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.

CZ OPENSCREEN National Infrastructure for Chemical Biology Institute of Molecular Genetics Czech Academy of Sciences 142 00 Prague Czech Republic

Department of Paediatrics and Inherited Metabolic Disorders 1st Faculty of Medicine Charles University and General University Hospital Prague Ke Karlovu 455 2 128 08 Prague Czech Republic

Section on Nephrology Wake Forest School of Medicine Winston Salem NC 27103 USA

Zobrazit více v PubMed

Krijt M., Souckova O., Baresova V., Skopova V., Zikanova M. Metabolic Tools for Identification of New Mutations of Enzymes Engaged in Purine Synthesis Leading to Neurological Impairment. Folia Biol. 2019;65:152–157. PubMed

Marie S., Heron B., Bitoun P., Timmerman T., van den Berghe G., Vincent M.F. AICA-ribosiduria: A novel, neurologically devastating inborn error of purine biosynthesis caused by mutation of ATIC. Am. J. Hum. Genet. 2004;74:1276–1281. doi: 10.1086/421475. PubMed DOI PMC

Pelet A., Skopova V., Steuerwald U., Baresova V., Zarhrate M., Plaza J.M., Hnizda A., Krijt M., Souckova O., Wibrand F., et al. PAICS deficiency, a new defect of de novo purine synthesis resulting in multiple congenital anomalies and fatal outcome. Hum. Mol. Genet. 2019;28:3805–3814. doi: 10.1093/hmg/ddz237. PubMed DOI

Ramond F., Rio M., Heron B., Imbard A., Marie S., Billemaz K., Denomme-Pichon A.S., Kuentz P., Ceballos I., Piraud M., et al. AICA-ribosiduria due to ATIC deficiency: Delineation of the phenotype with three novel cases, and long-term update on the first case. J. Inherit. Metab. Dis. 2020;43:1254–1264. doi: 10.1002/jimd.12274. PubMed DOI

Jurecka A., Zikanova M., Kmoch S., Tylki-Szymanska A. Adenylosuccinate lyase deficiency. J. Inherit. Metab. Dis. 2015;38:231–242. doi: 10.1007/s10545-014-9755-y. PubMed DOI PMC

Stone T.W., Roberts L.A., Morris B.J., Jones P.A., Ogilvy H.A., Behan W.M., Duley J.A., Simmonds H.A., Vincent M.F., van den Berghe G. Succinylpurines induce neuronal damage in the rat brain. Adv. Exp. Med. Biol. 1998;431:185–189. doi: 10.1007/978-1-4615-5381-6_36. PubMed DOI

Jaeken J., van den Bergh F., Vincent M.F., Casaer P., van den Berghe G. Adenylosuccinase deficiency: A newly recognized variant. J. Inherit. Metab. Dis. 1992;15:416–418. doi: 10.1007/BF02435992. PubMed DOI

Jaeken J., van den Berghe G. An infantile autistic syndrome characterised by the presence of succinylpurines in body fluids. Lancet. 1984;2:1058–1061. PubMed

Zikanova M., Skopova V., Hnizda A., Krijt J., Kmoch S. Biochemical and structural analysis of 14 mutant adsl enzyme complexes and correlation to phenotypic heterogeneity of adenylosuccinate lyase deficiency. Hum. Mutat. 2010;31:445–455. doi: 10.1002/humu.21212. PubMed DOI

Krijt J., Kmoch S., Hartmannova H., Havlicek V., Sebesta I. Identification and determination of succinyladenosine in human cerebrospinal fluid. J. Chromatogr. B Biomed. Sci. Appl. 1999;726:53–58. doi: 10.1016/S0378-4347(99)00024-9. PubMed DOI

Adenylosuccinate Lyase Deficiency. [(accessed on 28 March 2022)]. Available online: https://www.adenylosuccinatelyasedeficiency.com/

Race V., Marie S., Kienlen-Campard P., Hermans E., Octave J.N., van den Berghe G., Vincent M.F. Adenylosuccinate lyase deficiency: Study of physiopathologic mechanism(s) Nucleosides Nucleotides Nucleic Acids. 2004;23:1227–1229. doi: 10.1081/NCN-200027491. PubMed DOI

