Browse
Submit Data
Databases
API
Help

Dataset Information

25 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability.

ABSTRACT: Thermal unfolding methods are commonly used as a predictive technique by tracking the protein's physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a 'Gold Standard' for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins.

SUBMITTER: Kopra K

PROVIDER: S-EPMC9266822 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Rapid Evaluation of Small Molecule Cellular Target Engagement with a Luminescent Thermal Shift Assay.

Project description:Determination of target engagement for candidate drug molecules in the native cellular environment is a significant challenge for drug discovery programs. The cellular thermal shift assay (CETSA) has emerged as a powerful tool for determining compound target engagement through measurement of changes to a protein's thermal stability upon ligand binding. Here, we present a HiBiT thermal shift assay (BiTSA) that deploys a quantitative peptide tag for determination of compound target engagement in the native cellular environment using a high throughput, plate-based luminescence readout. We demonstrate that BiTSA can rapidly assess cellular target engagement of small molecule ligands against their cognate targets and highlight two applications of BiTSA for differentiating small molecules targeting mutant KRAS and TP53.

| S-EPMC8366000 | biostudies-literature

Analysis of protein stability and ligand interactions by thermal shift assay.

Project description:Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye.

| S-EPMC4332540 | biostudies-literature

Homogeneous Assay for Target Engagement Utilizing Bioluminescent Thermal Shift.

Project description:Protein thermal shift assays (TSAs) provide a means for characterizing target engagement through ligand-induced thermal stabilization. Although these assays are widely utilized for screening libraries and validating hits in drug discovery programs, they can impose encumbering operational requirements, such as the availability of purified proteins or selective antibodies. Appending the target protein with a small luciferase (NanoLuc) allows coupling of thermal denaturation with luminescent output, providing a rapid and sensitive means for assessing target engagement in compositionally complex environments such as permeabilized cells. The intrinsic thermal stability of NanoLuc is greater than mammalian proteins, and our results indicate that the appended luciferase does not alter thermal denaturation of the target protein. We have successfully applied the NanoLuc luciferase thermal shift assay (NaLTSA) to several clinically relevant protein families, including kinases, bromodomains, and histone deacetylases. We have also demonstrated the suitability of this assay method for library screening and compound profiling.

| S-EPMC6004564 | biostudies-literature

POT1 stability and binding measured by fluorescence thermal shift assays.

Project description:The protein POT1 (Protection of Telomeres 1) is an integral part of the shelterin complex that protects the ends of human chromosomes from degradation or end fusions. It is the only component of shelterin that binds single-stranded DNA. We describe here the application of two separate fluorescent thermal shift assays (FTSA) that provide quantitative biophysical characterization of POT1 stability and its interactions. The first assay uses Sypro Orange™ and monitors the thermal stability of POT1 and its binding under a variety of conditions. This assay is useful for the quality control of POT1 preparations, for biophysical characterization of its DNA binding and, potentially, as an efficient screening tool for binding of small molecule drug candidates. The second assay uses a FRET-labeled human telomeric G-quadruplex structure that reveals the effects of POT1 binding on thermal stability from the DNA frame of reference. These complementary assays provide efficient biophysical approaches for the quantitative characterization of multiple aspects of POT1 structure and function. The results from these assays provide thermodynamics details of POT1 folding, the sequence selectivity of its DNA binding and the thermodynamic profile for its binding to its preferred DNA binding sequence. Most significantly, results from these assays elucidate two mechanisms for the inhibition of POT1 -DNA interactions. The first is by competitive inhibition at the POT1 DNA binding site. The second is indirect and is by stabilization of G-quadruplex formation within the normal POT1 single-stranded DNA sequence to prevent POT1 binding.

| S-EPMC8009405 | biostudies-literature

Quantitation of protein-protein interactions by thermal stability shift analysis.

