Crystal structure of the class A extended-spectrum beta-lactamase CTX-M-96 in complex with relebactam at 1.03 Angstrom resolution (Comunicación Oral)

BÁRBARA GHIGLIONE; MARÍA M. RODRÍGUEZ; ANNETTE CANCIO-BELLO GONZÁLEZ; PEDRO PENZOTTI; GABRIEL GUTKIND; ROBERT A. BONOMO; SEBASTIÁN KLINKE; PABLO POWER

Atlanta

Congreso; ASM Microbe 2023; 2023

American Society for Microbiology

The use of beta-lactam/beta-lactamase inhibitors (BL-BLI) constitutes one strategy to counteract beta-lactamases in MDR bacteria. Recent reports described ceftazidime/avibactam (CZA) resistant isolates producing CTX-M variants including different substitutions (e.g. P167S, L169Q, S130G). Relebactam (REL) proved efficacy against Enterobacteriaceae expressing ESBLs, serine-carbapenemases and AmpCs when combined with imipenem. The objective of this work was to obtain a structural complex and evaluate the inhibitory efficacy of relebactam (REL) against CTX-M-96, a CTX-M-15-type variant. Steady-state kinetic parameters and inhibition constants were determined on pure CTX-M-96. Crystals of apo CTX-M-96 were grown by the hanging drop vapor diffusion method. The REL adduct was obtained by soaking. Native X-ray diffraction data were collected at 100 K at the PROXIMA 2A beamline at the SOLEIL Synchrotron, France. Structure determination was achieved by molecular replacement through Phaser using the apo CTX-M-96 structure (PDB 3ZNY). Refinement and model building were performed with Phenix and Coot. Models were visualized with PyMOL.The CTX-M-96 structure was obtained in complex with REL at 1.03 Å resolution (PDB 8EHH). REL is covalently bound to the S70-O-gamma atom upon cleavage of the C7-N6 bond. Compared to apo CTX-M-96, binding of REL forces a slight displacement of the deacylating water inwards the active site (0.98 Å), making E166 and N170 side chains shift. REL displaces the Y105 ring 0.8 Å outwards the active site core which provokes a shift of other residues in the hydrophobic patch, including P107 and Y129. A significant disturbance of  about 4 Å of the N104 side chain outwards the active site core was observed due to the presence of the terminal piperidine ring of REL. As in other CTX-M/AVI complexes, the side chain of K73 points towards S130 instead of E166 and S70 as observed in the apo structures, likely supporting the role of K73 in the proton-relay pathway in beta-lactams catalysis, and of E166 as the general base in the deacylation step upon protonation and activation of the deacylating water molecule. Our data supports REL effectiveness against CTX-M-producing Gram-negative pathogens. Binding of REL disturbs the hydrophobic patch formed by Y105, P107 and Y129, likely due to the piperidine ring of REL that creates clashes with these residues. Also, a remarkable change in the positioning of the N104 side chain is also affected by the piperidine ring. Imipenem/REL remains a choice for dealing with MDR isolates co-producing CTX-M with other beta-lactamases.