Volume 24, Issue 1 - Special issue: 15th International Symposium on Pteridines and Folates, Antalya, Turkey, May 6–13, 2012

The key precursor, N 2 -( N,N -dimethylaminomethylene)-6-hydroxymethyl-8-methyl-3-[2-(4-nitrophenyl)ethyl]-7-xanthopterin ( 16 ) was efficiently prepared from 2,5-diamino-6-methylamino-3 H -pyrimidin-4-one ( 5 ) and ethyl 3-( tert -butyldimethylsilyloxy)-2-oxopropionate ( 12 ), followed by the protection of the pteridine ring. Glycosylation of 16 with 1- O -acetyl-2,3,5-tri- O -benzoyl-β-D-ribofuranose ( 18 ) in the presence of tin(IV) chloride yielded the corresponding β-D-ribofuranoside. Successive removal of the protecting groups of the resulting D-ribofuranoside provided asperopterin A ( 4b ).

Current knowledge distinguishes three classes of tetrahydrobiopterin-dependent enzymes as based on protein sequence similarity. These three protein sequence clusters hydroxylate three types of substrate atoms and use three different forms of iron for catalysis. The first class to be discovered was the aromatic amino acid hydroxylases, which, in mammals, include phenylalanine hydroxylase, tyrosine hydroxylase, and two isoforms of tryptophan hydroxylases. The protein sequences of these tetrahydrobiopterin-dependent aromatic amino acid hydroxylases are significantly similar, and all mammalian aromatic amino acid hydroxylases require a non-heme-bound iron atom in the active site of the enzyme for catalysis. The second classes of tetrahydrobiopterin-dependent enzymes to be characterized were the nitric oxide synthases, which in mammals occur as three isoforms. Nitric oxide synthase protein sequences form a separate cluster of homologous sequences with no similarity to aromatic amino acid hydroxylase protein sequences. In contrast to aromatic amino acid hydroxylases, nitric oxide synthases require a heme-bound iron for catalysis. The alkylglycerol monooxygenase protein sequence was the most recent to be characterized. This sequence shares no similarity with aromatic amino acid hydroxylases and nitric oxide synthases. Motifs contained in the alkylglycerol monooxygenase protein sequence suggest that this enzyme may use a di-iron center for catalysis.

Ischemia-reperfusion injury is a primarily non-allospecific event leading to the depletion of the essential nitric oxide synthase cofactor and potent antioxidant tetrahydrobiopterin. Suboptimal concentrations of tetrahydrobiopterin result in a reduced biosynthesis of nitric oxide leading to vascular endothelial dysfunction. Tetrahydrobiopterin supplementation has been shown to protect from this pathological state in a plethora of cardiovascular diseases including transplant-related ischemia-reperfusion injury. Even though still controversially discussed, there is increasing evidence emerging from both human as well as animal studies that tetrahydrobiopterin-mediated actions rely on its nitric oxide synthase cofactor activity rather than on its antioxidative properties. Herein, we review the current literature regarding the role of tetrahydrobiopterin in ischemia-reperfusion injury including our experience acquired in a murine pancreas transplantation model.

Almost all current standard treatment of care for patients with chronic inflammatory diseases such as rheumatoid arthritis (RA) includes the folate antagonist methotrexate (MTX). Despite the proven efficacy of MTX in RA treatment, it is anticipated that further improvement of antifolate-based therapies can be achieved by integrating the continuously extending knowledge of parameters underlying drug efficacy. Herein, an overview will be presented of strategies that may assist in the future design of rationalized and personalized targeted therapies with a folate antagonist. Issues that will be discussed include (i) early detection of arthritis, (ii) genomic studies of folate/MTX pathway genes for MTX response predictions, (iii) identification of MTX resistance modalities, (iv) exploration of second/third generation of antifolates which may substitute for MTX, and (v) folate (food) supplementation.