Project description:Thermal stability shift analysis is a powerful method for examining binding interactions in proteins. We demonstrate that under certain circumstances, protein-protein interactions can be quantitated by monitoring shifts in thermal stability using thermodynamic models and data analysis methods presented in this work. This method relies on the determination of protein stabilities from thermal unfolding experiments using fluorescent dyes such as SYPRO Orange that report on protein denaturation. Data collection is rapid and straightforward using readily available real-time polymerase chain reaction instrumentation. We present an approach for the analysis of the unfolding transitions corresponding to each partner to extract the affinity of the interaction between the proteins. This method does not require the construction of a titration series that brackets the dissociation constant. In thermal shift experiments, protein stability data are obtained at different temperatures according to the affinity- and concentration-dependent shifts in unfolding transition midpoints. Treatment of the temperature dependence of affinity is, therefore, intrinsic to this method and is developed in this study. We used the interaction between maltose-binding protein (MBP) and a thermostable synthetic ankyrin repeat protein (Off7) as an experimental test case because their unfolding transitions overlap minimally. We found that MBP is significantly stabilized by Off7. High experimental throughput is enabled by sample parallelization, and the ability to extract quantitative binding information at a single partner concentration. In a single experiment, we were able to quantify the affinities of a series of alanine mutants, covering a wide range of affinities (∼ 100 nM to ∼ 100 μM).

| S-EPMC3189529 | biostudies-literature

EDTA aggregates induce SYPRO orange-based fluorescence in thermal shift assay.

Project description:Ethylenediaminetetraacetic acid (EDTA) is widely used in the life sciences as chelating ligand of metal ions. However, formation of supramolecular EDTA aggregates at pH > 8 has been reported, which may lead to artifactual assay results. When applied as a buffer component at pH ≈ 10 in differential scanning fluorimetry (TSA) using SYPRO Orange as fluorescent dye, we observed a sharp change in fluorescence intensity about 20°C lower than expected for the investigated protein. We hypothesized that this change results from SYPRO Orange/EDTA interactions. TSA experiments in the presence of SYPRO Orange using solutions that contain EDTA-Na+ but no protein were performed. The TSA experiments provide evidence that suggests that at pH > 9, EDTA4- interacts with SYPRO Orange in a temperature-dependent manner, leading to a fluorescence signal yielding a "denaturation temperature" of ~68°C. Titrating Ca2+ to SYPRO Orange and EDTA solutions quenched fluorescence. Ethylene glycol tetraacetic acid (EGTA) behaved similarly to EDTA. Analytical ultracentrifugation corroborated the formation of EDTA aggregates. Molecular dynamics simulations of free diffusion of EDTA-Na+ and SYPRO Orange of in total 27 μs suggested the first structural model of EDTA aggregates in which U-shaped EDTA4- arrange in an inverse bilayer-like manner, exposing ethylene moieties to the solvent, with which SYPRO Orange interacts. We conclude that EDTA aggregates induce a SYPRO Orange-based fluorescence in TSA. These results make it relevant to ascertain that future TSA results are not influenced by interference between EDTA, or EDTA-related molecules, and the fluorescent dye.

| S-EPMC5417642 | biostudies-literature

High-Throughput Cellular Thermal Shift Assay Using Acoustic Transfer of Protein Lysates.

Project description:Cellular thermal shift assay (CETSA) is a valuable method to confirm target engagement within a complex cellular environment, by detecting changes in a protein's thermal stability upon ligand binding. The classical CETSA method measures changes in the thermal stability of endogenous proteins using immunoblotting, which is low-throughput and laborious. Reverse-phase protein arrays (RPPAs) have been demonstrated as a detection modality for CETSA; however, the reported procedure requires manual processing steps that limit throughput and preclude screening applications. We developed a high-throughput CETSA using an acoustic RPPA (HT-CETSA-aRPPA) protocol that is compatible with 96- and 384-well microplates from start-to-finish, using low speed centrifugation to remove thermally destabilized proteins. The utility of HT-CETSA-aRPPA for guiding structure-activity relationship studies was demonstrated for inhibitors of lactate dehydrogenase A. Additionally, a collection of kinase inhibitors was screened to identify compounds that engage MEK1, a clinically relevant kinase target.

