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Giuseppe Pizzorno - One of the best experts on this subject based on the ideXlab platform.
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differential expression of Uridine Phosphorylase in tumors contributes to an improved fluoropyrimidine therapeutic activity
Molecular Cancer Therapeutics, 2011Co-Authors: Deliang Cao, Giuseppe Pizzorno, James L Mccabe, Laxiang Wan, Ruilan Yan, Amy Ziemba, Bradford Kim, Michael H Gach, Stuart D FlynnAbstract:Abrogation of Uridine Phosphorylase (UPase) leads to abnormalities in pyrimidine metabolism and host protection against 5-fluorouracil (5-FU) toxicity. We elucidated the effects on the metabolism and antitumor efficacy of 5-FU and capecitabine (N4-pentyloxycarbonyl-5′-deoxy-5-fluorocytidine) in our UPase knockout ( UPase−/− ) model. Treatment with 5-FU (85 mg/kg) or capecitabine (1,000 mg/kg) five days a week for four weeks caused severe toxicity and structural damage to the intestines of wild-type (WT) mice, but not in UPase−/− animals. Capecitabine treatment resulted in a 70% decrease in blood cell counts of WT animals, with only a marginal effect in UPase−/− mice. UPase expressing colon 38 tumors implanted in UPase−/− mice revealed an improved therapeutic efficacy when treated with 5-FU and capecitabine because of the higher maximum tolerated dose for fluoropyrimidines achievable in UPase−/− mice. 19F-MRS evaluation of capecitabine metabolism in tumors revealed similar activation of the prodrug in UPase−/− mice compared with WT. In WT mice, approximately 60% of capecitabine was transformed over three hours into its active metabolites, whereas 80% was transformed in tumors implanted in UPase−/− mice. In UPase−/− mice, prolonged retention of 5′dFUR allowed a proportional increase in tumor tissue. The similar presence of fluorinated catabolic species confirms that dihydropyrimidine dehydrogenase activity was not altered in UPase−/− mice. Overall, these results indicate the importance of UPase in the activation of fluoropyrimidines, the effect of Uridine in protecting normal tissues, and the role for tumor-specific modulation of the phosphorolytic activity in 5-FU or capecitabine-based chemotherapy. Mol Cancer Ther; 10(12); 2330–9. ©2011 AACR .
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a novel structural mechanism for redox regulation of Uridine Phosphorylase 2 activity
Journal of Structural Biology, 2011Co-Authors: Tarmo P Roosild, Samantha Castronovo, Adelbert Villoso, Amy Ziemba, Giuseppe PizzornoAbstract:Uridine Phosphorylase (UPP) catalyzes the reversible conversion of Uridine to uracil and ribose-1-phosphate and plays an important pharmacological role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil and capecitabine. Most vertebrate animals, including humans, possess two homologs of this enzyme (UPP1 & UPP2), of which UPP1 has been more thoroughly studied and is better characterized. Here, we report two crystallographic structures of human UPP2 (hUPP2) in distinctly active and inactive conformations. These structures reveal that a conditional intramolecular disulfide bridge can form within the protein that dislocates a critical phosphate-coordinating arginine residue (R100) away from the active site, disabling the enzyme. In vitro activity measurements on both recombinant hUPP2 and native mouse UPP2 confirm the redox sensitivity of this enzyme, in contrast to UPP1. Sequence analysis shows that this feature is conserved among UPP2 homologs and lacking in all UPP1 proteins due to the absence of a necessary cysteine residue. The state of the disulfide bridge has further structural consequences for one face of the enzyme that suggest UPP2 may have additional functions in sensing and initiating cellular responses to oxidative stress. The molecular details surrounding these dynamic aspects of hUPP2 structure and regulation provide new insights as to how novel inhibitors of this protein may be developed with improved specificity and affinity. As Uridine is emerging as a promising protective compound in neuro-degenerative diseases, including Alzheimer's and Parkinson's, understanding the regulatory mechanisms underlying UPP control of Uridine concentration is key to improving clinical outcomes in these illnesses.
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abstract 4390 role of Uridine Phosphorylase 2 in fluoropyrimidine activation and host toxicity
Cancer Research, 2011Co-Authors: Amy Ziemba, Steven Brotman, Yang Gao, Michio Hirano, Giuseppe PizzornoAbstract:Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Uridine Phosphorylase (UPP) and thymidine Phosphorylase (TPP) both catalyze the reversible conversion of Uridine, deoxyUridine and thymidine to the corresponding free bases and ribose or deoxyribose-1-phosphate. They play a pivotal role in the metabolism of pyrimidine analogs used in cancer chemotherapy. Recently, a novel pyrimidine Phosphorylase has been identified, named Uridine Phosphorylase 2 (UPP-2). In human tissues, UPP-2 is predominantly expressed in kidney cells, while in mouse tissue it is predominately expressed in the liver. We have recently screened a panel of human kidney and liver tissues by real-time PCR. The data concurred with high expression in kidney, but also a significant level of the UPP2 transcript was identified in human liver, albeit 10% of the level in kidney. It is critical for chemotherapeutic agents to be selective for tumor tissue sparing the surrounding normal tissues from toxic anti-proliferative effects, yet few agents have been designed to be selectively activated in tumor tissue. Capecitabine and 5′-deoxy-5-fluoroUridine (5′-DFUR) are two successful prodrugs that are metabolized to 5-FU mainly in tumor tissue due to an increased phosphorolytic activity because of an elevated presence of UPP and TPP. Several organs and tissues, including liver, express the same phosphorolytic enzymes resulting in the activation to 5-FU with consequent toxic effects. We previously determined that the UPP-2 isoform is overexpressed 4-fold in the liver of UPP/TPP double-knockout mice (DKO) and contemplated what role UPP2 may play in 5′-DFUR activation and toxicity. Both the physiological role of UPP-2 in nucleoside metabolism and the pyrimidine homeostatic regulation of UPP-1 and UPP-2 in liver are presently unclear. Wild-type (WT) and DKO mice were treated with 500 or 750 mg/kg of 5′-DFUR daily Monday through Friday for up to 6 weeks. A 1,000 mg/kg dosage group was included for DKO animals only, as WT mice display severe toxicity after only a few days of treatment at this high dosage. Both WT treatment groups had significant weight loss after the first week of treatment, while DKO animals across all treatment groups did not display weight loss until the third week of 5′-DFUR administration. 5-FU levels in mouse serum were evaluated 3 hours after 5′-DFUR administration. Interestingly, circulating 5-FU was detectable within the DKO treatment group. This indicates UPP-2 may contribute to 5’DFUR conversion into 5-FU. 5-Benzyl-acyclo-Uridine (BAU) is a potent inhibitor of UPP1. We therefore evaluated its effect on UPP2 activity, and by in vivo 19F-NMR identified a significant inhibitory activity against UPP2 by nullifying the activation of 5′-DFUR. It will be important to further quantify and discern the role of UPP-2 in prodrug activation and in normal tissue toxicities since we were not able to identify any tumor type expressing this second Uridine phosphorolytic activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4390. doi:10.1158/1538-7445.AM2011-4390
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activation of stat1 irf 1 and nf κb is required for the induction of Uridine Phosphorylase by tumor necrosis factor α and interferon γ
Nucleosides Nucleotides & Nucleic Acids, 2010Co-Authors: Laxiang Wan, Deliang Cao, Amy Ziemba, Jianmin Zeng, Giuseppe PizzornoAbstract:Uridine Phosphorylase (UPase) has been shown to be induced in various human and murine tumors and could potentially serve as a specific target for the modulation of tumor-selectivity of fluoropyrimidines. However, the signaling mechanisms underlying the regulation of UPase gene expression have not been determined. In this study, we investigated the effects of IFN-γ on the regulation of TNF-α-induced UPase activity and have uncovered the molecular mechanisms of this potentiation, utilizing murine EMT6 breast cancer cells. Our data has shown that IFN-γ can significantly increase UPase mRNA expression and the enzymatic activity induced by TNF-α in a dose-dependent manner, resulting in an enhanced sensitivity to 5-fluorouracil (5-FU) and 5′-Deoxy-5-fluoroUridine (5′DFUR). We have previously shown that TNF-α activates NF-κB through increased translocation of NF-κB p65 from the cytoplasm into the nuclei. Exposure to IFN-γ mainly affects nuclear IRF-1 and STAT1 in EMT6, but inhibits NF-κB p65 activity, indicatin...
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implications of the structure of human Uridine Phosphorylase 1 on the development of novel inhibitors for improving the therapeutic window of fluoropyrimidine chemotherapy
BMC Structural Biology, 2009Co-Authors: Tarmo P Roosild, Samantha Castronovo, Michael Fabbiani, Giuseppe PizzornoAbstract:Uridine Phosphorylase (UPP) is a key enzyme of pyrimidine salvage pathways, catalyzing the reversible phosphorolysis of ribosides of uracil to nucleobases and ribose 1-phosphate. It is also a critical enzyme in the activation of pyrimidine-based chemotherapeutic compounds such a 5-fluorouracil (5-FU) and its prodrug capecitabine. Additionally, an elevated level of this enzyme in certain tumours is believed to contribute to the selectivity of such drugs. However, the clinical effectiveness of these fluoropyrimidine antimetabolites is hampered by their toxicity to normal tissue. In response to this limitation, specific inhibitors of UPP, such as 5-benzylacycloUridine (BAU), have been developed and investigated for their ability to modulate the cytotoxic side effects of 5-FU and its derivatives, so as to increase the therapeutic index of these agents. In this report we present the high resolution structures of human Uridine Phosphorylase 1 (hUPP1) in ligand-free and BAU-inhibited conformations. The structures confirm the unexpected solution observation that the human enzyme is dimeric in contrast to the hexameric assembly present in microbial UPPs. They also reveal in detail the mechanism by which BAU engages the active site of the protein and subsequently disables the enzyme by locking the protein in a closed conformation. The observed inter-domain motion of the dimeric human enzyme is much greater than that seen in previous UPP structures and may result from the simpler oligomeric organization. The structural details underlying hUPP1's active site and additional surfaces beyond these catalytic residues, which coordinate binding of BAU and other acycloUridine analogues, suggest avenues for future design of more potent inhibitors of this enzyme. Notably, the loop forming the back wall of the substrate binding pocket is conformationally different and substantially less flexible in hUPP1 than in previously studied microbial homologues. These distinctions can be utilized to discover novel inhibitory compounds specifically optimized for efficacy against the human enzyme as a step toward the development of more effective chemotherapeutic regimens that can selectively protect normal tissues with inherently lower UPP activity.
A. M. Mikhailov - One of the best experts on this subject based on the ideXlab platform.
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X-ray structures of Uridine Phosphorylase from Vibrio cholerae in complexes with Uridine, thymidine, uracil, thymine, and phosphate anion: Substrate specificity of bacterial Uridine Phosphorylases
Crystallography Reports, 2016Co-Authors: I.i. Prokofev, Vladislav V. Balaev, Azat Gabdulkhakov, T. A. Seregina, Alexander S. Mironov, Alexander A. Lashkov, Ch. Betzel, A. M. MikhailovAbstract:In many types of human tumor cells and infectious agents, the demand for pyrimidine nitrogen bases increases during the development of the disease, thus increasing the role of the enzyme Uridine Phosphorylase in metabolic processes. The rational use of Uridine Phosphorylase and its ligands in pharmaceutical and biotechnology industries requires knowledge of the structural basis for the substrate specificity of the target enzyme. This paper summarizes the results of the systematic study of the three-dimensional structure of Uridine Phosphorylase from the pathogenic bacterium Vibrio cholerae in complexes with substrates of enzymatic reactions—Uridine, phosphate anion, thymidine, uracil, and thymine. These data, supplemented with the results of molecular modeling, were used to consider in detail the structural basis for the substrate specificity of Uridine Phosphorylases. It was shown for the first time that the formation of a hydrogen-bond network between the 2′-hydroxy group of Uridine and atoms of the active-site residues of Uridine Phosphorylase leads to conformational changes of the ribose moiety of Uridine, resulting in an increase in the reactivity of Uridine compared to thymidine. Since the binding of thymidine to residues of Uridine Phosphorylase causes a smaller local strain of the β-N1-glycosidic bond in this the substrate compared to the Uridine molecule, the β-N1-glycosidic bond in thymidine is more stable and less reactive than that in Uridine. It was shown for the first time that the phosphate anion, which is the second substrate bound at the active site, interacts simultaneously with the residues of the β5-strand and the β1-strand through hydrogen bonding, thus securing the gate loop in a conformation
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modified 5 fluorouracil Uridine Phosphorylase inhibitor
Crystallography Reports, 2016Co-Authors: A A Lashkov, A A Shchekotikhin, Alexander A Shtil, S E Sotnichenko, A. M. MikhailovAbstract:5-Fluorouracil (5-FU) is a medication widely used in chemotherapy to treat various types of cancer. Being a substrate for the reverse reaction catalyzed by Uridine Phosphorylase (UPase), 5-FU serves as a promising prototype molecule (molecular scaffold) for the design of a selective UPase inhibitor that enhances the antitumor activity of 5-FU and exhibits intrinsic cytostatic effects on cancer cells. The chemical formula of the new compound, which binds to the uracil-binding site and, in the presence of a phosphate anion, to the phosphate-binding site of UPase, is proposed and investigated by molecular simulation methods.
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three dimensional structures of unligated Uridine Phosphorylase from yersinia pseudotuberculosis at 1 4 a resolution and its complex with an antibacterial drug
Crystallography Reports, 2015Co-Authors: Vladislav V. Balaev, Azat Gabdulkhakov, Ch. Betzel, A S Mironov, A A Lashkov, M V Dontsova, A. M. MikhailovAbstract:Uridine Phosphorylases play an essential role in the cellular metabolism of some antibacterial agents. Acute infectious diseases (bubonic plague, yersiniosis, pseudotuberculosis, etc., caused by bacteria of the genus Yersinia) are treated using both sulfanilamide medicines and antibiotics, including trimethoprim. The action of an antibiotic on a bacterial cell is determined primarily by the character of its interactions with cellular components, including those which are not targets (for example, with pyrimidine Phosphorylases). This type of interaction should be taken into account in designing drugs. The three-dimensional structure of Uridine Phosphorylase from the bacterium Yersinia pseudotuberculosis (YptUPh) with the free active site was determined for the first time by X-ray crystallography and refined at 1.40 A resolution (DPI = 0.062 A; ID PDB: 4OF4). The structure of the complex of YptUPh with the bacteriostatic drug trimethoprim was studied by molecular docking and molecular dynamics methods. The trimethoprim molecule was shown to be buffered by the enzyme YptUPh, resulting in a decrease in the efficiency of the treatment of infectious diseases caused by bacteria of the genus Yersinia with trimethoprim.
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structure of a complex of Uridine Phosphorylase from yersinia pseudotuberculosis with the modified bacteriostatic antibacterial drug determined by x ray crystallography and computer analysis
Crystallography Reports, 2015Co-Authors: Vladislav V. Balaev, T. A. Seregina, A G Gabdoulkhakov, A A Lashkov, M V Dontsova, A. M. MikhailovAbstract:Pseudotuberculosis and bubonic plague are acute infectious diseases caused by the bacteria Yersinia pseudotuberculosis and Yersinia pestis. These diseases are treated, in particular, with trimethoprim and its modified analogues. However, Uridine Phosphorylases (pyrimidine nucleoside Phosphorylases) that are present in bacterial cells neutralize the action of trimethoprim and its modified analogues on the cells. In order to reveal the character of the interaction of the drug with bacterial Uridine Phosphorylase, the atomic structure of the unligated molecule of Uridine-specific pyrimidine nucleoside Phosphorylase from Yersinia pseudotuberculosis (YptUPh) was determined by X-ray diffraction at 1.7 A resolution with high reliability (Rwork = 16.2, Rfree = 19.4%; r.m.s.d. of bond lengths and bond angles are 0.006 A and 1.005°, respectively; DPI = 0.107 A). The atoms of the amino acid residues of the functionally important secondary-structure elements—the loop L9 and the helix H8—of the enzyme YptUPh were located. The three-dimensional structure of the complex of YptUPh with modified trimethoprim—referred to as 53I—was determined by the computer simulation. It was shown that 53I is a pseudosubstrate of Uridine Phosphorylases, and its pyrimidine-2,4-diamine group is located in the phosphate-binding site of the enzyme YptUPh.
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Crystallization and preliminary X-ray study of Vibrio cholerae Uridine Phosphorylase in complex with 6-methyluracil
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2013Co-Authors: I.i. Prokofev, Azat Gabdulkhakov, T. A. Seregina, Alexander S. Mironov, Alexander A. Lashkov, M V Dontsova, Ch. Betzel, A. M. MikhailovAbstract:Uridine Phosphorylase catalyzes the phosphorolysis of ribonucleosides, with the nitrogenous base and ribose 1-phosphate as products. Additionally, it catalyzes the reverse reaction of the synthesis of ribonucleosides from ribose 1-phosphate and a nitrogenous base. However, the enzyme does not catalyze the synthesis of nucleosides when the substrate is a nitrogenous base substituted at the 6-position, such as 6-methyluracil (6-MU). In order to explain this fact, it is essential to investigate the three-dimensional structure of the complex of 6-MU with Uridine Phosphorylase. 6-MU is a pharmaceutical agent that improves tissue nutrition and enhances cell regeneration by normalization of nucleotide exchange in humans. 6-MU is used for the treatment of diseases of the gastrointestinal tract, including infectious diseases. Here, procedures to obtain the Uridine Phosphorylase from the pathogenic bacterium Vibrio cholerae (VchUPh), purification of this enzyme, crystallization of the complex of VchUPh with 6-MU, and X-ray data collection and preliminary X-ray analysis of the VchUPh–6-MU complex at atomic resolution are reported.
Vern L Schramm - One of the best experts on this subject based on the ideXlab platform.
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constrained bonding environment in the michaelis complex of trypanosoma cruzi Uridine Phosphorylase
Biochemistry, 2012Co-Authors: Rafael G Silva, Randal D Kipp, Vern L SchrammAbstract:The transition state for the Trypanosoma cruzi Uridine Phosphorylase (TcUP) reaction has an expanded S(N)2 character. We used binding isotope effects (BIE's) to probe Uridine distortion in the complex with TcUP and sulfate to mimic the Michaelis complex. Inverse 1'-(3)H and 5'-(3)H BIE's indicate a constrained bonding environment of these groups in the complex. Quantum chemical modeling identified a Uridine conformer whose calculated BIE's match the experimental values. This conformer differs in sugar pucker and uracil orientation from the unbound conformer and the transition-state structure. These results support ground-state stabilization in the Michaelis complex.
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Uridine Phosphorylase from trypanosoma cruzi kinetic and chemical mechanisms
Biochemistry, 2011Co-Authors: Rafael G Silva, Vern L SchrammAbstract:The reversible phosphorolysis of Uridine to generate uracil and ribose 1-phosphate is catalyzed by Uridine Phosphorylase and is involved in the pyrimidine salvage pathway. We define the reaction mechanism of Uridine Phosphorylase from Trypanosoma cruzi by steady-state and pre-steady-state kinetics, pH–rate profiles, kinetic isotope effects from Uridine, and solvent deuterium isotope effects. Initial rate and product inhibition patterns suggest a steady-state random kinetic mechanism. Pre-steady-state kinetics indicated no rate-limiting step after formation of the enzyme-products ternary complex, as no burst in product formation is observed. The limiting single-turnover rate constant equals the steady-state turnover number; thus, chemistry is partially or fully rate limiting. Kinetic isotope effects with [1′-3H]-, [1′-14C]-, and [5′-14C,1,3-15N2]Uridine gave experimental values of α-T(V/K)Uridine = 1.063, 14(V/K)Uridine = 1.069, and 15,β-15(V/K)Uridine = 1.018, in agreement with an ANDN (SN2) mechanism whe...
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Transition-State Analysis of Trypanosoma cruzi Uridine Phosphorylase-Catalyzed Arsenolysis of Uridine
Journal of the American Chemical Society, 2011Co-Authors: Rafael G Silva, Mathew J. Vetticatt, Emilio F. Merino, Maria B. Cassera, Vern L SchrammAbstract:Uridine Phosphorylase catalyzes the reversible phosphorolysis of Uridine and 2′-deoxyUridine to generate uracil and (2-deoxy)ribose 1-phosphate, an important step in the pyrimidine salvage pathway. The coding sequence annotated as a putative nucleoside Phosphorylase in the Trypanosoma cruzi genome was overexpressed in Escherichia coli, purified to homogeneity, and shown to be a homodimeric Uridine Phosphorylase, with similar specificity for Uridine and 2′-deoxyUridine and undetectable activity toward thymidine and purine nucleosides. Competitive kinetic isotope effects (KIEs) were measured and corrected for a forward commitment factor using arsenate as the nucleophile. The intrinsic KIEs are: 1′-14C = 1.103, 1,3-15N2 = 1.034, 3-15N = 1.004, 1-15N = 1.030, 1′-3H = 1.132, 2′-2H = 1.086, and 5′-3H2 = 1.041 for this reaction. Density functional theory was employed to quantitatively interpret the KIEs in terms of transition-state structure and geometry. Matching of experimental KIEs to proposed transition-stat...
A A Lashkov - One of the best experts on this subject based on the ideXlab platform.
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x ray structure and molecular dynamics study of Uridine Phosphorylase from vibrio cholerae in complex with 2 2 anhydroUridine
Crystallography Reports, 2020Co-Authors: P A Eistrikhheller, I.i. Prokofev, Azat Gabdulkhakov, A S Mironov, S V Rubinsky, A A LashkovAbstract:The high-resolution three-dimensional structure of Uridine Phosphorylase from the pathogenic bacterium Vibrio cholerae in complex with the competitive inhibitor 2,2'-anhydroUridine was determined by X-ray diffraction (RCSBPDB ID: 6RCA). The three-dimensional structure of this complex is compared with the previously determined structures of V. cholerae Uridine Phosphorylase in complex with the substrate (Uridine) and S. typhimurium Uridine Phosphorylase in complex with 2,2'-anhydroUridine. The protein–inhibitor and protein–substrate binding free energies were calculated by the free-energy perturbation method. The number of stable hydrogen bonds between the 2,2'-anhydroUridine molecule and the active site of the enzyme is smaller and these bonds are longer compared to the natural substrate of the enzyme (Uridine). However, calculations taking into account solvation energy of the molecule and the entropy effects showed that the binding of the inhibitor (2,2'-anhydroUridine) at the active site of the protein is energetically more favorable than the binding of the native substrate (Uridine). These results may be useful in the design of new inhibitors with a higher selectivity for the binding sites of Uridine Phosphorylases.
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modified 5 fluorouracil Uridine Phosphorylase inhibitor
Crystallography Reports, 2016Co-Authors: A A Lashkov, A A Shchekotikhin, Alexander A Shtil, S E Sotnichenko, A. M. MikhailovAbstract:5-Fluorouracil (5-FU) is a medication widely used in chemotherapy to treat various types of cancer. Being a substrate for the reverse reaction catalyzed by Uridine Phosphorylase (UPase), 5-FU serves as a promising prototype molecule (molecular scaffold) for the design of a selective UPase inhibitor that enhances the antitumor activity of 5-FU and exhibits intrinsic cytostatic effects on cancer cells. The chemical formula of the new compound, which binds to the uracil-binding site and, in the presence of a phosphate anion, to the phosphate-binding site of UPase, is proposed and investigated by molecular simulation methods.
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three dimensional structures of unligated Uridine Phosphorylase from yersinia pseudotuberculosis at 1 4 a resolution and its complex with an antibacterial drug
Crystallography Reports, 2015Co-Authors: Vladislav V. Balaev, Azat Gabdulkhakov, Ch. Betzel, A S Mironov, A A Lashkov, M V Dontsova, A. M. MikhailovAbstract:Uridine Phosphorylases play an essential role in the cellular metabolism of some antibacterial agents. Acute infectious diseases (bubonic plague, yersiniosis, pseudotuberculosis, etc., caused by bacteria of the genus Yersinia) are treated using both sulfanilamide medicines and antibiotics, including trimethoprim. The action of an antibiotic on a bacterial cell is determined primarily by the character of its interactions with cellular components, including those which are not targets (for example, with pyrimidine Phosphorylases). This type of interaction should be taken into account in designing drugs. The three-dimensional structure of Uridine Phosphorylase from the bacterium Yersinia pseudotuberculosis (YptUPh) with the free active site was determined for the first time by X-ray crystallography and refined at 1.40 A resolution (DPI = 0.062 A; ID PDB: 4OF4). The structure of the complex of YptUPh with the bacteriostatic drug trimethoprim was studied by molecular docking and molecular dynamics methods. The trimethoprim molecule was shown to be buffered by the enzyme YptUPh, resulting in a decrease in the efficiency of the treatment of infectious diseases caused by bacteria of the genus Yersinia with trimethoprim.
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structure of a complex of Uridine Phosphorylase from yersinia pseudotuberculosis with the modified bacteriostatic antibacterial drug determined by x ray crystallography and computer analysis
Crystallography Reports, 2015Co-Authors: Vladislav V. Balaev, T. A. Seregina, A G Gabdoulkhakov, A A Lashkov, M V Dontsova, A. M. MikhailovAbstract:Pseudotuberculosis and bubonic plague are acute infectious diseases caused by the bacteria Yersinia pseudotuberculosis and Yersinia pestis. These diseases are treated, in particular, with trimethoprim and its modified analogues. However, Uridine Phosphorylases (pyrimidine nucleoside Phosphorylases) that are present in bacterial cells neutralize the action of trimethoprim and its modified analogues on the cells. In order to reveal the character of the interaction of the drug with bacterial Uridine Phosphorylase, the atomic structure of the unligated molecule of Uridine-specific pyrimidine nucleoside Phosphorylase from Yersinia pseudotuberculosis (YptUPh) was determined by X-ray diffraction at 1.7 A resolution with high reliability (Rwork = 16.2, Rfree = 19.4%; r.m.s.d. of bond lengths and bond angles are 0.006 A and 1.005°, respectively; DPI = 0.107 A). The atoms of the amino acid residues of the functionally important secondary-structure elements—the loop L9 and the helix H8—of the enzyme YptUPh were located. The three-dimensional structure of the complex of YptUPh with modified trimethoprim—referred to as 53I—was determined by the computer simulation. It was shown that 53I is a pseudosubstrate of Uridine Phosphorylases, and its pyrimidine-2,4-diamine group is located in the phosphate-binding site of the enzyme YptUPh.
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in silico analysis of the three dimensional structures of the homodimer of Uridine Phosphorylase from yersinia pseudotuberculosis in the ligand free state and in a complex with 5 fluorouracil
Crystallography Reports, 2013Co-Authors: A A Lashkov, S E Sotnichenko, A. M. MikhailovAbstract:Pseudotuberculosis is an acute infectious disease characterized by a lesion of the gastrointestinal tract. A positive therapeutic effect can be achieved by selectively suppressing the activity of Uridine Phosphorylase from the causative agent of the disease Yersinia pseudotuberculosis. The synergistic effect of a combination of the chemotherapeutic agent 5-fluorouracil and antimicrobial drugs, which block the synthesis of pyrimidine bases, on the cells of pathogenic protozoa and bacteria is described in the literature. The three-dimensional structures of Uridine Phosphorylase from Yersinia pseudotuberculosis (YptUPh) both in the ligand-free state and in complexes with pharmacological agents are unknown, which hinders the search for and design of selective inhibitors of YptUPh. The three-dimensional structure of the ligand-free homodimer of YptUPh was determined by homology-based molecular modeling. The three-dimensional structure of the subunit of the YptUPh molecule belongs to α/β proteins, and its topology is a three-layer α/β/α sandwich. The subunit monomer of the YptUPh molecule consists of 38% helices and 24% β strands. A model of the homodimer structure of YptUPh in a complex with 5-FU was obtained by the molecular docking. The position of 5-FU in the active site of the molecule is very consistent with the known data on the X-ray diffraction structures of other bacterial Uridine Phosphorylases (the complex of Uridine Phosphorylase from Salmonella typhimurium (StUPh) with 5-FU, ID PDB: 4E1V and the complex of Uridine Phosphorylase from Escherichia coli (EcUPh) with 5-FU and ribose 1-phosphate, ID PDB: 1RXC).
Steven E. Ealick - One of the best experts on this subject based on the ideXlab platform.
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the crystal structure of streptococcus pyogenes Uridine Phosphorylase reveals a distinct subfamily of nucleoside Phosphorylases
Biochemistry, 2011Co-Authors: Timothy H Tran, Stig Christoffersen, Paula W Allan, William B Parker, Jure Piskur, Immacolata Serra, Marco Terreni, Steven E. EalickAbstract:Uridine Phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of Uridine or 2'-deoxyUridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside Phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes Uridine Phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside Phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 A resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an α/β monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUP showed that its substrate specificity is similar to that of EcUP, while EcUP is ∼7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.
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Glycal formation in crystals of Uridine Phosphorylase.
Biochemistry, 2010Co-Authors: Debamita Paul, Sean T. O’leary, Kanagalaghatta R. Rajashankar, Angela V. Toms, Ethan C. Settembre, Jennie M. Sanders, Tadhg P. Begley, Steven E. EalickAbstract:Uridine Phosphorylase is a key enzyme in the pyrimidine salvage pathway. This enzyme catalyzes the reversible phosphorolysis of Uridine to uracil and ribose 1-phosphate (or 2{prime}-deoxyUridine to 2{prime}-deoxyribose 1-phosphate). Here we report the structure of hexameric Escherichia coli Uridine Phosphorylase treated with 5-fluoroUridine and sulfate and dimeric bovine Uridine Phosphorylase treated with 5-fluoro-2{prime}-deoxyUridine or Uridine, plus sulfate. In each case the electron density shows three separate species corresponding to the pyrimidine base, sulfate, and a ribosyl species, which can be modeled as a glycal. In the structures of the glycal complexes, the fluorouracil O2 atom is appropriately positioned to act as the base required for glycal formation via deprotonation at C2{prime}. Crystals of bovine Uridine Phosphorylase treated with 2{prime}-deoxyUridine and sulfate show intact nucleoside. NMR time course studies demonstrate that Uridine Phosphorylase can catalyze the hydrolysis of the fluorinated nucleosides in the absence of phosphate or sulfate, without the release of intermediates or enzyme inactivation. These results add a previously unencountered mechanistic motif to the body of information on glycal formation by enzymes catalyzing the cleavage of glycosyl bonds.
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structural basis for inhibition of escherichia coli Uridine Phosphorylase by 5 substituted acycloUridines
Acta Crystallographica Section D-biological Crystallography, 2005Co-Authors: Ethan C. Settembre, Mahmoud El H Kouni, Steven E. EalickAbstract:Uridine Phosphorylase (UP) catalyzes the reversible phosphorolysis of Uridine to uracil and ribose 1-phosphate and is a key enzyme in the pyrimidine-salvage pathway. Escherichia coli UP is structurally homologous to E. coli purine nucleoside Phosphorylase and other members of the type I family of nucleoside Phosphorylases. The structures of 5-benzylacycloUridine, 5-phenylthioacycloUridine, 5-phenylselenenylacycloUridine, 5-m-benzyloxybenzyl acycloUridine and 5-m-benzyloxybenzyl barbituric acid acyclonucleoside bound to the active site of E. coli UP have been determined, with resolutions ranging from 1.95 to 2.3 A. For all five complexes the acyclo sugar moiety binds to the active site in a conformation that mimics the ribose ring of the natural substrates. Surprisingly, the terminal hydroxyl group occupies the position of the nonessential 5′-hydroxyl substituent of the substrate rather than the 3′-hydroxyl group, which is normally required for catalytic activity. Until recently, inhibitors of UP were designed with limited structural knowledge of the active-site residues. These structures explain the basis of inhibition for this series of acycloUridine analogs and suggest possible additional avenues for future drug-design efforts. Furthermore, the studies can be extended to design inhibitors of human UP, for which no X-ray structure is available.
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crystallization of Uridine Phosphorylase from e coli on earth macrogravity and microgravity
1997Co-Authors: Elena Blagova, A. M. Mikhailov, Ekaterina Morgunova, E A Smirnova, Sh R Armstrong, Ch Mao, Steven E. EalickAbstract:Uridine Phosphorylase from E. coli (Uph) has been crystallized on Earth by a vapour diffusion technique, at zero gravity in space (space station “Mir”), and in macrogravity (ultracentrifuge G-62). We compare results obtained under these three conditions. We show the advantages of macrogravity growth of Uph crystals, i.e. high stability, rate and reproducibility. Crystallization of this protein in the ultracentrifuge allowed us to choose the optimal conditions for the production of large crystals suitable for X-ray analysis. The protein structure was determined by the molecular replacement method, using crystals obtained in macrogravity and on Earth.
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atomic structure at 2 5 a resolution of Uridine Phosphorylase from e coli as refined in the monoclinic crystal lattice
FEBS Letters, 1995Co-Authors: Yu E Morgunova, A. M. Mikhailov, Steven E. Ealick, Elena Blagova, E A Smirnova, Sh R Armstrong, Ch Mao, A N Popov, B K Vainshtein, Andrey A KomissarovAbstract:Uridine Phosphorylase from E. coli (Upase) has been crystallized using vapor diffusion technique in a new monoclinic crystal form. The structure was determined by the molecular replacement method at 2.5 A resolution. The coordinates of the trigonal crystal form were used as a starting model and the refinement by the program XPLOR led to the R-factor of 18.6%. The amino acid fold of the protein was found to be the same as that in the trigonal crystals. The positions of flexible regions were refined. The conclusion about the involvement in the active site is in good agreement with the results of the biochemical experiments.
