Thomas F. Durand-Réville

Kinetics of Avibactam Inhibition against Class A, C, and D β-Lactamases

Background: Avibactam is a β-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. Avibactam is a non-β-lactam β-lactamase inhibitor with a spectrum of activity that includes β-lactamase enzymes of classes A, C, and selected D examples. In this work acylation and deacylation rates were measured against the clinically important enzymes CTX-M-15, KPC-2, Enterobacter cloacae AmpC, Pseudomonas aeruginosa AmpC, OXA-10, and OXA-48. The efficiency of acylation (k2/Ki) varied across the enzyme spectrum, from 1.1 × 101 m−1s−1 for OXA-10 to 1.0 × 105 for CTX-M-15. Inhibition of OXA-10 was shown to follow the covalent reversible mechanism, and the acylated OXA-10 displayed the longest residence time for deacylation, with a half-life of greater than 5 days. Across multiple enzymes, acyl enzyme stability was assessed by mass spectrometry. These inhibited enzyme forms were stable to rearrangement or hydrolysis, with the exception of KPC-2. KPC-2 displayed a slow hydrolytic route that involved fragmentation of the acyl-avibactam complex. The identity of released degradation products was investigated, and a possible mechanism for the slow deacylation from KPC-2 is proposed.

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 Although β-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 β-lactamase enzymes that are not affected by currently marketed β-lactam/β-lactamase inhibitor combinations. Avibactam is a novel, covalent, non-β-lactam β-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 β-lactamases. Here we describe the structures of two clinically important β-lactamase enzymes bound to avibactam, the class A CTX-M-15 extended-spectrum β-lactamase and the class C Pseudomonas aeruginosa AmpC β-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.

Molecular Basis of Selective Inhibition and Slow Reversibility of Avibactam against Class D Carbapenemases: A Structure-Guided Study of OXA-24 and OXA-48

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.

ETX2514 is a broad-spectrum β-lactamase inhibitor for the treatment of drug-resistant Gram-negative bacteria including Acinetobacter baumannii

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.

Discovery of Efficacious Pseudomonas aeruginosa-Targeted Siderophore-Conjugated Monocarbams by Application of a Semi-mechanistic Pharmacokinetic/Pharmacodynamic Model

To identify new agents for the treatment of multi-drug-resistant Pseudomonas aeruginosa, we focused on siderophore-conjugated monocarbams. This class of monocyclic β-lactams are stable to metallo-β-lactamases and have excellent P. aeruginosa activities due to their ability to exploit the iron uptake machinery of Gram-negative bacteria. Our medicinal chemistry plan focused on identifying a molecule with optimal potency and physical properties and activity for in vivo efficacy. Modifications to the monocarbam linker, siderophore, and oxime portion of the molecules were examined. Through these efforts, a series of pyrrolidinone-based monocarbams with good P. aeruginosa cellular activity (P. aeruginosa MIC90 = 2 μg/mL), free fraction levels (>20% free), and hydrolytic stability (t1/2 ≥ 100 h) were identified. To differentiate the lead compounds and enable prioritization for in vivo studies, we applied a semi-mechanistic pharmacokinetic/pharmacodynamic model to enable prediction of in vivo efficacy from in vitro data.

Discovery and Characterization of New Hydroxamate Siderophores, Baumannoferrin A and B, produced by Acinetobacter baumannii

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 strains growth under iron limitation depends on baumannoferrin, a novel hydroxamate that could play a role in its virulence.

Whole-Cell-Based Assay To Evaluate Structure Permeation Relationships for Carbapenem Passage through the Pseudomonas aeruginosa Porin OprD

The global emergence of antibiotic resistance, especially in Gram-negative bacteria, is an urgent threat to public health. Discovery of novel classes of antibiotics with activity against these pathogens has been impeded by a fundamental lack of understanding of the molecular drivers underlying small molecule uptake. Although it is well-known that outer membrane porins represent the main route of entry for small, hydrophilic molecules across the Gram-negative cell envelope, the structure-permeation relationship for porin passage has yet to be defined. To address this knowledge gap, we developed a sensitive and specific whole-cell approach in Escherichia coli called titrable outer membrane permeability assay system (TOMAS). We used TOMAS to characterize the structure porin-permeation relationships of a set of novel carbapenem analogues through the Pseudomonas aeruginosa porin OprD. Our results show that small structural modifications, especially the number and nature of charges and their position, have dramatic effects on the ability of these molecules to permeate cells through OprD. This is the first demonstration of a defined relationship between specific molecular changes in a substrate and permeation through an isolated porin. Understanding the molecular mechanisms that impact antibiotic transit through porins should provide valuable insights to antibacterial medicinal chemistry and may ultimately allow for the rational design of porin-mediated uptake of small molecules into Gram-negative bacteria.

SAR and Structural Analysis of Siderophore-Conjugated Monocarbam Inhibitors of Pseudomonas aeruginosa PBP3

A main challenge in the development of new agents for the treatment of Pseudomonas aeruginosa infections is the identification of chemotypes that efficiently penetrate the cell envelope and are not susceptible to established resistance mechanisms. Siderophore-conjugated monocarbams are attractive because of their ability to hijack the bacterias iron uptake machinery for transport into the periplasm and their inherent stability to metallo-β-lactamases. Through development of the SAR we identified a number of modifications to the scaffold that afforded active anti-P. aeruginosa agents with good physicochemical properties. Through crystallographic efforts we gained a better understanding into how these compounds bind to the target penicillin binding protein PBP3 and factors to consider for future design.

Enantioselective Synthesis and Profiling of Two Novel Diazabicyclooctanone β-Lactamase Inhibitors

The enantioselective synthesis of two novel cyclopropane-fused diazabicyclooctanones is reported here. Starting from butadiene monoxide, the key enone intermediate 7 was prepared in six steps. Subsequent stereoselective introduction of the cyclopropane group and further transformation led to compounds 1a and 1b as their corresponding sodium salt. The great disparity regarding their hydrolytic stability was rationalized by the steric interaction between the cyclopropyl methylene and urea carbonyl. These two novel β-lactamase inhibitors were active against class A, C, and D enzymes.