scholarly journals Evaluation of the InteraX™ System Technology in a High-Throughput Screening Environment

2005 ◽  
Vol 10 (5) ◽  
pp. 485-494 ◽  
Author(s):  
Frank H. Büttner ◽  
Renate Kumpf ◽  
Susanne Menzel ◽  
Dominique Reulle ◽  
Martin J. Valler
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Egf Receptor ◽  
Chimeric Proteins ◽  
System Technology ◽  
Protein Protein Interactions ◽  
A Cell ◽  
Well Assay ◽  
Beta Galactosidase

The authors have developed a cell-based high-throughput screening (HTS)-compatible assay tomeasure EGFRdimerization using the InteraX TMenzyme complementation technology of Applied Biosystems. The cells contain 2 chimeric proteins with complementing deletionmutants of the beta galactosidase enzyme, each fused to the extracellular and transmembrane part of EGFR. On binding of EGF, EGF receptor dimerizes and an active beta galactosidase is built. The authors used this homogeneous 384-well assay to screen about 20,000 diverse compounds. From 2 independent primary screen runs 239 hits were identified. For run 1, amean S/Bratio of 4.26 and ameanZβ factor of 0.74were obtained, for run 2 amean S/Bratio of 3.88 and amean Zβ factor of 0.71 were obtained. After hit confirmation, repeated 4 times, 112 hits remainedwith a confirmation rate of 48.9%. Thirty of the 112 could be identified as cytotoxic. Fifty-one of the remaining 82 compounds could be shown to be inhibitors of the beta galactosidase enzymeitself. In summary, 31 compounds remained as potential EGFRdimerization or EGF stimulation inhibitors. The authors conclude that the InteraX TMsystemtechnology is HTS capable and can detect smallmolecule inhibitors capable of inhibiting protein-protein interactions.

scholarly journals Inhibitors of Difficult Protein–Protein Interactions Identified by High-Throughput Screening of Multiprotein Complexes

2013 ◽  
Vol 8 (9) ◽  
pp. 1988-1997 ◽  
Author(s):  
Laura C. Cesa ◽  
Srikanth Patury ◽  
Tomoko Komiyama ◽  
Atta Ahmad ◽  
Erik R. P. Zuiderweg ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Protein Protein Interactions ◽  
Multiprotein Complexes

scholarly journals Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

2019 ◽  
Author(s):  
Christina K. Kim ◽  
Kelvin F. Cho ◽  
Min Woo Kim ◽  
Alice Y. Ting
Keyword(s):  
Transcription Factor ◽  
Small Molecule ◽  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Cellular Protein ◽  
Protein Protein Interactions ◽  
Lov Domain ◽  
Omics Technologies ◽  
Lower Background

Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested “AND” gate design of SPARK2—in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest—yields a lower-background system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.

scholarly journals A High-Throughput Screening Strategy to Identify Inhibitors of SSB Protein–Protein Interactions in an Academic Screening Facility

2017 ◽  
Vol 23 (1) ◽  
pp. 94-101 ◽  
Author(s):  
Andrew F. Voter ◽  
Michael P. Killoran ◽  
Gene E. Ananiev ◽  
Scott A. Wildman ◽  
F. Michael Hoffmann ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Bacterial Infections ◽  
Small Molecule Inhibitors ◽  
Klebsiella Pneumonia ◽  
Protein Protein Interactions ◽  
Response Curves ◽  
Single Stranded Dna

Antibiotic-resistant bacterial infections are increasingly prevalent worldwide, and there is an urgent need for novel classes of antibiotics capable of overcoming existing resistance mechanisms. One potential antibiotic target is the bacterial single-stranded DNA binding protein (SSB), which serves as a hub for DNA repair, recombination, and replication. Eight highly conserved residues at the C-terminus of SSB use direct protein–protein interactions (PPIs) to recruit more than a dozen important genome maintenance proteins to single-stranded DNA. Mutations that disrupt PPIs with the C-terminal tail of SSB are lethal, suggesting that small-molecule inhibitors of these critical SSB PPIs could be effective antibacterial agents. As a first step toward implementing this strategy, we have developed orthogonal high-throughput screening assays to identify small-molecule inhibitors of the Klebsiella pneumonia SSB-PriA interaction. Hits were identified from an initial screen of 72,474 compounds using an AlphaScreen (AS) primary screen, and their activity was subsequently confirmed in an orthogonal fluorescence polarization (FP) assay. As an additional control, an FP assay targeted against an unrelated eukaryotic PPI was used to confirm specificity for the SSB-PriA interaction. Nine potent and selective inhibitors produced concentration–response curves with IC50 values of <40 μM, and two compounds were observed to directly bind to PriA, demonstrating the success of this screen strategy.

