Aurintricarboxylic acid structure modifications lead to reduction of inhibitory properties against virulence factor YopH and higher cytotoxicity

Abstract

Yersinia sp. bacteria owe their viability and pathogenic virulence to the YopH factor, which is a highly active bacterial protein tyrosine phosphatase. Inhibition of YopH phosphatase results in the lack of Yersinia sp. pathogenicity. We have previously described that aurintricarboxylic acid inhibits the activity of YopH at nanomolar concentrations and represents a unique mechanism of YopH inactivation due to a redox process. This work is a continuation of our previous studies. Here we show that modifications of the structure of aurintricarboxylic acid reduce the ability to inactivate YopH and lead to higher cytotoxicity. In the present paper we examine the inhibitory properties of aurintricarboxylic acid analogues, such as eriochrome cyanine R (ECR) and pararosaniline. Computational docking studies we report here indicate that ATA analogues are not precluded to bind in the YopH active site and in all obtained binding conformations ECR and pararosaniline bind to YopH active site. The free binding energy calculations show that ECR has a stronger binding affinity to YopH than pararosaniline, which was confirmed by experimental YopH enzymatic activity studies. We found that ATA analogues can reversibly reduce the enzymatic activity of YopH, but possess weaker inhibitory properties than ATA. The ATA analogues induced inactivation of YopH is probably due to oxidative mechanism, as pretreatment with catalase prevents from inhibition. We also found that ATA analogues significantly decrease the viability of macrophage cells, especially pararosaniline, while ATA reveals only slight effect on cell viability.

Keywords: Aurintricarboxylic acid (ATA); Eriochrome cyanine R; Pararosaniline; Protein tyrosine phosphatases (PTPs); YopH.

Fig. 1

The structure of aurintricarboxylic acid (a) and its analogues: eriochrome cyanine R (b) and pararosaniline (c)

Fig. 2

Computational analysis of eriochrome cyanine R binding and interactions in YopH active site. a The site of binding for top 30 conformations of eriochrome cyanine R obtained from docking of ECR into the YopH structure. In each conformation ECR binds in the YopH active site. b The PLIF diagram for the best binding pose of ATA in the YopH binding site. In predicted binding pose, the carboxyl group of ECR is directed toward essential Cys403, Arg409 and Asp356 residues in the active site. There are electrostatic interactions between polar groups of ECR with Cys403, Arg409 and Asp356

Fig. 3

Computational analysis of binding and interactions of pararosaniline in YopH active site. a The site of binding for top 30 conformations of pararosaniline obtained from docking of pararosaniline into the YopH structure. In each conformation, pararosaniline binds in the YopH active site. b The PLIF diagram for the best binding pose of pararosaniline in the YopH binding site. In predicted binding pose, there are interactions with essential Asp356

Fig. 4

The binding affinity and inhibitory properties of ATA analogues: eriochrome cyanine R and pararosaniline to YopH. a The binding affinity of ECR and pararosaniline to YopH active site presented as binding free energy and its components for the YopH-ECR, and YopH-pararosaniline complexes by MM/GBSA methods (kcal/mol). b The inhibitory properties of ECR and pararosaniline to YopH presented as IC₅₀ values. IC₅₀ values were determined from a plot presenting inhibitor concentration versus percentage of the enzymatic activity measured as absorbance with pNPP substrate of recombinant YopH after 15 min incubation with inhibitors, at 2 mM substrate concentration equal to Km value, (n = 3), mean ± SD. c The YopH activity after treatment with 50 µM ECR and 50 µM pararosaniline in presence and in absence of catalase (0.3 mg/mL). Data presented as a percent of control, mean ± SD (n = 3). T-test analysis of variance, *significantly different from control (p < 0.001), **significantly different in pairs (p < 0.001)

Fig. 5

The viability assay of macrophage cells treated for 24 and 72 h with ATA analogous: a eriochrome cyanine R, b pararosaniline, and c ATA . Data presented as percent of the control viability (100 %, cells not treated), mean ± SD (n = 3). One-way Anova test. *Means were significantly different from control (p < 0.05). **Means were significantly different from control (p < 0.001)