Structural Insight into Potent Broad-Spectrum Inhibition with Reversible Recyclization Mechanism: Avibactam in Complex with CTX-M-15 and Pseudomonas aeruginosa AmpC β-Lactamases

Abstract

dAlthough ␤-lactams have been the most effective class of antibacterial agents used in clinical practice for the past half century, their effectiveness on Gram-negative bacteria has been eroded due to the emergence and spread of ␤-lactamase enzymes that are not affected by currently marketed ␤-lactam/␤-lactamase inhibitor combinations. Avibactam is a novel, covalent, non-␤-lactam ␤-lactamase inhibitor presently in clinical development in combination with either ceftaroline or ceftazidime. In vitro studies show that avibactam may restore the broad-spectrum activity of cephalosporins against class A, class C, and some class D ␤-lactamases. Here we describe the structures of two clinically important ␤-lactamase enzymes bound to avibactam, the class A CTX-M-15 extended-spectrum ␤-lactamase and the class C Pseudomonas aeruginosa AmpC ␤-lactamase, which together provide insight into the binding modes for the respective enzyme classes. The structures reveal similar binding modes in both enzymes and thus provide a rationale for the broad-spectrum inhibitory activity of avibactam. Identification of the key residues surrounding the binding pocket allows for a better understanding of the potency of this scaffold. Finally, avibactam has recently been shown to be a reversible inhibitor, and the structures provide insights into the mechanism of avibactam recyclization. Analysis of the ultra-high-resolution CTX-M-15 structure suggests how the deacylation mechanism favors recyclization over hydrolysis.

Targeting Antibiotic Resistance

“…It is a covalent, reversible inhibitor that has been shown to have a very fast “on” rate, as well as a very slow “off” rate for deacylation, with a half‐life measured in days 173. X‐ray crystal structures of avibactam in complex with three different β‐lactamases have provided important insight into the mode of action 174, 175, 176. Avibactam interacts with conserved key residues in a fairly rigid conformation, and the highly polar sulfate group (which mimics the β‐lactam carboxylic acid, Figure 37) engages in strong polar interactions with Arg261 (Figure 38).…”

One ring to rule them all: Current trends in combating bacterial resistance to the β‐lactams

“…2(A,B)]. 25 25,27 The authors propose that upon carbamylation by the motif i serine, the C7-N6 bond is cleaved liberating the N6 nitrogen which may be protonated by the motif i S/Y in an analogous fashion to the b-lactam amide nitrogen. Similar to the analogous b-lactam carboxylate, the avibactam N6 sulfate occupies an electropositive pocket formed in proximity to motif iii.…”

“…Notably, the water involved in deacylation is present in the CTX-M-15-avibactam carbamyl enzyme crystal structure suggesting that the inherent stability of the carbamyl bond and/or protonation of the E166 general base likely contribute to avibactam's avoidance of hydrolytic deacylation. 25 Recyclization is proposed to be catalyzed by deprotonation of the N6 nitrogen by the motif ii S/Y resulting in an intramolecular decarbamylation and subsequent release from the catalytic serine. 25 Future development of the DBOs should focus on extending their spectrum of activity to include a broader range of class D and B blactamases.…”

“…25 Recyclization is proposed to be catalyzed by deprotonation of the N6 nitrogen by the motif ii S/Y resulting in an intramolecular decarbamylation and subsequent release from the catalytic serine. 25 Future development of the DBOs should focus on extending their spectrum of activity to include a broader range of class D and B blactamases.…”

What is the role of the new β-lactam/β-lactamase inhibitors ceftolozane/tazobactam and ceftazidime/avibactam?

“…Notwithstanding this difference, the activity spectra of the two new BLBLI combinations overlap [5]. CAZ/AVI combines an established third-generation cephalosporin (ceftazidime) and avibactam, a novel synthetic non-β-lactam/β-lactamase inhibitor that has very limited intrinsic antimicrobial activity but is endowed with a peculiar mechanism of β-lactamase inhibition [19] that makes the combination more effective against resistant GNB versus other BLBLIs [20]. The main pharmacokinetic features of TOL/TAZ are the following: rapid tissue distribution, low protein binding, nonclinically significant drug-drug interactions via the cytochrome P450 (CYP450) enzymes, lung penetration, and extensive renal excretion [21].…”