The Effect of Substituted Benzene-Sulfonamides and Clinically Licensed Drugs on the Catalytic Activity of CynT2, a Carbonic Anhydrase Crucial for Escherichia coli Life Cycle

Abstract

Proteins are relevant antimicrobial drug targets, and among them, enzymes represent a significant group, since most of them catalyze reactions essential for supporting the central metabolism, or are necessary for the pathogen vitality. Genomic exploration of pathogenic and non-pathogenic microorganisms has revealed genes encoding for a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1). CAs catalyze the physiologically crucial reversible reaction of the carbon dioxide hydration to bicarbonate and protons. Herein, we investigated the sulfonamide inhibition profile of the recombinant β-CA (CynT2) identified in the genome of the Gram-negative bacterium Escherichia coli. This biocatalyst is indispensable for the growth of the microbe at atmospheric pCO₂. Surprisingly, this enzyme has not been investigated for its inhibition with any class of CA inhibitors. Here, we show that CynT2 was strongly inhibited by some substituted benzene-sulfonamides and the clinically used inhibitor sulpiride (K_Is in the range of 82-97 nM). This study may be relevant for identifying novel CA inhibitors, as well as for another essential part of the drug discovery pipeline, such as the structure-activity relationship for this class of enzyme inhibitors.

Keywords: Escherichia coli; antibacterials; carbonic anhydrase; inhibitors; protonography; stopped-flow assay; sulfonamides.

Figure 1

Analysis of the heterologous protein expression on the Coomassie Blue stained gel (SDS–PAGE) and Bromothymol Blue stained gel (Protonography). The purified recombinant CynT2 was eluted from the affinity resin by adding 250 mM imidazole. The yellow band on the protonogram (right of the figure) corresponds to the enzyme activity responsible for the drop of pH from 8.2 to the transition point of the dye in the control buffer. Lane STD, molecular markers (form bottom to the top: 20, 25, and 37 kDa); Lane STD, Molecular markers; Lane 1, purified CynT2; Lane 2, purified CynT2 subjected to protonography; Lane 3, commercial bovine CA (bCA) used as a positive control in the protonography.

Figure 2

Pairwise comparison of the CynT2 polypeptide chain with VchCAβ (A), PigCAβ (B), and HpyCAβ (C) amino acid sequences, respectively. The identical amino acid residues are indicated between the two aligned amino acid sequences (black bold). The amino acid residues participating in the coordination of the metal ion are indicated in red (Cys, His, and Cys), whereas the catalytic dyad involved in the activation of the water molecule coordinated to zinc (Asp–Arg) is shown in blue. The pairwise alignment was performed with the program Blast Global Align. The accession numbers of the aligned sequences are: EEW0221051.1, CynT2 from Escherichia coli; WP_002051193.1, VchCAβ from Vibrio cholerae; WP_012458351.1, PgiCAβ from Porphyromonas gingivalis; WP_000642991.1, HpyCAβ from Helicobacter pylori.

Figure 3

The 42 compounds used to study CynT2 inhibitory behavior. Forty-one sulfonamides and one sulfamate (TPM) were exploited. In red, the series 1–24; in gray, the clinically used drugs.