Metabolic profiles and fingerprints for the investigation of the influence of nitisinone on the metabolism of the yeast Saccharomyces cerevisiae

Abstract

Nitisinone (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione, NTBC) is considered a potentially effective drug for the treatment of various metabolic diseases associated with disorders of L-tyrosine metabolism however, side-effects impede its widespread use. This work aimed to broaden the knowledge of the influence of NTBC and its metabolites 2-amino-4-(trifluoromethyl)benzoic acid (ATFA), 2-nitro-4-(trifluoromethyl)benzoic acid (NTFA), and cyclohexane-1,3-dione (CHD) on the catabolism of L-tyrosine and other endogenous compounds in Saccharomyces cerevisiae. Based on a targeted analysis performed by LC-ESI-MS/MS, based on multiple reaction monitoring, it was found that the dissipation kinetics of the parent compound and its metabolites are compatible with a first-order reaction mechanism. Moreover, it has been proven that formed NTBC metabolites, such as CHD, cause a decrease in L-tyrosine, L-tryptophan, and L-phenylalanine concentrations by about 34%, 59% and 51%, respectively, compared to the untreated model organism. The overall changes in the metabolism of yeast exposed to NTBC or its derivatives were evaluated by non-targeted analysis via LC-ESI-MS/MS in the ion trap scanning mode. Based on principal components analysis, a statistically significant similarity between metabolic responses of yeast treated with ATFA or NTFA was observed. These findings facilitate further studies investigating the influence of NTBC on the human body and the mechanism of its action.

Figure 1

Tyrosine metabolic pathway highlighting the enzyme defects responsible and the site of action of nitisinone. i—tyrosine aminotransferase, ii—4-hydroxyphenylpyruvate dioxygenase, iii—homogentisate 1,2-dioxygenase, iv—maleylacetoacetate isomerase, v—fumarylacetoacetate hydrolase.

Figure 2

Biosynthesis of the aromatic amino acids tryptophan, tyrosine, and phenylalanine via a common pathway to chorismite in S. cerevisiae. i—chorismate mutase, ii—prephenate dehydratase, iii—transaminases, iv—transaminases, v—arogenate dehydratase, vi—arogenate dehydrogenase, vii—prephenate dehydrogenase, viii—transaminase.

Figure 3

Degradation rate of (a) NTBC and (b) NTFA during incubation. Data are expressed as mean (n = 3); error bars represent CVs.

Figure 4

Percentage changes in the concentrations of metabolites of l-TYR, l-TRF, and l-PHE in yeast exposed to NTBC, NTFA, ATFA, and CHD after 30 min (a) and after 120 min of incubation (b). Blank was taken as 0%. Data are expressed as mean (n = 6); error bars represent CVs.

Figure 5

PCA plots for the data obtained after 30 min of yeast incubation of (a) the major components 1 (D1: 35.4%) and 2 (D2: 23.8%) and (b) the major components 2 (D2: 23.8%) and 3 (D3: 20.9%). Observations that can be associated with a variable group are delineated (ellipses).

Figure 6

PCA plots for data obtained after 120 min of yeast incubation of (a) the major components 1 (D1: 29.9%) and 2 (D2: 28.6%) and (b) the major components 1 (D1: 29.9%) and 3 (D3: 23.1%). Observations that can be associated with a variable group are delineated (ellipses).