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2021 , Vol. 33 >Issue 6: 1035 - 1043

  DOI: https://doi.org/10.7536/PC200659

文章编号: 1608628823751-399889850  

文献标识码: A

综述

小分子荧光探针在绿色农药开发中的应用

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  • 1 天津大学药物科学与技术学院 天津市现代药物传递及功能高效化重点实验室 天津 300072
  • 2 天津大学分子+研究院 天津 300072

收稿日期：2020-06-22

  修回日期：2020-08-04

  网络出版日期：2020-12-22

基金资助

国家重点研发计划项目(2017YFD0201403)

收起

Application of Small-Molecule Fluorescent Probes in the Development of Green Pesticides

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  • 1 School of Pharmaceutical Science and Technology, Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, Tianjin University, Tianjin 300072, China
  • 2 Institute of Molecular Plus, Tianjin University,Tianjin 300072, China
* Corresponding author e-mail: (Shengnan Liu);
(Qingzhi Gao)

Received：22 Jun. 2020

  Revised：4 Aug. 2020

  Online：22 Dec. 2020

Fund

National Key Research and Development Project(2017YFD0201403)

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摘要

小分子荧光探针以其灵敏度高、特异性强、稳定性好、操作便捷和成本低等特点在生命科学、医药化学和环境科学等领域得到了广泛的应用。在农药化学领域,小分子荧光探针常被用作农药残留及重金属污染的检测手段。近年来随着全球开发绿色农药战略需求的不断增强,作为靶向型药物设计和高通量筛选的重要分子工具,荧光探针在绿色农药新产品研发领域的应用不断普及和深化。本文从探针分子的化学设计、靶点识别及药物筛选的角度出发,围绕不同类型的绿色农药重要生物靶点,综述了小分子荧光探针在绿色农药开发领域的研究现状,并对其未来的发展趋势和应用前景进行了展望。

关键词： 小分子荧光探针 ; 绿色农药 ; 鱼尼丁受体 ; 几丁质酶 ; 高通量筛选

中图分类号： O69 ()   TQ453.6 ()   X592 (农用化学物质、有毒化学物质污染及其防治)  

本文引用格式

侯晓涵 , 刘胜男 , 高清志 . 小分子荧光探针在绿色农药开发中的应用[J]. 化学进展, 2021 , 33(6) : 1035 -1043 . DOI: 10.7536/PC200659

Xiaohan Hou , Shengnan Liu , Qingzhi Gao . Application of Small-Molecule Fluorescent Probes in the Development of Green Pesticides[J]. Progress in Chemistry, 2021 , 33(6) : 1035 -1043 . DOI: 10.7536/PC200659

Abstract

Small-molecule fluorescent probes are widely applied in the fields of life science, medicinal chemistry and environmental science, due to their characteristics of high sensitivity and specificity, good stability and economic applicability. In pesticide chemistry, small-molecule fluorescent probes are frequently utilized in the detection of pesticide residues and heavy metal pollutions. With global strategic needs and rapid technological progress in green pesticide development, fluorescent probes are urgently desired as important molecular tools for design, screening and development of environmentally benign agrichemicals. This article aims to review the key updates of small-molecule fluorescent bioprobes in green pesticide R&D by covering their chemical design, molecular targeting, and screening mechanisms against different green pesticide biotargets, and to provide the current status on their research and application as well as future perspectives.

Contents

1 Introduction

2 Ryanodine receptor-targeted small-molecule fluorescent probes

2.1 Anthranilic diamide-based fluorescent probes

2.2 Phthalic diamide-based fluorescent probes

3 Chitin-targeted small molecule fluorescent probes

3.1 Chitin synthase-targeted fluorescent probes

3.2 Chitin related enzyme-targeted fluorescent probes

4 Type Ⅲ secretion system-targeted small-molecule fluorescent probes

5 γ-Aminobutyric acid receptor-targeted small-molecule fluorescent pesticides

6 Acetohydroxyacid synthase-targeted small-molecule fluorescent probes

7 Conclusion and outlook

Key words： small-molecule fluorescent probe ; green pesticides ; ryanodine receptor ; chitin synthase ; high-throughput screening

