Ureidopenicillins Are Potent Inhibitors of Penicillin-Binding Protein 2 from Multidrug-Resistant Neisseria gonorrhoeae H041

Abstract

Effective treatment of gonorrhea is threatened by the increasing prevalence of Neisseria gonorrhoeae strains resistant to the extended-spectrum cephalosporins (ESCs). Recently, we demonstrated the promise of the third-generation cephalosporin cefoperazone as an antigonococcal agent due to its rapid second-order rate of acylation against penicillin-binding protein 2 (PBP2) from the ESC-resistant strain H041 and robust antimicrobial activity against H041. Noting the presence of a ureido moiety in cefoperazone, we evaluated a subset of structurally similar ureido β-lactams, including piperacillin, azlocillin, and mezlocillin, for activity against PBP2 from H041 using biochemical and structural analyses. We found that the ureidopenicillin piperacillin has a second-order rate of acylation against PBP2 that is 12-fold higher than cefoperazone and 85-fold higher than ceftriaxone and a lower MIC against H041 than ceftriaxone. Surprisingly, the affinity of ureidopenicillins for PBP2 is minimal, indicating that their inhibitory potency is due to a higher rate of the acylation step of the reaction compared to cephalosporins. Enhanced acylation results from the combination of a penam scaffold with a 2,3-dioxopiperazine-containing R₁ group. Crystal structures show that the ureido β-lactams overcome the effects of resistance mutations present in PBP2 from H041 by eliciting conformational changes that are hindered when PBP2 interacts with the weaker inhibitor ceftriaxone. Overall, our results support the potential of piperacillin as a treatment for gonorrhea and provide a framework for the future design of β-lactams with improved activity against ESC-resistant N. gonorrhoeae.

Keywords: Neisseria gonorrhoeae; antimicrobial resistance; penicillin-binding protein 2; β-lactams.

Fig. 1:. Structures of cephalosporins and ureido β-lactams examined in this study.

Red indicates the ureido moiety present in cefoperazone and the ureido β-lactams, piperacillin, azlocillin and azlocillin.

Fig. 2:. Binding affinity of ureido β-lactams for tPBP2^H041-S310A.

Binding affinity (K_S) of each ureido β-lactam was determined using isothermal titration calorimetry. A) Heat evolution upon titration of 30 μM tPBP2^H041-S310A with a 500 μM stock of cefoperazone in 2.7 μl increments. B) Fitting of cefoperazone titration data by nonlinear regression to a single-site isotherm. C) Heat evolution resulting from titration of 30 μM of tPBP2^H041-S310A with 500 μM piperacillin. D) The piperacillin titration data could not be fit to a single-site isotherm model due to undetectable binding. The same was true for azlocillin (not shown). A minimum of three experiments were performed for each compound and representative plots for each are shown.

Fig. 3:. The crystal structures of tPBP2^H041 in complex with ureido β-lactams.

A) Complex with cefoperazone (CFP), where CFP is orange and tPBP2^H041 is yellow. B) Complex with piperacillin (PPC), where PPC is blue and tPBP2^H041 is cyan. C) Complex with azlocillin (AZC), where AZC is dark green and tPBP2^H041 is green. For all panels, only the active-site region is shown, where the protein rendered in ribbon format and secondary structure elements of PBP2 are labelled. |F_o|-|F_c| unbiased electron density corresponding to each ureido β-lactam is shown as a blue mesh and contoured at 3.0 sigma. Interacting residues are labeled and potential hydrogen bonds are shown as dashed lines. Waters are depicted as red spheres. Insets highlight interactions involving the β-lactam carboxylate and the side chain of threonines 498 and 500 on β3.

Fig. 4:. Superimposition of acylated and apo structures of tPBP2^H041.

The active site region of crystal structures of tPBP2^H041 acylated by cefoperazone (orange), piperacillin (blue), and azlocillin (green) is shown. The apo structure of tPBP2^H041 is shown in grey. Dashed lines in corresponding colors represent potential hydrogen bonds in each structure. Note the similarity in binding mode for the three ureido β-lactams, including high degree of overlap among R₁ groups. The inward position of the β3-β4 loop in the acylated structures in contrast to its outbent conformation in apo tPBP2^H041. The positions of the β3-β4 loop mutations F504L and N512Y are marked as black spheres.

Fig. 5:. Interactions made by the β-lactam carboxylate and threonines 498 and 500 in acylated structures of tPBP2^H041 and tPBP2^FA19.

A) The structure of tPBP2^H041 (yellow) acylated by cefoperazone (CFP, orange), where the carboxylate group of CFP interacts with T498 and T500 on β3, and S545 is rotated away from T498 to form a polar contact with the backbone carbonyl of T517 (arrow). B) The structure of tPBP2^FA19 (light grey) in complex with ceftriaxone (CRO, grey), where the carboxylate has similar positioning as for tPBP2^H041 with CFP and forms a hydrogen bond with T498. Note how in PBP2 from H041, Gly545 is mutated to Ser. C) The structure of tPBP2^H041 (light green) in complex with CRO (green) in which T498 is rotated away from the active site to hydrogen bond with S545 and no longer interacts with the β-lactam carboxylate. D) Overlay of the structures of tPBP2^H041 acylated by CFP (yellow), piperacillin (PPC, blue) and azlocillin (AZC, green) showing the interactions of T498 and T500 with the β-lactam carboxylates in all three structures. For all panels, PBP2 is shown in ribbon format, and the antibiotics, together with the side chains of interacting residues, are shown as bonds. Potential hydrogen bonds are represented by dashed lines.

Fig. 6:. Cefoperazone adopts a “susceptible” mode of binding in tPBP2^H041 compared to ceftriaxone.

A) Overlay of tPBP2^FA19 acylated by ceftriaxone (CRO, grey) with tPBP2^H041 acylated by cefoperazone (CFP, yellow/orange) showing rotation of T498 to contact the β-lactam carboxylate and the inward conformation of the β3-β4 loop when tPBP2^H041 is acylated by CFP. B) Overlay of tPBP2^H041 acylated by cefoperazone (yellow/orange) and tPBP2^H041 acylated by ceftriaxone (green), showing the “resistant” binding mode of the latter.

Fig. 7:. Relative positioning of the R₁ groups of ceftriaxone and cefoperazone in the structures of tPBP2^FA19 and tPBP2^H041 relative to Y422 and Y509.

A) In the structure of tPBP2^FA19 acylated by ceftriaxone (CRO), the 2-aminothiazole ring of the R₁ group shows staggered π-π stacking with Y422, where the aminothiazole is oriented below Y422. The latter, in turn, forms perpendicular π-π stacking with Y509. B) For tPBP2^H041 in complex with cefoperazone (CFP), an opposite arrangement is observed, where the 2,3-dioxopiperazine group of CFP stacks above Y422 and the ethylene group of the 2,3-dioxoopiperazine group forms aliphatic CH-π interactions with Y509. C) In tPBP2^H041 acylated by CRO, Y422 is below the 2-aminothiazole, but the relative positions of the two moieties are suboptimal for stacking. Note the very different position of Y509 in this structure due to the “outbent” conformation of the β3-β4 loop, compared to when the loop is inward (gray) (distance between the two positions of Tyr509 is indicated by a dashed line).