New Antimicrobial Bioactivity against Multidrug-Resistant Gram-Positive Bacteria of Kinase Inhibitor IMD0354

Abstract

Multidrug-resistant pathogens pose a serious threat to human health. For decades, the antibiotic vancomycin has been a potent option when treating Gram-positive multidrug-resistant infections. Nonetheless, in recent decades, we have begun to see an increase in vancomycin-resistant bacteria. Here, we show that the nuclear factor-kappa B (NF-κB) inhibitor N-[3,5-Bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (IMD0354) was identified as a positive hit through a Caenorhabditis elegans-methicillin-resistant Staphylococcus aureus (MRSA) infection screen. IMD0354 was a potent bacteriostatic drug capable of working at a minimal inhibitory concentration (MIC) as low as 0.06 µg/mL against various vancomycin-resistant strains. Interestingly, IMD0354 showed no hemolytic activity at concentrations as high as 16 µg/mL and is minimally toxic to C. elegans in vivo with 90% survival up to 64 µg/mL. In addition, we demonstrated that IMD0354's mechanism of action at high concentrations is membrane permeabilization. Lastly, we found that IMD0354 is able to inhibit vancomycin-resistant Staphylococcus aureus (VRSA) initial cell attachment and biofilm formation at sub-MIC levels and above. Our work highlights that the NF-κB inhibitor IMD0354 has promising potential as a lead compound and an antimicrobial therapeutic candidate capable of combating multidrug-resistant bacteria.

Keywords: IMD0354; high-throughput screening; vancomycin-resistant Staphylococcus aureus; vancomycin-resistant enterococci.

Figure 1

IMD0354 rescues C. elegans from MRSA infection. (a) Chemical structure of IMD0354. (b) Fifteen MRSA-infected C. elegans were treated with 7.14 µg/mL IMD0354, 0.1% dymethlyl sulfoxide (DMSO) (control) and 10 µg/mL vancomycin for 5 days. After staining dead worms with SYTOX Orange, brightfield (left) and fluorescence (right) images were obtained.

Figure 2

Time-killing curve shows IMD0354 is bacteriostatic against vancomycin-resistant strain VRS1. In total, 10⁶ CFU/mL of VRS1 overnight culture was treated with various concentrations of vancomycin and IMD0354. At times of 1, 2, 3, 4, and 24 h, samples we collected, serially diluted, and spot-plated in order to enumerate CFU/mL. Each sample was tested in triplicate. (n = 3, ± S.D.).

Figure 3

Dose toxicity studies show IMD0354 has cytotoxicity activity above MIC levels. Cytotoxicity testing of human liver cell line HepG2 and human kidney proximal tubular cell line HKC-8 at various concentrations of IMD0354. LC₅₀ of IMD0354 is 1.1 µg/mL and is 0.94 µg/mL, respectively. Each sample was tested in triplicate. (n = 3, ± S.D.).

Figure 4

IMD0354 shows minimal toxicity toward C. elegans. Survival of C. elegans treated with various concentrations of IMD0354, normalized to C. elegans treated with DMSO. H₂O₂ was used as positive control. (n = 3, ± S.D). Bottom error bars are omitted for clarity.

Figure 5

IMD0354 does not show hemolytic activity. Human red blood cells were incubated with serially diluted IMD0354 (0.0156–16μg/mL) and normalized to hemolysis of 1% Triton-X 100. Each sample was tested in triplicate (n = 3, ± S.D).

Figure 6

IMD0354 induces membrane permeabilization at high concentrations. VRS1 membrane permeabilization was measured spectrophotometrically by monitoring the uptake of SYTOX Green (excitation wavelength of 485 nm and an emission wavelength of 525 nm) during treatment with IMD0354 at various concentrations. The legend units are µg/mL. Each assay was tested in triplicate. (n = 3, ± S.D). Error bars are omitted for clarity.

Figure 7

IMD0354 shows dose-dependent inhibition of initial cell attachment and complete inhibition of biofilm formation. (a) VRS1 cells were incubated in Brain Heart Infusion broth (BHI) 0.5% glucose at an optical density at 600 nm (OD₆₀₀) of 1.0 for 1 h. Cells were washed three times with phosphate buffered saline (PBS) and then treated with XTT to measure cell attachment. Measurements were normalized to a non-treatment control (0 µg/mL). (b) VRS1 cells were incubated in BHI 0.5% glucose at an OD₆₀₀ of 0.05 and indicated concentrations of IMD0354 for 24 h. At 24 h biofilm were washed 3 times with PBS. Total biofilm mass was measured using 1% crystal violet (n = 3, ± S.D.).