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

几个世纪以来，人类一直使用植物和真菌的天然产物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。天然产品及其衍生物仍然至关重要，占目前市场上药物的很大比例。了解它们在自然界中是如何产生的（生物合成）为药物开发提供了新的机会。聚酮化合物是一类重要的天然产物，具有多种结构和生物活性，如抗菌、抗真菌、抗癌等。莫匹罗星是从荧光假单胞菌中分离出的四种假单胞菌酸（PA）的混合物，是具有有趣生物合成途径的聚酮化合物的一个例子。它在临床上很重要，对MRSA（耐甲氧西林金黄色葡萄球菌）有活性，但由于其不稳定性仅局部使用。在一个冒险的跨学科计划，涉及微生物分子遗传学，生物化学和化学，我们已经学到了很多关于PA生物合成有许多有趣的功能，但需要进一步的研究，以充分了解。在我们的新计划中，我们的目标是在具有已证实的生物活性的分子之间创造新的杂交体，以增加开发新抗生素的机会，从而克服与耐药性相关的问题。由海洋细菌天然产生的抗菌化合物的硫代马林醇家族由两种元素组成：一种是PA，与莫匹罗星相似（但不相同）;另一种是像一种不太了解的化合物的核心，全霉素（更一般地称为吡咯）。我们最近的研究证实，基因负责使thiomarinol类似于细菌荧光假单胞菌中的莫匹罗星和一个尚未表征的基因簇，从丝状细菌链霉菌clavuligerus，我们现在可以分配到holomycin生物合成。从这个新的基因簇中，我们可以推断出，全霉素是由一种不寻常的蛋白质工厂产生的，这种蛋白质工厂被称为非核糖体肽合成酶，它首先连接两个半胱氨酸分子。我们已经证明，硫代马利诺的两个元素是分别合成的，然后连接在一起。使用突变体，我们可以将全霉素加入莫匹罗星本身，使其克服其靶酶异亮氨酰tRNA合成酶中的莫匹罗星抗性。因此，这确定了一种方法，可以修改现有的抗生素，以克服临床上正在蔓延的耐药性。我们的目标之一是使用遗传操作来用其他化学物种取代吡咯，以创造有利的新特性。我们将使用不同的基因簇来确定什么可以加入PA，以及对一系列当前超级细菌和其他重要的医学重要目标具有什么生物活性。我们还将化学合成的底物喂给不产生吡咯的突变体，以产生新化合物的家族。此外，由于制造吡咯的基因是不寻常的，我们将详细研究它们，以防它们的特性教会我们新的方法来构建可能成为新型抗生素一部分的小分子。了解莫匹罗星和硫代马利诺生物合成中的关键步骤是如何控制的也是至关重要的-特别是特定生物转化的时机和机制。莫匹罗星生物合成途径是其类别的原型，并且很好地回答了适用于相关系统的许多一般性问题。最后，我们将研究抗生素与其靶酶的相互作用，模拟它们的结构，并将小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改，这将创造新的生物学特性，有效地对抗关键的细菌病原体

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