Design, chemical synthesis and antiviral evaluation of 2'-deoxy-2'-fluoro-2'-C-methyl-4'-thionucleosides.

Nucleoside analogues represent an historically accomplished class of antiviral drug. Notwithstanding this, new molecular scaffolds are required to overcome their limitations and evolve pharmacophore space within this established field. Herein, we develop concise synthetic access to a new 2'-deoxy-2'-fluoro-2'-C-methyl-4'-thionucleoside chemotype, including the ProTide form of the uridine analogue. Biological evaluation of these materials in the Hepatitis C replicon assay shows little activity for the canonical pyrimidine forms, but the phosphoramidate of 2'-deoxy-2'-fluoro-2'-C-methyl-ß-d-4'-thiouridine has an EC50 of 2.99 µM. Direct comparison to the established Hepatitis C drug Sofosbuvir shows a 100-fold drop in activity upon substituting the furanose chalcogen; the reasons for this are as yet unclear.

8-Mercaptoguanine-based inhibitors of Mycobacterium tuberculosis dihydroneopterin aldolase: synthesis, in vitro inhibition and docking studies.

The dihydroneopterin aldolase (DHNA, EC 4.1.2.25) activity of FolB protein is required for the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and glycolaldehyde (GA) in the folate pathway. FolB protein from Mycobacterium tuberculosis (MtFolB) is essential for bacilli survival and represents an important molecular target for drug development. S8-functionalized 8-mercaptoguanine derivatives were synthesised and evaluated for inhibitory activity against MtFolB. The compounds showed IC50 values in the submicromolar range. The inhibition mode and inhibition constants were determined for compounds that exhibited the strongest inhibition. Additionally, molecular docking analyses were performed to suggest enzyme-inhibitor interactions and ligand conformations. To the best of our knowledge, this study describes the first class of MtFolB inhibitors.

S-adenosylmethionine and methylthioadenosine boost cellular productivities of antibody forming Chinese hamster ovary cells.

The improvement of cell specific productivities for the formation of therapeutic proteins is an important step towards intensified production processes. Among others, the induction of the desired production phenotype via proper media additives is a feasible solution provided that said compounds adequately trigger metabolic and regulatory programs inside the cells. In this study, S-(5'-adenosyl)- l-methionine (SAM) and 5'-deoxy-5'-(methylthio)adenosine (MTA) were found to stimulate cell specific productivities up to approx. 50% while keeping viable cell densities transiently high and partially arresting the cell cycle in an anti-IL-8-producing CHO-DP12 cell line. Noteworthy, MTA turned out to be the chemical degradation product of the methyl group donor SAM and is consumed by the cells.

Recent Advances in the Chemical Synthesis and Evaluation of Anticancer Nucleoside Analogues.

Nucleoside analogues have proven to be highly successful chemotherapeutic agents in the treatment of a wide variety of cancers. Several such compounds, including gemcitabine and cytarabine, are the go-to option in first-line treatments. However, these materials do have limitations and the development of next generation compounds remains a topic of significant interest and necessity. Herein, we discuss recent advances in the chemical synthesis and biological evaluation of nucleoside analogues as potential anticancer agents. Focus is paid to 4'-heteroatom substitution of the furanose oxygen, 2'-, 3'-, 4'- and 5'-position ring modifications and the development of new prodrug strategies for these materials.

Synthesis of 4'-Thionucleosides as Antitumor and Antiviral Agents.

Many attempts have been made to synthesize structurally novel nucleoside derivatives in order to identify effective compounds for the treatment of tumors and virus-caused disease. At our laboratories, as part of our efforts to synthesize 4'-thionucleosides, we have identified and characterized biologically active nucleosides. During the course of our synthetic study, we developed the Pummerer-type thioglycosylation reaction. As a result, we synthesized a potent antineoplastic nucleoside, 1-(2-deoxy-2-fluoro-ß-D-4-thio-arabino-furanosyl)cytosine (4'-thioFAC), and several novel 4'-thionucleosides that possess antiherpes virus activities.

Design, synthesis, and anticancer activity of C8-substituted-4'-thionucleosides as potential HSP90 inhibitors.

