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

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

The mupirocin biosynthetic gene cluster is one of the archetypes of the 'atypical' type I polyketide synthases that have trans-acting Acyl Transferases which deliver the starter and extender (malonate) units. We have studied it for a approximately 10 years in order to understand what controls a number of key features - most notably beta branch insertion via the HMGCoASynthase cassette, pyran ring formation and synthesis of the hydroxyl fatty acid that is joined to the monic acid core via a labile ester bond. We have recently sequenced the genomes of two marine bacteria to reveal the biosynthetic genes for the related thiomarinol compounds. These are essentially a hybrid between pseudomonic acid C (a minor component of mupirocin which is mainly pseudomonic acid A) and a pyrrothine. The new genes have provided the basis of comparative genomics and gene swap experiments. By comparing all known acyl carrier proteins that are associated with beta-branch insertion via an HCS cassette we have identified a uniquely conserved tryptophan that must be a signature for this activity. We will test knockouts and knockins to generate a general strategy for polyketide backbone modification. We will add selected modules from other pathways to extend the backbone to create analogues lacking the ester bond that is one reason for rapid inactivation of mupirocin. Gene knockouts have identified the pseudomonic acid and the pyrrothine biosynthetic genes as well as a gene, tmlU, encoding an amide ligase that joins the two components together. We have shown that the addition of pyrrothine is critical to potentiate the activity of the pseudomonic acid against both Gram-postive and negative bacteria and in the new grant part of the work will seek to understand how this occurs and whether we can substitute other molecules for the pyrrothine in order to generate families of new molecules that may have desirable properties.

Our primary aim is to provide the basis for the development of a range of novel pharmaceuticals by our modification of the mupirocin and thiomarinol biosynthetic pathways. This will provide knowhow for others to modify diverse biosynthetic pathways using the novel generic methods that we shall elucidate. This fits with the stated BBSRC strategy of 're-designing existing natural biological systems for useful purposes'. Already our work has been featured in the BBSRC publication 'The Science Behind Tackling Superbugs'. Our proposal targets this through an interdisciplinary project that is directly engaged with industry so as to be guided by applied needs and to ensure that the knowledge generated is used effectively. Polyketides alone are worth billions of pounds to the World economy each year and this work will contribute to the development of this field. Methicillin Resistant Staphylococcus aureus (MRSA) has spread beyond hospitals into the wider community. In the USA MRSA is among the leading causes of death by a single infectious agent, surpassing fatalities due to tuberculosis and HIV/AIDS. Mupirocin is currently used widely as an antibiotic against MRSA but cannot be used systemically due to both chemical instability and adverse effects. We believe that by modification of mupirocin through knockouts and knockins of the beta-branching modification of the backbone as well as removal of the epoxide and the potentially labile epoxide bond we will generate more versatile and useful sets of derivatives that will be of interest to one of the producers of mupirocin - GSK, Centany, etc. If we are successful then this will have major health and economic impact. On thiomarinol we are working closely with the Japanese Pharmaceutical company Daiichi-Sankyo who have expressed a desire to develop and exploit its potential properties and have already funded a member of their staff to work with us for two years. They are interested in the possibility that thiomarinol or derivatives could be used against MRSA but they would also be open to alternative lines of development - for example, the possibility that compounds related to thiomarinol could be developed as anticancer agents if derivatives turned out to be toxic to human normal or cancerous cell lines. We have a Material Transfer and IPR agreement in place that allows the University of Birmingham a stake in any practical application derived from the studies of the thiomarinol-producing bacteria, which are owned by Daiichi-Sankyo. If such developments materialise this agreement provides a mechanism for the proper development and marketing of novel molecules as well as leading in the long term to financial reward coming to the UK. We already have experience of the speed with which decisions can be made having just been through such discussions in preparation for submitting our first paper on thiomarinol (to Science). The knowledge that we generate will impact on how secondary metabolic pathways, particularly the production of polyketides, are understood. The logic that underpins these biosynthetic systems allows a great deal of predictive biology to underpin our development work. These compounds are a topic in many industrial biotechnology courses so our work will impact on the university curriculum providing examples of how bacteria can be manipulated for the benefit of man. These will appear in reviews and eventually in text books. The topic also attracts students to an area that has direct benefit to society. It provides a context in which students not only learn about the details of the particular organisms and molecules being studied but they can be enthused by scientific research and its benefits. It therefore helps us to recruit good students, keep their interest and help them to graduate with skills that are important in future wealth generation from industrial biotechnology.