A Dual-Mechanism Antibiotic Kills Gram-Negative Bacteria and Avoids Drug Resistance

Abstract

The rise of antibiotic resistance and declining discovery of new antibiotics has created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. To characterize its MoA, we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline demonstrates that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments in killing methicillin-resistant Staphylococcus aureus (MRSA) persisters. Building on the molecular core of SCH-79797, we developed a derivative, Irresistin-16, with increased potency and showed its efficacy against Neisseria gonorrhoeae in a mouse vaginal infection model. This promising antibiotic lead suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to targeting challenging bacterial pathogens.

Keywords: Acinetobacter baumannii; Gram-negative pathogens; Neisseria gonorrhoeae; antibiotics; broad spectrum; dual-target drugs; folate metabolism; membrane disrupting.

Figure 1.. SCH-79797 Is a Broad-Spectrum, Bactericidal Antibiotic that Is Effective in an Animal Model and Has a Low Frequency of Resistance

(A) The MIC of SCH-79797 against Gram-negative (red) and Gram-positive (black) bacteria. The MICs of E. cloacae and P. aeruginosa were greater the maximal drug concentrations tested. See also Table S1. (B) The relative growth of E. coli lptD4213 after treatment with SCH-79797. Bacterial growth was measured as the optical density at 600 nm (OD₆₀₀) 14 h following inoculation. Each data point represents 2 biological replicates. Mean ± SD are shown. (C) Colony forming units (CFUs mL⁻¹) after 3-h treatment of E. coli lptD4213 with 1% DMSO (solvent control), 6.2 μg/mL SCH-79797 (2× MIC), 0.12 μg/mL ampicillin (2× MIC), or 0.48 μg/mL novobiocin (4× MIC). Data points at 1 × 10² CFU mL⁻¹ are below the level of detection. Each data point represents 3 biological replicates. Mean ± SD are shown. (D) The percent survival of G. mellonella wax worms infected with A. baumannii and concomitantly treated with 2 μL/larva 100% DMSO, 67 μg/larva SCH-79797, 67 μg/larva gentamicin, 67 μg/larva merapenem, or 67 μg/larva rifampicin. Data represents a typical cohort (n = 12) from a biological triplicate. p values are determined from a Mantel-Cox test using Prism (**p < 0.01; ***p < 0.001), and the pooled results are presented in the supplemental material (Figure S1G). For other Mantel-Cox comparisons, see Table S2. (E) Fold increase in resistance of S. aureus MRSA USA300 to SCH-79797, novobiocin, trimethoprim, or nisin after 25 days of serial passaging in each drug. Data represents one biological replicate and the data for a second replicate is shown in Figure S1B.

Figure 2.. Bacterial Cytological Profiling Indicates that SCH-79797 Functions by a Mechanism Distinct from Known Classes of Antibiotics

(A) Fluorescent images of E. coli lptD4213 cells treated with antibiotics representative of 5 different antibiotic classes. Cells were treated for 2 h with 5× MIC of each drug. Merged image channels are phase contrast (gray), FM4–64 (red), DAPI (blue), and SYTOX (green). Scale bar, 1 μm. (B) Comparison of cytological profiles of known antibiotics with the cytological profile of SCH-79797. Single-linkage clustered dendrogram from one-way MANOVA comparisons between antibiotic treatment groups compared to all other antibiotic treatment groups. Inset: structure of SCH-79797.

Figure 3.. Thermal Proteome Profiling Suggests that SCH-79797 Binds Dihydrofolate Reductase

(A) Schematic of the thermal shift assay that compares the thermal stability of the entire proteome with and without drug treatment. Protein samples are aliquoted, and each aliquot is heated to a different temperature. The relative fraction of soluble and insoluble proteins is then determined for each aliquot by ultracentrifugation and mass spectrometry. (B and C) The relative thermal stability of the soluble E. coli lptD4213 proteome after treatment of whole cell and cell lysate samples with SCH-79797 (B) or trimethoprim (C). Changes in thermal stability were determined by measuring changes in the abundance of soluble protein across 10 different temperatures ranging from 42°C–72°C and 4 drug concentrations and a vehicle control. For each point, the color indicates the maximal effect size across all temperatures and the largest change in abundance across all concentrations. Squares represent the proteins with a change in abundance of at least 25% at three or more temperatures. To be considered consistent, the change in abundance of a protein had to show the same sign at least 90% of the time and have an effect size of at least 2-fold in either whole cells or cell lysates. Triangles represent a milder effect where at least one temperature had a change in abundance of at least 25% in both whole cell and cell lysate treatments.

Figure 4.. SCH-79797 Targets Folate Metabolism by Competitively Inhibiting Dihydrofolate Reductase

(A) A partial representation of the folate synthesis pathway. Where the E. coli and B. subtilis differ, the E. coli names are listed in parentheses. (B) The growth of CRISPRi B. subtilis knockdown mutants (Peters et al., 2016) involved in folate synthesis relative to a DMSO-treated control after SCH-79797 or trimethoprim treatment. OD₆₀₀ of each condition 14 h after inoculation was plotted against drug concentration. Each data point represents 2 biological replicates. Mean ± SD are shown. (C) Metabolomic analysis of E. coli NCM3722 cells treated with 13.9 μg/mL SCH-79797 (1× MIC) or 0.15 μg/mL trimethoprim (1× MIC). Samples were taken 0, 5, 10, and 15 min after drug treatment. Folate metabolite abundance at each time point was quantified relative to the DMSO-treated control samples at the initial time point. Each data point represents 3 independent replicates. Mean ± SD are shown. (D and E) E. coli dihydrofolate reductase (FolA) activity is reduced in the presence of SCH-79797, IRS-10, or trimethoprim. Activity is relative to the standard condition of 60 μM NADPH and 100 μM DHF. (D) IC₅₀ values were derived from fits to the Hill equation for reactions performed at 60 μM NADPH and 100 μM DHF. (E) FolA activity as a function of dihydrofolate concentration in the presence of 8.6 μM SCH-79797 (IC₅₀), 0.065 μM IRS-10 (IC₅₀), or 0.015 μM trimethoprim (IC₅₀). Kinetic parameters are derived from fits to the Michaelis-Menten model.

