CN107223663B - 春雷霉素及其衍生物为几丁质酶抑制剂的应用 - Google Patents
春雷霉素及其衍生物为几丁质酶抑制剂的应用 Download PDFInfo
- Publication number
- CN107223663B CN107223663B CN201710271627.4A CN201710271627A CN107223663B CN 107223663 B CN107223663 B CN 107223663B CN 201710271627 A CN201710271627 A CN 201710271627A CN 107223663 B CN107223663 B CN 107223663B
- Authority
- CN
- China
- Prior art keywords
- application
- derivative
- inhibitor
- chitinase
- kasugarnycin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 102000012286 Chitinases Human genes 0.000 title claims abstract description 23
- 108010022172 Chitinases Proteins 0.000 title claims abstract description 23
- 239000003112 inhibitor Substances 0.000 title claims abstract description 17
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 23
- 241000346285 Ostrinia furnacalis Species 0.000 claims abstract description 10
- 238000011161 development Methods 0.000 claims abstract description 7
- 241000238631 Hexapoda Species 0.000 claims description 13
- 102000004157 Hydrolases Human genes 0.000 claims description 8
- 108090000604 Hydrolases Proteins 0.000 claims description 8
- 229920002101 Chitin Polymers 0.000 claims description 7
- 125000003147 glycosyl group Chemical group 0.000 claims description 7
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 6
- 230000000694 effects Effects 0.000 claims description 5
- 241000207199 Citrus Species 0.000 claims description 4
- 235000020971 citrus fruits Nutrition 0.000 claims description 4
- 125000001424 substituent group Chemical group 0.000 claims description 4
- 241001672675 Adoxophyes Species 0.000 claims description 3
- 241000380450 Danaus melanippus Species 0.000 claims description 3
- 241000255967 Helicoverpa zea Species 0.000 claims description 3
- 241001531327 Hyphantria cunea Species 0.000 claims description 3
- 241001622642 Parnara bada Species 0.000 claims description 3
- 241000721451 Pectinophora gossypiella Species 0.000 claims description 3
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 3
- 235000014443 Pyrus communis Nutrition 0.000 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 210000001015 abdomen Anatomy 0.000 claims description 3
- 235000013399 edible fruits Nutrition 0.000 claims description 3
- 239000002532 enzyme inhibitor Substances 0.000 claims description 3
- 239000010977 jade Substances 0.000 claims description 3
- 229940125532 enzyme inhibitor Drugs 0.000 claims description 2
- 235000013311 vegetables Nutrition 0.000 claims description 2
- 241000511870 Brachmia Species 0.000 claims 1
- 241000510032 Ellipsaria lineolata Species 0.000 claims 1
- 241000721703 Lymantria dispar Species 0.000 claims 1
