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Updated: May 18, 2026
Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System
Published on: August 8, 2016
Inhibitors of Streptococcus pneumoniae surface endonuclease EndA discovered by high-throughput screening using a
Eliza J R Peterson1, Dmitri Kireev, Andrea F Moon
1Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Abstract:
The human commensal pathogen Streptococcus pneumoniae expresses a number of virulence factors that promote serious pneumococcal diseases, resulting in significant morbidity and mortality worldwide. These virulence factors may give S. pneumoniae the capacity to escape immune defenses, resist antimicrobial agents, or a combination of both. Virulence factors also present possible points of therapeutic intervention. The activities of the surface endonuclease, EndA, allow S. pneumoniae to establish invasive pneumococcal infection. EndA's role in DNA uptake during transformation contributes to gene transfer and genetic diversification. Moreover, EndA's nuclease activity degrades the DNA backbone of neutrophil extracellular traps (NETs), allowing pneumococcus to escape host immune responses. Given its potential impact on pneumococcal pathogenicity, EndA is an attractive target for novel antimicrobial therapy. Herein, we describe the development of a high-throughput screening assay for the discovery of nuclease inhibitors. Nuclease-mediated digestion of double-stranded DNA was assessed using fluorescence changes of the DNA dye ligand, PicoGreen. Under optimized conditions, the assay provided robust and reproducible activity data (Z'= 0.87) and was used to screen 4727 small molecules against an imidazole-rescued variant of EndA. In total, six small molecules were confirmed as novel EndA inhibitors, some of which may have utility as research tools for understanding pneumococcal pathogenesis and for drug discovery.
Insights
Researchers developed a high-throughput screening assay to find inhibitors of EndA, a key virulence factor in Streptococcus pneumoniae. Six novel EndA inhibitors were identified, offering potential for new antimicrobial therapies against pneumococcal diseases.
Area of Science:
- Microbiology
- Molecular Biology
- Drug Discovery
Background:
- * Streptococcus pneumoniae is a major cause of global morbidity and mortality.
- * Virulence factors, like the surface endonuclease EndA, are crucial for pneumococcal pathogenesis and immune evasion.
- * EndA degrades neutrophil extracellular traps (NETs), aiding bacterial survival and genetic exchange.
Purpose of the Study:
- * To develop a high-throughput screening assay for identifying EndA inhibitors.
- * To discover novel small molecules targeting EndA for potential antimicrobial therapy.
- * To explore the utility of EndA inhibitors as research tools for pneumococcal pathogenesis.
Main Methods:
- * Development of a fluorescence-based assay using PicoGreen to monitor double-stranded DNA digestion by EndA.
- * Optimization of assay conditions yielding robust and reproducible results (Z'= 0.87).
- * Screening of 4727 small molecules against an imidazole-rescued variant of EndA.
Main Results:
- * Identification of six novel small molecules that inhibit EndA nuclease activity.
- * Validation of the high-throughput screening assay's effectiveness and reproducibility.
- * Demonstration of EndA's critical role in pneumococcal virulence and immune evasion.
Conclusions:
- * The developed assay is effective for discovering EndA inhibitors.
- * Novel EndA inhibitors were identified, representing potential therapeutic leads.
- * These inhibitors could advance research into pneumococcal pathogenesis and drug discovery.
