CN111423359A - 一种草菇ubev2抑制剂及其筛选方法和用途 - Google Patents

一种草菇ubev2抑制剂及其筛选方法和用途 Download PDF

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CN111423359A
CN111423359A CN202010208904.9A CN202010208904A CN111423359A CN 111423359 A CN111423359 A CN 111423359A CN 202010208904 A CN202010208904 A CN 202010208904A CN 111423359 A CN111423359 A CN 111423359A
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ubev2
inhibitor
screening
compound
straw mushroom
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龚明
汪滢
万佳宁
鲍大鹏
尚俊军
邹根
郭婷
杨瑞恒
茅文俊
王莹
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Shanghai Guosen Biotechnology Co ltd
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Shanghai Guosen Biotechnology Co ltd
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Abstract

本发明提供了一种草菇UBEV2抑制剂。本发明利用已报道的UBE2T的别构位点区域，通过同源模建构建了UBEV2蛋白的小分子抑制剂别构位点，结合该药效团模型和类药性五规则，通过分子对接方法筛选出潜在的UBEV2抑制剂小分子化合物，结合计算验证，为草菇抗冻保鲜剂的研发提供先导化合物。本发明基于UBEV2介导的泛素化导致草菇“低温自溶”，利用UBEV2的抑制剂抑制泛素化过程从而间接削弱草菇低温自溶的原理，进而研发草菇抗冻保鲜剂。

Description

一种草菇UBEV2抑制剂及其筛选方法和用途
技术领域
本发明属于食品领域，涉及一种食品保鲜剂，具体来说一种草菇UBEV2抑制剂及其筛选方法和用途。
背景技术
草菇(Volvariella volvacea)被称为“中国菇”，是一种重要的食用菌。草菇不仅味道鲜美，而且具有重要的医疗保健作用，如抗氧化、调节免疫、抗肿瘤等。草菇肉质鲜美且生长周期短(大约10天左右)，本应成为经济效益很高的食用菌。然而，草菇菌丝的生长和结实要求比较高的温度(28–35℃)，尤其是其菌丝和子实体不耐低温，即使在常规低温4℃下，菌丝和子实体在短时间内便会软化、液化甚至腐烂，即通常所说的“低温自溶”。草菇“低温自溶”的独特性提出了保鲜难题。所以，一直以来草菇的货架期明显短于其它菇类，使其栽培和普及受到极大地限制，也间接影响了降解秸秆经济效益的发挥。
泛素结合酶E2(ubiquitin-conjugating enzyme，UBE2)家族以泛素结合酶方式起作用，直接控制下游底物的修饰类型，在泛素化修饰过程中起核心级联作用。泛素化修饰的蛋白参与信号传导，内吞作用，膜蛋白转运，DNA修复，蛋白降解等多个细胞活动。
近来研究发现一个特异的草菇泛素化结合酶E2(a specific ubiquitin-conjugating E2 enzyme，UBEV2)上调表达促进低温自溶，通过加入UBE2家族成员泛素结合酶E2T(UBE2T)的小分子抑制剂能够一定程度抑制低温自溶。因此，通过高通量虚拟筛选UBEV2特异性的小分子抑制剂就有可能开发针对草菇的抗冻保鲜剂。
发明内容
本发明的目的在于提供一种草菇UBEV2抑制剂及其筛选方法和用途，所述的这种草菇UBEV2抑制剂及其筛选方法和用途要解决现有技术中草菇的保鲜效果不佳的技术问题。
本发明提供了一种草菇UBEV2抑制剂，其结构式如下所示：
Figure BDA0002422129530000021
Figure BDA0002422129530000031
Figure BDA0002422129530000041
Figure BDA0002422129530000051
Figure BDA0002422129530000061
Figure BDA0002422129530000071
Figure BDA0002422129530000081
Figure BDA0002422129530000091
Figure BDA0002422129530000101
Figure BDA0002422129530000111