- 239000000843 powder Substances 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 abstract description 16
- FVIZARNDLVOMSU-UHFFFAOYSA-N ginsenoside K Natural products C1CC(C2(CCC3C(C)(C)C(O)CCC3(C)C2CC2O)C)(C)C2C1C(C)(CCC=C(C)C)OC1OC(CO)C(O)C(O)C1O FVIZARNDLVOMSU-UHFFFAOYSA-N 0.000 abstract description 11
- 238000012216 screening Methods 0.000 abstract description 7
- 230000000749 insecticidal effect Effects 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 6
- 239000000126 substance Substances 0.000 abstract description 3
- 241000607479 Yersinia pestis Species 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 8
- PVTHJAPFENJVNC-MHRBZPPQSA-N kasugamycin Chemical compound N[C@H]1C[C@H](NC(=N)C(O)=O)[C@@H](C)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)[C@@H]1O PVTHJAPFENJVNC-MHRBZPPQSA-N 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- 230000018109 developmental process Effects 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 229910000029 sodium carbonate Inorganic materials 0.000 description 4
- 240000008067 Cucumis sativus Species 0.000 description 3
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 230000003111 delayed effect Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 229930000044 secondary metabolite Natural products 0.000 description 3
- 235000016068 Berberis vulgaris Nutrition 0.000 description 2
- 241000335053 Beta vulgaris Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 244000017020 Ipomoea batatas Species 0.000 description 2
- 235000002678 Ipomoea batatas Nutrition 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 241001439019 Phthorimaea operculella Species 0.000 description 2
- 241000255969 Pieris brassicae Species 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 229930182478 glucoside Natural products 0.000 description 2
- 150000008131 glucosides Chemical class 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000009533 lab test Methods 0.000 description 2
- 230000001418 larval effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000000575 pesticide Substances 0.000 description 2
- 239000002574 poison Substances 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 240000007087 Apium graveolens Species 0.000 description 1
- 235000015849 Apium graveolens Dulce Group Nutrition 0.000 description 1
- 235000010591 Appio Nutrition 0.000 description 1
- 241001225321 Aspergillus fumigatus Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 240000008574 Capsicum frutescens Species 0.000 description 1
- 102100037328 Chitotriosidase-1 Human genes 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- 244000000626 Daucus carota Species 0.000 description 1
