Abstract
β-Lactamase-mediated resistance to β-lactam antibiotics has been significantly threatening the efficacy of these clinically important antibacterial drugs. Although some β-lactamase inhibitors are prescribed in combination with β-lactam antibiotics to overcome this resistance, the emergence of enzymes resistant to current inhibitors necessitates the development of novel β-lactamase inhibitors. In this study, we evaluated the inhibitory effect of dinucleotides on an extended-spectrum class C β-lactamase, AmpC BER. Of the dinucleotides tested, NADPH, a cellular metabolite, decreased the nitrocefin-hydrolyzing activity of the enzyme with a Ki value of 103 μM in a non-covalent competitive manner. In addition, the dissociation constant (KD) between AmpC BER and NADPH was measured to be 40 μM. According to our in vitro susceptibility study based on growth curves, NADPH restored the antibacterial activity of ceftazidime against a ceftazidime-resistant Escherichia coli BER strain producing AmpC BER. Remarkably, a single dose of combinatory treatment with NADPH and ceftazidime conferred marked therapeutic efficacy (100% survival rate) in a mouse model infected by the E. coli BER strain although NADPH or ceftazidime alone failed to prevent the lethal bacterial infection. These results may offer the potential of the dinucleotide scaffold for the development of novel β-lactamase inhibitors.
Highlights
The β-lactam antibiotics are the mainstay in the treatment of serious bacterial infections in clinical use (Liu et al, 2015)
Our studies demonstrated that guanosine monophosphate (GMP) and inosine monophosphate (IMP) are noncovalent competitive inhibitors (Na et al, 2017) of class C βlactamases and acAMP exerted its inhibitory effect through the covalent attachment of its AMP moiety to the nucleophilic serine residue of class C β-lactamases (Kim et al, 2017)
The AMP moiety and GMP/IMP bind to R1 and R2 subsites in the active site of class C β-lactamases, respectively, they have identical structures except for subtle differences in the purine base; the R1 and R2 subsites structurally refer to the distinct active site regions that accommodate the R1 side chain at the position C7 and the R2 side chain at the position C3 of β-lactam antibiotics on substrate binding, respectively (Kim et al, 2006)
Summary
The β-lactam antibiotics are the mainstay in the treatment of serious bacterial infections in clinical use (Liu et al, 2015). Several β-lactamase inhibitors, including β-lactam inhibitors (clavulanate, sulbactam, and tazobactam) and non-β-lactam inhibitors (avibactam and vaborbactam), have been developed and clinically used to treat bacterial infections in combination with β-lactam antibiotics (e.g., amoxicillin/clavulanate, ticarcillin/clavulanate, ampicillin/sulbactam, cefoperazone/sulbactam, piperacillin/tazobactam, ceftazidime/avibactam, and meropenem/vaborbactam) (Harris et al, 2015; Cho et al., 2018). The combination of acAMP and ceftazidime, a third-generation cephalosporin, significantly reduced the growth of antibiotic resistant bacteria (Kim et al, 2017). These results suggest that nucleotide scaffolds could be useful candidates to develop novel β-lactamase inhibitors. Through in vitro assays and a mouse infection model, we characterized the inhibitory mechanism and effect of NADPH on an extended-spectrum class C β-lactamase, AmpC. These studies might facilitate the identification and design of new and high effective inhibitors targeting class C β-lactamases
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