Unconjugated Pterins Research Articles

Identification of new potential inhibitors of pteridine reductase-1 (PTR1) via biophysical and biochemical mechanism-based approaches: Step towards the treatment of Leishmaniasis

Leishmaniasis is a parasitic disease, which spreads from the bite of an infected Phlebotomine fly to human hosts. The disease is characterized by a number of clinical manifestations, such as ulcerative lesions at the site of sandfly bite (cutaneous form), inflammation of mucosal membranes (mucosal leishmaniasis) or the deadly visceral form. This study was aimed to target pteridine reductase-1 (PTR1), a member of short chain dehydrogenases, which accounts for the reduction of conjugated and unconjugated pterins in Leishmania parasite. The ptr1-pET28a+-tev construct was expressed using BL21 (DE3) cells, followed by two tandem purification steps including affinity and gel permeation chromatography. In the next phase, functional studies of PTR1 were performed via screening of an in-house library of 500 compounds. The biochemical-mechanism based assay of PTR1 identified 11 hits that were also found to be non-cytotoxic against human fibroblast cell line (BJ) (except compound 6), and thus further studied via computational technique and saturation transfer difference-nuclear magnetic resonance (STD-NMR) spectroscopy. These high throughput techniques identified six compounds 2, 4, 5, 7, 9, and 11 as active, which were then assessed via in-vitro assay. Among them, compounds 2, 4, and 7 showed substantial leishmanicidal activity, comparable to the standard drug, miltefosine (IC50 value: 31.8 ± 0.2 μM). These results narrowed down the search to 3 compounds as potential leads, with prominent protein-ligand interaction profiles. Hence, the respective compounds can be further assessed for their therapeutic potential against leishmaniasis.

Front Cover: Unveiling Photodegradation and Photosensitization Mechanisms of Unconjugated Pterins (Chem. Eur. J. 29/2023)

Pterins can be considered as biological jacks-of-all-trades. Firstly identified as pigments in butterfly wings, they are nowadays well-known cofactors in the synthesis of NO, nucleotides, and amino acids, markers in cell-mediated immune responses. Yet, not all of their properties are beneficial. Some of these pterins contribute to the photochemical evolution of vitiligo, a depigmentation disorder. Enhanced oxidative stress and cytotoxicity lead to melanocyte destruction and tissue damage. More information can be found in the Research Article by E. M. Arpa and I. Corral (DOI: 10.1002/chem.202300519).

Unveiling Photodegradation and Photosensitization Mechanisms of Unconjugated Pterins

Invited for the cover of this issue are Enrique M. Arpa (Linköping University) and Inés Corral (Universidad Autónoma de Madrid). The image depicts two examples where pterin chemistry is relevant, the wing coloration of some butterflies and the cytotoxic action in vitiligo. Read the full text of the article at 10.1002/chem.202300519.

Unveiling Photodegradation and Photosensitization Mechanisms of Unconjugated Pterins.

Unconjugated pterins are ubiquitous molecules participating in countless enzymatic processes and potentially involved in the photosensitization of singlet oxygen, amino acids, and nucleotides. Following electronic excitation with UV-A light, some of these pterins degrade, producing hydrogen peroxide as the main side product. This process, that is known to take place in vivo, contributes to oxidative stress and melanocyte destruction in vitiligo. In this work, we present for the first time mechanistic insight to the formation of transient triplet species which simultaneously trigger Type I and Type II photosensitizing processes and the initiation of degradation processes. Our calculations reveal that photodegradation of 6-biopterin, which accumulates in the skin of vitiligo patients, leads to 6-formylpterin via retro-aldol reaction, and subsequently to 6-carboxypterin through a water-mediated aldehyde oxidation. Additionally, we show that the changes in the photosensitizing potential of these systems with pH come from the modulation of their excited-state redox potentials.

