Design, synthesis and testing of novel phytopathogenic fungicides.

Enzymes are proteins that facilitate the reactions that enable living organisms to acquire energy for growth, reproduction and maintenance. A key challenge in understanding the structure-function relationship of one such group of enzymes, the alternative oxidases (AOX), rests upon the identification of its substrate and inhibitor-binding site and its mechanism of action. A detailed knowledge of the nature of this binding site is important since it will reveal whether or not there is a common architecture that can be applied to substrate and inhibitor-binding sites in general and hence provide an insight into the mechanism of binding. More importantly, this knowledge will assist in the suitable rational design of phytopathogenic and anti-parasitic drugs that are specifically targeted to the alternative oxidase. The alternative oxidase is not only present in fungi and plants but also widespread amongst human parasites such as Trypanosoma brucei (the causative agent of African sleeping sickness), intestinal parasites such as Cryptosporidium parvum (responsible for an airborne intestinal infection cryptosporidiosis) and opportunistic human pathogens such as Candida albicans (causes candidiasis or 'thrush'). With respect to the role of AOX in fungi, the development of resistance to agrochemicals by plant fungal pathogens is an international problem that affects all major crops. Indeed fungicide resistance is an important factor in the successful cultivation of cereals in the UK. It is estimated that the UK market for fungicides in cereals is approximately £250M (worldwide $3bn) with winter wheat being the main crop. Fungicides are used against a number of diseases, the major one of winter wheat being caused by Septoria tritici. The main chemical classes of fungicides used to treat UK cereals include the sterol biosynthesis inhibitors. The most important and successful group of these fungicides that have proved effective in the control of plant pathogens are the strobilurin fungicides which are specifically targeted to the mitochondrial respiratory chain (Qo site) thereby inhibiting fungal respiration. Unfortunately resistance to this fungicide often develops resulting in an inability to control fungal pathogens through continued application. Although the mechanism for conferring resistance to Qo fungicides is still controversial there is growing evidence to suggest that the addition of inhibitors, such as azoxystrobin, to fungal pathogens results in a strong induction of the alternative oxidase (AOX). AOX is a mitochondrial terminal oxidase which by-passes the Qo site and is induced in all plants, fungal pathogens and protists following stress induction. We have previously demonstrated that fungal plant pathogens such as Septoria tritici, a fungus that causes major leaf spot diseases in wheat & the wheat "Take-all" fungus, Gaeumannomyces graminis var. tritici have the capacity to express AOX when treated with respiratory inhibitors thereby allowing a strobilurin-resistant respiratory pathway to develop which may account for the varying efficacy of strobilurin fungicides. The major objective of this study is to identify formulations containing novel compounds which inhibit the fungal alternative oxidase and/or the main respiratory chain thereby providing a technique for the effective control of fungal diseases in cereal crops. We have already designed a number of these compounds (PCT/GB2015/050148 & PCT/GB2013/051030) which have proved to be potent inhibitors of the Ash dieback fungus and have demonstrated that these compounds are also effective against Septoria tritici. We will express the fungal rAOX enzyme in a yeast which will enable us to not only to quickly screen the compounds inhibitory effectiveness but also through genetic manipulation of the fungal enzyme ascertain the extent to which the enzyme may become resistant to the compounds we have synthesised.

The research outlined in this proposal builds upon our continued research programme on the characterisation of the quinone-binding and active-site of the alternative oxidases. Outcome of this research to date confirms that the quinone-binding site of the alternative oxidase is a suitable and promising novel target in the treatment of fungal pathogens. A major milestone and breakthrough of this study was the determination of the first crystal structure of an AOX protein both in the presence and absence of a number of stoichiometric inhibitors. A knowledge of the structure of AOX in the presence of a number of inhibitors now places us in a very powerful position to undertake some further rational fungicidal molecular design which we predict will result in the production of a library of compounds that have the capacity to act as phytopathogenic fungicides specifically targeted not only at the AOX but additionally at the cytochrome bc1 complex.

