Novel In Vitro Protein Fragment Complementation Assay Applicable to High-Throughput Screening in a 1536-Well Format

Abstract

Protein-protein interactions (PPIs) play key roles in all cellular processes and hence are useful as potential targets for new drug development. To facilitate the screening of PPI inhibitors as anticancer drugs, the authors have developed a highthroughput screening (HTS) system using an in vitro protein fragment complementation assay (PCA) with monomeric Kusabira-Green fluorescent protein (mKG). The in vitro PCA system was established by the topological formation of a functional complex between 2 split inactive mKG fragments fused to target proteins, which fluoresces when 2 target proteins interact to allow complementation of the mKG fragments. Using this assay system, the authors screened inhibitors for TCF7/β-catenin, PAC1/PAC2, and PAC3 homodimer PPIs from 123,599 samples in their natural product library. Compound TB1 was identified as a specific inhibitor for PPI of PAC3 homodimer. TB1 strongly inhibited the PPI of PAC3 homodimer with an IC 50 value of 0.020 µM and did not inhibit PPI between TCF7/β-catenin and PAC1/PAC2 even at a concentration of 250 µM. The authors thus demonstrated that this in vitro PCA system applicable to HTS in a 1536-well format is capable of screening for PPI inhibitors from a huge natural product library. (Journal of Biomolecular Screening 2009:970-979)

Interaction between TIM-1 and NPC1 Is Important for Cellular Entry of Ebola Virus

“…After 24 h, cells with fluorescent signals derived from complemented mKG molecules accumulated in cells were counted using flow cytometry. Complemented mKG molecules have been shown to be little dissociated in cells (40,41). For visualization of early endosomes and Golgi compartments, 293T cells transfected with the mKG fusion plasmids were incubated with CellLight Early Endosomes-RFP (red fluorescent protein) reagents (C10587; Life Technologies) or CellLight Golgi-RFP reagents (C10593; Life Technologies) for 16 h at 37°C.…”

Bimolecular Fluorescence Complementation (BiFC) Analysis: Advances and Recent Applications for Genome-Wide Interaction Studies

“…This study uncovered an intramolecular interaction within p40 phox as well as interactions between p67 phox and adaptor proteins, p47 phox and p40 phox [42]. The mKG-based BiFC system has also been used in high-throughput screening for PPI inhibitors using a large collection of natural product library [44]. By screening 123,599 samples of the natural product library in a 1536-well format, a specific inhibitor for PPI of PAC3 homodimer was identified.…”

“…In another recent study, BiFC technology was used to screen anti-influenza A virus drug candidates that could reduce autophagy induction by affecting Beclin-Bcl2 heterodimer dissociation [118]. As discussed earlier, to facilitate anticancer drug discovery, mKG-based BiFC system was used to screen for PPI inhibitors in a natural product library [44]. These studies demonstrate that BiFC assays have great potential for high-throughput screening in drug discovery.…”

Using proteomics to identify the HBx interactome in hepatitis B virus: how can this inform the clinic?

“…Their interactions are captured with antibody against FLAG, and determined by performing an Renilla luciferase enzymatic assay [154] Protein-fragment complementation assay • Protein-fragment complementation assays rely on reconstituting a wide range of 'reporters', such as monomeric Kusabira-Green protein, wherein reporter proteins are split into two inactive fragments that are fused to bait and prey. An interaction between the fused bait and prey restores the function of the fragmented reporter protein, and the fluorescence emitted due to monomeric Kusabira-Green protein complementation by target protein interaction is measured [155]. Protein-fragment complementation assays can be used in the identification of PPIs between proteins of any molecular weight and expressed at their endogenous levels, and can be performed in any living cell, multicellular organism [156] Akt1 RAC-alpha serine/threonine-protein kinase Co-IP [49] CEBPa CCAAT/enhancer-binding protein alpha Co-IP…”