CN107194205B - 具有杀菌活性的jak2激酶抑制剂及其虚拟筛选方法 - Google Patents

具有杀菌活性的jak2激酶抑制剂及其虚拟筛选方法 Download PDF

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CN107194205B
CN107194205B CN201710399061.3A CN201710399061A CN107194205B CN 107194205 B CN107194205 B CN 107194205B CN 201710399061 A CN201710399061 A CN 201710399061A CN 107194205 B CN107194205 B CN 107194205B
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jak2 kinase
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赵金浩
姚停停
程敬丽
李中珊
赵洋
董晓武
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Zhejiang University ZJU
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Abstract

本发明公开了一种快速、高效的JAK2激酶抑制剂虚拟筛选方法，包括如下步骤：1)、下载农业真菌JAK2激酶的氨基酸序列，然后，从Protein data bank中获取农业真菌JAK2激酶的同源蛋白，并通过同源模建构建农业真菌的JAK2激酶的三维结构，最后，依据Protein data bank晶体结构中的抑制剂确定分子对接活性口袋；2)、根据设定的活性口袋，利用分子对接程序，对小分子化合物库中的分子进行对接打分；3)、根据对接打分结果进行排序，初步筛选出与农业真菌的JAK2激酶结合好的先导化合物。利用上述方法筛选得到的活性成分为具有杀菌活性的JAK2激酶抑制剂，能用于防治农业病原菌。

Description

具有杀菌活性的JAK2激酶抑制剂及其虚拟筛选方法
技术领域
本发明涉及农药杀菌剂领域，更具体地，涉及JAK2激酶抑制剂及其虚拟筛选方法，以及该虚拟筛选获得的几类化合物作为活性成分在农业和园艺杀真菌剂方面的用途。
背景技术
在农业生产中，植物病菌的危害是难以避免的，因而农用杀菌剂的开发和使用一直是保障农业生产的重要措施。目前已商品化的农用杀菌剂主要包括三唑类、吡唑类、苯胺基嘧啶胺类、噁唑啉酮类、甲氧基丙烯酸酯、氨基酸衍生物和酰胺类化合物。然而，现有商品化杀菌剂的不合理使用给植物病菌带来了巨大的选择压力，加之病原菌自身极易发生突变，使得植物病菌的抗性水平迅速发展。因此，针对目前严峻的抗性形势，开发作用机理独特、广谱高效和选择性的新型杀菌剂将是未来杀菌剂创制的主要研究方向。
植物病菌在生长过程会通过信号转导系统对外界刺激进行识别并产生应答。当信号转导任一环节受到阻碍，就可能导致病原菌的死亡。因此，信号转导系统及其关键蛋白是杀菌剂开发的重要靶标。JAK2-STAT通路是一个重要的潜在杀菌剂作用靶标。研究表明该通路参与细胞的增殖、分化、凋亡等生物学过程，在生命活动中具有极为重要的作用。JAK2-STAT信号转导途径如下：首先，细胞因子与相应的受体结合后引起受体分子的二聚化或寡聚化，从而激活JAK2的激酶活性。活化的JAK2催化受体上的酪氨酸残基发生磷酸化修饰，为下游信号分子提供“停泊位点”，同时含有SH2结构域的STAT蛋白被招募到“停泊位点”。最后， JAK2激酶催化STAT蛋白发生磷酸化修饰，活化的STAT蛋白与靶基因结合而调控基因的转录。因此，当JAK2激酶的磷酸化活性受到抑制时，其底物的正常磷酸化就会受阻，细胞信号传导通路就会中断。鉴于JAK2激酶在信号转导中的关键作用，针对JAK2激酶设计抑制剂可作为尝试开发新型、高效、广谱杀菌剂的重要切入点。
