5-Iodotubercidin inhibits SARS-CoV-2 RNA synthesis.

Coronavirus disease 2019 (COVID-19) is a newly emerged infectious disease caused by a novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid global emergence of SARS-CoV-2 highlights the importance and urgency for potential drugs to control the pandemic. The functional importance of RNA-dependent RNA polymerase (RdRp) in the viral life cycle, combined with structural conservation and absence of closely related homologs in humans, makes it an attractive target for designing antiviral drugs. Nucleos(t)ide analogs (NAs) are still the most promising broad-spectrum class of viral RdRp inhibitors. In this study, using our previously developed cell-based SARS-CoV-2 RdRp report system, we screened 134 compounds in the Selleckchemicals NAs library. Four candidate compounds, Fludarabine Phosphate, Fludarabine, 6-Thio-20-Deoxyguanosine (6-Thio-dG), and 5-Iodotubercidin, exhibit remarkable potency in inhibiting SARS-CoV-2 RdRp. Among these four compounds, 5-Iodotubercidin exhibited the strongest inhibition upon SARS-CoV-2 RdRp, and was resistant to viral exoribonuclease activity, thus presenting the best antiviral activity against coronavirus from a different genus. Further study showed that the RdRp inhibitory activity of 5-Iodotubercidin is closely related to its capacity to inhibit adenosine kinase (ADK).

S-Adenosylmethionine and methylthioadenosine inhibit ß-catenin signaling by multiple mechanisms in liver and colon cancer.

S-Adenosylmethionine (SAMe), the principal methyl donor that is available as a nutritional supplement, and its metabolite methylthioadenosine (MTA) exert chemopreventive properties against liver and colon cancer in experimental models. Both agents reduced ß-catenin expression on immunohistochemistry in a murine colitis-associated colon cancer model. In this study, we examined the molecular mechanisms involved. SAMe or MTA treatment in the colitis-associated cancer model lowered total ß-catenin protein levels by 47 and 78%, respectively. In an orthotopic liver cancer model, increasing SAMe levels by overexpressing methionine adenosyltransferase 1A also reduced total ß-catenin levels by 68%. In both cases, lower cyclin D1 and c-Myc expression correlated with lower ß-catenin levels. In liver (HepG2) and colon (SW480, HCT116) cancer cells with constitutively active ß-catenin signaling, SAMe and MTA treatment inhibited ß-catenin activity by excluding it from the nuclear compartment. However, in liver (Huh-7) and colon (RKO) cancer cells expressing wild-type Wnt/ß-catenin, SAMe and MTA accelerated ß-catenin degradation by a glycogen synthase kinase 3-ß-dependent mechanism. Both agents lowered protein kinase B activity, but this was not mediated by inhibiting phosphoinositide 3-kinase. Instead, both agents increased the activity of protein phosphatase 2A, which inactivates protein kinase B. The effect of MTA on lowering ß-catenin is direct and not mediated by its conversion to SAMe, as blocking this conversion had no influence. In conclusion, SAMe and MTA inhibit Wnt/ß-catenin signaling in colon and liver cancer cells regardless of whether this pathway is aberrantly induced, making them ideal candidates for chemoprevention and/or chemotherapy in these cancers.

Preclinical studies of methylthioadenosine for the treatment of multiple sclerosis.

BACKGROUND: Methylthioadenosine (MTA) is a natural metabolite with immunomodulatory properties. MTA improves the clinical course and pathology of the animal model of multiple sclerosis, even when therapy is started after disease onset. OBJECTIVE: Our aim was to compare the efficacy of MTA in ameliorating experimental autoimmune encephalomyelitis (EAE) compared with first line approved therapies, to develop an oral formulation of MTA and to assess its pharmacokinetic profile. METHODS: EAE was induced in C57BL/6 mice by immunization with MOG(35-55) peptide in Freund's Adjuvant. Animals were treated with MTA, interferon-beta or glatiramer acetate starting the day of immunization and the clinical score was collected blind. Pharmacokinetic studies were performed in Sprague Dawley rats by administering MTA by intraperitoneal injection and orally, and collecting blood at different intervals. MTA levels were measured by high-performance liquid chromatography. RESULTS: We found that MTA ameliorated EAE in a dose-response manner. Moreover, the highest dose of MTA (60 mg/kg) was more efficacious than mouse interferon-beta or glatiramer acetate. We developed a salt of MTA for oral administration, with similar dose-response effect in the EAE model. Combination therapy assays between MTA and interferon-beta or glatiramer acetate were more effective than the individual therapies. Finally, oral MTA half-life was 20 min, with a C(max) of 80 mg/L and without signs of obvious toxicity (animal death, behavioural changes, liver enzymes). CONCLUSIONS: In the EAE model MTA is more efficacious than first line therapies for multiple sclerosis, with a dose- response effect and higher efficacy when combined with interferon-beta or glatiramer acetate. Oral MTA was also effective in the animal model of multiple sclerosis.

