Water-soluble and cell-permeant zwitterionic rhodamine dyes enable quantitative imaging of drug target engagement

2021 ◽  
Author(s):  
Antonio R. Montaño ◽  
Lei G. Wang ◽  
Allison Solanki ◽  
Nathan P. McMahon ◽  
Marcus Kwon ◽  
...  
Keyword(s):  
Drug Target ◽  
Quantitative Imaging ◽  
Water Soluble ◽  
Target Engagement ◽  
Rhodamine Dyes

scholarly journals Mechanism-based inhibitors of SIRT2: structure–activity relationship, X-ray structures, target engagement, regulation of α-tubulin acetylation and inhibition of breast cancer cell migration

2020 ◽  
Author(s):  
Alexander L. Nielsen ◽  
Nima Rajabi ◽  
Norio Kudo ◽  
Kathrine Lundø ◽  
Carlos Moreno-Yruela ◽  
...  
Keyword(s):  
Drug Target ◽  
Water Soluble ◽  
Target Engagement ◽  
X Ray ◽  
Tubulin Acetylation ◽  
Structure Activity ◽  
Complex Functions ◽  
Lysine Residues ◽  
Chain Acyl ◽  
Single Compound

ABSTRACTSirtuin 2 (SIRT2) is a protein deacylase enzyme that removes acetyl groups and longer chain acyl groups from post-translationally modified lysine residues. It affects diverse biological functions in the cell and has been considered a drug target in relation to both neurodegenerative diseases and cancer. Therefore, access to well-characterized and robust tool compounds is essential for the continued investigation of the complex functions of this enzyme. Here, we report a collection of probes that are potent, selective, stable in serum, water-soluble, amenable to cell culture experiments, and inhibit both SIRT2 deacetylation and demyristoylation. Compared to the current landscape of SIRT2 inhibitors, this is a unique ensemble of features built into a single compound. We expect the developed chemotypes to find broad application in the interrogation of SIRT2 functions in both healthy and diseased cells, and to provide a foundation for the development of future therapeutics.

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.

Monitoring Drug-Target Interactions through Target Engagement-Mediated Amplification on Arrays and in situ

2021 ◽  
Author(s):  
Rasel Al-Amin ◽  
Lars Johansson ◽  
Eldar Abdurakhmanov ◽  
Nils Landegren ◽  
Liza Löf ◽  
...  
Keyword(s):  
Drug Target ◽  
Kinase Inhibitors ◽  
Expression Profiles ◽  
Clinical Samples ◽  
Target Engagement ◽  
Rolling Circle Replication ◽  
Target Proteins ◽  
Dna Conjugates

Abstract Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to a panel of fixed adherent cells agreed with expectations from expression profiles of the cells. These findings were corroborated by competition experiments using kinase inhibitors with overlapping and non-overlapping target specificities, and translated to pathology tissue sections. We also introduce a proximity ligation variant of TEMA in which these drug-DNA conjugates are combined with antibody-DNA conjugates to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug-target interactions at spatial resolution in protein arrays, cells and tissues.

Rapid discovery of drug target engagement by isothermal shift assay

2019 ◽  
Author(s):  
Kristofor J. Webb ◽  
Kerri A. Ball ◽  
Stephen J. Coleman ◽  
Jeremy Jacobsen ◽  
Michael H.B. Stowell ◽  
...  
Keyword(s):  
Drug Target ◽  
Drug Targets ◽  
Kinase Inhibitors ◽  
Rapid Identification ◽  
Living Cells ◽  
Experimental Designs ◽  
Living Systems ◽  
Target Engagement ◽  
Protein Targets ◽  
Drug Molecules

Identifying protein targets directly bound by drug molecules within living systems remains challenging. Here we present the isothermal shift assay, iTSA, for rapid identification of drug targets. Compared with thermal proteome profiling, a prevailing method for target engagement, iTSA offers a simplified workflow, 4-fold higher throughput, and multiplexed experimental designs with higher replication. We demonstrate application of iTSA to identify targets for several kinase inhibitors in lysates and living cells.

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 Accurate MS-based Rab10 Phosphorylation Stoichiometry Determination as Readout for LRRK2 Activity in Parkinson's Disease

2020 ◽  
Vol 19 (9) ◽  
pp. 1546-1560 ◽  
Author(s):  
Özge Karayel ◽  
Francesca Tonelli ◽  
Sebastian Virreira Winter ◽  
Phillip E. Geyer ◽  
Ying Fan ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Drug Target ◽  
Patient Stratification ◽  
Leucine Rich Repeat ◽  
Target Engagement ◽  
Mutation Carriers ◽  
Lrrk2 G2019s ◽  
Before And After ◽  
Inhibitor Treatment

Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) are the predominant genetic cause of Parkinson's disease (PD). They increase its activity, resulting in augmented Rab10-Thr73 phosphorylation and conversely, LRRK2 inhibition decreases pRab10 levels. Currently, there is no assay to quantify pRab10 levels for drug target engagement or patient stratification. To meet this challenge, we developed an high accuracy and sensitivity targeted mass spectrometry (MS)-based assay for determining Rab10-Thr73 phosphorylation stoichiometry in human samples. It uses synthetic stable isotope-labeled (SIL) analogues for both phosphorylated and nonphosphorylated tryptic peptides surrounding Rab10-Thr73 to directly derive the percentage of Rab10 phosphorylation from attomole amounts of the endogenous phosphopeptide. The SIL and the endogenous phosphopeptides are separately admitted into an Orbitrap analyzer with the appropriate injection times. We test the reproducibility of our assay by determining Rab10-Thr73 phosphorylation stoichiometry in neutrophils of LRRK2 mutation carriers before and after LRRK2 inhibition. Compared with healthy controls, the PD predisposing mutation carriers LRRK2 G2019S and VPS35 D620N display 1.9-fold and 3.7-fold increased pRab10 levels, respectively. Our generic MS-based assay further establishes the relevance of pRab10 as a prognostic PD marker and is a powerful tool for determining LRRK2 inhibitor efficacy and for stratifying PD patients for LRRK2 inhibitor treatment.

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