Design of bioactive sesquiterpene-based chemical signals with enhanced stability

自然界中生物之间的许多相互作用是由外部化学信号介导的，这些信号通常被称为半化学物质，它们是典型的低分子量亲脂化合物。这些相互作用涉及微生物（如细菌）、藻类、高等植物和动物，包括人类。一旦被发射生物释放，这些信号可以通过改变受体生物的行为或发育而在种内或种间起作用。每当有关识别信号或开发其实际用途（即管理有害生物）的工作报告时，“为什么不设计结构类似物？”的问题被提出。这种方法可以潜在地解决与使用半化学物质相关的许多问题，例如不稳定性和挥发性，这些问题导致效力降低。然而，目前还没有合理的方法来进行结构活性关系研究，以设计具有更高稳定性和性能的类似物。引用的原因是受体系统，例如动物的外周感觉神经元，外部信号识别具有更高的选择性，因为需要从巨大的多样性和外部化学物质的浓度范围中进行选择，而生物体内的受体系统可以很容易地设计类似物。倍半萜家族，即由3 x 5碳单位组成的天然化学物质，在自然界中种类繁多，可以具有各种信号作用。卡迪夫实验室已经证明，参与倍半萜合成的酶（萜烯合成酶）依赖于一个三维结构来转化前体（法尼酯二磷酸酯，FDP），这是所有倍半萜合成酶共享的。合成酶活性位点的可塑性，即进行转化的酶的区域，能够通过使用合成底物类似物和在进化过程中酶活性位点组成的细微改变来生产许多萜类化合物。据推测，在实验室中，活性部位的细微改变，即合并倍半萜合成酶的化学空间，将允许引入和操纵FDP的类似物，并导致生产天然存在的倍半萜的“非天然”类似物。卡迪夫和洛桑的目标是利用(S)-germacrene D来验证这一关于倍半萜合成酶活性位点改变的假设。(S)-germacrene D被洛桑鉴定为一种有效的蚜虫（世界主要作物害虫）的符号化学物质，作为模型外部符号化学物质。预计修饰后的germacrene D合成酶酶位点的化学空间在结构上与野生型GDS接近，因此类似物在结构上与(S)-germacrene D非常匹配，从而具有较高的半化学活性。因此，该项目的总体目标将是生产稳定的，具有生物活性的(S)-微生物烯D类似物，代表生物活性天然产物的活性类似物的第一个设计。具体目标包括：1)使用已建立的实验室细菌系统生产纯化酶；2)开发新的化学方法来合成可添加到酶制剂中的FDP类似物；3)利用未修饰的(S)-germacrene D合成酶将合成的FDP底物转化为(S)-germacrene D类似物；4)进行定点诱变，以产生修饰的(S)- germacrene D合成酶，并用于将合成的FDP底物转化为类似物5)使用昆虫触角的电记录（电生理学）和实验室行为分析，测量与一系列经济上重要的蚜虫物种产生的(S)- germacrene D类似物的活性；6)完善假设，设计新的底物和饲料来修饰GDS，并评估精制类似物的电生理和行为活性。

Many interactions between organisms in nature are mediated by external chemical signals, generally termed semiochemicals, that are typically low molecular weight lipophilic compounds. These interactions involve microbes (e.g. bacteria), algae, higher plants and animals, including human beings. Upon release by an emitting organism, such signals can act intra- or interspecifically by modifying either the behaviour or the development of recipient organisms. Whenever work is reported on identifying the signals or developing their practical use, i.e. managing pest organisms, the question 'why not design structural analogues?' is raised. Such an approach could potentially solve many problems associated with the use of semiochemicals, such as instability and volatility, which result in reduced efficacy. However, no rational approach has emerged by which to conduct structure activity relationship studies, to design analogues with greater stability and performance. The reason cited is that receptor systems, e.g. the animal peripheral sensory neurons, for external signal recognition are more highly selective, as a consequence of a need to select from an enormous diversity and concentration range of external chemicals, than receptor systems within organisms for which analogues can readily be designed. The family of sesquiterpenes, i.e. naturally-occurring chemicals that comprise of 3 x 5-carbon units, is widely diverse in nature and can have various signalling roles. The Cardiff laboratory has shown that enzymes (terpene synthases), which are involved in sesquiterpene production, rely on a three-dimensional structure for conversion of a precursor (farnesyl diphospate, FDP), which is shared by all sesquiterpene synthases. Plasticity in the active site of the synthases, i.e. the region of the enzyme that performs the conversion, enables the production of many terpenoids through the use of synthetic substrate analogues and subtle alterations in the composition of the active site of enzymes during evolution. It is hypothesized that subtle alterations in the active site, i.e. incorporating the chemical space of sesquiterpene synthases, in the laboratory will allow the introduction and manipulation of analogues of FDP, and lead to the production of 'non-natural' analogues of naturally-occurring sesquiterpenes. Together, Cardiff and Rothamsted aim to test this hypothesis regarding active site alteration of sesquiterpene synthases using (S)-germacrene D, which is identified by Rothamsted as a potent semiochemical for aphids, major world crop pests, as the model external semiochemical. It is also expected that the chemical space of the enzyme site in modified germacrene D synthase will be structurally close to that of the wild type GDS, and, therefore, the analogues will closely match (S)-germacrene D in terms of structure, thereby exhibiting high semiochemical activity. Thus, the overall aim of the project will be to produce stable, biologically active analogues of (S)-germacrene D, representing the first design of active analogues of a biologically active natural product. The specific objectives include: 1) production of the purified enzymes using an established laboratory bacterial system; 2) develop novel chemistry to produce synthetic FDP analogues that can be added to enzyme preparations; 3) convert synthetic FDP substrates to (S)-germacrene D analogues using unmodified (S)-germacrene D synthase; 4) Perform site-directed mutagenesis for the creation of modified (S)- germacrene D synthases, and use to convert synthetic FDP substrates to analogues 5) use electrical recordings of the antennae of insects (electrophysiology), and laboratory behavioural assays, to measure the activity of generated (S)- germacrene D analogues with a range of economically important aphid species; 6) refine the hypothesis, design new substrates and feed to modified GDS, and assess the electrophysiological and behavioural activity of the refined analogues.