A series of C8-substituted-4'-thioadenosine analogs 3a-3g, 15, and 17 and their truncated derivatives 4a-4j, 23-25, and 27 have been successfully synthesized from d-ribose and d-mannose, respectively, employing Pummerer type or Vorbrüggen condensation reactions and the functionalization at the C8-position of nucleobase via Stille coupling or nucleophilic aromatic substitution reactions as key steps. All the synthesized compounds were assayed for their HSP90 inhibitory activity, but they were found to be inactive up to 100µM. However, the 8-iodo derivatives 15, 17, and 27 exhibited potent anticancer activity, indicating that different mechanism of action might be involved in their biological activity.

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.

Immunomodulatory metabolites released by the frog-killing fungus Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis is a fungal pathogen in the phylum Chytridiomycota that causes the skin disease chytridiomycosis. Chytridiomycosis is considered an emerging infectious disease linked to worldwide amphibian declines and extinctions. Although amphibians have well-developed immune defenses, clearance of this pathogen from the skin is often impaired. Previously, we showed that the adaptive immune system is involved in the control of the pathogen, but B. dendrobatidis releases factors that inhibit in vitro and in vivo lymphocyte responses and induce lymphocyte apoptosis. Little is known about the nature of the inhibitory factors released by this fungus. Here, we describe the isolation and characterization of three fungal metabolites produced by B. dendrobatidis but not by the closely related nonpathogenic chytrid Homolaphlyctis polyrhiza. These metabolites are methylthioadenosine (MTA), tryptophan, and an oxidized product of tryptophan, kynurenine (Kyn). Independently, both MTA and Kyn inhibit the survival and proliferation of amphibian lymphocytes and the Jurkat human T cell leukemia cell line. However, working together, they become effective at much lower concentrations. We hypothesize that B. dendrobatidis can adapt its metabolism to release products that alter the local environment in the skin to inhibit immunity and enhance the survival of the pathogen.

Antiherpesvirus activities of two novel 4'-thiothymidine derivatives, KAY-2-41 and KAH-39-149, are dependent on viral and cellular thymidine kinases.

The emergence of drug-resistant herpesviruses represents a significant problem in clinical practice, primarily in immunocompromised patients. Furthermore, effective antiviral therapies against gammaherpesvirus-associated diseases are lacking. Here, we present two thiothymidine derivatives, KAY-2-41 and KAH-39-149, with different spectra of antiviral activity from those of the reference antiherpetic drugs, showing inhibitory activities against herpes simplex virus, varicella-zoster virus (VZV), and particularly against Epstein-Barr virus, with high selectivity in vitro. While KAY-2-41- and KAH-39-149-resistant herpesviruses were found to harbor mutations in the viral thymidine kinase (TK), these mutations conferred only low levels of resistance to these drugs but high levels to other TK-dependent drugs. Also, antiviral assays in HeLa TK-deficient cells showed a lack of KAY-2-41 and KAH-39-149 activities against herpes simplex virus 1 (HSV-1) and HSV-2 TK-deficient mutants. Furthermore, enzymatic TK assays showed the ability of HSV-1 TK, VZV TK, and cellular TK1 and TK2 to recognize and phosphorylate KAY-2-41 and KAH-39-149. These results demonstrate that the compounds depend on both viral and host TKs to exert antiviral activity. Additionally, the antiviral efficacy of KAH-39-149 proved to be superior to that of KAY-2-41 in a mouse model of gammaherpesvirus infection, highlighting the potential of this class of antiviral agents for further development as selective therapeutics against Epstein-Barr virus.