Figure BDA0002422129530000121
Figure BDA0002422129530000131
Figure BDA0002422129530000141
Figure BDA0002422129530000151
Figure BDA0002422129530000161
Figure BDA0002422129530000171
Figure BDA0002422129530000181
Figure BDA0002422129530000191
Figure BDA0002422129530000201
Figure BDA0002422129530000202
或者
Figure BDA0002422129530000203
本发明还提供了上述的草菇UBEV2抑制剂小分子的筛选方法，包括如下步骤：
1)同源建模：依据UBEV2蛋白序列，在RCSB PDB库中进行BLAST搜索，得到同源度最高的蛋白是来自人源的UBE2T，UBE2T在UniProtKB数据库登陆号为Q9NPD8，所述的UBE2T蛋白包含一个别构位点抑制剂EM04，将抑制剂EM04周围的氨基酸定义为别构位点；
2)虚拟筛选：基于同源模建得到的UBEV2的三维空间结构，从ChemDiv小分子数据库中筛选UBEV2小分子抑制剂，选取步骤1)的别构位点作为小分子抑制剂虚拟筛选的结合位点，利用sybyl-X2.1药物设计平台的Surflex分子对接模块进行第一次筛选，第一次筛选的规则为：化合物的分子量小于700道尔顿；化合物的脂水分配系数的对数值在-4–6范围内；化合物中氢键受体数量不多于15；化合物中氢键给体数量小于等于6；化合物中可旋转键数量小于11；
然后再采用Sybyl-X2.1软件中的Surflex模块程序实现分子对接，在第一次筛选的化合物中筛选出分子打分数值在排名前1％的小分子化合物，参数为：“每个碎片的最大构象”为10，“每个分子可旋转键的最大数目”为50，“每个配体的最大姿势数”为3，取消默认的“Per-Dock最小化”和“Post-Dock最小化”的选项，其它参数采用默认值，在首轮虚拟筛选到的排名前1％的结果上，恢复到默认参数，进行第二轮虚拟筛选；
3)人工筛选：对二轮虚拟筛选得到的靶中化合物进行人工筛选，标准是：与UBEV2形成稳定的氢键和疏水相互作用，最终从Chemdiv小分子数据库中选取了化合物作为潜在的UBEV2抑制剂。
本发明提供了上述的抑制剂在削弱草菇低温自溶中的用途。
本发明利用文献报道的泛素缀合E2酶家族成员UBE2T的别构位点的EM04抑制剂结合区域，通过同源模建构建了UBEV2蛋白的小分子抑制剂别构位点，基于此，进行UBEV2抑制剂小分子的虚拟筛选。所述的UBEV2小分子抑制剂虚拟筛选的方法是片段筛选。所述的UBEV2小分子抑制剂虚拟筛选为二轮虚拟筛选。然后手动筛选考察二轮虚拟筛选获得的化合物的结合亲和力，筛选标准为是否形成多条氢键，化合物的分子结构的亲疏水性与活性位点的匹配程度，化合物的结构是否有足够的刚性，即可旋转键的数量不能太多。
本发明利用已报道的UBE2T的别构位点区域，通过同源模建构建了UBEV2蛋白的小分子抑制剂别构位点，结合该药效团模型和类药性五规则，通过分子对接方法筛选出潜在的UBEV2抑制剂小分子化合物，结合计算验证，为草菇抗冻保鲜剂的研发提供先导化合物。
本发明基于UBEV2介导的泛素化导致草菇“低温自溶”，利用UBEV2的抑制剂抑制泛素化过程从而间接削弱草菇低温自溶的原理，进而研发草菇抗冻保鲜剂。
本发明和已有技术相比，其技术效果是积极和明显的。采用草菇UBEV2抑制剂小分子的虚拟筛选方法，本发明具有以下优点：
(1)较好的解决了UBEV2序列过长，而绝大部分泛素缀合E2酶家族成员序列较短，无法使用现有晶体结构进行UBEV2同源建模的限制；
(2)获得了150个UBEV2的抑制剂小分子化合物。
附图说明
图1显示了UBEV2和模板(PDB号5NGZ)的序列比对。方框代表UBEV2的别构位点序列比对的起止位置。
图2显示了UBEV2蛋白模型评估的拉氏图。
图3显示了UBEV2的Verify-3D图。
图4显示了UBEV2的三维结构及其比对。(A)UBEV2的三维结构(氨基酸范围7-156)；(B)同源模建出的UBEV2(深灰色cartoon)与其模板蛋白UBE2T(PDB号5NGZ，灰色cartoon)的蛋白三维空间叠合图。UBE2T的别构位点抑制剂EM04以箭头所指的棍棒(stick)标出。
图5显示了UBEV2抑制剂的虚拟筛选工作流程。
图6显示了化合物E2i与UBEV2相互作用模式图。图中箭头所指的stick表示靶中的化合物E2i。其它stick表示UBEV2上的关键氨基酸残基，也正是这些氨基酸参与了与小分子化合物E2i的结合。
图7-21为150个抑制剂小分子化合物的结构式。
图22显示了UBEV2和模板(PDB号4CCG)的序列比对。
具体实施方式
实施例1
①同源建模