1 引言

全面推进和发展生态文明建设是我国“五位一体”总体布局的重要组成部分,其中重点强调了我国发展生态农业和推广绿色防控实现农药零增长的现代农业发展方向。农药的安全性和农药在食用农产品中的残留是危害人类健康的重要因素之一,早在20世纪末,我国就针对传统化学农药引起的农田污染和食品安全等问题提出了重视发展生态经济和“绿色农药”的概念[1⇓~3]。绿色农药,顾名思义就是能选择性抑制或杀伤农作物害虫及病原菌但对人、畜及害虫天敌健康无害、不破坏生态环境的靶向型农药产品[4],主要由杀虫剂、杀菌剂和除草剂等组成。绿色农药研发需要解决的关键科学问题是针对病原菌和农业害虫特异性生物靶点的探索和发掘以及基于这些绿色靶标开展合理药物设计和高效药物筛选[5]。在此过程中,传统的方法主要是借助同位素标记的靶蛋白或小分子药物实施筛选以获得活性先导化合物。由于同位素标记具有合成成本高、放射性污染和普及较困难等问题,因此制约了生产企业的科研创新活动。随着药物设计技术和光学成像技术的飞速发展以及农药筛选技术的更新换代,以绿色农药生物靶标为导向的小分子荧光探针成为研究农药作用机制、设计与筛选新型农药分子、加速靶向型绿色农药研究与开发的重要工具。本文就小分子荧光探针在绿色农药研发领域的前沿成果和最新动态进行概述,同时对小分子荧光探针在促进绿色农业发展的未来趋势进行展望。

2 靶向鱼尼丁受体的小分子荧光探针

鱼尼丁受体(Ryanodine receptor, RyR)是控制钙离子(Ca2+)从内质网储存释放并调节细胞内Ca2+浓度的重要离子通道,它因能与植物碱鱼尼丁发生高亲合性结合而得名[6]。由于哺乳动物的RyR与昆虫类RyR的氨基酸仅有40%~50%的同源性,因此昆虫RyR成为开发绿色杀虫剂的理想靶标,该类杀虫剂对甜菜夜蛾、小菜蛾、黏虫、二化螟以及稻纵卷叶螟等鳞翅目害虫具有很好的防效。近年来,由国内外各大农药公司及研究机构开发上市及即将上市的两大类RyR靶向型绿色杀虫剂:邻氨基苯甲酰胺类(Anthranilic diamide)和邻苯二甲酰胺类(Phthalic diamide)达八个品种之多,分别是氟苯虫酰胺[7]、氯虫苯甲酰胺[8]、溴氰虫酰胺[9]、四氯虫酰胺[10]、氯氟氰虫酰胺[11]、环溴虫酰胺[12]、氟氰虫酰胺[13]和Broflanilide[14,15]。该类型杀虫剂因活性高、作用机制新颖且对哺乳动物安全等特点,迅速取代了许多传统杀虫剂而在农药市场中占绝对份额[16]。这也使得RyR成为绿色农药开发过程中最重要的研究靶点之一。

2.1 邻氨基苯甲酰胺类RyR荧光探针

邻氨基苯甲酰胺类昆虫RyR靶向型绿色杀虫剂主要包括日本农药和杜邦公司的氯虫苯甲酰胺(Chlorantraniliprole, Chlo)[17]、先正达公司的溴氰虫酰胺(Cyantraniliprole, Cyan)[18]、沈阳化工研究院的四氯虫酰胺(Tetrachlorantraniliprole)[19]、日本开发的环溴虫酰胺(Cyclaniliprole)[20]以及拜尔公司的氟氰虫酰胺(Tetraniliprole)[21]等。鱼尼丁作为RyR的天然底物其本身对昆虫和哺乳动物并不具有选择性,因此鱼尼丁本身不能作为绿色杀虫剂使用。这些邻氨基苯甲酰胺类绿色杀虫剂被证实与天然产物鱼尼丁具有不同的结合位点,因此设计和开发邻氨基苯甲酰胺类似物小分子荧光探针不仅能够用于该类杀虫剂的分子机制研究,还有助于建立基于荧光探针的高通量筛选体系用于新型绿色杀虫剂的设计和高效筛选。
Liu等[22]以Chlo和Cyan两个绿色杀虫剂的分子母核为基础,通过“点击化学”将羟基香豆素荧光团与邻氨基苯甲酰胺杀虫剂相连接,设计并合成了稳定性好、特异性强的RyR特异性荧光探针ChloF和CyanF(图1)。利用探针化合物与鱼尼丁放射性配体及对应的放射性农药分子进行竞争结合试验,发现该探针能够抑制放射性氯虫苯甲酰胺([3H]Chlo)农药分子与受体的结合(IC50 = 34~39 nmol/L),同时发现该探针尚可刺激天然鱼尼丁放射性配体([3H]Ry)与RyR的结合(EC50 = 5~7 nmol/L)。上述结果验证了该探针与鱼尼丁天然配体在昆虫 RyR中不共享相同的结合模式但与Chlo具有相同的结合位点。该现象也同时验证了Chlo以及ChloF探针作为RyR的“变构调节”配体(Allosteric Regulator)能够间接影响和调控RyR钙离子通道,对理解RyR通道的结构复杂性具有重要意义。尽管探针分子较Chlo和Cyan相比,对小菜蛾幼虫的杀虫活性有所减弱(LC50 = 2.6~7.6 ppm)。但因其良好的特异性和荧光稳定性,在以RyR为靶点的新型农药分子设计和筛选中有望取代放射性探针发挥重要作用。
图1 (a) 靶向RyR的小分子荧光探针的设计原理示意图;(b) RyR靶向型荧光探针ChloF和鱼尼丁在受体中不同的结合位点