S-Adenosylmethionine and methylthioadenosine inhibit cellular FLICE inhibitory protein expression and induce apoptosis in colon cancer cells.

S-Adenosylmethionine (SAMe) and its metabolite 5'-methylthioadenosine (MTA) inhibit mitogen-induced proliferative response in liver and colon cancer cells. SAMe and MTA are also proapoptotic in liver cancer cells by selectively inducing Bcl-x(S) expression. The aims of this work were to assess whether these agents are proapoptotic in colon cancer cells, and if so, to elucidate the molecular mechanisms. We found that both SAMe and MTA are proapoptotic in HT-29 and RKO cells in a dose- and time-dependent manner. Gene microarray uncovered down-regulation of cellular FLICE inhibitory protein (cFLIP). SAMe and MTA treatment led to a decrease in the mRNA and protein levels of both the long and short cFLIP isoforms. This required de novo RNA synthesis and was associated with activation of procaspase-8, Bid cleavage, and release of cytochrome c from the mitochondria. Inhibiting caspase 8 activity or overexpression of cFLIP protected against apoptosis, whereas supplementing with polyamines did not. SAMe and MTA treatment sensitized RKO cells to tumor necrosis factor alpha-related apoptosis-inducing ligand-induced apoptosis. Although SAMe and MTA are proapoptotic in colon cancer cells, they have no toxic effects in NCM460 cells, a normal colon epithelial cell line. In contrast to liver cancer cells, SAMe and MTA had no effect on Bcl-x(S) expression in colon cancer cells. In conclusion, SAMe and MTA are proapoptotic in colon cancer cells but not normal colon epithelial cells. One molecular mechanism identified is the inhibition of cFLIP expression. SAMe and MTA may be attractive agents in the chemoprevention and treatment of colon cancer.

Structure-activity relationship of thiopyrimidines as mGluR5 antagonists.

Structure-activity relationship investigations of the thiopyrimidine (1), an HTS hit with micromolar activity as a metabotropic glutamate receptor 5 (mGluR5) antagonist, led to compounds with sub-micromolar activity.

EFA (9-beta-D-erythrofuranosyladenine) is an effective salvage agent for methylthioadenosine phosphorylase-selective therapy of T-cell acute lymphoblastic leukemia with L-alanosine.

The deficiency of methylthioadenosine phosphorylase (MTAP) in T-cell acute lymphoblastic leukemia (T-ALL) and other cancers, while constitutively expressed in normal cells, allows for selective therapy using L-alanosine, an inhibitor of de novo AMP synthesis. We demonstrate that MTAP- T-ALL cells obtained at relapse are as sensitive to L-alanosine toxicity as diagnosis samples. The therapeutic index of L-alanosine can be increased by the use of a MTAP substrate, which protects MTAP+ normal cells. Since MTAP substrates MTA and 5'deoxyadenosine are prone to toxicities associated with adenosine, we synthesized and evaluated a potentially nontoxic MTAP substrate, 9-beta-D-erythrofuranosyladenine (EFA). The cytotoxicity of EFA to hematopoietic progenitors erythroid burst-forming units (BFU-Es) and granulocyte-macrophage colony-forming units (CFU-GMs) was at least 26- to 41-fold less than that of MTA. In addition, EFA selectively rescued MTAP+ MOLT-4 cells from L-alanosine toxicity at 25 microM with negligible toxicity even at 100 microM. As for MTA, significant, albeit incomplete, rescue was achieved at 12.5 microM, but higher concentrations were toxic. EFA at 20 microM or less rescued primary MTAP+ T-ALL cells and normal lymphocytes from L-alanosine toxicity. Collectively, these data indicate that EFA is an effective agent for salvaging MTAP+ cells from L-alanosine toxicity and is superior to MTA due to lower cytotoxicity.

Isolation and properties of arginase from a shade plant, ginseng (Panax ginseng C.A. Meyer) roots.

Arginase (EC 3.5.3.1) was purified to homogeneity from root tissues of three-year-old ginseng (Panax ginseng C.A. Meyer), shade plant, and was found to be an extraordinarily large molecule relatively stable to heat. The enzyme was decameric having a molecular mass of 352,000 Da, with an optimal temperature and pH of 60 degrees C and 9.5, respectively. Analogues of arginine could not replace it as substrate, and a cysteine residue is at or near the active site. Maximum activity was obtained with Mn(2+) and Co(2+) also activated the proteins, whereas, both agmatine and 5'-deoxy-methylthioadenosine were inhibitors. Specific activities of the enzyme in sliced ginseng roots were increased by plant hormones such as GA(3), IAA, kinetin and putrescine, whereas the activities of the purified enzyme were unaffected by putrescine. Increases in arginase activities by these plant hormones could affect metabolism of polyamine intracellularly.

