Targeting Multidrug-Resistant Acinetobacter spp.: Sulbactam and the Diazabicyclooctenone β-Lactamase Inhibitor ETX2514 as a Novel Therapeutic Agent

Abstract

Multidrug-resistant (MDR) Acinetobacter spp. poses a significant therapeutic challenge in part due to the presence of chromosomally encoded β-lactamases, including class C Acinetobacter-derived cephalosporinases (ADC) and class D oxacillinases (OXA), as well as plasmid-mediated class A β-lactamases. Importantly, OXA-like β-lactamases represent a gap in the spectrum of inhibition by recently approved β-lactamase inhibitors such as avibactam and vaborbactam. ETX2514 is a novel, rationally designed, diazabicyclooctenone inhibitor that effectively targets class A, C, and D β-lactamases. We show that addition of ETX2514 significantly increased the susceptibility of clinical Acinetobacterbaumannii isolates to sulbactam. AdeB and AdeJ were identified to be key efflux constituents for ETX2514 in A. baumannii The combination of sulbactam and ETX2514 was efficacious against A. baumannii carrying bla_TEM-1, bla_ADC-82, bla_OXA-23, and bla_OXA-66 in a neutropenic murine thigh infection model. We also show that, in vitro, ETX2514 inhibited ADC-7 (k₂/K_i 1.0 ± 0.1 × 10⁶ M^-1 s^-1) and OXA-58 (k₂/K_i 2.5 ± 0.3 × 10⁵ M^-1 s^-1). Cocrystallization of ETX2514 with OXA-24/40 revealed hydrogen bonding interactions between ETX2514 and residues R261, S219, and S128 of OXA-24/40 in addition to a chloride ion occupied in the active site. Further, the C3 methyl group of ETX2514 shifts the position of M223. In conclusion, the sulbactam-ETX2514 combination possesses a broadened inhibitory range to include class D β-lactamases as well as class A and C β-lactamases and is a promising therapeutic candidate for infections caused by MDR Acinetobacter spp.IMPORTANCE The number and diversity of β-lactamases are steadily increasing. The emergence of β-lactamases that hydrolyze carbapenems poses a significant threat to our antibiotic armamentarium. The explosion of OXA enzymes that are carbapenem hydrolyzers is a major challenge (carbapenem-hydrolyzing class D [CHD]). An urgent need exists to discover β-lactamase inhibitors with class D activity. The sulbactam-ETX2514 combination demonstrates the potential to become a treatment regimen of choice for Acinetobacter spp. producing class D β-lactamases.

Keywords: Acinetobacter; DBO; ETX2514; beta-lactamases; beta-lactams; diazabicyclooctanone; diazabicyclooctenone; sulbactam; β-lactamase inhibitor.

FIG 1

Structures of the DBOs, avibactam, ETX2514, and ETX2514 analog compound 3.

FIG 2

Susceptibility summary of 98 A. baumannii clinical isolates.

FIG 3

Mass spectra of ADC-7 and OXA-58 β-lactamases and ETX2514. Mass accuracy is within 5 Da.

FIG 4

Mass spectra of ETX2514 acyl transfer from OXA-58 using KPC-2 as the recipient β-lactamase. Mass accuracy is within 5 Da.

FIG 5

ETX2514 in the active site of OXA-24/40. (A) Shown is an unbiased omit |Fo| − |Fc| electron density with ETX2514 removed from refinement and map calculations of OXA-24/40. The inhibitor is shown in blue carbon atom stick representation, whereas the protein is depicted in gray carbon atom stick representation. Electron density is contoured at the 3σ level. The K84 side chain was observed in two conformations; one carbamylated and one noncarbamylated (0.6 and 0.4 occupancy conformations labeled a and b, respectively). A chloride ion with 0.4 occupancy was also refined in the active site (green sphere labeled Cl). (B) Hydrogen bonds (dashed lines) between ETX2514 and OXA-24/40. Water molecules are depicted as red spheres, and the partially occupied chloride ion is depicted as a green sphere.

FIG 6

Superposition of ETX2514 and avibactam OXA-24/40 complexes. The avibactam OXA-24/40 complex (PDB ID 4wm9 [38]) is colored orange; the ETX2514 complex is colored as described in the legend to Fig. 5. The C3 atom for ETX2514 is labeled; the two conformations of K84 are labeled a and b. Crystallographic water molecules observed in the two structures are shown as spheres (colored red and orange for the ETX2514 and avibactam OXA-24/40 complexes, respectively).

FIG 7

Superpositioning of ETX2514 and compound 3 OXA-24/40 complexes. The compound 3 OXA-24/40 complex (PDB ID 5vfd [2]) is colored magenta; the ETX2514 complex is colored as in Fig. 5. The differentiating methyl substituents on the DBO ring are labeled C4 (for compound 3) and C3 for ETX2514.