Insights into Molecular Structure of Pterins Suitable for Biomedical Applications

Pterins are an inseparable part of living organisms. Pterins participate in metabolic reactions mostly as tetrahydropterins. Dihydropterins are usually intermediates of these reactions, whereas oxidized pterins can be biomarkers of diseases. In this review, we analyze the available data on the quantum chemistry of unconjugated pterins as well as their photonics. This gives a comprehensive overview about the electronic structure of pterins and offers some benefits for biomedicine applications: (1) one can affect the enzymatic reactions of aromatic amino acid hydroxylases, NO synthases, and alkylglycerol monooxygenase through UV irradiation of H4pterins since UV provokes electron donor reactions of H4pterins; (2) the emission properties of H2pterins and oxidized pterins can be used in fluorescence diagnostics; (3) two-photon absorption (TPA) should be used in such pterin-related infrared therapy because single-photon absorption in the UV range is inefficient and scatters in vivo; (4) one can affect pathogen organisms through TPA excitation of H4pterin cofactors, such as the molybdenum cofactor, leading to its detachment from proteins and subsequent oxidation; (5) metal nanostructures can be used for the UV-vis, fluorescence, and Raman spectroscopy detection of pterin biomarkers. Therefore, we investigated both the biochemistry and physical chemistry of pterins and suggested some potential prospects for pterin-related biomedicine.

Crystal structure of the ternary complex of Leishmania major pteridine reductase 1 with the cofactor NADP+/NADPH and the substrate folic acid.

Pteridine reductase 1 (PTR1) is a key enzyme of the folate pathway in protozoan parasites of the genera Leishmania and Trypanosoma and is a valuable drug target for tropical diseases. This enzyme is able to catalyze the NADPH-dependent reduction of both conjugated (folate) and unconjugated (biopterin) pterins to their tetrahydro forms, starting from oxidized- or dihydro-state substrates. The currently available X-ray structures of Leishmania major PTR1 (LmPTR1) show the enzyme in its unbound, unconjugated substrate-bound (with biopterin derivatives) and inhibitor-bound forms. However, no structure has yet been determined of LmPTR1 bound to a conjugated substrate. Here, the high-resolution crystal structure of LmPTR1 in complex with folic acid is presented and the intermolecular forces that drive the binding of the substrate in the catalytic pocket are described. By expanding the collection of LmPTR1 structures in complex with process intermediates, additional insights into the active-site rearrangements that occur during the catalytic process are provided. In contrast to previous structures with biopterin derivatives, a small but significant difference in the orientation of Asp181 and Tyr194 of the catalytic triad is found. This feature is shared by PTR1 from T. brucei (TbPTR1) in complex with the same substrate molecule and may be informative in deciphering the importance of such residues at the beginning of the catalytic process.

Effect of pterin impurities on the fluorescence and photochemistry of commercial folic acid

Folic acid, or pteroyl‑l‑glutamic acid (PteGlu) is a conjugated pterin derivative that is used in dietary supplementation as a source of folates, a group of compounds essential for a variety of physiological functions in humans. Photochemistry of PteGlu is important because folates are not synthesized by mammals, undergo photodegradation and their deficiency is related to many diseases. We have demonstrated that usual commercial PteGlu is unpurified with the unconjugated oxidized pterins 6‑formylpterin (Fop) and 6‑carboxypterin (Cap). These compounds are in such low amounts that a normal chromatographic control would not detect any pterinic contamination. However, the fluorescence of PteGlu solutions is due to the emission of Fop and Cap and the contribution of the PteGlu emission, much lower, is negligible. This is because the fluorescence quantum yield (ΦF) of PteGlu is extremely weak compared to the ΦF of Fop and Cap. Likewise, the PteGlu photodegradation upon UV-A radiation is an oxidation photosensitized by oxidized unconjugated pterins present in the solution, and not a process initiated by the direct absorption of photons by PteGlu. In brief, the fluorescence and photochemical properties of PteGlu solutions, prepared using commercially available solids, are due to their unconjugated pterins impurities and not to PteGlu itself. This fact calls into question many reported studies on fluorescence and photooxidation of this compound.

