Inhibition of Amido Phosphoribosyltransferase by Antifolates in Mouse L1210 Leukaemia Cells

Sant, Melissa E.; Lyons, Stephen D.; Phillips, Leonidas; Szabados, Eve; Christopherson, Richard I.

doi:10.1515/pteridines.1991.3.12.133

Cited by 12 publications

(26 citation statements)

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“…The pronounced accumulation of ZMP despite a minimal increase in dUMP after prolonged exposure to low concentrations of MTX suggests that low drug doses can be used to selectively target the accumulation of the anti-inflammatory TABLE 2 Inhibition constants (K i ) of enzymes involved in nucleotide biosynthesis for MTX and its polyglutamates (Allegra et al, 1985a,b;Drake et al, 1987;Baram et al, 1988;Sant et al, 1992) (Allegra et al, 1985b;Baram et al, 1988;Sant et al, 1992) Selective Accumulation of ZMP with Low-Dose Methotrexate mediator without significantly inhibiting TS activity (Fig. 7).…”

Section: Discussionmentioning

confidence: 99%

“…The pharmacodynamic hypothesis is based on the notion that the polyglutamates of MTX are the biologically active form of the drug and, therefore, may be predictive of pharmacological activity. Metabolism of MTX to its polyglutamated forms has been found to result in increasingly potent inhibition of enzymes of nucleotide biosynthesis, including thymidylate synthase (TS), phosphoribosylglycinamide formyltransferase (GART), aminoimidazole carboxamide ribonucleotide transformylase (AICART), and amido phosphoribosyltransferase (ATase) (Allegra et al, 1985a,b;Baram et al, 1988;Sant et al, 1992). However, such a correlation between MTX polyglutamation and inhibition of these enzymes in a cellular system has not been experimentally evaluated.…”

Section: Introductionmentioning

confidence: 99%

“…Specifically, the antiproliferative activity of MTX is primarily attributed to inhibition of purine and pyrimidine nucleotide biosynthesis through depletion of intracellular reduced-folate cofactors, via inhibition of dihydrofolate reductase (DHFR), and direct inhibition of TS and enzymes of de novo purine biosynthesis, including ATase, GART, and AICART (Borsa and Whitmore, 1969b;Pinedo et al, 1976). In addition, inhibition of DHFR by MTX results in the cellular accumulation of dihydrofolate (DHF), which also has varying potency in inhibition of these enzymes (Allegra et al, 1985bBaram et al, 1988;Chu et al, 1990;Sant et al, 1992). The anti-inflammatory mechanism of MTX activity remains controversial, but current evidence supports the inhibition of the purine synthesis pathway through direct inhibition of AICART ( Fig.…”

Section: Introductionmentioning

confidence: 99%

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Low-Dose Methotrexate Results in the Selective Accumulation of Aminoimidazole Carboxamide Ribotide in an Erythroblastoid Cell Line

Funk

Haandel

Becker

et al. 2013

J Pharmacol Exp Ther

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Therapeutic and toxic response to low-dose methotrexate (MTX) in the treatment of autoimmune disease continues to be highly variable, resulting in a critical need to identify predictive biomarkers of response. Biomarker development has been hampered by an incomplete understanding of the molecular pharmacology of low-dose MTX. To address this issue, accumulation of the substrates for aminoimidazole carboxamide ribonucleotide transformylase (AICART) and thymidylate synthase (TS) was measured as markers of pharmacological activity of MTX in an erythroblastoid cell line. A 115-fold increase in the AICART substrate and anti-inflammatory mediator, 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranosyl 59-monophosphate (ZMP), was observed following exposure to 10 nM MTX but subsequently decreased with increasing MTX concentrations, declining to baseline levels with 1000 nM MTX. In contrast, the TS substrate, 29-deoxyuridine 59-monophosphate disodium salt (dUMP), displayed concentration-dependent accumulation, increasing 29-, 342-, and 471-fold over baseline with 10, 100, and 1000 nM MTX, respectively. Cellular levels of dUMP correlated with levels of the parent drug (MTX-PG 1 ; r 5 0.66, P , 0.001) and its polyglutamates (MTX-PG 2-6 ) (r 5 0.81, P , 0.001), whereas cellular levels of ZMP were only moderately correlated with MTX-PG 1 (r 5 0.34, P , 0.01). In contrast, accumulation of ZMP at 10 nM MTX was associated with a 2.9-fold increase in the AICART inhibitor dihydrofolate (DHF), represented primarily by long-chain DHF polyglutamates. Selectivity, defined as the ratio of ZMP to dUMP, was maximal following exposure to 6 nM MTX. Characterizing the range of MTX concentrations that selectively promote ZMP accumulation while preserving pyrimidine biosynthesis may lead to optimization of low-dose MTX therapy.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Low-Dose Methotrexate Results in the Selective Accumulation of Aminoimidazole Carboxamide Ribotide in an Erythroblastoid Cell Line

