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

Alexandra A Bouza,Emilia Caselli,Hollister C Swanson,Bradley J Wallar,Alison L Vandine,Magdalena A Taracila,Kali A Smolen,Robert A Bonomo,Rachel A Powers,Fabio Prati,Chiara Romagnoli

doi:10.1021/acsinfecdis.7b00152

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 Δ Tm (+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.

Highlights

Acinetobacter baumannii is a multidrug resistant pathogen that infects more than 12 000 patients each year in the US
There are many mechanisms that contribute to the resistance, such as efflux pumps and porins, β-lactamases are the primary cause of resistance to β-lactam antimicrobials.[1] β-Lactamases function by hydrolyzing the amide bond in the lactam ring of β-lactam antibiotics, preventing β-lactam inhibition of its original cellular targets, the transpeptidases. 2,7 A significant portion of the β-lactamase-mediated resistance in Acinetobacter results from a unique family of class C enzymes, the Acinetobacter-derived cephalosporinases (ADCs).[2,8,9]
In order to assess the potency of the inhibitors in whole cells, Minimum Inhibitory Concentrations (MICs) and disc susceptibility assays were performed using E. coli DH10B expressing wtADC-7 and the R340A variant

Summary

Introduction

Acinetobacter baumannii is a multidrug resistant pathogen that infects more than 12 000 patients each year in the US. Novel non-β-lactam-based BLIs have been identified and characterized, including the phosphonates,[14] hydroxamates,[15] and diazabicyclooctanes (DBOs).[16,17] In 2015, the FDA approved the combination therapy known as Avycaz, which pairs the third-generation cephalosporin ceftazidime with the novel DBO inhibitor avibactam.[18] Another class of novel BLIs are the boronic acid transition state analog inhibitors (BATSIs) that have long been known to inhibit class A and C β-lactamases (Figure 1).[10,19,20,12] Previous work in our group suggested that BATSIs offer excellent potential as inhibitors of ADC enzymes in A. baumannii, ADC-7, with Ki values in the nanomolar range.[10] the X-ray crystal structure of ADC-7 in complex with the BATSI S02030 (Protein Data Bank, PDB 4U0X10) provided insight into ways this series might be optimized

Objectives

Methods

Results