The design and synthesis of Novel AOX and cytochrome bc1 complex inhibitors which act as phytopathogenic fungicides

Misselbrook, James (2019) The design and synthesis of Novel AOX and cytochrome bc1 complex inhibitors which act as phytopathogenic fungicides. Doctoral thesis (PhD), University of Sussex.

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Abstract

The metabolic pathway for oxidative phosphorylation has remained a reliable target for the development of novel fungicides. The reliance on single target site fungicides within this pathway has increased the selection pressure for point mutations within a number of key complexes, including the cytochrome bc1 complex. The alternative oxidase (AOX) provides an alternative route for respiration, introducing a mechanism by which selection pressure and pathogenicity can be increased. In particular, the fungal pathogen Septoria tritici has developed a highly fungicide resistant strain. The design and synthesis of inhibitors targeting the fungal AOX, or the AOX and the cytochrome bc1 complex, represents a new class of fungicides improving crop yield outcomes.

Investigation into the AOX found in S. tritici (StAOX), led to new techniques to fully characterise and overexpress the protein in a haem deficient Escherichia coli strain, which has then been compared to that of the well-studied protozoan AOX, Trypanosoma brucei brucei (TAO). The enzymatic activity of StAOX was found to be significantly lower in comparison to AOXs from other species, but responded dramatically to the nucleotide regulators, GMP and IMP. The purified protein has also been shown to be sensitive to its lipid environment with full enzymatic recovery following re-introduction into a lipid membrane.

The natural quinol analogue, ascofuranone, displays selectivity towards the AOX and was selected for further design and lead modification. Several ascofuranone derivatives were synthesised according to a new synthetic route. A variety of new methods were utilised to analyse the inhibitors providing IC50, KD, thermodynamic and cytotoxicity data. Two of the newly synthesised compounds provided selectivity for TAO over the cytochrome bc1 complex, but failed to show selectivity to the StAOX. In vitro data suggests a single phenylalanine residue restricts inhibitor’s tail length to within an 8-carbon chain length, supported by an in-silico docking screen.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QP Physiology > QP0501 Animal biochemistry > QP0550 Organic substances > QP0551 Proteins, amino acids, etc. > QP0552 Special proteins (other than amino acids, enzymes, or hormones), A-Z > QP0552.C94 Cytochromes
T Technology > TP Chemical technology > TP0200 Chemicals > TP0247 Organic chemicals and preparations > TP0248 Special, A-Z > TP0248.F79 Fungicides
Depositing User: Library Cataloguing
Date Deposited: 13 Jun 2019 10:00
Last Modified: 10 Mar 2020 12:11
URI: http://sro.sussex.ac.uk/id/eprint/84220

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