Use of Differential Scanning Fluorimetry as a High-Throughput Assay to Identify Nuclear Receptor Ligands

DeSantis, Kara A.; Reed, Aaron; Rahhal, Raneen; Reinking, Jeff

doi:10.1621/nrs.10002

Cited by 35 publications

(26 citation statements)

References 16 publications

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“…DSF allows the identification of a compound binding to its target protein through the observed thermal shift (ΔTm) of the protein [47–49]. APE1 was incubated with C10 at 37°C for 30 min and then the fluorescence dye was added.…”

Section: Resultsmentioning

confidence: 99%

An APE1 inhibitor reveals critical roles of the redox function of APE1 in KSHV replication and pathogenic phenotypes

Zhong

Wang

et al. 2017

PLoS Pathog

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APE1 is a multifunctional protein with a DNA base excision repair function in its C-terminal domain and a redox activity in its N-terminal domain. The redox function of APE1 converts certain transcription factors from inactive oxidized to active reduced forms. Given that among the APE1-regulated transcription factors many are critical for KSHV replication and pathogenesis, we investigated whether inhibition of APE1 redox function blocks KSHV replication and Kaposi’s sarcoma (KS) phenotypes. With an shRNA-mediated silencing approach and a known APE-1 redox inhibitor, we demonstrated that APE1 redox function is indeed required for KSHV replication as well as KSHV-induced angiogenesis, validating APE1 as a therapeutic target for KSHV-associated diseases. A ligand-based virtual screening yielded a small molecular compound, C10, which is proven to bind to APE1. C10 exhibits low cytotoxicity but efficiently inhibits KSHV lytic replication (EC50 of 0.16 μM and selective index of 165) and KSHV-mediated pathogenic phenotypes including cytokine production, angiogenesis and cell invasion, demonstrating its potential to become an effective drug for treatment of KS.

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Section: Resultsmentioning

confidence: 99%

An APE1 inhibitor reveals critical roles of the redox function of APE1 in KSHV replication and pathogenic phenotypes

Zhong

Wang

et al. 2017

PLoS Pathog

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“…The temperature of the samples were changed from 25 to 95 °C at a heating rate of 0.5 °C/min, and the fluorescence was monitored by Mx3005P qPCR instrument (Agilent). The melting point (Tm) was calculated as the lowest point of the first derivative plot (DeSantis et al, 2012). …”

Section: Methodsmentioning

confidence: 99%

Structure of the Mtb CarD/RNAP β-Lobes Complex Reveals the Molecular Basis of Interaction and Presents a Distinct DNA-Binding Domain for Mtb CarD

Gulten

Sacchettini

2013

Structure

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SUMMARY CarD from Mycobacterium tuberculosis (Mtb) is an essential protein thought to be involved in stringent response through downregulation of rRNA and ribosomal protein genes. CarD interacts with the β-subunit of RNAP and this interaction is vital for Mtb’s survival during the persistent infection state. We have determined the crystal structure of CarD in complex with the RNAP β-subunit β1 and β2 domains at 2.1 Å resolution. The structure reveals the molecular basis of CarD/RNAP interaction, providing a basis to further our understanding of RNAP regulation by CarD. The structural fold of the CarD N-terminal domain is conserved in RNAP interacting proteins such as TRCF-RID and CdnL, and displays similar interactions to the predicted homology model based on the TRCF/RNAP β1 structure. Interestingly, the structure of the C-terminal domain, which is required for complete CarD function in vivo, represents a novel DNA binding fold.

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“…The shift is dependent on the affinity and also on the concentration of compound. An assay based on this stabilization effect on proteins has been designed and is termed the Fluorescent Thermal Shift Assay (FTSA, also called ThermoFluor Ò [1] and differential scanning fluorimetry [2][3][4][5], DSF) [6][7][8] when protein unfolding is followed by the intrinsic tryptophan fluorescence or extrinsic fluorescence of a solvatochromic probe such as 1,8-anilino-naphthalenesulfonate (ANS) [9,10] or Sypro Orange [11,12].…”

Section: Introductionmentioning

confidence: 99%

Thermodynamic characterization of human carbonic anhydrase VB stability and intrinsic binding of compounds

Kasiliauskaitė

Časaitė

Juozapaitienė

et al. 2015

J Therm Anal Calorim

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The thermodynamics of low molecular weight synthetic sulfonamide inhibitor binding to carbonic anhydrase (CA) VB was determined by the isothermal titration calorimetry (ITC) and the fluorescent thermal shift assay (FTSA). ITC provided the enthalpic and entropic contributions to the binding affinity of ethoxzolamide to CA VB. FTSA is a high-throughput assay that measures protein thermal stabilization by added ligands. FTSA enabled determination of extremely high affinity of several compounds binding to CA VB. CA VB is one of two isoforms that are expressed in mitochondria, participate in carbon metabolism and pH homeostasis and are implicated in diseases such as obesity. Therefore CA VB is a drug target. Here a series of para-substituted tetrafluoro benzenesulfonamides were investigated as high affinity inhibitors of CA VB. Thermodynamic equilibrium binding measurements such as ITC and FTSA provide only the observed parameters. Dissection of binding-linked reactions is necessary to obtain the intrinsic parameters that in turn could be correlated with the chemical structure of the inhibitors. Intrinsic dissociation constants of the inhibitors were estimated and they reached 1 pM, one of the strongest binding reactions observed between any protein-ligand binding.

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Use of Differential Scanning Fluorimetry as a High-Throughput Assay to Identify Nuclear Receptor Ligands

Cited by 35 publications

References 16 publications

An APE1 inhibitor reveals critical roles of the redox function of APE1 in KSHV replication and pathogenic phenotypes

An APE1 inhibitor reveals critical roles of the redox function of APE1 in KSHV replication and pathogenic phenotypes

Structure of the Mtb CarD/RNAP β-Lobes Complex Reveals the Molecular Basis of Interaction and Presents a Distinct DNA-Binding Domain for Mtb CarD

Thermodynamic characterization of human carbonic anhydrase VB stability and intrinsic binding of compounds

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