Digital Commons@Becker Digital Discovery of antivirulence agents against methicillin-resistant Discovery of antivirulence agents against methicillin-resistant staphylococcus aureus staphylococcus aureus

Antivirulence agents inhibit the production of disease-causing virulence factors but are neither bacteriostatic nor bactericidal. Antivirulence agents against methicillin-resistant Staphylococcus aureus (MRSA) strain USA300, the most widespread commu-nity-associated MRSA strain in the United States, were discovered by virtual screening against the response regulator AgrA, which acts as a transcription factor for the expression of several of the most prominent S. aureus toxins and virulence factors involved in pathogenesis. Virtual screening was followed by similarity searches in the databases of commercial vendors. The small-molecule compounds discovered inhibit the production of the toxins alpha-hemolysin and phenol-soluble modulin (cid:1) in a dose-dependent manner without inhibiting bacterial growth. These antivirulence agents are small-molecule biaryl compounds in which the aromatic rings either are fused or are separated by a short linker. One of these compounds is the FDA-approved nonsteroidal anti-inﬂammatory drug diﬂunisal. This represents a new use for an old drug. Antivirulence agents might be useful in prophylaxis and as adjuvants in antibiotic therapy for MRSA infections. to Asp 59 on the N-terminal domain of the response regulator protein AgrA. Phosphorylated AgrA undergoes a conformational change to form a dimer, which enables its C-terminal DNA-binding domains to bind to promoter P2 to activate AIP transcription in an autocatalytic fashion. When the AIP concentration reaches a certain threshold, AgrA also binds to the 10-fold-weaker promoter P3, which drives the transcriptionofRNAIII,amasterregulatorofexpressionofaseriesoftoxinsandvirulencefactorsinthepost-exponential-growthphase(6). RNAIIIencodesthetoxindelta-hemolysin(Hld). ThedrugdiscoverytargetinthisworkistheinhibitionofphosphoryltransfertoAgrA,asindicatedbythelargeshadedXatthetopcenter. Rabbit blood hemolytic assay. The rabbit blood hemolytic assay is a functional assay measuring the release of hemoglobin from erythrocytes due to the hemolytic activity of Hla. Cultures of MRSA strain USA300 were grown at 37°C for 16 to 18 h with shaking to the post-exponential-growth phase (OD 600 , 2.5; equivalent to 1 (cid:4) 10 9 CFU/ml). Compounds were added at various concentrations and were incubated for 6 h at37°C. Samples (100 (cid:2) l) of bacterial culture were ﬁltered and were added to 900 (cid:2) l hemolysin buffer (0.145 M NaCl, 0.02 M CaCl 2 ) and 25 (cid:2) l of deﬁ-brinated rabbit blood (Hemostat Laboratories, Dixon, CA). The solution was incubated for 15 min at 37°C. Unlysed blood cells were pelleted by centrifugation (5,500 (cid:4) g , room temperature, 1 min). The hemolytic activity of the supernatant was determined by measuring the optical density at 541 nm. Sterile culture medium served as the standard for 0% hemolysis, and a bacterial culture supernatant devoid of any inhibitor (control) was designated as the standard for 100% hemolysis. The percentage of hemolysis inhibition was calculated by comparison with the control culture. Assays were performed in triplicate.