Assessing the Potency of β-Lactamase Inhibitors with Diverse Inactivation Mechanisms against the PenA1 Carbapenemase from Burkholderia multivorans

Abstract

Burkholderia cepacia complex (Bcc) poses a serious health threat to people with cystic fibrosis or compromised immune systems. Infections often arise from Bcc strains, which are highly resistant to many classes of antibiotics, including β-lactams. β-Lactam resistance in Bcc is conferred largely via PenA-like β-lactamases. Avibactam was previously shown to be a potent inactivator of PenA1. Here, we examined the inactivation mechanism of PenA1, a class A serine carbapenemase from Burkholderia multivorans using β-lactamase inhibitors (β-lactam-, diazabicyclooctane-, and boronate-based) with diverse mechanisms of action. In whole cell based assays, avibactam, relebactam, enmetazobactam, and vaborbactam restored susceptibility to piperacillin against PenA1 expressed in Escherichia coli. The rank order of potency of inactivation in vitro based on k inact/K I or k 2/K values (range: 3.4 × 102 to 2 × 106 M–1 s–1) against PenA1 was avibactam > enmetazobactam > tazobactam > relebactam > clavulanic acid > vaborbactam. The contribution of selected amino acids (S70, K73, S130, E166, N170, R220, K234, T237, and D276) in PenA1 toward inactivation was evaluated using site-directed mutagenesis. The S130A, R220A, and K234A variants of PenA1 were less susceptible to inactivation by avibactam. The R220A variant was purified and assessed via steady-state inhibition kinetics and found to possess increased K i‑app values and decreased k inact/K I or k 2/K values against all tested inhibitors compared to PenA1. Avibactam was the most affected by the alanine replacement at 220 with a nearly 400-fold decreased acylation rate. The X-ray crystal structure of the R220A variant was solved and revealed loss of the hydrogen bonding network between residues 237 and 276 leaving a void in the active site that was occupied instead by water molecules. Michaelis–Menten complexes were generated to elucidate the molecular contributions of the poorer in vitro inhibition profile of vaborbactam against PenA1 (k 2/K, 3.4 × 102 M–1 s–1) and was compared to KPC-2, a class A carbapenemase that is robustly inhibited by vaborbactam. The active site of PenA1 is larger than that of KPC-2, which impacted the ability of vaborbactam to form favorable interactions, and as a result the carboxylate of vaborbactam was drawn toward K234/T235 in PenA1 displacing the boronic acid from approaching the nucleophilic S70. Moreover, in PenA1, the tyrosine at position 105 compared to tryptophan in KPC-2, was more flexible rotating more than 90°, and as a result PenA1’s Y105 competed for binding with the cyclic boronate vs the thiophene moiety of vaborbactam, further precluding inhibition of PenA1 by vaborbactam. Given the 400-fold decreased k 2/K for the R220A variant compared to PenA1, acyl–enzyme complexes were generated via molecular modeling and compared to the PenA1-avibactam crystal structure. The water molecules occupying the active site of the R220A variant are unable to stabilize the T237 and D276 region of the active site altering the ability of avibactam to form favo...

Coming from the Wild: Multidrug Resistant Opportunistic Pathogens Presenting a Primary, Not Human-Linked, Environmental Habitat

“…Like several other bacteria, Bcc presents in its genome genes encoding different AR determinants, including efflux pumps and inducible class A and class C β-lactamases, as PenB [ 275 ] (formerly dubbed PenA [ 276 ]) or AmpC [ 277 ], respectively, which expression is coregulated [ 278 ]. Recent work suggests that β-lactam inhibitors such as relebactam, enmetazobactam, avibactam or vaborbactam, might be useful for increasing the susceptibility to β-lactams of Bcc isolates [ 279 ].…”

Biomolecular Simulations with the Three-Dimensional Reference Interaction Site Model with the Kovalenko-Hirata Closure Molecular Solvation Theory

“…The 3D-RISM-KH based water site prediction is implemented in the MOE © suite [ 81 ]. Some examples of successful applications of the 3D-RISM-KH theory in exploiting the explicit role of water maps are reported for in-drug design and protein aggregation studies [ 82 , 83 , 84 ]. A very recent modification of the 3D-RISM theory in mapping solvation sites in enzyme active site was reported by Nguyen et al [ 85 ].…”

Secondary Structure and Conformational Stability of the Antigen Residues Making Contact with Antibodies

“…In contrast, the active site residues displayed low B-factors. In carbapenemase, an enzyme to dissociate βlactamase antibiotics, 53 an outer loop and a large lid to hold the substrate are flexible. These regions showed large B-factor values in both X-ray crystallography and computer simulation.…”