Van den Bergh F., Vincent M.F., Jaeken J., van den Berghe G. Radiochemical assay of adenylosuccinase: Demonstration of parallel loss of activity toward both adenylosuccinate and succinylaminoimidazole carboxamide ribotide in liver of patients with the enzyme defect. Anal. Biochem. 1991;193:287–291. doi: 10.1016/0003-2697(91)90023-M. PubMed DOI

Van den Berghe G., Jaeken J. Adenylosuccinase deficiency. Adv. Exp. Med. Biol. 1986;195 Pt A:27–33. doi: 10.1007/978-1-4684-5104-7_4. PubMed DOI

Van den Bergh F., Vincent M.F., Jaeken J., van den Berghe G. Functional studies in fibroblasts of adenylosuccinase-deficient children. J. Inherit. Metab. Dis. 1993;16:425–434. doi: 10.1007/BF00710293. PubMed DOI

Lee H., DeLoache W.C., Dueber J.E. Spatial organization of enzymes for metabolic engineering. Metab. Eng. 2012;14:242–251. doi: 10.1016/j.ymben.2011.09.003. PubMed DOI

An S., Kumar R., Sheets E.D., Benkovic S.J. Reversible compartmentalization of de novo purine biosynthetic complexes in living cells. Science. 2008;320:103–106. doi: 10.1126/science.1152241. PubMed DOI

Baresova V., Skopova V., Sikora J., Patterson D., Sovova J., Zikanova M., Kmoch S. Mutations of ATIC and ADSL affect purinosome assembly in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency. Hum. Mol. Genet. 2012;21:1534–1543. doi: 10.1093/hmg/ddr591. PubMed DOI

Deng Y., Gam J., French J.B., Zhao H., An S., Benkovic S.J. Mapping protein-protein proximity in the purinosome. J. Biol. Chem. 2012;287:36201–36207. doi: 10.1074/jbc.M112.407056. PubMed DOI PMC

Chan C.Y., Zhao H., Pugh R.J., Pedley A.M., French J., Jones S.A., Zhuang X., Jinnah H., Huang T.J., Benkovic S.J. Purinosome formation as a function of the cell cycle. Proc. Natl. Acad. Sci. USA. 2015;112:1368–1373. doi: 10.1073/pnas.1423009112. PubMed DOI PMC

Proschel M., Detsch R., Boccaccini A.R., Sonnewald U. Engineering of Metabolic Pathways by Artificial Enzyme Channels. Front. Bioeng. Biotechnol. 2015;3:168. doi: 10.3389/fbioe.2015.00168. PubMed DOI PMC

Spurr I.B., Birts C.N., Cuda F., Benkovic S.J., Blaydes J.P., Tavassoli A. Targeting tumour proliferation with a small-molecule inhibitor of AICAR transformylase homodimerization. ChemBioChem. 2012;13:1628–1634. doi: 10.1002/cbic.201200279. PubMed DOI PMC

Van den Berghe G., Vincent M.F., Jaeken J. Inborn errors of the purine nucleotide cycle: Adenylosuccinase deficiency. J. Inherit. Metab. Dis. 1997;20:193–202. doi: 10.1023/A:1005304722259. PubMed DOI

Vincent M.F., van den Berghe G. Influence of succinylpurines on the binding of adenosine to a particulate fraction of rat cerebral cortex. Adv. Exp. Med. Biol. 1989;253:441–445. doi: 10.1007/978-1-4684-5676-9_65. PubMed DOI

Garcia D., Shaw R.J. AMPK: Mechanisms of Cellular Energy Sensing and Restoration of Metabolic Balance. Mol. Cell. 2017;66:789–800. doi: 10.1016/j.molcel.2017.05.032. PubMed DOI PMC

Hardie D.G. The AMP-activated protein kinase pathway—New players upstream and downstream. Pt 23J. Cell Sci. 2004;117:5479–5487. doi: 10.1242/jcs.01540. PubMed DOI

Jose C., Hebert-Chatelain E., Bellance N., Larendra A., Su M., Nouette-Gaulain K., Rossignol R. AICAR inhibits cancer cell growth and triggers cell-type distinct effects on OXPHOS biogenesis, oxidative stress and Akt activation. Biochim. Biophys. Acta. 2011;1807:707–718. doi: 10.1016/j.bbabio.2010.12.002. PubMed DOI

