Activator Protein 1 (AP-1) is a basic leucine zipper (bZIP) transcription factor that is formed from the dimerisation of cJun and cFos proteins to form a coiled coil (CC). As AP-1 is implicated in the pathways of multiple cancers (such as breast cancer, cervical cancer, and colon cancer) this α-helical peptide-peptide interface represents an interesting target for the inhibition of AP-1 formation and transcriptional activity. Peptidomimetic sequences based on cFos are able to preferentially bind to cJun. Three methods were explored to develop and optimise the use of in silico screening methods in the design of cJun antagonists. Firstly, in cellulo screening techniques were modelled computationally and used to design a stable and specific cJun antagonist. Secondarily, this method was further expanded in cellulo with Protein-fragment Complementation Assay screening, with the aim of validating the peptide library generated. Both peptides derived from these techniques showed similarly high levels of stability with cJun and promoted instability in negative and off-target complexes. The in cellulo derived sequence showed additional co-compatability with a previously characterised cFos antagonist and allowed for specific targeting of both AP-1 components. Finally, a set of heptapeptide “cassettes” were synthesised and characterised in both a linear and cyclic form, using b-f lactam bridges. The variances in helicity seen in the lactamised forms highlighted differences in constraint tolerance. This data was coupled with computational screening data to modularly select a combination of cassettes predicted to bind to cJun. Full-length peptides selected computationally were shown to have decreased homodimeric and heterodimeric stability, whereas the combined in-silico/in-vitro selections showed high levels of stability and were able to successfully target cJun. This provides novel insights into the tolerance of constraints and the design of peptides for stability and helicity. Overall, the methods developed (and the peptides derived) represent a step forward in the design of specific peptide therapies capable of targeting AP-1.
Helical constraints and in silico library screening to develop novel antagonists of oncogenic Activator Protein-1: (Alternative Format Thesis)
Lathbridge, A. (Author). 19 Feb 2020
Student thesis: Doctoral Thesis › PhD