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The electrostatic potential map of the PBP2 active site is shown as generated using APBS in PyMOL. This modeling and superpositioning were done using COOT. 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). Experiments were performed in duplicate (a representative curve is depicted). The derivative of the change in fluorescence is plotted versus temperature.
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(A) DSF thermal shift assay of WCK 5153 and zidebactam binding to PBP2. Pseudomonas aeruginosa antibiotic resistance penicillin-binding proteins structural biology.ĭifferential scanning fluorimetry (DSF) measurement of WCK 5153 and zidebactam binding to PBP2 and modeling of zidebactam. aeruginosa PBP2 to be inhibited by β-lactam antibiotics, and provide insights that could be used for further antibiotic development. These structures and analyses help define the antibiotic properties of these inhibitors, explain the decreased susceptibility of P. 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.
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Diazabicyclooctane inhibitors such as zidebactam possess this potential as they readily inactivate penicillin-binding proteins, yet cannot be degraded by β-lactamases. Developing novel antibiotics that overcome known resistance mechanisms is highly desired. IMPORTANCE Antibiotic resistance is a significant clinical problem. 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. 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. Even though the DBOs show covalent binding to PBP3, they destabilized PBP3. aeruginosa PBP3 was explored crystallographically. To aid in the design of DBOs that can inhibit multiple PBPs, the ability of three DBOs to interact with P. Both DBOs increase the melting temperature of PBP2, affirming their stabilizing interactions. Modeling of zidebactam in the active site of PBP2 reveals a similar binding mode. 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. WCK 5153 forms an inhibitory covalent bond with the catalytic S327 of PBP2. aeruginosa PBP2 in complex with WCK 5153. aeruginosa PBP2 is less susceptible to inhibition by β-lactam antibiotics compared to the Escherichia coli PBP2, we determined the crystal structure of P. To structurally probe their mode of PBP2 inhibition as well as investigate why P. 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. A major mechanism of resistance expressed by MDR pathogens is β-lactamase-mediated degradation of β-lactam antibiotics. This tight regulatory mechanism is consistent with the cell’s need to ensure appropriate use of the limited pool of lipid II.Multidrug-resistant (MDR) pathogens pose a significant public health threat.
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All together the results suggest that FtsW interacts with lipid II preventing its polymerization by PBP1b unless PBP3 is also present, indicating that PBP3 facilitates lipid II release and/or its transfer to PBP1b after transport across the cytoplasmic membrane. Moreover, we found that FtsW, but not the other flippase candidate MurJ, impairs lipid II polymerization and peptide cross-linking activities of PBP1b, and that PBP3 relieves these inhibitory effects. We also show that the large loop between transmembrane helices 7 and 8 of FtsW is important for the interaction with PBP3. We show that FtsW interacts with PBP1b and lipid II and that PBP1b, FtsW and PBP3 co-purify suggesting that they form a trimeric complex. Yet, the exact molecular mechanisms of their function in complexes are largely unknown. coli, the lipid II transporter candidate FtsW is thought to work in concert with the PG synthases penicillin-binding proteins PBP3 and PBP1b. The divisome controls septal PG synthesis and separation of daughter cells. Bacteria utilize specialized multi-protein machineries to synthesize the essential peptidoglycan (PG) cell wall during growth and division.