A screen for compounds that modulate the activity of the Staphylococcus aureus MgrA protein
A global transcriptional regulator, MgrA, was identified previously as a key virulence determinant in Staphylococcus aureus. A fluorescence anisotropy (FA)-based high-throughput screen was performed that identified 5, 5-methylenedisalicylic acid (MDSA) as capable of blocking the DNA-binding activity of MgrA. MDSA represses the expression of alpha-toxin that is normally stimulated by MgrA and more ..
BioActive Compounds: 105
A global transcriptional regulator, MgrA, was identified previously as a key virulence determinant in Staphylococcus aureus. A fluorescence anisotropy (FA)-based high-throughput screen was performed that identified 5, 5-methylenedisalicylic acid (MDSA) as capable of blocking the DNA-binding activity of MgrA. MDSA represses the expression of alpha-toxin that is normally stimulated by MgrA and activates transcription of the protein A gene, which is normally suppressed by MgrA. MDSA alters bacterial antibiotic susceptibilities via an MgrA-dependent pathway. A mouse model of infection indicated that MDSA can attenuate S. aureus virulence. This work demonstrates that small molecules can block protein-DNA interactions and effect biological regulation at the transcriptional level.
Labeled oligonucleotide (5'-6-F-TAAACAACAAGTTGTCCAAA-3') was prepared by incorporating 5'- fluorescein phosphoramidite (6-FAM) (Glen Research, Inc.) at the 5' end during solid-phase synthesis. The labeled oligonucleotide was then purified by denaturing polyacrylamide gel electrophoresis and annealed with its complementary strand before use in the screening assay. For purification of His6-MgrA, E. coli (BL21star (DE3)) carrying pet28a::mgrA in LB was grown at 37 degrees C. After reaching mid-log phase (OD600 ~ 0.6), cells were induced with 1 mM of IPTG for 4 hr at 30 degrees C. Cells were harvested and stored at -78 degrees C before use. His6-MgrA was purified on a Ni-NTA column following the manufacturer's instructions (GE Healthcare). Protein purity was determined by SDS-PAGE, and the protein was desalted to remove imidazole before use in the screening assay.
30 microL of His6-MgrA protein (700 nM in 10 mM Tris pH 7.4 and 25 mM NaCl) was added to all wells of black, polystyrene 384-well plates (Corning 3575), except for column 24, to which 30 microL of control buffer (10 mM Tris pH 7.4 and 25 mM NaCl) was added. Test compounds (100 nL of 5 mg/mL stock in 100% DMSO) were added into all wells, except columns 23 (negative controls) and 24. Assay plates were incubated at room temperature for 20 minutes, after which 20 microL of DNA (50 nM stock) was added to all wells, followed by incubation for another 20 minutes. The final assay concentrations of His6-MgrA and DNA were 400 nM and 20 nM, respectively.
Positive control: Wells in column 24 received buffer containing DNA but no His6-MgrA (10 mM Tris pH 7.4, 25 mM NaCl, 20 nM DNA).
Negative control: Wells in column 23 received His6-MgrA and DNA in buffer (10 mM Tris pH 7.4, 25 mM NaCl, 400 nM His6-MgrA, and 20 nM DNAy) but no experimental compounds.
The Envision plate reader (475 nm excitation/525 nm emission) was used to detect fluorescence polarization values.
Percent inhibition for each replicate was calculated by subtracting well fluorescence polarization (FP) from the plate average negative control FP, dividing by the difference between plate average negative and positive control FP, and multiplying by 100. Wells were considered active if the % inhibition > 50% for at least one replicate. Activity scores were calculated using replicate average % inhibition. Average % inhibition <= 0 was scored as 0 for activity; average % inhibition >= 100 was scored as 100 for activity. Average % inhibition between 0 and 100 was used to generate activity scores for intermediate values.
Data Table (Concise)