title: Inhibitors of Streptococcus pneumoniae surface endonuclease EndA discovered by high-throughput screening using a PicoGreen fluorescence assay.
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.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Eliza J R
authors.initials: EJ
authors.lastname: Peterson
authors.forename: Dmitri
authors.initials: D
authors.lastname: Kireev
authors.forename: Andrea F
authors.initials: AF
authors.lastname: Moon
authors.forename: Marika
authors.initials: M
authors.lastname: Midon
authors.forename: William P
authors.initials: WP
authors.lastname: Janzen
authors.forename: Alfred
authors.initials: A
authors.lastname: Pingoud
authors.forename: Lars C
authors.initials: LC
authors.lastname: Pedersen
authors.forename: Scott F
authors.initials: SF
authors.lastname: Singleton
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D005453:Fluorescence; D057166:High-Throughput Screening Assays / Q000379:methods*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000037:antagonists & inhibitors / Q000378:metabolism; D009930:Organic Chemicals / Q000737:chemistry; D015203:Reproducibility of Results; D013296:Streptococcus pneumoniae / Q000187:drug effects* / Q000201:enzymology* / Q000378:metabolism; D037521:Virulence Factors / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009930:Organic Chemicals; C099476:PicoGreen; D037521:Virulence Factors; D004247:DNA; D004706:Endodeoxyribonucleases; C064668:endA protein, bacteria; D008836:Micrococcal Nuclease