Thermal Shift Assay for Inhibitors of FLuc (Firefly Luciferase, Photinus pyralis) in Presence 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 , and has been independently demonstrated to be a highly potent inhibitor of FLuc . 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 . ..more
BioActive Compounds: 8
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 , and has been independently demonstrated to be a highly potent inhibitor of FLuc . 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 .
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 . 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 . 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 presence of ATP. See related BioAssay "Thermal Shift Assay for Inhibitors of FLuc (Firefly Luciferase, Photinus pyralis) in the Absence of ATP"
See  for more details on this assay and related studies.
 Welch, et al. PTC124 targets genetic disorders caused by nonsense mutations. Nature 447 (2007) 87-91.
 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.
 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.
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.
 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.
 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.
1.8 uM FLuc was tested in Tris-acetate buffer (pH 7.6) or phosphate buffer saline (pH 7.4). ATP final concentration was 2 mM. 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)
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.
** Test Concentration.
Data Table (Concise)