Fig.1 (a) Design concept of RyR-targeted specific fluorescent probe.(b) Different binding location of ChloF and Ryanodine in RyR

Chen及Liu等[23]于2019年通过应用RyR蛋白色氨酸残基与上述荧光探针分子之间的荧光共振能量转移(FRET)技术研究了绿色农药Chlo对哺乳动物鱼尼丁受体RyR1的影响,并进一步考察了Chlo与RyR之间的结合位点。研究结果显示,杀虫剂Chlo与哺乳动物RyR1的亲合力较昆虫RyR相比低约100~200倍。此项研究不仅确认了该类杀虫剂的安全性,还进一步阐明了Chlo与昆虫RyR之间的结合模式与其他所有RyR激动剂或抑制剂,例如:咖啡因、鱼尼丁、阻断剂钌红等均有所不同,解释了该类绿色杀虫剂针对昆虫RyR的高选择性[24]。

2.2 邻苯二甲酰胺类RyR荧光探针

氟苯虫酰胺(Flubendiamide, Flu)是拜耳公司于2007年开发上市的另一类昆虫RyR靶向型绿色杀虫剂,其属于邻苯二甲酰胺类化合物。与Chlo类似,Flu也是有效的钙通道激动剂,通过激活昆虫RyR促进内质网内Ca2+持续释放,最终导致昆虫肌肉麻痹、呕吐、回流,影响进食而死亡[25]。然而,研究证明邻苯二甲酰胺和邻氨基二酰胺类化合物在RyR靶点中的结合位点略有不同并且与鱼尼丁互不重叠[26,27]。为了确认Flu在昆虫组织中的吸收、分布以及明确Flu与靶点RyR的结合位点等问题,本课题组[28]利用基于药效团模型的计算机辅助设计技术将荧光团7-氨基-4-甲基香豆素与Flu进行结构融合,设计并合成了一系列具有杀虫活性和荧光特性的RyR靶向荧光探针(图2)。通过钙流成像研究发现,这些探针能够诱导小菜蛾RyR转染的Sf9细胞中钙离子的释放,引起胞质Ca2+瞬变,其中以探针FluF作用效果最明显(图2)。随后,经共聚焦荧光显微镜观察探针与Flu的竞争性结合,验证了FluF富集在Sf9细胞的内质网表面,并通过与Flu的竞争结合试验证实了探针化合物与Flu具有相同的RyR结合模式,为建立高通量新药筛选奠定了基础。最后,通过小菜蛾幼虫模型的体内实验探究FluF在昆虫体内的吸收与分布情况,发现探针化合物蓄积在富含RyR的神经节和中肠内。由于中肠是小菜蛾多种生理活动的重要器官,该研究结果间接验证了Flu类绿色农药能够很好地抑制小菜蛾生存的生物学机理。集荧光特征与杀虫活性为一体的探针化合物FluF作为分子工具有望取代放射性同位素,进行昆虫对杀虫剂的抗性机制研究、杀虫剂对不同物种鱼尼丁受体的结合差异探索以及用于靶向昆虫RyR新型绿色农药的荧光高通量筛选。
图2 基于Flu设计的RyR靶向荧光探针用于体内外成像研究[28]

Fig.2 RyR-targeted fluorescent probe based on Flu for in vitro and in vivo imaging studies[28]

鉴于昆虫RyR是目前和将来最受关注的绿色农药生物靶点之一,2020年Liu等[29]将近红外罗丹明荧光团引入Flu,设计并合成了含有罗丹明取代的Flu-R-L探针,开发了一种基于Flu-R-L荧光探针的荧光偏振检测方法(图3)。Flu-R-L与RyR上的Flu结合位点具有很强的结合力(KD < 25 nmol/L),且在RyR工作缓冲液中具有很高的荧光量子产率以及良好的溶解性。在12.5 nmol/L的浓度条件下,并非RyR上的所有特异性结合位点都被Flu-RL分子占据,此时在RyR上结合的探针对其他目标化合物的竞争较敏感。基于此探针分子建立的荧光偏振药物筛选体系,对与RyR上Flu活性位点具有潜在结合活性的化合物进行了筛选测试研究,结果发现了9种具有较强Flu类似活性的衍生物。与其他测定法相比,荧光偏振法具有许多关键优势,例如:它在液相中进行,非放射性,不需要分离结合和游离的配体,读数快等 [30,31]。这些合理设计的小分子荧光探针对建立新型杀虫剂候选药物的体外筛选方法,特别是高通量筛选,具有重要的意义。
图3 基于Flu设计的RyR靶向近红外荧光探针[29]

Fig.3 RyR-targeted NIR fluorescent probe based on Flu[29]

