MAC-545496

MAC-545496 shows inhibition of mprF expression in a concentration-dependent manner; the half-maximal inhibitory concentration (IC50) is 0.0376 µg/mL—matching with the concentration range of the synergistic effect with cefuroxime. MAC-545496 only inhibits the citrate-induced biofilm formation in the wild type in a concentration-dependent manner, suggesting additional potential as a lead for drug discovery. Treatment of S. aureus-infected macrophages with different doses of MAC-545496 added 30 min after phagocytosis shows that the MAC-545496 halts S. aureus replication within macrophages starting at 0.5 µg/mL.^[1].

Stationary-phase cells are washed and diluted in serum-free RPMI (SF-RPMI) and used for infection of RAW macrophages at a multiplicity of infection of ten. One set of replicates for S. aureus USA300 contains MAC-545496 (20 µg/mL) so that the drug is present throughout every step of the experiment. Macrophages exposed to bacteria are centrifuged at 277g to synchronize infection and after 30 min at 37 °C in 5% CO₂, the medium is replaced with SF-RPMI containing gentamicin (100 µg/mL) for 1 h. Here, after phagocytosis, another set of S. aureus USA300 replicates are exposed to MAC-545496 along with gentamicin so that the drug is added after the bacteria are phagocytosed. For wells already treated with MAC-545496, the drug is added too. After gentamicin treatment, one set of wells are lysed by treatment with 0.5 ml of 0.1% Triton X-100 after washing with PBS. The remaining well for each infection is incubated with RPMI containing 5% (vol/vol) non-heat-inactivated FBS until 12 h after infection when the cells are lysed.

MAC-545496 activity is evidenced by increased survival of the drug-treated larvae as compared to infected untreated ones. This corresponds to concentration-dependent killing of S. aureus in the hemolymph of the larvae observed from the CFUs recovered from the hemolymph 200 min after infection. This matchs with the attenuated virulence of the graR::Tn mutant relative to the wild type in Galleria. Treatment with MAC-545496 recapitulates the phenotype of ΔgraR and ΔgraS mutants, effectively inhibiting intracellular S. aureus growth.^[1].