Funk

Haandel

Becker

et al. 2013

J Pharmacol Exp Ther

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“…Dihydrofolate polyglutamates at these concentrations are capable of inhibiting the first committed step of purine biosynthesis catalyzed by PPAT and the two transformylase reactions catalyzed by GART and AICART [22]. In addition to dihydrofolate polyglutamates, methotrexate polyglutamates have also been implicated as effectors of inhibition of these three steps of de novo purine synthesis [22], [33], [34], [54].…”

Section: Discussionmentioning

confidence: 99%

LD-Aminopterin in the Canine Homologue of Human Atopic Dermatitis: A Randomized, Controlled Trial Reveals Dosing Factors Affecting Optimal Therapy

Zebala

Mundell²,

Messinger³

et al. 2014

PLoS ONE

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BackgroundOptions are limited for patients with atopic dermatitis (AD) who do not respond to topical treatments. Antifolate therapy with systemic methotrexate improves the disease, but is associated with adverse effects. The investigational antifolate LD-aminopterin may offer improved safety. It is not known how antifolate dose and dosing frequency affect efficacy in AD, but a primary mechanism is thought to involve the antifolate-mediated accumulation of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). However, recent in vitro studies indicate that AICAR increases then decreases as a function of antifolate concentration. To address this issue and understand how dosing affects antifolate efficacy in AD, we examined the efficacy and safety of different oral doses and schedules of LD-aminopterin in the canine model of AD.Methods and FindingsThis was a multi-center, double-blind trial involving 75 subjects with canine AD randomized to receive up to 12 weeks of placebo, once-weekly (0.007, 0.014, 0.021 mg/kg) or twice-weekly (0.007 mg/kg) LD-aminopterin. The primary efficacy outcome was the Global Score (GS), a composite of validated measures of disease severity and itch. GS improved in all once-weekly cohorts, with 0.014 mg/kg being optimal and significant (43%, P<0.01). The majority of improvement was seen by 8 weeks. In contrast, GS in the twice-weekly cohort was similar to placebo and worse than all once-weekly cohorts. Adverse events were similar across all treated cohorts and placebo.ConclusionsOnce-weekly LD-aminopterin was safe and efficacious in canine AD. Twice-weekly dosing negated efficacy despite having the same daily and weekly dose as effective once-weekly regimens. Optimal dosing in this homologue of human AD correlated with the concentration-selective accumulation of AICAR in vitro, consistent with AICAR mediating LD-aminopterin efficacy in AD.

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“…The cellular effects of these drugs likely vary due to their degree of specificity for a particular enzyme. For example, methotrexate targets three enzymes in the purine de novo synthesis pathway (Sant et al 1992), whereas DDATHF targets a single enzyme (Christopherson et al 2002). In addition, the effects of inhibitors vary with cell type, leading to death or reversible arrest, depending on the presence or absence of a G 1 checkpoint, respectively (Zhang et al 1998).…”

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confidence: 99%

A Link Between Impaired Purine Nucleotide Synthesis and Apoptosis in Drosophila melanogaster

Holland¹,

Lipsett²,

Clark

2011

Genetics

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The biosynthetic pathways and multiple functions of purine nucleotides are well known. However, the pathways that respond to alterations in purine nucleotide synthesis in vivo in an animal model organism have not been identified. We examined the effects of inhibiting purine de novo synthesis in vivo and in cultured cells of Drosophila melanogaster. The purine de novo synthesis gene ade2 encodes phosphoribosylformylglycinamidine synthase (EC 6.3.5.3). An ade2 deletion, generated by P-element transposon excision, causes lethality in early pupal development, with darkening, or necrosis, of leg and wing imaginal disc tissue upon disc eversion. Together with analysis of a previously isolated weaker allele, ade2 4 , and an allele of the Prat gene, which encodes an enzyme for the first step in the pathway, we determined that the lethal arrest and imaginal disc phenotypes involve apoptosis. A transgene expressing the baculovirus caspase inhibitor p35, which suppresses apoptosis caused by other stresses such as DNA damage, suppresses both the imaginal disc tissue darkening and the pupal lethality of all three purine de novo synthesis mutants. Furthermore, we showed the presence of apoptosis at the cellular level in both ade2 and Prat mutants by detecting TUNEL-positive nuclei in wing imaginal discs. Purine de novo synthesis inhibition was also examined in tissue culture by ade2 RNA interference followed by analysis of genome-wide changes in transcript levels. Among the upregulated genes was HtrA2, which encodes an apoptosis effector and is thus a candidate for initiating apoptosis in response to purine depletion.

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Inhibition of Amido Phosphoribosyltransferase by Antifolates in Mouse L1210 Leukaemia Cells

Cited by 12 publications

References 0 publications

Low-Dose Methotrexate Results in the Selective Accumulation of Aminoimidazole Carboxamide Ribotide in an Erythroblastoid Cell Line

Low-Dose Methotrexate Results in the Selective Accumulation of Aminoimidazole Carboxamide Ribotide in an Erythroblastoid Cell Line

LD-Aminopterin in the Canine Homologue of Human Atopic Dermatitis: A Randomized, Controlled Trial Reveals Dosing Factors Affecting Optimal Therapy

A Link Between Impaired Purine Nucleotide Synthesis and Apoptosis in Drosophila melanogaster

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