Aminoimidazole Carboxamide Ribonucleotide Transformylase Research Articles

Beneficial effect of methotrexate on a case of Nakajo-Nishimura syndrome

Nakajo-Nishimura syndrome (NNS) is a very rare autosomal recessively-inherited autoinflammatory disorder that onsets in infancy with pernio-like rashes and gradually develops into partial lipodystrophy, accompanied with remittent fever and nodular skin eruptions. This disease is caused by a unique mutation of the PSMB8 gene, which not only impairs an enzymatic activity of the encoding beta5i subunit, but also disturbs formation of the immunoproteasome complex. As the pathogenesis for NNS, cellular accumulation of ubiquitinated and oxidized proteins due to immunoproteasome deficiency is considered to cause MAP kinase activation with nuclear accumulation of phosphorylated p38 and following IL-6 production. The treatment for Nakajo-Nishimura syndrome has not been established. Inflammatory attacks can temporarily respond to the oral administration of high-dose corticosteroid, but they easily recur by tapering the dose of corticosteroid. Furthermore, the high-dose corticosteroid therapy has various side effects such as growth failure in infancy. In our child case of NNS (Kunimoto et al, Dermatology 2013), additional administration of methotrexate (MTX) significantly decreased a frequency of febrile attacks, in comparison to the treatment with oral corticosteroid alone. Notably, effectiveness of MTX was previously described on some infant cases of CANDLE syndrome, another PSMB8-mutated proteasome-associated autoinflammatory syndrome (PRAAS). MTX is known to execute anti-inflammatory effects through inhibition of folic acid-dependent enzymes, including dihydrofolate reductase (DHFR) and aminoimidazole carboxamide ribonucleotide transformylase (ATIC). ATIC inhibition causes accumulation of intracellular aminoimidazole carboxamide ribonucleotide and inhibits AMP deaminase, to increase the production of adenosine. Adenosine can inhibit superoxide production of neutrophils and their attachment to endothelial cells. As preliminary results, increased ROS production has been observed in primary neutrophils of NNS patients, suggesting one of the points that MTX affects.

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

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-β-d-ribofuranosyl 5'-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, 2'-deoxyuridine 5'-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-PG1; r = 0.66, P < 0.001) and its polyglutamates (MTX-PG2-6) (r = 0.81, P < 0.001), whereas cellular levels of ZMP were only moderately correlated with MTX-PG1 (r = 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.

Targeting Tumour Proliferation with a Small‐Molecule Inhibitor of AICAR Transformylase Homodimerization

Aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is a bifunctional homodimeric enzyme that catalyzes the last two steps of de novo purine biosynthesis. Homodimerization of ATIC, a protein-protein interaction with an interface of over 5000 Å(2), is required for its aminoimidazole carboxamide ribonucleotide (AICAR) transformylase activity, with the active sites forming at the interface of the interacting proteins. Here, we report the development of a small-molecule inhibitor of AICAR transformylase that functions by preventing the homodimerization of ATIC. The compound is derived from a previously reported cyclic hexapeptide inhibitor of AICAR transformylase (with a K(i) of 17 μM), identified by high-throughput screening. The active motif of the cyclic peptide is identified as an arginine-tyrosine dipeptide, a capped analogue of which inhibits AICAR transformylase with a K(i) value of 84 μM. A library of nonnatural analogues of this dipeptide was designed, synthesized, and assayed. The most potent compound inhibits AICAR transformylase with a K(i) value of 685 nM, a 25-fold improvement in activity from the parent cyclic peptide. The potential for this AICAR transformylase inhibitor in cancer therapy was assessed by studying its effect on the proliferation of a model breast cancer cell line. Using a nonradioactive proliferation assay and live cell imaging, a dose-dependent reduction in cell numbers and cell division rates was observed in cells treated with our ATIC dimerization inhibitor.