依据UBEV2蛋白序列(图1)，在RCSB PDB库中进行BLAST搜索，得到同源度最高的蛋白是来自人源的UBE2T(UniProtKB数据库登陆号为Q9NPD8)，同源度是38.27％。使用UBE2T的晶体结构5NGZ，5NGZ的优势在于存在一个别构位点抑制剂EM04，可在后续的虚拟筛选过程中，为选择抑制剂活性位点提供帮助。UBE2T别构位点的氨基酸序列在UBEV2序列中也存在，相同度为23.718％(线圈中的氨基酸序列比对)，高度保守(图1)。因此，我们采用UBE2T晶体结构5NGZ模构出UBEV2蛋白相对应的小分子抑制剂别构位点，则可以基于该别构位点的空间结构信息开展UBEV2蛋白全新小分子抑制剂的虚拟筛选。
②模型评价
Top 5的模型打分值显示模型VVO-00268.03.pdb具有最大的GA341值，最小的molpdf值，最大的DOPE值，表明这个模型是最优的3D结构(表1)。因此，该模型被作为进一步分析的UBEV2模型。
Figure BDA0002422129530000231
表1
UBEV2模型的拉氏图显示在最青睐的区域有85.7％的残基，在允许的区域有12.7％的残基，在允许的区域有1.6％的残基。模型中99.5％的蛋白质残基的二面角都在合理的范围之内，符合立体化学能量规则(图2)。Verify_3D评估结果显示，UBEV2蛋白模型中，有82.67％的氨基酸得分≥0.2，表明模型中氨基酸侧链构象合理(图3)。
同源模建出的UBEV2蛋白的3D结构如下图4A所示。三维空间叠合图显示UBEV2和模板蛋白UBE2T两者的同源度很高，三维空间构象相似度也很高(图4B)，因此该模型被作为进一步对接分析的模型。
实施例2
①虚拟筛选
基于同源模建得到的UBEV2的三维空间结构，从ChemDiv小分子数据库中筛选UBEV2小分子抑制剂，选取步骤1)的别构位点作为小分子抑制剂虚拟筛选的结合位点，利用sybyl-X2.1药物设计平台的Surflex分子对接模块进行第一次虚拟筛选，由于ChemDiv小分子数据库中化合物数量较多(大于160万)，利用“化合物过滤”对该数据库进行类药性五规则预处理，挑选出108万左右的“理想化合物”。类药性五规则具体为：化合物的分子量小于700道尔顿；化合物的脂水分配系数的对数值在-4–6范围内；化合物中氢键受体数量不多于15；化合物中氢键给体数量不多于6；化合物中可旋转键数量小于11。步骤如图5所示。
然后再采用Sybyl-X2.1软件中的Surflex模块程序实现分子对接，在第一次筛选的化合物中筛选出分子打分数值在排名前1％的小分子化合物。具体参数为：“每个碎片的最大构象”为10，“每个分子可旋转键的最大数目”为50，“每个配体的最大姿势数”为3，取消默认的“Per-Dock最小化”和“Post-Dock最小化”的选项，其他参数采用默认值。在首轮虚拟筛选到的得分排名前1％结果的基础上，恢复到默认参数，进行第二轮虚拟筛选，得到500个靶中化合物。
②人工筛选
对二轮虚拟筛选得到的500个靶中化合物进行人工筛选，具体标准：可与UBEV2形成稳定相互作用(氢键和疏水相互作用)，即可与UBEV2的Thr73的主链氨基，Gly77的主链氨基，Ile78的主链羰基或者氨基，Phe84的主链羰基，Gly85的主链羰基，Asp86的主链羰基，Tyr87的主链羰基；Asp79的侧链等形成多条氢键相互作用；可与UBEV2上的多个疏水性氨基酸Ile78，Leu83，Phe84，Asp86(侧链的疏水部分)，His138，Ile141等有较强的疏水相互作用。化合物的结构相对来说要有足够的刚性，即可旋转键的数量不能太多。最终从Chemdiv小分子数据库中选取了150个化合物作为潜在的UBEV2抑制剂。150个化合物与UBEV2对接打分值最高为8.06，最低为6.58。
③化合物列表和结构
表2列出了150个抑制剂小分子化合物的名称，分子对接打分，酯水分配系数(clogP表示化合物的脂溶性和水溶性的。数值越大表示脂溶性越好，越小甚至是负值表示水溶性越好)等性质。CLOGP是sybyl软件平台中的一个模块“clogP”，它是用来计算化合物的logP的。也就是说clogP的数值表示了化合物的酯水分配系数logP。而CLOGP_ERROR是clogP在计算logP时得到的error值(误差值)，这个数值越小，说明预测越准确可信。溶剂可及表面(PSA)数字越大，说明水溶性会好些。
图7-21展示了150个抑制剂小分子化合物的结构。本发明的150个化合物结构均可以在市场上购买得到，或者采用常规的有机合成方法制备，这是本领域的技术人员都熟知的，在此不再赘述。
对比例1
(1)依据UBEV2(VVO_00268)蛋白序列经过同源模建序列搜索，得到同源度最高的蛋白是来自人源的UBE2T(UniProtKB数据库登陆号为Q9NPD8)，同源度是38.27％。
(2)序列比对发现，两个蛋白的同源部分的重叠较少(图22)，就无法用UBE2T的晶体结构(PDB号4CCG)来模建这个UBEV2的全部600多个氨基酸的三维结构(图22)。
