AID	Name	Type	Comment
422	HTS for 14-3-3 protein interaction modulators	screening	Primary screen preceding this dose response confirmation assay.

NIH Molecular Libraries Screening Centers Network [MLSCN]
Emory Chemical Biology Discovery Center in MLSCN
MLSCN Grant: 1 X01MH78953-01

The 14-3-3 proteins are the prototype for a novel class of protein modules that can recognize phosphoserine/threonine (pS/T)-containing motifs in a variety of signaling proteins. To date, 14-3-3 proteins have been reported to bind more than 200 client proteins. Through these interactions, 14-3-3 proteins play important roles in a wide range of vital regulatory processes, such as Bad-induced apoptosis, Raf-1-mediated cell proliferation, and Cdc25-regulated cell cycle progression. In addition to their participation in diverse physiological processes, 14-3-3 proteins have been implicated in a number of clinically important pathological conditions, such as neurodegenerative disorders and cancers. Thus, such studies on the 14-3-3/client-protein interactions may provide tremendous opportunities for therapeutic interventions. Therefore, chemical tools would allow pharmacological probing of 14-3-3 function under various conditions.

To develop small-molecule modulators of 14-3-3 proteins, a highly sensitive fluorescence polarization (FP)-based 14-3-3 assay was designed and optimized. In this assay, the interaction of 14-3-3 with a fluorescently labeled phosphopeptide from Raf-1 was used as a model system. A simple 1-step "mix-and-measure" method was achieved for analyzing 14-3-3 proteins. This is a solution-based, versatile method that can be used to monitor the binding of 14-3-3 with a variety of client proteins. The 14-3-3 FP assay is highly stable and has achieved a robust performance in a 384-well format with a demonstrated signal-to-noise ratio of above 10 and a Z' factor of above 0.7. Because of its simplicity and high sensitivity, this assay is used for high-throughput screening of 14-3-3 modulators.

Compounds identified as active from the primary screen, AID 422 - HTS for 14-3-3 protein interaction modulators, were selected for testing in this assay.

Each compound was tested at 6 concentrations, 1:2 serial dilutions starting at a nominal test concentration of 50 micromolar. Each concentration was tested in triplicate. In some cases, compounds were retested in 6 concentration, 1:2 serial dilutions starting at 25 micromolar or lower, to obtain the final IC50. These cases are noted in the PUBCHEM_ASSAYDATA_COMMENT field.

The recombinant hexaHis-tagged 14-3-3gamma were expressed and purified as previously described (Fu et al, PNAS, 1993). The peptide sequence containing phosphorylated S259 of Raf-1 was synthesized and labeled with 5/6 carboxytetramethylrhodamine. HEPES buffer (10mM, pH 7.4), NaCl (150mM), Tween-20 (0.05%), and DTT (0.5 mM) was used through the screening.

The 14-3-3 FP assay was carried out in black 384-well uplates in a total volume of 50uL in each well. 49uL of assay reaction buffer (1uM of His-14-3-3gamma and 2nM of TMR-pS259-Raf peptide in HEPES buffer) were dispensed to 384-well black plate. For each assay plate, column 1, 2 and 23, 24 contain controls: TMRpS259-Raf peptide only (blank) and TMR-pS259-Raf peptide with His-14-3-3gamma (negative control); R18 peptide was added to wells with TMR-pS259-Raf peptide and His-14-3-3gamma and serve as positive control. Each compound was tested in triplicate at the noted concentrations. Plates were incubated at room temperature for 2 hr and the fluorescence polarization value in millipolarization (mP) units was measured with an Analyst HT reader. An excitation filter at 545nm and an emission filter at 610 to 675nm were used with a dichroic mirror of 565 nm.

Assay data was analyzed using BioAssay software from CambridgeSoft. Percentage of control was calculated by dividing mP (subtracting blank) value from compound wells by the average mP (subtracting blank) from negative controls (His-14-3-3gamma and peptide) in each plate.

For each compound, a 4 parameter sigmoidal dose-response curve was fitted using BioAssay software from CambridgeSoft. The reported IC50 values were generated from fitted curves by solving for X-intercept at the 50% level of Y-intercept.

When the highest concentration tested (50 micromolar) did not result in greater than 50% inhibition, the IC50 is reported as greater than 50 micromolar. Similarly, when the lowest concentration tested (1.5625 micromolar) resulted in greater than 50% inhibition, the IC50 is reported as less than 1.5625 micromolar.

Compounds with IC50 values of greater than 30 micromolar were considered inactive, compounds with an IC50 equal or less than 30 micromolar are considered active.

The activity score was calculated based on the observed range of the IC50 values obtained, according to the following formula:

PUBCHEM_ACTIVITY_SCORE = 100 * (1 - ((IC50 - LowestIC50) / (HighestIC50 - LowestIC50)))

1. In some cases where the IC50 was found to be lower than the lowest tested concentration, compounds were retested in 6 point, 1:2 serial dilutions starting at 25 micromolar or less, to obtain the final IC50. These cases are noted in the PUBCHEM_ASSAYDATA_COMMENT field.

2. All data reported were normalized on a per-plate basis.

3. Possible interference in this assay, but are not limited to, compounds with auto-fluorescence, compound quenching fluorescence, or precipitate.