| S-EPMC10026039 | biostudies-literature

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

Project description:Solute carriers (SLCs) are transmembrane proteins that transport various nutrients, metabolites, and drugs across cellular membranes. Despite the relevance of SLCs to cell homeostasis, metabolism, and disease states, for the majority of SLCs we lack experimental evidence regarding the nature of the cognate ligands, whether endobiotic or xenobiotic. Moreover, even for the roughly 20 SLCs for which inhibitors have been characterized, engagement assays in cells are limited to the accessibility of radiolabeled or fluorescent probes. The cellular thermal shift assay (CETSA) has been introduced as a powerful method to assess target engagement by monitoring ligand-induced changes in the thermal stability of cellular proteins. We addressed the question of whether CETSA could be modified to become routinely applicable to membrane transporters such as SLCs. We used SLC16A1 (MCT1) and SLC1A2 (EAAT2) as targets to establish robust conditions by which chemical engagement of SLCs can be detected. Using immunoblotting, we demonstrate that treatment with the SLC16A1 inhibitors AZD3965 and AR-C155858 stabilized endogenous SLC16A1 in HEK293 cell lysates as well as intact cells. In addition, the high-affinity ligand of SLC16A1, l-lactate, and the low-affinity ligand, formate, resulted in strong and weak stabilization of SLC16A1, respectively. Moreover, we observed stabilization of SLC1A2 upon treatment with the selective inhibitor WAY-213613. We propose that the experimental approach presented here should be generally and easily applicable for monitoring the engagement of chemical ligands by SLCs in cellular settings and thus assisting in their deorphanization.

| S-EPMC6067815 | biostudies-literature

TSA-CRAFT: A Free Software for Automatic and Robust Thermal Shift Assay Data Analysis.

Project description:Thermal shift assay (TSA) is an increasingly popular technique used for identifying protein stabilizing conditions or interacting ligands in X-ray crystallography and drug discovery applications. Although the setting up and running of TSA reactions is a relatively simple process, the subsequent analysis of TSA data, especially in high-throughput format, requires substantial amount of effort if conducted manually. We therefore developed the Thermal Shift Assay-Curve Rapid and Automatic Fitting Tool (TSA-CRAFT), a freely available software that enable automatic analysis of TSA data of any throughput. TSA-CRAFT directly reads real-time PCR instrument data files and displays the analyzed results in a web browser. This software features streamlined data processing and Boltzmann equation fitting, which is demonstrated in this study to provide more accurate data analysis than the commonly used first-derivative method. TSA-CRAFT is freely available as a cross-operating system-compatible standalone tool ( https://sourceforge.net/projects/tsa-craft/ ) and also as a freely accessible web server ( http://tbtlab.org/tsacraft.html ).

| S-EPMC6537141 | biostudies-literature

A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase.

Project description:Assessment of the interactions between a drug and its protein target in a physiologically relevant cellular environment constitutes a major challenge in the pre-clinical drug discovery space. The Cellular Thermal Shift Assay (CETSA) enables such an assessment by quantifying the changes in the thermal stability of proteins upon ligand binding in intact cells. Here, we present the development and validation of a homogeneous, standardized, target-independent, and high-throughput (384- and 1536-well formats) CETSA platform that uses a split Nano Luciferase approach (SplitLuc CETSA). The broad applicability of the assay was demonstrated for diverse targets, and its performance was compared with independent biochemical and cell-based readouts using a set of well-characterized inhibitors. Moreover, we investigated the utility of the platform as a primary assay for high-throughput screening. The SplitLuc CETSA presented here enables target engagement studies for medium and high-throughput applications. Additionally, it provides a rapid assay development and screening platform for targets where phenotypic or other cell-based assays are not readily available.

| S-EPMC6013488 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data