CN108358852B - Sglt2蛋白的抑制剂以及应用 - Google Patents

Sglt2蛋白的抑制剂以及应用 Download PDF

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CN108358852B
CN108358852B CN201810237107.6A CN201810237107A CN108358852B CN 108358852 B CN108358852 B CN 108358852B CN 201810237107 A CN201810237107 A CN 201810237107A CN 108358852 B CN108358852 B CN 108358852B
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于磊
徐巧枝
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Abstract

一种SGLT2蛋白的抑制剂以及化合物在制备治疗糖尿病的药物的应用、以及抑制剂的筛选方法。其中，所述抑制剂包括具有式(1)所示结构的化合物：
Figure DDA0001604268370000011
该抑制剂可作为活性的先导物治疗糖尿病。

Description

SGLT2蛋白的抑制剂以及应用
技术领域
本发明涉及与糖尿病治疗相关的领域，进一步涉及一种SGLT2(Na+-glucosecotransporter 2，OMIM：*182381,也称为SLC5A2)蛋白的抑制剂，还涉及一种化合物在治疗糖尿病的应用，还涉及SGLT2蛋白抑制剂的筛选方法。
背景技术
肾脏在调节血糖水平中具有重要作用，肾脏中存在两类葡萄糖转运蛋白，其中一类是钠依赖的葡萄糖转运蛋白(Na+-glucose cotransporter，SGLT)，消耗能量以主动方式逆浓度梯度转运葡萄糖。Wells等从人肾脏的cDNA文库发现了一种被称作SGLT2(OMIM:*182381,亦为SLC5A2)的转运蛋白。
家族性肾性糖尿(Familial renal Glucosuria,FRG)是以空腹血糖及糖耐量试验正常，伴有持续尿糖为主要特点的近端肾小管葡萄糖再吸收功能减低的疾病，且病人大都不伴有肾小管功能及结构的异常，预后良好。现有技术中对家族性肾性糖尿患者肾组织中进行了表达研究，发现野生型及突变型SGLT2蛋白均表达在肾小管上皮细胞中，其中野生型SGLT2可正常的表达，而SGLT2突变型蛋白在细胞膜上出现了异常的分布和表达，从而重新确定了家族性肾性糖尿应定义为共显性遗传，而既往所认为的不完全外显考虑与SGLT2野生型蛋白的代偿有关。
近年研究发现糖尿病病人的SGLT2的表达及功能明显上调，而家族性肾性糖尿患者预后良好，因此，特异阻滞近曲小管SGLT2转运蛋白，进而抑制葡萄糖重吸收来控制血糖水平成为了目前研究的热点。根皮苷(phlorizin)是1835年法国化学家从苹果树皮中分离出的第一个SGLT抑制剂，在随后的许多糖尿病动物研究中发现根皮苷可以降低血糖，恢复胰岛素的敏感性，但是由于口服生物利用度比较低，特异性不高，其代谢产物可能抑制GLUT1等蛋白的功能，在中枢神经系统以及其他系统都有一些副作用，所以没有应用于糖尿病的临床治疗。
发明内容
(一)要解决的技术问题
有鉴于此，本发明的目的在于提供一种SGLT2蛋白的抑制剂以及化合物在制备治疗糖尿病的药物的应用，以及SGLT2蛋白抑制剂的筛选方法，以解决以上所述的至少一项技术问题。
(二)技术方案
根据本发明的一方面，提供一种SGLT2蛋白的抑制剂，包括具有式(1)所示结构的化合物：
Figure BDA0001604268350000021
其中，R1独立为：
Figure BDA0001604268350000022
R2独立为：
Figure BDA0001604268350000031
R3独立为H或甲基。
根据本发明的又一方面，提供一种化合物在制备治疗糖尿病的药物中的应用，其中，所述化合物具有以上式(1)所示结构：
根据本发明的再一方面，提供一种SGLT2蛋白抑制剂的筛选方法，包括：
设置SGLT2蛋白与化合物的对接条件；