Neopterin and biopterin as biomarkers of immune system activation associated with castration in piglets1

Recent reports have shown that stressful situations may affect the production of unconjugated pterins (neopterin and biopterin). The aim of the study was to investigate the effect of castration on neopterin and biopterin plasma concentrations in piglets, using 2 groups of 12 piglets allocated to castrated and uncastrated (control) groups. Pterin concentrations were determined by HPLC with fluorescence detection. Blood samples were also analzyed for leukocyte profiles and plasma cortisol concentrations. A time × treatment interaction (P < 0.05) was detected for neopterin concentrations, such that neopterin was greater (P < 0.01) at 1 h after surgery in castrated piglets compared with precastration concentrations, and neopterin was greater (P = 0.05) in castrated than in control piglets at 1 h. Castration had no effect on biopterin concentration (P > 0.1). Time effects (P < 0.05) for neutrophil and lymphocyte concentrations and neutrophil-to-lymphocyte ratios were found. A time × treatment interaction (P < 0.01) was detected for plasma cortisol concentrations, such that cortisol was greater (P < 0.01) at 1 and 24 h after surgery in castrated piglets compared with precastration concentrations and was greater (P < 0.01) in castrated than in control piglets at 1 and 24 h. This study showed that castration activated the immune system of piglets as demonstrated by an increase in plasma neopterin concentrations.

Mechanism of photooxidation of folic acid sensitized by unconjugated pterins

Folic acid, or pteroyl-l-glutamic acid (PteGlu), is a precursor of coenzymes involved in the metabolism of nucleotides and amino acids. PteGlu is composed of three moieties: a 6-methylpterin (Mep) residue, a p-aminobenzoic acid (PABA) residue, and a glutamic acid (Glu) residue. Accumulated evidence indicates that photolysis of PteGlu leads to increased risk of several pathologies. Thus, a study of PteGlu photodegradation can have significant ramifications. When an air-equilibrated aqueous solution of PteGlu is exposed to UV-A radiation, the rate of the degradation increases with irradiation time. The mechanism involved in this "auto-photo-catalytic" effect was investigated in aqueous solutions using a variety of tools. Whereas PteGlu is photostable under anaerobic conditions, it is converted into 6-formylpterin (Fop) and p-aminobenzoyl-l-glutamic acid (PABA-Glu) in the presence of oxygen. As the reaction proceeds and enough Fop accumulates in the solution, a photosensitized electron-transfer process starts, where Fop photoinduces the oxidation of PteGlu to Fop, and H(2)O(2) is formed. This process also takes place with other pterins as photosensitizers. The results are discussed with the context of previous mechanisms for processes photosensitized by pterins, and their biological implications are evaluated.

Quenching of the Fluorescence of Aromatic Pterins by Deoxynucleotides

Steady-state and time-resolved studies of the fluorescence of four aromatic unconjugated pterins (pterin (Ptr), 6-(hydroxymethyl)pterin (Hmp), 6-methylpterin (Mep), and 6,7-dimethylpterin (Dmp)) in aqueous solutions in the presence of different nucleotides (2'-deoxyguanosine 5'-monophosphate (dGMP), 2'-deoxyadenosine 5'-monophosphate (dAMP), and 2'-deoxycytosine 5'-monophosphate (dCMP)) have been performed using the single-photon counting technique. The singlet excited states of acid forms of pterins are deactivated by purine nucleotides (dGMP and dAMP) via a combination of dynamic and static processes. The efficiency of the dynamic quenching is high, independently of the nature of the purine base of the nucleotide and of the chemical structure of the substituents linked to the pterin moiety. Analysis of the static quenching indicates that ground-state association between pterins and purine nucleotides takes place, but the formation of the corresponding complexes is significant only at relatively high reactant concentrations. The quenching of the fluorescence of acid forms of pterin derivatives by dCMP, a pyrimidine nucleotide, is slightly less efficient than the quenching by purine nucleotides and is purely dynamic. In alkaline media, the fluorescence quenching is much less efficient than in acidic media, the deactivation by purine nucleotides being purely dynamic, whereas quenching by dCMP is negligible. Possible mechanisms for the quenching of fluorescence of pterin derivatives by the different nucleotides are discussed.

