Aspartate α-decarboxylase a new therapeutic target in the fight against Helicobacter pylori infection

Abstract

Effective eradication therapy for Helicobacter pylori is a worldwide demand. Aspartate α-decarboxylase (ADC) was reported as a drug target in H. pylori, in an in silico study, with malonic acid (MA) as its inhibitor. We evaluated eradicating H. pylori infection through ADC inhibition and the possibility of resistance development. MA binding to ADC was modeled via molecular docking. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of MA were determined against H. pylori ATCC 43504, and a clinical H. pylori isolate. To confirm selective ADC inhibition, we redetermined the MIC in the presence of products of the inhibited enzymatic pathway: β-alanine and pantothenate. HPLC was used to assay the enzymatic activity of H. pylori 6x-his tagged ADC in the presence of different MA concentrations. H. pylori strains were serially exposed to MA for 14 passages, and the MICs were determined. Cytotoxicity in different cell lines was tested. The efficiency of ADC inhibition in treating H. pylori infections was evaluated using a Sprague-Dawley (SD) rat infection model. MA spectrum of activity was determined in different pathogens. MA binds to H. pylori ADC active site with a good docking score. The MIC of MA against H. pylori ranged from 0.5 to 0.75 mg/mL with MBC of 1.5 mg/mL. Increasing β-alanine and pantothenate concentrations proportionally increased MA MIC. The 6x-his tagged ADC activity decreased by increasing MA concentration. No resistance to ADC inhibition was recorded after 14 passages; MA lacked cytotoxicity in all tested cell lines. ADC inhibition effectively eradicated H. pylori infection in SD rats. MA had MIC between 0.625 to 1.25 mg/mL against the tested bacterial pathogens. In conclusion, ADC is a promising target for effectively eradicating H. pylori infection that is not affected by resistance development, besides being of broad-spectrum presence in different pathogens. MA provides a lead molecule for the development of an anti-helicobacter ADC inhibitor. This provides hope for saving the lives of those at high risk of infection with the carcinogenic H. pylori.

Keywords: Helicobacterpylori; aspartate α-decarboxylase; broad-spectrum; drug target; malonic acid.

Figure 1

Interaction of the binding site of aspartate α-decarboxylase with different ligands. (A) The co-crystalized ligand ((N-2-(2-amino-1-methyl-2-oxoethylidene)asparaginate)). (B,C) Aspartate (Two-D structures and three-D structures). (D,E) Malonate (Two-D structures and three-D structures). Images were generated by Molecular Operating Environment (MOE, 2015.10) software.

Figure 2

Minimum inhibitory concentration (MIC) of malonic acid in presence of the products of aspartate α-decarboxylase catalyzed enzymatic reaction. MIC of malonic acid in the presence of increasing concentrations of (A) β-alanine and (B) pantothenate, using both agar dilution and broth microdilution methods, against both Helicobacter pylori ATCC 43504 and the clinical H. pylori isolate (HPM001). The correlation between the MIC of malonic acid and the concentrations of (C) β-alanine, and (D) pantothenate.

Figure 3

Cloning and expression of Helicobacter pylori ATCC 43504 aspartate α-decarboxylase. (A) Schematic diagram generated by BioEdit (7.2.5., 2015) of pET22b + vector containing the panD insert with the positions of different primers highlighted. (B) Polymerase chain reaction (PCR) amplicons produced by different primer combinations performed on H. pylori ATCC 43504 DNA (panD), empty pET22b(+) plasmid vector and the recombinant plasmid vector pET22b(+) containing the insert panD, (C) Crude protein extract from: lane 1: Escherichia coli BL21, lane 2: E.coli BL21/RecPl induced by 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), lane 3: E. coli BL21/RecPl induced by 0.75 mM IPTG (D) The wash, bind and elute of Ni-NTA columns purification of the recombinant protein from E. coli BL21/RecPl.

Figure 4

Selective inhibition of Helicobacter pylori aspartate α-decarboxylase (ADC) by malonic acid. β-alanine (Black line) produced from the action of (A) crude enzyme extract and (B) purified 6x-His tagged ADC, of Isopropyl β-D-1-thiogalactopyranoside induced Escherichia coli BL21/RecPl, in the presence of increasing concentrations of malonic acid. The red lines depict the correlation between the two variables.

Figure 5

Lack of resistance development in Helicobacter pylori by repeated exposure to aspartate α-decarboxylase inhibition using malonic acid. Fold increase in minimum inhibitory concentration (MIC) of malonic acid and clarithromycin against H. pylori ATCC 43504 and the clinical H. pylori HPM001 isolate, following 14 serial passages.

Figure 6

Inhibition of aspartate-α-decarboxylase successfully treated Helicobacter pylori infected rats. (A) Weekly follow up of the number of rats that cleared infection with either H. pylori ATCC 43504 or the clinical H. pylori HPM001 strains as indicated by H. pylori Stool Antigen (HpSA) test during the 3 weeks treatment period. (B) The total H. pylori count in the stomachs of malonic acid treated and untreated (control) groups of rats at the end of the 3 weeks treatment period. OD, once daily; BID, twice daily; LD₅₀, lethal dose 50; Control, rat groups that received sterile water instead of malonic acid.