Polymyxin B₃–Tobramycin Hybrids with <i>Pseudomonas aeruginosa</i>-Selective Antibacterial Activity and Strong Potentiation of Rifampicin, Minocycline, and Vancomycin

There is an urgent need to develop novel antibacterial agents able to eradicate drug-resistant Gram-negative pathogens such as <i>Pseudomonas aeruginosa</i>. Antimicrobial hybrids have emerged as a promising strategy to combat bacterial resistance, as a stand-alone drug but also as an adjuvant in combination with existing antibiotics. Herein, we report for the first time the synthesis and biological evaluation of polymyxin-aminoglycoside heterodimers composed of polymyxin B₃ covalently linked to tobramycin via an aliphatic hydrocarbon linker. The polymyxin B₃–tobramycin hybrids demonstrate potent activity against carbapenem-resistant as well as multidrug- or extensively drug-resistant (MDR/XDR) <i>P. aeruginosa</i> clinical isolates. Furthermore, the most potent hybrid was able to synergize with currently used antibiotics against wild-type and MDR/XDR <i>P. aeruginosa</i> but also against <i>Acinetobacter baumannii</i> as well. The promising biological activity described herein warrants additional studies into design and development of new antimicrobial hybrids able to surmount the problem of antimicrobial resistance

Absolute Quantitation of Glycoforms of Two Human IgG Subclasses Using Synthetic Fc Peptides and Glycopeptides

International audienceImmunoglobulins, such as immunoglobulin G (IgG), are of prime importance in the immune system. Polyclonal human IgG comprises four subclasses, of which IgG1 and IgG2 are the most abundant in healthy individuals. In an effort to develop an absolute MALDI-ToF-MS quantitative method for these subclasses and their Fc N-glycoforms, (glyco)peptides were synthesized using a solid-phase approach and used as internal standards. Tryptic digest glycopeptides from monoclonal IgG1 and IgG2 samples were first quantified using EEQYN(GlcNAc)STYR and EEQFN(GlcNAc)STFR standards, respectively. For IgG1, a similar glycopeptide where tyrosine (Y) was isotopically labelled was used to quantify monoclonal IgG1 that had been treated with the enzyme Endo-F2, i.e., yielding tryptic glycopeptide EEQYN(GlcNAc)STYR. The next step was to quantify single subclasses within polyclonal human IgG samples. Although ion abundances in the MALDI spectra often showed higher signals for IgG2 than IgG1, depending on the spotting solvent used, determination of amounts using the newly developed quantitative method allowed to obtain accurate concentrations where IgG1 species were predominant. It was observed that simultaneous analysis of IgG1 and IgG2 yielded non-quantitative results and that more success was obtained when subclasses were quantified one by one. More experiments served to assess the respective extraction and ionization efficiencies of EEQYNSTYR/EEQFNSTFR and EEQYN(GlcNAc)STYR/EEQFN(GlcNAc)STFR mixtures under different solvent and concentration conditions. Graphical Abstract ᅟ

A Tobramycin Vector Enhances Synergy and Efficacy of Efflux Pump Inhibitors against Multidrug-Resistant Gram-Negative Bacteria

Drug efflux mechanisms interact synergistically with the outer membrane permeability barrier of Gram-negative bacteria, leading to intrinsic resistance that presents a major challenge for antibiotic drug development. Efflux pump inhibitors (EPIs) which block the efflux of antibiotics synergize antibiotics, but the clinical development of EPI/antibiotic combination therapy to treat multidrug-resistant (MDR) Gram-negative infections has been challenging. This is in part caused by the inefficiency of current EPIs to penetrate the outer membrane and resist efflux. We demonstrate that conjugation of a tobramycin (TOB) vector to EPIs like NMP, paroxetine, or DBP enhances synergy and efficacy of EPIs in combination with tetracycline antibiotics against MDR Gram-negative bacteria including <i>Pseudomonas aeruginosa</i>. Besides potentiating tetracycline antibiotics, TOB–EPI conjugates can also suppress resistance development to the tetracycline antibiotic minocycline, thereby providing a strategy to develop more effective adjuvants to rescue tetracycline antibiotics from resistance in MDR Gram-negative bacteria

Amphiphilic Tobramycin–Lysine Conjugates Sensitize Multidrug Resistant Gram-Negative Bacteria to Rifampicin and Minocycline

Chromosomally encoded low membrane permeability and highly efficient efflux systems are major mechanisms by which <i>Pseudomonas aeruginosa</i> evades antibiotic actions. Our previous reports have shown that amphiphilic tobramycin–fluoroquinolone hybrids can enhance efficacy of fluoroquinolone antibiotics against multidrug-resistant (MDR) <i>P. aeruginosa</i> isolates. Herein, we report on a novel class of tobramycin–lysine conjugates containing an optimized amphiphilic tobramycin-C12 tether that sensitize Gram-negative bacteria to legacy antibiotics. Combination studies indicate the ability of these conjugates to synergize rifampicin and minocycline against MDR and extensively drug resistant (XDR) <i>P. aeruginosa</i> isolates and enhance efficacy of both antibiotics in the <i>Galleria mellonella</i> larvae <i>in vivo</i> infection model. Mode of action studies indicate that the amphiphilic tobramycin–lysine adjuvants enhance outer membrane cell penetration and affect the proton motive force, which energizes efflux pumps. Overall, this study provides a strategy for generating effective antibiotic adjuvants that overcome resistance of rifampicin and minocycline in MDR and XDR Gram-negative bacteria including <i>P. aeruginosa</i>