This will be achieved through;

Strand 1. The design and synthesis of novel AOX inhibitors. We will build upon the progress outlined in PCT/GB2015/050148 & PCT/GB2013/051030 to synthesize a library of compounds which will then be tested on membrane-bound and purified fungal rAOX protein expressed in E. coli.

Strand 2. Random and site-specific mutagenesis studies to identify additional amino acid residues that may lead to inhibitor-resistance.

Strand 3. Kinetic characterisation of wt and mutant forms of AOX using polarographic and spectrophotometric techniques to measure activity and inhibition kinetics.

Strand 4. Antifungal susceptibility will be determined using two methods to test the sensitivity of fungal cells to the compounds generated under Strand 1: namely the agarose disc diffusion method and a plate assay system using strains of S. cereviseae in which the cytochrome b gene has been mutated.

Strand 5. Structurally characterise the inhibitor-binding sites through modelling and crystallography.

1 Who will benefit from this research?
The alternative oxidase is a respiratory enzyme that decreases the efficiency of mitochondrial energy conservation but which is poorly understood. Our research, which is aimed at improving the structural and mechanistic understanding of the alternative oxidase, will contribute to the elucidation of the molecular basis of the energy metabolism of fungi and plants, clearly a key biological process in both organisms. The overall aim of our research is to effectively control fungal diseases in cereal crops through the development of inhibitors which are specifically targetted at the fungal alternative oxidase. In
addition to researchers in the immediate professional circle carrying out similar research, other wider beneficiaries include:
Within the commercial private sector those in the agrochemical (for treatment of plant fungal pathogens such as DowAgroSciences, Syngenta, BASF etc) and pharmaceutical industries in the development of drugs to treat trypanosomiasis and cryptosporidiosis - parasites which also contain an identical alternative oxidase;
Those working in infectious and tropical disease institutes- in particular those working with intestinal parasites such as Blastocystis and opportunistic human pathogens such as Candida;
Potential policy-makers, within international, national, local or devolved government and government agencies who are actively involved in delivering financial aid in developing countries, such as DfID, for the treatment of the above diseases;
Other beneficiaries within the public sector who might use the results to their advantage include organisations such as the Eden Project and Kew Gardens who are keen to get publicly-funded scientists to disseminate the impact of their results to a wider audience. Results from this type of project are of particular interest since information such as this informs the public how research from plants and fungi can be used to generate drugs to treat human diseases.

2 How will they benefit from this research?
The fundamental knowledge that is gained by the project could very well find industrial application, since the alternative oxidase has been implicated as a potential target for both phytopathogenic fungicides and certain anti-parasitic pharmaceuticals. The rational design and development of such compounds, particularly dual-mode anti-fungals, will benefit through an enhanced molecular insight of the alternative oxidase structure and catalysis. Furthermore information on the substrate and inhibitor binding sites will prove invaluable in the design of rational inhibitors.
Should the project prove successful and result in the identification of additional fungicides which specifically target the alternative oxidase the research has the real potential to impact on the nation's health, wealth and culture. Furthermore it has the potential to impact upon the economic competitiveness of the United Kingdom through the development of drugs to treat trypanosomiasis and cryptosporidiosis by pharmaceutical companies and fungicides, by agrochemical companies, to treat plant pathogens which attack economically important cereals thereby increasing the UK's global economic
performance. It is estimated that the UK market for fungicides in cereals is approximately £250M with winter wheat being the main crop.

3 Timescales
The design and development of suitable phytopathogenic fungicides and anti-parasitic pharmaceuticals will take a considerable time (probably >10 yrs) but nevertheless should this prove successful then it will have considerable impact upon the UK's global economic performance since, for instance, the current global annual expenditure on the above fungicides is in excess of $10bn.

4 Staff Development
The type of research and professional skills which staff working on this project will develop include:the ability to multitask,
work in a team with non-scientists and those within the agrochemical community.