Structural Characterization of Diazabicyclooctane β-Lactam "Enhancers" in Complex with Penicillin-Binding Proteins PBP2 and PBP3 of Pseudomonas aeruginosa

Abstract

Multidrug-resistant (MDR) pathogens pose a significant public health threat. A major mechanism of resistance expressed by MDR pathogens is β-lactamase-mediated degradation of β-lactam antibiotics. The diazabicyclooctane (DBO) compounds zidebactam and WCK 5153, recognized as β-lactam "enhancers" due to inhibition of Pseudomonas aeruginosa penicillin-binding protein 2 (PBP2), are also class A and C β-lactamase inhibitors. To structurally probe their mode of PBP2 inhibition as well as investigate why P. aeruginosa PBP2 is less susceptible to inhibition by β-lactam antibiotics compared to the Escherichia coli PBP2, we determined the crystal structure of P. aeruginosa PBP2 in complex with WCK 5153. WCK 5153 forms an inhibitory covalent bond with the catalytic S327 of PBP2. The structure suggests a significant role for the diacylhydrazide moiety of WCK 5153 in interacting with the aspartate in the S-X-N/D PBP motif. Modeling of zidebactam in the active site of PBP2 reveals a similar binding mode. Both DBOs increase the melting temperature of PBP2, affirming their stabilizing interactions. To aid in the design of DBOs that can inhibit multiple PBPs, the ability of three DBOs to interact with P. aeruginosa PBP3 was explored crystallographically. Even though the DBOs show covalent binding to PBP3, they destabilized PBP3. Overall, the studies provide insights into zidebactam and WCK 5153 inhibition of PBP2 compared to their inhibition of PBP3 and the evolutionarily related KPC-2 β-lactamase. These molecular insights into the dual-target DBOs advance our knowledge regarding further DBO optimization efforts to develop novel potent β-lactamase-resistant, non-β-lactam PBP inhibitors.IMPORTANCE Antibiotic resistance is a significant clinical problem. Developing novel antibiotics that overcome known resistance mechanisms is highly desired. Diazabicyclooctane inhibitors such as zidebactam possess this potential as they readily inactivate penicillin-binding proteins, yet cannot be degraded by β-lactamases. In this study, we characterized the inhibition by diazabicyclooctanes of penicillin-binding proteins PBP2 and PBP3 from Pseudomonas aeruginosa using protein crystallography and biophysical analyses. These structures and analyses help define the antibiotic properties of these inhibitors, explain the decreased susceptibility of P. aeruginosa PBP2 to be inhibited by β-lactam antibiotics, and provide insights that could be used for further antibiotic development.

Keywords: Pseudomonas aeruginosa; antibiotic resistance; penicillin-binding proteins; structural biology.

FIG 1

Structures of DBO inhibitors and P. aeruginosa PBP2. (A) Chemical structures of DBOs zidebactam, WCK 5153, WCK 4234, and avibactam. The R1-groups of the DBOs are shaded gray. (B) Co-crystal structure of P. aeruginosa PBP2 in complex with WCK 5153 (the latter is shown in stick representation with cyan-colored carbon atoms). The PBP2 anchor (red), head (blue), linker (green), and catalytic (orange) domains are labeled, as well as most of the secondary structure elements. (C) Superpositioning of the P. aeruginosa PBP2:WCK 5153 complex and E. coli PBP2 structure. The domain color coding for P. aeruginosa PBP2 is the same as in panel B with WCK 5153 depicted in spheres with cyan-colored carbon atoms. E. coli PBP2 is shown in similar but paler colors for its respective domains.

FIG 2

WCK 5153 bound in the PBP2 active site. (A) |Fo|-|Fc| electron density difference map contoured at 2.5 σ level. WCK 5153 is depicted with cyan-colored carbon atoms. The unbiased density map was calculated after removing the ligand from the coordinates and performing 10 cycles of crystallographic refinement using Refmac. (B) Interactions of WCK 5153 in the active site of PBP2. Hydrogen bonds are depicted as dashed lines, and distances are shown (in Å). The following moieties of WCK 5153 are labeled: carbonyl oxygen (1), sulfate (2), piperidine ring of the DBO scaffold (3), diacylhydrazide (4), and the pyrrolidine ring (5). WCK 5153 is covalently attached to the catalytic S327. Key secondary structure elements are labeled.

