Influence of the α-Methoxy Group on the Reaction of Temocillin with Pseudomonas aeruginosa PBP3 and CTX-M-14 β-Lactamase

Abstract

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. Temocillin is the 6-α-methoxy analogue of ticarcillin, a carboxypenicillin with well-characterized antipseudomonal properties. The α-methoxy modification confers resistance to serine β-lactamases, yet temocillin is ineffective against P. aeruginosa growth. The origins of temocillin’s inferior antibacterial properties against P. aeruginosa have remained relatively unexplored. 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. We show that the 6-α-methoxy group perturbs the stability of the PBP3 acyl-enzyme, which manifests in an elevated off-rate constant (koff) in biochemical assays comparing temocillin with ticarcillin. Complex crystal structures with PBP3 reveal similar binding modes of the two drugs but with important differences. 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. This hypothesis is supported by the observation that the acyl-enzyme complex of temocillin has reduced thermal stability compared with ticarcillin. 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. 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.

Reduced In Vitro Susceptibility of Streptococcus pyogenes to β-Lactam Antibiotics Associated with Mutations in the pbp2x Gene Is Geographically Widespread

“…The structure of this protein has been well studied by several investigators because of its importance in betalactam resistance in this common human pathogen(12)(13)(14)16). The variant amino acids at positions 342, 599, 600, and 601 mapped to regions known to influence structure-function relationships(20,21). This determination of influence was derived from overlaying PBP2X from S. pneumoniae with the clinically relevant and structurally similar P. aeruginosa PBP3 (PDB: 6UN3) and S. aureus PBP2a (PDB: 1VQQ, chain A).…”

High-Throughput Crystallography Reveals Boron-Containing Inhibitors of a Penicillin-Binding Protein with Di- and Tricovalent Binding Modes

“…Examination of the reported PaPBP3 structures ,,− reveals the β5-α11 loop (residues 528–539, Figure S10) adopts different conformations. The β-lactam-containing PaPBP3 inhibitors (e.g., amoxicillin, aztreonam, ceftazidime, and piperacillin , ) induce formation of a “hydrophobic wall” composed of Tyr533, Phe532, and residues within the β5-α11 loop together with Tyr503 .…”

“…Inhibition plots with meropenem and ceftazidime were done to ensure the correct parameters for BOCILLIN FL binding were used. Progress curves were analyzed with KinTek, USA, as described; , reported errors represent the standard error. The acylation of a PBP by BOCILLIN FL (Figure S1) can be described by a simple one-step model .…”

Drug Discovery in the Field of β-Lactams: An Academic Perspective

“…For many bacteria, the value of each PBP for drug discovery is only starting to be unraveled. For example, some studies have identified PBP3 as an essential transpeptidase for P. aeruginosa growth and therefore a key β-lactam target (Figure 4) [171][172][173], while PBP2 may also represent a good therapeutic target for novel antibiotic discovery [128]. Other researchers used Bacillus subtilis cells to visualize and study PBP inhibition profiles of β-lactam antibiotics in live cells, which allowed the determination of PBPs essential for the growth of the microorganism and their roles in antibiotic resistance [174].…”