Keller K.E., Tan I.S., Lee Y.S. SAICAR stimulates pyruvate kinase isoform M2 and promotes cancer cell survival in glucose-limited conditions. Science. 2012;338:1069–1072. doi: 10.1126/science.1224409. PubMed DOI PMC

Liu X., Chhipa R.R., Pooya S., Wortman M., Yachyshin S., Chow L.M., Kumar A., Zhou X., Sun Y., Quinn B., et al. Discrete mechanisms of mTOR and cell cycle regulation by AMPK agonists independent of AMPK. Proc. Natl. Acad. Sci. USA. 2014;111:E435–E444. doi: 10.1073/pnas.1311121111. PubMed DOI PMC

Lopez J.M., Santidrian A.F., Campas C., Gil J. 5-Aminoimidazole-4-carboxamide riboside induces apoptosis in Jurkat cells, but the AMP-activated protein kinase is not involved. Pt 3Biochem. J. 2003;370:1027–1032. doi: 10.1042/bj20021053. PubMed DOI PMC

Racanelli A.C., Rothbart S.B., Heyer C.L., Moran R.G. Therapeutics by cytotoxic metabolite accumulation: Pemetrexed causes ZMP accumulation, AMPK activation, and mammalian target of rapamycin inhibition. Cancer Res. 2009;69:5467–5474. doi: 10.1158/0008-5472.CAN-08-4979. PubMed DOI PMC

Su C.C., Hsieh K.L., Liu P.L., Yeh H.C., Huang S.P., Fang S.H., Cheng W.C., Huang K.H., Chiu F.Y., Lin I.L., et al. AICAR Induces Apoptosis and Inhibits Migration and Invasion in Prostate Cancer Cells through an AMPK/mTOR-Dependent Pathway. Int. J. Mol. Sci. 2019;20:1647. doi: 10.3390/ijms20071647. PubMed DOI PMC

Ali E.S., Sahu U., Villa E., O’Hara B.P., Gao P., Beaudet C., Wood A.W., Asara J.M., Ben-Sahra I. ERK2 Phosphorylates PFAS to Mediate Posttranslational Control of De Novo Purine Synthesis. Mol. Cell. 2020;78:1178–1191.e6. doi: 10.1016/j.molcel.2020.05.001. PubMed DOI PMC

Goswami M.T., Chen G., Chakravarthi B.V., Pathi S.S., Anand S.K., Carskadon S.L., Giordano T.J., Chinnaiyan A.M., Thomas D.G., Palanisamy N., et al. Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer. Oncotarget. 2015;6:23445–23461. doi: 10.18632/oncotarget.4352. PubMed DOI PMC

Chakravarthi B., Rodriguez Pena M.D.C., Agarwal S., Chandrashekar D.S., Hodigere Balasubramanya S.A., Jabboure F.J., Matoso A., Bivalacqua T.J., Rezaei K., Chaux A., et al. A Role for De Novo Purine Metabolic Enzyme PAICS in Bladder Cancer Progression. Neoplasia. 2018;20:894–904. doi: 10.1016/j.neo.2018.07.006. PubMed DOI PMC

Chakravarthi B.V., Goswami M.T., Pathi S.S., Dodson M., Chandrashekar D.S., Agarwal S., Nepal S., Hodigere Balasubramanya S.A., Siddiqui J., Lonigro R.J., et al. Expression and Role of PAICS, a De Novo Purine Biosynthetic Gene in Prostate Cancer. Prostate. 2017;77:10–21. doi: 10.1002/pros.23243. PubMed DOI

Jiang T., Sanchez-Rivera F.J., Soto-Feliciano Y.M., Yang Q., Song C.Q., Bhuatkar A., Haynes C.M., Hemann M.T., Xue W. Targeting the De Novo Purine Synthesis Pathway through Adenylosuccinate Lyase Depletion Impairs Liver Cancer Growth by Perturbing Mitochondrial Function. Hepatology. 2021;74:233–247. doi: 10.1002/hep.31685. PubMed DOI PMC