FIG 3

Superpositioning of P. aeruginosa PBP2:WCK 5153 complex with the E. coli PBP2:avibactam complex structure. The P. aeruginosa PBP2 structure (orange) and the E. coli PBP2 structure (gray, PDB ID 6G9F) are shown with their respective ligands shown in ball-and-stick representation. The large conformational difference of the N-terminal part of the helix α21 and connecting loop in the P. aeruginosa structure from that in the E. coli PBP2 structure is depicted by the blue arrow.

FIG 4

Superpositioning of WCK 5153 inhibited complexes of PBP2 and KPC-2 β-lactamase. PBP2 is depicted in orange and KPC-2 in green. The WCK 5153 molecules are shown in a ball-and-stick model; equivalent key active-site residues of PBP2 and KPC-2 are labeled and shown in stick model. The Ω-loop present in KPC-2 is indicated; this loop is not present in PBPs. Conformational shifts between the α20 and α21 helices in PBP2 and their respective corresponding helices in KPC-2 are indicated by purple arrows. The moieties of WCK 5153 are labeled as in Fig. 2B. The active site Cα atoms of PBP2 residues 325 to 334 (α12 helix region containing catalytic S327), 381 to 389 (α13 region), 535 to 540 (β18), and 566 to 570 (β19) were superpositioned onto their equivalent atoms of KPC-2 β-lactamase (residues 68 to 77, 127 to 135, 231 to 236, and 244 to 248, respectively), yielding an RMSD of 0.87 Å for 30 Cα atoms.

FIG 5

Differential scanning fluorimetry (DSF) measurement of WCK 5153 and zidebactam binding to PBP2 and modeling of zidebactam. (A) DSF thermal shift assay of WCK 5153 and zidebactam binding to PBP2. The derivative of the change in fluorescence is plotted versus temperature. Experiments were performed in duplicate (a representative curve is depicted). (B) Modeling of zidebactam in P. aeruginosa PBP2 active site. The coordinates of zidebactam were obtained by transplanting most of the atom coordinates from the similar WCK 5153 when bound to PBP2, yet with the pyrrolidine ring being replaced by the piperidine ring of zidebactam using the zidebactam piperidine conformation when complexed to KPC-2 (11). This modeling and superpositioning were done using COOT. The electrostatic potential map of the PBP2 active site is shown as generated using APBS in PyMOL. Zidebactam in shown in ball-and-stick representation, and the individual moieties are labeled as in Fig. 2B with the noted change that 5 now represents the (larger) piperidine ring of the R1-group.

FIG 6

Crystal structure of P. aeruginosa PBP3 in complex with WCK 4234. The noncatalytic N-terminal domain and catalytic TP domain are colored cyan and gray, respectively. Bound WCK 4234 is shown in spheres with green carbon atoms. Secondary structure elements near the active site are labeled (numbering as in reference 24).

FIG 7

Crystal structures of WCK 4234, zidebactam, and avibactam bound to P. aeruginosa PBP3. (A, C, and E) Electron difference density for WCK 4234, zidebactam, and avibactam, respectively, contoured at 2.75 σ level. (B, D, and F) Interactions of DBO ligands in the active site of PBP3 for WCK 4234, zidebactam, and avibactam, respectively.

FIG 8

Superposition of WCK 5153 inhibited complex of PBP2 with zidebactam complexed to PBP3. PBP2 and bound WCK 5153 are depicted in orange, and PBP3 is in gray with its bound zidebactam ligand in teal carbon atoms. The moieties of WCK 5153 and zidebactam are labeled as in Fig. 2B. The active site Cα atoms of PBP2 residues 325 to 333 (α12 helix region containing catalytic S327), 364 to 367 (includes β15), 378 to 387 (α13 region), 447 to 452 (α17), and 537 to 542 (β18) were superpositioned onto their equivalent atoms in PBP3 (residues 292 to 300, 330 to 333, 343 to 352, 404 to 409, and 483 to 488, respectively), yielding an RMSD of 0.81 Å for 35 Cα atoms.

FIG 9

DSF thermal shift measurement of DBOs binding to PBP3. (A) Zidebactam, WCK 5153, and WCK 4234 binding to PBP3 at the indicated concentrations. The compounds were dissolved in DMSO with a final DMSO concentration of 2% in the DSF experiment; a PBP3 2% DMSO control experiment is therefore also included. For comparison, ceftazidime is a positive control since it showed a large increase in T_m as previously published (29). Experiments were done in duplicate, and a representative curve is shown for each experiment. (B) Avibactam binding to PBP3. Data are plotted similarly as in Fig. 5. Avibactam was dissolved in water. Experiments were done in triplicate, and a representative curve is shown for each experiment.