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

点击翻译按钮获取中文摘要

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