The proinflammatory cytokine interferon (IFN) γ activates GTP-cyclohydrolase I. In turn, the production of neopterin in human monocytic cells and of 5,6,7,8-tetrahydrobiopterin (BH 4 ) in other human cells and cells of other species is markedly upregulated. BH 4 is cofactor for the biosynthesis of the neurotransmitters 5-hydroxytryptamine (serotonin) and the catecholamines dopamine, epinephrine (adrenaline), and norepinephrine (noradrenaline). The finding of increased neopterin concentrations in patients with viral infections, autoimmune syndromes, malignant tumors, and during treatment with specific cytokines corresponds well with its immunobiological background. However, there is no clear information about BH 4 concentrations in these patients. Furthermore, higher blood phenylalanine (Phe)-to-tyrosine (Tyr) ratios have been described in patients with ovarian cancer, after multiple trauma and with sepsis, in patients with HIV-1 infection, in elderly individuals, and in patients with HCV infection under IFN-α therapy. Recent studies already showed that the alterations of Phe metabolism are associated with mood changes and depression. Results point to an impaired hydroxylation of Phe when the enzyme phenylalanine 4-hydroxylase (PAH) is less efficient. As the decrease of PAH activity might result from a diminished availability of BH 4 , the determination of the Phe/Tyr ratio may serve as an indirect measure of BH4 availability.

Increased neopterin concentrations and altered tryptophan degradation are observed in diseases concomitant with cellular immune activation. This may be involved in the pathogenesis of several age-related disorders such as neurodegenerative disorders, autoimmune diseases, cardiovascular system disorders and malignancies. Therefore, in the present study, the evaluation of immune system activation by determination of tryptophan degradation and serum neopterin levels was carried out in volunteers aged ≥65 and <65 years old. The kynurenine-to-tryptophan ratio was calculated to estimate indoleamine-(2,3)-dioxygenase (IDO) activity. Tryptophan levels in the elderly (53.1±1.6 μmol/L) were lower than individuals under 65 years (61.4±2.2 μmol/L), whereas kynurenine concentrations in geriatrics and adults were 5.0±0.2 μmol/L and 4.3±0.2 μmol/L, respectively (both p<0.05). The kynurenine-to-tryptophan ratio was also significantly higher in geriatrics (92.1±3.2) than adults (73.5±2.8) (p<0.05). Neopterin levels were slightly higher in geriatrics compared to adults under 65 years old (p>0.05). Effects of gender, smoking habit, pathology and drug use on measured parameters were also evaluated. In conclusion, our findings show that aging is associated with immune activation, and immune activation may be induced by the number of existing pathologies as well as the number of drugs used.

In transfusions, safety measures are used for the health of both receivers and donors. In this regard, early and sensitive markers are strongly needed. The aim of this study was to evaluate neopterin profile and tryptophan degradation in a group of Turkish blood donors. The effect of age, gender, blood type and Rh factor were also assessed. Neopterin was measured by ELISA kits. Serum tryptophan and kynurenine were determined by high performance liquid chromatography. Kynurenine/tryptophan (kyn/trp) was calculated to estimate tryptophan degradation. Tryptophan, kynurenine and kyn/trp levels were found to be higher in males compared with females (all p<0.05). Neopterin and kynurenine levels tended to increase with age in age subgroups (all p<0.05). On the contrary, smoking, Rh factor and blood types were all observed to have no effect on measured parameters (all p>0.05). In routine analysis, early and reliable biomarkers are needed. In parallel with current markers, neopterin can be used to select safe donors and to maintain the safety of transfusion. In the face of our results, we recommend that donations with neopterin levels above 19 nmol/L should be excluded from the program because increased neopterin levels indicate activation of the cellular immune system.

