title: Assessing the Efficacy of Mdm2/Mdm4-Inhibiting Stapled Peptides Using Cellular Thermal Shift Assays.
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.
authors.affiliation: p53 Laboratory, A*STAR, Singapore.
authors.forename: Ban Xiong
authors.initials: BX
authors.lastname: Tan
authors.affiliation: p53 Laboratory, A*STAR, Singapore.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Brown
authors.affiliation: p53 Laboratory, A*STAR, Singapore.
authors.forename: Fernando J
authors.initials: FJ
authors.lastname: Ferrer
authors.affiliation: Institute of Chemical and Engineering Sciences, A*STAR.
authors.forename: Tsz Ying
authors.initials: TY
authors.lastname: Yuen
authors.affiliation: p53 Laboratory, A*STAR, Singapore.
authors.forename: Soo Tng
authors.initials: ST
authors.lastname: Quah
authors.affiliation: p53 Laboratory, A*STAR, Singapore.
authors.forename: Boon Hong
authors.initials: BH
authors.lastname: Chan
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Anna E
authors.initials: AE
authors.lastname: Jansson
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Hsiang Ling
authors.initials: HL
authors.lastname: Teo
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Pär
authors.initials: P
authors.lastname: Nordlund
authors.affiliation: p53 Laboratory, A*STAR, Singapore.
authors.forename: David P
authors.initials: DP
authors.lastname: Lane
mesh_terms: D001681:Biological Assay / Q000379:methods; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D045325:HCT116 Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; C482775:MDM4 protein, human; D009687:Nuclear Proteins; D010455:Peptides; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2