随着结构生物学的发展，JAK激酶的结构和功能被不断阐明。与其他激酶相比，JAK2 激酶家族具有独特的结构特征：在激酶C端存在两个相似的结构域，分别是激酶结构域JH1 和假激酶结构域JH2，这两个结构域能够被N段的FERM结构域调控，而对于SH2结构域的功能目前尚在研究中。近年来，JAK2激酶抑制剂的研发主要集中于靶向抗肿瘤药物领域，主要针对JH1激酶结构域。该类抑制剂通常为ATP竞争性抑制剂，以杂环作为骨架结构占据ATP结合口袋的嘌呤区，而取代基占据旁边的疏水区，模拟ATP和激酶的结合模式，与铰链区形成两个氢键，这些研究为基于JAK2激酶结构筛选新型、高效、广谱杀菌剂先导化合物提供了研究基础。
农用杀菌剂先导化合物的常规筛选方法是随机合成筛选、类同合成法，该方法需要合成或收集大量化合物，利用建立的生物筛选模型进行大量筛选，以期从中发现有苗头的化合物进入大田试验阶段。据文献报道，平均随机筛选一两万个化合物，才能发现一两个有希望的先导化合物。因此，随机筛选的效率低、成本高、盲目性大。因此，本领域迫切需要高效、快速的筛选方法，以满足JAK2激酶抑制剂在农用杀菌剂开发方面的需要。
随着计算机技术在药物研发领域的应用，计算机虚拟筛选为药物先导化合物的发现提供了一种快速、高效的筛选技术。该技术针对重要疾病特定靶标生物大分子的三维结构或药效团模型，从现有小分子数据库中，搜寻与靶标结合或符合药效团模型的化合物，进行预筛选，集中研究目标，大大降低实验筛选化合物数量，缩短研发周期和节约开发成本。因此，建立一种快速、高效的JAK2激酶抑制剂的虚拟筛选技术，对于新型杀菌剂先导化合物的发现具有重要意义。
发明内容
本发明要解决的技术问题是提供一种快速、高效的JAK2激酶抑制剂虚拟筛选方法，以及虚拟筛选获得的几类化合物，及这些化合物作为农用杀真菌剂的用途。
为了解决上述技术问题，本发明提供一种快速、高效的JAK2激酶抑制剂虚拟筛选方法，包括如下步骤：
1)、从NCBI网站下载农业真菌JAK2激酶的氨基酸序列，然后，从Protein databank中获取农业真菌JAK2激酶的同源蛋白，并通过同源模建构建农业真菌的JAK2激酶的三维结构，最后，依据Protein data bank晶体结构中的抑制剂确定分子对接活性口袋；
即，从Protein data bank中获取JAK2激酶与抑制剂共晶的三维结构，从NCBI网站下载农业真菌JAK2激酶的氨基酸序列，并通过同源模建获得农业真菌的JAK2激酶的三维结构，依据Protein data bank晶体结构中的抑制剂确定活性口袋；
2)、根据设定的活性口袋，利用分子对接程序，对小分子化合物库中的分子进行对接打分；
3)、根据对接打分结果进行排序，初步筛选出与农业真菌的JAK2激酶结合好(较好) 的先导化合物。
作为本发明的快速、高效的JAK2激酶抑制剂虚拟筛选方法的改进，还包括如下步骤4)：
4)、在步骤3)基础上，基于命中的先导化合物进行相似性检索，快速搜索与先导化合物具有相似结构的分子。
本步骤4)是对步骤3)的延伸，目的是在第3)步所获先导的基础上，根据结构决定活性的原理，通过分子相似性检索，快速获得二次先导化合物，缩短新农药的研发周期。
作为本发明的快速、高效的JAK2激酶抑制剂虚拟筛选方法的进一步改进：
步骤1)中农业真菌为如下之一(即，选用的农业真菌JAK2激酶的氨基酸序列，其来源可以使如下真菌之一)：立枯丝核菌(Rhizoctoniasolani)、稻瘟病菌(Pyriculariaoryzae)、恶苗病 (Gibberella Fujikuroi)、叶条病(Pyrenophora graminea)、网斑病(Pyrenophora teres)、恶苗病 (Gibberella zeae)、条锈病(Puccinia striiformis)、杆锈病(P.graminis)、褐锈病(P.recondita)、褐锈病(P.hordei)、颖枯病(Leptosphaerianoclorum)、葡萄白粉病(Uncinula necator)、炭疽病 (Elsinoe ampelina)、无花果炭疽病(Glomer ella cingulata)、锈病(Phakopsora ampelopsidis)、苹果白粉病(Podosphaeraleucotricha)、黑星病(Venturia inaequalis)、轮斑落叶病(Alternariamali)、锈病(Gymnosporangiunm yamadae)、花腐病(Sclerotinia mali)、梨黑斑(Alternaria kikuchiana)、黑星病(Venturia nashicola)、锈病(Gymnospora ngiumnaraeanum)、桃褐腐病 (Sclerotinia cinerea)、角斑病(Cercospora Kaki；Mycosphaerella nawae)、甜瓜白粉病(Sph aerotheca fuliginea)、炭疽病(ColletotriChum Lagenarium)、蔓枯病 (Mycosphaerella melonis)、番茄早疫病(AlternariaSolani)、叶霉病(Cladosporium fulvam)、白粉病(Erysiphe cichoracoarum)、灰霉病(Botrytis cinerea)、菌核病(Sclerotinia sclerotiornm)。