对比例2
(1)E2i是一种细胞选择透过性泛素结合酶E2Ubc13-Uev1A抑制剂，研究证实了该化合物溶液能够一定程度减缓草菇冻伤程度。采用实施例1分子对接方法进行E2i和UBEV2的分子对接。
(2)蛋白对接分析显示E2i可与UBEV2的Thr73的主链氨基，Asp86的主链羰基， Gly85的主链羰基形成了3条氢键相互作用(图6)。E2i也同时与结合位点的Val75，His76， Ile78，Phe84等氨基酸形成较强的疏水相互作用(图6)。可见，氢键和疏水相互作用共同维 持了化合物E2i与靶点UBEV2的结合。化合物E2i与UBEV2蛋白分子对接打分值为5.1713，低 于虚拟筛选出的150个抑制剂小分子化合物的打分值(表2-3)。
Name Total_Score MW CLOGP CLOGP_ERROR RBC PSA
S787_0059 8.06 403.5167 3.017 30 8 145.277
CM4495_5415 8.05 352.4699 4.8907 0 5 89.336
C620_0571 8.01 353.3719 3.5681 0 7 136.827
Y041_6425 7.94 436.3443 5.4902 0 4 41.89
J107_0806 7.93 387.4344 2.6156 0 5 127.779
J107_0077 7.81 338.3755 3.9772 0 4 58.763
SA95_0307 7.68 393.5251 2.782 41 7 26.464
C620_0590 7.68 399.4418 5.7799 0 7 97.208
8640_0548 7.67 351.4421 3.0362 0 6 117.692
J107_0654 7.65 364.4839 2.862 0 5 60.063
G621_0412 7.58 423.5279 2.8121 0 8 98.065
SA95_0176 7.57 419.5161 3.843 41 6 43.254
J107_0116 7.54 402.4425 3.8496 0 4 122.12
F129_0176 7.45 347.3657 2.8306 0 8 146.008
6267_0066 7.37 419.4564 2.6975 59 4 146.938
E959_1672 7.3 377.3718 0.7543 0 6 205.76
SA95_0269 7.29 421.5352 1.8487 30 8 28.08
S579_0658 7.28 344.4231 2.7883 40 6 48.449
8019_7541 7.24 573.5997 4.7274 41 8 181.492
8018_6693 7.23 406.4345 4.057 30 7 183.342
8009_1221 7.23 408.4702 1.996 30 8 154.046
D347_4045 7.2 419.5591 4.096 30 8 46.489
5623_0098 7.16 370.3743 2.9074 10 8 160.09
C445_0308 7.14 477.5388 2.0115 0 8 198.225
S635_0065 7.11 341.4473 -0.387 30 5 139.056
SA95_0288 7.1 379.4986 2.563 41 7 38.476
5608_0200 7.1 358.4546 2.8522 30 8 149.414
D658_0291 7.08 435.4724 0.8085 30 8 178.737
S720_2189 7.08 365.4256 2.7881 0 6 134.644
E518_1323 7.08 308.3743 1.525 0 6 115.829
F062_0979 7.08 408.515 2.1491 0 8 94.847
Y200_5911 7.07 442.571 5.512 30 7 170.842
D302_0195 7.07 482.5553 1.1337 30 7 210.74
P937_3310 7.06 349.4295 2.0143 0 6 85.696
J107_0074 7.06 320.385 3.6662 0 4 60.916
E959_1804 7.06 381.7909 1.6163 0 5 189.297
S629_1323 7.06 368.4263 1.4955 30 7 127.991
J107_0606 7.05 355.406 2.508 0 4 90.331
J107_0693 7.04 376.4516 1.489 0 3 82.058
L345_0044 7.04 272.3422 2.0368 41 4 64.245