- 235000002767 Daucus carota Nutrition 0.000 description 1
- 241000272168 Laridae Species 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000244206 Nematoda Species 0.000 description 1
- 241001147397 Ostrinia Species 0.000 description 1
- 241000382353 Pupa Species 0.000 description 1
- 241000607715 Serratia marcescens Species 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 240000006394 Sorghum bicolor Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 241000187214 Streptomyces kasugaensis Species 0.000 description 1
- 241001655322 Streptomycetales Species 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 239000002647 aminoglycoside antibiotic agent Substances 0.000 description 1
- 229940091771 aspergillus fumigatus Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000001390 capsicum minimum Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 108010057052 chitotriosidase Proteins 0.000 description 1
- 210000001136 chorion Anatomy 0.000 description 1
- -1 compound Compound Chemical class 0.000 description 1
- 230000002508 compound effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000002050 diffraction method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 229930000226 fungal secondary metabolite Natural products 0.000 description 1
- 230000000855 fungicidal effect Effects 0.000 description 1
- 239000000417 fungicide Substances 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- FDZZZRQASAIRJF-UHFFFAOYSA-M malachite green Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](C)C)C=C1 FDZZZRQASAIRJF-UHFFFAOYSA-M 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000029052 metamorphosis Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
- A01N43/40—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom six-membered rings
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本发明公开一种春雷霉素及其衍生物作为几丁质酶抑制剂的应用，所述抑制剂的结构通式如I所示，通过对所筛选化合物的抑制效果、选择性和杀虫活性评价对化合物的抑制活性的研究，结果表明，化合物Kasugamycin对OfChtI具有抑制效果，在使用的终浓度为不低于50μM时，此浓度下测得的抑制率为86.4％，半数抑制浓度IC50值为14μM。本发明所述的春雷霉素及其衍生物在生物学和化学生物学等领域有广泛的应用前景，尤其是在延缓亚洲玉米螟发育方面有较好的应用前景。
Description
技术领域
本发明属于生物技术领域，具体涉及一种真菌次级代谢产物及其衍生物作为几丁质酶的抑制剂的应用。
背景技术
几丁质(Chitin)是以N-乙酰-β-D-葡萄糖胺(GlcNAc)为基本单元，通过β-1,4糖苷键连接的天然线性直链多糖。作为重要的结构组成部分，几丁质大量存在于真菌和硅藻的细胞壁、软体动物的外壳、线虫的卵壳以及甲壳类生物和昆虫的外骨骼中，其合成和水解的动态平衡对于这些生物的生长发育具有极其重要的作用。来自糖基水解酶18家族的昆虫几丁质酶 ChtI(chitinase I)是昆虫蜕皮和变态发育中不可或缺的几丁质水解酶。下调或干扰ChtI基因表达水平将导致昆虫蜕皮不正常而死亡。ChtI在生理功能上是特异的表皮几丁质降解酶，功能区别于人类的同源酶，因此，将18家族几丁质酶作为农药设计的靶点，开发高效的几丁质酶抑制剂对于农业害虫的防治具有非常重要的意义。近十年来，昆虫ChtI的研究在生物学和化学生物学等领域取得了突出进展。其中，来源于农业害虫亚洲玉米螟—ChtI(OfChtI)的晶体结构的解析取得重要进展，为针对ChtI的农用小分子理性设计提供理论基础。