作为本发明的快速、高效的JAK2激酶抑制剂虚拟筛选方法的进一步改进：
所述步骤2)中，分子对接采用的软件为Discovery Studio 2.5中LigandFit对接模块，采用优选的打分函数进行排序。
作为本发明的快速、高效的JAK2激酶抑制剂虚拟筛选方法的进一步改进：
所述步骤2)中，对接方案验证及打分函数选择方法：从Binding database中随机选择骨架结构多样的JAK2激酶抑制剂作为阳性化合物，再从Maybridge数据库中随机抽取无活性化合物；将上述抽取的化合物进行对接打分，对LigandFit模块的7个打分函数(LigScore1_Dreiding,LigScore2_Dreiding,-PLP1,-PLP2,Jain,-PMF,DOCK_SCORE)进行分布分析和ROC分析，选择最优的打分函数作为步骤2)中的排序依据。
作为本发明的快速、高效的JAK2激酶抑制剂虚拟筛选方法的进一步改进：
步骤4)中，训练集和测试集选取Binding database数据库中活性好(较好)、骨架结构丰富的JAK2激酶抑制剂。
本发明还同时提供了利用上述方法筛选得到的活性成分(具有杀菌活性的JAK2激酶抑制剂)，活性成分的结构式为如下任一所述：
Figure GDA0002575242640000041
即，活性成分为结构I～VII所述的化合物、及其光学异构体或其农业上可接受的盐、酯、溶剂化合物。
上述活性成分的用途是：用于防治农业病原菌。所述农业病原菌包括稻瘟病(Pyricularia oryzae)、胡麻斑病(Cochliobolus miyabeanus)、纹枯病(Rhizoctoniasolani)、恶苗病 (Gibberella Fujikuroi)、叶条病(Pyrenophoragraminea)、网斑病(Pyrenophora teres)、恶苗病 (Gibberella zeae)、条锈病(Pucciniastriiformis)、杆锈病(P.graminis)、褐锈病(P.recondita)、褐锈病(P.hordei)、颖枯病(Leptosphaeria noclorum)、葡萄白粉病(Uncinula necator)、炭疽病 (Elsinoeampelina)、无花果炭疽病(Glomer ella cingulata)、锈病(Phakopsora ampelopsidis)、苹果白粉病(Podosphaeraleucotricha)、黑星病(Venturia inaequalis)、轮斑落叶病(Alternaria mali)、锈病(Gymnosporangiunm yamadae)、花腐病(Sclerotinia mali)、梨黑斑(Alternaria kikuchiana)、黑星病(Venturia nashicola)、锈病(Gymnospora ngiumnaraeanum)、桃褐腐病 (Sclerotinia cinerea)、角斑病(Cercospora Kaki；Mycosphaerella nawae)、甜瓜白粉病(Sph aerotheca fuliginea)、炭疽病(ColletotriChum Lagenarium)、蔓枯病 (Mycosphaerella melonis)、番茄早疫病(AlternariaSolani)、叶霉病(Cladosporium fulvam)、白粉病(Erysiphe cichoracoarum)、灰霉病(Botrytis cinerea)和菌核病(Sclerotinia sclerotiornm)。
本发明的设计构思：首先，利用同源模建方法构建农业真菌JAK2激酶的三维结构，利用分子对接程序对小分子化合物库中的化合物进行虚拟筛选，挑选出其中打分较高的分子，再通过生物活性测定进行验证，筛选出具有杀菌活性的JAK2激酶抑制剂先导化合物。即，本发明提供了一种全新作用机制的杀菌剂开发靶标；本发明还提供了一种杀菌剂先导化合物的快速筛选方法。