Quantitative proteomics analysis reveals possible anticancer mechanisms of 5'-deoxy-5'-methylthioadenosine in cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) is an aggressive cancer originating from bile duct epithelium, particularly prevalent in Asian countries with liver fluke infections. Current chemotherapy for CCA often fails due to drug resistance, necessitating novel anticancer agents. This study investigates the potential of 5'-deoxy-5'-methylthioadenosine (MTA), a naturally occurring nucleoside, against CCA. While MTA has shown promise against various cancers, its effects on CCA remain unexplored. We evaluated MTA's anticancer activity in CCA cell lines and drug-resistant sub-lines, assessing cell viability, migration, invasion, and apoptosis. The potential anticancer mechanisms of MTA were explored through proteomic analysis using LC-MS/MS and bioinformatic analysis. The results show a dose-dependent reduction in CCA cell viability, with enhanced effects on cancer cells compared to normal cells. Moreover, MTA inhibits growth, induces apoptosis, and suppresses cell migration and invasion. Additionally, MTA enhanced the anticancer effects of gemcitabine on drug-resistant CCA cells. Proteomics revealed the down-regulation of multiple proteins by MTA, affecting various molecular functions, biological processes, and cellular components. Network analysis highlighted MTA's role in inhibiting proteins related to mitochondrial function and energy derivation, crucial for cell growth and survival. Additionally, MTA suppressed proteins involved in cell morphology and cytoskeleton organization, important for cancer cell motility and metastasis. Six candidate genes, including ZNF860, KLC1, GRAMD1C, MAMSTR, TANC1, and TTC13, were selected from the top 10 most down-regulated proteins identified in the proteomics results and were subsequently verified through RT-qPCR. Further, KLC1 protein suppression by MTA treatment was confirmed through Western blotting. Additionally, based on TCGA data, KLC1 mRNA was found to be upregulated in the tissue of CCA patients compared to that of normal adjacent tissues. In summary, MTA shows promising anticancer potential against CCA by inhibiting growth, inducing apoptosis, and suppressing migration and invasion, while enhancing gemcitabine's effects. Proteomic analysis elucidates possible molecular mechanisms underlying MTA's anticancer activity, laying the groundwork for future research and development of MTA as a treatment for advanced CCA.

Thermodynamic analysis of transition-state features in picomolar inhibitors of human 5'-methylthioadenosine phosphorylase.

Human 5'-methylthioadenosine phosphorylase (MTAP) is solely responsible for 5'-methylthioadenosine (MTA) metabolism to permit S-adenosylmethionine salvage. Transition-state (TS) analogues of MTAP are in development as anticancer candidates. TS analogues of MTAP incorporate a cationic nitrogen and a protonated 9-deazaadenine leaving group, which are mimics of the ribocation transition state. MT-ImmA and MT-DADMe-ImmA are two examples of these TS analogues. Thermodynamic analysis of MTA, inhibitor, and phosphate binding reveals the cationic nitrogen to provide -2.6 and -3.6 kcal/mol binding free energy for MT-ImmA and MT-DADMe-ImmA, respectively. The protonated deazaadenine provides an additional -1.3 (MT-ImmA) to -1.7 kcal/mol (MT-DADMe-ImmA). MT-DADMe-ImmA is a better match in TS geometry than MT-ImmA and is thermodynamically favored. Binding of TS analogues to the MTAP/phosphate complex is fully entropic, in contrast to TS analogue binding to the related human purine nucleoside phosphorylase/phosphate complex, which is fully enthalpic (Guan, R., Ho, M. C., Brenowitz, M., Tyler, P. C., Evans, G. B., Almo, S. C., and Schramm, V. L. (2011) Biochemistry 50, 10408-10417). The binding thermodynamics of phosphate or TS analogues alone to MTAP are fully dominated by enthalpy. Phosphate anchored in the catalytic site forms an ion pair with the cationic TS analogue to cause stabilization of the enzyme structure in the ternary complex. The ternary-induced conformational changes convert the individual enthalpic binding energies to entropy, resulting in a presumed shift of the protein architecture toward the transition state. Formation of the ternary TS analogue complex with MTAP induces a remarkable increase in thermal stability (ΔTm 28 °C). The enthalpic, entropic, and protein-stability features of TS analogue binding to human MTAP are resolved in these studies.

Structure-Activity Relationship of Truncated 2,8-Disubstituted-Adenosine Derivatives as Dual A2A/A3 Adenosine Receptor Antagonists and Their Cancer Immunotherapeutic Activity.