按照对接条件在数据库中进行虚拟筛选；
根据类药性原则进行对虚拟筛选结果进一步筛选和打分，获得设定分数以上的化合物作为糖尿病治疗的先导化合物；
其中，设置SGLT2蛋白与化合物的对接条件包括：
设定化合物与SGLT2的作用是通过两部分通过强相互作用维系，一方面是亲水性较强的糖基部分与口袋朝外部分的氨基酸残基形成较稳定氢键；另一方面是小分子芳香环部分与口袋较深处的疏水口袋的疏水相互作用和π-π相互作用；化合物的脂水分配系数的对数值logP为-2～5、分子量为200～500、氢键给体0～5、氢键受体0～10、可旋转键数目为0～10、重原子数量为20～70以及电荷值为-2～2。
(三)有益效果
通过上述方案，可知本发明的有益效果在于：
通过本发明的SGLT2蛋白的抑制剂中的化合物，可作为活性的先导化合物治疗糖尿病。
本发明的SGLT2蛋白的抑制剂中的化合物包括亲水基团与疏水口袋，与SGLT2蛋白活性位点的极性口袋、水上口袋相互作用，抑制SGLT2蛋白的表达。
本发明的化合物可以应用于制备糖尿病药物。
附图说明
图1是本发明实施例SGLT2蛋白的抑制剂的化合物。
图2是本发明实施例抑制剂的化合物与SGLT2蛋白作用原理图。
图3是本发明实施例家族性肾性糖尿患者中发现的突变位点。
图4是本发明实施例化合物与SGLT2蛋白的构效关系图。
图5是本发明具体实例1中的标准曲线。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本发明作进一步的详细说明。
由于现有技术中既往的SGLT2表达及功能研究仅发现了其中的2种机制，本申请发明人发现其余的4种机制均在家族性肾性糖尿发病过程中具有重要作用，并明确了相关位点。发明人进一步发现c.294C>A/p.(F98L),c.736C>T/p.(P246S)为糖尿病药物(SGLT2蛋白抑制剂)筛查的理想靶位点。由于对SGLT2确切的3D结构仍然不是很清楚，药物研发都是寻找根皮苷的衍生物或者类似物，在抑制SGLT2蛋白功能的同时可能对其他的葡萄糖转运蛋白也造成不同程度的影响，产生了不良作用，在药物的研发过程中造成了一定的困难。
根据本发明实施例的一方面，提供一种SGLT2蛋白的抑制剂，包括如下化合物：
Figure BDA0001604268350000051
其中，R1独立为：
Figure BDA0001604268350000052
R2独立为：
Figure BDA0001604268350000053
Figure BDA0001604268350000061
R3独立为氢或甲基。
如图1所示式(1)的化合物，为三个小分子的对接结构，参照图1所示，其包括左右两侧的亲水基团和疏水基团(虚线方框内标注)，以及中间的连接部分(中间虚线方框)，(结合图2所示，SGLT2作为标靶蛋白，其活性位点有明显的极性口袋(例如图2中的残基Ser460)和疏水口袋(例如Trp289)。
图2中，这些化合物与SGLT2的结合方式主要有三部分：1、化合物的亲水基团与Ser460等残基形成氢键相互作用；2、亲水基团和疏水基团的连接部分通过残基Trp289形成氢键作用来稳定化合物的结合；3、化合物的疏水基团进入疏水口袋，形成疏水作用；尤其当疏水基团为芳香环时，与Trp289残基形成较强的π-π键，此外L286V突变可使小分子的苯甲基部分(图4中圆圈内结构)有足够的空间位置与蛋白相互作用。
通过上述抑制剂，能够特异性地抑制SGLT2蛋白功能，有效的降低空腹及餐后血糖，成为糖尿病治疗的新途径。
上述抑制剂可以粉末为原料，做成片状等结构的药物。
在进一步的实施例中，所述R1可以独立为
Figure BDA0001604268350000071
Figure BDA0001604268350000072
Figure BDA0001604268350000073
优选的，R1可以独立为
Figure BDA0001604268350000074
更优选的R1可以独立为
Figure BDA0001604268350000075
在一些实施例中，所述R2独立为
Figure BDA0001604268350000081
Figure BDA0001604268350000082
优选的R2独立为
