Safer Aminoglycoside Therapeutics by Biosynthetic Engineering

Lead Research Organisation: University of Cambridge
Department Name: Biochemistry

Abstract

Bacterial infections in hospital patients can lead to sepsis, in which an overwhelming infection of the bloodstream by toxin-producing bacteria becomes life-threatening. Very few new antibiotics are being developed, and so the established antibiotic gentamicin, discovered over 40 years ago, is likely to remain a vital mainstay in efforts to combat sepsis. Unfortunately there are real dangers associated with its use: a large percentage of patients treated with gentamicin, or related antibiotics, develop acute kidney failure (nephrotoxicity). The drug can also cause irreversible hearing loss (ototoxicity). Treatment is very costly because patients have to be closely monitored to minimise these severe side-effects.
Sustained efforts are being made to research ways of minimising the side-effects of gentamicin, by altering the dosing strategy for example; and to understand the biochemical mechanisms by which the kidney and the inner ear are damaged. Unfortunately all the gentamicin used clinically is a mixture of compounds and different batches of commercial gentamicin have different amounts of each component. The individual components can be separated on a small scale but it has not been commercially viable to do this on the scale needed. It was believed that all components were equally effective and equally toxic, but in 2006 researchers in the USA re-tested each of the main components (known as C1, C1a, C2, and C2a) and showed that, surprisingly, purified component C2 is fully effective as an antibiotic but not nephrotoxic at all (at least in rats).
The aim of this project is to build on the success of our previous MRC-supported research aimed at deciphering all of the individual steps in the late stages of gentamicin biosynthesis. We now think that the best prospect for obtaining C2 by fermentation is to use as a feedstock the widely commercially available precursor called G418. However, we still need to deconvolute the role of key individual enzymes in this part of the pathway. The ready availability of such monocomponent gentamicins would encourage potentially safer formulations of the antibiotic to be tested. Since gentamicins and related aminoglycosides are also promising agents for the correction of certain human genetic diseases, such as cystic fibrosis and the muscle-wasting disease Duchenne muscular dystrophy, there could be wider medical benefits too.

Technical Summary

Gentamicin and related aminoglycosides are valuable broad-spectrum antibiotics, active against a broad spectrum of pathogenic bacteria. Gentamicin itself is used increasingly to combat hospital-acquired sepsis, despite the well-known danger of associated kidney damage and hearing loss. Unfortunately studies aimed at clarifying the mechanisms of toxicity have not taken into account that gentamicin as used clinically is a mixture of four main components with subtle structural differences. In a recent study, component C2 was reported not to cause kidney damage while retaining its antibacterial effectiveness. A monocomponent gentamicin with a greater margin of safety might bring significant patient benefit. In previous MRC-funded research we have developed methods of analysing the individual roles of the genes and enzymes that govern the biosynthesis of gentamicin in the bacterium Micromonospora echinospora. We have shown that pyridoxal-phosphate enzymes GenB2, GenB3, and GenB4 are the key to our attempts to design and construct a strain that can deliver single component gentamicins, especially C2. The first aim of this project is to use our panel of characterised mutant strains to isolate late-stage intermediates and use them to study these unusual enzymes, which bring about the loss of hydroxy groups that creates the distinctive gentamicin C structures. We aim then to reconstruct the entire pathway in vitro from the (readily commercially-available) precursor called G418 (geneticin) to gentamicin C2. This will provide the basis on which to construct and test recombinant strains to bioconvert G418 into single components of the gentamicin C complex, especially C2. Success in this would allow future toxicology studies to be carried out on pure components instead of mixtures, and if the results are promising would provide the basis for a sustainable process for manufacturing the safer drug.

Planned Impact

Who will benefit? A range of academic and industrial research groups who are working in natural product drug discovery
Companies including Novartis, Astellas, Sanofi-Aventis and Eisai who are following up natural product leads.
Leading producers of aminoglycosides such as Schering (gentamicin producer)
SMEs and biotechnology companies (Isomerase Therapeutics, Cubist, Novacta, WarpDrive Bio, Evolva) who are developing biosynthetic engineering strategies for expanding the diversity of natural products and for optimising the properties of lead compounds.
Public-private partnerships aiming to deliver drugs to developing countries (Oneworld Health, Gates, DNDi)
Both academic and industrial end-users will benefit through free access to publications on fundamental insights on individual enzyme mechanisms.
How will they benefit?
To ensure that the full benefit of the research is realised, we will protect IP arising from the project using the established mechanisms in the University of Cambridge. We will seek academic and industrial partners in order to test rigorously the pharmacological and toxicological properties of individual gentamicins. If the safety profile of an individual gentamicin is confirmed to offer potential for patient benefit, it could have a highly significant impact in an area of real clinical need.
Strategic impact?
Synthetic biology is an umbrella term for a range of activities in design and redesign of biological systems. It is rightly open to challenge from civil society. The kind of work we propose, if successful, could be among the first practical fruits of synthetic biology and help to establish this type of work as acceptable and worthy of public support and trust. It has been identified as a key technology for the UK, but the expected economic benefits will not materialise if we do not act prudently and transparently.

Publications

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Description Gentamicin biosynthesis 
Organisation Wuhan University
Department State Key Laboratory for Combinatorial Biosynthesis
Country China 
Sector Academic/University 
PI Contribution We will conduct all in vitro experiments in investigation of the biosynthetic pathway to gentamicin antibiotics; and undertake the heterologous expression of (subsets) of gentamicin biosynthetic genes in other actinomycete hosts; as well as refactoring the gene cluster to produce monocomponent gentamicins of lower nephrotoxicity
Collaborator Contribution Wuhan is conducting the wholesale genetic knockouts of individual genes for gentamicin biosynthesis, construction and analysis of double mutants, and attempts to generate high-yielding strains by conventional mutagenesis. All mutants are being scrutinised for (novel) metabolites and purified metabolites supplied to Cambridge for in vitro work.
Impact The Chinese Government has agreed to fund travel and subsistence for members of the Cambridge team to visit Wuhan for periods of two months each over the next three years, for joint work and scientific discussions. The first two mss arising from this work are in preparation and the first of these will be submitted to Chemistry and Biology within the next two weeks. We are also considering whether the discoveries we have made warrant the lodging of a patent application before the publication is made.
Start Year 2016
 
Description SEPARATION OF GENTAMICINS BY CCC 
Organisation Brunel University London
Department Brunel Institute for Bioengineering
Country United Kingdom 
Sector Academic/University 
PI Contribution WE SUPPLY BRUNEL WITH FERMENTATION SUPERNATANTS CONTAINING GENTAMICINS AND RELATED METABOLITES
Collaborator Contribution DR IAN GARRARD AND COLLEAGUES USE THEIR PROPRIETARY CENTRIFUGAL COUNTER-CURRENT CHROMATOGRAPHY TO SEPARATE HYDROPHILIC NATURAL PRODUCTS SUCH AS THE GENTAMICINS
Impact The chief output is a supply of homogenous gentamicin samples comprising all the four major components of clinically-used gentamicin mixtures. In the near future (1st/2nd Q 2014) we expect to start separating novel metabolites obtained from blocked mutants
Start Year 2016