Arg220 and diazabicyclooctane (DBO) inhibitors: understanding structure activity relationships (SAR) in the PER beta-lactamase, a unique class A enzyme (Póster)

MELINA RUGGIERO; KRISZTINA M. PAPP-WALLACE; FLORENCIA BRUNETTI; GABRIEL GUTKIND; SEBASTIÁN KLINKE; ROBERT A. BONOMO; PABLO POWER

Evento online debido a la pandemia de coronavirus

Congreso; ASM Microbe Online; 2020

American Society for Microbiology (ASM)

Background: DBO inhibitors demonstrate potent activity against serine-beta-lactamases. Relebactam (REL) and avibactam (AVI) effectively inhibit clinically-important beta-lactamases (e.g., TEM, CTX-M and KPC). PER beta-lactamases are unique among class A enzymes as they possess enhanced oxyimino-cephalosporinase activity. The presence of an inverted omega loop and enlarged beta-3 strand that expands the active site by 2-fold is believed to be responsible for this singular activity. Previously, it was observed that substitutions at Ambler position R220 in PER impact in the activity of mechanism-based inhibitors. Our goal was to provide structural and kinetic insights into the ability of REL (in comparison to AVI) to inhibit PER-2 beta-lactamase, and to evaluate the effect of the R220G substitution on the inhibition mechanism. Methods: Inhibition constants (Ki, k2/K) were determined using nitrocefin as reporter substrate. Crystals of apo PER-2 and PER-2/R220G, and the corresponding complexes with both DBOs were generated by the hanging drop vapor diffusion method at 20 degree. X-ray diffraction was measured at the Soleil synchrotron (France) under cryogenic conditions (100 K). Indexing, integration and scaling were performed with XDS, and the structure was solved by molecular replacement with Phaser under Phenix platform. Results: REL possessed a higher Ki than AVI for both PER-2 and PER-2/R220G (Ki = 5 vs. 20 micromolar; 21 vs 350 micromolar for REL and AVI, respectively), which correlated with k2/K determinations (11,200 vs. 2,200 M^(-1).s^(-1); and 3,100 vs 200 M^(-1).s^(-1), respectively). PER-2/R220G displayed up to 17-fold higher Ki and 4-11-fold lower k2/K values compared to PER-2 for both DBOs. Structures were obtained at high resolution (1.5-2.1 A; Rfree: 0.2034-0.2995). REL is oriented in equivalent position compared to CTX-M-15 and KPC-2 except for the piperidine ring that is rotated up to 90 degree likely due to the presence of H170. In R220G variant, T237 is oriented away from the active site, lacking the HB with the AVI sulfate.  Conclusion: In this structural analysis the binding of REL favors the interaction between H170 and T104, not observed in AVI complex (Figure). Interestingly, R220G substitution results in a structural disturbance in the loop connecting alpha-6 with beta-3, drawing P218 towards the DBO sulfate. Moreover, a unique orientation of the REL piperidine ring near H170 is present. Structural differences created by substitutions at R220 have unanticipated effects in the efficacy of both DBOs.