Lv Y., Wang X., Li X., Xu G., Bai Y., Wu J., Piao Y., Shi Y., Xiang R., Wang L. Nucleotide de novo synthesis increases breast cancer stemness and metastasis via cGMP-PKG-MAPK signaling pathway. PLoS Biol. 2020;18:e3000872. doi: 10.1371/journal.pbio.3000872. PubMed DOI PMC

Meng M., Chen Y., Jia J., Li L., Yang S. Knockdown of PAICS inhibits malignant proliferation of human breast cancer cell lines. Biol. Res. 2018;51:24. doi: 10.1186/s40659-018-0172-9. PubMed DOI PMC

Zhang H., Xia P., Liu J., Chen Z., Ma W., Yuan Y. ATIC inhibits autophagy in hepatocellular cancer through the AKT/FOXO3 pathway and serves as a prognostic signature for modeling patient survival. Int. J. Biol. Sci. 2021;17:4442–4458. doi: 10.7150/ijbs.65669. PubMed DOI PMC

Zhou S., Yan Y., Chen X., Wang X., Zeng S., Qian L., Wei J., Yang X., Zhou Y., Gong Z., et al. Roles of highly expressed PAICS in lung adenocarcinoma. Gene. 2019;692:1–8. doi: 10.1016/j.gene.2018.12.064. PubMed DOI

Baresova V., Krijt M., Skopova V., Souckova O., Kmoch S., Zikanova M. CRISPR-Cas9 induced mutations along de novo purine synthesis in HeLa cells result in accumulation of individual enzyme substrates and affect purinosome formation. Mol. Genet. Metab. 2016;119:270–277. doi: 10.1016/j.ymgme.2016.08.004. PubMed DOI

Furukawa J., Inoue K., Maeda J., Yasujima T., Ohta K., Kanai Y., Takada T., Matsuo H., Yuasa H. Functional identification of SLC43A3 as an equilibrative nucleobase transporter involved in purine salvage in mammals. Sci. Rep. 2015;5:15057. doi: 10.1038/srep15057. PubMed DOI PMC

Pastor-Anglada M., Perez-Torras S. Emerging Roles of Nucleoside Transporters. Front. Pharmacol. 2018;9:606. doi: 10.3389/fphar.2018.00606. PubMed DOI PMC

Zikanova M., Krijt J., Hartmannova H., Kmoch S. Preparation of 5-amino-4-imidazole-N-succinocarboxamide ribotide, 5-amino-4-imidazole-N-succinocarboxamide riboside and succinyladenosine, compounds usable in diagnosis and research of adenylosuccinate lyase deficiency. J. Inherit. Metab. Dis. 2005;28:493–499. doi: 10.1007/s10545-005-0493-z. PubMed DOI

Madrova L., Krijt M., Baresova V., Vaclavik J., Friedecky D., Dobesova D., Souckova O., Skopova V., Adam T., Zikanova M. Mass spectrometric analysis of purine de novo biosynthesis intermediates. PLoS ONE. 2018;13:e0208947. doi: 10.1371/journal.pone.0208947. PubMed DOI PMC

Scientific Instrument Services (SIS) by Adaptas Solutions. [(accessed on 28 March 2022)]. Available online: https://www.sisweb.com/mstools/isotope.htm.

Hayes D.P. Nutritional hormesis. Eur. J. Clin. Nutr. 2007;61:147–159. doi: 10.1038/sj.ejcn.1602507. PubMed DOI

Mouchegh K., Zikanova M., Hoffmann G.F., Kretzschmar B., Kuhn T., Mildenberger E., Stoltenburg-Didinger G., Krijt J., Dvorakova L., Honzik T., et al. Lethal fetal and early neonatal presentation of adenylosuccinate lyase deficiency: Observation of 6 patients in 4 families. J. Pediatr. 2007;150:57–61.e2. doi: 10.1016/j.jpeds.2006.09.027. PubMed DOI

Bonan C.D. Ectonucleotidases and nucleotide/nucleoside transporters as pharmacological targets for neurological disorders. CNS Neurol. Disord. Drug Targets. 2012;11:739–750. doi: 10.2174/187152712803581092. PubMed DOI

Keller K.E., Doctor Z.M., Dwyer Z.W., Lee Y.S. SAICAR induces protein kinase activity of PKM2 that is necessary for sustained proliferative signaling of cancer cells. Mol. Cell. 2014;53:700–709. doi: 10.1016/j.molcel.2014.02.015. PubMed DOI PMC

