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，通常被称为信号化学物质，通常是低分子量亲油化合物。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。这种信号一旦被发射有机体释放，就可以通过改变受体有机体的行为或发育而在特定范围内或之间发挥作用。每当有关于识别信号或开发其实际用途的工作被报道时，即管理害虫生物体，问题就会出现：为什么不设计结构类似物？都被养大了。这种方法可能会解决与使用信号化学品有关的许多问题，如不稳定和挥发性，这会导致药效降低。然而，还没有出现合理的方法来进行结构活性关系研究，设计具有更高稳定性和性能的类似物。引用的原因是，由于需要从外部化学物质的巨大多样性和浓度范围中进行选择，用于外部信号识别的受体系统，例如动物外周感觉神经元，比生物体内易于设计类似物的受体系统具有更高的选择性。倍半萜家族，即由3×5碳单元组成的自然产生的化学物质，在性质上有广泛的多样性，可以有不同的信号作用。卡迪夫实验室已经证明，参与倍半萜生产的酶(萜烯合酶)依赖于一个三维结构来转换前体(法尼基二磷酸盐，FDP)，这是所有倍半萜合酶所共有的。合成酶活性部位的可塑性，即执行转换的酶区域，能够通过使用合成底物类似物和进化过程中酶活性部位组成的细微变化来产生许多萜类化合物。据推测，活性部位的细微变化，即在实验室中加入倍半萜合成酶的化学空间，将允许引入和操纵FDP的类似物，并导致自然产生的倍半萜类似物的产生。加的夫和Rothamsted共同致力于使用(S)-Germacene D来检验这一关于倍半萜合成酶活性位点变化的假说，Germacene D被Rothamsted确定为对世界主要农作物害虫--蚜虫的一种有效的信息化学物质，作为模型外部信息化学物质。还预计修饰的杰马烯D合成酶的酶中心的化学空间将在结构上接近野生型GDS，因此，类似物在结构上将与(S)-杰马林D紧密匹配，从而显示出高的信号化学活性。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)-杰马烯D类似物，这是生物活性天然产品的活性类似物的第一个设计。具体目标包括：1)利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产生可添加到酶制剂中的合成的fdp类似物；3)使用未修饰的(S)-germacene D合成酶将合成的fdp底物转化为(S)-germacene D类似物；4)进行定点突变以产生修饰的(S)-germacene 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，以测量产生的(S)-germacene 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，设计新的底物和饲料来改良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.