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

K. M. PAPP-WALLACE; S. KLINKE; F. BRUNETTI; R. A. BONOMO; M. RUGGIERO; G. GUTKIND; P. POWER

Evento online debido a la pandemia de coronavirus

Congreso; ASM Microbe 2020; 2020

Background: Relebactam (REL) and avibactam (AVI) have potent activity against serine-β-lactamases. PER β-lactamases possess enhanced oxyimino-cephalosporinase activity due to the presence of an inverted Ω loop and enlarged β3 strand that expand the active site. Previously, it was observed that substitutions at position R220 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, and to evaluate the role of the R220G mutation on 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. X-ray diffraction was measured at the Soleil synchrotron (France). Data process was performed with XDS, and the structure was solved by molecular replacement with Phenix. Results: REL showed lower Ki than AVI for both PER-2 (5 vs. 20 μM) and PER-2/R220G (21 vs 350 μM), which correlated with higher k2/K (PER-2: 11,200 vs. 2,200 M-1.s-1; PER-2/R220G: 3,100 vs 200 M-1.s-1). 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 at high resolution (1.5-2.1 Å) showed that, although REL is oriented in equivalent position compared to CTX-M-15 and KPC-2, the piperidine ring is rotated up to 90°, likely due to the presence of H170. In PER-2/R220G, T237 points away from the active site, lacking the HB with the AVI sulfate. Conclusion: The binding of REL favors the interaction between H170 and T104, not observed in AVI complex (Figure). R220G mutation results in a structural disturbance in the α6-β3 loop, drawing P218 towards the DBO sulfate. Moreover, a unique orientation of the REL piperidine near H170 is present. Structural differences created by substitutions at R220 have unanticipated effects in the efficacy of both DBOs.