Increased efficacy of antileishmanial antisense phosphorothioate oligonucleotides in Leishmania amazonensis overexpressing ribonuclease H.

Ribonuclease H (RNase H), an enzyme that cleaves an RNA sequence base-paired with a complementary DNA sequence, is proposed to be the mediator of antisense phosphorothioate oligonucleotide (S-oligo) lethality in a cell. To understand the role of RNase H in the killing of the parasitic protozoan Leishmania by antisense S-oligos, we expressed an episomal copy of the Trypanosoma brucei RNase H1 gene inside L. amazonensis promastigotes and amastigotes that constitutively express firefly luciferase. Our hypothesis was that S-oligo-directed degradation of target mRNA is facilitated in a cell that has higher RNase H activity. Increased inhibition of luciferase mRNA expression by anti-luciferase S-oligo and by anti-miniexon S-oligo in these stably transfected promastigotes overexpressing RNase H1 was correlated to the higher activity of RNase H in these cells. The efficiency of killing of the RNase H overexpressing amastigotes inside L. amazonensis-infected macrophages by anti-miniexon S-oligo was higher than in the control cells. Thus, RNase H appears to play an important role in the antisense S-oligo-mediated killing of Leishmania. Chemical modification of S-oligos that stimulate RNase H and/or co-treatment of cells with an activator of RNase H may be useful for developing an antisense approach against leishmaniasis. The transgenic Leishmania cells overexpressing RNase H should be a good model system for the antisense-mediated gene expression ablation studies in these parasites.

Synthesis and antiviral activity of new carbonylphosphonate 2',3'-dideoxy-3'-thiacytidine conjugates.

The synthesis of new potential PFA-BCH-189 conjugate analogues is described and their molecular structure clearly identified through NMR and mass spectra techniques. The anti-HIV-1 activity was determined according to the inhibition of syncytium formation in MT-4 cells, while the anti-HBV activity was determined in infected duck hepatocytes. Both antiviral activities of the PFA-BCH-189 conjugates were much lower than those of the parent BCH-189 (2',3'-dideoxy-3'-thiacytidine) (1). Whereas a prodrug effect, following cleavage and release of the free BCH-189 and PFA, cannot be ruled out, poor cellular permeation of the drug seems to be the most likely reason for the reduced activities against HIV and DHBV. The presence of the PFA moiety appears to be detrimental for both the anti-HIV and anti-DHBV activity of PFA-BCH-189 cases.

Effects of dietary polyamine precursors on the metabolism and tissue concentrations of amino acids in the rat.

Experiments were conducted to determine the effects of dietary supplements of amino acid precursors of polyamines on amino acid metabolism in the rat in order to better understand comparative aspects of polyamine metabolism. Rats were fed isonitrogenous combinations of methionine, ornithine, arginine and 2-difluoromethylornithine in casein-based diets. It was observed that hepatic concentrations of methionine, 5'-deoxy-5'-methylthioadenosine and decarboxylated S-adenosylmethionine were more easily influenced by diet than were arginine and metabolites. It was concluded that rats may be more refractory to exogenous polyamine precursor amino acids than are chicks because of the presence of a functional urea cycle.

Inhibition of human and rat platelet aggregation by extracts of Mo-er (Auricularia auricula).

Hot water extracts of Mo-er (1 gm by 15 ml of water), an oriental food (Auricularia auricula), inhibit strongly both human and rat platelet ADP-induced aggregation. HPLC analysis of two varieties of Mo-er, A. auricula and A. polytricha (a black tree fungus), shows that they contain adenosine (Ado), 133 and 154 micrograms per gram of dry fungus, respectively. The inhibition of ADP-induced platelet aggregation by Mo-er extracts and by Ado was compared. Mo-er extracts caused a more rapid onset and a longer duration of inhibition that produced by equivalent amounts of Ado. Furthermore, Mo-er extract treated with adenosine deaminase to degrade the Ado retained the capacity to inhibit platelet aggregation. The inhibitory effects of Mo-er extracts of ADP-induced human platelet aggregation are greatly potentiated by the inhibitors of cyclic AMP phosphodiesterase such as oxagrelate (phthalazinol) and papaverine. The inhibition of platelet aggregation is only partially blocked by 2',5'-dideoxy-adenosine (DDA), an inhibitor of platelet adenylate cyclase and 5'-deoxy, 5'-methylthioadenosine (MTA), an antagonist of ADO receptors. ADP-induced rat platelet aggregation is strongly inhibited by Mo-er extracts, but not by Ado. This inhibition is not reversed by either DDA or MTA. These findings indicate that Mo-er extracts contain an agent (or agents) in addition to Ado, that blocks platelet aggregation by a mechanism that does not involve the platelet cyclic AMP system.