Design of bioactive sesquiterpene-based chemical signals with enhanced stability

自然界中生物体之间的许多相互作用是由外部化学信号介导的，通常称为化学信息素，其通常是低分子量亲脂性化合物。这些相互作用涉及微生物（例如细菌）、藻类、高等植物和动物，包括人类。在由发射生物体释放时，这种信号可以通过改变受体生物体的行为或发育而在种内或种间起作用。每当有关于识别信号或开发其实际用途的工作报告时，即管理有害生物，问题是“为什么不设计结构类似物？”被提升。这种方法可以潜在地解决与使用化学信息素相关的许多问题，例如导致功效降低的不稳定性和挥发性。然而，没有合理的方法已经出现，进行结构活性关系的研究，设计类似物具有更大的稳定性和性能。所引用的理由是，由于需要从大量多样性和浓度范围的外部化学物质中进行选择，因此用于外部信号识别的受体系统，例如动物外周感觉神经元，比生物体内的受体系统具有更高的选择性，生物体内的受体系统可以容易地设计类似物。倍半萜家族，即包含3 X 5-碳单元的天然存在的化学物质，在性质上是广泛多样的，并且可以具有各种信号传导作用。卡迪夫实验室已经表明，参与倍半萜烯生产的酶（萜烯脱氢酶）依赖于三维结构来转化所有倍半萜烯脱氢酶共有的前体（法呢基二磷酸，FDP）。酶的活性位点（即进行转化的酶区域）的可塑性使得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产生许多萜类化合物。据推测，在实验室中，活性位点的细微改变，即掺入倍半萜烯脱氢酶的化学空间，将允许引入和操纵FDP的类似物，并导致天然存在的倍半萜烯的“非天然”类似物的产生。卡迪夫和Rothamsted共同致力于使用（S）-大根香叶烯D作为外部信息素模型来检验关于倍半萜烯脱氢酶的活性位点改变的这一假设，大根香叶烯D被Rothamsted鉴定为蚜虫（主要世界作物害虫）的有效信息素。还预期修饰的大根香叶烯D合酶中酶位点的化学空间在结构上接近于野生型GDS的化学空间，因此，类似物在结构上与（S）-大根香叶烯D紧密匹配，从而表现出高的化学信息素活性。因此，该项目的总体目标是生产稳定的、具有生物活性的（S）-大根香叶烯D类似物，这是首次设计具有生物活性的天然产物的活性类似物。具体目标包括：1）使用已建立的实验室细菌系统生产纯化的酶; 2）开发新的化学方法以生产可添加到酶制剂中的合成的FDP类似物; 3）使用未修饰的（S）-大根香叶烯D合酶将合成的FDP底物转化为（S）-大根香叶烯D类似物; 4）进行定点突变以产生修饰的（S）-吉马烯D合酶，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）和实验室行为测定，以测量所产生的（S）-大根香叶烯D类似物与一系列经济上重要的蚜虫物种的活性; 6）完善假设，设计新的底物并进料至经修饰的GDS，并评估经完善的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.