Patterns of interaction between genetic and nongenetic attributes and methotrexate efficacy in rheumatoid arthritis

The contribution of low-penetrance single nucleotide polymorphisms to methotrexate efficacy in rheumatoid arthritis (RA) is inconsistent between studies. We sought to elucidate architecture of methotrexate response in three cohorts of patients with RA treated with methotrexate. Single nucleotide polymorphism frequencies in genes from folate, purine, and pyrimidine pathways were measured to develop a model of gene-gene interactions using multifactor dimensionality reduction in 439 patients who received methotrexate in the USA and The Netherlands. A third cohort of 530 patients with RA from Sweden was used to replicate the findings. Methotrexate efficacy was assessed using the European League Against Rheumatism criteria in the majority of patients. Nonlinear patterns of gene-gene interactions between variants in aminoimidazole carboxamide ribonucleotide transformylase (C347G), reduced-folate carrier (G80A) and inosine-triphosphate pyrophosphatase (C94A) revealed a predisposing genetic attribute significantly associated with methotrexate response in the USA and Dutch cohorts [odds ratio (OR)=2.9, 95% confidence interval (CI): 1.9-4.2; P<0.001]. Although the finding was not replicated in the Swedish cohort (OR=0.9; 95% CI: 0.64-1.37; P=0.74) a multifactor dimensionality reduction analysis superimposing the predisposing genetic attribute with patient's age, sex, and anticitrullinated peptide antibodies positivity (ACPA) revealed a pattern of interaction significant in all three cohorts (OR=2.2, 95% CI: 1.6-2.9; P<0.01). The selective advantage toward response in the presence of the predisposing genetic attribute was lost in females and ACPA-positive patients, whereas older and male ACPA-negative patients tended to exhibit a greater likelihood of response in the absence of the predisposing genetic attribute. Gene-gene interactions together with nongenetic attributes may contribute to methotrexate efficacy in RA.

Influence of Reduced Folate Carrier and Aminoimidazole Carboxamide Ribonucleotide Transformylase gene polymorphisms on the efficacy of methotrexate in juvenile idiopathic arthritis

Methods Ninety seven JIA patients with a polyarticular course were enrolled into the study. All patients received methotrexate treatment for at least for 6 months. The efficacy of treatment was judged by improvement in physician’s global assesment, the number of affected joints, erythrocyte sedimentation rate and C-reactive protein. RFC and ATIC gene polymorphisms were determined by polymerase chain reactionrestriction fragment length polymorphism ( PCR-RFLP) analysis. Results The median age of the patients was 72 (12-92) months. The response rate to MTX was 45%. There was no significant association between RFC and ATIC polymorphisms and efficacy of MTX treatment (p: 0.254, p: 0.098 respectively).

Gene–gene interactions in folate and adenosine biosynthesis pathways affect methotrexate efficacy and tolerability in rheumatoid arthritis

As no single nucleotide polymorphism has emerged as pivotal to predict the lack of efficacy and dose-limiting toxicities to methotrexate (MTX), we evaluated the contribution of gene-gene interactions to the effects of this prodrug in rheumatoid arthritis. A total of 255 patients treated with MTX for at least 3 months were evaluated with efficacy assessed using the European League Against Rheumatism response criteria or a physician's assessment of patient's response to MTX visual analog scale. Gastrointestinal and neurological idiosyncrasies were recorded in 158 patients. Fourteen single nucleotide polymorphisms in folate and adenosine biosynthesis pathways were measured and detection of gene-gene interactions was performed using multifactor-dimensionality reduction, a method that reduces high-dimensional genetic data into a single dimension of predisposing or risk-genotype combinations. Efficacy to MTX (53% responders) was associated with high-order epistasis among variants in inosine-triphosphate pyrophosphatase, aminoimidazole-carboxamide ribonucleotide transformylase, and reduced folate carrier genes. In the absence of predisposing genotype combinations, a 3.8-fold lower likelihood of efficacy was observed (vs. in their presence, 95% confidence interval: 2.2-6.4; P<0.001). Increasing MTX polyglutamate concentrations tended to partially overcome this selective disadvantage. Idiosyncrasies occurred in 29% of patients. In the presence of risk-genotype combinations among variants in methylene tetrahydrofolate reductase, γ-glutamyl-hydrolase, thymidylate synthase, serine hydroxymethyltransferase, and inosine-triphosphate pyrophosphatase genes, an 8.9-fold higher likelihood to exhibit toxicities was observed (vs. in their absence, 95% confidence interval: 3.6-21.9; P<0.001). False-positive report probabilities were below 0.2, thereby indicating that true signals were likely detected in this cohort. These data indicate that gene-gene interactions impact MTX efficacy and tolerability in rheumatoid arthritis.

