A theoretical approach for the acylation/deacylation mechanisms of avibactam in the reversible inhibition of KPC-2

Abstract

Klebsiella pneumoniae carbapenemase (KPC-2) is the most commonly encountered class A β-lactamase variant worldwide, which confer high-level resistance to most available antibiotics. In this article we address the issue by a combined approach involving molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations. The study contributes to improve the understanding, at molecular level, of the acylation and deacylation stages of avibactam involved in the inhibition of KPC-2. The results show that both mechanisms, acylation and deacylation, the reaction occur via the formation of a tetrahedral intermediate. The formation of this intermediate corresponds to the rate limiting stage. The activation barriers are 19.5 kcal/mol and 23.0 kcal/mol for the acylation and deacylation stages, respectively. The associated rate constants calculated, using the Eyring equation, are 1.2 × 10^-1 and 3.9 × 10^-4 (s^-1). These values allow estimating a value of 3.3 × 10^-3 for the inhibition constant, in good agreement with the experimental value.

Keywords: Avibactam; Inhibition; KPC-2; QM/MM.

Fig. 1

Representative structure of the active site taken at 65 ns for the neutral form

Scheme 1

Proposed inhibition mechanisms for class A β-lactamases

Fig. 2

3-D view of the potential energy surface for the first stage of mechanism A

Fig. 3

Structure of the transition state TS-A1 in the active site

Fig. 4

Structure of the tetrahedral intermediate INT

Fig. 5

3-D view of the PES-A2 corresponding to the second stage of the mechanism A

Fig. 6

3-D view of the PES-B for the acylation of avibactam according to the mechanism B

Fig. 7

Structure of the transition state for the acylation of avibactam according to the mechanism B

Fig. 8

Structure of the acylated avibactam in the active site

Fig. 9

Potential energy profile for the avibactam acylation pathway. Green: mechanism A; light blue: mechanism B

Fig. 10

Orientations of the Ser129(O–H)–(N)Lys72 and N6–H–(O)Ser129 angles during the simulation

Fig. 11

Clustering analysis in terms of the dihedral angles α, β and γ. The red dot represents the centroids

Fig. 12

Structure corresponding to the centroid taken at 29 ns

Scheme 2

Mechanism of recyclization of avibactam from its complex with KPC-2

Fig. 13

3-D view of the PES-C1, corresponding to the formation of the tetrahedral intermediate

Fig. 14

3-D view of the tetrahedral intermediate INT

Fig. 15

3-D view of the transition state TS-C1

Fig. 16

2-D view of the PES-C2, corresponding to the recyclization of avibactam

Fig. 17

3-D view of the transition state TS-C2

Fig. 18

Potential energy profile along the avibactam deacylation pathway

Scheme 3

Representation of the acylation/deacylation cycle of avibactam with KPC-2