Purine de novo purine synthesis involves 10 reactions catalysed by six enzymes, including phosphoribosylformyglycinamidine synthase (PFAS). To date, genetic defects of three of these enzymes, namely ATIC, ADSL and PAICS, have been characterised in humans. Here, we report for the first time two individuals with PFAS deficiency. Probands were identified through metabolic and genetic screening of neurologically impaired individuals. The pathogenicity of the variants was established by structural and functional studies. Probands C1 and C2 presented with prematurity, short stature, recurrent seizures and mild neurological impairment. C1 had elevated urinary levels of formylglycineamide riboside (FGAr) and bi-allelic PFAS variants encoding the NP_036525.1:p.Arg811Trp substitution and the NP_036525.1:p.Glu228_Ser230 in-frame deletion. C2 is a 20-year-old female with a homozygous NP_036525.1:p.Asn264Lys substitution. These amino acid changes are predicted to affect the structural stability of PFAS. Accordingly, C1 skin fibroblasts showed decreased PFAS content and activity, with impaired purinosome formation that was restored by transfection with pTagBFP_PFAS_wt. The enzymatic activities of the corresponding recombinant mutant PFAS proteins were also reduced, and none of them, after transfection, corrected the elevated FGAR/r levels in PFAS-deficient HeLa cells. While genetic defects in purine de novo synthesis are typically considered in patients with severe neurological impairment, these disorders, especially PFAS deficiency, should also be considered in milder phenotypes.

• Keywords
• FGAR, PFAS deficiency, formylglycinamide riboside, metabolic disorder, purine de novo synthesis, purinosome,
• MeSH
• Humans MeSH
• Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor * genetics   deficiency   metabolism   MeSH
• Young Adult MeSH
• Mutation MeSH
• Purine-Pyrimidine Metabolism, Inborn Errors * genetics   MeSH
• Child, Preschool MeSH
• Purines * biosynthesis   MeSH
• Check Tag
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Names of Substances
• Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor * MeSH
• phosphoribosylformylglycinamidine synthetase MeSH Browser
• purine MeSH Browser
• Purines * MeSH

Deoxycytidine analogues (dCas) are widely used for the treatment of malignant diseases. They are commonly inactivated by cytidine deaminase (CDD), or by deoxycytidine monophosphate deaminase (dCMP deaminase). Additional metabolic pathways, such as phosphorylation, can substantially contribute to their (in)activation. Here, a new technique for the analysis of these pathways in cells is described. It is based on the use of 5-ethynyl 2'-deoxycytidine (EdC) and its conversion to 5-ethynyl 2'-deoxyuridine (EdU). Its use was tested for the estimation of the role of CDD and dCMP deaminase in five cancer and four non-cancer cell lines. The technique provides the possibility to address the aggregated impact of cytidine transporters, CDD, dCMP deaminase, and deoxycytidine kinase on EdC metabolism. Using this technique, we developed a quick and cheap method for the identification of cell lines exhibiting a lack of CDD activity. The data showed that in contrast to the cancer cells, all the non-cancer cells used in the study exhibited low, if any, CDD content and their cytidine deaminase activity can be exclusively attributed to dCMP deaminase. The technique also confirmed the importance of deoxycytidine kinase for dCas metabolism and indicated that dCMP deaminase can be fundamental in dCas deamination as well as CDD. Moreover, the described technique provides the possibility to perform the simultaneous testing of cytotoxicity and DNA replication activity.

• MeSH
• Cytidine * metabolism   MeSH
• Cytidine Deaminase metabolism   MeSH
• DCMP Deaminase * MeSH
• Deoxycytidine MeSH
• Deoxycytidine Kinase genetics   metabolism   MeSH
• Metabolic Networks and Pathways MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytidine * MeSH
• Cytidine Deaminase MeSH
• DCMP Deaminase * MeSH
• Deoxycytidine MeSH
• Deoxycytidine Kinase MeSH

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,
• Publication type
• Journal Article MeSH

Three genetically determined enzyme defects of purine de novo synthesis (PDNS) have been identified so far in humans: adenylosuccinate lyase (ADSL) deficiency, 5-amino-4-imidazole carboxamide-ribosiduria (AICA-ribosiduria), and deficiency in bifunctional enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS). Clinical signs of these defects are mainly neurological, such as seizures, psychomotor retardation, epilepsy, autistic features, etc. This work aims to describe the metabolic changes of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual steps of PDNS to better understand known and potential defects of the pathway in humans. High-performance liquid chromatography coupled with mass spectrometry was used for both targeted and untargeted metabolomic analyses. The statistically significant features from the untargeted study were identified by fragmentation analysis. Data from the targeted analysis were processed in Cytoscape software to visualize the most affected metabolic pathways. Statistical significance of PDNS intermediates preceding deficient enzymes was the highest (p-values 10 × 10-7-10 × 10-15) in comparison with the metabolites from other pathways (p-values of up to 10 × 10-7). Disturbed PDNS resulted in an altered pool of adenine and guanine nucleotides. However, the adenylate energy charge was not different from controls. Different profiles of acylcarnitines observed among deficient cell lines might be associated with a specific enzyme deficiency rather than global changes related to the PDNS pathway. Changes detected in one-carbon metabolism might reduce the methylation activity of the deficient cells, thus affecting the modification state of DNA, RNA, and proteins.

• Keywords
• HeLa cells, mass spectrometry, metabolomics, purine de novo synthesis, rare metabolic disorders,
• Publication type
• Journal Article MeSH

Adenylosuccinate lyase (ADSL) functions in de novo purine synthesis (DNPS) and the purine nucleotide cycle. ADSL deficiency (ADSLD) causes numerous neurodevelopmental pathologies, including microcephaly and autism spectrum disorder. ADSLD patients have normal serum purine nucleotide levels but exhibit accumulation of dephosphorylated ADSL substrates, S-Ado, and SAICAr, the latter being implicated in neurotoxic effects through unknown mechanisms. We examined the phenotypic effects of ADSL depletion in human cells and their relation to phenotypic outcomes. Using specific interventions to compensate for reduced purine levels or modulate SAICAr accumulation, we found that diminished AMP levels resulted in increased DNA damage signaling and cell cycle delays, while primary ciliogenesis was impaired specifically by loss of ADSL or administration of SAICAr. ADSL-deficient chicken and zebrafish embryos displayed impaired neurogenesis and microcephaly. Neuroprogenitor attrition in zebrafish embryos was rescued by pharmacological inhibition of DNPS, but not increased nucleotide concentration. Zebrafish also displayed phenotypes commonly linked to ciliopathies. Our results suggest that both reduced purine levels and impaired DNPS contribute to neurodevelopmental pathology in ADSLD and that defective ciliogenesis may influence the ADSLD phenotypic spectrum.

• Keywords
• ADSL, ADSLD, DNA damage, SAICAR, cell biology, chicken, cilia, developmental biology, human, microcephaly, zebrafish,
• MeSH
• Adenylosuccinate Lyase deficiency   metabolism   MeSH
• Aminoimidazole Carboxamide analogs & derivatives   metabolism   MeSH
• Autistic Disorder metabolism   MeSH
• Cell Line MeSH
• Cell Cycle MeSH
• Ciliopathies metabolism   MeSH
• Zebrafish metabolism   MeSH
• Phenotype MeSH
• Phosphoproteins metabolism   MeSH
• Chickens metabolism   MeSH
• Humans MeSH
• Microcephaly metabolism   MeSH
• Neurogenesis * MeSH
• Autism Spectrum Disorder metabolism   MeSH
• Purine-Pyrimidine Metabolism, Inborn Errors metabolism   MeSH
• DNA Damage MeSH
• Microtubule-Associated Proteins metabolism   MeSH
• Cell Cycle Proteins metabolism   MeSH
• Purines metabolism   MeSH
• Ribonucleotides metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Adenylosuccinate Lyase MeSH
• Aminoimidazole Carboxamide MeSH
• CCP110 protein, human MeSH Browser
• Phosphoproteins MeSH
• Microtubule-Associated Proteins MeSH
• Cell Cycle Proteins MeSH
• purine MeSH Browser
• Purines MeSH
• Ribonucleotides MeSH
• SAICAR MeSH Browser

BACKGROUND: Metabolomic analyses from our group and others have shown that tumors treated with glutamine antagonists (GA) exhibit robust accumulation of formylglycinamide ribonucleotide (FGAR), an intermediate in the de novo purine synthesis pathway. The increase in FGAR is attributed to the inhibition of the enzyme FGAR amidotransferase (FGAR-AT) that catalyzes the ATP-dependent amidation of FGAR to formylglycinamidine ribonucleotide (FGAM). While perturbation of this pathway resulting from GA therapy has long been recognized, no study has reported systematic quantitation and analyses of FGAR in plasma and tumors. OBJECTIVE: Herein, we aimed to evaluate the efficacy of our recently discovered tumor-targeted GA prodrug, GA-607 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)hexanamido)-6-diazo-5-oxohexanoate), and demonstrate its target engagement by quantification of FGAR in plasma and tumors. METHODS: Efficacy and pharmacokinetics of GA-607 were evaluated in a murine EL4 lymphoma model followed by global tumor metabolomic analysis. Liquid chromatography-mass spectrometry (LC-MS) based methods employing the ion-pair chromatography approach were developed and utilized for quantitative FGAR analyses in plasma and tumors. RESULTS: GA-607 showed preferential tumor distribution and robust single-agent efficacy in a murine EL4 lymphoma model. While several metabolic pathways were perturbed by GA-607 treatment, FGAR showed the highest increase qualitatively. Using our newly developed sensitive and selective LC-MS method, we showed a robust >80- and >10- fold increase in tumor and plasma FGAR levels, respectively, with GA-607 treatment. CONCLUSION: These studies describe the importance of FGAR quantification following GA therapy in cancer and underscore its importance as a valuable pharmacodynamic marker in the preclinical and clinical development of GA therapies.