D271_0729 7.03 498.5266 1.0339 30 8 228.416
E787_0023 7.02 406.5173 4.2913 41 6 65.578
D347_2509 7.01 413.4867 5.558 10 5 35.714
Y050_0732 7 334.3901 1.9606 41 7 181.665
D011_5349 7 322.4439 5.527 0 6 23.994
D399_1307 6.99 368.4693 3.549 41 8 109.777
Y050_2070 6.98 286.3473 1.2506 0 7 181.28
Y080_0049 6.97 296.3636 2.693 0 7 66.247
6665_0362 6.97 310.3902 3.187 0 7 61.381
5225_5979 6.97 433.5029 3.3029 30 7 126.274
SA95_0228 6.96 395.5377 3.151 30 8 52.456
CM3759_3552 6.96 414.498 0.623 0 7 152.834
Y041_6424 6.95 343.4216 4.3568 0 3 49.927
D074_0258 6.94 411.4525 3.8439 30 5 196.702
D510_0955 6.93 469.5747 5.2679 10 7 84.585
8019_2599 6.92 387.4742 3.553 0 8 62.961
5617_0751 6.89 427.4768 5.4925 41 8 197.246
D011_0942 6.89 463.9311 5.691 10 6 31.107
V019_3001 6.89 385.4518 5.375 41 6 65.99
F432_0263 6.89 467.6036 5.793 0 7 106.775
E959_1807 6.88 327.3562 0.0195 0 8 215.813
D271_0728 6.88 498.5266 1.0339 30 8 221.035
E959_1697 6.88 411.8169 1.3428 0 6 213.358
S219_0721 6.87 370.4024 2.0146 41 8 76.531
7162_0307 6.87 384.4919 5.4015 0 8 121.421
S901_0543 6.87 432.4967 1.3823 59 7 121.378
8020_0459 6.86 288.3416 -0.044 0 8 103.24
D347_2685 6.86 460.3657 5.749 10 4 34.973
S579_0597 6.85 427.5381 3.6743 40 7 70.95
D347_2790 6.85 399.4601 5.029 10 4 37.815
M735_1094 6.84 405.5325 4.2854 0 7 50.009
6190_0037 6.84 461.4335 2.4029 59 8 323.275
J107_0095 6.83 304.3856 3.681 41 3 49.456
F129_0175 6.83 351.399 4.1442 41 6 114.362
D011_0941 6.81 429.4861 4.978 10 6 29.037
D347_2517 6.81 409.5228 5.914 10 5 35.804
D271_0719 6.81 418.4668 -0.1481 10 7 237.542
4772_0584 6.81 464.5798 5.0931 59 8 121.249
5225_5965 6.8 449.5023 2.7229 30 8 141.307
表2
Name Total_Score MW CLOGP CLOGP_ERROR RBC PSA
D392_0055 6.79 450.5102 2.067 30 7 257.739
Y080_0674 6.79 395.4962 5.385 10 4 36.467
D494_0331 6.79 338.4499 4.8177 0 8 71.551
S219_0178 6.78 390.4351 2.752 42 6 56.314
J107_0154 6.78 361.4121 3.982 41 3 82.677
D042_0332 6.78 380.4634 4.2861 0 5 104.973
8019_2263 6.77 324.4168 3.836 0 6 65.22
Y200_4164 6.77 417.4174 2.886 30 6 295.897
T160_0441 6.76 405.5541 3.0735 0 8 75.96
