CN114681449B - 一种氨基甲酸酯类化合物作为谷氨酸脱氢酶抑制剂的应用 - Google Patents
一种氨基甲酸酯类化合物作为谷氨酸脱氢酶抑制剂的应用 Download PDFInfo
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Abstract
本发明提供了一种氨基甲酸酯类化合物（R）‑（1‑（环己基氨基）‑3‑（1H‑吲哚‑3‑基）‑1‑氧丙烷‑2‑基）氨基甲酸酯作为谷氨酸脱氢酶（Glutamate dehydrogenase,GDH）抑制剂的应用；该化合物在微摩尔浓度条件下对谷氨酸脱氢酶（Glutamate dehydrogenase,GDH）活性的有效抑制作用。谷氨酸脱氢酶是一种线粒体酶，是谷氨酸进入三羧酸循环产生ATP的关键酶。GDH获得功能突变是导致新生儿患有先天性高胰岛素血症（Congenital hyperinsulinism，CHI）的原因之一。随着研究的发展，越来越多的证据表明，GDH与肿瘤细胞的增殖、迁移、入侵等活动密切相关，在许多肿瘤细胞中都存在表达异常的现象。本发明提供的GDH抑制剂能够很好的抑制GDH的活性，进而达到治疗因GDH突变引起的CHI以及寻找有效癌症治疗药物的作用。
Description
技术领域
本发明属于生物医药技术领域，本发明涉及一种氨基甲酸酯类化合物(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯作为谷氨酸脱氢酶抑制剂的应用。
背景技术
先天性高胰岛素血症(Congenital hyperinsulinism，CHI)是由于胰岛素分泌过多，血胰岛素浓度增加，从而导致的顽固性持久性的低血糖症且难以治愈。引起低血糖的原因复杂(已经发现了至少11个致病基因)，主要致病基因包括：
1)氨基酸代谢关键酶，谷氨酸脱氢酶(GDH)获得功能突变；
2)葡萄糖代谢关键酶，葡萄糖激酶(GCK)获得功能突变；
3)ATP依赖钾离子通道(SUR1和Kir6.2)失去功能突变；
4)脂肪酸代谢关键酶(SCHAD)失去功能突变等。
谷氨酸脱氢酶(Glutamate dehydrogenase,GDH)获得功能突变是CHI的其中一个亚型，GDH的突变位点如果干扰了对内源性抑制物的敏感性，就会表现出功能的增强，GDH在胰岛β细胞的功能增强直接导致胰岛素的不恰当分泌，进而导致胰岛素释放过量以及低血糖症。病人不仅患有低血糖，还会有肝肾功能损害造成的高氨血症及脑功能损害造成的癫痫和脑发育延迟，学习能力障碍等，是一个全身的系统性疾病，急需要一个针对突变GDH的全身性的抑制剂。
目前关于CHI的治疗手段和药物非常有限，一线药物二氮嗪只针对三分之一的新生儿低血糖病人，而且对脑功能障碍及肝肾损害没有任何治疗办法，并且会引起水钠潴留和高血糖等副作用，极大限制了对这些非遗传病的低血糖患儿的治疗。对二氮嗪无效的病人，需要手术切除胰腺，手术切除胰腺不仅难于在中国开展，同时也直接引发糖尿病，需要终身注射胰岛素，因此病人急需新的治疗药物，GDH抑制剂的开发为治疗CHI提供了一种新的途径。
目前在许多类型的癌症中发现了GDH基因转录的增加，如乳腺癌、胶质瘤、结直肠癌以及卵巢癌等。肿瘤细胞基因突变，导致葡萄糖有氧糖代谢途径的明显改变，在氧气充足条件下仍将绝大部分葡萄糖进行无氧酵解，产生大量ATP维持自身代谢需求。为满足生物大分子合成的需求，肿瘤细胞摄入大量谷氨酰胺，在线粒体内经谷氨酰胺酶催化生成谷氨酸，GDH催化谷氨酸产生的α-酮戊二酸(α-ketoglutarate,α-KG)参与三羧酸循环(tricarboxylic acid cycle,TCA cycle)，为肿瘤细胞提供能量来源，因此，抑制该途径中GDH的活性可达到抑制肿瘤细胞生长的目的。目前GDH在逐渐成为癌症治疗方面具有潜力的作用靶点，有关GDH抑制剂的筛选已经成为研究热点。
发明内容
为了克服现有针对先天性高胰岛素血症的药物数量较少的不足，以及发展GDH在作为癌症治疗靶点中的作用，本发明提供一种GDH抑制剂，该在微摩尔浓度的条件下，在GDH的酶动力学试验中表现出一定的针对GDH活性的抑制效果。
本发明采取以下方式实现：
化合物(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯作为谷氨酸脱氢酶抑制剂的应用，其特征在于将以上化合物组分用于抑制谷氨酸脱氢酶的活性；
该化合物组分化学式为：
进一步地，上述GDH抑制剂用于抑制GDH的活性，所述组分半抑制浓度IC50为26.39μM。
本发明第二个方面，提供一种GDH抑制剂的应用方法，其特征在于，化合物(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯作为谷氨酸脱氢酶抑制剂，使用上述GDH抑制剂抑制GDH的活性；
进一步地，该应用方法将上述抑制剂应用于降低胰岛素释放，升高血糖，缓解胰岛压力。
进一步地，该应用方法将上述抑制剂应用于先天性高胰岛素血症和癌症治疗。
