Thymidine esters as substrate analogue inhibitors of angiogenic enzyme thymidine phosphorylase in vitro

Thymidine phosphorylase (TP) catalyzes the cleavage of thymidine into thymine and 2-deoxy-α-d-ribose-1-phosphate. Elevated activity of TP prevents apoptosis, and induces angiogenesis which ultimately leads to tumor growth and metastasis. Critical role of TP in cancer progression makes it a valid target in anti-cancer research. Discovery of small molecules as TP inhibitors is vigorously pursued in cancer therapy. In the present study, we functionalized thymidine as benzoyl ester to synthesize compounds 3–16. In vitro evaluation of thymidine esters for their thymidine phosphorylase inhibition activity was subsequently carried out. Compounds 4, 10, 14, and 15 showed good activities with lower IC₅₀ values than the standard, 7-deazaxanthine (IC₅₀ = 41.0 ± 1.63 μM). Among them, compound 14 showed five folds higher activity (IC₅₀ = 7.5 ± 0.8 μM), while 4 (IC₅₀ = 18.5 ± 1.0 μM) and 10 (IC₅₀ = 18.8 ± 1.2 μM) showed two folds higher activity than the standard. Compound 15 showed slightly better activity (IC₅₀ = 33.3 ± 1.5 μM) to the standard. Potent compounds were further subjected to kinetic and molecular docking studies to identify their mode of inhibition, and to study their interactions with the protein at atomic level, respectively. All active compounds were non-cytotoxic to mouse fibroblast 3T3 cell line. These results identify thymidine esters as substrate analogue (substrate-like) inhibitors of angiogenic enzyme thymidine phosphorylase for further studies.     

Synthesis,thymidine phosphorylase,angiogenic inhibition and molecular docking study of isoquinoline derivatives

Isoquinoline analogues (KA-1 to 16) have been synthesized and evaluated for their E. coli thymidine phosphorylase inhibitory activity. Except compound 11, all other analogs showed outstanding thymidine inhibitory potential ranging in between 4.40 ± 0.20 to 69.30 ± 1.80 µM when compared with standard drug 7-Deazaxanthine (IC₅₀ = 38.68 ± 4.42 µM). Structure Activity Relationships has been established for all compounds, mainly based on substitution pattern on phenyl ring. All analogs were characterized by various spectroscopic techniques such as ¹H NMR, ¹³C NMR and EI-MS. The binding interactions of isoquinoline analogues with the active site of TP enzyme, the molecular docking studies were performed. Furthermore, the angiogenic inhibitory potentials of isoquinoline analogues (KA-1-9, 14, 12 and 16) were determined in the presence of standard drug Dexamethasone based on percentage inhibitions at various concentrations. Herein this work analogue KA-12, 14 and 16 emerged with most potent angiogenic inhibitory potentials among the synthesized analogues.     

Regulation of the synthesis and activity of thymidine phosphorylase in Lactobacillus casei

Abstract: Lactobacillus casei cells grown on excess thymine or on folic acid contained low levels of thymidine phosphorylase. On the other hand, thymine starved cells and also cells of a thymidine-monophosphate-kinase-defective mutant grown on excess thymine, possessed derepressed levels. These results suggest that the synthesis of thymidine phosphorylase is regulated by the end product of the thymidine-triphosphate-biosynthetic pathway. L. casei cells lacked 2-deoxyribose-1-phosphate-mutase activity and did not grow on 2-deoxyribose or thymidine as the sole-carbon source. Growth in the presence of thymidine did not result in induction of thymidine-phosphorylase synthesis, probably due to the inability of the cell to convert it to 2-deoxyribose-5-phosphate, which is known to act as an inducer in E. coli cells. Thymidine triphosphate inhibited non-competitively the activity of thymidine phosphorylase. It was also inhibited by dihydrofolic acid.     