E551_0228 6.76 388.4672 0.3082 0 7 244.547
J107_0603 6.76 337.4155 2.197 0 4 96.136
D072_0257 6.76 418.8939 5.4291 0 6 80.626
Y080_1079 6.75 251.7551 2.8185 0 5 71.358
5224_0087 6.75 374.3944 0.1043 0 8 218.056
J107_0795 6.75 367.3769 1.9757 0 4 127.294
Y070_3872 6.74 441.4355 1.511 0 5 181.662
C090_0497 6.74 419.473 5.9984 0 6 154.709
7706_0382 6.73 387.4329 2.4725 10 8 95.757
5891_0063 6.73 342.4336 5.734 41 5 18.907
5267_0356 6.72 296.3636 3.003 0 6 56.749
C875_0512 6.72 375.4866 5.1257 30 6 41.607
D563_0046 6.71 472.534 1.7709 0 7 257.491
C630_0376 6.71 413.4867 5.528 10 4 38.016
Y080_1180 6.7 316.7821 3.162 0 6 67.37
S629_1436 6.7 342.3923 0.5749 30 6 137.421
D011_6109 6.69 323.3889 1.995 0 7 67.717
M735_1091 6.68 405.5325 4.2854 0 7 55.147
Y070_4060 6.68 484.5 2.286 30 8 194.224
SA02_0096 6.68 436.5035 1.067 41 7 143.72
J107_0650 6.68 354.4212 2.628 0 4 101.202
C200_6911 6.68 404.4815 5.9578 0 6 94.466
D399_2308 6.67 372.4332 3.193 41 8 110.205
D072_0809 6.67 440.4905 5.0032 0 8 115.444
6959_0308 6.66 484.4569 2.003 41 6 219.381
S980_0033 6.66 380.4833 0.5299 30 7 98.107
5225_5951 6.66 453.9214 3.5169 30 7 129.08
D271_0724 6.66 480.5362 0.8909 30 8 221.681
T158_1004 6.65 344.4512 1.5985 0 8 45.376
C090_0502 6.65 500.5027 4.3994 0 8 203.738
8585_0121 6.65 292.3749 3.293 0 6 50.372
SA95_0237 6.64 367.5276 4.801 0 6 24.693
Y080_0013 6.64 310.3902 3.692 0 6 52.981
8019_2521 6.64 395.4962 5.385 10 4 38.22
8009_7732 6.64 423.5015 2.551 20 4 163.354
V019_2999 6.64 415.4778 5.4648 41 7 76.107
F677_0594 6.64 487.6165 3.6469 41 7 62.419
D347_2799 6.64 460.3657 5.749 10 4 34.305
D399_2304 6.64 358.4066 2.664 41 7 112.703
5055_3945 6.63 479.9354 5.8012 59 3 133.937
E135_0481 6.63 375.3742 3.3353 59 3 187.099
SA95_0199 6.63 429.4779 4.802 41 6 18.084
D347_2500 6.63 413.4867 5.558 10 5 35.644
C875_0235 6.63 363.4511 5.3997 30 5 37.17
C063_0013 6.63 361.4833 5.5052 0 7 88.584
F432_0271 6.62 483.603 5.5716 0 8 199.752
Y080_0047 6.61 312.363 2.398 0 7 83.034
8018_5667 6.61 365.4538 1.8516 0 6 177.221
V010_4864 6.6 450.3213 5.9098 41 6 66.459
F129_0205 6.6 317.3828 3.8332 0 6 103.931