3 靶向几丁质受体的小分子荧光探针

几丁质受体,又称几丁质酶。它在甲壳纲动物、真菌和昆虫的围食膜降解、蜕皮、身体免疫防御、细胞增殖以及生长发育过程中起重要作用[32]。几丁质合成酶(Chitin synthase, CHS)是几丁质合成中最关键的酶,抑制CHS的几丁质合成能够有效阻止真菌生长或阻止昆虫幼虫和蛹的脱皮达到杀虫效果。由于植物和哺乳动物不存在CHS基因和几丁质代谢系统[33],因此设计和筛选几丁质合成酶抑制剂有望开发出对人低毒无害的抗真菌药物和绿色杀虫剂,几丁质合成酶被公认为是特异性强、安全性好的绿色农药开发关键靶点之一[34⇓~36]。

3.1 几丁质合成酶小分子荧光探针

由于几丁质合成酶本身的三维结构以及其催化合成几丁质的生物学机制尚不完全明确,因此针对几丁质合成酶的抑制剂设计、荧光探针开发都具有非常大的挑战性。放射性氚标记的尿苷-5'-二磷酸-N-乙酰基-葡糖胺(3H-UDP-GlcNAc)和13C标记的尿苷-5'-二磷酸-N-乙酰基-葡糖胺(13C-UDP-GlcNAc)[37]是最早被开发用于CHS活性测试的放射性标记底物,其中,UDP-GlcNAc是CHS生物合成几丁质过程中所必需的糖供体。虽然放射性标记底物可定量检测CHS的活性,但开发一种非放射性的CHS靶向型荧光探针可避免放射性危害且操作便捷、能实现快速高效的通量活性筛选,因此具有重要的科学价值和广阔的应用前景。Bahmed等[38]在放射性底物部分结构N-乙酰氨基葡萄糖(GlcNAc)的基础上设计并合成了一种含丹磺酰荧光团的CHS底物小分子荧光探针6-O-丹磺酰基-N-乙酰氨基葡萄糖(DNAG),通过定量检测DNAG与CHS结合后荧光的减弱强度,可以较准确地评估CHS的活性,且与通过放射性方法获得的结果一致(图4)。
图4 (a) 几丁质合成酶(CHS)催化合成几丁质的过程;(b) 传统的放射性探针及丹磺酸荧光探针[38]

Fig.4 (a) CHS catalyzed chitin synthesis.(b) Traditional radio-labeled substrate and the fluorescent probe[38]

针对几丁质合成酶抑制剂的设计与合成研究,大连理工大学Yang等[39]常年致力于该研究领域的前沿工作,创造出了多种具有CHS抑制活性的先导化合物,对引领我国在几丁质靶向绿色农药领域的发展具有重要的作用。

3.2 其他几丁质酶小分子荧光探针

几丁质的合成过程不仅涉及关键的几丁质合成酶,还需要己糖胺酶、几丁质水解酶等一系列几丁质代谢关键酶的参与。因此,抑制其中的任何一种代谢酶的功能都有望发现有效防治动植物致病真菌、寄生虫以及农业害虫的绿色农药化合物。4-甲基伞形乙酰基-β-D-氨基葡萄糖苷(4-MU-GlcNAc)及4-硝基苯基-N-乙酰基-β-D-氨基葡萄糖苷(PNP-GlcNAc)是最常用的检测几丁质水解酶以及己糖胺酶的荧光底物[40]。正由于它们具有普适性,因此利用这些荧光底物探针进行药物筛选并不是理想的选择。Zhang等[41]于2019年设计并开发了用于评估小分子抑制剂和具有活体细胞成像功能的己糖胺酶选择性荧光探针:萘二甲酰亚胺类荧光探针Probe-1C(图5)。与传统的4-MU-GlcNAc和PNP-GlcNAc相比,Probe-1C具有水溶性高、灵敏度好和荧光强度大等特点。该探针不仅能够用于高通量酶标仪测试系统,还能通过视觉化鉴别实施批量样品的己糖胺酶抑制剂快速筛选。
图5 (a) 用于几丁质酶底物的广谱荧光探针;(b) 高选择性己糖胺酶小分子荧光探针[41]

Fig.5 (a) Traditional chitinase fluorescent substrates.(b) Hexosaminidase selective fluorescent probe[41]