The Phosphatidylinositol 3-Kinase/Akt Cassette Regulates Purine Nucleotide Synthesis

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is highly conserved throughout evolution and regulates cell size and survival and cell cycle progression. It regulates the latter by stimulating procession through G(1) and the G(1)/S phase transition. Entry into S phase requires an abundant supply of purine nucleotides, but the effect of the PI3K/Akt pathway on purine synthesis has not been studied. We now show that the PI3K/Akt cassette regulates both de novo and salvage purine nucleotide synthesis in insulin-responsive mouse mesenchymal cells. We found that serum and insulin stimulated de novo purine synthesis in serum-starved cells largely through PI3K/Akt signaling, and pharmacologic and genetic inhibition of PI3K/Akt reduced de novo synthesis by 75% in logarithmically growing cells. PI3K/Akt regulated early steps of de novo synthesis by modulating phosphoribosylpyrophosphate production by the non-oxidative pentose phosphate pathway and late steps by modulating activity of the bifunctional enzyme aminoimidazole-carboxamide ribonucleotide transformylase IMP cyclohydrolase, an enzyme not previously known to be regulated. The effects of PI3K/Akt on purine nucleotide salvage were likely through regulating phosphoribosylpyrophosphate availability. These studies define a new mechanism whereby the PI3K/Akt cassette functions as a master regulator of cellular metabolism and a key player in oncogenesis.

Discovery of AICAR Tfase inhibitors that disrupt requisite enzyme dimerization

The discovery of a new class of aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) inhibitors through screening peptidomimetic libraries (>40,000 compounds) that act by inhibiting requisite enzyme dimerization is disclosed. In addition to defining key structural features of the lead compounds responsible for the activity, kinetic analysis of the remarkably small inhibitors established that they act as noncompetitive, dissociative inhibitors of AICAR Tfase with the prototypical lead ( A1B3, Cappsin 1) exhibiting a K i of 3.1 ± 0.3 μM. Thus, the studies define a unique approach to selectively targeting AICAR Tfase over all other folate-dependent enzymes, and it represents only one of a few enzymes for which inhibition achieved by disrupting requisite enzyme dimerization has emerged from screening unbiased combinatorial libraries.

Design, synthesis, and biological evaluation of 10-methanesulfonyl-DDACTHF, 10-methanesulfonyl-5-DACTHF, and 10-methylthio-DDACTHF as potent inhibitors of GAR Tfase and the de novo purine biosynthetic pathway

The synthesis and evaluation of 10-methanesulfonyl-DDACTHF (1), 10-methanesulfonyl-5-DACTHF (2), and 10-methylthio-DDACTHF (3) as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) are reported. The compounds 10-methanesulfonyl-DDACTHF (1, Ki=0.23μM), 10-methanesulfonyl-5-DACTHF (2, Ki=0.58μM), and 10-methylthio-DDACTHF (3, Ki=0.25μM) were found to be selective and potent inhibitors of recombinant human GAR Tfase. Of these, 3 exhibited exceptionally potent, purine sensitive growth inhibition activity (3, IC50=100nM) against the CCRF–CEM cell line being 3-fold more potent than Lometrexol and 30-fold more potent than the parent, unsubstituted DDACTHF, whereas 1 and 2 exhibited more modest growth inhibition activity (1, IC50=1.0μM and 2, IC50=2.0μM).