Figure BDA0001604268350000083
更优选的R2独立为
Figure BDA0001604268350000084
本发明实施例还提供一种化合物在制备治疗糖尿病的药物中的应用，其中，所述化合物具有以上所述式(1)所示结构。
根据本发明实施例的另一方面，提供一种SGLT2蛋白抑制剂的筛选方法，包括：
设置SGLT2蛋白与化合物的对接条件；
按照对接条件在数据库中进行虚拟筛选；
根据类药性原则进行对虚拟筛选结果进一步筛选和打分，获得高分的化合物作为糖尿病治疗的先导化合物。
本发明实施例中，以内蒙古地区家族性肾性糖尿患者中发现的SGLT2基因突变为基础，进一步对SGLT2从基因突变到蛋白表达、功能进行深入的研究，对于揭示SGLT2在家族性肾性糖尿发病机制中的关键作用及其作用机制提供临床和基础研究证据和理论依据(参见图3所示)，通过虚拟筛选，根据类药性原理，我们排除了部分筛选结果，最后获得小分子化合物。通过小分子与受体蛋白的相互作用关系，我们分析挑选小分子可用于后期的生物实验验证。进一步通过细胞及动物水平验证，为糖尿病治疗药物的设计、筛查及新药物研发提供有意义的靶标和重要的线索及依据。
筛选过程包括：1、药物虚拟筛选：利用功能研究的结果确定药物筛选的靶点，进一步筛查治疗糖尿病的药物。基于已知拮抗剂根皮苷及其衍生物或者类似物进行SGLT2蛋白功能的抑制实验的结果，建立定量构效关系模型，采用三维药效基团模型搜寻的方法，在化合物数据库(商业性的小分子化合物库ACD-SC、MDDR和中国天然产物数据库CNPD)中进行虚拟筛选，寻找有活性的先导化合物。并根据类药性原则进行进一步筛选，打分，最终得到几个高分化合物作为糖尿病治疗的先导化合物，为药物研发提供新的线索及依据。2、虚拟筛选方案：通过蛋白与三个药物分子的柔性对接测试，我们初步可以判定：成药小分子与蛋白的作用很可能是通过两部分较强相互作用维系，一方面是亲水性较强的糖基部分与口袋朝外部分的氨基酸残基形成较稳定氢键；另一方面是小分子芳香环部分与口袋较深处的疏水口袋的疏水相互作用和π-π相互作用。按照设置好的对接条件做虚拟筛选，用北京市计算中心的虚拟筛选平台进行筛选，选择的小分子库为成药数据库和可供应数据库C。3、药效团筛选过程，具体包括：小分子的挑选：a/小分子具有≥4个药物分子的共性，建出的药效团能全数筛出5个小分子；b/小分子对SGLT2和SGLT1有特异性区别识别位点。药效团建立与测试，能筛选出5个药物；再设置一个decoy test组，包含5个小分子，测试筛出全5个药物为最优。
药效团筛选上市药物库全部和可供应数据库c的对接打分top30％虚拟筛选工作。
选出药效团打分较高的小分子，根据可视化小分子与氨基酸残基结合的特异性挑选出本发明实施例的小分子。
图4是本发明实施例化合物与SGLT2蛋白的构效关系图。将已有的三个药物分子对接到SGLT2蛋白中，可以发现，三个小分子的对接结果有一定的规律。如图4所示，从蛋白的膜内区向蛋白深处看，三个小分子的糖基部分是重叠的，此外小分子在口袋较深处的疏水区均有多个疏水官能团。比例模型显示了SGLT2与SGLT1在口袋位置有突变的氨基酸残基，，初步分析发现，三个药物特异性结合到SGLT2口袋可能是因为小分子更易与疏水口袋形成氢键相互作用，此外L286V突变可使小分子的苯甲基部分有足够的空间位置与蛋白相互作用(参见图4圆圈部分)。
以下将列举具体实例1，在细胞水平验证本发明实施例的化合物的效果，寻找糖尿病治疗的先导化合物，方法如下：
细胞水平验证：
一、实验仪器及试剂
Figure BDA0001604268350000101
二、实验方法
①、细胞准备
1.用0.25％胰蛋白酶消化细胞，制备细胞悬液，调整细胞密度为2×105个/ml。
2.在24孔板中接种细胞悬液(500μL/孔)，将培养板放在培养箱中培养(37℃,5％CO2)过夜。
3.选择性加转染质粒(SGLT-2或GFP)，通过转染试剂P-transter形成转染质粒细胞。
4.将培养板放在培养箱中培养(培养环境37℃，5％CO2)48h。
②、药物处理
1.48h后，换成无血清培养基于培养箱中培养(培养环境：37℃，5％CO2)，过夜。
2.更换为无血清无糖培养基，可以选择性加入对应的抑制剂，在培养箱中培养(培养环境：37℃，5％CO2)40min。这里的抑制剂具有式(1)所示结构：
