CN115340535A - 具有dj-1蛋白稳定剂作用的化合物及其应用 - Google Patents
具有dj-1蛋白稳定剂作用的化合物及其应用 Download PDFInfo
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Abstract
本发明公开了具有DJ‑1蛋白稳定剂作用的化合物及其应用。本发明通过计算机辅助药物设计的方法，基于DJ‑1蛋白晶体结构，对化合物数据库进行虚拟筛选，再通过体外活性评价，发现一组与DJ‑1蛋白有较好亲和力的化合物。本发明是以与多种氧化应激诱导疾病的发生发展相关的DJ‑1蛋白为研究对象，筛选得到的化合物具有调控DJ‑1蛋白的氧化水平的活性，作为DJ‑1蛋白稳定剂，具有良好的药物开发前景。
Description
技术领域
本发明属于医药技术领域，具体涉及具有DJ-1蛋白稳定剂作用的化合物及其应用。
背景技术
DJ-1蛋白于1997年首次在小鼠NH3T3细胞中鉴定为肿瘤相关蛋白，后期研究表明该蛋白可作为内源性抗氧化剂，对抗氧化应激(OS)和调控体内正常的活性氧(ROS)水平。
DJ-1蛋白包括189个高度保存的氨基酸序列，具有螺旋-折叠-螺旋的三明治结构，并形成二聚体晶体结构。DJ-1结构中包含11个β折叠(β1-β11)和8个α螺旋(αA-αH)。αA螺旋位于二聚体结构的中心，αH螺旋位于DJ-1的羧基端，可限制DJ-1表面的催化位点并调节酶活性，OS时可因催化活性改变而导致构象改变，第106位半胱氨酸、126位组氨酸和18位谷氨酸残基对于催化活性的调节均具有重要作用。当DJ-1蛋白表达发生改变时，DJ-1的内在折叠方式将会发生改变，成为非折叠结构而无法形成二聚体晶体结构。DJ-1主要存在于细胞质，也可存在于细胞核，与线粒体功能密切相关，参与线粒体形态、功能及稳态的调节及自噬，这可能反应了一种机体自我保护机制，但具体机制尚不完全清楚。在OS状态下，广泛存在于细胞质的DJ-1迁移至细胞核和线粒体，通过氨基端的12个氨基酸残基定位于线粒体，与线粒体复合物I的亚基结合并调剂其活性。DJ-1的过表达可以增加线粒体的聚集而不会破坏线粒体的形态，在DJ-1过表达细胞中，线粒体复合体I以及5’-三磷酸腺苷的活性增加，进而影响线粒体功能。DJ-1蛋白包含三个半胱氨酸，Cys46，Cys53和Cys106，半胱氨酸的巯基(-SH)可以被ROS氧化成次磺酸基(-SOH)，亚磺酸基(-SO2H)，磺酸基(-SO3H)，国内外多项研究表明，次磺酸基及亚磺酸基是DJ-1中的活性基团，具有抗氧化作用，而磺酸基则会使DJ-1彻底失活，因此，这三个半胱氨酸残基对OS的调节具有重要作用。有研究认为体内DJ-1的氧化态水平影响其抗氧化活性，DJ-1的氧化态处于较低水平，才能维持其二聚体结构，保持其抗氧化活性。
随着DJ-1抗OS、线粒体功能保护重要作用的不断深入研究与发现，其在OS和线粒体功能障碍相关的多种疾病(弱精症、帕金森病、脑梗死、缺血再灌注损伤等)中均可能成为新的潜在治疗靶点。因此，开发能够调控DJ-1的氧化态水平，稳定DJ-1的氧化态水平，促进其二聚体结构稳定的化合物可能具有重要的意义和广泛的临床应用价值。
随着分子生物学、X射线晶体学的发展，大量与疾病相关的生物大分子的三维结构被确定；计算科学的迅速崛起使得数据挖掘、机器学习等技术快速发展。在这两方面的推动下，计算机辅助药物设计应运而生，并渗透到新药研发的各个环节。CADD可以提高药物研发的成功率，降低研发成本，缩短研发周期，是目前创新药物研究的核心技术之一。如今虚拟筛选方法逐渐发展壮大，已经成为一类通过计算机辅助药物设计的核心技术，虚拟筛选是基于靶标生物大分子的三维结构或基于小分子活性配体构建的药效团模型，对已知化合物库进行筛选，选取打分较高的化合物作为备选先导化合物，进行生物活性筛选等深入开发。虚拟筛选极大地减少了化合物合成及药物筛选的工作量、时间与资金投入，提高了筛选的效率和准确性。因此，虚拟筛选在新药开发研究中得到越来越广泛的运用。
