Ring-fused 2-pyridones effective against multidrug-resistant Gram-positive pathogens and synergistic with standard-of-care antibiotics

Abstract

The alarming rise of multidrug-resistant Gram-positive bacteria has precipitated a healthcare crisis, necessitating the development of new antimicrobial therapies. Here we describe a new class of antibiotics based on a ring-fused 2-pyridone backbone, which are active against vancomycin-resistant enterococci (VRE), a serious threat as classified by the Centers for Disease Control and Prevention, and other multidrug-resistant Gram-positive bacteria. Ring-fused 2-pyridone antibiotics have bacteriostatic activity against actively dividing exponential phase enterococcal cells and bactericidal activity against nondividing stationary phase enterococcal cells. The molecular mechanism of drug-induced killing of stationary phase cells mimics aspects of fratricide observed in enterococcal biofilms, where both are mediated by the Atn autolysin and the GelE protease. In addition, combinations of sublethal concentrations of ring-fused 2-pyridones and standard-of-care antibiotics, such as vancomycin, were found to synergize to kill clinical strains of VRE. Furthermore, a broad range of antibiotic resistant Gram-positive pathogens, including those responsible for the increasing incidence of antibiotic resistant healthcare-associated infections, are susceptible to this new class of 2-pyridone antibiotics. Given the broad antibacterial activities of ring-fused 2-pyridone compounds against Gram-positive (GmP) bacteria we term these compounds GmPcides, which hold promise in combating the rising tide of antibiotic resistant Gram-positive pathogens.

Keywords: VRE; antibiotic resistance; antibiotic synergy; multidrug-resistant pathogens.

Fig. 1.

First generation 2-pyridone compounds have bactericidal activity against E. faecalis OG1RF and clinical isolates. (A) Structure of the 2-pyridone scaffold. (B) First-generation compounds EC240 and EC305, and second-generation compound PS757. MICs (see Materials and Methods) of EC240, EC305, and PS757 for E. faecalis OGR1RF are shown. (C) Net fold change (see Materials and Methods) in CFU/mL of clinical enterococcal isolates treated with DMSO (green), 100 µM EC240 (orange), and 100 µM EC305 relative to the CFU/mL of the untreated initial inoculum. Black bars represent SDs of replicates. Statistical analysis by two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 2.

Second-generation 2-pyridone PS757 has bacteriostatic activity on exponential phase cultures and Atn autolysin-mediated bactericidal activity on nongrowing stationary phase cultures. (A) Growth of exponential phase cultures in the presence of 1/2–4× MIC (3 to 24 µM) of PS757. Exponential phase cultures (OD₆₀₀= 0.2 to 0.3) of OG1RF were split and left untreated (green), treated with DMSO as a vehicle control (orange), and treated with 3 µM (yellow), 6 µM (light green), 12 µM (pink), and 24 µM (purple) PS757 at time 0. The OD₆₀₀ was monitored at 15-, 30-, 60-, and 1,080-min (18 h) time points. To account for change in the OD₆₀₀ due to compound addition, OD₆₀₀ values were normalized by subtracting the change in OD₆₀₀ at t = 0 from t = 15. Black bars represent SD of replicates. (B) Time course killing curves of exponential phase cells. Exponential phase cultures (OD₆₀₀ = 0.2 to 0.3) of OG1RF were split and left untreated (green), treated with DMSO as a vehicle control (orange), and treated with 100 µM (purple) or 50 µM (pink) PS757 at time 0. The CFU/mL were enumerated at time 0, and 2 hr, 4 hr, 6 hr, and 24 hr timepoints posttreatment. (C) Time course killing curves of stationary phase cells. Stationary phase cultures (18 h) of OG1RF were split and left untreated (green), treated with DMSO as a vehicle control (orange), and treated with 100 µM (purple) or 50 µM (pink) PS757 at time 0. The CFU/mL were enumerated at time 0 and 2 hr, 4 hr, 6 hr, and 24 hr time points posttreatment. Black bars represent the SD of the replicates. (D, E) Spot titer assays of PS757-treated mutant cultures. Exponential phase (OD₆₀₀ = 0.2 to 0.3) or stationary phase (18 h) cultures of indicated strains were treated with the vehicle control DMSO (−) or 100 µM PS757 (+) for 24 h. The serial dilution is indicated.

Fig. 3.

Sublethal concentrations of PS757 acts in combination with sublethal levels of gentamicin, vancomycin, and ciprofloxacin to kill OG1RF, V583, and VRE clinical isolates. Sublethal concentrations of PS757 combined with sublethal concentrations of gentamicin or vancomycin results in cell death. (A) Overnight cultures of OG1RF were diluted into fresh media and treated with either DMSO or sublethal concentrations of PS757 (1.56 μM or 3.2 μM) alone or in combination with a sublethal concentration of gentamicin (8 μg/mL). (B) Overnight cultures of V583 were diluted into fresh media and treated with either DMSO or sublethal concentrations of PS757 (1.56 μM or 3.2 μM) alone or in combination with a sublethal concentration of vancomycin (8 μg/mL). (A, B) Net fold change (see Materials and Methods) is presented as the average change in final CFU/mL relative to the CFU/mL of the untreated initial inoculum. Black bars represent SD of replicates. Statistical analysis by one-way ANOVA (*P < 0.05, **P < 0.01, ****P < 0.0001, ns: not significant). (C) Sums of the FIC derived from a checkerboard analysis (see Materials and Methods) of PS757 with gentamicin, vancomycin, and ciprofloxacin in clinical VRE strains. Strains OG1RF, V583, 1332, and 1333 are antibiotic resistant E. faecalis and strains 1379, 1381, 1386, and 1388 are antibiotic resistant E. faecium. FIC of ≤ 0.5 was considered synergistic (“Syn.”), >0.5 to 2.0 additive (“Add.”) and >2.0 antagonistic (“Antag.”), as indicated by the dashed lines.

Fig. 4.

Sublethal concentrations of PS757 increase influx and decrease efflux in OG1RF. (A) EtBr accumulation in OG1RF cells treated with vehicle (DMSO, green) or PS757 (pink). EtBr accumulation results are plotted as fold-change of EtBr fluorescence over a 30-min time course. (B) Relative fluorescence of EtBr depletion of OG1RF cells pretreated with EtBr and i) vehicle (DMSO, green); or ii) PS757 (pink). (C) Relative fluorescence of PS757 depletion of OG1RF cells pretreated with EtBr and i) vehicle (DMSO, green); or ii) PS757 (pink). EtBr and PS757 depletion results are plotted as % relative EtBr fluorescence (respective to the initial fluorescence, 100% at t = 0) over a 30-min time course. Cells were pretreated with EtBr (1 µg/mL), DMSO (0.05%) and/or PS757 (5 µM). The fluorescence signals emitted from PS757 (λEx = 400 nm and λEm = 440 nm) and EtBr (λEx = 544 nm and λEm = 590 nm) were measured with SpectraMax iD3 microplate reader (Molecular Devices). All measurements were performed in triplicate and results were plotted using GraphPad Prism (version 9.3.1). Black bars represent SDs of replicates.