Synthesis and biological evaluation of N-{4-[5-(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pentyl]benzoyl}- l-glutamic acid as a potential inhibitor of GAR Tfase and the de novo purine biosynthetic pathway

The synthesis and evaluation of N-{4-[5-(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pentyl]benzoyl}- l-glutamic acid ( 2) as an inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) are reported. The inhibitor 2 was prepared in a convergent synthesis involving C-alkylation of methyl 4-(4,4,4-trifluoro-3-dimethylhydrazonobutyl)benzoate with 1-chloro-3-iodopropane followed by construction of the pyrimidinone ring. Compound 2 was found to be an effective inhibitor of recombinant human GAR Tfase ( K i = 0.50 μM), whereas it was inactive ( K i > 100 μM) against E. coli GAR Tfase as well as recombinant human AICAR Tfase. Compound 2 exhibited modest, purine-sensitive growth inhibitory activity against the CCRF-CEM cell line (IC 50 = 6.0 μM).

Polyglutamation of methotrexate with common polymorphisms in reduced folate carrier, aminoimidazole carboxamide ribonucleotide transformylase, and thymidylate synthase are associated with methotrexate effects in rheumatoid arthritis.

Methotrexate (MTX) enters cells through the reduced folate carrier (RFC-1) and exerts part of its effects through polyglutamation to MTX polyglutamates (MTXPGs) and inhibition of 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (ATIC) and thymidylate synthase (TS). We investigated the contribution of common genetic polymorphisms in RFC-1 (G80A), ATIC (C347G), and TS (28-bp tandem repeats located in the TS enhancer region [TSER*2/*3]) and of MTXPGs to the effect of MTX in patients with rheumatoid arthritis. The study was cross-sectional. All patients received MTX for at least 3 months. The numbers of tender and swollen joints, the Visual Analog Scale (VAS) scores for the physician's global assessment of disease activity, and the modified Health Assessment Questionnaire scores were collected. Using the VAS score for the physician's assessment of patient's response to MTX, the population of patients was dichotomized into responders to MTX (VAS score < or =2 cm) and nonresponders to MTX (VAS score >2 cm). A pharmacogenetic index was calculated as the sum of homozygous variant genotypes (RFC-1 AA + ATIC 347GG + TSER *2/*2) carried by the patients. MTXPG concentrations were measured in red blood cells (RBCs) by high-performance liquid chromatography. The dose of MTX was not associated with the effects of MTX (P > 0.05). In contrast, increased RBC long-chain MTXPG concentrations (median 40 nmoles/liter; range <5-131 nmoles/liter) and an increased pharmacogenetic index were associated with a lower number of tender and swollen joints (P < 0.05) and a lower score for the physician's global assessment of disease activity (P < or = 0.001). Patients with RBC MTXPG levels of >60 nmoles/liter and carriers of a homozygous variant genotype were 14.0-fold (95% confidence interval [95% CI] 3.6-53.8) and 3.7-fold (95% CI 1.7-9.1), respectively, more likely to have a good response to MTX (P <or = 0.01). These data suggest that measuring RBC MTXPG levels and/or the common polymorphisms in the folate-purine-pyrimidine pathway may help in monitoring MTX therapy.

10-Formyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid (10-Formyl-DDACTHF): A potent cytotoxic agent acting by selective inhibition of human GAR Tfase and the de novo purine biosynthetic pathway

