nucleotides and protein. Therefore, its specific inhibitors might be beneficial to the treatment of tuberculosis. We established an effective and stable screening platform by coupling DHFR with a substrate that produces fluorescence upon reaction. As a result of HTS camping, three hits were identified and one of which displayed a true specificity for DHFR.
Key words: Type 2 diabetes mellitus, G protein-coupled receptor 119, agonist, positive allosteric modulator, high-throughput screening, glucocorticoid receptor, antagonist, Mycobacterium tuberculosis, dihydrofolate reductase
目录
缩略词 III
中文摘要 V
英文摘要 VII
第一章 GPR119小分子激动剂的发现与药理学研究 1
第一节 前言 1
第二节 基于萤光素酶报告基因的GPR119高通量筛选模的优化与应用 10
一、材料与方法 10
二、实验结果 14
第三节 MW1219的体内外药效学初步研究 34
一、材料与方法 34
二、实验结果 39
讨论与总结 53
参考文献 57
第二章 糖皮质激素受体非甾体拮抗剂的体外药理学研究 65
第一节 前言 65
第二节 糖皮质激素受体拮抗剂NC3327及其衍生物的药理学研究 68
一、 材料与方法 68
二、实验结果 75
讨论与总结 91
参考文献 93
第三章 二氢叶酸还原酶高通量筛选模型的建立及应用 97
第一节 前言 97
第二节 基于二氢叶酸还原酶的高通量筛选模型的建立及应用 99
一、材料与方法 99
二．实验结果 103
讨论与总结 109
参考文献 111
在学期间发表的论文 114
致 谢 115
缩略词
AC Adenylate cyclase 腺苷酸环化酶
cAMP Cyclic adenosine monophosphate 环磷酸腺苷
CDT-FBS Charcoal/dextran treated FBS 活性炭/葡聚糖处理的胎牛血清
CV Coefficient of variation 变异系数
DBD DNA binding domain DNA结合区域
Dex Dexamethasone 地塞米松
DHFR Dihydrofolate reductase 二氢叶酸还原酶
DMEM Dulbecco’s modified Eagle’s medium DMEM培养基
DPP-IV Dipeptidyl peptidase IV 二肽基肽酶IV
EC50 Half maximal effective concentration 半数有效浓度
ELISA Enzyme-linked immunosorbent assay 酶联免疫吸附检测
HTRF Homogeneous time-resolved fluorescence 均相时间分辨荧光
GIP Glucose-dependent insulinotropic polypeptide 葡萄糖依赖性促胰岛素分泌肽
GLP-1 Glucagon-like peptide-1 胰高血糖素样肽-1
GPCR G protein-coupled receptor G蛋白偶联受体
GPR119 G protein-coupled receptor 119 G蛋白偶联受体119
GR Glucocoiticoid receptor 糖皮质激素受体
GRE Glucocoiticoid response element 糖皮质激素应答元件
HA Hydroxyapatite 羟基磷灰石
HbA1c Hemoglobin A1c 糖化血红蛋白
HEK293 cell Human embryonic kidney 293 cell 人胚胎肾293细胞
HTS High-throughput screening 高通量筛选
IC50 Half maximal inhibitory concentration 半数抑制浓度
IPGTT Intraperitoneal glucose tolerance test 腹腔注射糖耐量检测
IR Insulin resistance 胰岛素抵抗
LBD Ligand binding domain 配体结合域
MMP-9 Matrix metalloproteinase-9 基质金属酶蛋白酶-9
Mtb Mycobacterium tuberculosis 结核分枝杆菌
MTX Methotrexate 甲氨蝶呤
NFκB Nuclear factor κB 核因子κB
PAM Positive allosteric modulator 正向变构调节剂
PKA Protein kinase A 蛋白激酶A
PLC Phospholipase C 磷脂酶C
PMA Phorbol myristate acetate 佛波醇乙酯
S/B Signal/background ratio 信号本底比
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