Competitive inhibitors of the CphA metallo-beta-lactamase from Aeromonas hydrophila

Abstract

Various inhibitors of metallo-beta-lactamases have been reported; however, none are effective for all subgroups. Those that have been found to inhibit the enzymes of subclass B2 (catalytically active with one zinc) either contain a thiol (and show less inhibition towards this subgroup than towards the dizinc members of B1 and B3) or are inactivators behaving as substrates for the dizinc family members. The present work reveals that certain pyridine carboxylates are competitive inhibitors of CphA, a subclass B2 enzyme. X-ray crystallographic analyses demonstrate that pyridine-2,4-dicarboxylic acid chelates the zinc ion in a bidentate manner within the active site. Salts of these compounds are already available and undergoing biomedical testing for various nonrelated purposes. Pyridine carboxylates appear to be useful templates for the development of more-complex, selective, nontoxic inhibitors of subclass B2 metallo-beta-lactamases.

FIG. 1.

Structures of 2-picolinic acid and its derivatives, 2,3-PDCA, 2,4-PDCA, 2,5-PDCA, 2,6-PDCA (dipicolinic acid), and 3,4-PDCA, which were tested as MBL inhibitors.

FIG. 2.

Hanes linearization for the inhibition of CphA by 2,4-PDCA at 0, 10, and 20 μM 2,4-PDCA, depicted by graphs of filled circles, open diamonds, and open squares, respectively.

FIG. 3.

pH dependence of the log₁₀ of the competitive inhibition constant K_i with 2,4-PDCA and 2-picolinic acid, depicted by graphs of filled circles and open squares, respectively.

FIG. 4.

Active site of CphA-Zn(II) in complex with the pyridine-2,4-dicarboxylate inhibitor (carbon atoms colored in orange). The conformational change upon inhibitor binding is represented by superimposition of the wild-type Gly232 and Asn233 residues (green). The zinc ion is represented as a blue sphere. This figure was prepared using the program PYMOL.

FIG. 5.

Difference electron density map (F_complex − F_wt) at contoured 2σ (orange) corresponding to the area of the complex structure where the pyridine-2,4-dicarboxylate inhibitor was modeled. (F_complex and F_wt are the structure factor amplitudes of the CphA crystal soaked in solution containing 2,4-PDCA and of the uncomplexed crystals, respectively.) The final refined coordinates of the inhibitor molecule, zinc ion, and D120, C221, H263, and V67 side chains have been superimposed. The distance between V67 (CG2) and 2,4-PDCA (C-41) is indicated in angstroms.