The synthesis of 10-formyl-DDACTHF (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) is reported. Aldehyde 3, the corresponding γ- and α-pentaglutamates 21 and 25 and related agents were evaluated for inhibition of folate-dependent enzymes including GAR Tfase and AICAR Tfase. The inhibitors were found to exhibit potent cytotoxic activity (CCRF-CEM IC50 for 3=60nM) that exceeded their enzyme inhibition potency [Ki (3)=6 and 1μM for Escherichia coli GAR and human AICAR Tfase, respectively]. Cytotoxicity rescue by medium purines, but not pyrimidines, indicated that the potent cytotoxic activity is derived from selective purine biosynthesis inhibition and rescue by AICAR monophosphate established that the activity is derived preferentially from GAR versus AICAR Tfase inhibition. The potent cytotoxic compounds including aldehyde 3 lost activity against CCRF-CEM cell lines deficient in the reduced folate carrier (CCRF-CEM/MTX) or folylpolyglutamate synthase (CCRF-CEM/FPGS−) establishing that their potent activity requires both reduced folate carrier transport and polyglutamation. Unexpectedly, the pentaglutamates displayed surprisingly similar Ki's versus E. coli GAR Tfase and only modestly enhanced Ki's versus human AICAR Tfase. On the surface this initially suggested that the potent cytotoxic activity of 3 and related compounds might be due simply to preferential intracellular accumulation of the inhibitors derived from effective transport and polyglutamation (i.e., ca. 100-fold higher intracellular concentrations). However, a subsequent examination of the inhibitors against recombinant human GAR Tfase revealed they and the corresponding γ-pentaglutamates were unexpectedly much more potent against the human versus E. coli enzyme (Ki for 3, 14nM against rhGAR Tfase versus 6μM against E. coli GAR Tfase) which also accounts for their exceptional cytotoxic potency.

Abenzyl 10-formyl-trideazafolic acid (abenzyl 10-formyl-TDAF): An effective inhibitor of glycinamide ribonucleotide transformylase

The synthesis of N-[7-(2- amino-3,4-dihydro-4-oxo-quinazolin-6-yl)-6-formyl-1-oxo-heptyl]- l-glutamic acid ( 2, abenzyl 10-formyl-5,8,10-trideazafolic acid) as a potential enzyme-assembled tight binding inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) or aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) is reported. The inhibitor was prepared by a convergent synthesis utilizing the sequential alkylations of acetaldehyde dimethylhydrazone with 6 and 8. The agent exhibited effective inhibition of GAR Tfase ( K i = 4.5 ± 0.3 μM) and more modest inhibition of AICAR Tfase ( K i = 42 ± 11 μM).

10-formyl-5,8,10-trideazafolic acid (10-formyl-TDAF): A potent inhibitor of glycinamide ribonucleotide transformylase

The synthesis of 10-formyl-5,8,10-trideazafolic acid ( 3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) is reported. The target compound was prepared by a convergent synthesis utilizing the alkylation of hydrazone 5 with benzylic bromide 6 to construct the core heterocycle 7. The aldehyde 3 and related agents were evaluated as inhibitors of purN GAR Tfase and avian AICAR Tfase. Compound 3 exhibited potent inhibition of GAR Tfase with a K i of 0.26 ± 0.05 μM. In contrast, 3 exhibited more moderate inhibition of aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase), with K i of 7.6 ± 1.5 μM.

Reexamination of the Intracellular Localization of de Novo Purine Synthesis in Cowpea Nodules.

Sucrose and Percoll density gradient centrifugation were used to separate organelles from the central zone tissue of cowpea (Vigna unguiculata L. Walp. cv Vita 3: Bradyrhizobium strain CB 756) nodules. Enzyme activity analysis has shown that both plastids and mitochondria have a full complement of enzymes for de novo purine synthesis. In vitro activities of individual component enzymes (glycinamide ribonucleotide synthetase, EC 6.3.4.13; glycinamide ribonucleotide transformylase, EC 2.1.2.2; aminoimidazole ribonucleotide synthetase, EC 6.3.3.1; aminoimidazole carboxamide ribonucleotide transformylase, EC 6.3.2.6; and adenylosuccinate-AMP lyase, EC 4.3.2.2) as well as of the whole purine pathway (from ribose-5-phosphate to inosine monophosphate) were similar in the two organelles. No significant cytosolic or bacteroidal activity of any of the purine pathway enzymes was detected on assay. These findings are contrary to earlier studies (M.J. Boland, K.R. Schubert [1983] Arch Biochem Biophys 220: 179-187; B.J. Shelp C.A. Atkins, P.J. Storer, D.T. Canvin [1983] Arch Biochem Biophys 224: 429-441) that concluded that enhanced expression of purine synthesis in nodules of ureide-forming species is localized to plastids. Significantly increased recovery of activity of key pathway enzymes (particularly of labile aminoimidazole ribonucleotide synthetase) coupled with improved assay methods and the use of Percoll in addition to sucrose for gradient centrifugation have together contributed to much higher reaction rates and more definitive analyses of particulate fractions.