春雷霉素(kasugamycin)由春日链霉菌(Streptomyces kasugaensis)所产生，属氨基糖苷类抗生素。春雷霉素在亚洲和南美洲被广泛应用于农业，对水稻稻瘟病(包括叶瘟、稻头瘟、谷瘟)等的防治尤为显著，一般可达到80％以上。不仅如此，春雷霉素的适用作物还包括马铃薯、黄瓜、芹菜、高粱、辣椒、菜豆、柑橘等，可防治甜菜上的甜菜生尾孢、马铃薯上的胡萝卜软鸥文氏菌、菜豆上的栖菜豆假单孢菌、黄瓜上的流泪假单孢菌、番茄叶霉病、黄瓜细菌性角斑病等。另外因春雷霉素对人畜无毒、无残留、无污染，符合现代环保要求，被中国绿色食品发展中心推荐为AA级绿色食品生产资料，被农业部列为无公害农产品生产推荐农药，被上海市列为蔬菜标准化工程首推杀菌剂。以其为基础开发新的药学用途可以大大节约研发周期和成本,“老药新用”被认为是新药开发中最快捷、最有效的策略之一。春雷霉素及其衍生物针对18家族糖基水解酶的生物活性还没有报导，因此，研究春雷霉素作为18 家族糖基水解酶抑制剂对于扩展其应用有非常积极的意义。
发明内容
在防治农业害虫的研究中，为了找到高效的18家族糖基水解酶抑制剂，本发明筛选了大量的化合物，通过抑制效果、选择性和杀虫活性评价对化合物的抑制活性进行了抑制效果评价研究，最终筛选到了春雷霉素及其衍生物。
首先，本发明公开春雷霉素及其衍生物作为几丁质酶抑制剂的应用，其结构通式如I所示，即春雷霉素及其衍生物。
R1基团选自以下取代基：
R2基团选自以下取代基：
本发明还公开结构通式如I所示的抑制剂和/或其衍生物在抑制18家族几丁质酶活性中的应用。具体的，所述抑制剂和/或其衍生物在抑制18家族几丁质酶活性中使用的终浓度为 50μM。
本发明还公开所述的抑制剂在防治农业害虫方面的应用。
具体的所述的农业害虫是鳞翅目昆虫，包括麦蛾、棉红铃虫、马铃薯块茎蛾、甘薯麦蛾、棉褐带卷蛾、大豆食心虫、梨大食心虫、亚洲玉米螟、大造桥虫、菜粉蝶、棉铃虫、舞毒蛾、玉带风蝶、金凤蝶、舟形毛虫、黄腹灯蛾、红腹灯蛾、美国白蛾、葡萄天蛾和稻苞虫等。
本发明提供了评价结构通式如I所示的化合物(Kasugamycin)抑制活性所获得的数据，包括抑制剂筛选、IC50值测定和选择性测定所获得的数据。结果表明，在筛选的所有556个微生物次级代谢产物中，化合物Kasugamycin(终浓度50μM)对几丁质酶OfChtI的抑制率为86.4％，IC50值为14μM。对化合物的适用对象测定表明，在50μM终浓度下，化合物Kasugamycin对几丁质酶OfChtI表现出较高的抑制活性，对于其他来源的几丁质酶(除了粘质沙雷氏菌来源的SmChiB、SmChiC之外)也同样表现出一定的抑制活性。综上，化合物Kasugamycin针对不同物种来源的几丁质酶均能表现出抑制活性，同时根据IC50值可以推断，当化合物Kasugamycin的浓度≥20μM时，对几丁质酶OfChtI有较强抑制活性。
本发明化合物杀虫活性的方法为：用H2O作为溶剂溶解化合物，采用注射方式直接将化合物导入研究对象体内。经过研究化合物Kasugamycin在50μM浓度下对亚洲玉米螟的杀虫活性可以发现，相较于对照组，实验组幼虫的发育被延缓。
由本发明实施例3的体内试验数据证明，春雷霉素及其衍生物在防治农业害虫方面有良好的应用前景。尤其是在延缓亚洲玉米螟发育方面有较好的效果；优选情况下，所述的农业害虫是鳞翅目昆虫。具体的，所述的鳞翅目昆虫为麦蛾、棉红铃虫、马铃薯块茎蛾、甘薯麦蛾、棉褐带卷蛾、大豆食心虫、梨大食心虫、亚洲玉米螟、大造桥虫、菜粉蝶、棉铃虫、舞毒蛾、玉带风蝶、金凤蝶、舟形毛虫、黄腹灯蛾、红腹灯蛾、美国白蛾、葡萄天蛾或稻苞虫。
附图说明
图1为化合物Kasugamycin对OfChtI的IC50值测定示意图。其中横坐标Log[Kasugamycin Conc(μM)]表示以10为底的化合物浓度对数值，浓度单位为μM；纵坐标Inhibition rate表示抑制率；图中曲线对应不同的底物浓度下，Inhibition rate随化合物浓度的变化而变化的趋势，曲线上纵坐标为0.5的点所对应的横坐标数值即为化合物对OfChtI的半数抑制浓度IC50值，为14μM。
图2是50μM终浓度下化合物Kansugamycin对不同物种来源的18家族几丁质酶(GH18) 的抑制活性示意图。图中横坐标表示抑制活性，纵坐标表示不同来源的几丁质酶。
图3为50μM化合物Kasugamycin对亚洲玉米螟的杀虫活性示意图，其中A为对照组，B为实验组。A、B图横坐标表示统计时间，纵坐标表示相应表型的亚洲玉米螟所占的百分比；■代表正常幼虫，▲代表死亡幼虫，●代表正常蛹，◆代表不正常蛹。
具体实施方式
下述非限制性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何方式限制本发明。任何熟悉本技术领域的技术人员在本发明披露的技术范围内，根据本发明的技术方案及其发明构思进行等同替换或改变均属于本发明保护范畴。
本发明实施例中所用的酶OfChtI如下述参考文献给出：Lei Chen,T.L.,YongZhou,Qi Chen,Xu Shen and Qing Yang,Structural characteristics of an insectgroup I chitinase,an enzyme indispensable to moulting.Acta CrystallographicaSection D Biological Crystallography 2013, ISSN,1399-0047.
实施例1
将几丁质酶OfChtI作为靶标，对556个微生物次级代谢产物进行抑制剂筛选。具体步骤如下：
正对照：设置2组平行正对照。在30℃反应温度，100μL反应体系的条件下，2nmol/L糖基水解酶OfChtI和50μmol/L底物(MU-(GlcNAc)2)在20mmol/L的pH 6.0的磷酸盐缓冲液中孵育30min，之后加入100μL 0.5mol/L碳酸钠溶液终止反应，反应液用360nm波长的激发光进行激发后测定450nm波长下的吸光度值。
实验组：设置3组平行实验组。在30℃反应温度，100μL反应体系的条件下，2nmol/L糖基水解酶OfChtI和50μmol/L底物(MU-(GlcNAc)2)以及50μM化合物在20mmol/L的pH 6.0的磷酸盐缓冲液中孵育30min，之后加入100μL 0.5mol/L碳酸钠溶液终止反应，反应液用360nm波长的激发光进行激发后测定450nm波长下的吸光度值。
根据以下公式计算抑制活性
抑制百分数＝(正对照-实验组)/正对照*100