进一步地，该应用方法将上述抑制剂应用于治疗高胰岛素-高血氨综合征治疗。
本发明第三个方面，提供一种用于先天性高胰岛素血症治疗药物，其特征在于，该药物包含化合物(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯，该化合物被用来作为谷氨酸脱氢酶的抑制剂。
本发明第四个方面，提供一种用于高胰岛素-高血氨综合征治疗药物，其特征在于，该药物包含化合物(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯，该化合物被用来作为谷氨酸脱氢酶的抑制剂。
本发明的有益效果为：
本发明提供了一种化合物(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯在谷氨酸脱氢酶获得功能突变治疗中改善低血糖的应用。
微摩尔浓度的(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯能够有效抑制GDH的活性，这为解决现有的针对罕见病CHI的药物稀少的问题提供有效尝试，可显著改善目前的CHI治疗现状，打破GDH获得功能突变病人无药可用的被动局面，提高患者生活质量，同时筛选并研制能有效抑制肿瘤细胞中GDH活性的药物也将具有广阔的应用前景。
附图说明
图1为GDH酶动力学检测实验测定结果图；化合物(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯(以AR-Z-003表示)在微摩尔含量条件下对GDH的活性有抑制作用曲线。
图2为胰岛孵育实验结果；在谷氨酰胺和亮氨酸组中加入不同浓度化合物(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯(以AR-Z-003表示)的胰岛素释放量柱形图。
具体实施方式
为使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施方式，进一步阐述发明。
实施例一
化合物(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯作为谷氨酸脱氢酶抑制剂的应用，将以上化合物组分用于抑制谷氨酸脱氢酶的活性：
该化合物组分化学式为：
上述GDH抑制剂用于抑制GDH的活性，所述组分半抑制浓度IC50为26.39μM。
实施例二
一种GDH抑制剂的应用方法，其特征在于，化合物(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯作为谷氨酸脱氢酶抑制剂，使用上述GDH抑制剂抑制GDH的活性；
上述抑制剂应用于降低胰岛素释放，升高血糖，缓解胰岛压力。
上述抑制剂应用于先天性高胰岛素血症和癌症治疗。
上述抑制剂应用于治疗高胰岛素-高血氨综合征治疗。
实施例三
一种用于先天性高胰岛素血症治疗药物，其特征在于，该药物包含实施例一所述的化合物(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯，该化合物被用来作为GDH抑制剂。
实施例四
一种用于高胰岛素-高血氨综合征治疗药物，其特征在于，该药物包含实施例一所述的化合物(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯，该化合物被用来作为GDH抑制剂。
以下详述本发明实验过程，以进一步说明本发明原理、特征和优点。
1、虚拟高通量药物筛选：
(1)选定GDH的3D蛋白结构。本专利中选择牛野生型GDH的晶体为做筛选的3D结构，PDB的code号为6DHL。
(2)处理蛋白靶点，建立对接网格。
运用Maestro软件中的ProteinPreparationWizard模块对GDH蛋白进行加氢，除水分，能量优化处理。ADP对GDH有刺激作用，但是ECG对GDH有抑制作用；由此可以得出与GDH中ADP(或ECG)位点的口袋结合的化合物可能具备刺激作用或是抑制作用。本发明中以ECG的位点作为高通量筛选的位点，并通过Maestro软件中的ReceptorGridGeneration生产对接网格。
(3)整理小分子数据库，建立对接数据库。将数据库文件导入Maestro软件，并通过LigPrep板块对小分子进行构象搜索，能量优化。最终建立对接Ligands数据库。
(4)运用对接软件进行高通量虚拟筛选。运用Maestro软件中的LigandDocking板块中的HTVS模式高通量筛选。对打分结果进行排序分析，并对排名考前的化合物再次进行SP对接分析，选定本发明化合物。
2.GDH酶动力学检测：
(1)分析缓冲液及预混液的配制：
分析缓冲液配置：称取1212mg Trisma和820mg Sodium Acetate,加入到500mL的蒸馏水中，待全部溶解后再加入20μL的500mM EDTA,定容至1L，最后将pH调至8.0。
预混液配置：将10mg NADH溶于6.4ml NH4Cl，用分析缓冲液进行4倍稀释得到预混液。
