Toolbox of Diverse Linkers for Navigating the Cellular Efficacy Landscape of Stapled Peptides

Abstract

Stapled peptides have great potential as modulators of protein–protein interactions (PPIs). However, there is a vast landscape of chemical features that can be varied for any given peptide, and identifying a set of features that maximizes cellular uptake and subsequent target engagement remains a key challenge. Herein, we present a systematic analysis of staple functionality on the peptide bioactivity landscape in cellular assays. Through application of a “toolbox” of diversified dialkynyl linkers to the stapling of MDM2-binding peptides via a double-click approach, we conducted a study of cellular uptake and p53 activation as a function of the linker. Minor changes in the linker motif and the specific pairing of linker with peptide sequence can lead to substantial differences in bioactivity, a finding which may have important design implications for peptide-based inhibitors of other PPIs. Given the complexity of the structure–activity relationships involved, the toolbox approach represents a generalizable strategy for optimization when progressing from in vitro binding assays to cellular efficacy studies.

Strategies to expand peptide functionality through hybridisation with a small molecule component

“…In an example where peptide-small molecule hybrids were investigated in a biological context, Spring and co-workers found a small molecule carrier (SMoC) group incorporated through the cyclising linker can enhance the cellular uptake of a series MDM2binding peptides. 118 SMoCs are molecules that possess guanidinium cations linked to a lipophilic biphenyl system; they were designed by Selwood and co-workers to be a smaller and potentially more proteolytically stable mimic of guanidine-rich cell-penetrating peptides. 119 Higher uptake for the SMoC-bearing MDM2-binding peptide was visualised by confocal microscopy, which correlated with an improvement in p53 activation in cells.…”

Designing stapled peptides to inhibit protein‐protein interactions: An analysis of successes in a rapidly changing field

“…[ 142 ] Only a few studies have directly compared the effects of different types of staple chemistry on similar sequences, therefore it is difficult to generalise about the impact of staples on factors such as helicity, or cell uptake. [ 31 ] Maximising helicity has been one of the principle aims of many chemical studies, but it has been shown that flexibility in macrocyclic structures is often critical in allowing a peptide to cross the cellular lipid membrane. [ 145 ]…”

“…Functionalisation is not limited to fluorescence, and Wu et al ., describe a library of functionalised dialkynyl linkers compatible with double CuAAC stapling of peptides (Figure 9B); these include linkers with additional non‐peptidic polyamines and nuclear targeting peptides. [ 31 ] It was shown that in vitro binding affinity for MDM2 was not significantly affected by the linker chemistry; but positively charged residues enhanced both cellular uptake and target engagement in cell reporter assays.…”

“…[ 17–23 ] In some instances, only very subtle differences in the staple chemistry, or stereochemistry, trigger a significant change in the binding affinity, the pharmaceutical profile of the peptide, or give added functionality. [ 21,24–29 ] Despite multiple publications and reviews describing reliable methods for the synthesis of stapled peptides [ 30–35 ] and the commercialisation of the most common unnatural amino acids, their synthesis remains costly and until a few years ago, non‐automated. Consequently, the synthesis and screening of stapled peptides have usually been restricted to a few dozen peptides, limiting access to extensive libraries.…”

Stapled Peptide Inhibitors of Autophagy Adapter LC3B

“…Notably, most peptide stapling strategies are limited to helical motifs. Because LIR motifs bind LC3B in an extended conformation, we applied an innovative diversity‐oriented stapling strategy that does not assume any specific secondary structure …”