• Keywords
• Glutamine antagonist, LC-MS., biomarker, cancer, formylglycinamide ribonucleotide, formylglycinamidine ribonucleotide, purine synthesis,
• MeSH
• Biomarkers, Pharmacological analysis   metabolism   MeSH
• Chromatography, Liquid methods   MeSH
• Glutamine antagonists & inhibitors   MeSH
• Glycine analogs & derivatives   analysis   metabolism   MeSH
• Mass Spectrometry methods   MeSH
• Metabolic Networks and Pathways drug effects   MeSH
• Mice MeSH
• Biomarkers, Tumor analysis   metabolism   MeSH
• Neoplasms * drug therapy   metabolism   MeSH
• Ribonucleotides * analysis   metabolism   MeSH
• Drug Development methods   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Biomarkers, Pharmacological MeSH
• Glutamine MeSH
• Glycine MeSH
• Biomarkers, Tumor MeSH
• phosphoribosyl-N-formylglycineamide MeSH Browser
• Ribonucleotides * MeSH

In humans, GART [phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) / phosphoribosylglycinamide synthetase (EC 6.3.4.13) / phosphoribosylaminoimidazole synthetase (EC 6.3.3.1)] is a trifunctional protein which catalyzes the second, third, and fifth reactions of the ten step de novo purine synthesis (DNPS) pathway. The second step of DNPS is conversion of phosphoribosylamine (5-PRA) to glycineamide ribonucleotide (GAR). 5-PRA is extremely unstable under physiological conditions and is unlikely to accumulate in the absence of GART activity. Recently, a HeLa cell line null mutant for GART was constructed via CRISPR-Cas9 mutagenesis. This cell line, crGART, is an important cellular model of DNPS inactivation that does not accumulate DNPS pathway intermediates. In the current study, we characterized the crGART versus HeLa transcriptomes in purine-supplemented and purine-depleted growth conditions. We observed multiple transcriptome changes and discuss pathways and ontologies particularly relevant to Alzheimer disease and Down syndrome. We selected the Cluster of Differentiation (CD36) gene for initial analysis based on its elevated expression in crGART versus HeLa as well as its high basal expression, high log2 value, and minimal P-value.

• MeSH
• Phosphoribosylglycinamide Formyltransferase metabolism   genetics   MeSH
• HeLa Cells MeSH
• Humans MeSH
• Metabolome * MeSH
• Purines * metabolism   biosynthesis   MeSH
• Transcriptome * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Phosphoribosylglycinamide Formyltransferase MeSH
• purine MeSH Browser
• Purines * MeSH

Adenylosuccinate lyase (ADSL) catalyzes two steps in de novo purine synthesis (DNPS). Mutations in ADSL can result in inborn errors of metabolism characterized by developmental delay and disorder phenotypes, with no effective treatment options. Recently, SAICAR, a metabolic substrate of ADSL, has been found to have alternative roles in the cell, complicating the role of ADSL. crADSL, a CRISPR KO of ADSL in HeLa cells, was constructed to investigate DNPS and ADSL in a human cell line. Here we employ this cell line in an RNA-seq analysis to initially investigate the effect of DNPS and ADSL deficiency on the transcriptome as a first step in establishing a cellular model of ADSL deficiency. We report transcriptome changes in genes relevant to development, vascular development, muscle, and cancer biology, which provide interesting avenues for future research.

• Keywords
• Adenylosuccinate lyase, Purine synthesis, RNA-seq, Transcriptome, adenosine monophosphate, (AMP), adenylosuccinate lyase, (ADSL), aminoimidazole carboxamide ribonucleotide, (AICAR), de novo purine synthesis, (DNPS), differentially expressed gene, (DEG), false discovery rate, (FDR), fetal calf macroserum, (FCM), fragments per kilobase of exon per million reads mapped, (FPKM), gene ontology, (GO), guanosine monophosphate, (GMP), minus adenine crADSL to minus adenine WT comparison, (MM), phosphoribosyl pyrophosphate, (PRPP), phosphoribosylaminoimidazolesuccinocarboxamide, (SAICAR), plus adenine crADSL to plus adenine WT comparison, (PP), succinyladenosine monophosphate, (SAMP),
• Publication type
• Journal Article MeSH