Tetrahydrobiopterin (BH4) is a major endogenous vasoprotective agent that improves endothelial function by increasing nitric oxide (NO) synthesis and scavenging of superoxide and peroxynitrite. Accordingly, administration of BH4 is considered as a promising therapy of cardiovascular diseases associated with endothelial dysfunction and oxidative stress. In a recent study we identified a novel function of BH4 that might contribute to the beneficial vascular effects of the pteridine. As demonstrated with cultured porcine aortic endothelial cells, oxidative inactivation of soluble guanylate cyclase with nitroglycerin or the heme-site inhibitor ODQ (1H-[1,2,4]-oxadiazolo[4,3-a]quinoxaline-1-one) resulted in a decrease in NO-induced cGMP accumulation that was insensitive to scavengers of reactive oxygen species but prevented upon supplementation of the cells with BH4. Tetrahydroneopterin had the same effect and virtually identical results were obtained with RFL-6 fibroblasts, suggesting that our observation reflects a general feature of tetrahydropteridines that is unrelated to NO synthase function and not limited to endothelial cells. Protection of soluble guanylate cyclase against oxidative inactivation may contribute to the known beneficial effects of BH4 in cardiovascular disorders associated with oxidative stress.

Higher serum neopterin concentrations and kynurenine-to-tryptophan (Kyn/Trp) ratios are associated with increased mortality in patients with coronary artery disease (CAD). Preferentially, Th1-type cytokine interferon-γ stimulates tryptophan breakdown and neopterin production by GTP cyclohydrolase I (GCH-I) in parallel in monocyte-derived macrophages and dendritic cells. In other cells, activation of GCH-I leads to the formation of 5,6,7,8-tetrahydrobiopterin (BH4), the necessary cofactor of amino acid hydroxylases such as phenylalanine 4-hydroxylase (PAH) and nitric oxide synthases. In 31 CAD patients (70.3±9.9 years; 21 males, 10 females), we determined serum concentrations of phenylalanine, tyrosine, and Kyn/Trp by HPLC, neopterin by ELISA, and nitrite by the colorimetric Griess assay. The phenylalanine-to-tyrosine ratio (Phe/Tyr) served as an estimate of PAH enzyme activity. Elevated Phe/Tyr concentrations were detected in a subgroup of CAD patients and correlated with Kyn/Trp (r=0.396, p<0.05) and neopterin (r=0.354, p<0.05) and inversely with nitrite (r=–0.371, p<0.05) concentrations. Higher Phe/Tyr in patients is associated with immune activation and indicates subnormal PAH activity that might be involved in the precipitation of neuropsychiatric symptoms in CAD patients.

Thymidylate synthase (TS) is a valid target for treatment of non-small cell lung cancer (NSCLC) and mesothelioma (MPM). The TS inhibitor pemetrexed (PMX) is now commonly used in combination with cisplatin (CDDP) or carboplatin, in the first-line setting for both of these lethal diseases. A combination of another TS inhibitor, Tomudex with CDDP, demonstrated similar activity in MPM patients. However, the efficacy of TS inhibitors is limited by uptake, metabolism and target affinity. While uptake of most antifolates is mediated by the reduced folate carrier, PMX is also a good substrate for the proton-coupled folate transporter (PCFT), which displays optimal activity at the acidic pH of the tumor microenvironment. NSCLC and MPM have a variable expression of PCFT, which might be caused by the differential methylation status of the PCFT promoter, resulting in decreased anticancer activity. PMX and TDX activity also depends on polyglutamylation, catalyzed by folylpolyglutamate synthetase (FPGS), which results in intracellular retention and thus enhanced cytotoxicity. We demonstrated that resistance to the classical antifolate methotrexate in human leukemia cells with a marked loss of FPGS activity was associated with impaired splicing of FPGS pre-mRNA. Moreover, we recently showed that the sensitivity of MPM patients to PMX-carboplatin is related to tumor TS expression. Patients with low TS mRNA levels had a significantly longer overall survival (20 vs. 7 months) compared with patients with high expression. Intriguingly, recent trials demonstrated that histology plays an important role in the sensitivity of NSCLC patients, and PMX-CDDP is now approved only for adenocarcinoma patients. These results might be partly explained by the higher TS expression detected in lung tumors of squamous histology. In summary, the clinical success of TS inhibitors may depend on biomarker-driven patient selection, and further studies should evaluate the genetic/epigenetic factors involved in the modulation of these biomarkers in the preclinical and clinical setting.

