The PBP3 superimposition with the vaborbactam PBP3 structure resulted in a RMSD of 0.78 Å for 473 Cα atoms. D, meropenem-bound PBP3 (PDBid 3PBR, meropenem is shown with dark green-colored carbon atoms). Moieties, if present as in A, are labeled. The PBP3 superimposition with the vaborbactam PBP3 structure resulted in a RMSD of 1.76 Å for 473 Cα atoms. C, cefoperazone-bound PBP3 (PDBid 5DF8, cefoperazone is shown with gold-colored carbon atoms). In addition to the same moieties a- c as in A, also labeled are the five-membered aminothiazole ring ( d), and ceftazidime’s 2-carboxypropan-2-yl group ( e). The PBP3 superimposition with the vaborbactam PBP3 structure resulted in a RMSD of 0.45 Å for 473 Cα atoms. B, ceftazidime-bound PBP3 (PDBid 3PBO, ceftazidime is shown with green-colored carbon atoms).
The ligand’s oxygen located in the oxyanion hole ( a), the carboxyl ( b), the amide ( c), and the five-membered thiophene ( d) are labeled. These insights could form the basis for developing more potent novel cyclic boronate-based PBP inhibitors to inhibit these targets and overcome β-lactamases-mediated resistance mechanisms.Ī, vaborbactam-bound PBP3 structure with ligand in ball-and-stick representation with cyan-colored carbon atoms. The active site of the vaborbactam-bound PBP3 harbors the often observed ligand-induced formation of the aromatic wall and hydrophobic bridge, yet the residues involved in this wall and bridge display much higher temperature factors compared to PBP3 structures bound to high-affinity β-lactams. The thiophene ring and cyclic boronate ring of vaborbactam form hydrophobic interactions, including with V333 and Y503. The carboxyl group of vaborbactam hydrogen bonds with T487, S485, and S349. The amide moiety of vaborbactam hydrogen bonds with N351 and the backbone oxygen of T487. Crystallographic analysis of the PBP3:vaborbactam complex reveals that vaborbactam forms a covalent bond with the catalytic S294. We found that this cyclic boronate, vaborbactam, inhibited PBP3 (IC50 of 262 μM), and its binding to PBP3 increased the protein thermal stability by about 2☌.
Based on the structural similarities between the evolutionary related serine β-lactamases and PBPs, we investigated whether the potent β-lactamase inhibitor, vaborbactam, could also form an acyl-enzyme complex with Pseudomonas aeruginosa PBP3. Developing novel PBP inhibitors with a non-β-lactam scaffold could potentially evade this resistance mechanism. β-Lactamases, periplasmic enzymes that are widely distributed in the bacterial world, protect penicillin-binding proteins (PBPs), the major cell wall synthesizing enzymes, from inactivation by β-lactam antibiotics. The results suggest that the α-methoxy group prevents hydrolysis by locking the compound into an unexpected conformation that impedes access of the catalytic water to the acyl-enzyme adduct.Antimicrobial resistance (AMR) mediated by β-lactamases is the major and leading cause of resistance to penicillins and cephalosporins among Gram-negative bacteria. Furthermore, we explore temocillin's mechanism of β-lactamase inhibition with a high-resolution complex structure of CTX-M-14 class A serine β-lactamase. This hypothesis is supported by the observation that the acyl-enzyme complex of temocillin has reduced thermal stability compared with ticarcillin. Most notably, the 6-α-methoxy group disrupts a high-quality hydrogen bond with a conserved residue important for ligand binding while also being inserted into a crowded active site, possibly destabilizing the active site and enabling water molecule from bulk solvent to access and cleave the acyl-enzyme bond. Complex crystal structures with PBP3 reveal similar binding modes of the two drugs but with important differences. aeruginosa We show that the 6-α-methoxy group perturbs the stability of the PBP3 acyl-enzyme, which manifests in an elevated off-rate constant ( k off ) in biochemical assays comparing temocillin with ticarcillin. Here, we analyze the reaction kinetics, protein stability, and binding conformations of temocillin and ticarcillin with penicillin-binding protein 3 (PBP3), an essential PBP in P. aeruginosa have remained relatively unexplored. The origins of temocillin's inferior antibacterial properties against P. The α-methoxy modification confers resistance to serine β-lactamases, yet temocillin is ineffective against P. Temocillin is the 6-α-methoxy analogue of ticarcillin, a carboxypenicillin with well-characterized antipseudomonal properties. The prevalence of multidrug-resistant Pseudomonas aeruginosa has led to the reexamination of older "forgotten" drugs, such as temocillin, for their ability to combat resistant microbes.