Bookmark and Share
BioAssay: AID 2229

Thermal Shift Assay for Inhibitors of FLuc (Firefly Luciferase, Photinus pyralis) in Absence of ATP

Firefly (Photinus pyralis) luciferase (FLuc) is widely used as a reporter in biological assays. PTC124 (Ataluren , SID 85852879) is a small molecule clinical candidate that was optimized in FLuc based assays [1], and has been independently demonstrated to be a highly potent inhibitor of FLuc [2]. PTC124 and related inhibitors of FLuc have been shown to stabilize luciferase and increase enzyme levels leading to an apparent nonspecific activation, which can lead to false positive results in FLuc-based assays [3]. ..more
_
   
 Tested Compounds
 Tested Compounds
All(4)
 
 
Active(4)
 
 
 Tested Substances
 Tested Substances
All(4)
 
 
Active(4)
 
 
AID: 2229
Data Source: NCGC (FLUC202)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-01

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 4
Related Experiments
Show more
AIDNameTypeComment
2228Thermal Shift Assay for Inhibitors of FLuc (Firefly Luciferase, Photinus pyralis) in Presence of ATPScreeningdepositor-specified cross reference: Thermal Shift Assay for Inhibitors of FLuc (Firefly Luciferase, Photinus pyralis) in Presence of ATP
2309Probe Summary for Inhibitors and Stabilizers of Firefly LuciferaseSummarydepositor-specified cross reference: Probe Summary for Inhibitors and Stabilizers of Firefly Luciferase
411qHTS Assay for Inhibitors of Firefly LuciferaseConfirmatorysame project related to Summary assay
588342qHTS profiling assay for firefly luciferase inhibitor/activator using purifed enzyme and Km concentrations of substrates (counterscreen for miR-21 project)Confirmatorysame project related to Summary assay
602357FLuc inhibitory activity for the follow-up compounds in a biochemical assay with Km concentrations of substrateConfirmatorysame project related to Summary assay
602358FLuc inhibitory activity for the follow-up compounds in a biochemical assay with Km concentrations of substrate and 500microM CoASHConfirmatorysame project related to Summary assay
602364FLuc inhibitory activity for the follow-up compounds in a biochemical assay with 1mM ATPConfirmatorysame project related to Summary assay
602365FLuc inhibitory activity for the follow-up compounds in a biochemical assay with 1mM D-luciferinConfirmatorysame project related to Summary assay
602474FLuc inhibitory activity for the follow-up compounds in a biochemical assay with a commercial detection reagent - BriteliteTM Plus (PerkinElmer)Confirmatorysame project related to Summary assay
602475FLuc inhibitory activity for the follow-up compounds in a biochemical assay with an in-house formulation of detection reagentConfirmatorysame project related to Summary assay
602476FLuc inhibitory activity for the follow-up compounds in a cell-based translational read-through assayConfirmatorysame project related to Summary assay
602477FLuc inhibitory activity for the follow-up compounds in a cell-based assay to assess the activity of miR-21Confirmatorysame project related to Summary assay
602478FLuc inhibitory activity for the follow-up compounds in a cell-based translational read-through assay (72 hour incubation)Confirmatorysame project related to Summary assay
624030Biochemical firefly luciferase enzyme assay for NPCConfirmatorysame project related to Summary assay
652016qHTS Assay for Inhibitors of Firefly Luciferase from the GSK Published Protein Kinase Inhibitor SetConfirmatorysame project related to Summary assay
Description:
Firefly (Photinus pyralis) luciferase (FLuc) is widely used as a reporter in biological assays. PTC124 (Ataluren , SID 85852879) is a small molecule clinical candidate that was optimized in FLuc based assays [1], and has been independently demonstrated to be a highly potent inhibitor of FLuc [2]. PTC124 and related inhibitors of FLuc have been shown to stabilize luciferase and increase enzyme levels leading to an apparent nonspecific activation, which can lead to false positive results in FLuc-based assays [3].

In order to determine if FLuc thermal stabilization parallels inhibitor potency, a fluorescence-based thermal denaturation assay was developed in this present study. An environmentally sensitive fluorescence dye, SYPRO Orange (Invitrogen, presently Life Technologies) was utilized to monitor the fluorescence change as a function of temperature. The dye preferentially binds to the hydrophobic regions of the protein, and its high signal-to-noise ratio, large Stokes shift and high excitation wavelength make it ideal for thermal shift application [4]. In this assay, the melting temperature (Tm) (the inflection point of the protein melt transition) was assessed for each sample tested. Differences in the Tm between controls and compound-containing samples were determined (the thermal shift or delta Tm), and compounds were ranked by the degree of their thermal shift at the top concentration (200 uM). Positive shifts suggest enhanced protein stability in the presence of the compound and thus support binding.

FLuc catalyzes light production in bioluminescent organisms [5]. The reaction requires luciferin, ATP and O2 as substrate, and it includes the formation of a luciferyl-AMP intermediate and the subsequent production of light, AMP, oxyluciferin and CO2. In this study, PTC124 and analogues were evaluated for their potential to stabilize FLuc in the absence and presence of ATP. This particular BioAssay is in the absence of ATP. See related BioAssay "Thermal Shift Assay for Inhibitors of FLuc (Firefly Luciferase, Photinus pyralis) in the Presence of ATP"

See [6] for more details on this assay and related studies.

[1] Welch, et al. PTC124 targets genetic disorders caused by nonsense mutations. Nature 447 (2007) 87-91.

[2] D.S. Auld, N. Thorne, W.F. Maguire, and J. Inglese, Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression. Proc Natl Acad Sci U S A 106 (2009) 3585-90.

[3] D.S. Auld, N. Thorne, D.T. Nguyen, and J. Inglese, A specific mechanism for nonspecific activation in reporter-gene assays. ACS Chem Biol 3 (2008) 463-70.

[4]F.H. Niesen, H. Berglund, and M. Vedadi, The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2 (2007) 2212-21.

[5] J.R. de Wet, K.V. Wood, M. DeLuca, D.R. Helinski, and S. Subramani, Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7 (1987) 725-37.

[6] Auld, et al. Molecular Basis for the High Affinity Binding and Stabilization of Firefly Luciferase by PTC124. Proc Natl Acad Sci U S A, accepted.
Protocol
Assay Protocol:

1.8 uM FLuc was tested in Tris-acetate buffer (pH 7.6) or phosphate buffer saline (pH 7.4). Compounds were tested at 200 uM. Data was captured on an iQ5 Real Time PCR Detection system (Bio-Rad) using a temperature range from 20 to 95C in increments of 1C and a ramping rate of 0.1C/s. Fluorescence intensity changes (Ex 490/Em 575 nm) were monitored with a charge-coupled device (CCD) camera.

Buffer: 50 mM Tris-acetate, pH 7.6 (with or without 2 mM ATP); 1X PBS, pH 7.4 (with or without 2 mM ATP)
Comment
Compound Ranking:

1. Compounds that gave positive thermal shift (delta_Tm) were declared as "Active".

2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, the score is delta_Tm*10 rounded to closest to integer. The higher the Tm shift, the higher the PUBCHEM_ACTIVITY_SCORE. Note that the score is not normalized and can be greater than 100.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1delta_Tm (200μM**)thermal shift in degrees CelciusFloatCelcius
2Compound_QCsource of compound QCString

** Test Concentration.
Additional Information
Grant Number: U54

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
PageFrom: