Differential scanning fluorimetry as secondary screening platform for small molecule inhibitors of Bcl-X<sub>L</sub>

Wan, Kah Fei; Wang, Sifang; Brown, Christopher J.; Yu, Victor C.; Entzeroth, Michael; Lane, David P.; Lee, May Ann

doi:10.4161/cc.8.23.10114

Cited by 29 publications

(26 citation statements)

References 30 publications

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“…This contrasts with the stabilizing effect observed (Grasberger et al, 2005;Wan et al, 2009) for small-molecule ligand binding to many proteins (e.g., G␣ o ; Supplemental Fig. 2).…”

Section: Discussioncontrasting

confidence: 45%

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Reversible, Allosteric Small-Molecule Inhibitors of Regulator of G Protein Signaling Proteins

Blazer¹,

Roman²,

Chung³

et al. 2010

Mol Pharmacol

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Regulators of G protein signaling (RGS) proteins are potent negative modulators of G protein signaling and have been proposed as potential targets for small-molecule inhibitor development. We report a high-throughput time-resolved fluorescence resonance energy transfer screen to identify inhibitors of RGS4 and describe the first reversible small-molecule inhibitors of an RGS protein. Two closely related compounds, typified by CCG-63802 [((, inhibit the interaction between RGS4 and G␣ o with an IC 50 value in the low micromolar range. They show selectivity among RGS proteins with a potency order of RGS 4 Ͼ 19 ϭ 16 Ͼ 8 Ͼ Ͼ 7. The compounds inhibit the GTPase accelerating protein activity of RGS4, and thermal stability studies demonstrate binding to the RGS but not to G␣ o . On RGS4, they depend on an interaction with one or more cysteines in a pocket that has previously been identified as an allosteric site for RGS regulation by acidic phospholipids. Unlike previous small-molecule RGS inhibitors identified to date, these compounds retain substantial activity under reducing conditions and are fully reversible on the 10-min time scale. CCG-63802 and related analogs represent a useful step toward the development of chemical tools for the study of RGS physiology.

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“…This contrasts with the stabilizing effect observed (Grasberger et al, 2005;Wan et al, 2009) for small-molecule ligand binding to many proteins (e.g., G␣ o ; Supplemental Fig. 2).…”

Section: Discussioncontrasting

confidence: 45%

“…In most instances, proteins with endogenous small-molecule ligands (e.g., G␣ proteins) are stabilized by the presence of their ligand (Grasberger et al, 2005;Wan et al, 2009). This notion was recently borne out by the recent crystallization of several GPCRs (Cherezov et al, 2007;Jaakola et al, 2008;Scheerer et al, 2008;Warne et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

Reversible, Allosteric Small-Molecule Inhibitors of Regulator of G Protein Signaling Proteins

Blazer¹,

Roman²,

Chung³

et al. 2010

Mol Pharmacol

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“…The experiments reveal typical TSA curves [36, 37] of the relative change in fluorescence intensity in the presence of EDTA, with an inflection point at 68°C, whereas no increase in the fluorescence signal with increasing temperature was observed in the absence of EDTA (Fig 1). These results demonstrate an interference of EDTA and SYPRO Orange that leads to a temperature-dependent change in fluorescence intensity resembling that observed when a protein unfolds.…”

Section: Resultsmentioning

confidence: 98%

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

et al. 2017

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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.

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“…The well documented successes in expediting protein stabilization, drug discovery (especially in less well financed laboratories) and crystallization 10,[23][24][25] have made it an attractive method for initial screening of compounds. Compounds added to proteins show a clear dose dependent increase in the apparent melting temperature 7,9 .…”

Section: Discussionmentioning

confidence: 99%

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

Vivoli

Novak

Littlechild

et al. 2014

JoVE

122

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A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

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Differential scanning fluorimetry as secondary screening platform for small molecule inhibitors of Bcl-X_L

Cited by 29 publications

References 30 publications

Reversible, Allosteric Small-Molecule Inhibitors of Regulator of G Protein Signaling Proteins

Reversible, Allosteric Small-Molecule Inhibitors of Regulator of G Protein Signaling Proteins

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

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

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Differential scanning fluorimetry as secondary screening platform for small molecule inhibitors of Bcl-XL

Cited by 29 publications

References 30 publications

Reversible, Allosteric Small-Molecule Inhibitors of Regulator of G Protein Signaling Proteins

Reversible, Allosteric Small-Molecule Inhibitors of Regulator of G Protein Signaling Proteins

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

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

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Product

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Differential scanning fluorimetry as secondary screening platform for small molecule inhibitors of Bcl-X_L