Identification of 1,2,4-triazoles as new thymidine phosphorylase inhibitors: Future anti-tumor drugs

Thymidine phosphorylase (TP) is over expressed in several solid tumors and its inhibition can offer unique target suitable for drug discovery in cancer. A series of 1,2,4-triazoles 3a–3l has been synthesized in good yields and subsequently inhibitory potential of synthesized triazoles 3a–3l against thymidine phosphorylase enzyme was evaluated. Out of these twelve analogs five analogues 3b, 3c, 3f, 3l and 3l exhibited a good inhibitory potential against thymidine phosphorylase. Inhibitory potential in term of IC₅₀ values were found in the range of 61.98 ± 0.43 to 273.43 ± 0.96 μM and 7-Deazaxanthine was taken as a standard inhibitor with IC₅₀ = 38.68 ± 4.42 μM. Encouraged by these results, more analogues 1,2,4-triazole-3-mercaptocarboxylic acids 4a–4g were synthesized and their inhibitory potential against thymidine phosphorylase was evaluated. In this series, six analogues 4b–4g exhibited a good inhibitory potential in the range of 43.86 ± 1.11–163.43 ± 2.03 μM. Angiogenic response of 1,2,4-triazole acid 4d was estimated using the chick chorionic allantoic membrane (CAM) assay. In the light of these findings, structure activity relationship and molecular docking studies of selected triazoles to determine the key binding interactions was discussed. Docking studies demonstrate that synthesized analogues interacted with active site residues of thymidine phosphorylase enzyme through π-π stacking, thiolate and hydrogen bonding interactions.     

Rapid disappearance of deoxyribose-1-phosphate in platelet derived endothelial cell growth factor/thymidine phosphorylase overexpressing cells

Platelet derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) catalyzes the phosphorolysis of thymidine (TdR) to thymine and deoxyribose-1-phosphate (dR-1-P) and has a pro-angiogenic effect for which dR-1-P may be responsible. Using a purine nucleoside phosphorylase based assay it was found that TdR incubation did not increase dR-1-P accumulation in colon cancer cell line Colo320 and its PD-ECGF/TP transfected variant Colo320TP1. The assay was linear up to 25,000pmol dR-1-P with complete recovery of dR-1-P from cellular extracts. There was a huge discrepancy between thymine production and the measured dR-1-P level, 0.05% of the expected value for dR-1-P was found, indicating that there was a rapid disappearance of dR-1-P. However, in cellular extracts, TdR incubation increased dR-1-P, measurable by trapping, which was inhibited by a thymidine phosphorylase inhibitor. dR-1-P directly added to cellular extracts disappeared within 5-10min. In conclusion, large amounts of dR-1-P are produced by Colo320TP1 cells, which rapidly disappear thus not resulting in a net accumulation of dR-1-P in these cells.     

The role of blood platelets in nucleoside metabolism: regulation of platelet thymidine phosphorylase

Blood platelets are the smallest cellular elements in mammalian blood. Because of their small size, platelets have an usually large surface area: volume ratio and are exquisitely sensitive to a multitude of physiologica and environmental stimuli. Platelets lack nuclei, but most possess functional mitochondria and remain capable of both anaerobic and aerobic energy metabolism, for which they utilise a variety of substrates including many which are cytotoxic and genotoxic for other (nucleated) cells. Nucleic acid precursors are amongst the potentially genotoxic compounds for which platelets have an apparently insatiable appetite. In particular platelets actively scavenge adenine and adenosine, which they convert to nucleotides and use in energy metabolism, but they also rapidly phosphorylyse thymidine and liberate thymine into the extracellular medium. In addition, platelets contain non-metabolisable membrane-bound pools of adenine nucleotides which they secrete in response to strong agonists. Taken together, these observations suggest that blood platelets play an important role in nucleic acid precursor metabolism.

In the previous paper we have shown that most thymidine phosphorylase activity present in normal human blood resides in the cytoplasm of platelets. Here we demonstrate that this enzyme activity can be modulated in a dose-dependent fashion, not only by substances recognised as platelet agonists and antagonists, but also by some compounds which are considered to be toxic, mutagenic and/or carcinogenic. The data which we present provide additional support for our previous suggestion that platelets regulate thymidine homeostasis and further imply that this is the normal, physiological, platelet function. Preliminary results suggest that assays of blood platelet thymidine metabolism may provide data with a wide variety of applications.     

