AlphaScreen Technology for High Throughput Screening of 14-3-3/Raf-1 Disruptors
Principal Investigator/Assay Provider:
Haian Fu, PhD, Emory University
Isolation of small molecule compounds that can disrupt the interaction of 14-3-3 with its client proteins
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.