筛选来源的微生物次级代谢产物经由各类真菌发酵产生，数目较大(556个)，种类丰富，结构差异明显，主要有抗生素、激素、生物碱、各类毒素及维生素等，其中化合物Kasugamycin 属于氨基糖苷类化合物。对抑制剂进行筛选时，先进行长时间的初筛，在初筛的基础上对初筛获得的阳性结果进行进一步的复筛(具体步骤同上)确认后获得最终数据，结果表明，化合物Kasugamycin对OfChtI具有抑制效果，在50μM终浓度下对OfChtI抑制率分别为86.4％。
实施例2
1)半数抑制浓度IC50测定
OfChtI：MU-(GlcNAc)2作为底物，底物浓度为30μM。在相同底物浓度下取终浓度分别为 200、100、50、25、12.5、6.25、3.125、1.5625μM八组化合物浓度梯度进行抑制活性测定。反应体系为100μL，缓冲环境为20mM磷酸盐缓冲液，pH6.0，酶终浓度为2nM，反应温度30℃，反应时间30min，之后加入100μL浓度为0.5M的碳酸钠溶液终止反应，释放的MU经由360nm 激发光激发后于450nm波长下测定其吸光度值。数据拟合后得IC50＝14μM，结果如图2所示。
2)化合物的适用对象测定
GH18：选择7个不同物种来源的18家族几丁质酶对化合物Kasugamycin的抑制活性进行评价，6个几丁质酶分别为来源于亚洲玉米螟的OfChtI，来源于人的壳三糖酶HsChit1，来源于烟曲霉的AfChiB1以及来源于粘质沙雷氏菌的SmChiA、SmChiB和SmChiC。反应设置2组平行对照组和3组平行实验组。MU-(GlcNAc)2作为底物，终浓度为30μM，化合物Kasugamycin终浓度为50μM。反应体系为100μL，缓冲环境为20mM磷酸盐缓冲液，pH6.0，各几丁质酶终浓度为2nM，反应温度30℃，反应时间30min，之后加入100μL浓度为0.5M的碳酸钠溶液终止反应，释放的MU经由360nm激发光激发后于450nm波长下测定其吸光度值。根据实施例1中公式计算抑制活性。
实施例3
化合物Kasugamycin的杀虫活性评价具体步骤如下：
实验选取五龄第四天的健康幼虫作为实验材料，设置对照组和实验组。化合物Kasugamycin用H2O溶解，浓度为500μM。对照组幼虫注射2μL的H2O，实验组幼虫注射2μL 浓度为500μM的化合物Kasugamycin。注射后的幼虫于26℃，相对湿度70％-90％，每天16 小时光照，8小时黑暗的条件下培养至全部化蛹为止，期间每天对正常幼虫、死亡幼虫、正常蛹和不正常蛹的数量和表型进行统计。统计结果绘制成图3，其中A为对照组，B为实验组。结果表明，实验组幼虫的发育被延缓。
Claims (6)
1.一种春雷霉素及其衍生物作为几丁质酶的抑制剂的应用，其特征在于：所述几丁质酶抑制剂的结构通式如I所示：
R1基团选自以下取代基：
R2基团取自以下取代基：
2.如权利要求1所述的抑制剂和/或其衍生物在抑制糖基水解酶18家族几丁质酶活性中的应用。
3.根据权利要求1或2所述的应用，其特征在于：结构通式I所示的几丁质酶抑制剂使用的终浓度为不低于20μM。
4.如权利要求1所述的抑制剂在防治鳞翅目昆虫方面的应用。
5.根据权利要求4所述的应用，其特征在于：所述的鳞翅目昆虫为麦蛾、棉红铃虫、马铃薯块茎蛾、甘薯麦蛾、棉褐带卷蛾、大豆食心虫、梨大食心虫、亚洲玉米螟、大造桥虫、菜粉蝶、棉铃虫、舞毒蛾、玉带风蝶、金凤蝶、舟形毛虫、黄腹灯蛾、红腹灯蛾、美国白蛾、葡萄天蛾和稻苞虫。
6.如权利要求4所述的应用，其特征在于：所述在防治鳞翅目昆虫方面的应用是在延缓亚洲玉米螟发育方面的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710271627.4A CN107223663B (zh) | 2017-04-24 | 2017-04-24 | 春雷霉素及其衍生物为几丁质酶抑制剂的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710271627.4A CN107223663B (zh) | 2017-04-24 | 2017-04-24 | 春雷霉素及其衍生物为几丁质酶抑制剂的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107223663A CN107223663A (zh) | 2017-10-03 |
| CN107223663B true CN107223663B (zh) | 2019-10-29 |
Family
ID=59934043
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710271627.4A Expired - Fee Related CN107223663B (zh) | 2017-04-24 | 2017-04-24 | 春雷霉素及其衍生物为几丁质酶抑制剂的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107223663B (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3668511A4 (en) * | 2017-08-17 | 2021-05-05 | Brown University | METHODS AND COMPOSITIONS FOR TREATMENT OF FIBROSE |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107832577B (zh) * | 2017-10-30 | 2021-07-13 | 中国农业大学 | 一种筛选几丁质酶OfChtⅠ抑制剂的方法 |
| CN108467395B (zh) * | 2018-05-09 | 2020-12-11 | 大连理工大学 | 一种几丁质酶抑制剂及其应用 |
| CN113180043B (zh) * | 2021-04-02 | 2022-05-13 | 大连理工大学 | 一种几丁质水解酶抑制剂在调节昆虫生长活性中的应用 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103766343A (zh) * | 2014-01-10 | 2014-05-07 | 大连理工大学 | 一种作为几丁质酶抑制剂的3-7聚葡萄糖胺单体 |
