Restoring and Enhancing the Potency of Existing Antibiotics against Drug-Resistant Gram-Negative Bacteria through the Development of Potent Small-Molecule Adjuvants

Abstract

The rapid and persistent emergence of drug-resistant bacteria poses a looming public health crisis. The possible task of developing new sets of antibiotics to replenish the existing ones is daunting to say the least. Searching for adjuvants that restore or even enhance the potency of existing antibiotics against drug-resistant strains of bacteria represents a practical and cost-effective approach. Herein, we describe the discovery of potent adjuvants that extend the antimicrobial spectrum of existing antibiotics and restore their effectiveness toward drug-resistant strains including mcr-1-expressing strains. From a library of cationic compounds, MD-100, which has a diamidine core structure, was identified as a potent antibiotic adjuvant against Gram-negative bacteria. Further optimization efforts including the synthesis of ∼20 compounds through medicinal chemistry work led to the discovery of a much more potent compound MD-124. MD-124 was shown to sensitize various Gram-negative bacterial species and strains, including multidrug resistant pathogens, toward existing antibiotics with diverse mechanisms of action. We further demonstrated the efficacy of MD-124 in an ex vivo skin infection model and in an in vivo murine systemic infection model using both wild-type and drug-resistant Escherichia coli strains. MD-124 functions through selective permeabilization of the outer membrane of Gram-negative bacteria. Importantly, bacteria exhibited low-resistance frequency toward MD-124. In-depth computational investigations of MD-124 binding to the bacterial outer membrane using equilibrium and steered molecular dynamics simulations revealed key structural features for favorable interactions. The very potent nature of such adjuvants distinguishes them as very useful leads for future drug development in combating bacterial drug resistance.

Keywords: LPS binding; antibiotic adjuvants; effective in vivo; extending the antimicrobial spectrum; overcoming multi-drug resistance.

Fig. 1.. MD-124 sensitizes Gram-negative bacteria towards various antibiotics.

(A) 5 μg/ml MD-124 sensitized E. coli towards rifampicin (Rif). E. coli was cultured with rifampicin at various concentrations in the presence or absence of 5 μg/ml MD-124 for 24 h at 37 °C. Then bacterial growth density was determined by measuring OD₆₀₀. Values are means ± SD. n = 3. (B) 5 μg/ml MD-124 itself showed no inhibition effect on bacterial growth. E. coli (5 × 10⁵ CFUs/ml) was cultured in the absence or presence of 5 μg/ml MD-124 and OD₆₀₀ values at different time points were recorded. Values are means ± SD. n = 3. (C) Checkerboard assay of MD-124 (0 to 100 μg/ml) and rifampicin on E. coli. From red bar to blue bar: decreasing bacterial growth density. (D) Checkerboard assay of MD-124 (0 to 10 μg/ml) and rifampicin on E. coli. (E) MD-124 sensitizes various Gram-negative bacterial species towards rifampicin (Rif). Sensitization fold = MIC of rifampicin only /MIC of rifampicin with MD-124. (F) Comparison among MD-124, pentamidine and PMBN for their sensitization effects on E. coli towards rifampicin. (G) 5 μg/ml MD-124 sensitizes E. coli towards a broad range of antibiotics. All results shown represent biologically independent triplicates.

Fig. 2.. MD-124 sensitization of wild-type A. baumannii, K. pneumoniae and drug-resistant Gram-negative strains towards existing antibiotics.

(A and B) Checkerboard assays showed sensitization of A. baumannii by MD-124 towards rifampicin and novobiocin. (C and D) Checkerboard assays showed sensitization of K. pneumoniae by MD-124 towards rifampicin and clarithromycin. (E) Sensitization of an NDM-1-expressing strain of E. coli by MD-124 towards rifampicin. (F) Sensitization of an mcr-1-expressing strain of E. coli by MD-124 towards rifampicin. (G) Sensitization of MDR K. pneumoniae by MD-124 towards rifampicin. (H) Sensitization of MDR S. Typhimurium by MD-124 towards rifampicin. All experiments were done at least in biologically independent duplicate.

Fig. 3.. Mechanistic studies revealed that MD-100 and MD-124 sensitize E. coli by disrupting the outer membrane and increasing antibiotics uptake through binding to LPS.