4 靶向细菌Ⅲ型分泌系统的小分子荧光探针

细菌Ⅲ型分泌系统(Type Ⅲ secretion system, T3SS)是许多有害病原菌,例如辣椒炭疽病菌、烟草赤星病菌、番茄灰霉病菌以及水稻黄单胞杆菌等共有的由蛋白复合体构成的跨膜分子装置,能够将病原菌分泌的致病相关效应子注入宿主细胞从而引发感染[42]。传统的抗菌药物,多是通过影响细菌的DNA复制、RNA转录、蛋白质合成及细胞壁合成等生长过程,达到抗菌的目的。然而,抗菌药物的滥用造成了大规模耐药性的产生[43]。以T3SS为靶点开发的抑制剂,可以干扰病原菌侵染宿主的途径而不影响细菌存活,所以不会导致病原细菌耐药性的产生[44]。因此,靶向T3SS被视为开发新型抗病原菌微生物药物的新策略。近十年来,已有多种天然产物显示出T3SS抑制活性,并有大量的活性小分子被筛选出来[45⇓~47]。它们中有些是在基因水平上调控T3SS,有些是以T3SS的装配组分为靶点[48]。目前大多数T3SS抑制剂精确的生物学靶标和作用机制尚不完全清楚,因此开发靶向T3SS体系的小分子荧光探针在新型病原菌抑制剂的开发、药物活性筛选和作用机理研究等方面具有重要意义。先前,大部分的T3SS抑制剂的高通量筛选体系是通过利用荧光素酶报告系统[49]和β-内酰胺酶报告系统[50]来实现的。2010年,Hang等[51]利用羧肽酶G2(CPG2)能够选择性地从小分子代谢产物中裂解谷氨酸(Glu)而大多数细菌或高等真核生物中的CPG2谷氨酰羧肽酶没有活性这一特性,发现了一种可用于荧光可见的检测革兰氏阴性病原菌中Ⅲ型蛋白的分泌的新型羧肽酶G2(CPG2):Glu-CyFur报告系统(图6a)。其中,Glu-CyFur化合物是基于谷氨酸(Glu)和荧光分子2-二氰基亚甲基-3-氰基-2,5-二氢呋喃(CyFur)设计合成的,是CPG2的底物型荧光分子探针,该探针的成功开发为评价影响蛋白质毒素分泌的细胞因子、效应因子、及新型杀菌剂的发现提供了一种更简单、更快速高效的方法。例如,Hang等[52,53]为了评估传统药用植物对鼠伤寒沙门氏菌Ⅲ型蛋白分泌的活性,应用该报告系统筛选了120种具有潜在抗感染活性的中药[54],并且揭示了黄酮类多酚儿茶素具有抗细菌感染作用的机理。如图6b所示,该测定法使用CPG2作为酶报道分子,当CPG2被分泌出来,便会通过裂解荧光底物Glu-CyFur快速而特异性地报道Ⅲ型蛋白的分泌,这为监测靶向T3SS的小分子的抑制活性提供了灵敏的方法。利用该方法,Tsou等成功地发现了儿茶素没食子酸酯(CG)和表儿茶素没食子酸酯(ECG)等多酚儿茶素可以完全抑制T3SS的报告基因活性,而儿茶素和没食子酸在25 μmol/L时的抑制活性却不到20%。由此可见,该筛选系统的应用可能为探索新型抗病原菌绿色植物代谢物提供新的平台。
图6 (a) 基于病原菌Ⅲ型蛋白分泌系统的荧光探针检测技术;(b) Glu-CyFur荧光探针体系用于高通量活性筛选[51⇓⇓~54]

Fig.6 (a) T3SS-targeted specific fluorescence probe.(b) Glu-CyFur reporter system for high-throughput screening[51⇓⇓~54]

5 靶向GABA受体的小分子荧光农药+探针

γ-氨基丁酸(GABA)受体(GABAR)是目前研究较为深入的哺乳动物及昆虫体内重要的抑制性神经递质,参与介导肌肉和神经细胞中突触的快速传递,与神经抑制性生理活性密切相关[55],因此是杀虫剂作用的重要靶点之一。靶向GABA受体的苯并吡唑类杀虫剂如氟虫腈,通过抑制昆虫GABA门控氯化物通道(GABACls),干扰中枢神经系统的正常功能,从而对多种农作物害虫如水稻昆虫、白蚁、家蝇等表现出高效的杀虫活性[56,57]。然而,该类农药的不足之处是不具备韧皮部渗透性,很难抑制吸吮性昆虫[58];另一方面,由于农药在环境中会逐渐发生降解和代谢,某些降解或代谢产物长期暴露于环境中对非靶标生物具有较高的危险[59]。因此改善苯基吡唑类农药的非韧皮部吸收,并揭示其在环境中的降解途径迫在眉睫。其中,氟虫腈及其有毒代谢产物因光降解性差造成了农药残留和土壤污染等问题。Chen等[60]为了增强氟虫腈的韧皮部流动性和光降解性,采用一锅加热法(将氟虫腈与相应的酰氯衍生物进行加成-消除反应)将共轭结构和有机生色团引入氟虫腈的母体结构中,合成了一系列新型“荧光杀虫剂”-苯并吡唑酰胺衍生物(PAs)(图7a)。在模拟太阳辐射的作用下(图7b),这些PAs通过扩展的共轭体系不但增强了其韧皮部迁移性,而且增强了其在天然辐射下的光吸收能力,从而表现出良好的光降解性。光降解后,PAs可以重新转化为氟虫腈母体片段,从而增强杀虫活性。此外,PAs的荧光量子产率几乎是氟虫腈的4倍,可作为高效的荧光探针用以监测农药在韧皮部的输导,并追踪昆虫进食植物后对农药的摄取、体内运输途径和分布(图7b)。随着荧光标记技术的飞速发展,该类兼具荧光探针及杀虫性能的创新型小分子探针的开发为农药残留物的荧光检测奠定了基础,也为绿色农药的高效筛选和开发提供了新策略。
图7 (a) 基于氟虫腈设计的韧皮输导性新型荧光杀虫剂;(b) PAs荧光杀虫剂的作用机制示意图[60]