Reactivity of Conjugated and Unconjugated Pterins with Singlet Oxygen (O2(1Δg)): Physical Quenching and Chemical Reaction†

Pterins (PTs) belong to a class of heterocyclic compounds present in a wide range of living systems. They participate in relevant biological functions and are involved in different photobiological processes. We have investigated the reactivity of conjugated PTs (folic acid [FA], 10-methylfolic acid [MFA], pteroic acid [PA]) and unconjugated PTs (PT, 6-hydroxymethylpterin [HPT], 6-methylpterin [MPT], 6,7-dimethylpterin [DPT], rhamnopterin [RPT]) with singlet oxygen (1O2) in aqueous solutions, and compared the efficiencies of chemical reaction and physical quenching. The chemical reactions between 1O2, produced by photosensitization, and PT derivatives were followed by UV-visible spectrophotometry and high-performance liquid chromatography, and corresponding rate constants (k(r)) were evaluated. Whenever possible, products were identified and quantified. Rate constants of 1O2 total quenching by the PT derivatives investigated were obtained from steady-state 1O2 luminescence measurements. Results show that the behavior of conjugated PTs differs considerably from that of unconjugated derivatives, and the mechanisms of 1O2 physical quenching by these compounds and of their chemical reaction with 1O2 are discussed in relation to their structural features.

Biopterin and neopterin in human saliva

Presence of biopterin and neopterin in human saliva was investigated by HPLC after iodine oxidation in acidic medium. Concentrations of biopterin and neopterin (M±SEM) were 1.27±0.254 and 0.358±0.075 ng per ml, respectively, in saliva of apparently healthy young male adults, ages 20 to 22 years ( n=9). Nearly identical value of the neopterin/biopterin ratio (0.29±0.07) was obtained for each of these specimens. Monapterin, the L- threo -isomer of neopterin (0.084±0.022 ng per ml saliva), and other unconjugated pterins such as xanthopterin, 6-hydroxymethylpterin and pterin were also found in the saliva. These pterins were all detectable in saliva of young female adults with similar levels to those of male saliva. Another fluorescent compound which was identical with 7-iso biopterin in retention time on HPLC was observed in all specimens of normal saliva examined.

Unconjugated Pterins in Neurobiology; Basic and Clinical Aspects. (Topics in Neurochemistry &amp; Neuropharmacology Vol 1).

Unconjugated Pterins in Neurobiology; Basic and Clinical Aspects. (Topics in Neurochemistry &amp; Neuropharmacology Vol 1).

Pterins: pigments, cofactors and signal connections in cell interactions

Pteridines were originally described as pigments of insects and lower vertebrates. The electron-donating function of tetrahydrobiopterin for aromatic amino acid hydroxylation and thus, for neurotransmitter biosynthesis adduced the participation of unconjugated pterins in cellular metabolism. There has been increasing evidence moreover that they are signal molecules for intercellular recognition in primitive eucaryotes, as well as modifiers of signal polypeptides in higher vertebrates.

Interferon-gamma enhances biosynthesis of pterins in peripheral blood mononuclear cells by induction of GTP-cyclohydrolase I activity.

In a recent publication, evidence was presented that cellular immune responses are associated with increased in vivo and in vitro excretion of neopterin. Our study aimed at investigating the biosynthesis of unconjugated pterins in highly purified human macrophages and T lymphocytes before and during stimulation with supernatants of activated T cells or with recombinant human interferon-gamma (IFN-gamma) by monitoring the following parameters: substrate concentration (GTP, guanosine triphosphate), activity of the enzyme initiating the biosynthesis of pterins (GTP-cyclohydrolase I) and product concentrations of total neopterin, biopterin, and pterin. In contrast to T cells and other tissues, macrophages were unable to produce tetrahydrobiopterin. This was indicated by our failure to detect biopterin and pterin. Instead, products of the first biosynthetic step accumulated, which were measured as total neopterin. We concluded that in macrophages the other enzymes required for biosynthesis of tetrahydrobiopterin are limiting. GTP concentration correlated with GTP cyclohydrolase I activity. An increase in both was induced by IFN-gamma and suppressed by neutralization of T-cell supernatants with monoclonal antibodies having specificity for IFN-gamma. Addition of tetrahydrobiopterin to the culture medium only led to a suppressed increase in GTP cyclohydrolase I activity and neopterin, but not in GTP concentration. Thus, it appears that IFN-gamma selectively stimulates the early steps of pterin biosynthesis in macrophages, thereby leading to accumulation and excretion of dihydroneopterin and neopterin. Although the physiological role of this phenomenon remains obscure, the fact that it seems to reflect endogenous release of IFN-gamma deserves particular attention.

