Metabolites of De Novo Purine Synthesis: Metabolic Regulators and Cytotoxic Compounds

Abstract

Cytotoxicity of de novo purine synthesis (DNPS) metabolites is critical to the pathogenesis of three known and one putative autosomal recessive disorder affecting DNPS. These rare disorders are caused by biallelic mutations in the DNPS genes phosphoribosylformylglycineamidine synthase (PFAS), phosphoribosylaminoimidazolecarboxylase/phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS), adenylosuccinate lyase (ADSL), and aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) and are clinically characterized by developmental abnormalities, psychomotor retardation, and nonspecific neurological impairment. At a biochemical level, loss of function of specific mutated enzymes results in elevated levels of DNPS ribosides in body fluids. The main pathogenic effect is attributed to the accumulation of DNPS ribosides, which are postulated to be toxic to the organism. Therefore, we decided to characterize the uptake and flux of several DNPS metabolites in HeLa cells and the impact of DNPS metabolites to viability of cancer cell lines and primary skin fibroblasts. We treated cells with DNPS metabolites and followed their flux in purine synthesis and degradation. In this study, we show for the first time the transport of formylglycinamide ribotide (FGAR), aminoimidazole ribotide (AIR), succinylaminoimidazolecarboxamide ribotide (SAICAR), and aminoimidazolecarboxamide ribotide (AICAR) into cells and their flux in DNPS and the degradation pathway. We found diminished cell viability mostly in the presence of FGAR and AIR. Our results suggest that direct cellular toxicity of DNPS metabolites may not be the primary pathogenetic mechanism in these disorders.

Keywords: ADSL; AICAR; AIR; ATIC; FGAR; PAICS; PFAS; SAICAR; cytotoxicity; purine synthesis.

Figure 1

Purine metabolism. De novo purine synthesis (DNPS). Phosphoribosylpyrophosphate (PRPP) is processed by amidophosphoribosyl transferase (PPAT) to phosphoribosylamine (PRA). The 2nd, 3rd, and 5th steps are catalyzed by the trifunctional enzyme glycineamide ribonucleotide synthetase/aminoimidazole ribonucleotide synthetase/glycineamide ribonucleotide transformylase (GART). PRA results in glycineamide ribotide (GAR), from which formylglycineamide ribotide (FGAR) is created. The enzyme phosphoribosylformylglycineamidine synthase (PFAS) catalyzes transformation into formylglycineamidine ribotide (FGAM), which then forms aminoimidazole ribotide (AIR). AIR is processed by the bifunctional enzyme phosphoribosylaminoimidazolecarboxylase/phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS) into carboxyaminoimidazole ribotide (CAIR) and succinylaminoimidazolecarboxyamide ribotide (SAICAR), respectively. The bifunctional enzyme adenylosuccinate lyase (ADSL) catalyzes the reaction of SAICAR to aminoimidazolecarboxamide ribotide (AICAR) in DNPS and succinyl-AMP (SAMP) to adenosine monophosphate (AMP) in the purine nucleotide cycle. The bifunctional enzyme aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) catalyzes the last two steps that result in formamidoimidazolecarboxamide ribotide (FAICAR) and inosine monophosphate (IMP). IMP is precursor for synthesis of guanosine monophosphate (GMP) and AMP. Defects in DNPS enzymes lead to accumulation of ribosides, the dephosphorylated DNPS metabolites: FGAr, AIr, SAICAr, AICAr, succinyladenosine (SAdo). Enzymes connected with DNPS disorders are distinguished by colors: green for PFAS deficiency, purple for PAICS deficiency, red for ADSL deficiency, blue for AICAribosiduria. The main salvage enzyme of purines—hypoxanthineguaninephosphoribosyl transferase (HGPRT)—catalyzes conversion of hypoxanthine into IMP and guanine into GMP (not shown). Degradation of purines goes through IMP to inosine, hypoxanthine, xanthine to the final product uric acid.

Figure 2

Accumulation of isotopically labeled DNPS metabolites (FGAR*, AIR*, SAICAR*, AICAR*). Vertical dashed line indicates which DNPS metabolite was added. The graphs (A–D) represent the concentration of detected isotopically labeled metabolites in HeLa control cell lysate after the addition of FGAR*, AIR*, SAICAR*, and AICAR* (red), and in ATIC KO cell lysate after addition of FGAR*, AIR*, SAICAR*, and AICAR*(dark green). The graphs (E–H) represent the concentration of detected isotopically labeled metabolites in HeLa control cell growth medium after the addition of FGAR*, AIR*, SAICAR*, and AICAR* (red), and in ATIC KO cell growth medium after addition of FGAR*, AIR*, SAICAR*, and AICAR* (dark green). The graphs (I–L) display results for detected labeled metabolites in blank medium (medium without cells) after addition of FGAR*, AIR*, SAICAR*, and AICAR* (light green). The results for untreated cells were zero or approaching zero, and are not shown in the graph. Each data point represents the mean of a single experiment measured in duplicate. Vertical bars represent S.D. (n = 2). List of all measured isotopically labeled metabolites is shown in Table S1.

Figure 3

Cytotoxicity of DNPS metabolites (FGAR/r, AIR/r, SAICAR/r, AICAR/r) in HeLa, CAD 5, HepG2, K562 cells and skin fibroblasts. Cells were cultured in normal growth medium containing corresponding phosphorylated (squares, red dotted line) or dephosphorylated (black circles, blue dashed line) DNPS metabolites at 14 concentrations ranging from 1.7 μmol/l to 1 mmol/L, and cell viability was evaluated 72 h later. Viability curves of (A) FGAR and FGAr, (B) AIR and AIr, (C) SAICAR and SAICAr, and (D) AICAR and AICAr were established. The X-axis displays the concentration of the DNPS metabolite [μM] and the Y-axis displays the cell viability [%]. The viability level of untreated cells was set as reference and data were normalized to untreated cells and IC₅₀ values (Table 1) were calculated using GraphPad Prism software. Each data point represents the mean of 3 independent experiments. Vertical bars represent S.D. (n = 3).

Figure 4

AICAR restores the viability of HeLa GART KO cells. The viability of HeLa GART KO cells was measured after (A) 1 day of incubation, (B) 3 days of incubation, (C) 5 days of incubation in purine-depleted medium with FGAR (blue), AIR (green), SAICAR (orange), AICAR (red), IMP (grey) in concentration range from 5 μmol/L to 500 μmol/L. (D) HeLa control, GART KO, ADSL KO, GART-ADSL KO viability measurement after 72 h incubation in normal medium containing no metabolite (grey) or 200 μM AICAR (red). Each data point represents the mean of 3 measurements. Vertical bars represent S.D. (n = 3).