High-Throughput Screening in the Discovery of Small-Molecule Inhibitors of Protein-Protein Interactions

2018 ◽  
pp. 29-51
Author(s):  
Chunlin Zhuang ◽  
Chunquan Sheng
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Protein Protein Interactions

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

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 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.

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