Structure-Based Analysis of Boronic Acids as Inhibitors of Acinetobacter-Derived Cephalosporinase-7, a Unique Class C β-Lactamase

Abstract

Acinetobacter baumannii is a multidrug resistant pathogen that infects more than 12 000 patients each year in the US. Much of the resistance to β-lactam antibiotics in Acinetobacter spp. is mediated by class C β-lactamases known as Acinetobacter-derived cephalosporinases (ADCs). ADCs are unaffected by clinically used β-lactam-based β-lactamase inhibitors. In this study, five boronic acid transition state analog inhibitors (BATSIs) were evaluated for inhibition of the class C cephalosporinase ADC-7. Our goal was to explore the properties of BATSIs designed to probe the R1 binding site. Ki values ranged from low micromolar to subnanomolar, and circular dichroism (CD) demonstrated that each inhibitor stabilizes the β-lactamase–inhibitor complexes. Additionally, X-ray crystal structures of ADC-7 in complex with five inhibitors were determined (resolutions from 1.80 to 2.09 Å). In the ADC-7/CR192 complex, the BATSI with the lowest Ki (0.45 nM) and greatest ΔTm (+9 °C), a trifluoromethyl substituent, interacts with Arg340. Arg340 is unique to ADCs and may play an important role in the inhibition of ADC-7. The ADC-7/BATSI complexes determined in this study shed light into the unique recognition sites in ADC enzymes and also offer insight into further structure-based optimization of these inhibitors.

Exploring Additional Dimensions of Complexity in Inhibitor Design for Serine β-Lactamases: Mechanistic and Intra- and Inter-molecular Chemistry Approaches

“…S02030 is very potent at inhibiting β-lactamases observed in Klebsiella pneumoniae and E. coli species. Developing boronic acid β-lactamase inhibitors is a promising approach as has been shown for a number of different β-lactamases (Tondi et al, 2010 , 2014 ; Powers et al, 2014 ; Bouza et al, 2017 ; Werner et al, 2017 ; Caselli et al, 2018 ).…”

Structural Insights into Inhibition of the Acinetobacter-Derived Cephalosporinase ADC-7 by Ceftazidime and Its Boronic Acid Transition State Analog

“…The O2 hydroxyl is also observed to be modified with the addition of a phosphate group, which has been documented in other ADC-7 complexes with BATSIs lacking an R2 group and appears to be an artifact of the crystallization conditions. 29 We had also explored the alternative possibility that the inhibitor and phosphate ion were not covalently attached and separately occupy the active site with partial occupancies.…”

“…This residue has previously been shown to interact with carboxylates and tetrazoles of other boronic acid inhibitors and contributes to their high affinity binding to ADC-7. 26,29…”

“…MICs were performed as previously described [28][29][30][31][32] and according to Clinical and Laboratory Standards Institute (CLSI) guidelines, using a 6 x 104 cfu/mL inoculum.…”

α‐Triazolylboronic Acids: A Promising Scaffold for Effective Inhibitors of KPCs

“…Using nitrocefin (NCF) as a substrate of KPC-2, boronic acids 1 a-n and avibactam and vaborbactam were tested as previously described. [25][26][27] The measurements of the initial velocities were performed with the addition of 100 μM NCF after a 5 min pre-incubation of the enzyme (2 nM) with increasing concentration of the inhibitor. The average velocities (v 0 ) were then fitted to equation:…”