Session: P060 News on relebactam and vaborbactam

Category: 5a. Mechanisms of action, preclinical data & pharmacology of antibacterial

agents

24 April 2017, 12:30 - 13:30

P1284

Relebactam (REL) is a potent inhibitor of the KPC-2 beta-lactamase and restores the
susceptibility of imipenem (IMI) against KPC-producing Enterobacteriaceae

Krisztina Papp-Wallace*1, Melissa Barnes2, Jim Alsop3, Chris Bethel4, Scott Becka4, David
Van Duin5, Barry Kreiswirth6, Robert A. Bonomo7

1Lscdvamc

2Louis Stokes Cleveland Vamc and Cwru

3University of Minnesota

4Louis Stokes Cleveland Vamc

5University of North Carolina

6New Jersey Medical School, Rutgers The State University of New Jersey ; Public Health
Research Institute Centre

7Case Western Reserve University

Background: REL is a bridged diazabicyclooctane non-β-lactam β-lactamase inhibitor (BLI). IMI-REL

combination is a potential treatment regimen for infections caused by Enterobacteriaceae possessing
complex β-lactamase backgrounds. We investigated the inactivation of Klebsiella pneumoniae
carbapenemase (KPC-2), the most widespread class A carbapenemase, by REL and performed
susceptibility testing with IMI-REL using KPC-producing clinical isolates of Enterobacteriaceae.

Material/methods: KPC-2 β-lactamase was purified from E. coli Origami 2 DE3 pET24a+ producing
the KPC-2 protein without its signal peptide. Purified KPC-2 was analyzed via steady-state inhibitor
kinetics and mass spectrometry using REL. Minimal inhibitory concentration (MIC) measurements
using agar dilution methods according to Clinical and Laboratory Standards Institute for IMI and IMI-
REL (REL was maintained at 4 mg/L) was performed on 101 clinical isolates of KPC-producing
Enterobacteriaceae (Klebsiella pneumoniae, K. oxytoca, Enterobacter cloacae, E. aerogenes,
Citrobacter freundii, C. koseri, and Escherichia coli).
Results: REL inhibited KPC-2 with a k2/K value of 24,750 M-1s-1 and demonstrated a slow koff of
0.0002 s-1 (Table 1a and Figure 1a and 1b). Mass spectrometry revealed that the KPC-2-REL acyl-
enzyme complex was stable for up to 24 hours; desulfation of REL was not observed using mass
spectrometry. In vitro, KPC-producing clinical isolates of Enterobacteriaceae were highly susceptible
(100% of isolates) to IMI-REL (MIC ≤ 2 mg/L) (Table 1b).

Table 1a. Kinetic parameters for KPC-2 with REL.

Parameter Value
NCF Km and REL Ki app (µM) 8 ± 1 and 2.3 ± 0.3
k2/K (M-1s-1) 24,750 ± 2,475
koff (s )
-1 0.00020 ± 0.00002
kcat/kinact 1
Table 1b. MICs in mg/L for selected KPC-producing Enterobacteriaceae clinical
isolates and control strains.
Strain IMI IMI-REL
E. coli DH10B 0.5 0.5
E. coli DH10B pBR322-catI blaKPC-2 8 0.5
MIC90 = 64 MIC90 = 0.5
KPC-producing Enterobacteriaceae
MIC50 = 8 MIC50 = 0.25

Conclusions: The favorable inhibitory kinetic profile of REL against KPC-2 is in agreement with the
robust activity profile vs. clinical isolates of KPC-producing Enterobacteriaceae. These findings
suggest that IMI-REL is a worthwhile candidate for in vivo studies of infections caused by KPC-
producing Enterobacteriaceae.