Structural analysis of the boronic acid β-lactamase inhibitor vaborbactam binding to Pseudomonas aeruginosa penicillin-binding protein 3

Abstract

Antimicrobial resistance (AMR) mediated by β-lactamases is the major and leading cause of resistance to penicillins and cephalosporins among Gram-negative bacteria. 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. Developing novel PBP inhibitors with a non-β-lactam scaffold could potentially evade this resistance mechanism. 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. 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°C. Crystallographic analysis of the PBP3:vaborbactam complex reveals that vaborbactam forms a covalent bond with the catalytic S294. The amide moiety of vaborbactam hydrogen bonds with N351 and the backbone oxygen of T487. The carboxyl group of vaborbactam hydrogen bonds with T487, S485, and S349. The thiophene ring and cyclic boronate ring of vaborbactam form hydrophobic interactions, including with V333 and Y503. 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. 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.

Fig 1. Chemical structures of vaborbactam and β-lactams, a monobactam and a DBO inhibitor.

Vaborbactam, ceftazidime, cefoperazone, meropenem, aztreonam, and zidebactam are represented. The R₁-and R₂-groups for ceftazidime are indicated.

Fig 2. Bocillin™ competition assay probing inhibition of P. aeruginosa PBP3 by vaborbactam.

SDS PAGE gel fluorescence scan of PBP3 reacted with the fluorescent Bocillin^TM reporter β-lactam in the presence of varying concentrations of the vaborbactam inhibitor.

Fig 3. Differential Scanning Fluorimetry (DSF)/thermal shift assay probing protein stabilization of P. aeruginosa PBP3 by vaborbactam.

The rate of change in SYPRO orange fluorescence is plotted versus temperature for PBP3 in the presence of varying concentrations of vaborbactam. Duplicate measured T_m values are as follows: PBP3 (with DMSO): 47.2 and 47.8°C; PBP3 with 0.5 mM vaborbactam (49.0 and 48.7°C), with 1.0 mM vaborbactam (49.0 and 49.0°C), and 2 mM vaborbactam (49.6 and 49.9°C).

Fig 4. Structure and electron density of vaborbactam bound covalently in the active site of P. aeruginosa PBP3.

A, simplified diagram of the crystal structure of PBP3 complexed to vaborbactam. The catalytic domain is colored grey; the N-terminal domain is in green. Vaborbactam is depicted in spheres with the carbon atoms in cyan. The catalytic S294 to which vaborbactam is covalently bonded is shown in stick model (labeled ‘*’). The N- and C-termini are labeled, and the residue numbers at the ends of the large missing region in the N-terminal domain are labeled. B, Unbiased omit Fo-Fc difference density is obtained after removing vaborbactam from the model and subsequently performing 10 cycles of crystallographic refinement before map calculation. Vaborbactam is shown in a stick model with cyan carbon atoms. The difference density is contoured at the 2.75 σ level. C, same as B, but the view is rotated about 90°.

Fig 5. Figure showing vaborbactam binding in the active site of P. aeruginosa PBP3.

A, Close-up view of vaborbactam in the PBP3 active site. Hydrogen bonds are depicted as dashed lines. Atom coloring is as in Fig 4. The two conformations of the aminothiazole containing moiety of vaborbactam are indicated by labels ‘a’ and ‘b’ near their respective sulfur atoms. A crystallographically observed water molecule interacting with vaborbactam is shown as a red sphere (labeled W#1). B, same as A but view is rotated about 90°.

Fig 6. Comparisons of vaborbactam-bound PBP3 with the P. aeruginosa PBP3-β-lactam, monobactam, and DBO complexes.

A, vaborbactam-bound PBP3 structure with ligand in ball-and-stick representation with cyan-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. B, ceftazidime-bound PBP3 (PDBid 3PBO, [30]; ceftazidime is shown with green-colored carbon atoms). The PBP3 superimposition with the vaborbactam PBP3 structure resulted in a RMSD of 0.45 Å for 473 Cα 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). C, cefoperazone-bound PBP3 (PDBid 5DF8, [42]; cefoperazone is shown with gold-colored carbon atoms). The PBP3 superimposition with the vaborbactam PBP3 structure resulted in a RMSD of 1.76 Å for 473 Cα atoms. Moieties, if present as in A, are labeled. D, meropenem-bound PBP3 (PDBid 3PBR, [30]; meropenem is shown with dark green-colored carbon atoms). The PBP3 superimposition with the vaborbactam PBP3 structure resulted in a RMSD of 0.78 Å for 473 Cα atoms. Moieties, if present as in A, are labeled. E, aztreonam-bound PBP3 (PDBid 3PBS, [30]; aztreonam is shown with pink-colored carbon atoms). The PBP3 superimposition with the vaborbactam PBP3 structure resulted in a RMSD of 0.66 Å for 473 Cα atoms. Moieties, if present as in B, are labeled with b now indicating the sulfo moiety. F, zidebactam-bound PBP3 (PDBid 7KIW, [18]; zidebactam is shown with teal-colored carbon atoms). Moieties, if present as in E, are labeled.

Fig 7. Superimposition of vaborbactam-bound PBP3 onto KPC-2 β-lactamase bound vaborbactam structure.

The colors and representation of PBP3 and its bound vaborbactam are as in Fig 6A. KPC-2 (PDBid 6V7I is shown in green backbone trace and green carbon atoms; its bound vaborbactam is depicted in ball-and-stick with green carbon atoms. Shifts in the β3 and β4 strands are indicated by arrows. The active site Cα atoms of PBP3 residues 294–301, 346–353, 481–486, and 505–509, were superimpositioned onto their equivalent atoms in KPC-2 (residues 70–77, 127–134, 231–236, and 244–248) yielding an RMSD of 0.97 Å for 27 Cα atoms.

Fig 8. Vaborbactam interactions with the active site of PBP3 during MD simulations.

A, Vaborbactam PBP3 active site interactions during MD simulations with K297 in the protonated state analyzed using the Schrodinger Simulation Interactions Diagram program. Hydrogen bonds with side chains (pink dashed arrow) and main chains (pink solid arrow), and salt bridge interactions (purple-pink line) are indicated, and their percentage present during the simulation is shown. B, same as A but with K297 kept neutral during the simulation.