Cellular thermal shift and clickable chemical probe assays for the determination of drug-target engagement in live 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.

Download Full-text

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

Analytical Chemistry ◽

10.1021/acs.analchem.1c02225 ◽

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

Download Full-text

Aza-SAHA Derivatives are Selective Histone Deacetylase 10 Chemical Probes That Inhibit Polyamine Deacetylation

10.33774/chemrxiv-2021-37shs ◽

2021 ◽

Author(s):

Raphael R. Steimbach ◽

Corey J. Herbst-Gervasoni ◽

Glynis Klinke ◽

Magalie Géraldy ◽

Gergely Tihanyi ◽

...

Keyword(s):

Histone Deacetylase ◽

Target Engagement ◽

Target Validation ◽

Chemical Probes ◽

Combination Cancer Therapy ◽

Selective Chemical ◽

First Time ◽

Double Digit ◽

Novel Therapeutic ◽

Thermal Shift

We report the first selective chemical probes for histone deacetylase 10 (HDAC10) with unprecedented selectivity over other HDAC isozymes. HDAC10 deacetylates polyamines and has a distinct substrate specificity, making it unique among the 11 zinc-dependent HDAC hydrolases. Taking inspiration from HDAC10 polyamine substrates, we systematically inserted an amino group (“aza-scan”) into the hexyl linker moiety of the approved drug Vorinostat (SAHA). This one atom replacement (C-->N) transformed SAHA from an unselective pan-HDAC inhibitor into a specific HDAC10 inhibitor. Optimization of the aza-SAHA structure yielded DKFZ-748, which has a double-digit nanomolar IC50 against HDAC10 in cells and >500-fold selectivity over the closest relative HDAC6 as well as the Class I enzymes (HDAC1, 2, 3, 8). Potency of our aza-SAHA derivatives is rationalized with HDAC10 co-crystal structures and demonstrated by cellular and biochemical target-engagement, as well as thermal-shift, assays. Treatment of cells with DKFZ-748, followed by quantification of selected polyamines, confirmed for the first time the suspected cellular function of HDAC10 as a poly-amine deacetylase. Selective HDAC10 chemical probes provide a valuable pharmacological tool for target validation and will enable further studies on the enigmatic biology of HDAC10 and acetylated polyamines. HDAC10-selective aza-SAHA derivatives are not cytotoxic, which opens the doors to novel therapeutic applications as immunomodulators or in combination cancer therapy.

Download Full-text

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

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555219897256 ◽

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.

Download Full-text