Cytoplasmic Delivery of an Antibiotic, Trimethoprim, with a Simple Bidentate Catechol Analog as a Siderophore Mimetic

Abstract

Concerns about antibiotic-resistant Gram-negative pathogens are escalating, and accordingly siderophore-based intracellular antibiotic delivery is attracting more attention as an effective means to overcome these infections. Despite the successful clinical translation of this strategy, the delivery potential of siderophores has been limited to periplasm targeting, and this has appreciably restricted the repertoire of applicable antibiotics. To overcome this shortcoming of the current technology, this study focused on investigating the capability of simple bidentate catechol analogs to function as vehicles for cytoplasmic antibiotic delivery. Specifically, by employing trimethoprim, an inhibitor of dihydrofolate reductase located in the cytoplasm, as a model antibiotic, a chemical library of chelator-antibiotic conjugates featuring four different catechol analogs was prepared. Then, their various pharmacological properties and antimicrobial activities were evaluated. Analysis of these characterization data led to the identification of the active conjugates exhibiting notable iron-and trimethoprim-dependent potency against Escherichia coli. Further characterization of these hit molecules using E. coli mutant strains revealed that 2,3dihydroxybenzoate could effectively deliver several corresponding conjugates to the cytoplasm by exploiting the siderophore uptake machineries present across the outer and inner membranes, originally designated for the native siderophore of E. coli, enterobactin. Considering the synthetic simplicity, such a catechol analog could have appreciable usage in potentiating cytoplasm-active antibiotics against recalcitrant Gram-negative pathogens.

Conjugation to Native and Nonnative Triscatecholate Siderophores Enhances Delivery and Antibacterial Activity of a β-Lactam to Gram-Negative Bacterial Pathogens

“…We also observed sensitization of the fepA and fepA iroN mutants to Cfdc (Figure S6b), which similarly could be due to an increased reliance on Cir in the absence of FepA and IroN, leading to enhanced import and activity. This type of phenomenon has also been observed by others for catechol-based conjugates tested in Ec OM receptor mutants. , …”

“…This type of phenomenon has also been observed by others for catecholbased conjugates tested in Ec OM receptor mutants. 38,70 A few minor differences in the antibacterial activity profiles of TC-Amp and Ent-Amp do suggest slightly different effects on the receptor mutants: the STm fepA single mutant showed no change in susceptibility to Ent-Amp 32 while a ∼10-fold decrease in susceptibility was observed for TC-Amp, potentially indicating a greater reliance on FepA for the transport of TC-Amp. Additionally, TC-Amp inhibited the growth of the fepA iroN and cirA fepA iroN strains at 10 −6 Μ.…”

Opportunities and challenges of microbial siderophores in the medical field

“…The conjugate was four times more potent against P. aeruginosa and Burkholderia pseudomallei than ciprofloxacin alone. On the other hand, a conjugate was prepared between a library of catechol-type siderophore analogs and the antibiotic trimethoprim, which showed activity only against E. coli (Kim et al 2023 ).…”

Exploring the Localization of Siderophore-Mediated Cargo Delivery in Gram-Negative Bacteria Using 3-Hydroxypyridin-4(1H)-one-Fluorescein Probes

“…The conjugation of siderophores to antibiotics that target periplasmic proteins acting on the cell wall or cell membrane as an antibacterial cargo has demonstrated greater efficacy over the past few decades. The minimum inhibitory concentration (MIC) value of some conjugates even decreased 1000-fold against Gram-negative pathogens (Figure A). − In contrast, the antibacterial activity of the conjugates that employ antimicrobial agents targeting cytoplasmic proteins was found to be generally equivalent to or inferior to that of the parent antimicrobial agents (Figure B). ,− Considering that antibacterial drugs must reach the site of action and interact with the target proteins to exert antibacterial activity, the antibacterial activity of siderophore-antibiotic conjugates may be related to the final distribution of the conjugates after their entry into bacteria. Therefore, elucidating the general rules of siderophore delivery of antibiotics into Gram-negative bacteria and the distribution of siderophore-antibiotic conjugates in bacteria will hopefully shed light on the possible causes of this phenomenon and provide a reference for the future design of antimicrobial agents based on the Trojan horse strategy.…”