Cofactor role for 10-formyldihydrofolic acid.

10-Formyl-7,8-dihydrofolic acid (10-HCO-H2folate) was prepared by controlled air oxidation of 10-formyl-5,6,7,8-tetrahydrofolic acid (10-HCO-H4folate). The UV spectra of the 10-HCO-H2folate preparation has lambda max. 234, 333 nm and lambda min. 301 nm at pH 7.4, and lambda max. 257, 328 nm and lambda min. 229, 307 nm at pH 1. 1H-NMR spectroscopy of 10-HCO-H2folate (in 2H2O; 300 MHz) suggested a pure compound and gave resonances for one formyl group proton, two protons on C-7 and C-9, and no evidence for a C-6 proton, which is consistent with the structure proposed. The spectral properties indicated that the 10-HCO-H2folate preparation is not appreciably contaminated with 10-HCO-H4folate, 5,10-methenyltetrahydrofolic acid (5,10-CH = H4folate) or 10-formylfolic acid (10-HCO-folate). The above data establish that the 10-HCO-H2folate prepared here is authentic. In contrast, a folate with a UV spectrum having lambda max. 272 nm and lambda min. 256 nm at pH 7, which was prepared by 2,6-dichloro-indophenol oxidation of 10-HCO-H4folate and reported to be 97% pure [Baram, Chabner, Drake, Fitzhugh, Sholar and Allegra (1988) J. Biol. Chem. 263, 7105-7111], is apparently not 10-HCO-H2folate. 10-HCO-H2folate is utilized by Jurkat-cell (human T-cell leukaemia) and chicken liver aminoimidazolecarboxamide ribonucleotide transformylase (AICAR T'ase; EC 2.1.2.3) in the presence of excess 5-amino-imidazole-4-carboxamide ribotide (AICAR) resulting in the appearance of approximately 1 mol of H2folate product for each mol of AICAR formylated. The present 10-HCO-H2folate preparation had a kinetic advantage over 10-HCO-H4folate resulting from a difference of approx. 5-fold in K(m) values when both folates were used as cofactors for Jurkat-cell and rat bone marrow AICAR T'ase. No substantial kinetic advantage was observed using chicken liver AICAR T'ase. 10-HCO-H2folate had little or no activity with Jurkat-cell or chicken liver glycinamide ribonucleotide transformylase (GAR T'ase, EC 2.1.2.2). The existence in vivo of 10-HCO-H2folate is suggested in mammals by several reports of detectable amounts of radiolabelled 10-HCO-folate in bile and urine after administration of radiolabelled folic acid.

Novel pyrrolo[2,3-d]pyrimidine antifolate TNP-351: cytotoxic effect on methotrexate-resistant CCRF-CEM cells and inhibition of transformylases of de novo purine biosynthesis