(A) Proposed sensitization mechanism of MD-124 and MD-100. (B) MD-124 showed decreased ability to sensitize E. coli strain NR698 (a mutant with a “leaky” outer membrane) towards clarithromycin. Values are means ± SD. n = 3. (C) Bacterial sensitizers facilitated E. coli lysis by lysozyme. Poly B and Penta are short for polymyxin B and pentamidine, respectively. As control, E. coli was incubated with various bacterial sensitizers at the same concentration in the absence of lysozyme (pink bar). Values are means ± SD. n = 3. P values were determined using unpaired two-tailed Student’s t-tests. ***: P < 0.001 compared with vehicle group. (D) LPS decreased the sensitization ability of MD-124 on E. coli in a concentration-dependent manner. (E) Mg²⁺ and Ca²⁺ decreased the sensitization ability of MD-124 in a concentration-dependent manner. (F) Dansyl-PMBN (DP) displacement assay. 10 μM Dansyl-PMBN was added to E. coli (OD₆₀₀ = 0.3) in HEPES buffer (pH = 7.4), and the fluorescent intensity was recorded as F1 (Ex = 340 nm; Em = 520 nm). Then 200 μM compounds were added, and the fluorescent intensity was recorded as Fx. Fluorescent intensity/% = (F1-Fx)/F1. The fluorescent intensity of 10 μM Dansyl-PMBN with E. coli (F1) was marked as 100 %. Values are means ± SD. n = 3. P values were determined using unpaired two-tailed Student’s t-tests. ***: P < 0.001 compared with vehicle group.

Fig. 4.. Molecular dynamics simulations of the interactions between bacterial sensitizers and E. coli outer membrane (OM).

(A) MD-124 interacts with phosphate groups of LPS. A representative state for one of the two copies of MD-124 present in the E. coli OM simulation. Hydrogen bonds are indicated by dotted lines. Purple spheres are Ca²⁺ ions. (B) d1 is defined as the distance between the two positively charged diamidine groups. (C) MD-124 and MD-126 adopt similar geometries in water in terms of the distance between the two amidine groups (d1). (D) MD-126 diamidine groups come closer together than those of MD-124 when they directly interact with the LPS layer of the OM. (E and F) Steered molecular dynamics (SMD) simulations (pulling speed of 0.25 Å/ns) show large differences in the d1 values between MD-124 and MD-126 in the hydrophilic part (Lipid A sugars and core sugars) of the LPS layer. Sim1 and Sim2 are short for simulation 1 and simulation 2.

Fig. 5.. Validation of MD-124 efficacy in an ex-vivo skin infection model (A, B, C) and in-vivo systemic infection model in mice (D, E, F).

(A) Combination of MD-124 and novobiocin inhibited wild-type (WT) E. coli growth. Concentration (w/w) for polymyxin B (poly B), novobiocin (Novo) and MD-124 were 1‰, 4‰ and 1.5‰. The same concentration was used for novobiocin and MD-124 in the combination treatment groups (Novo + MD-124). (B) Combination of MD-124 and clindamycin inhibited the growth of NDM-1-expressing E. coli. Concentration (w/w) for polymyxin B (poly B), clindamycin (Clind) and MD-124 were 1‰, 3‰ and 1.5‰. The same concentration was used for clindamycin and MD-124 in the combination groups (Clind + MD-124). (C) Combination of MD-124 and polymyxin B inhibited the growth of mcr-1-expressing E. coli. Concentration (w/w) for polymyxin B (poly B) and MD-124 were 3‰ and 1.5‰. The same concentration was used for polymyxin B and MD-124 in the combination groups (Poly B + MD-124). For Fig. A, B, C, values are means ± SEM. n = 5, P values were determined using unpaired two-tailed Student’s t-tests. ***P < 0.001 vs antibiotics or MD-124 alone group. (D) Schematic illustration of the experimental procedures of a systemic infection model in mice. (E and F) MD-124 and novobiocin combinations significantly increased the survival rates of mice after infection by WT E. coli (E) and NDM-1-expressing E. coli (F). Arrows in Fig. E and F indicate the treatment time. The concentration for MD-124 and novobiocin is 10 mg/kg and 80 mg/kg respectively. The same concentration was used for novobiocin and MD-124 in the combination treatment groups. For Fig. E and F, n = 15 biologically independent animals per group. ***P < 0.001 vs antibiotics or MD-124 alone group. Statistical analysis using Log-rank (Mantel-Cox) test.

Scheme 1.

Discovery of MD-100 and MD-124.

Scheme 2.

Class I and II analogs of MD-100.

Scheme 3.

Class III analogs of MD-100.