Red-light modulated ortho-chloro azobenzene photoswitch for peptide stapling via aromatic substitution

Acknowledgements: M. K. acknowledges EPSRC for funding (SynTech EP/S024220/1). F. J. P. A. gratefully acknowledges both postdoctoral fellowships from Fundación Ramón Areces (reference BEVP31A6160) and Marie Skłodowska-Curie Individual Fellowships (MSCA-IF-2020, grant number 101025271). N. A. acknowledges a studentship from AstraZeneca. T. S. acknowledges EU funding by the H2020-MSCA-RISE-2020 through the ALISE project (grant 101007256). The Spring group research was supported by grants from UKRI. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript (AAM) version arising. Authors are also grateful to Dr Rohan Eapen for the purification of MDM2 protein, Dr Katherine Stott for interpretation of CD spectra and the NMR team at Cambridge for measuring advanced NMR experiments (Andrew Mason, Duncan Howe and Peter Gierth).The application of peptide stapling using photoswitchable linkers has gained notable interest for potential therapeutic applications. However, many existing methodologies of photoswitching still rely on the use of tissue-damaging and weakly skin-penetrating UV light. Herein, we describe the development of a tetra-ortho-chloro azobenzene linker that was successfully used for cysteine-selective peptide stapling via SNAr. This linker facilitates precise photocontrol of peptide structure via trans to cis isomerisation under red light irradiation. As a proof-of-concept, we applied the developed peptide stapling platform to a modified PMI peptide, targeting the inhibition of MDM2/p53 protein-protein interaction (PPI). Biophysical characterisation of the photoswitchable peptide by competitive fluorescence polarisation showed a significant difference in affinity between the trans and cis isomer for the p53-interacting domain of the human MDM2. Remarkably, the cis isomer displayed a >240-fold higher potency. To the best of our knowledge, this is the highest reported difference in binding affinity between isoforms of a photoswitchable therapeutic peptide. Overall, our findings demonstrate the potential of this novel photoswitchable peptide stapling system for tuneable, selective modulation of PPIs via visible-light isomerisation with deeply-tissue penetrating red light

Protein folding in the secretory pathway of animal cells

The exit of newly-synthesized proteins from the lumen of the endoplasmic reticulum (ER) is the rate-determining step in protein secretion. Only correctly-folded and fully-assembled proteins exit the ER and progress along the secretory pathway. Folding and assembly in the ER are mediated by a variety of factors including folding catalysts and molecular chaperones. The properties of these factors, and the nature of their interactions with folding substrates, are beginning to be clarified. Little work has been done to characterize these processes and these factors in cell lines employed for large-scale cell culture. Manipulation of these process may permit improvement in yield or productivity of recombinant proteins by cultured animal cells