发明内容
为了克服现有技术的不足，本发明的目的在于提供具有DJ-1蛋白稳定剂作用的化合物及其应用。本发明通过基于DJ-1蛋白晶体结构的计算机辅助虚拟技术、药物化学知识和药理活性筛选发现一组具有维持DJ-1蛋白的氧化态的稳定性的活性化合物。
本发明的目的通过以下技术方案实现。
具有DJ-1蛋白稳定剂作用的化合物，具体化合物如下：：
N-(3-甲基苯基)-2-[3-(噻吩-2-羰基)-1H-吲哚-1-基]乙酰胺(化合物1)
2-(4-氟苯氧基)-N-{3-甲基-1-[(萘-1-基)甲基]-1H-吡唑-5-基}乙酰胺(化合物2)
2-[6-甲氧基-3-(4-甲氧基苯甲酰基)-4-氧代-1，4-二氢喹啉-1-基]-N-(3-甲氧基苯基)乙酰胺(化合物3)
1-(4-氟苯基)-7-(3，4，5-三甲氧基苯基)-1H，4H，5H，6H，7H-咪唑并[4，5-B]吡啶-5-酮(化合物4)。
通过体外药理活性实验发现，明确了这些化合物对DJ-1蛋白的亲和力，并且可以调节DJ-1蛋白氧化态水平，对抗氧化环境下DJ-1蛋白的氧化，有利于维持DJ-1的抗氧化应激活性。
以上所述的具有DJ-1蛋白稳定剂作用的化合物在制备DJ-1蛋白相关的抗氧化应激的药物中的应用。
优选的，所述化合物的浓度为1±0.1μM，进一步优选为1μM。
优选的，所述化合物为
2-[6-甲氧基-3-(4-甲氧基苯甲酰基)-4-氧代-1，4-二氢喹啉-1-基]-N-(3-甲氧基苯基)乙酰胺。
优选的，所述药物为治疗弱精症、帕金森氏病的药物。
一种筛选具有DJ-1蛋白稳定剂作用的化合物的方法，包括以下步骤：
利用Schrodinger 2015和Discovery Studio 3.5软件，基于DJ-1的蛋白晶体结构，确定氨基酸Cys106、His126为活性中心位点，以其范围的
建立对接格点，进行一系列的虚拟筛选与药物设计，获得有利于DJ-1蛋白稳定的活性化合物。
本发明基于对DJ-1在体内的形态与功能之间的关系的理解，运用基于靶标结构的药物设计方法中的虚拟筛选方法和技术，构建以Cys106和His126为核心的结合口袋，对现有化合物库进行一系列的筛选，并结合相关化合物的活性文献报道，最终确定未在文献中报道有抗氧化相关活性研究的化合物。再通过体外活性测试，发现了具有良好的DJ-1蛋白稳定作用的活性化合物，具有良好的药物开发前景。
与现有技术相比，本发明具有如下优点和有益效果：
本发明的化合物1、2、3、4能结合DJ-1蛋白，并能对抗氧化压力下DJ-1的过度氧化，维持DJ-1的氧化态蛋白水平稳定，这对于维持DJ-1的抗氧化应激作用具有重要意义，增强DJ-1蛋白的抗氧化作用，在治疗弱精症、帕金森氏病等多个疾病中具有潜在的应用价值。
附图说明
图1为本发明化合物1、2、3、4与DJ-1结合的表面等离子共振(SPR)的实验结果图，可以获得化合物对DJ-1的解离常数。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1
基于数据库的虚拟筛选获得化合物1、2、3、4。
(1)通过分子对接筛选基于DJ-1蛋白结构的潜在活性化合物。
选择亚磺酸型DJ-1蛋白(PDB：6afc)进行加氢优化，能量最小化，确定Cys106、His126为活性中心位点，以其范围的
