Assessing the Efficacy of Mdm2/Mdm4-Inhibiting Stapled Peptides Using Cellular Thermal Shift Assays.

Ban Xiong Tan,Anna E Jansson,David P Lane,Fernando J Ferrer,Boon Hong Chan,Tsz Ying Yuen,Hsiang Ling Teo,Pär Nordlund,Soo Tng Quah,Christopher J Brown

doi:10.1038/srep12116

Abstract

Previous publications on stapled peptide inhibitors against Mdm2/Mdm4-p53 interactions have established that this new class of drugs have the potential to be easily optimised to attain high binding affinity and specificity, but the mechanisms controlling their cellular uptake and target engagement remain elusive and controversial. To aid in understanding the rules of peptide and staple design, and to enable rapid optimisation, we employed the newly-developed cellular thermal shift assay (CETSA). CETSA was able to validate stapled peptide binding to Mdm2 and Mdm4, and the method was also used to determine the extent of cellular uptake, cellular availability, and intracellular binding of the endogenous target proteins in its native environment. Our data suggest that while the stapled peptides engage their targets intracellularly, more work is needed to improve their cellular entry and target engagement efficiency in vivo. CETSA now provides a valuable tool to optimize such in vivo properties of stapled peptides.

Highlights

Dubbed the ‘guardian of the genome’, the tumour suppressor p53 has been found to be mutated in half of all cancers[1,2], and with more than 70 000 publications describing the gene, it is undoubtedly one of the most important and well-studied cancer-related genes
Stapled peptides bind to Mdm[2] and Mdm[4] at high affinity in CETSA
Stapled peptides based on the N-terminal Mdm2-binding domain on p53 have been shown to successfully inhibit Mdm[2] and Mdm[4] interacting with p53 in in vitro thermofluor assays, as well function in cell based p53 reporter based activation assays

Summary

Introduction

Dubbed the ‘guardian of the genome’, the tumour suppressor p53 has been found to be mutated in half of all cancers[1,2], and with more than 70 000 publications describing the gene, it is undoubtedly one of the most important and well-studied cancer-related genes. Blocking the interactions between p53 and Mdm2/Mdm[4] prevents the occlusion of the p53 transactivation domain, as well as preventing p53 nuclear export, ubiquitination and proteasomal degradation[4,5] This results in the accumulation of p53, allowing both p53 transcription-dependent and independent activity, fulfilling its role as a tumour suppressor. The amount of thermal shift in the presence of ligands correlates well with ligand affinity[17], and can be used to approximate binding affinities between drugs and their target proteins These assays require the use of purified proteins, representing a challenge in the case of proteins that are difficult to isolate. The stapled peptides were found to induce large thermal shifts in the stability of their target proteins in cell lysates largely in concordance to their affinities estimated using other techniques with purified proteins, though discrepancies may arise from the use of different approaches to estimate binding affinities. This study provided evidence that CETSA is an immensely useful tool for drug design and optimisation, not just in target affinity, and in cell entry and availability and is capable of dealing with novel drug classes such as larger peptide molecules

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