Leucovorin-modulated 5-fluorouracil (5-FU) plus irinotecan (FOLFIRI) is the most common treatment of metastatic colorectal cancer (CRC). 5-FU inhibits thymidylate synthase (TS) and irinotecan topoisomerase I, leading to inhibition of DNA replication and repair. FOLFIRI efficacy suggested that other TS inhibitors might synergize with irinotecan, and Phase I/II studies for second-line treatment showed promising results of combinations with the multitargeted antifolate pemetrexed (PMX), which exerts its effects primarily via TS inhibition. However, a randomized Phase II trial of PMX + irinotecan (ALIRI) showed similar efficacy and safety, but significantly shorter progression-free survival (PFS) compared with FOLFIRI in locally advanced or metastatic CRC. In our previous aCGH study, we evaluated genome-wide copy number variations, whereas in the current study we evaluated relationships between functional polymorphisms and PFS. Candidate polymorphisms were studied by restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) (TSER-2R/3R) or Taqman-PCR ( MTHFR-1958G>A, MTR-2756A>G, MTHFR-1298A>C, SHMT1-1420C>T, ATIC-347C>G, AMPD-134C>T, MTRR-66A>G and SLC-19A180G>A ) in 84 patients (40 FOLFIRI, 44 ALIRI). The Kaplan-Meier method was used to plot PFS, and the log-rank test to compare curves. At univariate analysis the homozygous variants of both MTR-2756A>G and SHMT1-1420C>T were associated with significantly shorter PFS. Conversely, a significantly longer PFS (7.3 months) was observed when ATIC-347C>G CC+CG genotypes were grouped vs. GG . At multivariate analysis the genotypes MTR-2756A>G AA+AG, SHMT1-1420C>T TT+CT and ATIC-347C>G CC+CG emerged as significant predictors for PFS. Because MTR, SHMT1 and ATIC are all involved in folate pathways, we further explored the effect of a combination of their risk genotypes on PFS, showing that patients carrying two risk genotypes had a significantly shorter PFS (3.9 months, p<0.001). The correlations of polymorphisms in genes with clinical outcome underscore the importance of a candidate gene-based approach. Ultimately, the validation of the role of these polymorphisms in prospective multicenter trials might optimize currently available treatments in selected CRC patients (e.g., FOLFIRI) or PMX-based treatments in other tumor types.

Sensitivity to antifolates can be decreased by endogenous or exogenous folates. Leucovorin protects cancer patients against toxicity of the dihydrofolate reductase inhibitor methotrexate (MTX), while folic acid is used to protect rheumatoid arthritis patients against MTX. Folates and antifolates can be effluxed from the cell by ABC transporters multidrug resistance protein 1 (MRP1), 2 and 3. We previously demonstrated in 2008 ovarian cancer cells that MRP overexpression reduced cellular folate content by 40%, while folate depletion increased expression of MRP1. As MRPs mediate resistance to several unrelated drugs, we investigated whether folate status would affect sensitivity to doxorubicin, daunorubicin, etoposide and vincristine. Ovarian cancer 2008 cells and its MRP1 transfected variant (2008/MRP1) were adapted from normal folate medium [2.3 μM; high folate (HF) cells] to short-term folate depletion (up to 7 days) (low folate cells); drugs were added after 2 days and sensitivity was tested by the MTT test after 3 additional days. The effect on folate homeostasis was evaluated by measurement of intracellular homocysteine using high-performance liquid chromatography and glutathione using a kit. MRP expression of wild-type (WT) 2008 cells did not increase homocysteine pools in 2008/MRP1 cells. Three day folate depletion increased homocysteine pools 23-fold in 2008 cells and 8.6-fold in the MRP variant. Folate depletion increased glutathione 20%–40% in 2008/WT and 2008/MRP1. In 2008 HF cells MRP1 expression did not affect sensitivity to MTX, but induced 4- to 10-fold resistance to doxorubicin, daunorubicin, etoposide and vincristine. Folate depletion decreased 50% growth inhibition (IC 50 ) for MTX in both 2008 variants 25- to 4-fold, but that to doxorubicin and daunorubicin approximately 2-fold. Sensitivity to etoposide and vincristine was not affected. In conclusion, folate depletion markedly increased homocysteine, but moderately increased glutathione. Folate depletion increased MTX sensitivity, but effects on other drugs were most pronounced in WT cells, probably because MRP expression is already high in transfected variants.

