1,2,4‐Triazole‐3‐thione Compounds as Inhibitors of Dizinc Metallo‐β‐lactamases

Abstract

Metallo-β-lactamases (MBLs) cause resistance of Gram-negative bacteria to β-lactam antibiotics and are of serious concern, because they can inactivate the last-resort carbapenems and because MBL inhibitors of clinical value are still lacking. We previously identified the original binding mode of 4-amino-2,4-dihydro-5-(2-methylphenyl)-3H-1,2,4-triazole-3-thione (compound IIIA) within the dizinc active site of the L1 MBL. Herein we present the crystallographic structure of a complex of L1 with the corresponding non-amino compound IIIB (1,2-dihydro-5-(2-methylphenyl)-3H-1,2,4-triazole-3-thione). Unexpectedly, the binding mode of IIIB was similar but reverse to that of IIIA. The 3 D structures suggested that the triazole-thione scaffold was suitable to bind to the catalytic site of dizinc metalloenzymes. On the basis of these results, we synthesized 54 analogues of IIIA or IIIB. Nineteen showed IC values in the micromolar range toward at least one of five representative MBLs (i.e., L1, VIM-4, VIM-2, NDM-1, and IMP-1). Five of these exhibited a significant inhibition of at least four enzymes, including NDM-1, VIM-2, and IMP-1. Active compounds mainly featured either halogen or bulky bicyclic aryl substituents. Finally, some compounds were also tested on several microbial dinuclear zinc-dependent hydrolases belonging to the MBL-fold superfamily (i.e., endonucleases and glyoxalase II) to explore their activity toward structurally similar but functionally distinct enzymes. Whereas the bacterial tRNases were not inhibited, the best IC values toward plasmodial glyoxalase II were in the 10 μm range.

Structural and biochemical analysis of the metallo‐β‐lactamase L1 from emerging pathogen Stenotrophomonas maltophilia revealed the subtle but distinct di‐metal scaffold for catalytic activity

“…L1 is the only B class β‐lactamase (BBL) that forms a tetramer and the formation of a tetramer is critical for the catalytic activity and/or substrate profile of L1 as shown by mutational study . All L1 MBL high‐resolution structures in this study, including the native form (L1‐Native), ligand‐bound forms, and ethylenediaminetetraacetic acid (EDTA)‐treated apo form (L1‐E) are virtually identical to those previously reported L1 structures with or without ligands bound . Root mean square deviations (RMSDs) on 264 or 265 Cα atoms are 0.3–0.4 Å, only a few residues deviate more than 1.0 Å (<2.0 Å) are at the surface of the protein away from the active site.…”

“…As shown in the reported structures, the active site is made of two Zn +2 (or Cd +2 or Cu +2 ) ions and the protein side chains coordinating metal ions at the bottom of the binding pocket. All the following atomic distances are observed in L1‐Native.…”

“…31 All L1 MBL high-resolution structures in this study, including the native form (L1-Native), ligand-bound forms, and ethylenediaminetetraacetic acid (EDTA)-treated apo form (L1-E) are virtually identical to those previously reported L1 structures with or without ligands bound. [17][18][19][20][21][22][23][24] Root mean square deviations (RMSDs) on 264 or 265 Cα atoms are 0.3-0.4 Å, only a few residues deviate more than 1.0 Å (<2.0 Å) are at the surface of the protein away from the active site. This indicates that the protein is not required to change significantly to bind ligands (hydrolyzed antibiotics, inhibitors, and different metals) in various crystalline conditions.…”

“…S. maltophilia L1 is a subclass B3 MBL, one of two chromosome‐encoded β‐lactamases (L2 belongs to a class A) . Several crystal structures of L1 and other B3 MBLs have been reported in the complexes with hydrolyzed antibiotics and inhibitors, which are supported by multiple biochemical analysis . B3 MBLs share a relatively low sequence identity of 23–35%; however, they are structurally well‐conserved particularly their di‐nuclear zinc active sites.…”

“…16 Several crystal structures of L1 [17][18][19][20][21] and other B3 MBLs have been reported in the complexes with hydrolyzed antibiotics and inhibitors, which are supported by multiple biochemical analysis. [17][18][19][20][21][22][23][24][25][26][27][28][29][30] B3 MBLs share a relatively low sequence identity of 23-35%; however, they are structurally well-conserved particularly their di-nuclear zinc active sites. Here, we extend the L1 studies and report several high-resolution structures of L1, the two zinc bound form (native form), the metal-free form (apo form), the captopril inhibitor bound form, several complexes with hydrolyzed antibiotics (imipenem, moxalactam, meropenem, and penicillin G) and with different metal ions (Zn +2 , Cd +2 , and Cu +2 ).…”