Esterase-Catalyzed Siderophore Hydrolysis Activates an Enterobactin–Ciprofloxacin Conjugate and Confers Targeted Antibacterial Activity

Abstract

Enteric Gram-negative bacteria, including Escherichia coli, biosynthesize and deploy the triscatecholate siderophore enterobactin (Ent) in the vertebrate host to acquire iron, an essential nutrient. We report that Ent-Cipro, a synthetic siderophore-antibiotic conjugate based on the native Ent platform that harbors an alkyl linker at one of the catechols with a ciprofloxacin cargo attached, affords targeted antibacterial activity against E. coli strains that express the pathogen-associated iroA gene cluster. Attachment of the siderophore to ciprofloxacin, a DNA gyrase inhibitor and broad-spectrum antibiotic that is used to treat infections caused by E. coli, generates an inactive prodrug and guides the antibiotic into the cytoplasm of bacteria that express the Ent uptake machinery (FepABCDG). Intracellular hydrolysis of the siderophore restores the activity of the antibiotic. Remarkably, Fes, the cytoplasmic Ent hydrolase expressed by all E. coli, does not contribute to Ent-Cipro activation. Instead, this processing step requires IroD, a cytoplasmic hydrolase that is expressed only by E. coli that harbor the iroA gene cluster and are predominantly pathogenic. In the uropathogenic E. coli UTI89 and CFT073, Ent-Cipro provides antibacterial activity comparable to unmodified ciprofloxacin. This work highlights the potential of leveraging and targeting pathogen-associated microbial enzymes in narrow-spectrum antibacterial approaches. Moreover, because E. coli include harmless gut commensals as well as resident microbes that can contribute to disease, Ent-Cipro may provide a valuable chemical tool for strain-selective modulation of the microbiota.

Microbial esterases and ester prodrugs: An unlikely marriage for combating antibiotic resistance

“…(a) Decomposition of the ciprofloxacin–enterobactin conjugate 3 . IroD‐catalyzed hydrolysis breaks apart the siderophore, leaving behind the monocatecholate product 4 (Neumann, Sassone‐Corsi, Raffatellu, & Nolan, ). (b) Ester prodrugs of the metalloproteinase proinhibitor PY‐2: methyl ester 7 , benzyl ether linked 8 , and catechol linked 9 .…”

Trimethyl Lock: A Multifunctional Molecular Tool for Drug Delivery, Cellular Imaging, and Stimuli‐Responsive Materials

“…[31] Siderophores are small-molecule iron chelators produced, secreted, and taken up by bacteria to satisfy their supply of iron, which is an essential growth factor for them.G iven that bacterial resistance to established antibiotic is increasing worldwide, [32] the development of siderophore-drug conjugates has been identified as apromising Trojan horse strategy especially to address Gram-negative bacteria by linking antimicrobial drugs to siderophores, thereby facilitating the uptake anda ccumulation of antimicrobial drugs in the pathogens. [34][35][36] In their report, Miller and Ji link the Nterminus of ciprofloxacin 22 (Scheme 6) through an amide bond to the carboxylic acid of aT ML core and attach desferrioxamin B( 23), as iderophore and xenosiderophore of several Gram-positive and Gram-negative bacteria, through as hort succinyl spacer to the phenolic hydroxy group of aT ML moiety.R esulting desferrioxaminciprofloxacin conjugate 21 shows increased growth inhibitory activity against the Gram-negative pathogen Pseudomonasa er-uginosa in an agar diffusion assay compared to the parent ciprofloxacin and noncleavablea nalogue 24. [34][35][36] In their report, Miller and Ji link the Nterminus of ciprofloxacin 22 (Scheme 6) through an amide bond to the carboxylic acid of aT ML core and attach desferrioxamin B( 23), as iderophore and xenosiderophore of several Gram-positive and Gram-negative bacteria, through as hort succinyl spacer to the phenolic hydroxy group of aT ML moiety.R esulting desferrioxaminciprofloxacin conjugate 21 shows increased growth inhibitory activity against the Gram-negative pathogen Pseudomonasa er-uginosa in an agar diffusion assay compared to the parent ciprofloxacin and noncleavablea nalogue 24.…”

“…[49][50][51] Scheme7.Esterase-sensitiveT ML prodrugs of hydrogen sulfide. [50] Furthermore, desymmetrization of rhodamine-110 (35)t hrough the attachment of only one TML acetate gives the opportunity to functionalize the second amino group for the development of fluorogenic chemicalp robes (Scheme 9). PLE = porcinel iver esterase, PBS = phosphate-buffered saline.…”

“…PLE = porcinel iver esterase, PBS = phosphate-buffered saline. [53] Fortunately,s ubstitutiono fo ne amino functionb yau rea linkage leads to fluorescenceq uantum yields that are significantly higher than those obtained with parent rhodamine-110 (35). [52] Although fluorescence maskingb ya cetylation is also possible for phenol fluorophores, the corresponding esters tend to be very labile against spontaneoush ydrolysis.…”

Diglycine Enables Rapid Intrabacterial Hydrolysis for Activating Anbiotics against Gram‐negative Bacteria

“…[2c] Thus,t here is an urgent need for developing new antimicrobial approaches against MDR bacterial pathogens.Besides investigating novel agents against new targets in bacteria [3] via previously unexplored mechanisms,a nother strategy is to enhance the efficacya nd safety of the existing antibiotics via drug combinations or increasing the specificity.F or example, Kahne et al demonstrated that the use of novobiocin analogues greatly lowers the dose of polymyxins required to treat Gram-negative bacteria by inhibiting DNAg yrase,b inding LptB,a nd disrupting the outer membrane. [4] Yanetalreported nanoaggregates of ciprofloxacin for inhibiting E. coli. [4] Yanetalreported nanoaggregates of ciprofloxacin for inhibiting E. coli.…”