scholarly journals Sensitive Measurement of Drug-Target Engagement by a Cellular Thermal Shift Assay with Multiplex Proximity Extension Readout

2021 ◽  
Author(s):  
Rasel A. Al-Amin ◽  
Caroline J. Gallant ◽  
Phathutshedzo M. Muthelo ◽  
Ulf Landegren
Keyword(s):  
Drug Target ◽  
Target Engagement ◽  
Thermal Shift Assay ◽  
Cellular Thermal Shift Assay ◽  
Thermal Shift

Monitoring Drug Target Engagement in Cells and Tissues Using the Cellular Thermal Shift Assay

Science ◽  
2013 ◽  
Vol 341 (6141) ◽  
pp. 84-87 ◽  
Author(s):  
Daniel Martinez Molina ◽  
Rozbeh Jafari ◽  
Marina Ignatushchenko ◽  
Takahiro Seki ◽  
E. Andreas Larsson ◽  
...  
Keyword(s):  
Drug Target ◽  
Drug Transport ◽  
Drug Binding ◽  
Target Engagement ◽  
Tissue Samples ◽  
Target Proteins ◽  
Thermal Shift Assay ◽  
Cellular Thermal Shift Assay ◽  
Thermal Shift ◽  
In Cells

The efficacy of therapeutics is dependent on a drug binding to its cognate target. Optimization of target engagement by drugs in cells is often challenging, because drug binding cannot be monitored inside cells. We have developed a method for evaluating drug binding to target proteins in cells and tissue samples. This cellular thermal shift assay (CETSA) is based on the biophysical principle of ligand-induced thermal stabilization of target proteins. Using this assay, we validated drug binding for a set of important clinical targets and monitored processes of drug transport and activation, off-target effects and drug resistance in cancer cell lines, as well as drug distribution in tissues. CETSA is likely to become a valuable tool for the validation and optimization of drug target engagement.

scholarly journals Drug Target Engagement Using Coupled Cellular Thermal Shift Assay—Acoustic Reverse-Phase Protein Array

2019 ◽  
Vol 25 (2) ◽  
pp. 207-214
Author(s):  
Adrien Herledan ◽  
Marine Andres ◽  
Aurore Lejeune-Dodge ◽  
Florence Leroux ◽  
Alexandre Biela ◽  
...  
Keyword(s):  
Drug Target ◽  
Small Sample ◽  
Protein Array ◽  
Reverse Phase Protein Array ◽  
Target Engagement ◽  
Reverse Phase ◽  
Thermal Shift Assay ◽  
Phase Protein ◽  
Cellular Thermal Shift Assay ◽  
Thermal Shift

In the last 5 years, cellular thermal shift assay (CETSA), a technology based on ligand-induced changes in protein thermal stability, has been increasingly used in drug discovery to address the fundamental question of whether drug candidates engage their intended target in a biologically relevant setting. To analyze lysates from cells submitted to increasing temperature, the detection and quantification of the remaining soluble protein can be achieved using quantitative mass spectrometry, Western blotting, or AlphaScreen techniques. Still, these approaches can be time- and cell-consuming. To cope with limitations of throughput and protein amount requirements, we developed a new coupled assay combining the advantages of a nanoacoustic transfer system and reverse-phase protein array technology within CETSA experiments. We validated the technology to assess engagement of inhibitors of insulin-degrading enzyme (IDE), an enzyme involved in diabetes and Alzheimer’s disease. CETSA—acoustic reverse-phase protein array (CETSA-aRPPA) allows simultaneous analysis of many conditions and drug–target engagement with a small sample size, in a rapid, cost-effective, and biological material-saving manner.

scholarly journals A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1

2017 ◽  
Vol 23 (1) ◽  
pp. 34-46 ◽  
Author(s):  
Dean E. McNulty ◽  
William G. Bonnette ◽  
Hongwei Qi ◽  
Liping Wang ◽  
Thau F. Ho ◽  
...  
Keyword(s):  
High Throughput ◽  
Dose Response ◽  
Drug Target ◽  
Rank Order ◽  
Target Engagement ◽  
Suspension Cells ◽  
Persistent Problem ◽  
Thermal Shift Assay ◽  
Cellular Thermal Shift Assay ◽  
Thermal Shift

A persistent problem in early small-molecule drug discovery is the frequent lack of rank-order correlation between biochemical potencies derived from initial screens using purified proteins and the diminished potency and efficacy observed in subsequent disease-relevant cellular phenotypic assays. The introduction of the cellular thermal shift assay (CETSA) has bridged this gap by enabling assessment of drug target engagement directly in live cells based on ligand-induced changes in protein thermal stability. Initial success in applying CETSA across multiple drug target classes motivated our investigation into replacing the low-throughput, manually intensive Western blot readout with a quantitative, automated higher-throughput assay that would provide sufficient capacity to use CETSA as a primary hit qualification strategy. We introduce a high-throughput dose-response cellular thermal shift assay (HTDR-CETSA), a single-pot homogenous assay adapted for high-density microtiter plate format. The assay features titratable BacMam expression of full-length target proteins fused to the DiscoverX 42 amino acid ePL tag in HeLa suspension cells, facilitating enzyme fragment complementation–based chemiluminescent quantification of ligand-stabilized soluble protein. This simplified format can accommodate determination of full-dose CETSA curves for hundreds of individual compounds/analyst/day in replicates. HTDR-CETSA data generated for substrate site and alternate binding mode inhibitors of the histone-lysine N-methyltransferase SMYD3 in HeLa suspension cells demonstrate excellent correlation with rank-order potencies observed in cellular mechanistic assays and direct translation to target engagement of endogenous Smyd3 in cancer-relevant cell lines. We envision this workflow to be generically applicable to HTDR-CETSA screening spanning a wide variety of soluble intracellular protein target classes.

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