Novel hybrid anti-MRSA antibiotics from manipulation of the mupirocin and thiomarinol biosynthetic pathways

几个世纪以来，人类一直将天然产品用于植物和真菌的粗提物以及纯化形式，作为治疗疾病的药物。许多这样的化合物已经成为家喻户晓的名字，例如杀死细菌的青霉素抗生素。天然产品及其衍生品仍然至关重要，占目前市场上药物的很大比例。了解它们是如何在自然界中制造的(生物合成)为药物开发打开了新的机会。聚酮类化合物是一类重要的天然产物，具有抗菌、抗真菌、抗癌等多种结构和生物活性。莫匹罗星是从荧光假单胞菌中分离出来的四种假单胞酸(PAS)的混合物，它是聚酮的一个例子，具有有趣的生物合成途径。它在临床上很重要，对耐甲氧西林金黄色葡萄球菌(MRSA)有活性，但由于其不稳定，仅用于局部。在一个涉及微生物分子遗传学、生物化学和化学的冒险交叉学科项目中，我们学到了许多关于PA生物合成的知识，它具有许多有趣的特征，但需要进一步研究才能完全理解。在我们的新计划中，我们的目标是在具有已被证明的生物活性的分子之间创造新的杂交，以增加开发能够克服与耐药性相关的问题的新抗生素的机会。由海洋细菌自然产生的硫代马林酚抗菌化合物家族由两个元素组成：一个是PA，与莫匹罗星相似(但不完全相同)；另一个类似于一种不太为人所知的化合物的核心，全息霉素(更普遍地称为吡咯氨酸)。我们最近的研究证实，负责制造硫代马林醇的基因既类似于荧光假单胞菌中的莫匹罗星基因，也类似于丝状细菌棒状链霉菌的一个尚未确定的基因簇，我们现在可以将其归类为全霉素的生物合成。根据这个新的基因簇，我们可以推断，全息霉素是由一种名为非核糖体多肽合成酶的不寻常形式的蛋白质工厂产生的，它是从连接两个氨基酸半胱氨酸分子开始的。我们已经证明，硫代马利诺的两种元素是分开制造的，然后连接在一起。使用突变体，我们可以将全息霉素与莫匹罗星本身连接起来，使其在其靶酶异亮氨酰tRNA合成酶中克服莫匹罗星的耐药性。因此，这确定了一种方法，可以对现有的抗生素进行修改，以克服正在临床传播的耐药性。我们的目标之一是使用基因操作来用其他化学物种取代吡咯烷，以创造有利的新性质。我们将使用不同的基因簇来确定PA可以与哪些基因结合，以及具有哪些生物活性来对抗一系列当前的超级细菌和其他重要的医学目标。我们还将把化学合成的底物喂给不产生吡咯氨酸的突变体，以产生一系列新的化合物。此外，由于制造吡咯氨酸的基因不同寻常，我们将对它们进行详细研究，以防它们的性质教会我们制造小分子的新方法，这些小分子可能成为新型抗生素的一部分。了解如何控制莫匹罗星和硫代马林醇生物合成的关键步骤--特别是特定生物转化的时间和机制也是至关重要的。莫匹罗星生物合成途径是其类别的原型，很好地回答了一些适用于相关系统的一般问题。最后，我们将研究抗生素与其目标酶的相互作用，对其结构进行建模，并将小型抗生素与蛋白质对接，以预测分子的哪些部分是重要的，以及为什么。这应该有助于指导所需的修饰，从而创造出有效对抗关键细菌病原体的新的生物学特性

For centuries mankind has used natural products in crude extracts from plants and fungi as well as in purified form as remedies to cure disease. Many such compounds have become house-hold names e.g. the penicillin antibiotics which kill bacteria. Natural products and their derivatives are still vitally important, representing a large proportion of drugs currently on the market. Understanding of how they are made in nature (biosynthesis) opens new opportunities in drug development. Polyketides, an important class of natural products, exhibit huge diversity of structures and biological activities e.g. antibacterial, antifungal and anticancer. Mupirocin, a mixture of four pseudomonic acids (PAs) isolated from the bacterium Pseudomonas fluorescens, is an example of a polyketide with an intriguing biosynthetic pathway. It is clinically important, being active against MRSA (methicillin resistant Staphylococcus aureus), but is only used topically due to its instability. In an adventurous interdisciplinary programme involving microbial molecular genetics, biochemistry and chemistry we have learnt much about PA biosynthesis which has many intriguing features but requires further study for a full understanding. In our new programme we aim to create new hybrids between molecules with proven biological activity to increase the chance of developing new antibiotics that can overcome problems associated with resistance. The thiomarinol family of antibacterial compounds produced naturally by marine bacteria consists of two elements: one, a PA is similar (but not identical) to mupirocin; the other is like the core of a less well understood compound, holomycin (more generically called pyrrothine). Our recent studies confirm that the genes responsible for making thiomarinol resemble both those for mupirocin in the bacterium Pseudomonas fluorescens and an as yet un-characterised gene cluster from the filamentous bacterium Streptomyces clavuligerus which we can now assign to holomycin biosynthesis. From this new gene cluster we can deduce that holomycin is produced by an unusual form of a protein factory called a non-ribosomal peptide synthetase and starts by joining two molecules of the amino acid cysteine. We have shown that both elements of thiomarinol are made separately and then linked together. Using mutants we can join holomycin to mupirocin itself allowing it to overcome mupirocin resistance in its target enzyme, isoleucyl tRNA synthetase. This therefore identifies a way that an existing antibiotic can be modified to overcome the resistance that is spreading clinically. One of our aims is to use genetic manipulation to replace the pyrrothine by other chemical species to create favourable new properties. We will use diverse gene clusters to determine what can be joined to PA and with what biological activity against a range of current superbugs and other important medically important targets. We will also feed chemically synthesised substrates to mutants that do not make the pyrrothine in order to generate families of new compounds. Also, because the genes that make the pyrrothine are unusual we will study them in detail in case their properties teach us new ways to build small molecules that could become part of novel antibiotics. It is also vital to understand how key steps in mupirocin and thiomarinol biosynthesis are controlled - particularly the timing and mechanism of specific biotransformations. The mupirocin biosynthetic pathway is an archetype of its class and is well placed to answer a number of general questions that apply to related systems. Finally we will study the interaction of the antibiotics with their target enzymes, modelling their structures and docking the small antibiotics with the proteins to predict which parts of the molecules are important and why. This should help direct desirable modifications that will create novel biological properties effective against key bacterial pathogens