建立对接格点，使用Schrodinger 2015版的Glide模块对Chemdiv数据库(上海陶素生化科技有限公司免费提供)中的120万个小分子进行柔性对接，依据对接得分，与蛋白的作用位置及与活性位点附近氨基酸的作用模式，筛选出具有潜在活性的若干个小分子进行后续的验证。
(2)运用Discovery Studio 3.5毒性预测软件TOPKAT，对设计所得化合物的毒理学性质进行预测，剔除具有潜在毒性的化合物。
(3)对化合物的吸收、分布、代谢、排泄(ADME)性质进行预测，剔除具有潜在不良药代动力学性质的化合物。
(4)通过人工筛选，综合各项虚拟评价结果、分子结构多样性、文献检索情况优选得到4个小分子化合物进入体外活性评价，结构式如下：
N-(3-甲基苯基)-2-[3-(噻吩-2-羰基)-1H-吲哚-1-基]乙酰胺(化合物1)
2-(4-氟苯氧基)-N-{3-甲基-1-[(萘-1-基)甲基]-1H-吡唑-5-基}乙酰胺(化合物2)
2-[6-甲氧基-3-(4-甲氧基苯甲酰基)-4-氧代-1，4-二氢喹啉-1-基]-N-(3-甲氧基苯基)乙酰胺(化合物3)
1-(4-氟苯基)-7-(3，4，5-三甲氧基苯基)-1H，4H，5H，6H，7H-咪唑并[4，5-B]吡啶-5-酮(化合物4)。
实施例2
化合物1、2、3、4与DJ-1结合能力的体外活性评价。
运用表面等离子共振(SPR)技术对化合物1、2、3、4进行体外活性测定，实验所用仪器为BIACORE T200，实验所用材料有：CM5传感器芯片；重组DJ-1蛋白；化合物1、2、3、4；PBS-P+缓冲液；醋酸盐(pH 4.0、4.5、5.0、5.5)；50mM NaOH；氨基偶联试剂(EDC/NHC/乙醇胺)；分析纯DMSO；无盖1.5mL EP管。
具体实验步骤如下：
(1)预富集：用不同pH的醋酸盐缓冲液配制蛋白配体溶液，设置进样时间为180s，流速10μL/min，再生溶液为50mM NaOH。选择响应最高的pH为5.0的醋酸盐缓冲液配制配体。
(2)蛋白偶联：用合适pH的10mM醋酸盐溶液配制DJ-1配体溶液为20μg/mL。通过氨基法将重组DJ-1固定在CM5芯片上，其中Fc-2作为测试通道，Fc-1作为参比通道，流速10μL/min，根据配体偶联水平公式计算固定的水平应控制在8000RU左右。RL＝Rmax/Sm×M配体/M分析物(Rmax一般设为100RU；Sm表示分析物和配体的化学计量比，未知时选择Sm为1；RL为配体偶联水平，实际偶联量为1.5RL)。
(3)运行缓冲液和化合物样品准备：化合物运行缓冲液选用含5％DMSO的1×PBS-P+，用4.5％和5.8％校正母液配置5％DMSO浓度校正曲线(4-8个梯度浓度点)。采用倍半稀释的方法将化合物配置成含5％DMSO的5个梯度浓度(12.5μM，25μM，50μM，100μM，200μM)。间隔设置一个重复浓度，增加一个0浓度。另外由于化合物3在高浓度在5％DMSO中溶解度较差，因此设置了更小浓度梯度(0.78μM，1.56μM，3.125μM，6.25μM，12.5μM)。
(4)多循环动力学检测：在LNW kinetics模块进行动力学检测。将梯度浓度的分析物置于样品架上，选择通道Fc 2-1，进样时间为60s，解离时间为120s，流速为30μL/min进行动力学检测。
(5)数据分析：利用Biacore T200 Evaluation Software进行数据分析。首先在solvent corecction模块进行溶剂校正分析。溶剂校正曲线一般要求落在-500到+1000RU，两条竖线落在矫正曲线范围内，拟合的Chi2小于2。之后在Kinetics/Affinity中进行亲和力拟合分析。选择Affinity中的Steady State Affinity进行拟合分析得到亲和力KD数据。所有数据重复三次。