Fig.7 (a) Fipronil-based fluorescent phloem-mobile insecticides.(b) Mechanism of fluorescent PAs insecticides[60]

6 靶向乙酰羟酸合成酶的小分子荧光探针

乙酰羟酸合成酶(Acetohydroxyacid Synthase, AHAS)又称乙酰乳酸合成酶(ALS),是催化缬氨酸、亮氨酸和异亮氨酸这三种支链氨基酸生物合成的关键酶[61],广泛分布于植物、细菌、真菌和藻类中[62,63],且不存在于哺乳动物体内,是广泛应用于多种农作物除草剂的作用靶点[64⇓~66]。目前,乙酰羟酸合成酶抑制剂类除草剂主要有五大类:磺酰脲类(SU)[67]、咪唑啉酮类(IMI)[68]、三唑并嘧啶类(TP)[69]、嘧啶基苯甲酸类(PYB)[70]和磺酰胺类(SCT)[71,72],它们因具极强的除草活性和哺乳动物安全性而被全世界广泛使用[73,74]。
随着AHAS抑制剂类除草剂的长期和大量使用,许多杂草表现出高水平的耐药性及交叉抗性[75⇓~77]。因此开发具有抗耐药性的新型AHAS抑制剂是发展绿色除草剂的重要方向之一。传统的AHAS类抑制剂筛选方法是通过不连续比色法[78]测试AHAS天然催化底物丙酮酸在333 nm处的吸光度变化[79]而实施的,该方法灵敏度低、不具有连续性且无法实现高通量,因此筛选效率低。为了实现针对AHAS靶标的高通量绿色除草剂筛选,Xie等[80]基于AHAS的酶催化反应机制,利用苯甲醛衍生物既能作为AHAS识别底物又能参与AHAS催化反应过程的特性,成功设计了分子内电荷转移(ICT)荧光致变型小分子荧光探针。如图8所示,作为AHAS的识别底物,对位给电子基团取代的苯甲醛衍生物荧光探针probe-1,在参与AHAS介导的丙酮酸催化过程后,由于醛羰基对分子共轭体系的吸电子效应被打破(图8b,c)产生荧光蓝移从而构建了适于快速发现AHAS靶向型除草剂的高通量筛选体系。
图8 基于苯甲醛衍生物荧光探针1的AHAS抑制剂筛选原理[80]

Fig.8 New AHAS inhibitor screening method based on aldehyde-containing fluorescent probe 1[80]

7 结论和展望

本文围绕新型绿色农药的研发,从分子设计原理和靶标特异性的角度系统地概述了最前沿小分子荧光探针在绿色农药领域的开发和应用现状。在荧光染料的选择上,用于靶向探针设计的荧光染料涵盖了从短波长蓝色香豆素到近红外罗丹明等具有不同光学性能的荧光团。一方面,从高通量筛选的角度出发,除了荧光染料自身的荧光强度、荧光效率及光化学稳定性等因素以外,荧光探针与水性筛选体系(细胞、酶)的相容性非常重要。另一方面,就荧光探针的靶向选择性而言,由于不同的靶标在目标测试体系中存在的位置与环境不同,例如,鱼尼丁受体存在于昆虫细胞内质网膜而几丁质合成酶以及病原菌分泌系统属于跨膜分子装置,因此在设计荧光探针的过程中,除了需要评估荧光染料自身的水溶特性(与筛选体系的相容性)以外,更重要的是如何从微观和宏观两个方面满足靶向型荧光探针设计的需要,即:在微观上所设计的探针分子需要针对目标靶点有特异性识别,以提高筛选结果的靶向选择性,在宏观上整个探针分子既需要与筛选体系具有相容性又需要到达测试体系中靶标分布的空间位置。总之,高选择性、高特异性、高灵敏度、安全廉价的小分子荧光探针,在新型绿色农药的设计、筛选与开发、促进绿色农业发展、保障人类健康及生态平衡等领域越来越发挥非常重要的作用。目前,已经有大量的小分子探针被应用于农药、重金属及溶剂残留的检测,然而,针对绿色农药生物靶点的荧光探针研究才刚刚开始,具有理想应用价值的研究成果仍然较少。如上所述,在荧光染料选择和探针设计理念上除了需要注重靶点导向之外,还有诸多的功能性因素需要我们去思考和挑战。例如,如何使小分子荧光探针高特异性标记害虫不同生长发育阶段的特异性生物靶标帮助寻找新的绿色农药靶点?如何将小分子荧光探针用于研究各类昆虫的生殖与代谢过程?如何利用小分子荧光探针研究和揭示病原菌及害虫对农药的耐药机制?面临这些挑战,设计和构建用于不同目的的特异性更强、灵敏度更高的分子探针是全世界科学家们需要协同攻关的方向。

