Mutation of an active site-adjacent residue in VIM indirectly dictates interactions with and blunts inhibition by D-captopril

Abstract

Activity assays and X-ray crystallographic studies were undertaken to elucidate the inhibitory mechanism of captopril stereoisomers on Verona integron-encoded metallo-β-lactamases, specifically VIM-20, VIM-31, and VIM-15. All three VIM-2-like variants (VIM-20, VIM-31, and VIM-15) and VIM-2 expressed in Escherichia coli exhibited catalytic activity with comparable steady-state kinetic parameters. Among the tested thiol drugs (L- and D-captopril, D,L-thiorphan, and 2,3-dimercaprol), IC₅₀ analyses indicated that D-captopril and 2,3-dimercaprol were more potent inhibitors against the VIM enzymes examined in this study. Notably, the IC₅₀ value of D-captopril against VIM-31 was an exception, closely resembling that of L-captopril. To elucidate this exceptional inhibitory potency of D-captopril and its binding mode in the active site of VIM-31, high-resolution crystal structures of VIM-20, VIM-31, and VIM-15 in complex with both L- and D-captopril are reported. These findings will help evaluate whether the identified potent inhibitor D-captopril could be further developed as a pan inhibitor targeting the VIM-family enzymes.

Keywords: Antibiotic resistance; Enzyme inhibition; Metallo-β-lactamase; Protein crystallography.

Figure 1:

Chemical structures of (A) the chromogenic cephalosporin substrate nitrocefin and (B) the thiol-containing compounds tested as MBL inhibitors in this study.

Figure 2:

IC₅₀ plots for (A) L-captopril, (B) D-captopril, (C) DL-thiorphan, and (D) 2,3-dimercaprol, respectively, taking nitrocefin as a substrate and enzyme concentration at 5.4 nM. All data points represent the average slope of three experiments, and error bars indicate the standard deviation.

Figure 3:

Captopril isomers as inhibitors against VIM enzymes, with X-ray crystallographic electron density omit maps for (A) L-captopril (salmon) with VIM-20 (lime green), (B) D-captopril (grey) with VIM-20 (forest green), (C) L-captopril (salmon) with VIM-31 (cyan), (D) D-captopril (grey) with VIM-31 (dark teal), and (E) L-captopril (salmon) with VIM-15 (in yellow). The F_o-F_c electron density omit maps were calculated using POLDER and are drawn at 3.0 σ throughout, with positive density in black and negative density in red.

Figure 4:

X-ray crystal structures of VIM enzymes in complex with D-captopril or L-captopril. (A) Overlay of VIM-2 (pink) in complex with D-captopril (grey) and VIM-2 (purple) in complex with L-captopril (salmon). (B) Overlay of VIM-20 (lime green) in complex with D-captopril (grey) and VIM-20 (forest green) in complex with L-captopril (salmon). (C) Overlay of VIM-31 (dark teal) in complex with D-captopril (grey) and VIM-31 (cyan) in complex with L-captopril (salmon). (D)VIM-15 (yellow) in complex with L-captopril (salmon).

Figure 5:

An overlay of X-ray crystallographic structures featuring VIM-20 and VIM-31 with L-captopril and D-captopril. (A) VIM-20 with D-captopril and VIM-3 with D-captopril; and (B) rotated view of A highlighting the Tyr-201-His mutation and altered position of Ser-207. In each panel, VIM-31 is colored dark teal, D-captopril bound to VIM-31 is colored dark grey, VIM-20 is colored lime green, and D-captopril bound to VIM-20 is colored light grey.