TY - JOUR

T1 - Siderophore conjugation with cleavable linkers boosts the potency of RNA polymerase inhibitors against multidrug-resistant E. coli

AU - Peukert, Carsten

AU - Vetter, Anna C.

AU - Fuchs, Hazel L.S.

AU - Harmrolfs, Kirsten

AU - Karge, Bianka

AU - Stadler, Marc

AU - Brönstrup, Mark

N1 - Funding Information: This work was funded by the Joint Program Initiative on Antimicrobial Resistance (JPI AMR, grant number: 01KI1825), and the Helmholtz International Lab for Anti-infectives. C. P. thanks the “Fonds der chemischen Industrie” for a scholarship. We thank Linn Müggenburg, Christel Kakoschke and Monika Rettstadt for the measurement of NMR samples and Ulrike Beutling for the mass spectrometric measurements. We are thankful to Silke Reinecke, Kerstin Schobert, Kathrin Wittstein, Rolf Jansen, Tanja Tinius and Birthe Sandargo (Department of Microbial Drugs, Helmholtz Centre for Infection Research) for providing us with sorangicin A and corallopyronins. We acknowledge the licensed usage of biorender® for figures in the main text and the ESI.

PY - 2023/4/26

Y1 - 2023/4/26

N2 - The growing antibiotic resistance, foremost in Gram-negative bacteria, requires novel therapeutic approaches. We aimed to enhance the potency of well-established antibiotics targeting the RNA polymerase (RNAP) by utilizing the microbial iron transport machinery to improve drug translocation across their cell membrane. As covalent modifications resulted in moderate-low antibiotic activity, cleavable linkers were designed that permit a release of the antibiotic payload inside the bacteria and unperturbed target binding. A panel of ten cleavable siderophore-ciprofloxacin conjugates with systematic variation at the chelator and the linker moiety was used to identify the quinone trimethyl lock in conjugates 8 and 12 as the superior linker system, displaying minimal inhibitory concentrations (MICs) of ≤1 μM. Then, rifamycins, sorangicin A and corallopyronin A, representatives of three structurally and mechanistically different natural product RNAP inhibitor classes, were conjugated via the quinone linker to hexadentate hydroxamate and catecholate siderophores in 15-19 synthetic steps. MIC assays revealed an up to 32-fold increase in antibiotic activity against multidrug-resistant E. coli for conjugates such as 24 or 29 compared to free rifamycin. Experiments with knockout mutants in the transport system showed that translocation and antibiotic effects were conferred by several outer membrane receptors, whose coupling to the TonB protein was essential for activity. A functional release mechanism was demonstrated analytically by enzyme assays in vitro, and a combination of subcellular fractionation and quantitative mass spectrometry proved cellular uptake of the conjugate, release of the antibiotic, and its increased accumulation in the cytosol of bacteria. The study demonstrates how the potency of existing antibiotics against resistant Gram-negative pathogens can be boosted by adding functions for active transport and intracellular release.

AB - The growing antibiotic resistance, foremost in Gram-negative bacteria, requires novel therapeutic approaches. We aimed to enhance the potency of well-established antibiotics targeting the RNA polymerase (RNAP) by utilizing the microbial iron transport machinery to improve drug translocation across their cell membrane. As covalent modifications resulted in moderate-low antibiotic activity, cleavable linkers were designed that permit a release of the antibiotic payload inside the bacteria and unperturbed target binding. A panel of ten cleavable siderophore-ciprofloxacin conjugates with systematic variation at the chelator and the linker moiety was used to identify the quinone trimethyl lock in conjugates 8 and 12 as the superior linker system, displaying minimal inhibitory concentrations (MICs) of ≤1 μM. Then, rifamycins, sorangicin A and corallopyronin A, representatives of three structurally and mechanistically different natural product RNAP inhibitor classes, were conjugated via the quinone linker to hexadentate hydroxamate and catecholate siderophores in 15-19 synthetic steps. MIC assays revealed an up to 32-fold increase in antibiotic activity against multidrug-resistant E. coli for conjugates such as 24 or 29 compared to free rifamycin. Experiments with knockout mutants in the transport system showed that translocation and antibiotic effects were conferred by several outer membrane receptors, whose coupling to the TonB protein was essential for activity. A functional release mechanism was demonstrated analytically by enzyme assays in vitro, and a combination of subcellular fractionation and quantitative mass spectrometry proved cellular uptake of the conjugate, release of the antibiotic, and its increased accumulation in the cytosol of bacteria. The study demonstrates how the potency of existing antibiotics against resistant Gram-negative pathogens can be boosted by adding functions for active transport and intracellular release.

UR - http://www.scopus.com/inward/record.url?scp=85157984639&partnerID=8YFLogxK

U2 - 10.1039/d2sc06850h

DO - 10.1039/d2sc06850h

M3 - Article

AN - SCOPUS:85157984639

VL - 14

SP - 5490

EP - 5502

JO - Chemical science

JF - Chemical science

SN - 2041-6520

IS - 20

ER -