Urinary excretion levels of unconjugated pterins in cancer patients and normal individuals

Urinary excretion levels of seven unconjugated pterins in healthy individuals and in cancer patients, most of whom were undergoing chemotherapy, were measured utilizing a newly developed high-pressure liquid Chromatographie system. Excretion of pterins in the control group appears to be under strict metabolic control as the values obtained were confined within a small range. When the mean excretion levels in control subjects were compared with those in cancer patients, we found a significant increase in the excretion of xanthopterin, neopterin and pterin and a significant decrease in isoxanthopterin by cancer patients. Biopterin levels, on the other hand, were found only slightly but not significantly increased, whereas pterin-6-carboxylic acid and 6-hydroxymethylpterin were found to be excreted in approximately equal amounts in both groups. Urinary excretion levels of pterins were monitored for a period of nine months in a patient being treated with chemotherapy for metastatic ovarian carcinomatosis. We found that the excretion pattern of pterins appeared to correlate with the clinical status of the patient. These results indicate that a definite imbalance in pterin, and possibly folate metabolism, is associated with the presence of malignant diseases.

Neopterin and biopterin levels in patients with atypical forms of phenylketonuria.

The pattern of unconjugated pterins in liver tissue and in urine from patients with atypical forms of phenylketonuria with hyperphenylalaninemia (HPA) has been investigated with a high performance liquid chromatographic technique. Two patients with defects in the biosynthesis of biopterin have been shown to have higher than normal levels of neopterin and lower than normal levels of biopterin. In contrast, a patient with HPA due to a deficiency of dihydropteridine reductase has the reverse urinary pattern, i.e., high biopterin, low neopterin. These results indicate that the ratio of neopterin to biopterin in urine can be of value in discriminating between HPA due to a deficiency of phenylalanine hydroxylase (classic PKU), HPA due to dihydropteridine reductase deficiency, and HPA due to a block in the biosynthesis of biopterin.

Occurrence of unconjugated pterins in a higher plant,Stizolobium hassjoo

3 unconjugated pterins, D-erythro-neopterin, 6-hydroxymethylpterin and isoxanthopterin, were isolated from the pericarps of a leguminous plant,Stizolobium hassjoo.

Analysis of unconjugated pterins in food resources and human urine.

A sensitive and simple method for separation of eight unconjugated pterins has been developed and used to analyze banana, seeds, nuts, cereals, leaves, royal jelly and human urine. Acid extraction, a simple clean-up using Florisil, and reversed-phase high-performance liquid chromatography with a fluorescence detector were employed. The eight pterins analyzed were: 6-carboxypterin, erythro-neopterin, threo-neopterin, xanthopterin, biopterin, isoxanthopterin, 6-hydroxymethylpterin and pterin. Lumazine compounds, 6-hydroxymethyllumazine and lumazine, were tentatively identified. Over ten ng of the unconjugated pterins/g of sample were quantified. Characteristic patterns of pterins were observed in each product. It was also shown that this analytical method was useful in diagnosis of malignant hyperphenylalaninemia.

Quantitative determination of unconjugated pterins in urine by gas chromatography/mass fragmentography

A gas chromatographic/mass fragmentographic method is described which permits the determination of unconjugated pterins in urine. After the addition of 6,7-dimethylpterin as an internal standard, the acidified urine samples are purified by liquid chromatography on Dowex-50 and Dowex-1 columns. The pterins are then converted to their corresponding trimethylsilyl derivatives and the base peaks of biopterin ( m e 409 ), neopterin ( m e 409 ) and 6,7-dimethylpterin ( solm e 320 ) are determined. The method is sensitive and specific and permits the processing of large numbers of samples. By means of this method, the urinary excretion of biopterin and neopterin from 9 healthy subjects has been determined.