D009_0259 6.6 494.5594 5.592 10 8 80.443
D186_0393 6.6 379.4953 2.944 0 8 85.417
Y050_0481 6.6 326.4112 2.2236 0 6 187.225
S629_1613 6.59 357.4036 -0.0065 30 6 168.343
SA95_0260 6.59 403.5167 2.628 30 8 23.991
F677_0116 6.59 473.5899 3.1479 41 7 60.828
S564_0464 6.59 350.4176 0.9704 0 5 94.633
E551_0109 6.59 374.4407 -0.1908 0 7 244.83
J107_0813 6.58 387.4344 2.6156 0 5 107.219
Y080_0048 6.58 296.3636 2.808 0 6 65.467
V029_3925 6.58 438.5607 4.85 30 8 30.123
8019_2711 6.58 337.4155 2.977 0 8 100.418
表3
鉴于E2i溶液能够一定程度减缓草菇的冻伤，较低的化合物E2i分子对接打分值提示150个化合物具有较强的抑制效果，为进一步抗冻保鲜剂的研发提供了明确的先导化合物。
对本领域的技术人员来说，可根据以上描述的技术方案以构思，做出其它各种相应的改变以及形变，而所有的这些改变以及形变都应该属于本发明权利要求的保护范围之内。

Claims (3)

1.一种草菇UBEV2抑制剂，其特征在于其结构式如下所示：
Figure FDA0002422129520000011
Figure FDA0002422129520000021
Figure FDA0002422129520000031
Figure FDA0002422129520000041
Figure FDA0002422129520000051
Figure FDA0002422129520000061
Figure FDA0002422129520000071
Figure FDA0002422129520000081
Figure FDA0002422129520000091
Figure FDA0002422129520000101
Figure FDA0002422129520000111
Figure FDA0002422129520000121
Figure FDA0002422129520000131
Figure FDA0002422129520000141
Figure FDA0002422129520000151
Figure FDA0002422129520000161
Figure FDA0002422129520000171
Figure FDA0002422129520000181
Figure FDA0002422129520000191
或者
Figure FDA0002422129520000192
2.权利要求1所述的一种草菇UBEV2抑制剂小分子的筛选方法，其特征在于包括如下步骤：
1)同源建模：依据UBEV2蛋白序列，在RCSB PDB库中进行BLAST搜索，得到同源度最高的蛋白是来自人源的泛素结合酶E2T，人源的泛素结合酶E2T在UniProtKB数据库登陆号为Q9NPD8，所述的泛素结合酶E2T蛋白包含一个别构位点抑制剂EM04，将抑制剂EM04周围的氨基酸定义为别构位点；
2)虚拟筛选：基于同源模建得到的UBEV2的三维空间结构，从ChemDiv小分子数据库中筛选UBEV2小分子抑制剂，选取步骤1)的别构位点作为小分子抑制剂虚拟筛选的结合位点，利用sybyl-X2.1药物设计平台的Surflex分子对接模块进行第一次筛选，第一次筛选的规则为：化合物的分子量小于700道尔顿；化合物的脂水分配系数的对数值在-4–6范围内；化合物中氢键受体数量不多于15；化合物中氢键给体数量小于等于6；化合物中可旋转键数量小于11；
然后再采用Sybyl-X2.1软件中的Surflex模块程序实现分子对接，在第一次筛选的化合物中筛选出分子打分数值在排名前1％的小分子化合物，参数为：“每个碎片的最大构象”为10，“每个分子可旋转键的最大数目”为50，“每个配体的最大姿势数”为3，取消默认的“Per-Dock最小化”和“Post-Dock最小化”的选项，其它参数采用默认值，在首轮虚拟筛选到的排名前1％的结果上，恢复到默认参数，进行第二轮虚拟筛选；
3)人工筛选：对二轮虚拟筛选得到的靶中化合物进行人工筛选，标准是：与UBEV2形成稳定的氢键和疏水相互作用，最终从Chemdiv库中选取了化合物作为潜在的UBEV2抑制剂。
3.权利要求1所述的一种抑制剂在削弱草菇低温自溶中的用途。
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