Biochemical and biophysical characterization of the OXA-48-like carbapenemase OXA-436.

The crystal structure of the class D ß-lactamase OXA-436 was solved to a resolution of 1.80 Å. Higher catalytic rates were found at higher temperatures for the clinically important antibiotic imipenem, indicating better adaptation of OXA-436 to its mesophilic host than OXA-48, which is believed to originate from an environmental source. Furthermore, based on the most populated conformations during 100 ns molecular-dynamics simulations, it is postulated that the modulation of activity involves conformational shifts of the α3-α4 and ß5-ß6 loops. While these changes overall do not cause clinically significant shifts in the resistance profile, they show that antibiotic-resistance enzymes exist in a continuum. It is believed that these seemingly neutral differences in the sequence exist on a path leading to significant changes in substrate selectivity.

ZN148 Is a Modular Synthetic Metallo-ß-Lactamase Inhibitor That Reverses Carbapenem Resistance in Gram-Negative Pathogens In Vivo.

Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (ß-lactamases able to inactivate carbapenems) have been identified in both serine ß-lactamase (SBL) and metallo-ß-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 µM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

The biological assembly of OXA-48 reveals a dimer interface with high charge complementarity and very high affinity.

Many class D ß-lactamases form dimers in solution. The functional basis of the dimerization of OXA-48-like class D ß-lactamases is not known, but in order to understand the structural requirements for dimerization of OXA-48, we have characterized the dimer interface. Size exclusion chromatography, small angle X-ray scattering (SAXS), and nuclear magnetic resonance (NMR) were used to confirm the oligomeric state of OXA-48 in solution. X-ray crystallographic structures were used to elucidate the key interactions of dimerization. In silico residue scanning combined with site-directed mutagenesis was used to probe hot spots of dimerization. The affinity of dimerization was quantified using microscale thermophoresis, and the overall thermostability was investigated using differential scanning calorimetry. OXA-48 was consistently found to be a dimer in solution regardless of the method used, and the biological assembly found from the SAXS envelope is consistent with the dimer identified from the crystal structures. The buried chloride that interacts with Arg206 and Arg206' at the dimer interface was found to enhance the thermal stability by > 4 °C and crystal structures and mutations (R189A, R189A/R206A) identified several additional important ionic interactions. The affinity for OXA-48 R206A dimerization was in the picomolar range, thus revealing very high dimer affinity. In summary, OXA-48 has a very stable dimer interface, facilitated by noncovalent and predominantly charged interactions, which is stronger than the dimer interfaces previously described for other class D ß-lactamases. PDB CODES: The oxacillinase-48 (OXA-48) R206A structure has PDB ID: 5OFT and OXA-48 R189A has PDB ID: 6GOA.

Structure, activity and thermostability investigations of OXA-163, OXA-181 and OXA-245 using biochemical analysis, crystal structures and differential scanning calorimetry analysis.

The first crystal structures of the class D ß-lactamases OXA-181 and OXA-245 were determined to 2.05 and 2.20 Šresolution, respectively; in addition, the structure of a new crystal form of OXA-163 was resolved to 2.07 Šresolution. All of these enzymes are OXA-48-like and have been isolated from different clinical Klebsiella pneumoniae strains and also from other human pathogens such as Pseudomonas aeruginosa and Escherichia coli. Here, enzyme kinetics and thermostability studies are presented, and the new crystal structures are used to explain the observed variations. OXA-245 had the highest melting point (Tm = 55.8°C), as determined by differential scanning calorimetry, compared with OXA-163 (Tm = 49.4°C) and OXA-181 (Tm = 52.6°C). The differences could be explained by the loss of two salt bridges in OXA-163, and an overall decrease in the polarity of the surface of OXA-181 compared with OXA-245.

Role of Residues W228 and Y233 in the Structure and Activity of Metallo-ß-Lactamase GIM-1.

Metallo-ß-lactamases (MBLs) hydrolyze virtually all ß-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative ß-lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with ß-lactam substrates were modeled in silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the ß-lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1.

Discovery of Novel Inhibitor Scaffolds against the Metallo-ß-lactamase VIM-2 by Surface Plasmon Resonance (SPR) Based Fragment Screening.

Metallo-ß-lactamase (MBL) inhibitors can restore the function of carbapenem antibiotics and therefore help to treat infections of antibiotic resistant bacteria. In this study, we report novel fragments inhibiting the clinically relevant MBL Verona integron-encoded metallo-ß-lactamase (VIM-2). The fragments were identified from a library of 490 fragments using an orthogonal screening approach based on a surface plasmon resonance (SPR) based assay combined with an enzyme inhibition assay. The identified fragments showed IC50 values between 14 and 1500 µM and ligand efficiencies (LE) between 0.48 and 0.23 kcal/mol per heavy atom. For two of the identified fragments, crystal structures in complex with VIM-2 were obtained. The identified fragments represent novel inhibitor scaffolds and are good starting points for the design of potent MBL inhibitors. Furthermore, the established SPR based assay and the screening approach can be adapted to other MBLs and in this way improve the drug discovery process for this important class of drug targets.