Background:Avibactam is a ␤-lactamase inhibitor with a broad spectrum of activity. Results: Kinetic parameters of inhibition as well as acyl enzyme stability are reported against six clinically relevant enzymes. Conclusion: Inhibition efficiency is highest against class A, then class C, and then class D. Significance: These base-line inhibition values across enzyme classes provide the foundation for future structural and mechanistic enzymology experiments.

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.

Multidrug-resistant (MDR) bacterial infections are a serious threat to public health. Among the most alarming resistance trends is the rapid rise in the number and diversity of β-lactamases, enzymes that inactivate β-lactams, a class of antibiotics that has been a therapeutic mainstay for decades. Although several new β-lactamase inhibitors have been approved or are in clinical trials, their spectra of activity do not address MDR pathogens such as Acinetobacter baumannii. This report describes the rational design and characterization of expanded-spectrum serine β-lactamase inhibitors that potently inhibit clinically relevant class A, C and D β-lactamases and penicillin-binding proteins, resulting in intrinsic antibacterial activity against Enterobacteriaceae and restoration of β-lactam activity in a broad range of MDR Gram-negative pathogens. One of the most promising combinations is sulbactam-ETX2514, whose potent antibacterial activity, in vivo efficacy against MDR A. baumannii infections and promising preclinical safety demonstrate its potential to address this significant unmet medical need.

The Class D (or OXA-type) β-lactamases have expanded to be the most diverse group of serine β-lactamases with a highly heterogeneous β-lactam hydrolysis profile and are typically resistant to marketed β-lactamase inhibitors. Class D enzymes are increasingly found in multidrug resistant (MDR) Acinetobacter baumannii, Pseudomonas aeruginosa, and various species of the Enterobacteriaceae and are posing a serious threat to the clinical utility of β-lactams including the carbapenems, which are typically reserved as the drugs of last resort. Avibactam, a novel non-β-lactam β-lactamase inhibitor, not only inhibits all class A and class C β-lactamases but also has the promise of inhibition of certain OXA enzymes, thus extending the antibacterial activity of the β-lactam used in combination to the organisms that produce these enzymes. X-ray structures of OXA-24 and OXA-48 in complex with avibactam revealed the binding mode of this inhibitor in this diverse class of enzymes and provides a rationale for selective inhibition of OXA-48 members. Additionally, various subunits of the OXA-48 structure in the asymmetric unit provide snapshots of different states of the inhibited enzyme. Overall, these data provide the first structural evidence of the exceptionally slow reversibility observed with avibactam in class D β-lactamases. Mechanisms for acylation and deacylation of avibactam by class D enzymes are proposed, and the likely extent of inhibition of class D β-lactamases by avibactam is discussed.

Acinetobacter baumannii AYE does not produce acinetobactin but grows under iron limitation. Accordingly, analyses of AYE iron-restricted culture supernatants resulted in the isolation of two fractions, which contained only hydroxamates and showed siderophore activity. Structural analyses identified baumannoferrin A and baumannoferrin B, which differ only by a double bond. These siderophores are composed of citrate, 1,3-diaminopropane, 2,4-diaminobutyrate, decenoic acid, and α-ketoglutarate. Analysis of the AYE genome showed the presence of a 12-gene cluster coding for proteins similar to those involved in the production and utilization of the hydroxamate siderophores acinetoferrin and achromobactin. As A. baumannii AYE does not produce acinetobactin and harbors only one gene cluster encoding the production and utilization of a siderophore, this strain's growth under iron limitation depends on baumannoferrin, a novel hydroxamate that could play a role in its virulence.

OmpA is a conserved, abundantly expressed outer membrane porin in Acinetobacter baumannii whose presumed role in antibiotic permeation has not been clearly demonstrated. In this report, we use a titratable heterologous expression system to express OmpA in isolation and demonstrate selective passage of small molecule antibiotics through OmpA. ETX2514, a recently discovered broad-spectrum β-lactamase inhibitor, in combination with sulbactam, is currently in clinical testing for the treatment of drug-resistant A. baumannii infections. We demonstrate that ETX2514 permeates OmpA and potentiates the activity of sulbactam in an OmpA-dependent manner. In addition, we show that small modifications in the structure of ETX2514 differentially affect its passage through OmpA, revealing unique structure-porin-permeation relationships. Finally, we confirm the contribution of OmpA to bacterial fitness using a murine thigh model of A. baumannii infection. These results, combined with the high sequence homology of OmpA across Acinetobacter spp., suggest that optimization of antibiotic entry through OmpA may prove to be a feasible medicinal chemistry design strategy for future antibacterial discovery efforts.

Multidrug resistant Gram-negative bacterial infections are an increasing public health threat due to rapidly rising resistance toward β-lactam antibiotics. The hydrolytic enzymes called β-lactamases are responsible for a large proportion of the resistance phenotype. β-Lactamase inhibitors (BLIs) can be administered in combination with β-lactam antibiotics to negate the action of the β-lactamases, thereby restoring activity of the β-lactam. Newly developed BLIs offer some advantage over older BLIs in terms of enzymatic spectrum but are limited to the intravenous route of administration. Reported here is a novel, orally bioavailable diazabicyclooctane (DBO) β-lactamase inhibitor. This new DBO, ETX1317, contains an endocyclic carbon–carbon double bond and a fluoroacetate activating group and exhibits broad spectrum activity against class A, C, and D serine β-lactamases. The ester prodrug of ETX1317, ETX0282, is orally bioavailable and, in combination with cefpodoxime proxetil, is currently in development as an oral therapy for multidrug resistant and carbapenem-resistant Enterobacterales infections.

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