<i>In Situ</i> Target Engagement Studies in Adherent Cells

Axelsson, Hanna; Almqvist, Helena; Otrocka, Magdalena; Vallin, Michaela; Lundqvist, Sara; Hansson, Pia; Karlsson, Ulla; Lundbäck, Thomas; Seashore‐Ludlow, Brinton

doi:10.1021/acschembio.7b01079

Cited by 24 publications

(28 citation statements)

References 30 publications

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“…Starting from cells in suspension, after drug treatment and subsequent CETSA heating in PCR-tubes, cells were washed and collected by centrifugation before being transferred to clear bottom plates where they were fixed and stained (anti-tubulin and Hoechst). After we had developed this CETSA imaging format, two articles describing related methods were published on CETSA drug screening on adherent cells, demonstrating the feasibility of the image-based detection of target engagement 29,30 . Our method is, however, more versatile and allows for studies of clinical samples of cell suspensions such as fine needle aspirates.…”

Section: Resultsmentioning

confidence: 99%

CETSA-based target engagement of taxanes as biomarkers for efficacy and resistance

Langebäck

Bacanu

Laursen

et al. 2019

Sci Rep

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The use of taxanes has for decades been crucial for treatment of several cancers. A major limitation of these therapies is inherent or acquired drug resistance. A key to improved outcome of taxane-based therapies is to develop tools to predict and monitor drug efficacy and resistance in the clinical setting allowing for treatment and dose stratification for individual patients. To assess treatment efficacy up to the level of drug target engagement, we have established several formats of tubulin-specific Cellular Thermal Shift Assays (CETSAs). This technique was evaluated in breast and prostate cancer models and in a cohort of breast cancer patients. Here we show that taxanes induce significant CETSA shifts in cell lines as well as in animal models including patient-derived xenograft (PDX) models. Furthermore, isothermal dose response CETSA measurements allowed for drugs to be rapidly ranked according to their reported potency. Using multidrug resistant cancer cell lines and taxane-resistant PDX models we demonstrate that CETSA can identify taxane resistance up to the level of target engagement. An imaging-based CETSA format was also established, which in principle allows for taxane target engagement to be accessed in specific cell types in complex cell mixtures. Using a highly sensitive implementation of CETSA, we measured target engagement in fine needle aspirates from breast cancer patients, revealing a range of different sensitivities. Together, our data support that CETSA is a robust tool for assessing taxane target engagement in preclinical models and clinical material and therefore should be evaluated as a prognostic tool during taxane-based therapies.

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Section: Resultsmentioning

confidence: 99%

CETSA-based target engagement of taxanes as biomarkers for efficacy and resistance

Langebäck

Bacanu

Laursen

et al. 2019

Sci Rep

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“…CETSA HT would benefit greatly from endpoints allowing greater amplification of signal when quantifying low levels of endogenous thermostable protein, allowing a larger assay window and reducing requirements for cell numbers. Recent reports of identification of individual antibodies capable of selectively quantifying thermostable protein for p38α 10 and Chk1 11 have enabled application of CETSA with an imaging endpoint for these targets. This endpoint both allows single-cell resolution of target engagement and reduces requirements for cell numbers, though the successful application of this approach to measure binding to targets with low expression remains to be seen.…”

Section: Discussionmentioning

confidence: 99%

“…5,6 While such assays can be constructed using tagged proteins overexpressed in cells, [7][8][9] assays that measure label-free target engagement with endogenous protein by high-throughput CETSA (CETSA HT) have only been reported for a handful of targets to date. 5,[10][11][12] These studies have demonstrated that this technology might be used for compound screening to identify hits that are able to access and engage the target of interest in live cells, and to determine a measure of apparent potency of intracellular binding to rank compounds and inform on structure-activity relationships (SARs). 5,9,13 Variations in the assay setup have also been shown to differentiate compounds based on the kinetics of intracellular target engagement.…”

Section: Introductionmentioning

confidence: 99%

Positioning High-Throughput CETSA in Early Drug Discovery through Screening against B-Raf and PARP1

et al. 2019

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Methods to measure cellular target engagement are increasingly being used in early drug discovery. The Cellular Thermal Shift Assay (CETSA) is one such method. CETSA can investigate target engagement by measuring changes in protein thermal stability upon compound binding within the intracellular environment. It can be performed in high-throughput, microplate-based formats to enable broader application to early drug discovery campaigns, though high-throughput forms of CETSA have only been reported for a limited number of targets. CETSA offers the advantage of investigating the target of interest in its physiological environment and native state, but it is not clear yet how well this technology correlates to more established and conventional cellular and biochemical approaches widely used in drug discovery. We report two novel high-throughput CETSA (CETSA HT) assays for B-Raf and PARP1, demonstrating the application of this technology to additional targets. By performing comparative analyses with other assays, we show that CETSA HT correlates well with other screening technologies and can be applied throughout various stages of hit identification and lead optimization. Our results support the use of CETSA HT as a broadly applicable and valuable methodology to help drive drug discovery campaigns to molecules that engage the intended target in cells.

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“…This result could indicate, for example, that the compound induced a conformational change that increased thermal stability, while also masking the epitope binding site used for immunofluorescence detection. A second imaging-based CETSA was published concurrently with HCIF-CETSA: 21 Axelsson et al examined p38α aggregation in adherent cells and screened a kinase-focused collection of 1120 compounds, yielding 14 hits that stabilized the protein >30%. The high-content imaging approaches provide several advantages over traditional CETSA.…”

Section: Introductionmentioning

confidence: 99%

High-Throughput Cellular Thermal Shift Assays in Research and Drug Discovery

et al. 2020

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Thermal shift assays (TSAs) can reveal changes in protein structure, due to a resultant change in protein thermal stability. Since proteins are often stabilized upon binding of ligand molecules, these assays can provide a readout for protein target engagement. TSA has traditionally been applied using purified proteins and more recently has been extended to study target engagement in cellular environments with the emergence of cellular thermal shift assays (CETSAs). The utility of CETSA in confirming molecular interaction with targets in a more native context, and the desire to apply this technique more broadly, has fueled the emergence of higher-throughput techniques for CETSA (HT-CETSA). Recent studies have demonstrated that HT-CETSA can be performed in standard 96-, 384-, and 1536-well microtiter plate formats using methods such as beta-galactosidase and NanoLuciferase reporters and AlphaLISA assays. HT-CETSA methods can be used to select and characterize compounds from high-throughput screens and to prioritize compounds in lead optimization by facilitating dose–response experiments. In conjunction with cellular and biochemical activity assays for targets, HT-CETSA can be a valuable addition to the suite of assays available to characterize molecules of interest. Despite the successes in implementing HT-CETSA for a diverse set of targets, caveats and challenges must also be recognized to avoid overinterpretation of results. Here, we review the current landscape of HT-CETSA and discuss the methodologies, practical considerations, challenges, and applications of this approach in research and drug discovery. Additionally, a perspective on potential future directions for the technology is presented.

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In Situ Target Engagement Studies in Adherent Cells

Cited by 24 publications

References 30 publications

CETSA-based target engagement of taxanes as biomarkers for efficacy and resistance

CETSA-based target engagement of taxanes as biomarkers for efficacy and resistance

Positioning High-Throughput CETSA in Early Drug Discovery through Screening against B-Raf and PARP1

High-Throughput Cellular Thermal Shift Assays in Research and Drug Discovery

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