Identification of inhibitors for single-stranded DNA-binding proteins in eubacteria

Abstract

Objectives: The increasing threat of drug-resistant bacteria establishes a continuing need for the development of new strategies to fight infection. We examine the inhibition of the essential single-stranded DNA-binding proteins (SSBs) SSBA and SSBB as a potential antimicrobial therapy due to their importance in DNA replication, activating the SOS response and promoting competence-based mechanisms of resistance by incorporating new DNA.Methods: Purified recombinant SSBs from Gram-positive (Staphylococcus aureus and Bacillus anthracis) and Gram-negative (Escherichia coli and Francisella tularensis) bacteria were assessed in a high-throughput screen for inhibition of duplex DNA unwinding by small molecule inhibitors. Secondary electrophoretic mobility shift assays further validated the top hits that were then tested for MICs using in vitro assays. Results:We have identified compounds that show cross-reactivity in vitro, as well as inhibition of both F. tularensis and B. anthracis SSBA. Five compounds were moderately toxic to at least two of the four bacterial strains in vivo, including two compounds that were selectively non-toxic to human cells, 9-hydroxyphenylfluoron and purpurogallin. Three of the SSBA inhibitors also inhibited S. aureus SSBB in Gram-positive bacteria. Conclusions:Results from our study support the potential for SSB inhibitors as broad-spectrum antibacterial agents, with dual targeting capabilities against Gram-positive bacteria.

SAAV2152 is a single-stranded DNA binding protein: the third SSB inStaphylococcus aureus

“…Some compounds are known to inhibit ssDNA-binding activity of SaSsbA [ 40 ]. For example, ssDNA-binding ability of SaSsbA can be completely suppressed by the presence of 100 μM 9-methyl-2,3,7-trihydroxy-6-fluorone (NSC5426) [ 40 ], a tricyclic planar compound (Figure 13A ).…”

“…Some compounds are known to inhibit ssDNA-binding activity of SaSsbA [ 40 ]. For example, ssDNA-binding ability of SaSsbA can be completely suppressed by the presence of 100 μM 9-methyl-2,3,7-trihydroxy-6-fluorone (NSC5426) [ 40 ], a tricyclic planar compound (Figure 13A ). The amino acid sequence of SaSsbC shares 36% identity with that of SaSsbA (Figure 1 ), particularly within the first 110 aa, which is the ssDNA-binding domain, and the model structure of SaSsbC resembles the crystal structure of SaSsbA possessing OB-folds (Figure 10 ).…”

“…S. aureus , a Gram-positive pathogen, exhibits a remarkable ability to develop antibiotic resistance, and few therapies are effective against methicillin-resistant S. aureus (MRSA) [ 45 ]. Considering that SSB is essential for all DNA-dependent cellular processes and that nucleic acid metabolism is one of the most basic biological functions, SSB should be a prime target in antibiotic development [ 46 , 47 ]; therefore, SSBs become promising targets in antibiotic development [ 40 ]. Unlike E. coli , which produces only one type of SSB, S. aureus was indicated to express at least three SSBs in this study.…”

Targeting DNA Replication and Repair for the Development of Novel Therapeutics against Tuberculosis

“…Several recent high-throughput screens have been successful in identifying small-molecule inhibitors of SSB-protein interactions (Lu et al, 2010 ; Marceau et al, 2013 ; Glanzer et al, 2016 ). These include an attempt to identify inhibitors of SSB that might disrupt both DNA replication and SOS-mediated resistance pathways within Gram-positive and Gram-negative bacteria (Glanzer et al, 2016 ): following in vitro screening, six molecules were identified which successfully inhibited a broad range of bacterial SSBs, with a further four exhibiting species-specific activity—thereby establishing the potential for both broad-spectrum and species-targeted use. Notably, five of the six compounds were found to have whole-cell activity against a variety of the tested species, of which a single compound, 9-hydroxyphenylfluoron, was associated with minimal activity against the human SSB homolog.…”

Characterization of single-stranded DNA-binding protein SsbB fromStaphylococcus aureus: SsbB cannot stimulate PriA helicase

“…13 Some SSB inhibitors as broad-spectrum antibacterial agents targeting S. aureus and other pathogens have been discovered. 12,14 SsbA is referred to as a counterpart of EcSSB. SsbA and SsbB are essential for genome maintenance and transformational recombination, respectively.…”