Elevated plasma deoxyuridine in patients with thymidine phosphorylase deficiency

Mutations in the nuclear gene encoding thymidine phosphorylase (TP) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disease with mitochondrial dysfunction and mitochondrial DNA abnormalities. We have demonstrated alterations of thymidine (dThd) metabolism in MNGIE patients. Here, we report the accumulation of another substrate of TP, deoxyuridine (dUrd), whose circulating levels ranged from 5.5 to 24.4 microM (average 14.2) in MNGIE and were undetectable (<0.05 microM) in both TP mutation carriers and controls. The dramatic accumulation of dUrd may contribute to nucleotide pool imbalances and, together with the increased levels of dThd, is likely to contribute to the pathogenesis of MNGIE.     

Structures of native human thymidine phosphorylase and in complex with 5-iodouracil

Thymidine phosphorylase (TP) first identified as platelet derived endothelial cell growth factor (PD-ECGF) plays a key role in nucleoside metabolism. Human TP (hTP) is implicated in angiogenesis and is overexpressed in several solid tumors. Here, we report the crystal structures of recombinant hTP and its complex with a substrate 5-iodouracil (5IUR) at 3.0 and 2.5 Å, respectively. In addition, we provide information on the role of specific residues in the enzymatic activity of hTP through mutagenesis and kinetic studies.     

Synthesis,molecular docking study and in vitro thymidine phosphorylase inhibitory potential of oxadiazole derivatives

We have synthesized oxadiazole derivatives (1–16), characterized by ¹H NMR, ¹³C NMR and HREI-MS and screened for thymidine phosphorylase inhibitory potential. All derivatives display varied degree of thymidine phosphorylase inhibition in the range of 1.10 ± 0.05 to 49.60 ± 1.30 μM when compared with the standard inhibitor 7-Deazaxanthine having an IC₅₀ value 38.68 ± 1.12 μM. Structure activity relationships (SAR) has been established for all compounds to explore the role of substitution and nature of functional group attached to the phenyl ring which applies imperious effect on thymidine phosphorylase activity. Molecular docking study was performed to understand the binding interaction of the most active derivatives with enzyme active site.     

Synthesis,thymidine phosphorylase inhibition and molecular modeling studies of 1,3,4-oxadiazole-2-thione derivatives

Thymidine phosphorylase (TP) inhibitors have attracted great attention due to their ability to suppress the tumors formation. In our ongoing research, a series of 1,3,4-oxadiazole-2-thione (1–12) has been synthesized under simple reaction conditions in good to excellent yields (86–98%) and their TP inhibition potential has also been evaluated. The majority of synthesized compounds showed moderate thymidine phosphorylase inhibitory activity with IC50 values ranging from 38.24 ± 1.28 to 258.43 ± 0.43 μM, and 7-deazaxanthine (7DX) was used as a reference compound (IC50 38.68 ± 4.42). The TP activity was very much dependent on the C-5 substituents; among this series the compound 6 bearing 4-hydroxyphenyl group was found to be the most active with IC50 38.24 ± 1.28 μM. Molecular docking studies revealed their binding mode.     

Development and validation of a quantitative method for thymidine phosphorylase activity in peripheral blood mononuclear cells

Abstract

The enzyme thymidine phosphorylase (TP) is important for activation of capecitabine and 5-fluorouracil. Assessment of TP phenotype might be suitable for identification of patients at risk of fluoropyrimidine-induced toxicity. In this paper, we describe the development and validation an assay for TP activity in peripheral blood mononuclear cells (PBMCs). The assay was based on ex vivo conversion of the TP substrate thymidine to thymine. The amount of thymine formed was determined by high-performance liquid chromatography – ultraviolet detection (HPLC-UV) with 5-bromouracil as internal standard. Lymphocytes and monocytes were purified from isolated PBMCs to examine cell-specific TP activity. TP activity in PBMCs demonstrated Michaelis-Menten kinetics. The lower limit of quantification was 2.3?µg PBMC protein and assay linearity was demonstrated up to 22.7?µg PBMC protein. Within-day and between-day precisions were ≤9.2% and ≤6.0%, respectively. Adequate stability TP activity was demonstrated after long-term storage of PBMC dry pellets and lysates at ?80?°C. In monocytes, TP activity was approximately 3 times higher than in lymphocytes. Clinical applicability was demonstrated in samples that were collected from five cancer patients. A simple, precise and sensitive HPLC-UV assay for quantification of TP activity in PBMCs was developed that can be applied for clinical research.     