本发明所建立的JAK2激酶抑制剂虚拟筛选方法及其筛选化合物具有如下技术优势：
1、通过虚拟筛选，降低实验筛选化合物数量，缩短研发周期和节约成本；
2、通过体外酶活抑制试验发现，确认虚拟筛选命中化合物I～VII对JAK2激酶抑制活性明显，并且具有防治农业真菌的作用；
3、虚拟筛选命中化合物骨架新颖，可用于进一步开发新型JAK2激酶抑制剂型杀菌剂，其推荐使用剂量为50～250g a.i./hm2。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1为1LigandFit打分函数区别JAK2激酶抑制剂能力：
图1的 (a)～图1的 (g)为打分函数LigScore1_Dreiding,LigScore2_Dreiding,-PLP1,-PLP2,Jain, -PMF,DOCK_SCORE的分布情况，
图1的 (h)为上述其中打分函数的ROC曲线。
图2为虚拟筛选命中化合物I～VII的化学结构图。
图3为虚拟筛选命中化合物I～VII的体外JAK2激酶抑制活性图。
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此。
实施例1、一种JAK2激酶抑制剂虚拟筛选方法构建以及对SPCES数据库21万个化合物进行筛选的应用。
虚拟筛选步骤如下：
1)、通过NCBI数据库搜索(https://www.ncbi.nlm.nih.gov/protein/)，检索到由W.Daniel等报道的立枯丝核菌JAK激酶的氨基酸序列(CUA67419.1)；从Protein Data Bank中获取参考蛋白结构(登录号：4QAC、4UEU、3PPZ)，采用Discovery Studio 2.5软件BuildHomologyModels模块进行同源模建，获得立枯丝核菌的JAK2激酶结构，并拷贝4QAC共结晶抑制剂；采用Find site as volume of selected ligand工具，根据拷贝的抑制剂定义立枯丝核菌JAK2激酶的活性口袋；
2)、构建测试集验证分子对接虚拟筛选流程的可靠性，从Binding database中随机选择239 个JAK2激酶抑制剂作为阳性化合物，再从Maybridge数据库中随机抽取10000个无活性化合物，构建包含10239个化合物的测试集；采用Discovery Studio 2.5软件的LigandFit模块，将测试集化合物对接到步骤1)所得的立枯丝核菌JAK2激酶活性口袋中，采用LigandFit模块的7个打分函数(LigScore1_Dreiding,LigScore2_Dreiding,-PLP1,-PLP2,Jain,-PMF, DOCK_SCORE)进行分布分析和ROC分析，结果表明识别JAK2激酶抑制剂能力最强的打分函数是LigScore1_Dreiding(图1)。
3)、在确认筛选流程准确可靠后，采用LigandFit模块对SPCES数据库中21万个化合物进行对接(与立枯丝核菌JAK2激酶对接)打分，依据LigScore1_Dreiding进行排序，打分越高表明小分子与立枯丝核菌JAK2激酶结合越紧密，通过分子对接筛选出打分靠前的3000个分子；在分析结构新颖性和类农药性的基础上，最终挑选并购买了40个小分子化合物。
4)、体外JAK2激酶活性抑制测定：在V形底384孔板上进行测定。最终测定体积是30μl，由15μl酶和底物(荧光肽以及ATP)以及15μl溶于测定缓冲液(100mM HEPES(pH 7.4)、10mM MgCl2，25mMβ-甘油磷酸、0.015％Brij35及4mM DTT)的待测化合物构成。通过将 JAK2与底物以及测试化合物混合来开始反应。将反应混合物在室温孵育60分钟，然后通过向每种样品中加入45μl浓度为35mM的EDTA来终止。通过对荧光底物和磷酸化产物进行电泳分离在Caliper LabChip3000上对反应混合物进行分析。通过与无酶对照反应混合物的100％抑制和仅含媒介物的反应混合物的0％抑制进行比较来计算抑制数据。初筛浓度设定为20 μg/mL，初筛活性超过60％的化合物，根据活性按照3～4倍继续稀释7个浓度，产生剂量反应曲线，以确定抑制50％激酶活性所需的浓度(IC50)。