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DOI: 10.1021/jm201542d   PMID: 22364528

本文引用 [1] 摘要
The discovery of 1,3,8-triazaspiro[4.5]decane-2,4-diones (spirohydantoins) as a structural class of pan-inhibitors of the prolyl hydroxylase (PHD) family of enzymes for the treatment of anemia is described. The initial hit class, spirooxindoles, was identified through affinity selection mass spectrometry (AS-MS) and optimized for PHD2 inhibition and optimal PK/PD profile (short-acting PHDi inhibitors). 1,3,8-Triazaspiro[4.5]decane-2,4-diones (spirohydantoins) were optimized as an advanced lead class derived from the original spiroindole hit. A new set of general conditions for C-N coupling, developed using a high-throughput experimentation (HTE) technique, enabled a full SAR analysis of the spirohydantoins. This rapid and directed SAR exploration has resulted in the first reported examples of hydantoin derivatives with good PK in preclinical species. Potassium channel off-target activity (hERG) was successfully eliminated through the systematic introduction of acidic functionality to the molecular structure. Undesired upregulation of alanine aminotransferese (ALT) liver enzymes was mitigated and a robust on-/off-target margin was achieved. Spirohydantoins represent a class of highly efficacious, short-acting PHD1-3 inhibitors causing a robust erythropoietin (EPO) upregulation in vivo in multiple preclinical species. This profile deems spirohydantoins as attractive short-acting PHDi inhibitors with the potential for treatment of anemia. © 2012 American Chemical Society
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The inhibition of chitinase activity is considered of great importance for the development of novel antifungal agents. Here we explore 4-hydroxycoumarins as a novel natural product-derived scaffold for inhibiting chitinases. A new series of 4-hydoxycoumarin derivatives containing Schiffs base motif in the 3 position was synthesized and computationally predicted for chitinase binding affinity. Docking simulation study was carried out using the built homology models of specified fungal species and the top ranked molecules with promising binding affinity were subjected to further molecular dynamic simulation to evaluate their binding stability. The top compounds were then tested in vitro against three phytopathogenic fungi, including Fusarium solani, Fusarium oxysporium and Aspergillus niger in addition to three candida species including C albicans, C. tropicalis and C krusei. The active antifungal candidates were further assessed for chitinase inhibition effect. Most of the tested compounds displayed promising antifungal effects. The 2,4-dichlorophenyl Schiffs base 2e exhibited a wide range of antifungal activity against most of the tested fungi and yeast while the 3-fluorophenyl Schiff's base 2a showed the highest anti-candidal effects. Both Schiffs bases 2a and 2e displayed the highest chitinase inhibition effects (IC50=1.0 mM) having the same potential as the previously described chitinase inhibitor CI-4 (IC50=1.2 mM). The target compounds have high ligand efficiency and binding stability, and as such are promising leads for future development of chitinase inhibitors. (C) 2018 Elsevier B.V.
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DOI: S0048-3575(16)30076-1   PMID: 28187835

本文引用 [1] 摘要
Bradysia odoriphaga Yang and Zhang is the primary insect pest that affects Chinese chive in northern China. Nevertheless, very few studies have been conducted on the use of chitin synthesis inhibitors (CSIs) for the control of B. odoriphaga. Here, lethal and sublethal effects of the CSI chlorfluazuron on B. odoriphaga were studied to explore the use for integrated pest management (IPM) of B. odoriphaga. A contact and ingestion toxicity bioassay showed that chlorfluazuron was more active against B. odoriphaga than three other CSIs, with a 72h LC of 0.1593mg/L. Treatment with the LC dose of chlorfluazuron decreased both the intrinsic and finite rates of increase of B. odoriphaga, in addition to reproduction rate, survival rate, and fecundity, and the mean generation time, total preovipositional period and larval development duration were shortened, compared with those of the control and LC groups. The mean generation time, total preovipositional period and larval development duration were all also markedly decreased by treatment with chlorfluazuron at the LC. Furthermore, chlorfluazuron inhibited the feeding of the final instar larvae for a short period. Glutathione S-transferase and microsomal mixed function oxidase activities increased after exposure to the chemical. These results showed that chlorfluazuron at the sublethal LC treatment inhibited B. odoriphaga population growth, whereas the danger of causing rapid population growth by using a lower sublethal concentration was demonstrated with the sublethal LC treatment. Therefore, chlorfluazuron should be used with caution in an IPM program for B. odoriphaga. Copyright © 2016 Elsevier Inc. All rights reserved.
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Traditional Chinese Medicines (TCMs) have been historically used to treat bacterial infections. However, the molecules responsible for these anti-infective properties and their potential mechanisms of action have remained elusive. Using a high-throughput assay for type III protein secretion in Salmonella enterica serovar Typhimurium, we discovered that several TCMs can attenuate this key virulence pathway without affecting bacterial growth. Among the active TCMs, we discovered that baicalein, a specific flavonoid from Scutellaria baicalensis, targets S. Typhimurium pathogenicity island-1 (SPI-1) type III secretion system (T3SS) effectors and translocases to inhibit bacterial invasion of epithelial cells. Structurally related flavonoids present in other TCMs, such as quercetin, also inactivated the SPI-1 T3SS and attenuated S. Typhimurium invasion. Our results demonstrate that specific plant metabolites from TCMs can directly interfere with key bacterial virulence pathways and reveal a previously unappreciated mechanism of action for anti-infective medicinal plants.
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PMID: 6360995