Boison D. Adenosine as a neuromodulator in neurological diseases. Curr. Opin. Pharmacol. 2008;8:2–7. doi: 10.1016/j.coph.2007.09.002. PubMed DOI PMC

Fumagalli M., Lecca D., Abbracchio M.P., Ceruti S. Pathophysiological Role of Purines and Pyrimidines in Neurodevelopment: Unveiling New Pharmacological Approaches to Congenital Brain Diseases. Front. Pharmacol. 2017;8:941. doi: 10.3389/fphar.2017.00941. PubMed DOI PMC

Burnstock G. Pathophysiology and therapeutic potential of purinergic signaling. Pharmacol. Rev. 2006;58:58–86. doi: 10.1124/pr.58.1.5. PubMed DOI

Lara D.R., Dall’Igna O.P., Ghisolfi E.S., Brunstein M.G. Involvement of adenosine in the neurobiology of schizophrenia and its therapeutic implications. Prog. Neuropsychopharmacol. Biol. Psychiatry. 2006;30:617–629. doi: 10.1016/j.pnpbp.2006.02.002. PubMed DOI

Ipata P.L., Camici M., Micheli V., Tozz M.G. Metabolic network of nucleosides in the brain. Curr. Top. Med. Chem. 2011;11:909–922. doi: 10.2174/156802611795347555. PubMed DOI

Pastor-Anglada M., Felipe A., Casado F.J. Transport and mode of action of nucleoside derivatives used in chemical and antiviral therapies. Trends Pharmacol. Sci. 1998;19:424–430. doi: 10.1016/S0165-6147(98)01253-X. PubMed DOI

Douillet D.C., Pinson B., Ceschin J., Hurlimann H.C., Saint-Marc C., Laporte D., Claverol S., Konrad M., Bonneu M., Daignan-Fornier B. Metabolomics and proteomics identify the toxic form and the associated cellular binding targets of the anti-proliferative drug AICAR. J. Biol. Chem. 2019;294:805–815. doi: 10.1074/jbc.RA118.004964. PubMed DOI PMC

Scudiero O., Nigro E., Monaco M.L., Oliviero G., Polito R., Borbone N., D’Errico S., Mayol L., Daniele A., Piccialli G. New synthetic AICAR derivatives with enhanced AMPK and ACC activation. J. Enzyme Inhib. Med. Chem. 2016;31:748–753. doi: 10.3109/14756366.2015.1063622. PubMed DOI

Menze M.A., Chakraborty N., Clavenna M., Banerjee M., Liu X.H., Toner M., Hand S.C. Metabolic preconditioning of cells with AICAR-riboside: Improved cryopreservation and cell-type specific impacts on energetics and proliferation. Cryobiology. 2010;61:79–88. doi: 10.1016/j.cryobiol.2010.05.004. PubMed DOI PMC

Gadalla A.E., Pearson T., Currie A.J., Dale N., Hawley S.A., Sheehan M., Hirst W., Michel A.D., Randall A., Hardie D.G., et al. AICA riboside both activates AMP-activated protein kinase and competes with adenosine for the nucleoside transporter in the CA1 region of the rat hippocampus. J. Neurochem. 2004;88:1272–1282. doi: 10.1046/j.1471-4159.2003.02253.x. PubMed DOI

Young M.E., Radda G.K., Leighton B. Activation of glycogen phosphorylase and glycogenolysis in rat skeletal muscle by AICAR—An activator of AMP-activated protein kinase. FEBS Lett. 1996;382:43–47. doi: 10.1016/0014-5793(96)00129-9. PubMed DOI

Estramareix B., David S. Biosynthesis of thiamine: Origin of the methyl carbon atom of the pyrimidine moiety in Salmonella typhimurium. Biochem. Biophys. Res. Commun. 1986;134:1136–1141. doi: 10.1016/0006-291X(86)90369-4. PubMed DOI

Ali I., Lone M.N., Aboul-Enein H.Y. Imidazoles as potential anticancer agents. MedChemComm. 2017;8:1742–1773. doi: 10.1039/C7MD00067G. PubMed DOI PMC

Expanding clinical spectrum of PAICS deficiency: Comprehensive analysis of two sibling cases