5)、高活性化合物杀菌活性测定：供试菌种(菌核病、纹枯病、稻瘟病、白粉病、霜霉病、灰霉病)由浙江大学农药与环境毒理研究所提供。实验过程中所用锥形瓶、培养皿、量筒和接种针头等均经过蒸汽灭菌。无菌操作台、打孔器等使用前均先用75％酒精擦拭，再经过30min紫外灯灭菌。首先，将待测化合物配制成浓度为1mg/mL的DMSO溶液母液，用 DMSO稀释成不同的浓度。等体积加入PDA培养基，得到含药200，100，50，25，12.5，6.25， 3.125，1.56mg/mL。待培养基冷却至室温并凝固，取上步菌饼接入培养基中，同时测试上述 6种供试农业真菌，每个处理设置3个平行。以等量DMSO为空白对照。菌饼放于培养皿中央，菌丝朝上，完成后倒置养皿，25℃恒温黑暗下进行培养4d。采用十字交叉发测量菌落直径，抑菌活性计算方法：菌丝生长抑制率＝(1-药剂处理菌落直径/空白菌落直径)×100。使用 SPSSv17.0软件计算EC50值，以商品化SDHI类杀菌剂噻呋酰胺为阳性对照。
6)、按照步骤4)所述测试方法测定虚拟筛选命中化合物对JAK2激酶的抑制活性，结果如图2和图3：虚拟筛选命中化合物I～VII对于JAK2激酶抑制活性较好，并且这些命中化合物骨架结构新颖。其中是化合物III和IV对于JAK2激酶抑制IC50分别达到2.7和9.6μg/ml。
7)、按照步骤5)所述测试方法测定虚拟筛选命中化合物对于水稻纹枯病菌丝生长速率的抑制活性，结果如表1所示，虚拟筛选命中化合物对于代表性农业真菌的菌丝生长具有优异的抑制活性。表1为虚拟筛选命中化合物I～VII的对接打分、以及对代表性农业真菌的菌丝生长抑制活性。
表1
Figure GDA0002575242640000071
a SPECS数据库中化合物的货号；
b 虚拟筛选的对接打分Ligscore1_Dreding；
c 虚拟筛选命中化合物对于供试农业真菌的菌丝抑制EC50值。
最后，还需要注意的是，以上列举的仅是本发明的若干个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。

Claims (2)

1.利用JAK2激酶抑制剂虚拟筛选方法筛选得到的活性成分的用途，其特征是：用于防治农业病原菌；
活性成分的结构式为如下任一所述：
Figure FDA0002575242630000011
2.根据权利要求1所述的活性成分的用途，其特征是：
所述农业病原菌包括稻瘟病Pyricularia oryzae、胡麻斑病Cochliobolusmiyabeanus、纹枯病Rhizoctoniasolani、恶苗病Gibberella Fujikuroi、叶条病Pyrenophora graminea、网斑病Pyrenophora teres、恶苗病Gibberella zeae、条锈病Puccinia striiformis、杆锈病P.graminis、褐锈病P.recondita、褐锈病P.hordei、颖枯病Leptosphaeria noclorum、葡萄白粉病Uncinula necator、炭疽病Elsinoe ampelina、无花果炭疽病Glomer ella cingulata、锈病Phakopsora ampelopsidis、苹果白粉病Podosphaeraleucotricha、黑星病Venturia inaequalis、轮斑落叶病Alternaria mali、锈病Gymnosporangiunm yamadae、花腐病Sclerotinia mali、梨黑斑Alternariakikuchiana、黑星病Venturia nashicola、锈病Gymnospora ngium naraeanum，桃褐腐病Sclerotinia cinerea、角斑病Cercospora Kaki；Mycosphaerella nawae、甜瓜白粉病Sphaerotheca fuliginea、炭疽病Colletotri Chum Lagenarium、蔓枯病Mycosphaerellamelonis、番茄早疫病Alternaria Solani、叶霉病Cladosporium fulvam、白粉病Erysiphecichoracoarum、灰霉病Botrytis cinerea和菌核病Sclerotinia sclerotiornm。
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