Directing Drugs to Bugs: Antibiotic-Carbohydrate Conjugates Targeting Biofilm-Associated Lectins of Pseudomonas aeruginosa

Abstract

Chronic infections by Pseudomonas aeruginosa are characterized by biofilm formation, which effectively enhances resistance toward antibiotics. Biofilm-specific antibiotic delivery could locally increase drug concentration to break antimicrobial resistance and reduce the drug's peripheral side effects. Two extracellular P. aeruginosa lectins, LecA and LecB, are essential structural components for biofilm formation and thus render a possible anchor for biofilm-targeted drug delivery. The standard-of-care drug ciprofloxacin suffers from severe systemic side effects and was therefore chosen for this approach. We synthesized several ciprofloxacin-carbohydrate conjugates and established a structure-activity relationship. Conjugation of ciprofloxacin to lectin probes enabled biofilm accumulation in vitro, reduced the antibiotic's cytotoxicity, but also reduced its antibiotic activity against planktonic cells due to a reduced cell permeability and on target activity. This work defines the starting point for new biofilm/lectin-targeted drugs to modulate antibiotic properties and ultimately break antimicrobial resistance.

Figure 1

The lectin inhibitors 1 and 2 are conjugated to the antibiotic ciprofloxacin (3) resulting in pathogen-specific, lectin-targeted antibiotics. These compounds target the biofilm-associated lectins LecA and LecB and therefore increase local antibiotic concentration at the site of infection, resulting in fewer side effects caused by unspecific distribution and tissue accumulation. Blue arrows display growth vectors used in this work.

Scheme 1. Chemical Synthesis of the (A) LecA-Targeting (11–14) and (B) LecB-Targeting (19) Probes and (C) Alkyne Ciprofloxacin Derivatives 20 and 21

Reagents and conditions: (a) p-nitrothiophenol, BF₃·Et₂O, CH₂Cl₂, 0 °C to r.t., 16 h; (b) H₂, Pd/C, CH₂Cl₂, r.t., 24 h; (c) (i) Br(CH₂)_nCOHal, Et₃N, or K₂CO₃, DMF, 0 °C to r.t., 1–4 h, (ii) NaN₃, DMF, r.t., 4 h; (d) cat. NaOMe, MeOH, r.t., 1 h; (e) (i) PBr₃, CH₂Cl₂, 0 °C to r.t., 1 h, (ii) HSO₃Cl, CH₂Cl₂, 0 °C to r.t., 3 h; (f) crude 16, K₂CO₃, DMF, r.t., 5 h; (g) NaN₃, DMF, r.t., 5 h; (h) propargylbromide or 4-bromo-but-1-yne, Et₃N, DMF, 70 °C, 1–4 d.

Scheme 2. Assembly of the Lectin-Targeted Ciprofloxacin Conjugates

Reagents and conditions: (a) cat. CuSO₄, cat. sodium ascorbate, DMF/H₂O, r.t. 16 h, r.t. (for 11–14) or 40 °C (for 19).

Figure 2

Competitive binding assay of lectin-targeted ciprofloxacin conjugates 22–31, lectin probes 11–14 and 19, and control compounds with LecA, LecB_PAO1, and LecB_PA14. One representative titration of triplicates on one plate is shown for each compound (IC₅₀ in Table 1 and K_i in Table S1).

Figure 3

Effect of 20, 22, 23, 30, and ciprofloxacin (3) on gyrase-catalyzed DNA supercoiling. Propargylation (20) decreased the inhibitory concentration only by a factor of 3.5 compared to 3. Gyrase inhibition as a putative mode of action was confirmed as all conjugates inhibit gyrase-catalyzed DNA supercoiling. Mean and standard deviations were determined from three independent experiments. A representative titration of E. coli gyrase with 22 in a supercoiling inhibition assay is shown. Controls: plasmid without gyrase and inhibitor (leftmost band) and plasmid with gyrase and without inhibitor (rightmost band). ON, open circular/nicked plasmid; R, relaxed topoisomers; SC, supercoiled topoisomers of E. coli DNA.

Figure 4

Accumulation of 22 (targeting LecA) and 30 (targeting LecB) in P. aeruginosa PAO1 biofilm relative to ciprofloxacin (3). Each data point reflects the relative accumulation compared to ciprofloxacin of a single independent assay with at least three technical replicates. Bars show geometric mean and 95% confidence interval (see the Supporting Information for more detailed information, Figure S2).