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Abstract
The development of new aminoglycoside (AG) antibiotics has been required to overcome the resistance mechanism of AG-modifying enzymes (AMEs) of AG-resistant pathogens. The AG acetyltransferase, AAC(6′)-APH(2″), one of the most typical AMEs, exhibiting substrate promiscuity towards a variety of AGs and acyl-CoAs, was employed to enzymatically synthesize new 6′-N-acylated isepamicin (ISP) analogs, 6′-N-acetyl/-propionyl/-malonyl ISPs. They were all active against the ISP-resistant Gram-negative bacteria tested, and the 6′-N-acetyl ISP displayed reduced toxicity compared to ISP in vitro. This study demonstrated the importance of the modification of the 6′-amino group in circumventing AG-resistance and the potential of regioselective enzymatic modification of AG scaffolds for the development of more robust AG antibiotics.
Highlights
Aminoglycosides (AGs), one of the oldest classes of antibiotic agents, have a strong antibacterial activity against Gram-positive and Gram-negative bacterial pathogens because they interfere with the protein biosynthesis by acting on the bacterial ribosome [1,2]
Antibiotic-resistant E. coli strains for this study were provided by the National Biobank of the Kyungpook National University Hospital (KNUH; Daegu, Korea), a member of the KoreaBiobank Network-KNUH
Five clinical multi-drug resistant P. aeruginosa (MDRPA) isolates were obtained from patients at the KNUH [14]
Summary
Aminoglycosides (AGs), one of the oldest classes of antibiotic agents, have a strong antibacterial activity against Gram-positive and Gram-negative bacterial pathogens because they interfere with the protein biosynthesis by acting on the bacterial ribosome [1,2]. The primary resistance mechanism is the chemical modification of AG structures by AG-modifying enzymes (AMEs) [2,4,5] This led to substantial efforts to develop semi-synthetic AGs that can circumvent relevant AMEs and improve the pharmacological profile. N-acyl moieties at C1 or C6 positions are the common structures of the semi-synthetic antibiotics currently employed clinically, including the most recently approved antibiotic, plazomicin [8]. Escherichia coli BL21 (DE3) and plasmid pET-21c(+) (Novagen, Madison, WI, USA) were used for expression of the recombinant protein. The Ni-nitrilotriacetic acid (NTA) agarose (Qiagen, Valencia, CA, USA), PD10 column (GE Healthcare, Piscataway, NJ, USA), and an Amicon Ultracel 10 K molecular weight cut-off spin filter (Millipore, Bedford, MA, USA) were used to prepare the histidine-tagged recombinant protein. Approximately 10 mg of purified AAC(6 )-APH(2”) was obtained from 1 L of culture (Figure S1)
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