In silico binding analysis and SAR elucidations of newly designed benzopyrazine analogs as potent inhibitors of thymidine phosphorylase

Thymidine phosphorylase (TP) is up regulated in wide variety of solid tumors and therefore presents a remarkable target for drug discovery in cancer. A novel class of extremely potent TPase inhibitors based on benzopyrazine (1–28) has been developed and evaluated against thymidine phosphorylase enzyme. Out of these twenty-eight analogs eleven (11) compounds 1, 4, 14, 15, 16, 17, 18, 19, 20, 24 and 28 showed potent thymidine phosphorylase inhibitory potentials with IC₅₀ values ranged between 3.20 ± 0.30 and 37.60 ± 1.15 μM when compared with the standard 7-Deazaxanthine (IC₅₀ = 38.68 ± 4.42 μM). Structure-activity relationship was established and molecular docking studies were performed to determine the binding interactions of these newly synthesized compounds. Current studies have revealed that these compounds established stronger hydrogen bonding networks with active site residues as compare to the standard compound 7DX.     

Design,synthesis, in-vitro thymidine phosphorylase inhibition,in-vivo antiangiogenic and in-silico studies of C-6 substituted dihydropyrimidines

Thymidine phosphorylase (TP) is an angiogenic enzyme. It plays an important role in angiogenesis, tumour growth, invasion and metastasis. In current research work, we study the effect of structural modification of dihydropyrimidine-2-ones (DHPM-2-ones) on TP inhibition. A series of eighteen new derivatives of 3,4-dihydropyrimidone-2-one were designed and synthesized through the structural modification at C-6 position. All these new derivatives were then assessed for in-vitro inhibition of thymidine phosphorylase (TP) from E. coli. Oxadiazole derivatives 4a-e exhibited excellent TP-inhibition at low micromolar concentration levels better than standard drug 7-deazaxanthine (7-DX). Among all these compounds, 4b was found to be the most potent with IC₅₀ = 1.09 ± 0.004 μM. Anti-angiogenesis potential of representative compounds were also studied in a chorioallantoic membrane (CAM) assay. Here again, compound 4b was found to be the potent anti-angiogenesis compound in a CAM assay. Docking studies were also performed with Molecular Operating Environment (MOE) to further analyse the mode of inhibition of these compounds. Binding mode analysis of the most active inhibitors showed that these are well accommodated into the binding site of enzyme though stable hydrogen bonding and hydrophobic interactions.     

Facilitation of DNA damage-induced apoptosis by endoplasmic reticulum protein mitsugumin23

The endoplasmic reticulum (ER) emanates context-dependent signals, thereby mediating cellular response to a variety of stresses. However, the underlying molecular mechanisms have been enigmatic. To better understand the signaling capacity of the ER, we focused on roles played by mitsugumin23 (MG23), a protein residing predominantly in this organelle. Overexpression of MG23 in human embryonic kidney 293T cells specifically enhanced apoptosis triggered by etoposide, a DNA-damaging anti-cancer drug. Conversely, genetic deletion of MG23 reduced susceptibility of thymocytes to DNA damage-induced apoptosis, which was demonstrated by whole-body irradiation experiments. In this setting, induction of the tumor-suppressor gene p53 was attenuated in MG23-knockout thymocytes as compared with their wild-type counterparts, consistent with the elevated radioresistance. It is therefore suggested that MG23 is an essential component of ER-generated lethal signals provoked upon DNA damage, specifying cell fate under pathophysiological conditions.     

Synthesis and biological evaluation of novel oxadiazole derivatives: A new class of thymidine phosphorylase inhibitors as potential anti-tumor agents

Based on the fact that the thymidine phosphorylase inhibitors are considered potential anti-tumor agents, a range of novel oxadiazole derivatives 3a–3u was designed and synthesized by a simple and facile synthetic route. The biological assay revealed that majority of compounds displayed modest inhibitory activity against thymidine phosphorylase at low micromolar concentrations (IC₅₀ 173.23 ± 3.04 to 14.40 ± 2.45 μM). In the current study the most active compounds were 3h and 3q with IC₅₀ values 14.40 ± 2.45 and 17.60 ± 1.07 μM, respectively. Molecular docking studies were performed on the most active compounds (3h, 3k, 3o–3q) to show their binding mode.     

A kinetic,modeling and mechanistic re-analysis of thymidine phosphorylase and some related enzymes

Thymidine phosphorylase (TP) is an important target enzyme for cancer chemotherapy but currently available inhibitors lack in vivo potency. Related enzymes also are therapeutic targets. A greater understanding of enzyme structure and mechanism may help in the design of improved drugs and this work assists in that regard. Also important is the correct identification of the ionization states and tautomeric forms of substrates and products when bound to the enzyme and during the course of the reaction. Approximate methods for estimating some ΔpK_as between aqueous and protein-bound substrates are exemplified for nucleobases and nucleosides. The estimates demonstrate that carbonyl-protonated thymidine and hydroxy tautomers of thymine are not involved in TP's actions. Other estimates indicate that purine nucleoside phosphorylase binds inosine and guanosine as zwitterionic tautomers and that phosphorolysis proceeds through these forms. Extensive molecular modeling based on an X-ray structure of human TP indicates that TP is likely to be mechanistically similar to all other natural members of the class in proceeding through a α-oxacarbenium-like transition state or states.     

RNA polymerase II large subunit is cleaved by caspases during DNA damage-induced apoptosis

UV radiation induces DNA lesions that are repaired by the nucleotide excision repair (NER) pathway. Cells that are NER deficient such as those derived from xeroderma pigmentosum (XP) patients are susceptible to apoptosis after 10J/m(2) UV radiation, a dose largely survivable by repair proficient cells. Herein, we report that RNA polymerase II large subunit (RNAP II-LS) undergoes caspase-mediated cleavage, yielding a 140kDa C-terminal fragment in XP lymphoblasts but not NER proficient lymphoblasts after 10J/m(2) UV irradiation. Cleavage could also be induced by cisplatin or oxaliplatin, but not transplatin, an isomer of cisplatin that does not induce DNA adducts. The cleavage of RNAP II-LS was blocked by a panel of caspase inhibitors but not by proteasomal inhibitors or inhibitors of other proteases. In vitro cleavage with caspase 8 yielded the same 140kDa RNAP II-LS fragment observed in vivo. Using site-directed mutagenesis, the RNAP II-LS cleavage site was localized to an LETD sequence ending at residue 1339, which is near its C-terminal domain.     

Enzymatic activities of uridine and thymidine phosphorylase in normal and cancerous uterine cervical tissues

In this study, the preliminary analyses were conducted of enzymatic activities of uridine phosphorylase (UP) and thymidine phosphorylase (TP) in normal tissues and cancer tissues of the uterine cervix. The study was performed on 27 patients of cervical cancer, treated first in our hospital. Normal cervical tissues obtained from 15 patients undergoing hysterectomy for benign diseases were used as controls. The supernatant of the homogenated cervical tissues and the stroma (5-FU and ribose-1-P or deoxyribose-1-P) were analyzed by high performance liquid chromatography, and then the UP and TP activities calculated. TP activity was significantly greater than UP activity (P < 0.0001). Both UP and TP showed significantly greater activity in cancer tissues than in normal tissues (P < 0.0001). In the TP activity of the cancer tissues, there was no significant difference among the histological types, while the TP activity tended to be significantly higher in the cases with lymph node metastasis. These results showed that the TP-mediated route seemed important as the 5FU metabolic pathway in the uterine cervical tissues, and TP enzymatic activity might be associated with lymph node metastasis.     

Substrate specificity of Escherichia coli thymidine phosphorylase

Substrate specificity of Escherichia coli thymidine phosphorylase to thymidine derivatives modified at 5' -, 3' -, and 2' ,3' - positions of the sugar moiety was studied. Equilibrium and kinetic constants (K(m), K(I), k(cat)) of the phosphorolysis reaction have been determined for 20 thymidine analogs. The results are compared with X-ray and molecular dynamics data. The most important hydrogen bonds in the enzyme-substrate complex are revealed.