Crystal structures were solved of the binary complexes Trichinella spiralis and Caenorhabditis elegans thymidylate synthases with deoxyuridine monophosphate (dUMP), with crystals obtained by the vapor diffusion method in hanging drops. For the T. spiralis thymidylate synthase-dUMP complex, the diffraction data were collected at the BESSY Synchrotron to 1.9 Å resolution. The crystal belongs to the space group P1 with two dimers in the asymmetric unit (ASU). For the C. elegans TS-dUMP complex crystal, the diffraction data were collected at the BESSY Synchrotron to 2.48 Å resolution, and the crystal belongs to the space group P 32 2 1, with two monomers (one dimer) in the ASU. Structural comparisons were made of both structures and each of them with the corresponding mouse thymidylate synthase complex.

To solve the inhibition mechanism of thymidylate synthase (TS) by N 4 -hydroxy-dCMP (N 4 -OH-dCMP), crystallographic studies were undertaken. Structures of three mouse TS (mTS) complexes with the inhibitor were solved, based on crystals formed by the enzyme protein in the presence of either only N 4 -OH-dCMP [crystal A, belonging to the space group C 1 2 1, with two monomers in asymmetric unit (ASU), measured to 1.75 Å resolution] or both N 4 -OH-dCMP and N 5,10 - methylenetetrahydrofolate (mTHF) (crystals B and C, both belonging to the space group C 2 2 21, each with a single monomer in ASU, measured to resolution of 1.35 Å and 1.17 Å, respectively). Whereas crystal A-based structure revealed the mTS-N 4 -OH-dCMP binary complex, as expected, crystals B- and C-based structures showed the enzyme to be involved in a ternary complex with N 4 -OH-dCMP and noncovalently bound dihydrofolate (DHF), instead of expected mTHF, suggesting the inhibition to be a consequence of an abortive enzyme-catalyzed reaction, involving a transfer of the one-carbon group to a hitherto unknown site and oxidation of THF to DHF. Moreover, both C(5) and C(6) inhibitor atoms showed sp 3 hybridization, suggesting C(5) reduction, with no apparent indication of C(5) proton release. In accordance with our previous results, in all subunits of these structures the inhibitor molecule was identified as the anti rotamer of imino tautomer, forming, similar to deoxyuridine monophosphate, two hydrogen bonds with a conservative asparagine (mouse Asn220) side chain.

Alkylglycerol monooxygenase is a tetrahydrobiopterin-dependent enzyme that cleaves the O -alkyl-bond of alkylglycerols. It is an exceptionally unstable, hydrophobic membrane protein which has never been purified in active form. Recently, we were able to identify the sequence of alkylglycerol monooxygenase. TMEM195, the gene coding for alkylglycerol monooxygenase, belongs to the fatty acid hydroxylases, a family of integral membrane enzymes which have an 8-histidine motif crucial for catalysis. Mutation of each of these residues resulted in a complete loss of activity. We now extended the mutational analysis to another 25 residues and identified three further residues conserved throughout all members of the fatty acid hydroxylases which are essential for alkylglycerol monooxygenase activity. Furthermore, mutation of a specific glutamate resulted in an 18-fold decreased affinity of the protein to tetrahydrobiopterin, strongly indicating a potential important role in cofactor interaction. A glutamate residue in a comparable amino acid surrounding had already been shown to be responsible for tetrahydrobiopterin binding in the aromatic amino acid hydroxylases. Ab initio modelling of the enzyme yielded a structural model for the central part of alkylglycerol monooxygenase where all essential residues identified by mutational analysis are in close spatial vicinity, thereby defining the potential catalytic site of this enzyme.

