scholarly journals Detection of Chemical Engagement of Solute Carrier Proteins by a Cellular Thermal Shift Assay

2018 ◽  
Vol 13 (6) ◽  
pp. 1480-1486 ◽  
Author(s):  
Mari Hashimoto ◽  
Enrico Girardi ◽  
Ruth Eichner ◽  
Giulio Superti-Furga
Keyword(s):  
Carrier Proteins ◽  
Thermal Shift Assay ◽  
Solute Carrier ◽  
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 Determining direct binders of the Androgen Receptor using a high-throughput Cellular Thermal Shift Assay

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Joseph Shaw ◽  
Mathew Leveridge ◽  
Charlotta Norling ◽  
Jakob Karén ◽  
Daniel Martinez Molina ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
High Throughput ◽  
Thermal Shift Assay ◽  
Cellular Thermal Shift Assay ◽  
Thermal Shift

Identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay

2019 ◽  
Vol 11 (473) ◽  
pp. eaau3174 ◽  
Author(s):  
Jerzy M. Dziekan ◽  
Han Yu ◽  
Dan Chen ◽  
Lingyun Dai ◽  
Grennady Wirjanata ◽  
...  
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
Cysteine Proteinase ◽  
Antimalarial Drugs ◽  
Purine Nucleoside ◽  
Substantial Contribution ◽  
Cysteine Proteinase Inhibitor ◽  
Nucleoside Phosphorylase ◽  
Thermal Shift Assay ◽  
Cellular Thermal Shift Assay ◽  
Thermal Shift

Mechanisms of action (MoAs) have been elusive for most antimalarial drugs in clinical use. Decreasing responsiveness to antimalarial treatments stresses the need for a better resolved understanding of their MoAs and associated resistance mechanisms. In the present work, we implemented the cellular thermal shift assay coupled with mass spectrometry (MS-CETSA) for drug target identification inPlasmodium falciparum, the main causative agent of human malaria. We validated the efficacy of this approach for pyrimethamine, a folic acid antagonist, and E64d, a broad-spectrum cysteine proteinase inhibitor. Subsequently, we applied MS-CETSA to quinine and mefloquine, two important antimalarial drugs with poorly characterized MoAs. Combining studies in theP. falciparumparasite lysate and intact infected red blood cells, we foundP. falciparumpurine nucleoside phosphorylase (PfPNP) as a common binding target for these two quinoline drugs. Biophysical and structural studies with a recombinant protein further established that both compounds bind within the enzyme’s active site. Quinine binds to PfPNP at low nanomolar affinity, suggesting a substantial contribution to its therapeutic effect. Overall, we demonstrated that implementation of MS-CETSA forP. falciparumconstitutes a promising strategy to elucidate the MoAs of existing and candidate antimalarial drugs.

Sign in / Sign up

Export Citation Format

Share Document