scholarly journals Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Christina K Kim ◽  
Kelvin F Cho ◽  
Min Woo Kim ◽  
Alice Y Ting
Keyword(s):  
Transcription Factor ◽  
Small Molecule ◽  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Cellular Protein ◽  
Protein Protein Interactions ◽  
Lov Domain ◽  
Omics Technologies ◽  
Lower Background

Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested ‘AND’ gate design of SPARK2—in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest—yields a lower-background system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.

scholarly journals A Homogeneous 384-Well High-Throughput Binding Assay for a TNF Receptor Using Alphascreen Technology

2003 ◽  
Vol 8 (5) ◽  
pp. 522-532 ◽  
Author(s):  
Janet Wilson ◽  
Claudia Pena Rossi ◽  
Susanna Carboni ◽  
Christèle Fremaux ◽  
Dominique Perrin ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Binding Assay ◽  
Tumor Necrosis Factor Receptor ◽  
Receptor Activation ◽  
Affinity Constant ◽  
Protein Protein Interactions ◽  
Ligand Interaction ◽  
High Throughput Assay

To take advantage of the growing knowledge of cellular signaling pathways, modern-day drug discovery faces an increasing challenge to develop assays to screen for compounds that modulate protein-protein interactions. One bottleneck in achieving this goal is a lack of suitable and robust assay technologies amenable to a high-throughput format. In this report, we describe how we utilized Alphascreen™ technology to develop a high-throughput assay to monitor ligand binding to a member of the tumor necrosis factor receptor superfamily. We expressed a fusion protein consisting of the extracellular domain of the OX40 receptor with the constant domains of human IgG. In the presence of OX40 ligand, we determined a binding affinity constant consistent with reported values and optimized the protocol to develop a simple, homogeneous, and sensitive binding assay in a 384-well format. Finally, we assessed if this system could identify small peptides capable of inhibiting the OX40 receptor and ligand interaction. The results showed that the assay was able to detect such peptides and could be used to launch a high-throughput screening campaign for small molecules able to prevent OX40 receptor activation. ( Journal of Biomolecular Screening 2003:522-532)

A Phage-Assisted Continuous Selection Approach for Deep Mutational Scanning of Protein-Protein Interactions

2019 ◽  
Author(s):  
Julia Zinkus-Boltz ◽  
Craig Devalk ◽  
Bryan Dickinson
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Sequencing ◽  
Continuous Selection ◽  
Protein Protein Interactions ◽  
Design And Optimization ◽  
Molecular Determinants ◽  
A Cell ◽  
Key Drivers ◽  
Key Residues

Protein-protein interactions (PPIs) are critical for organizing molecules in a cell and mediating signaling pathways. Dysregulation of PPIs are often key drivers of disease. To better understand the biophysical basis of such disease processes – and to potentially target them - it is critical to understand the molecular determinants of PPIs. Deep mutational scanning (DMS) facilitates the acquisition of large amounts of biochemical data by coupling selection with high throughput sequencing (HTS). The challenging and labor-intensive design and optimization of a relevant selection platform for DMS, however, limits the use of powerful directed evolution and selection approaches. To address this limitation, we designed a versatile new phage assisted continuous selection (PACS) system using our proximity-dependent split RNA polymerase (RNAP) biosensors with the aim of greatly simplifying and streamlining the design of a new selection platform for PPIs. After characterization and validation using the model KRAS/RAF PPI, we generated a library of RAF variants and subjected them to PACS and DMS. Our HTS data revealed that amino acid (aa) positions 66, 84, and 89 on RAF, key residues in the KRAS/RAF PPI, are intolerant to mutations. We also identified a subset of residues with broad aa substitution tolerance, aa positions 52, 55, 76, and 79. Due to the plug and play nature of RNAP biosensors, this method can easily be extended to other PPIs. More broadly, this, and other methods under development, supports the application of evolutionary and high-throughput approaches to bear on biochemical problems, moving towards a more comprehensive understanding of sequence-function relationships in proteins.

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