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Background: Ceftazidime-avibactam was approved by the FDA to treat infections caused by Enterobacterales carrying blaKPC-2. However, variants of KPC-2 with amino acid substitutions at position 179 have emerged and confer resistance to ceftazidime-avibactam. Methods: The activity of imipenem-relebactam was assessed against a panel of 19 KPC-2 D179 variants. KPC-2 and the D179N and D179Y variants were purified for biochemical analyses. Molecular models were constructed with imipenem to assess differences in kinetic profiles. Results: All strains were susceptible to imipenem–relebactam, but resistant to ceftazidime (19/19) and ceftazidime-avibactam (18/19). KPC-2 and the D179N variant hydrolyzed imipenem, but the D179N variant’s rate was much slower. The D179Y variant was unable to turnover imipenem. All three β-lactamases hydrolyzed ceftazidime at varying rates. The acylation rate of relebactam for the D179N variant was ~2.5× lower than KPC-2. Poor catalytic turnover by the D179Y variant precluded the determination of inhibitory kinetic parameters. Acyl-complexes with imipenem and ceftazidime were less prevalent with the D179N variant compared to the D179Y variant, supporting the kinetic observations that the D179Y variant was not as active as the D179N variant. Relebactam was slower to form an acyl-complex with the D179Y variant compared to avibactam. The D179Y model with imipenem revealed that the catalytic water molecule was shifted, and the carbonyl of imipenem was not within the oxyanion hole. Conversely in the D179N model, imipenem was oriented favorably for deacylation. Conclusions: Imipenem–relebactam overcame the resistance of the D179 variants, suggesting that this combination will be active against clinical isolates harboring these derivatives of KPC-2.

Acinetobacter baumannii is a Gram-negative organism listed as an urgent threat pathogen by the World Health Organization (WHO). Carbapenem-resistant A. baumannii (CRAB), especially, present therapeutic challenges due to complex mechanisms of resistance to β-lactams. One of the most important mechanisms is the production of β-lactamase enzymes capable of hydrolyzing β-lactam antibiotics. Co-expression of multiple classes of β-lactamases is present in CRAB; therefore, the design and synthesis of “cross-class” inhibitors is an important strategy to preserve the efficacy of currently available antibiotics. To identify new, nonclassical β-lactamase inhibitors, we previously identified a sulfonamidomethaneboronic acid CR167 active against Acinetobacter-derived class C β-lactamases (ADC-7). The compound demonstrated affinity for ADC-7 with a Ki = 160 nM and proved to be able to decrease MIC values of ceftazidime and cefotaxime in different bacterial strains. Herein, we describe the activity of CR167 against other β-lactamases in A. baumannii: the cefepime-hydrolysing class C extended-spectrum β-lactamase (ESAC) ADC-33 and the carbapenem-hydrolyzing OXA-24/40 (class D). These investigations demonstrate CR167 as a valuable cross-class (C and D) inhibitor, and the paper describes our attempts to further improve its activity. Five chiral analogues of CR167 were rationally designed and synthesized. The structures of OXA-24/40 and ADC-33 in complex with CR167 and select chiral analogues were obtained. The structure activity relationships (SARs) are highlighted, offering insights into the main determinants for cross-class C/D inhibitors and impetus for novel drug design.

Boronic acid transition-state analog inhibitors (BATSIs) are partners with β-lactam antibiotics for the treatment of complex bacterial infections. Herein, microbiological, biochemical, and structural findings on four BATSIs with the FOX-4 cephamycinase, a class C β-lactamase that rapidly hydrolyzes cefoxitin, are revealed. FOX-4 is an extended-spectrum class C cephalosporinase that demonstrates conformational flexibility when complexed with certain ligands. Like other β-lactamases of this class, studies on FOX-4 reveal important insights into structure–activity relationships. We show that SM23, a BATSI, shows both remarkable flexibility and affinity, binding similarly to other β-lactamases, yet retaining an IC50 value < 0.1 μM. Our analyses open up new opportunities for the design of novel transition-state analogs of class C enzymes.