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Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b
Published on: November 11, 2016
Identification and evaluation of twin-arginine translocase inhibitors
Michael L Vasil1, Andrew P Tomaras, Arthur E Pritchard
1Department of Microbiology, University of Colorado School of Medicine, Aurora, CO, USA. mike.vasil@ucdenver.edu
Abstract:
The twin-arginine translocase (TAT) in some bacterial pathogens, including Pseudomonas aeruginosa, Burkholderia pseudomallei, and Mycobacterium tuberculosis, contributes to pathogenesis by translocating extracellular virulence determinants across the inner membrane into the periplasm, thereby allowing access to the Xcp (type II) secretory system for further export in Gram-negative organisms, or directly to the outside surface of the cell, as in M. tuberculosis. TAT-mediated secretion appreciably contributes to virulence in both animal and plant models of bacterial infection. Consequently, TAT function is an attractive target for small-molecular-weight compounds that alone or in conjunction with extant antimicrobial agents could become novel therapeutics. The TAT-transported hemolytic phospholipase C (PlcH) of P. aeruginosa and its multiple orthologs produced by the above pathogens can be detected by an accurate and reproducible colorimetric assay using a synthetic substrate that detects phospholipase C activity. Such an assay could be an effective indicator of TAT function. Using carefully constructed recombinant strains to precisely control the expression of PlcH, we developed a high-throughput screening (HTS) assay to evaluate, in duplicate, >80,000 small-molecular-weight compounds as possible TAT inhibitors. Based on additional TAT-related functional assays, purified PlcH protein inhibition experiments, and repeat experiments of the initial screening assay, 39 compounds were selected from the 122 initial hits. Finally, to evaluate candidate inhibitors for TAT specificity, we developed a TAT titration assay that determines whether inhibition of TAT-mediated secretion can be overcome by increasing the levels of TAT expression. The compounds N-phenyl maleimide and Bay 11-7082 appear to directly affect TAT function based on this approach.
Insights
The twin-arginine translocase (TAT) is crucial for bacterial virulence. Researchers screened over 80,000 compounds to identify novel TAT inhibitors, finding two promising candidates for therapeutic development.
Area of Science:
- Microbiology
- Molecular Biology
- Drug Discovery
Background:
- The twin-arginine translocase (TAT) system facilitates the transport of virulence factors in bacterial pathogens like Pseudomonas aeruginosa, Burkholderia pseudomallei, and Mycobacterium tuberculosis.
- TAT-mediated secretion is essential for bacterial pathogenesis in various infection models, making it a viable target for novel therapeutics.
- The hemolytic phospholipase C (PlcH) is a key TAT-transported virulence factor that can be assayed to assess TAT function.
Purpose of the Study:
- To develop and implement a high-throughput screening (HTS) assay for identifying small-molecule inhibitors of the twin-arginine translocase (TAT) system.
- To validate potential TAT inhibitors using functional assays and specificity testing.
- To discover novel compounds that can target bacterial TAT function for therapeutic intervention.
Main Methods:
- A high-throughput screening (HTS) assay was developed using recombinant bacterial strains and a colorimetric assay for phospholipase C activity to screen over 80,000 compounds for TAT inhibition.
- Initial hits were validated through secondary functional assays, purified protein inhibition experiments, and repeat screening.
- A TAT titration assay was employed to determine the specificity of candidate inhibitors by assessing if inhibition could be overcome by increased TAT expression.
Main Results:
- A high-throughput screening identified 122 initial hits from over 80,000 tested compounds.
- Further validation narrowed down the hits to 39 compounds with potential TAT inhibitory activity.
- The TAT titration assay indicated that N-phenyl maleimide and Bay 11-7082 directly inhibit TAT function.
Conclusions:
- The study successfully established a robust HTS assay for identifying TAT inhibitors.
- N-phenyl maleimide and Bay 11-7082 were identified as compounds that directly target bacterial TAT function.
- These findings provide a foundation for developing novel therapeutics against bacterial pathogens by inhibiting the TAT secretion system.
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