The use of adjuvants that rescue antibiotics against multidrug-resistant (MDR) pathogens is a promising combination strategy for overcoming bacterial resistance. While the combination of β-lactam antibiotics and β-lactamase inhibitors has been successful in restoring antibacterial efficacy in MDR bacteria, the use of adjuvants to restore fluoroquinolone efficacy in MDR Gram-negative pathogens has been challenging. We describe tobramycin-ciprofloxacin hybrid adjuvants that rescue the activity of fluoroquinolone antibiotics against MDR and extremely drug-resistant Pseudomonas aeruginosa isolates in vitro and enhance fluoroquinolone efficacy in vivo. Structure-activity studies reveal that the presence of both tobramycin and ciprofloxacin, which are separated by a C12 tether, is critical for the function of the adjuvant. Mechanistic studies indicate that the antibacterial modes of ciprofloxacin are retained while the role of tobramycin is limited to destabilization of the outer membrane in the hybrid.

Therapeutic interventions to treat multidrug-resistant (MDR) Pseudomonas aeruginosa infections are severely limited and often require the use of colistin as drug of last resort. The major challenges impeding the development of novel antipseudomonal agents are the lack of cell penetration and extensive efflux. We have discovered a tobramycin-moxifloxacin hybrid core structure which enhances outer membrane permeability and reduces efflux by dissipating the proton motive force of the cytoplasmic membrane in P. aeruginosa. The optimized hybrid protects Galleria mellonella larvae from the lethal effects of MDR P. aeruginosa. Attempts to select for resistance over a period of 25 days resulted in a 2-fold increase in the minimal inhibitory concentration (MIC) for the hybrid, while moxifloxacin or tobramycin resulted in a 16- and 512-fold increase in MIC. Although the hybrid possesses potent activity against MDR, P. aeruginosa isolates the activity that can be synergized when used in combination with other classes of antibiotics.

Amphiphilic aminoglycosides (AAGs) are an emerging source of antibacterials to combat infections caused by antibiotic-resistant bacteria. Mode-of-action studies indicate that AAGs predominately target bacterial membranes, thereby leading to depolarization and increased permeability. To assess whether AAGs also induce host-directed immunomodulatory responses, we determined the AAG-dependent induction of cytokines in macrophages in the absence or presence of lipopolysaccharide (LPS). Our results show for the first time that AAGs can boost the innate immune response, specifically the recruitment of immune cells such as neutrophils required for the resolution of infections. Moreover, AAGs can selectively control inflammatory responses induced in the presence of endotoxins to prevent septic shock. In conclusion, our study demonstrates that AAGs possess multifunctional properties that combine direct antibacterial activity with host-directed clearance effects reminiscent of those of host-defense peptides.

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Pseudaminic acid (Pse5Ac7Ac) is a nonmammalian sugar present on the cell surface of a number of bacteria including Pseudomonas aeruginosa, Campylobacter jejuni and Acinetobacter baumannii. However, the role Pse5Ac7Ac plays in host-pathogen interactions remains underexplored, particularly compared to its ubiquitous sialic acid analogue Neu5Ac. This is primarily due to a lack of access to difficult to prepare Pse5Ac7Ac glycosides. Herein we decribe the in vitro biocatalytic transfer of an activated Pse5Ac7Ac donor onto glycosyl acceptors enabling the enzymatic synthesis of Pse5Ac7Ac containing glycosides. In a chemoenzymatic approach, chemical synthesis initially afforded access to a late stage Pse5Ac7Ac biosynthetic intermediate, which was subsequently converted to the desired CMP-glycosyl donor in a one-pot-two-enzyme process using biosynthetic enzymes. Finally screening a library of 13 sialyltransferases (SiaT) with the unnatural substrate enabled the identification of a promiscuous inverting SiaT capable of turnover to afford β-Pse5Ac7Ac terminated glycosides. Figure 1 Nonulosonic acid structures; the pseudaminic acid Pse5Ac7Ac 1, and sialic acid Neu5Ac 2. Scheme 2 Chemical synthesis of 6-deoxy-L-AltdiNAc 4. a) i) NaOMe, MeOH. ii) Diphenyl borinate, DIPEA, BzCl, CH 3 CN, 80% over 2steps. b) i) Tf 2 O, Pyridine, DCM, 0 °C. ii) NaN 3 , DMF, 110 °C, 58% over 2-steps. c) NaOMe, MeOH, 95%. d) i) Tf 2 O, Pyridine, DCM, 0 °C. ii) TBANO 2 , CH 3 CN, 70 °C, 46% over 2-steps. e) i) PMe 3 , NaOH, THF, 60 °C. ii) Ac 2 O, Pyridine, 69% over 2-steps. f) NaOMe, MeOH, 81%. g) CAN, CH 3 CN:H 2 O (4:1 v/v), 94%, α:β 1:3. Scheme 1 Biosynthesis of CMP-Pse5Ac7Ac 3 via the key hexose intermediate 6-deoxy-ʟ-AltdiNAc 4.