RRC ID 58634
Author Nagasawa I, Muroi M, Kawatani M, Ohishi T, Ohba SI, Kawada M, Osada H.
Title Identification of a Small Compound Targeting PKM2-Regulated Signaling Using 2D Gel Electrophoresis-Based Proteome-wide CETSA.
Journal Cell Chem Biol
Abstract The cellular thermal shift assay (CETSA) has recently been devised as a label-free method for target validation of small compounds and monitoring the thermal stabilization or destabilization of proteins due to binding with the compound. Herein, we developed a modified method by combining the CETSA and proteomics analysis based on 2D gel electrophoresis, namely 2DE-CETSA, to identify the thermal stability-shifted proteins by binding with a new compound. We applied the 2DE-CETSA for analysis of a target-unknown compound, NPD10084, which exerts anti-proliferative activity against colorectal cancer cells in vitro and in vivo, and identified pyruvate kinase muscle isoform 2 (PKM2) as a candidate target protein. Interestingly, NPD10084 interrupted protein-protein interactions between PKM2 and β-catenin or STAT3, with subsequent suppression of downstream signaling. We thus demonstrate that our 2DE-CETSA method is applicable for identification of target compounds discovered by phenotypic screening.
Volume 27(2)
Pages 186-196.e4
Published 2020-2-20
DOI 10.1016/j.chembiol.2019.11.010
PII S2451-9456(19)30392-7
PMID 31813846
MeSH Animals Carbolines / chemistry* Carbolines / metabolism Carbolines / pharmacology Carbolines / therapeutic use Carrier Proteins / antagonists & inhibitors Carrier Proteins / genetics Carrier Proteins / metabolism* Cell Line, Tumor Cell Proliferation / drug effects Electrophoresis, Gel, Two-Dimensional / methods* Female Humans Membrane Proteins / antagonists & inhibitors Membrane Proteins / genetics Membrane Proteins / metabolism* Mice Mice, Nude Neoplasms / drug therapy Neoplasms / pathology Protein Binding / drug effects Proteome / analysis RNA Interference RNA, Small Interfering / metabolism STAT3 Transcription Factor / chemistry STAT3 Transcription Factor / metabolism Signal Transduction / drug effects Thyroid Hormones / genetics Thyroid Hormones / metabolism* Transplantation, Heterologous beta Catenin / chemistry beta Catenin / metabolism
IF 6.762
Times Cited 0
Human and Animal Cells HCT116(RCB2979) LoVo(RCB1639) COLO205(RCB2127) COLO-320(RCB1193) HeLa(RCB0007) MIA Paca2(RCB2094)