Derivation of β-amyloid and α-synuclein antagonists - We are developing peptides  that  can be used to effectively inhibit formation of toxic amyloid species. This will be  achieved by screening large peptide libraries using a 'Protein-fragment  Complementation Assay'. In this intracellular assay (shown left), an murine   dihydrofolate reductase (mDHFR) substitutes for the bacterial enzyme that is inhibited  by the addition of trimethoprim to the growth medium, conferring cell survival. One half  of mDHFR is fused at the genetic level to the amyloid protein, and the second half  fused to the peptide library a separate plasmid. Only library members that bind to   amyloid will bring the two halves of mDHFR into close proximity when co-expressed in  E. coli, render the enzyme active, and result in colony formation under selective  conditions.

Characterising the growth of β-amyloid and α-synuclein antagonists - We are also  developing methods to monitor the formation of amyloid as well as test the efficacy of  PCA selected peptides, using a variety of techniques that include ThT dye binding  assays, and imaging. One of these techniques, known as 'Total Internal Reflection  Microscopy' (TIRFM), will image single molecules of amyloid precursor, while a  concomitant cell viability assay, known as an MTT assay, will demonstrate which of  the ThT/TIRFM imaged intermediatary species is the most toxic. We can then image  the effect of mixing inhibitors with the precursor to establish their mechanism of action  and precisely how they reduce toxicity. The information will then feed-forward into the  second tranche of more effective inhibitors by serving as the template for a second  PCA library.