Avibactam and inhibitor-resistant SHV β-lactamases

Abstract

β-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of β-lactam antibiotics to treat infections. A novel non-β-lactam β-lactamase inhibitor with activity against many class A and C and some class D β-lactamase variants, avibactam, is now available in the clinic in partnership with ceftazidime. Here, we explored the activity of avibactam against a variety of characterized isogenic laboratory constructs of β-lactamase inhibitor-resistant variants of the class A enzyme SHV (M69I/L/V, S130G, K234R, R244S, and N276D). We discovered that the S130G variant of SHV-1 shows the most significant resistance to inhibition by avibactam, based on both microbiological and biochemical characterizations. Using a constant concentration of 4 mg/liter of avibactam as a β-lactamase inhibitor in combination with ampicillin, the MIC increased from 1 mg/liter for blaSHV-1 to 256 mg/liter for blaSHV S130G expressed in Escherichia coli DH10B. At steady state, the k2/K value of the S130G variant when inactivated by avibactam was 1.3 M(-1) s(-1), versus 60,300 M(-1) s(-1) for the SHV-1 β-lactamase. Under timed inactivation conditions, we found that an approximately 1,700-fold-higher avibactam concentration was required to inhibit SHV S130G than the concentration that inhibited SHV-1. Molecular modeling suggested that the positioning of amino acids in the active site of SHV may result in an alternative pathway of inactivation when complexed with avibactam, compared to the structure of CTX-M-15-avibactam, and that S130 plays a role in the acylation of avibactam as a general acid/base. In addition, S130 may play a role in recyclization. As a result, we advance that the lack of a hydroxyl group at position 130 in the S130G variant of SHV-1 substantially slows carbamylation of the β-lactamase by avibactam by (i) removing an important proton acceptor and donator in catalysis and (ii) decreasing the number of H bonds. In addition, recyclization is most likely also slow due to the lack of a general base to initiate the process. Considering other inhibitor-resistant mechanisms among class A β-lactamases, S130 may be the most important amino acid for the inhibition of class A β-lactamases, perhaps even for the novel diazabicyclooctane class of β-lactamase inhibitors.

FIG 1

(A) Proposed acylation mechanism of SHV-1 by clavulanic acid (4–7). (B) Proposed acylation mechanism of CTX-M-15 by avibactam (17).

FIG 2

(A) Timed inactivation of SHV-1 by increasing concentrations of avibactam. (B) Timed inactivation of SHV S130G by increasing concentrations of avibactam. The SHV S130G variant takes longer to plateau because of the lower NCF catalytic efficiency of this enzyme. An approximately 1,700-fold-higher concentration of avibactam was required to inhibit the SHV S130G variant compared to SHV-1.

FIG 3

Mass spectrometry of SHV-1 and SHV S130G at various time points, ranging from 1 min to after a 24-h incubation of avibactam-enzyme at a 1:1 ratio.

FIG 4

Overlay of the CTX-M-15–avibactam crystal structure (PDB ID 4HBT; purple) with the acyl enzyme model of SHV-1–avibactam (blue), showing significant movement of many of the important active site residues and an absence of water (Wat) molecules in the SHV-1–avibactam model.

FIG 5

(A) Michaelis-Menten complex model of SHV-1–avibactam. (B) SHV-1–avibactam acyl enzyme model. (C) Michaelis-Menten complex model of SHV S130G-avibactam. (D) SHV S130G-avibactam acyl enzyme model.

FIG 6

Proposed acylation mechanism of avibactam for the SHV β-lactamase (A and B) and the S130G variant enzyme (C and D).