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.

• Klíčová slova
• Glutamine antagonist, LC-MS., biomarker, cancer, formylglycinamide ribonucleotide, formylglycinamidine ribonucleotide, purine synthesis,
• MeSH
• biomarkery farmakologické analýza   metabolismus   MeSH
• chromatografie kapalinová metody   MeSH
• glutamin antagonisté a inhibitory   MeSH
• glycin analogy a deriváty   analýza   metabolismus   MeSH
• hmotnostní spektrometrie metody   MeSH
• metabolické sítě a dráhy účinky léků   MeSH
• myši MeSH
• nádorové biomarkery analýza   metabolismus   MeSH
• nádory * farmakoterapie   metabolismus   MeSH
• ribonukleotidy * analýza   metabolismus   MeSH
• vyvíjení léků metody   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biomarkery farmakologické MeSH
• glutamin MeSH
• glycin MeSH
• nádorové biomarkery MeSH
• phosphoribosyl-N-formylglycineamide MeSH Prohlížeč
• ribonukleotidy * 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.

• Klíčová slova
• ADSL, AICAR, AIR, ATIC, FGAR, PAICS, PFAS, SAICAR, cytotoxicity, purine synthesis,
• Publikační typ
• časopisecké články MeSH

The cellular pool of purines is maintained by de novo purine synthesis (DNPS), recycling and degradation. Mutations in genes encoding DNPS enzymes cause their substrates to accumulate, which has detrimental effects on cellular division and organism development, potentially leading to neurological impairments. Unspecified neurological symptoms observed in many patients could not be elucidated even by modern techniques. It is presumable that some of these problems are induced by dysfunctions in DNPS enzymes. Therefore, we determined the concentrations of dephosphorylated DNPS intermediates by LC-MS/MS as markers of yet unpublished mutations in PFAS and PAICS genes connected with dysfunctions of carboxylase/phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS) or phosphoribosylformylglycinamidine synthase (PFAS). We determined the criteria for normal values of metabolites and investigated 1,447 samples of urine and 365 dried blood spots of patients suffering from various forms of neurological impairment. We detected slightly elevated aminoimidazole riboside (AIr) concentrations in three urine samples and a highly elevated 5-formamidoimidazole-4-carboxamide riboside (FGAr) concentration in one urine sample. The accumulation of AIr or FGAr in body fluids can indicate PAICS or PFAS deficiency, respectively, which would be new disorders of DNPS caused by mutations in the appropriate genes. Measurement of DNPS intermediates in patients with neurological symptoms can uncover the cause of serious cellular and functional impairments that are otherwise inaccessible to detection. Further genetic and molecular analysis of these patients should establish the causal mutations for prenatal diagnosis, genetic consultation, and reinforce the DNPS pathway as a therapeutic target.

• MeSH
• lidé MeSH
• limita detekce MeSH
• metabolom MeSH
• metabolomika metody   MeSH
• moč MeSH
• mutace genetika   MeSH
• nemoci nervového systému genetika   metabolismus   MeSH
• puriny biosyntéza   krev   chemie   moč   MeSH
• test suché kapky krve MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• purine MeSH Prohlížeč
• puriny MeSH