测得化合物1与DJ-1之间的离解常数KD＝110μM，化合物2与DJ-1之间的离解常数KD＝29.9μM，化合物3与DJ-1之间的离解常数KD＝5.36μM，化合物4与DJ-1之间的离解常数KD＝223μM(见图1)。表明这4个化合物均与DJ-1有一定的亲和力，且以化合物3与DJ-1的亲和力最大。
上述结果提示可对这4个化合物开展更多的体外活性测试进行进一步的活性评估与优选。
实施例3
化合物1、2、3、4对DJ-1氧化态水平调控作用的体外活性评价。
采用等电聚焦电泳法(IEF)对化合物进行体外活性评价。参照IEF的一般操作流程进行。其中，选用SH-SY5Y细胞，分别与1μM的化合物1、2、3、4共孵化24h。然后，分别用0、100、200μM浓度的H2O2处理细胞10min，分离蛋白。参考文献(EMBO Rep，2004，5，213-218)，在pH5-8的范围内开展IEF，分别测定DJ-1氧化态与还原态水平。计算各实验组的氧化态DJ-1水平/总DJ-1水平，其结果见表1。
表1.化合物对SH-SY5Y细胞中DJ-1氧化态水平的调控作用
由表1可知，与空白对照组相比，4个化合物均能在一定程度上抑制由于H2O2导致的DJ-1过氧化，且化合物3的作用最明显。
总之，通过实施例2和3，可以看出化合物1-4与DJ-1的亲和力及维持DJ-1氧化态水平的活性，这有利于保持DJ-1的抗氧化应激活性，从而间接发挥抗氧化作用，对于氧化应激导致的多种疾病的治疗都具有潜在的临床应用价值。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其它的任何未背离本发明的精神实质与原理下所作的各种改动或修改，这些等价形式都包含在本发明的保护范围之内。
Claims (7)
1.具有DJ-1蛋白稳定剂作用的化合物，其特征在于，具体化合物如下：
N-(3-甲基苯基)-2-[3-(噻吩-2-羰基)-1H-吲哚-1-基]乙酰胺
2-(4-氟苯氧基)-N-{3-甲基-1-[(萘-1-基)甲基]-1H-吡唑-5-基}乙酰胺
2-[6-甲氧基-3-(4-甲氧基苯甲酰基)-4-氧代-1，4-二氢喹啉-1-基]-N-(3-甲氧基苯基)乙酰胺
1-(4-氟苯基)-7-(3，4，5-三甲氧基苯基)-1H，4H，5H，6H，7H-咪唑并[4，5-B]吡啶-5-酮。
2.权利要求1所述的具有DJ-1蛋白稳定剂作用的化合物在制备DJ-1蛋白相关的抗氧化应激的药物中的应用。
3.根据权利要求2所述的应用，其特征在于，所述化合物的浓度为1±0.1μM。
4.根据权利要求3所述的应用，其特征在于，所述化合物的浓度为1μM。
5.根据权利要求2所述的应用，其特征在于，所述化合物为
2-[6-甲氧基-3-(4-甲氧基苯甲酰基)-4-氧代-1，4-二氢喹啉-1-基]-N-(3-甲氧基苯基)乙酰胺。
6.根据权利要求2-5任一项所述的应用，其特征在于，，所述药物为治疗弱精症、帕金森氏病的药物。
7.一种筛选具有DJ-1蛋白稳定剂作用的化合物的方法，其特征在于，包括以下步骤：
利用Schrodinger 2015和Discovery Studio 3.5软件，基于DJ-1的蛋白晶体结构，确定氨基酸Cys106、His126为活性中心位点，以其范围的
建立对接格点，进行一系列的虚拟筛选与药物设计，获得有利于DJ-1蛋白稳定的活性化合物。
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