Agency for Science, Technology, and Research
Stapled Peptides from Bench to Bedside
Stapled peptides have been described since the landmark paper Greg Verdine in the year 2000. In 2019, four clinical trials of ALRN6924 are in progress as the first stapled peptide begins the long journey to clinical approval. Our own studies on stapled peptides have encompassed a number of targets and have given us great insight into the positive properties of this new class of molecules but also helped us to understand why they often fail and why the literature surrounding their use is so confused.
In the case of a study developing stapled peptides towards the eIF4E complex, we developed molecules that blocked the binding of eIF4G and 4EBP1 with exceptional affinity. However, very careful biological assays built around the nanoBret and nanoBit technologies demonstrated that these peptides completely failed to enter cells and access their targets. Permeabilising the cells allowed us to show that these molecules were capable of target engagement and potent biological activity so that the only barrier to their function was a failure of cell permeability.
In contrast, studies on inhibitors of the p53-MDM2 interaction yielded many molecules capable of biological activity both in tissue culture systems, and in whole animal models. The properties that allow the successful uptake of stapled peptides and their engagement with their target have been explored in extensive peptide series incorporating many unnatural amino acids and modified stapling chemistries. Eventually we have produced extraordinarily stable and potent inhibitors of MDM2. At the same time, we have realized that many published observations on stapled peptides have been overoptimistic in their interpretation of the data. Particular pitfalls include non-specific membrane engagement leading to cell lysis and the binding of the molecules to plasticware and other surfaces invoking false positive signals in a variety of biochemical assays.
Currently, the published RAS binding peptides fall into this category. This experience has allowed us to develop a series of critical technologies that can quickly discover the off-target effects of apparently promising molecules reflecting what has been seen for many years in the field of small molecule discovery where molecules aptly named as pan-assay interference compounds, PAINs, have long been seen as beguiling false positives.
Simon Nga, Yu-Chi Juangb, Arun Chandramohanb, Hung Yi Kristal Kaanb, Ahmad Sadruddinb, Tsz Ying Yuenc, Fernando J. Ferrera, Xue’Er Cheryl Leec, Liew Xic, Charles W. Johannesc, Christopher J. Browna, Srinivasaraghavan Kannand, Pietro G. Aronicad, Nils Berglundd, Chandra S. Vermad, Lijuan Liue, Alexander Stoecke, Tomi K. Sawyere, Anthony W. Partridgeb, 1, David P. Lanea, 1
a p53 Laboratory, A*STAR, Singapore
b MSD, Singapore
c Institute of Chemical and Engineering Sciences, A*STAR, Singapore
d Bioinformatics Institute, A*STAR, Singapore
e Merck & Co., Inc., Boston, MA, USA