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Acetohydroxyacid synthase I from Escherichia coli K-12 has been purified to near homogeneity. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two polypeptides, one with a molecular weight of 60,000 and one with a molecular weight of 9,500. These two polypeptides were present in constant proportion to each other and to enzyme activity. The molar ratio of the two polypeptides (Mr 9,500:60,000), estimated from stained polyacrylamide gels, was 1. Antisera raised against the 60,000 Mr polypeptide precipitated both the 60,000 and the 9,500 Mr polypeptides from extracts of cells labeled with [35S]methionine. The addition of sodium dodecyl sulfate before immunoprecipitation eliminated the smaller polypeptide, and only the larger one was recovered. The hydrodynamic properties of the native enzyme confirmed a previous report that the largest enzymatically active species has a molecular weight of about 200,000; this species contains both the 60,000- and 9,500-molecular-weight polypeptides.
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PMID: 12823560

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Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) catalyses the first step in branched-chain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides, which act as potent and specific inhibitors. Mutants of the enzyme have been identified that are resistant to particular herbicides. However, the selectivity of these mutants towards various sulfonylureas and imidazolinones has not been determined systematically. Now that the structure of the yeast enzyme is known, both in the absence and presence of a bound herbicide, a detailed understanding of the molecular interactions between the enzyme and its inhibitors becomes possible. Here we construct 10 active mutants of yeast AHAS, purify the enzymes and determine their sensitivity to six sulfonylureas and three imidazolinones. An additional three active mutants were constructed with a view to increasing imidazolinone sensitivity. These three variants were purified and tested for their sensitivity to the imidazolinones only. Substantial differences are observed in the sensitivity of the 13 mutants to the various inhibitors and these differences are interpreted in terms of the structure of the herbicide-binding site on the enzyme.
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PMID: 3407914

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Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, has received attention recently because of the finding that it is the site of action of several new herbicides. The most commonly used assay for detecting the enzyme is spectrophotometric involving an indirect detection of the product acetolactate. The assay involves the conversion of the end product acetolactate to acetoin and the detection of acetoin via the formation of a creatine and naphthol complex. There is considerable variability in the literature as to the details of this assay. We have investigated a number of factors involved in detecting AHAS in crude ammonium sulfate precipitates using this spectrophotometric method. Substrate and cofactor saturation levels, pH optimum, and temperature optimum have been determined. We have also optimized a number of factors involved in the generation and the detection of acetoin from acetolactate. The results of these experiments can serve as a reference for new investigators in the study of AHAS.
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PMID: 3907697

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A facile purification has been devised for recombinantly produced Salmonella typhimurium acetolactate synthase isozyme II. Purification of the enzyme was made possible by determining the complex set of factors that lead to loss of enzymic activity with this rather labile enzyme. When complexed with thiamin pyrophosphate, FAD, and magnesium, acetolactate synthase is subject to oxygen-dependent inactivation, a property not shared by the enzyme-FAD complex. When divorced from all of its tightly bound cofactors, losses of the enzymic activity are encountered at low ionic strength, especially at low protein concentrations. If purified and stored as the enzyme-FAD complex, acetolactate synthase is quite stable. The enzyme is composed of two types of subunits, a result that was not anticipated from previous studies of ilvG (the gene that codes for the large subunit of acetolactate synthase). These subunits were determined to be in equal molar ratio in the purified enzyme from the distribution of radioactivity between the two subunits after carboxymethylation with iodo[14C]acetate and their respective amino acid compositions. Besides the expected ilvG gene product (59.3 kDa), purified acetolactate synthase contained a smaller subunit (9.7 kDa; designated here as the ilvM gene product). On the basis of sequence homology of the small subunit with that coded for by the corresponding Escherichia coli gene sequence [Lawther, R. P., Calhoun, D. H., Adams, C. W., Hauser, C. A., Gray, J., & Hatfield, G. W. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 922-925], it is encoded by the region between ilvG and ilvE, beginning at base-pair (bp) 1914 (relative to the point of transcription initiation).(ABSTRACT TRUNCATED AT 250 WORDS)
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