Structure and mechanism of taniborbactam inhibition of the cefepime-hydrolyzing, partial R2-loop deletion Pseudomonas-derived cephalosporinase variant PDC-88.

Abstract

Pseudomonas aeruginosa is a major gram-negative pathogen responsible for a variety of infections and possessing an array of both intrinsic and acquired resistance mechanisms, including β-lactamases, like the chromosomal Pseudomonas-derived cephalosporinase (PDC). β-Lactams are the most widely prescribed class of antibiotics in the United States, and antipseudomonal cephalosporins (including cefepime) are important therapies (alone or combined with β-lactamase inhibitors) for P. aeruginosa infections. Taniborbactam is a novel, bicyclic boronate β-lactamase inhibitor with activity against all β-lactamase classes and is being developed in combination with cefepime. PDC-88 is an R2-loop deletion variant conferring resistance to cefepime and ceftazidime and elevating ceftolozane/tazobactam minimum inhibitory concentration (MIC). Herein, we elucidated PDC-88 resistance mechanisms and compared inhibition by taniborbactam and avibactam. In an isogenic background, PDC-88 increased cefepime MICs by 16-fold compared to PDC-3. In vitro, compared to PDC-3, PDC-88 had 8.3-fold higher catalytic efficiency for cefepime achieved by decreasing K_M 12.8-fold and decreasing k_cat 1.6-fold. This is supported by our crystallographic observation that the PDC-88 deletion enlarged the active site in the vicinity of the R2-loop, likely better accommodating cefepime. Taniborbactam and avibactam restored cefepime activity by inhibiting PDC-88. Compared to avibactam, taniborbactam had 4.1- and 9-fold lower K_{i app} values for PDC-3 and PDC-88, respectively, with higher k_on (k₂/K) and similar k_off for both enzymes. Structurally, taniborbactam positioned very similarly in the PDC-3 and PDC-88 active sites, interacting with many nearby residues. Based upon these data, cefepime-taniborbactam may represent an important alternative to ceftazidime-avibactam and ceftolozane-tazobactam for P. aeruginosa infections.