Nacubactam Enhances Meropenem Activity against Carbapenem-Resistant Klebsiella pneumoniae Producing KPC

Abstract

Carbapenem-resistant Enterobacteriaceae (CRE) are resistant to most antibiotics, making CRE infections extremely difficult to treat with available agents. Klebsiella pneumoniae carbapenemases (KPC-2 and KPC-3) are predominant carbapenemases in CRE in the United States. Nacubactam is a bridged diazabicyclooctane (DBO) β-lactamase inhibitor that inactivates class A and C β-lactamases and exhibits intrinsic antibiotic and β-lactam "enhancer" activity against Enterobacteriaceae In this study, we examined a collection of meropenem-resistant K. pneumoniae isolates carrying bla_KPC-2 or bla_KPC-3; meropenem-nacubactam restored susceptibility. Upon testing isogenic Escherichia coli strains producing KPC-2 variants with single-residue substitutions at important Ambler class A positions (K73, S130, R164, E166, N170, D179, K234, E276, etc.), the K234R variant increased the meropenem-nacubactam MIC compared to that for the strain producing KPC-2, without increasing the meropenem MIC. Correspondingly, nacubactam inhibited KPC-2 (apparent K_i [K_{i app}] = 31 ± 3 μM) more efficiently than the K234R variant (K_{i app} = 270 ± 27 μM) and displayed a faster acylation rate (k₂/K), which was 5,815 ± 582 M^-1 s^-1 for KPC-2 versus 247 ± 25 M^-1 s^-1 for the K234R variant. Unlike avibactam, timed mass spectrometry revealed an intact sulfate on nacubactam and a novel peak (+337 Da) with the K234R variant. Molecular modeling of the K234R variant showed significant catalytic residue (i.e., S70, K73, and S130) rearrangements that likely interfere with nacubactam binding and acylation. Nacubactam's aminoethoxy tail formed unproductive interactions with the K234R variant's active site. Molecular modeling and docking observations were consistent with the results of biochemical analyses. Overall, the meropenem-nacubactam combination is effective against carbapenem-resistant K. pneumoniae Moreover, our data suggest that β-lactamase inhibition by nacubactam proceeds through an alternative mechanism compared to that for avibactam.

Keywords: K234R; KPC; diazabicyclooctane (DBO); nacubactam; β-lactam; β-lactamase.

FIG 1

Structure of the diazabicyclooctanes (DBOs) avibactam and nacubactam.

FIG 2

MICs for 44 K. pneumoniae clinical isolates containing the KPC-2 or KPC-3 β-lactamase tested against meropenem (MERO) and meropenem combined with nacubactam (Nacu) at a 1:1 ratio.

FIG 3

Timed mass spectra of KPC-2 and the K234R variant (A) with nacubactam (B) or avibactam (C). KPC-2 (left) and the K234R variant (right) were incubated at a 1:1 molar ratio of enzyme-DBO for the indicated times. #KPC-2 (28,719 Da) and *K234R (28,745 Da) are the apo-β-lactamases. Acyl complexes with nacubactam are indicated with blue and green arrows. Acyl complexes with avibactam are indicated with orange and purple arrows.

FIG 4

Circular dichroism (CD) of KPC-2 (10 μM) (A) and the K234R variant (10 μM) (B) with nacubactam (Nacu; 100 μM) and avibactam (Avi; 100 μM). DBO binding to KPC-2 does not induce significant secondary structure changes (A). The binding of avibactam seems to partially restore the stability of the K234R variant, resulting in large secondary structure changes. Nacubactam induces only minor secondary structure changes in the K234R variant (B). θ, ellipticity.

FIG 5

Thermal denaturation of KPC-2 (10 μM) and the K234R variant (10 μM) measured alone (A, B) and with nacubactam (100 μM) or avibactam (100 μM) (B, C, D). The K234R variant is slightly less thermodynamically stable than wild-type KPC-2 (A, B). DBOs increase the thermal stability of KPC-2 (B, C), but only avibactam increases the thermal stability of K234R, by 2°C (B, D).

FIG 6

(A and B) Molecular modeling of KPC-2 (A) versus the K234R variant (B). S130 interacts with K234 but not R234. The K73-S70 interaction is also disrupted in the K234R variant, establishing a new hydrogen bond between S130 and K73. (C and D) Molecular docking of nacubactam in KPC-2 (C) and the K234R variant (D). R234 has a different conformation than K234 and forms an alternative network of bonds. The aminoethoxy tail of nacubactam forms additional interactions in K234R.