A Time-Resolved Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of 14-3-3 Protein–Protein Interaction Inhibitors

Abstract

Protein-protein interaction networks mediate diverse biological processes by regulating various signaling hubs and clusters. 14-3-3 proteins, a family of phosphoserine/threonine-binding molecules, serve as major interaction hubs in eukaryotic cells and have emerged as promising therapeutic targets for various human diseases. In order to identify chemical probes for mechanistic studies and for potential therapeutic development, we have developed highly sensitive bioassays to monitor the interaction of 14-3-3 with a client protein. In this study, we describe a homogenous time-resolved fluorescence resonance energy transfer (TR-FRET) assay to detect the interaction of 14-3-3 with Bad, a proapoptotic member of the Bcl-2 family. Through a series of titration studies in which europium-labeled 14-3-3 serves as an FRET donor and a Dy647-labeled phosphorylated Bad, the peptide acts as an FRET acceptor, we have achieved a robust TR-FRET assay that is suitable for high-throughput screening (HTS) with an excellent signal-to-background ratio of >20 and Z 0 values >0.7. This assay was further miniaturized to a 1,536-well format for ultra-HTS (uHTS), and exhibited a similar robust performance. The utility and performance of the assay for uHTS were validated by (i) known inhibitors, including peptide R18 and small molecule FOBISIN101, and (ii) screening of a 51,200 compound library. This simple and robust assay is generally applicable to detect the interaction of 14-3-3 with other client proteins. It provides a sensitive and easy-to-use tool to facilitate the discovery of 14-3-3 protein inhibitors as well as to study 14-3-3-mediated protein-protein interactions.

Stress Induces p38 MAPK-Mediated Phosphorylation and Inhibition of Drosha-Dependent Cell Survival

“…Procedures for TR-FRET assay were the same as previously described (Du et al, 2013; Du and Havel, 2012). Briefly, overexpressed proteins (Drosha-FLAG, FLAG-MKK3, GST-p38 and GST-MKK3) from various cell lysates after transfection were collected and diluted in FRET buffer.…”

Cables1 Complex Couples Survival Signaling to the Cell Death Machinery

“…The positive control Bad pS136 peptide and R18, which specifically bind to the amphipathic groove of 14-3-3 with high affinity, completely blocked the binding of GST-Cables1 with His-14-3-3γ at 10 μM. Next, we tested whether these Cables1 peptides can directly interact with 14-3-3 protein in a defined in vitro system using a homogenous TR-FRET assay (26). The TR-FRET assay provides a sensitive measurement for proximity based molecular interactions to evaluate the binding of the donor-fluorophore (Tb)-coupled 14-3-3 proteins with FITC-labeled Cables1 peptides.…”

“…Our published protocols for the TR-FRET assay were followed (26, 27). FITC-conjugated Cables1 T44 (FITC-Ahx-ENAPLRRCRTLSGSPR), T150 (FITC-Ahx-TNAFGARRNTIDSTSS), pT44 (FITC-Ahx-ENAPLRRCR (pT) LSGSPR) and pT150 (FITC-Ahx-TNAFGARRN (pT) IDSTSS) peptides were synthesized by Peptide 2.0 Inc (>80% purity).…”

Histone Deacetylase 6 (HDAC6) Promotes the Pro-survival Activity of 14-3-3ζ via Deacetylation of Lysines within the 14-3-3ζ Binding Pocket

“…21), these efforts have proven challenging at least in part due to difficulty in developing high-throughput screens for 14-3-3 activity (although recent progress has been made in this area (31)). This challenge motivated us to look for other ways to manipulate 14-3-3 binding, potentially via 14-3-3 regulatory mechanisms.…”