Molecular Features of Cephalosporins Important for Activity against Antimicrobial-Resistant Neisseria gonorrhoeae

Abstract

The increasing prevalence of Neisseria gonorrhoeae strains exhibiting decreased susceptibility to extended-spectrum cephalosporins (ESCs) presents a challenge for the successful treatment of gonorrhea infections. To address this challenge, we evaluated a panel of 23 cephalosporins against penicillin-binding protein 2 (PBP2) from the ESC-resistant (ESC^R) N. gonorrhoeae strain H041 to determine which molecular features are important for antimicrobial activity. Structure-activity relationships (SARs) developed from acylation rate constants against PBP2 and antimicrobial susceptibilities against the H041 strain of N. gonorrhoeae, and interpreted against docking models, reveal that cephalosporins possessing large, lipophilic R₁ side chains and electronegative R₂ side chains with planar groups are associated with higher acylation rates against PBP2, but also that these same amphipathic features can lower antimicrobial activity. Based on these studies, we tested cefoperazone, one of the most effective ESCs for targeting PBP2, in the female mouse model infected with H041 and showed that it was equally or more effective than ceftriaxone or gentamicin for clearing infections. Taken together, our results reveal that two U.S. Food and Drug Administration (FDA)-approved agents (cefoperazone, ceftaroline) and one FDA-qualified infectious disease product (ceftobiprole) have potential as first-line treatments for gonorrhea and provide a framework for the future design of cephalosporins with improved activity against ESC-resistant N. gonorrhoeae.

Keywords: Neisseria gonorrhoeae; cephalosporins; molecular docking; penicillin-binding protein 2; structure−activity relationship.

Figure 1:. Generations of cephalosporin antimicrobials.

Representative structures from each generation show new features discovered and adapted during each generation.

Figure 2:. Protein-ligand interaction fingerprints (PLIFs) for pharmacophore-constrained induced-fit docking of cephalosporins to tPBP2^H041.

A. Heat map of cephalosporin-tPBP2^H041 interactions colored by number of poses interacting with a given residue. B. Heat map of cephalosporin-tPBP2^H041 interactions, colored by the cephalosporin moiety interacting with a given residue. C. Consensus pharmacophore generated from all docked poses, set against a representative pose of ceftriaxone. The radius of the sphere indicates the variance of a given feature across all poses. Key: cefoperazone (CFP), ceftaroline (CPT), ceftobiprole (BPR), ceftriaxone (CRO, ceftizoximine (ZOX), cefotaxime (CTX), ceftazidime (CAZ), cefixime (CFM), cefepime (FEP), cefpodoxime (CPD), ceftolozane (TOL), cefdinir (CDR), cefaclor (CEC), ceftibuten (CTB), cefuroxime (CXM), cefmetazole (CMZ), cefoxitin (FOX), cefsulodin (CFS), cephalexin (LEX), cefazolin (CFZ), cephalothin (LOT), cephaloridine (LOR).

Figure 3:. Positioning of the R₁ and R₂ groups of ceftriaxone within the active site of tPBP2^H041.

A. The thiotriazinone (TTZ) R₂ of ceftriaxone (CRO, orange) makes contact with K361 (yellow) in a majority of docked poses. The predicted hydrogen bond between TZZ and Lys361 is shown with a dashed line. B. The ATAO R₁ group of CRO (orange) adopts two conformations in the docked poses. In each conformation, R₁ is in close proximity to either Y422 or Y544 (both colored yellow).

Figure 4:. Inspection of individual docked poses reveals two distinct predicted binding modes, representative poses of which are shown in Figure 5 and Figure S2.

A. Pie chart showing the relative abundance of the two predicted binding modes. B. Predicted binding mode abundance stratified by second-order acylation rate constant. C. Pooled energy scores for poses in each of the two predicted binding modes.

Figure 5:. Representative poses of ceftriaxone and cefoperazone.

A. In the major pose predicted by the pharmacophore-constrained docking protocol, the C4 carboxylate of ceftriaxone interacts with S483, its R₂ side chain interacts with K361, and the β-lactam ring system is not optimally oriented for nucleophilic attack by S310. B. In the minor pose, the C4 carboxylate interacts with the side chains of K313 and S310, and the β-lactam ring is available for attack by S310. C. Cefoperazone in the major pose. D. CFP in the minor pose. For all panels, ceftriaxone (CRO) and cefoperazone (CFP) are shown with orange bonds, and potential hydrogen bonds made involving the antibiotic are indicated by dashed lines. *Nucleophilic Oɣ of S310.

Figure 6:. Evaluation of decreasing doses of cefoperazone given three-times daily (TID) on one day in a murine model of gonococcal infection.

Mice were infected with N. gonorrhoeae strain H041 two days prior to treatment. Treatments began on day 0 and were cefoperazone (CFP), ceftriaxone (CRO) (comparator control), water (vehicle control), or gentamicin (GEN) (positive control). Each color represents a given treatment group as described in the legend. A. Percent of infected mice following administration of CFP. Differences were analyzed by the Log-rank (Mantel-Cox) test comparing all treatment and control groups. Mice that were culture negative (no H041 recovered) for 3 or more days were considered to have cleared infection. A significant Log-rank test for trend of CFP treatments indicated a dose response was observed. B. Bacterial burden recovered from each experimental group following administration of CFP. Data points are the average CFU/mL of vaginal swab suspension (log transformed) recovered from each group. Error bars indicate the standard error of the mean. The limit of detection (20 CFU) is denoted with a horizontal dashed line. For mice that had no recoverable CFU, a value of 20 was entered for analysis. Differences were compared by 2-way ANOVA with repeated measures with Bonferroni post-hoc analysis used for multiple comparisons.