(2)组织匀浆缓冲液配制：5mM Na2HPO4，5mM K2HPO4，1mM EDTA，1％Triton X-100。
(3)组织准备：在冰上的1.5mL离心管中的组织匀浆缓冲液(约200μL)中匀浆少量组织，然后在冰上静置20分钟。
(4)分别在384孔板中加入10L预混液，5μL DEM(20μM)，5μL样品(肝脏组织中0.1至0.2μg/μL蛋白)和5μL GTP，预读(5分钟)加入5μLα-KG，混合，再次读取(5分钟)。
3.小鼠的胰岛分离纯化及培养：
小鼠麻醉后，在胰腺导管内注射胶原酶(浓度：2mg/mL)，之后从腹腔内分离出胰腺，通过组织消化后(37℃不停震荡)分离出胰岛，纯化后得到纯度超过95％的胰岛组织，胰岛放置于细胞培养箱内，温度37℃，5％CO2，培养液为RPMI1640，添加10％胎牛血清和10mM葡萄糖。
4.高通量胰岛孵育胰岛素分泌的检验：
(1)孵育液Krebs-Ringer bicarbonate buffer(KRBB)配制：
·Buffer A：将107g NaCl溶于1000mL蒸馏水中。
·Buffer B：将5.96g KCl，32.256g NaHCO3和3.25g MgCl2-6H2O共同溶于1000mL蒸馏水中。
·Buffer C：将5.168g CaCl2-2H2O溶于1000mL蒸馏水中。
·Krebs buffer：分别取Buffer A，B和C各50mL，溶于蒸馏水，并加入2.0g BSA，1.906g HEPES，调节pH至7.4，定容至800mL，得到KRBB。
(2)刺激液配制：根据实验需求用KRBB配制不同的刺激液。
(3)手工挑选大小类似的胰岛，置于V型底的96孔平板，每孔放置1～5个胰岛，加入150μL KRBB。
(4)平板离心机离心(500r，10s)，移走上清，重复一次。
(5)加入150μL KRBB，置于37℃、5％CO2培养箱的摇床上，轻微摇动30分钟，离心(500r，10s)后移走上清。
(6)加入不同刺激液，再次置于37℃、5％CO2培养箱的摇床上，轻微摇动30分钟，离心(500r，10s)收集上清液，保存于-20℃冰箱直到激素测定，通过测定上清液的胰岛素分泌来确认刺激物或药物对胰岛素分泌的影响。
(7)取胰岛刺激实验后的上清液10μL，移入384孔板，之后按照试剂盒说明加入抗体，震荡混匀后置于室温孵育4小时后，用BMG的Clariostar酶标仪HTRF程序读数，根据标准曲线计算激素分泌值。胰岛素的检测是使用Cisbio的胰岛素检测试剂盒。
5.实验结果：
(1)运用计算机高通量筛选的方式从数据库中筛选出化合物(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯)对接分数为-6.639。
(2)GDH酶动力学检测实验测定结果如图1所示，结果显示，(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯能够抑制野生型小鼠肝脏中GDH的活性，IC50值为26.39μM。结果说明化合物(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯(以AR-Z-003标识)在微摩尔含量条件下，对GDH的活性有抑制作用。
(3)胰岛孵育实验结果如图2所示，从图中可以看出，与2mM谷氨酰胺(Glutamine，Q)组相比，添加10mM亮氨酸(Leu)后胰岛素分泌水平增加；同时结果显示，在谷氨酰胺和亮氨酸组中加入化合物(R)-(1-(环己基氨基)-3-(1H-吲哚-3-基)-1-氧丙烷-2-基)氨基甲酸酯在10μM和25μM时均能够有效降低胰岛素的释放，从而起到升高血糖的作用，缓解胰岛压力。
以上显示和描述了本发明的基本原理和主要特征和优点。本领域技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都属于本发明要求保护的范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (3)
1.一种氨基甲酸酯类化合物在制备疾病治疗药物中的应用，其特征在于，将所述氨基甲酸酯类化合物作为抑制谷氨酸脱氢酶的有效成分，用于制备所述药物，所述药物适用疾病为先天性高胰岛素血症和/或高胰岛素-高血氨综合征中的一种或两种，所述氨基甲酸酯类化合物为：
（R）-（1-（环己基氨基）-3-（1H-吲哚-3-基）-1-氧丙烷-2-基）氨基甲酸酯；
该化合物化学式为：
。
2.根据权利要求1所述的一种氨基甲酸酯类化合物在制备疾病治疗药物中的应用，其特征在于，所述氨基甲酸酯类化合物用于抑制GDH的活性，其半抑制浓度IC50为26.39μM。
3.根据权利要求1所述的一种氨基甲酸酯类化合物在制备疾病治疗药物中的应用，其特征在于：所述的氨基甲酸酯类化合物在所制备药物中作用为：降低胰岛素释放，升高血糖，缓解胰岛压力。
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