Heavy-Metal Trojan Horse: Enterobactin-Directed Delivery of Platinum(IV) Prodrugs toEscherichia coli

Abstract

The global crisis of untreatable microbial infections necessitates the design of new antibiotics. Drug repurposing is a promising strategy for expanding the antibiotic repertoire. In this study, we repurpose the clinically approved anticancer agent cisplatin into a targeted antibiotic by conjugating its Pt­(IV) prodrug to enterobactin (Ent), a triscatecholate siderophore employed by Enterobacteriaceae for iron (Fe) acquisition. The l-Ent-Pt­(IV) conjugate (l-EP) exhibits antibacterial activity against Escherichia coli K12 and the uropathogenic isolate E. coli CFT073. Similar to cisplatin, l-EP causes a filamentous morphology in E. coli and initiates lysis in lysogenic bacteria. Studies with E. coli mutants defective in Ent transport proteins show that Ent mediates the delivery of l-EP into the E. coli cytoplasm, where reduction of the Pt­(IV) prodrug releases the cisplatin warhead, causing growth inhibition and filamentation of E. coli. Substitution of Ent with its enantiomer affords the d-Ent-Pt­(IV) conjugate (d-EP), which displays enhanced antibacterial activity, presumably because d-Ent cannot be hydrolyzed by Ent esterases and thus Fe cannot be released from this conjugate. E. coli treated with l/d-EP accumulate ≥10-fold more Pt as compared to cisplatin treatment. By contrast, human embryonic kidney cells (HEK293T) accumulate cisplatin but show negligible Pt uptake after treatment with either conjugate. Overall, this work demonstrates that the attachment of a siderophore repurposes a Pt anticancer agent into a targeted antibiotic that is recognized and transported by siderophore uptake machinery, providing a design strategy for drug repurposing by siderophore modification and heavy-metal “trojan-horse” antibiotics.

Emerging Target-Directed Approaches for the Treatment and Diagnosis of Microbial Infections

“…131 In contrast, human embryonic kidney cells (HEK293T) treated with conjugates 40 and 41 showed negligible Pt uptake, corroborating the idea that conjugation of a Pt(IV) prodrug with a siderophore is a relevant approach to the development of a targeted antibiotic. 131 Very recently, a series of siderophore−methotrexate conjugates (Figure 18) with potent antibacterial activities have also been developed. 132 Methotrexate (Mtx) is an anticancer drug that is also capable of inhibiting bacterial dihydrofolate reductase (DHFR).…”

“…131 While both conjugates show an increased uptake of Pt in E. coli (≥10 fold increase when compared with cisplatin), conjugate 41 exhibits enhanced antibacterial activity, possibly because the D enantiomer of enterobactin is not hydrolyzed by the esterases and therefore the iron is not released from the siderophore. 131 In contrast, human embryonic kidney cells (HEK293T) treated with conjugates 40 and 41 showed negligible Pt uptake, corroborating the idea that conjugation of a Pt(IV) prodrug with a siderophore is a relevant approach to the development of a targeted antibiotic. 131 Very recently, a series of siderophore−methotrexate conjugates (Figure 18) with potent antibacterial activities have also been developed.…”

“…The conjugation of non-antimicrobial drugs with siderophores could be helpful in drug repurposing by allowing a more targeted delivery and potentially less toxicity or side effects. Based on this concept, Guo and Nolan developed two Trojan horses active against E. coli by conjugating enterobactin or its d enantiomer and a Pt­(IV) prodrug of the anticancer agent cisplatin (Figure ). The conjugates 40 and 41 enter the bacterial cells through the enterobactin transport proteins, and once in the cytoplasm the Pt­(IV) prodrug is reduced, releasing the active cisplatin .…”

“…Based on this concept, Guo and Nolan developed two Trojan horses active against E. coli by conjugating enterobactin or its d enantiomer and a Pt­(IV) prodrug of the anticancer agent cisplatin (Figure ). The conjugates 40 and 41 enter the bacterial cells through the enterobactin transport proteins, and once in the cytoplasm the Pt­(IV) prodrug is reduced, releasing the active cisplatin . While both conjugates show an increased uptake of Pt in E. coli (≥10 fold increase when compared with cisplatin), conjugate 41 exhibits enhanced antibacterial activity, possibly because the d enantiomer of enterobactin is not hydrolyzed by the esterases and therefore the iron is not released from the siderophore .…”

“…The conjugates 40 and 41 enter the bacterial cells through the enterobactin transport proteins, and once in the cytoplasm the Pt­(IV) prodrug is reduced, releasing the active cisplatin . While both conjugates show an increased uptake of Pt in E. coli (≥10 fold increase when compared with cisplatin), conjugate 41 exhibits enhanced antibacterial activity, possibly because the d enantiomer of enterobactin is not hydrolyzed by the esterases and therefore the iron is not released from the siderophore . In contrast, human embryonic kidney cells (HEK293T) treated with conjugates 40 and 41 showed negligible Pt uptake, corroborating the idea that conjugation of a Pt­(IV) prodrug with a siderophore is a relevant approach to the development of a targeted antibiotic …”