Figure 5.. SCH-79797 Is Distinct from Other Dihydrofolate Reductase Inhibitors and Disrupts Membrane Integrity

(A) The growth of wild-type (WT) and ΔthyA E. coli lptD4213 relative to a DMSO-treated control after SCH-79797 and trimethoprim treatment. Bacterial growth was measured for 14 h and the final OD₆₀₀ of each condition was plotted against drug concentration. Each data point represents 2 biological replicates. Mean ± SD are shown. (B) Schematic of flow cytometry data showing the expected results for each class of polarized, depolarized, permeable, and impermeable bacteria. (C) Flow cytometry analysis of the membrane potential and permeability of E. coli lptD4213 cells after 15 min incubation with 1% DMSO (solvent control), 5 μM CCCP, 25 μg/mL nisin (2× MIC), 0.8 μg/mL polymyxin B (2× MIC), 12.5 μg/mL SCH-79797 (2× MIC), or 2 μg/mL trimethoprim (10× MIC). The limits for the depolarized region were defined by comparing the values in the CCCP and solvent only controls. The limits for the permeabilized region were defined by comparing the nisin and solvent only controls.

Figure 6.. SCH-79797 Mimics Co-treatment with Folate Metabolism and Membrane Integrity Disruptors but Can Be More Effective Than Their Combination

(A) BCP analysis of E. coli lptD4213 cells after 30 min of treatment with 1% DMSO, 6.3 μg/mL SCH-79797 (1× MIC), 2 μg/mL trimethoprim (10× MIC), 25 μg/mL nisin (2× MIC), or the combination of 2 μg/mL trimethoprim (10× MIC) and 25 μg/mL nisin (2× MIC). Cytological profiles were clustered by the first three principal components that account for at least 90% of the variance between samples. Cells were stained with DAPI, FM4–64, and SYTOX Green. Shown here are the merged images of DAPI (blue) and FM4–64 (red). Scale bar, 2 μm. (B)The viability of E. coli lptD4213 cells measured in CFU mL⁻¹ after 2 h of treatment with 1% DMSO (solvent control), 2 μg/mL trimethoprim (10× MIC), 25 μg/mL nisin (2× MIC), the combination of 2 μg/mL trimethoprim (10× MIC) and 25 μg/mL nisin (2× MIC), 0.8 μg/mL polymixin B (2× MIC), the combination of 2 μg/mL trimethoprim (10× MIC) and 0.8 μg/mL polymixin B (2× MIC), or 3.1 μg/mL SCH-79797 (1× MIC). Each bar represents 3 biological replicates. Mean ± SD are shown. (C) Viability of S. aureus MRSA USA300 persister cells measured in CFU mL⁻¹ after 2 h of treatment with 1% DMSO (solvent control), 63 μg/mL trimethoprim (10× MIC), 100 μg/mL nisin (2× MIC), the combination of 63 μg/mL trimethoprim (10× MIC) and 50 μg/mL nisin (2× MIC), 63 μg/mL daptomycin (2× MIC), the combination of 63 μg/mL trimethoprim (10× MIC) and 63 μg/mL daptomycin (2× MIC), or 6.3 μg/mL SCH-79797 (1× MIC). Each bar represents 3 biological replicates. Mean ± SD are shown.

Figure 7.. Derivates of SCH-79797 Show Increased Potency and the Ability to Help Clear Infection in Mouse Vaginal N. gonorrhea Model

(A) Structures of SCH-79797, the pyrroloquinazolinediamine core lacking the side chains (IRS-10), and the pyrroloquinazolinediamine core with a biphenyl decoration (IRS-16). (B) The MICs of SCH-79797, IRS-10, and IRS-16 against a few selected species. For the MICs against additional strains, see Table S1. (C) The growth of CRISPRi B. subtilis knockdown mutants involved in folate synthesis relative to a DMSO-treated control after treatment with IRS-10 or IRS-16. Bacterial growth was measured for 14 h and the final optical density (OD₆₀₀) of each condition was plotted against drug concentration. Each data point represents 2 biological replicates. Mean ± SD are shown. (D) Flow cytometry analysis of the membrane potential and permeability of E. coli lptD4213 cells after 15 min incubation with 0.4 μg/mL IRS-10 (1× MIC) or 0.02 μg/mL IRS-16 (1× MIC). (E) Therapeutic index of SCH-79797 and IRS-16 was calculated by dividing the MIC of each drug for the indicated mammalian cell line by its MIC against E. coli lptD4213. The MIC of IRS-16 against PBMC was greater than the maximal drug concentrations tested. (F) The stability of IRS-16 was measured following incubation with mouse liver microsomes. Each data point represents 2 biological replicates. Mean ± SD are shown. (G) Treatment of mice with IRS-16 (10 mg/kg, i.v., twice a day [b.i.d.]) reduces the vaginal burden of N. gonorrhoeae 24 h after inoculation. p value from one-factor ANOVA.