N-[4-[3-(2,4-Diamino-7H-pyrrolo[2,3-d]pyrimidin-5- yl)propyl]benzoyl]-L-glutamic acid (TNP-351), characterized by a pyrrolo[2,3-d]pyrimidine ring, is a novel antifolate that exhibits potent antitumor activities against mammalian solid tumors. The mechanism of action of TNP-351 was evaluated using some methotrexate-resistant CCRF-CEM human lymphoblastic leukemia cell lines as well as partially purified enzymes folylpolyglutamate synthetase (FPGS), aminoimidazolecarboxamide ribonucleotide transformylase (AICARTFase), and glycinamide ribonucleotide transformylase (GARTFase) from parent CCRF-CEM cells. TNP-351 was found to inhibit the growth of L1210 and CCRF-CEM cells in culture, with the doses effective against 50% of the cells (ED50 values) being 0.79 and 2.7 nM, respectively. The growth inhibition caused by TNF-351 was reversed by leucovorin or a combination of hypoxanthine and thymidine. The methotrexate-resistant CCRF-CEM cell line, which has an impaired methotrexate transport, showed less resistance to TNP-351 than to methotrexate. TNP-351 was also found to be an excellent substrate for FPGS with a Michaelis constant (Km) of 1.45 microM and a maximum of velocity (Vmax) of 1,925 pmol h-1 mg-1. Inhibitory activities of TNF-351-Gn (n = 1-6) for AICARTFase were found to be significantly enhanced with increasing glutamyl chain length [inhibition constants (Ki): G1, 52 microM; G6, 0.07 microM]. Neither TNP-351 nor its polyglutamates were very strong inhibitors of GARTFase. These findings have significant implications regarding the mechanism of action of TNP-351.

Identification and biochemical properties of 10-formyldihydrofolate, a novel folate found in methotrexate-treated cells.

The folate compound 10-formyldihydrofolate (H2folate) has not been found as a component of intracellular folates in normal tissues but has been identified in the cytosol of methotrexate (MTX)-treated MCF-7 breast cancer cells and normal human myeloid precursor cells. Its identity was verified by coelution of this compound with a synthetic marker on high pressure liquid chromatography, its reduction to 10-formyltetrahydrofolate (H4folate) in the presence of dihydrofolate reductase, and its enzymatic deformylation to dihydrofolate in the presence of aminoimidazolecarboxamide ribonucleotide (AICAR) transformylase. Chemically synthesized monoglutamated or pentaglutamated 10-formyl-H2folate was examined for its interaction with three folate-dependent enzymes: AICAR transformylase, glucinamide ribotide (GAR) transformylase, and thymidylatesynthase. 10-Formyl-H2folate-Glu5 was a competitive inhibitor of thymidylate synthase (Ki = 0.16 microM with 5,10-methylene-H4folate-Glu1 as substrate and 1.6 microM with 5,10-methylene-H4folate-Glu5) and inhibited GAR transformylase (Ki = 2.0 microM). It acted as a substrate for AICAR transformylase (Km = 5.3 microM), and its efficiency was equal to that of the natural substrate 10-formyl-H4folate-Glu5. The inhibition of thymidylate synthase by 10-formyl-H2folate was highly dependent on the inhibitor's polyglutamation state, the -Glu5 derivative having a 52-85-fold greater affinity as compared to the affinity of -Glu1. Polyglutamation of 10-formyl-H2folate did not affect its inhibition of GAR transformylase. While the actual role of 10-formyl-H2folate contributing to the cytotoxicity of MTX has not been determined, this compound has the potential to enhance inhibition of GAR transformylase and thymidylate synthase, and at the same time provides additional substrate for AICAR transformylase. The MTX-induced intracellular accumulation of 10-formyl-H2folate and H2folate may play a role in the drug-related cytotoxicity through the contribution of these folates to the inhibition of thymidylate synthase and de novo purine synthesis.

Developmental changes in the folate-dependent enzymes of de novo purine biosynthesis in rat brain.

The activities of the two folate-dependent enzymes in the de novo purine biosynthetic pathway (e.g., glycinamide ribonucleotide transformylase and aminoimidazolecarboxamide ribonucleotide transformylase), have been evaluated as a function of age in crude extracts from rat brain, liver, kidney, and spleen. The activities of the enzymes in brain are similar to those found in liver and kidney. In all tissues the activity of both enzymes was higher during early development, more than nine times above adult levels. In the CNS the enzymatic activities are apparently related to the periods of increased nucleic acid synthesis, with different activities being found in different regions during development. Our findings lend strong support to the suggestion that folic acid-dependent metabolism plays an important role during early development of the brain.