Figure BDA0001604268350000111
R1为
Figure BDA0001604268350000112
R2为
Figure BDA0001604268350000113
R3为氢，该抑制剂标记为T。
③、加2-DG(2-deoxyglucose)
1.每孔加入20μl 2-DG。于培养箱中孵育(条件为：37℃,5％CO2)20min。
2.去掉培养基，用预冷的PBS洗3遍。
④、加2-DG
1.准备裂解缓冲液：加1％Triton X-100于NADP Extraction buffer(提取缓冲液)中。每孔准备220μl。
2.标准曲线：
a.准备5μM 2-DG6P(2-deoxyglucose 6-phosphate)：加入5μl 5 mM Standard(标准品)于4995μl蒸馏水中，混匀。
b.各标准样本稀释方法入下表1
表1
序号 Premix+H<sub>2</sub>O 2-DG6P(μM)
1 100μl+0μl 5.0
2 60μl+40μl 3.0
3 30μl+70μl 1.5
4 0μl+100μl 0
3.于24孔板的孔中加入200μl裂解缓冲液，摇床震荡5min。
4.分别吸取50μl细胞裂解液样本和50μl标准样本于1.5ml EP管，于80℃，10min。
5.加入50μl NADPH extraction buffer(提取缓冲液)于细胞样本管中，加入50μlH2O于标准样本管中，-20℃，5min。
6.准备WR1：加10μl Assay buffer(反应缓冲液)，1μl G6PDH(glucose 6-phosphate dehydrogenase)Enzyme，1μl NADP中。每管准备12μl。每管加入10μl WR1，于37℃，60min。
7.每管加入50μl NADPH extraction buffer，于80℃，15min。
8.每管加入50μl NADP extraction buffer，-20℃，5min。
9.细胞样本和标准样本中各吸取50μl，于一个黑色96孔板的孔中。
10.准备WR2：加45μl Assay buffer，1μl Enzyme A，1μl Enzyme B，10μl G6P(glucose 6-phosphate)Reagent，5μl Probe。
11.每孔加入50μl WR2，于530/585处分别测量0min和20min吸光值。
三、实验分组
实验分组情况如下表2，每个组做三个复孔。
表2
组别 详情 药物浓度
C1 转染质粒(GFP)细胞
C2 转染质粒(SGLT-2)细胞
C3 正常细胞
C4 转染质粒(SGLT-2)细胞+溶剂
T 转染质粒(SGLT-2)细胞+抑制剂T 1mM
四、实验结果及分析
1、标准曲线
表3
0(μM) 1.5(μM) 3(μM) 5(μM)
0min 13286 18528 20563 24000
20min 14764 24054 31384 42060
20-0min 1478 5526 10821 18060
上表为吸光值的结果，可拟合为图5所示的线性标准曲线。
2.葡萄糖摄取数据分析
表4 Tukey多重比较检验
Figure BDA0001604268350000131
Figure BDA0001604268350000141
对组别C1-C4以及T的的葡萄糖摄取量进行表4所示的Tukey多重比较校验，可以看出T组别的葡萄糖摄取量高于C1-C4组别的，具有较好的具有较好的抑制效果，可以做为糖尿病治疗的先导药物。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (1)

1.式(I)结构的以SGLT2蛋白为靶点的先导化合物在制备糖尿病药物中的应用，
Figure FDF0000012525880000011
其中，所述R1为
Figure FDF0000012525880000012
所述R2为
Figure FDF0000012525880000013
所述R3为氢。
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