| WO2016044655A2 (en) * | 2014-09-17 | 2016-03-24 | Spogen Biotech Inc. | Fusion proteins, recombinant bacteria, and methods for using recombinant bacteria |
-
2017
- 2017-04-24 CN CN201710271627.4A patent/CN107223663B/zh not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3668511A4 (en) * | 2017-08-17 | 2021-05-05 | Brown University | METHODS AND COMPOSITIONS FOR TREATMENT OF FIBROSE |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107223663A (zh) | 2017-10-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107223663B (zh) | 春雷霉素及其衍生物为几丁质酶抑制剂的应用 | |
| Godschalx et al. | Is protection against florivory consistent with the optimal defense hypothesis? | |
| CN108467395A (zh) | 一种几丁质酶抑制剂及其应用 | |
| CN106854144B (zh) | 一种抑制剂及其在抑制几丁质酶和己糖胺酶活性中的应用 | |
| Prasad et al. | Spatio-temporal dynamics of the parasitoid, Aenasius bambawalei Hayat (Hymenoptera: Encyrtidae) on mealybug, Phenacoccus solenopsis Tinsley in cotton based cropping systems and associated weed flora | |
| CN101480188A (zh) | 含有甲氨基阿维菌素苯甲酸盐和茚虫威的杀虫组合物 | |
| Du et al. | Evaluation of the safe use and dietary risk of beta-cypermethrin, pyriproxyfen, avermectin, diflubenzuron and chlorothalonil in button mushroom | |
| CN103081911A (zh) | 壳寡糖作为昆虫几丁质酶抑制剂及杀虫剂的应用 | |
| CN107094780B (zh) | 小檗碱及其衍生物作为己糖胺酶抑制剂的应用 | |
| CN103039536B (zh) | 一种防治线虫的农药组合物 | |
| CN102657235A (zh) | 含厚孢轮枝菌和淡紫拟青霉的杀线虫组合物及其制备方法 | |
| Pan et al. | Whorl-stage maize provides a microclimate refuge for predatory ladybeetles | |
| CN102440261A (zh) | 一种含氟啶虫胺腈和毒死蜱的杀虫组合物 | |
| Stygar et al. | Digestive enzymes activity in subsequent generations of Cameraria ohridella larvae harvested from horse chestnut trees after treatment with imidacloprid | |
| CN104705324B (zh) | 一种含中生菌素和壳寡糖的杀菌组合物 | |
| Decker et al. | Elevated atmospheric concentrations of CO2 increase endogenous immune function in a specialist herbivore | |
| CN113180043B (zh) | 一种几丁质水解酶抑制剂在调节昆虫生长活性中的应用 | |
| CN106993622A (zh) | 一种含有乙酰虫腈与嘧虫胺的杀虫组合物及其应用 | |
| CN104757014B (zh) | 一种含有异菌脲和噻虫嗪的农药组合物 | |
| AKINYOOLA et al. | Anti-insect potential of a lectin from the tuber, Dioscorea mangenotiana towards Eldana saccharina (Lepidoptera: Pyralidae) | |
| CN102440259A (zh) | 一种含氟啶虫胺腈和氯氰菊酯的杀虫组合物 | |
| CN101480185B (zh) | 一种具有增效杀虫作用的农药组合物及其应用 | |
| CN104770391A (zh) | 一种用于防治辣椒根结线虫的农药组合物 | |
| Zeng et al. | Application of natural aminopolysaccharide in seed film-coating for pest control and cotton growth | |
| CN103004842A (zh) | 啶虫脒和氟啶虫酰胺的杀虫剂复配组合物 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20191029 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |