Structure-Based Prototyping of Allosteric Inhibitors of Human Uridine/Cytidine Kinase 2 (UCK2).

Pyrimidine nucleotide biosynthesis in humans is a promising chemotherapeutic target for infectious diseases caused by RNA viruses. Because mammalian cells derive pyrimidine ribonucleotides through a combination of de novo biosynthesis and salvage, combined inhibition of dihydroorotate dehydrogenase (DHODH; the first committed step in de novo pyrimidine nucleotide biosynthesis) and uridine/cytidine kinase 2 (UCK2; the first step in salvage of exogenous nucleosides) strongly attenuates viral replication in infected cells. However, while several pharmacologically promising inhibitors of human DHODH are known, to date there are no reports of medicinally viable leads against UCK2. Here, we use structure-based drug prototyping to identify two classes of promising leads that noncompetitively inhibit UCK2 activity. In the process, we have identified a hitherto unknown allosteric site at the intersubunit interface of this homotetrameric enzyme. By reducing the kcat of human UCK2 without altering its KM, these new inhibitors have the potential to enable systematic dialing of the fractional inhibition of pyrimidine salvage to achieve the desired antiviral effect with minimal host toxicity.

Discovery of small molecule inhibitors of human uridine-cytidine kinase 2 by high-throughput screening.

Clinically relevant inhibitors of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in mammalian de novo pyrimidine synthesis, have strong antiviral and anticancer activity in vitro. However, they are ineffective in vivo due to efficient uridine salvage by infected or rapidly dividing cells. The pyrimidine salvage enzyme uridine-cytidine kinase 2 (UCK2), a ∼29 kDa protein that forms a tetramer in its active state, is necessary for uridine salvage. Notwithstanding the pharmacological potential of this target, no medicinally tractable inhibitors of the human enzyme have been reported to date. We therefore established and miniaturized an in vitro assay for UCK2 activity and undertook a high-throughput screen against a ∼40,000-compound library to generate drug-like leads. The structures, activities, and modes of inhibition of the most promising hits are described. Notably, our screen yielded non-competitive UCK2 inhibitors which were able to suppress nucleoside salvage in cells both in the presence and absence of DHODH inhibitors.

Crude Extracts, Flavokawain B and Alpinetin Compounds from the Rhizome of Alpinia mutica Induce Cell Death via UCK2 Enzyme Inhibition and in Turn Reduce 18S rRNA Biosynthesis in HT-29 Cells.

Uridine-cytidine kinase 2 is an enzyme that is overexpressed in abnormal cell growth and its implication is considered a hallmark of cancer. Due to the selective expression of UCK2 in cancer cells, a selective inhibition of this key enzyme necessitates the discovery of its potential inhibitors for cancer chemotherapy. The present study was carried out to demonstrate the potentials of natural phytochemicals from the rhizome of Alpinia mutica to inhibit UCK2 useful for colorectal cancer. Here, we employed the used of in vitro to investigate the effectiveness of natural UCK2 inhibitors to cause HT-29 cell death. Extracts, flavokawain B, and alpinetin compound from the rhizome of Alpinia mutica was used in the study. The study demonstrated that the expression of UCK2 mRNA were substantially reduced in treated HT-29 cells. In addition, downregulation in expression of 18S ribosomal RNA was also observed in all treated HT-29 cells. This was confirmed by fluorescence imaging to measure the level of expression of 18S ribosomal RNA in live cell images. The study suggests the possibility of MDM2 protein was downregulated and its suppression subsequently activates the expression of p53 during inhibition of UCK2 enzyme. The expression of p53 is directly linked to a blockage of cell cycle progression at G0/G1 phase and upregulates Bax, cytochrome c, and caspase 3 while Bcl2 was deregulated. In this respect, apoptosis induction and DNA fragmentation were observed in treated HT-29 cells. Initial results from in vitro studies have shown the ability of the bioactive compounds of flavokawain B and alpinetin to target UCK2 enzyme specifically, inducing cell cycle arrest and subsequently leading to cancer cell death, possibly through interfering the MDM2-p53 signalling pathway. These phenomena have proven that the bioactive compounds could be useful for future therapeutic use in colon cancer.

The pivotal role of uridine-cytidine kinases in pyrimidine metabolism and activation of cytotoxic nucleoside analogues in neuroblastoma.

Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine and cytidine as well as the pharmacological activation of several cytotoxic pyrimidine ribonucleoside analogues. In this study, we investigated the functional role of two isoforms of UCK in neuroblastoma cell lines. Analysis of mRNA coding for UCK1 and UCK2 showed that UCK2 is the most abundantly expressed UCK in a panel of neuroblastoma cell lines. Transient and stable overexpression of UCK2 in neuroblastoma cells increased the metabolism of uridine and cytidine as well as the cytotoxicity of 3-deazauridine. Knockdown of endogenous UCK2 as well as overexpression of UCK1 resulted in decreased metabolism of uridine and cytidine and protected the neuroblastoma cells from 3-deazauridine-induced toxicity. Subcellular localization studies showed that UCK1-GFP and UCK2-GFP were localized in the cell nucleus and cytosol, respectively. However, co-expression of UCK1 with UCK2 resulted in a nuclear localization of UCK2 instead of its normal cytosolic localization, thereby impairing its normal function. The physical association of UCK1 and UCK2 was further demonstrated through pull-down analysis using his-tagged UCK. The discovery that UCK2 is highly expressed in neuroblastoma opens the possibility for selectively targeting neuroblastoma cells using UCK2-dependent pyrimidine analogues, while sparing normal tissues.

In Silico Discovery of Potential Uridine-Cytidine Kinase 2 Inhibitors from the Rhizome of Alpinia mutica.

Uridine-cytidine kinase 2 is implicated in uncontrolled proliferation of abnormal cells and it is a hallmark of cancer, therefore, there is need for effective inhibitors of this key enzyme. In this study, we employed the used of in silico studies to find effective UCK2 inhibitors of natural origin using bioinformatics tools. An in vitro kinase assay was established by measuring the amount of ADP production in the presence of ATP and 5-fluorouridine as a substrate. Molecular docking studies revealed an interesting ligand interaction with the UCK2 protein for both flavokawain B and alpinetin. Both compounds were found to reduce ADP production, possibly by inhibiting UCK2 activity in vitro. In conclusion, we have identified flavokawain B and alpinetin as potential natural UCK2 inhibitors as determined by their interactions with UCK2 protein using in silico molecular docking studies. This can provide information to identify lead candidates for further drug design and development.

Thymidine kinase and uridine-cytidine kinase from Entamoeba histolytica: cloning, characterization, and search for specific inhibitors.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Although anti-amoebic drugs such as metronidazole, emetine, chloroquine and nitazoxanide are generally effective, there is always potential for development of drug resistance. In order to find novel targets to control E. histolytica proliferation we cloned, expressed and purified thymidine kinase (Eh-TK) and uridine-cytidine kinase (Eh-UCK) from E. histolytica. Eh-TK phosphorylates thymidine with a Km of 0.27 microm, whereas Eh-UCK phosphorylates uridine and cytidine with Km of 0.74 and 0.22 mM, respectively. For both enzymes, ATP acts as specific phosphate donor. In order to find alternative treatments of E. histolytica infection we tested numerous nucleoside analogues and related compounds as inhibitors and/or substrates of Eh-TK and Eh-UCK, and active compounds against E. histolytica in cell culture. Our results indicate that inhibitors or alternative substrates of the enzymes, although partially reducing protozoan proliferation, are reversible and not likely to become drugs against E. histolytica infections.

Metabolic effects of novel N-1-sulfonylpyrimidine derivatives on human colon carcinoma cells.

Novel N-1-sulfonylpyrimidine derivatives have a strong antiproliferative activity and an ability to induce apoptosis in treated tumor cells. The purpose of this study was to elucidate the effects of two N-1-sulfonylpyrimidine nucleobases on catalytic activity of tumor cells' enzymes involved in DNA and RNA synthesis, and in de novo and salvage pyrimidine and purine syntheses. Investigations were performed in vitro on colon carcinoma cells (Caco2). The biosynthetic activity of the tumor cells' enzymes was determined using sensitive radio-assays. Enzyme activity in treated cells was calculated relative to untreated control cells. Both of the investigated compounds, 1-(p-toluenesulfonyl) cytosine (TsC) and 5-bromo-1-(methanesulfonyl) uracil (BMsU) inhibited activities of specific enzymes involved in nucleic acid synthesis. BMsU strongly inhibited activities of DNA polymerase alpha (53%), thymidine kinase (68%), thymidilate synthase (43%), and ribonucleotide reductase (46%). De novo biosynthesis of pyrimidine and purine was reduced by 20%. TsC was able to inhibit RNA polymerase (37%), orotate phosphoribosyltransferase (39%), uridine kinase (44%), ribonucleotid reductase (47%), and de novo purine synthesis (61%). Antitumor activity of 1-(p-toluenesulfonyl) cytosine (TsC) and 5-bromo-1-(methanesulfonyl) uracil (BMsU) is closely associated with their inhibitory activity on enzymes that play an important role in the metabolism of tumor cells.

Mg2+ as activator of uridine phosphorylation in coordination with other cellular responses to growth factors.

The divalent cation ionophore A23187 facilitates the manipulation of intracellular Mg2+ without increasing the general permeability of the cell. The uptake of uridine into cells is limited by its rate of intracellular phosphorylation that increases within minutes after the addition of growth factors. In the experiments described here, the rate of uridine uptake in ionophore-treated cells stimulated by either serum or insulin depended on the extracellular and intracellular concentrations of Mg2+ and was independent of the extracellular Ca2+ concentration. In very high concentrations of Mg2+ (50 mM), ionophore-treated cells take up uridine as fast, in the absence of growth factors as in their presence, demonstrating that Mg2+ can replace the growth factor requirement for the stimulation of uridine uptake. In contrast, thymidine uptake, which also is limited by its rate of intracellular phosphorylation, showed no early response to either growth factors or Mg2+ concentration, which is consistent with the 10-fold lower Mg2+ requirement of thymidine kinase compared with uridine kinase. The feedback inhibition of uridine kinase by UTP and CTP in cell-free extracts was alleviated by increased Mg2+ concentration. The results support the thesis that the increased uptake of uridine in cells treated with growth factors is determined by a membrane-induced increase in intracellular free Mg2+. Such increase would also accelerate the rate of translation-initiation and other coordinate responses that, unlike increased uridine uptake, are essential for cell proliferation. The rate of uridine uptake is suggested as a direct indicator of free cytosolic Mg2+ that drives the shift from quiescence to proliferation.

Structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase.

Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine and cytidine and activates pharmacological ribonucleoside analogs. Here we present the crystal structures of human UCK alone and in complexes with a substrate, cytidine, a feedback inhibitor, CTP or UTP, and with phosphorylation products, CMP and ADP, respectively. Free UCK takes an alpha/beta mononucleotide binding fold and exists as a homotetramer with 222 symmetry. Upon inhibitor binding, one loop region was loosened, causing the UCK tetramer to be distorted. Upon cytidine binding, a large induced fit was observed at the uridine/cytidine binding site, which endows UCK with a strict specificity for pyrimidine ribonucleosides. The first UCK structure provided the structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase, which give clues for the design of novel antitumor and antiviral ribonucleoside analogs that inhibit RNA synthesis.

Cyclopentenyl uracil: an effective inhibitor of uridine salvage in vivo.

Cyclopentenyl uracil, a non-cytotoxic inhibitor of uridine kinase, was found to effectively block the salvage of circulating uridine by host and tumor tissues in the intact mouse. Dose-response characteristics of the inhibition were determined. Large doses (1 g/kg) of cyclopentenyl uracil were required, and the effect of a single dose fell rapidly over a 24-hr period. A sustained inhibition of uridine salvage of > 64-79% could be maintained by multiple doses of 1 g/kg given on an every 8-hr schedule. Mice given cyclopentenyl uracil (1 g/kg) every 8 hr for 5 days continued to gain weight and showed no signs of toxicity; however, the combination of cyclopentenyl uracil with a non-toxic dose of N-(phosphonacetyl)-L-aspartic acid (PALA; 200 mg/kg daily for 5 days) was lethal to mice, indicating that circulating uridine modifies the toxicity of agents that act on enzymes of the de novo pyrimidine pathway. Although the duration of action and potency of cyclopentenyl uracil are not ideal, this is the first demonstration of an effective inhibition of uridine salvage in the intact mouse with a non-cytotoxic agent. This makes possible the evaluation of concurrent inhibition of de novo and salvage routes to pyrimidine nucleotides as an approach to chemotherapy.

Inhibition of lymphocyte proliferation by liver arginase.

The activities of thymidine kinase and uridine kinase (enzymes for pyrimidine salvage pathway) in phytohemagglutinin (PHA)--prestimulated lymphocytes were inhibited by arginase in a similar pattern to the inhibition on thymidine incorporation. Further study revealed that arginase did not directly affect the activities of these enzymes in the cell-free system. Thymidine kinase and uridine kinase activities of PHA-prestimulated lymphocytes were inhibited by arginase making their activities as low as that cultured in arginine-free RPMI-1640 medium. These results suggest that arginine-depletion in the culture medium is the primary mode of action of arginase on the inhibition of mitogen-stimulated lymphocyte proliferation.

Uridine kinase inhibition is involved in the vasodilator effects of minoxidil in the rat.

1. Since minoxidil is a pyrimidine derivative, its actions on vascular smooth muscle may derive from structural relationships to the uridine nucleotides, which have been shown to be vasoconstrictive in the rat. 2. Minoxidil at a low vasodepressor dose of 0.03 mg/kg per min abolished the pressor response to uridine at doses from 2 to 8 mumol/kg per min, but did not reduce the responses to uridine monophosphate or uridine diphosphate in similar pressor doses, suggesting an action on either transport of uridine into cells or on uridine kinase which catalyses phosphorylation of uridine to uridine monophosphate, the mediator of uridine's vascular actions. 3. The active metabolite of minoxidil was found to inhibit rat liver uridine kinase in vivo using an HPLC technique. 4. Plasma uridine concentration was significantly higher in 11 hypertensive patients on minoxidil compared with pretreatment values, suggesting that uridine kinase inhibition is of a degree sufficient to increase the circulating pool of uridine. 5. The data is consistent with uridine kinase inhibition being a mechanism for the vasodilator actions of minoxidil.

Homogeneous uridine kinase from Ehrlich ascites tumor: substrate specificity and inhibition by bisubstrate analogs.

Uridine kinase has been purified to homogeneity from Ehrlich ascites tumor cells. For the phosphate acceptor site, the enzyme shows substrate specificity only for ribopyrimidine nucleosides and is active with various analogs that have limited structural alterations; both endocyclic and exocyclic substituents can be acceptable. Of nucleosides that have been used in the chemotherapy of cancer, 5-fluorouridine, 6-azauridine, and 3-deazauridine are good substrates, whereas arabinosylcytosine is a poor substrate. No analogs are better substrates than the physiological substrates uridine and cytidine. 5', 5''' -P1, P4-Bisnucleoside oligophosphate bisubstrate analogs (e.g., Ap4U, Ap5U) were synthesized and tested as inhibitors. The most effective compound was Ap4U; with a Ki of 197 microM, it bound more tightly than ATP but no better than uridine. Ap3A, Ap4A, and Ap5A were also tested, with the result that both Ap4A and Ap4U were most effective, suggesting that this size of bisubstrate analog most closely approaches the spacing of the catalytic site.

Inhibition of uridine kinase and the salvage of uridine by modified pyrimidine nucleosides.

Uridine kinase can play a crucial role in the provision of pyrimidine nucleotides for cellular nucleic acid synthesis, particularly when de novo synthesis is inhibited by chemotherapeutic agents. Therefore, uridine kinase is an attractive target for drug development. We examined a series of 29 analogs of uridine, most with modifications at the 5'-position, as inhibitors of uridine kinase in vitro and of uridine salvage by intact L1210 cells. Substitution at the 5'-position resulted in decreased efficacy as inhibitors of uridine kinase, particularly if the substituent was large. None of the analogs with 5'-position modifications effectively inhibited salvage of uridine by intact L1210 cells. Four carbocyclic pyrimidine nucleoside analogs (one series) were all effective competitive inhibitors of uridine kinase and of uridine salvage by intact L1210 cells. Cyclopentenyl uracil 19 shows promise for further development as it inhibits uridine salvage at nontoxic concentrations.

Multiple forms and inhibitors of uridine-cytidine kinase in neoplastic cells.

1. Two forms (isozymes) of uridine (urd)-cytidine (cyd) kinase are present in the 30-50% ammonium sulfate fraction of the cytosols of L1210 ascites leukemia cells and a human malignant lymphoma. 2. These findings confirm those which described multiple forms of urd-cyd kinase in tumors with rapid growth rate. 3. Studied of inhibitors (nucleoside analogs) of urd-cyd kinase derived from L1210 and 6410 leukemia cells resulted in the finding of four possible inhibitors of this enzyme.

Cytotoxicity of a new uridine analog, 4-hydroxy-1-(beta-D-ribofuranosyl)-pyridazine-6-one, and its interaction with uridine kinase.

A new uridine analog, 4-hydroxy-1-(beta-D-ribonfuranosyl)-pyridazin-6-one (3-deaza-6-azaUrd), inhibited the growth of L1210 cells in culture, with a concentration to reduce growth rate to 50% of control of 7 X 10(-5) M. After treatment for 24 or 48 h with 5 X 10(-4) M 3-deaza-6-azaUrd, 80% of the cells were unable to resume growth when the analog was removed from the cultures; also, 99% of the cells were killed, as determined by colony formation in soft agar. Studies on the prevention of the cytotoxic effects of 5 X 10(-4) M 3-deaza-6-azaUrd showed that uridine or cytidine gave complete protection. 2'-Deoxycytidine also gave partial protection, but orotic acid or thymidine had no effect on the growth inhibition by 3-deaza-6-azaUrd. These results suggested that growth inhibition by 3-deaza-6-azaUrd might be due to interference in pyrimidine biosynthesis. Activation of 3-deaza-6-azaUrd to its 5'-phosphate derivative appeared to be catalyzed by uridine kinase. 3-Deaza-6-azaUrd was shown to complete with uridine for phosphorylation (Ki = 4.7 mM) and, therefore, to be a possible alternative substrate for uridine kinase from mouse kidney (Km for uridine = 82 microM). The enzyme was partially purified by streptomycin sulfate precipitation, ammonium sulfate fractionation, and gel filtration. This preparation was found to be free of pyrimidine nucleoside phosphorylase and uridine monophosphate kinase.

Properties of uridine-cytidine kinase derived from L1210 leukemia cells.

Uridine-cytidine kinase isolated from murine L1210 leukemia cells exist in several isozymic forms, as indicted by isoelectric focusing and by column chromatography on Sepharose 6B. Of 39 compounds thus far examined as potential inhibitors of the phosphorylation of uridine by ATP, four were of significant activity: 5'-azido-5'-deoxycytidine, 5'-O-nitro-5-fluorouridine, 5'-O-nitrouridine, and 5'-azido-5'-deoxyuridine. 5'-Azido-5'-deoxycytidine was the most active (competitive with uridine) and exhibited a Ki of 37 x 10(-5) M. Other properties of uridine-cytidine kinase were examined, and the apparent Michaelis constants for uridine, cytidine, and adenosine 5'-triphosphate were 23 x 10(-5) M, 15 x 10(-5) M, and 34.9 x 10(-5) M, respectively.