The tetrahydrobiopterin-dependent degradation of ether lipids by alkylglycerol monooxygenase (AGMO) produces fatty aldehydes, which are toxic to cells. Therefore, it is of great physiological importance that these harmful compounds are converted into their corresponding, less toxic fatty acids by fatty aldehyde dehydrogenase (FALDH). Dysfunction of this enzyme causes Sjögren-Larsson syndrome. This severe inherited disorder is accompanied by symptoms such as ichthyosis, mental retardation and spasticity. Surprisingly, fatty alcohols and not fatty aldehydes were found to accumulate in fibroblasts of Sjögren-Larsson syndrome patients, suggesting that there can be wide-ranging alterations in the lipid composition of patient cells. In particular, this has to be considered when searching for possible treatment options for patients suffering from Sjögren-Larsson syndrome. For example, inhibition of fatty aldehyde producing ether lipid degradation would have multiple implications on ether lipid- and fatty alcohol-mediated signalling pathways.

Alkylglycerol monooxygenase (AGMO; EC 1.14.16.5) is the only enzyme known to cleave the O-alkyl ether bond of alkylglycerols in humans. It is an integral membrane protein with nine predicted transmembrane domains. We attempted to express and purify full-length and truncated forms of AGMO in Escherichia coli . Full-length AGMO could not be expressed in three different E. coli expression strains, three different expression vectors and several induction systems. We succeeded, however, in expression of three N-terminally strep-tagged truncated forms, named active sites 1, 2 and 3, with 205, 134 and 61 amino acids, respectively. Active site 1 fragment, containing two predicted transmembrane regions, a membrane associated region and all known amino acid residues important for catalytic activity, was not fully soluble even in 8 M urea. Active site 2 containing only one predicted membrane associated domain required 8 M urea for solubilisation and eluted in gel filtration in 1 M urea as a trimer. Active site 3 with no hydrophobic domain eluted in gel filtration in 1 M urea as monomer and dimer. These results show that even truncated forms of AGMO are barely soluble when expressed in E. coli and show a high tendency for aggregation.

Glutathione S-transferase omega (GSTO) genes in eukaryotic organisms encode proteins that are important for cell defense. However, the physiological roles of GSTOs have not been fully elucidated yet. Recently, genetic and molecular studies with Drosophila demonstrated that CG6781 is the structural gene of the eye color mutant sepia and that CG6673 is a novel genetic suppressor of the parkin mutant. These results provide valuable insight into the diverse functions of GSTOs in vivo. In this review, we briefly introduce recent studies and summarize the novel biological functions of GSTOs in Drosophila .

Trichinella spiralis is a parasitic nematode causing trichinellosis, a serious disease, and Caenorhabditis elegans is a free-living nematode, which is used as a model in parasitological studies. High levels of thymidylate synthase (EC 2.1.1.45; ThyA) and certain other enzymes involved in thymidylate biosynthesis were found throughout T. spiralis and C. elegans developmental cycles, including developmentally arrested forms, that is, T. spiralis muscle larva and C. elegans dauer larva. As ThyA activity is characteristic for cells that left the G 0 phase of the cell cycle, an exceptional regulation of the cell cycle in nematodes is suggested, manifested by a global cell cycle arrest in developmentally arrested larvae of the two species. ThyA immunolocalization during development of T. spiralis and C. elegans revealed the presence of high enzyme levels not only in the developing embryos, where it was expected, but also in gonad primordia, egg and sperm cells, nerve ring and secretory cells, opening to T. spiralis esophagus and C. elegans pharynx, where it may point to those cell populations remaining cell cycle arrested.