Use of Differential Scanning Fluorimetry to Identify Nuclear Receptor Ligands

Abstract

Identification of small molecules that interact specifically with the ligand-binding domains (LBDs) of nuclear receptors (NRs) can be accomplished using a variety of methodologies. Here, we describe the use of differential scanning fluorimetry to identify these ligands, a technique that requires no modification or derivatization of either the protein or the ligand, and uses an instrument that is becoming increasingly affordable and common in modern molecular biology laboratories, the quantitative, or real-time, PCR machine. Upon being introduced to specific ligands, nuclear receptors undergo structural and dynamic changes that tend to increase molecular stability, which can be measured by the resistance of the protein to heat denaturation. Differential scanning fluorimetry (DSF) uses a dielectric sensitive fluorescent dye to measure the thermal denaturation, or "melting" point (Tm) of a protein under different conditions, in this case in the absence and presence of a candidate ligand. Using DSF, multiple candidates can be screened at once, in numbers corresponding to plate size of the instrument used (e.g., 96- or 384-well), allowing significant throughput if a modest library of compounds needs to be tested.

Structure–Activity Relationship Studies of Trisubstituted Isoxazoles as Selective Allosteric Ligands for the Retinoic-Acid-Receptor-Related Orphan Receptor γt

“…A thermal shift assay (TSA) was performed as an orthogonal assay to investigate the effect of the compounds on the thermal denaturation of the RORγt protein (Table , Figure C). Ligand binding typically improves the thermal stability of a protein by stabilizing the protein fold, as indicated by the change in the melting temperature Δ T m . − The C-4 isoxazole derivatives 3–9 showed a thermal stabilization effect according to their potency, as observed in the TR-FRET assays. The most potent compounds 3 and 6 showed particularly high Δ T m values (4.9 and 6.4 °C resp.).…”

Structure–Activity Relationship Studies of Trisubstituted Isoxazoles as Selective Allosteric Ligands for the Retinoic-Acid-Receptor-Related Orphan Receptor γt

“…A thermal shift assay (TSA) was performed as an orthogonal assay to investigate the effect of the compounds on the thermal denaturation of the RORγt protein (Table , Figure C). Ligand binding typically improves the thermal stability of a protein by stabilizing the protein fold, as indicated by the change in the melting temperature Δ T m . − The C-4 isoxazole derivatives 3–9 showed a thermal stabilization effect according to their potency, as observed in the TR-FRET assays. The most potent compounds 3 and 6 showed particularly high Δ T m values (4.9 and 6.4 °C resp.).…”

Structural Basis for PPARs Activation by The Dual PPARα/γ Agonist Sanguinarine: A Unique Mode of Ligand Recognition

“…In contrast, sanguinarine had no effect on PPARδ for recruiting its co-activator motifs. Ligand binding usually improves the thermal stability of NRs via structural and dynamic changes of the protein fold [ 25 , 26 ]. The thermal shift assay (TSA) showed that sanguinarine increased the thermal stability of PPARα and PPARγ, but the Tm of PPARδ remained unchanged ( Figure 2 B,C).…”

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

“…In contrast, many nucleotide-binding proteins were captured with a relatively low specificity and, to a large degree, are probably false positives. Further validation of the specific binding to c-di-GMP using techniques such as DRaCALA 20 , UV cross-linking 15 , differential scanning fluorimetry (DSF) 21 , Microscale thermophoresis (MST) 22 , isothermal calorimetry (ITC) [23][24] … is thus necessary.…”

Structure–Activity Relationship Studies of Trisubstituted Isoxazoles as Selective Allosteric Ligands for the Retinoic-Acid-Receptor-Related Orphan Receptor γt

“…A thermal shift assay (TSA) was performed as an orthogonal assay to investigate the effect of the compounds on the thermal denaturation of the RORγt protein (Table , Figure C). Ligand binding typically improves the thermal stability of a protein by stabilizing the protein fold, as indicated by the change in the melting temperature Δ T m . − The C-4 isoxazole derivatives 3–9 showed a thermal stabilization effect according to their potency, as observed in the TR-FRET assays. The most potent compounds 3 and 6 showed particularly high Δ T m values (4.9 and 6.4 °C resp.).…”

Structural Basis for PPARs Activation by The Dual PPARα/γ Agonist Sanguinarine: A Unique Mode of Ligand Recognition

“…In contrast, sanguinarine had no effect on PPARδ for recruiting its co-activator motifs. Ligand binding usually improves the thermal stability of NRs via structural and dynamic changes of the protein fold [ 25 , 26 ]. The thermal shift assay (TSA) showed that sanguinarine increased the thermal stability of PPARα and PPARγ, but the Tm of PPARδ remained unchanged ( Figure 2 B,C).…”

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

“…In contrast, many nucleotide-binding proteins were captured with a relatively low specificity and, to a large degree, are probably false positives. Further validation of the specific binding to c-di-GMP using techniques such as DRaCALA 20 , UV cross-linking 15 , differential scanning fluorimetry (DSF) 21 , Microscale thermophoresis (MST) 22 , isothermal calorimetry (ITC) [23][24] … is thus necessary.…”

Structure–Activity Relationship Studies of Trisubstituted Isoxazoles as Selective Allosteric Ligands for the Retinoic-Acid-Receptor-Related Orphan Receptor γt

“…A thermal shift assay (TSA) was performed as an orthogonal assay to investigate the effect of the compounds on the thermal denaturation of the RORγt protein (Table , Figure C). Ligand binding typically improves the thermal stability of a protein by stabilizing the protein fold, as indicated by the change in the melting temperature Δ T m . − The C-4 isoxazole derivatives 3–9 showed a thermal stabilization effect according to their potency, as observed in the TR-FRET assays. The most potent compounds 3 and 6 showed particularly high Δ T m values (4.9 and 6.4 °C resp.).…”

Structural Basis for PPARs Activation by The Dual PPARα/γ Agonist Sanguinarine: A Unique Mode of Ligand Recognition

“…In contrast, sanguinarine had no effect on PPARδ for recruiting its co-activator motifs. Ligand binding usually improves the thermal stability of NRs via structural and dynamic changes of the protein fold [ 25 , 26 ]. The thermal shift assay (TSA) showed that sanguinarine increased the thermal stability of PPARα and PPARγ, but the Tm of PPARδ remained unchanged ( Figure 2 B,C).…”

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

“…In contrast, many nucleotide-binding proteins were captured with a relatively low specificity and, to a large degree, are probably false positives. Further validation of the specific binding to c-di-GMP using techniques such as DRaCALA 20 , UV cross-linking 15 , differential scanning fluorimetry (DSF) 21 , Microscale thermophoresis (MST) 22 , isothermal calorimetry (ITC) [23][24] … is thus necessary.…”