Based on hA2AAR structures, a hydrophobic C8-heteroaromatic ring in 5'-truncated adenosine analogues occupies the subpocket tightly, converting hA2AAR agonists into antagonists while maintaining affinity toward hA3AR. The final compounds of 2,8-disubstituted-N6-substituted 4'-thionucleosides, or 4'-oxo, were synthesized from d-mannose and d-erythrono-1,4-lactone, respectively, using a Pd-catalyst-controlled regioselective cross-coupling reaction. All tested compounds completely antagonized hA2AAR, including 5d with the highest affinity (Ki,A2A = 7.7 ± 0.5 nM). The hA2AAR-5d X-ray structure revealed that C8-heteroaromatic rings prevented receptor activation-associated conformational changes. However, the C8-substituted compounds still antagonized hA3AR. Structural SAR features and docking studies supported different binding modes at A2AAR and A3AR, elucidating pharmacophores for receptor activation and selectivity. Favorable pharmacokinetics were demonstrated, in which 5d displayed high oral absorption, moderate half-life, and bioavailability. Also, 5d significantly improved the antitumor effect of anti-PD-L1 in vivo. Overall, this study suggests that the novel dual A2AAR/A3AR nucleoside antagonists would be promising drug candidates for immune-oncology.

Preventative oral methylthioadenosine is anti-inflammatory and reduces DSS-induced colitis in mice.

Methylthioadenosine (MTA) is a precursor of the methionine salvage pathway and has been shown to have anti-inflammatory properties in various models of acute and chronic inflammation. However, the anti-inflammatory properties of MTA in models of intestinal inflammation are not defined. We hypothesized that orally administered MTA would be bioavailable and reduce morbidity associated with experimental colitis. We examined clinical, histological, and molecular markers of disease in mice provided oral MTA before (preventative) or after (therapy) the induction of colitis with 3% dextran sulfate sodium (DSS). We found a reduction in disease activity, weight loss, myeloperoxidase activity, and histological damage in mice given preventative MTA compared with DSS alone. We also found that equivalent supplementation with methionine could not reproduce the anti-inflammatory effects of MTA, and that MTA had no detectable adverse effects in control or DSS mice. Expression microarray analysis of colonic tissue showed several dominant pathways related to inflammatory cytokines/chemokines and extracellular matrix remodeling were upregulation by DSS and suppressed in MTA-supplemented mice. MTA is rapidly absorbed in the gastrointestinal tract and disseminated throughout the body, based on a time course analysis of an oral bolus of MTA. This effect is transient, with MTA levels falling to near baseline within 90 min in most organs. Moreover, MTA did not lead to increased blood or tissue methionine levels, suggesting that its effects are specific. However, MTA provided limited therapeutic benefit when administered after the onset of colitis. Our results show that oral MTA supplementation is a safe and effective strategy to prevent inflammation and tissue injury associated with DSS colitis in mice. Additional studies in chronic inflammatory models are necessary to determine if MTA is a safe and beneficial option for the maintenance of remission in human inflammatory bowel disease.

Down-regulation of methylthioadenosine phosphorylase (MTAP) induces progression of hepatocellular carcinoma via accumulation of 5'-deoxy-5'-methylthioadenosine (MTA).

Recently, we have shown that down-regulation of methylthioadenosine phosphorylase (MTAP) in hepatocellular carcinoma (HCC) cells enhances the invasive potential and the resistance against cytokines. Here, we aimed at investigating the molecular mechanism underlying this tumor-promoting effect and expanded the analysis to a large series of human HCC tissues. Liquid chromatography tandem mass spectrometry revealed that reduced MTAP expression resulted in higher intra- and extracellular concentrations of 5'-deoxy-5'-methylthioadenosine (MTA) in cultivated HCC cells and, concordantly, higher levels of MTA in HCC tissue. MTA induced matrix metalloproteinase (MMP) and interleukin-8 transcription in HCC cells in vitro, accompanied by enhanced proliferation and activation of the transcription factor NFκB. In addition, MTA secreted by HCC cells induced expression of fibroblast growth factor-2 and MMP1 in stromal myofibroblasts. In human HCC tissues, MTAP mRNA correlated inversely with MTA levels, and immunohistochemical analysis of a tissue microarray of 140 human HCCs revealed that low MTAP protein expression correlated with advanced tumor stages. In conclusion, MTAP deficiency results in accumulation of MTA, which is associated with increased tumorigenicity. These data further indicate MTAP as a tumor suppressor in HCC, and MTA as a potential biomarker for HCC progression.

A3 adenosine receptor agonist reduces brain ischemic injury and inhibits inflammatory cell migration in rats.

A3 adenosine receptor (A3AR) is recognized as a novel therapeutic target for ischemic injury; however, the mechanism underlying anti-ischemic protection by the A3AR agonist remains unclear. Here, we report that 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyl-4'-thioadenosine (LJ529), a selective A3AR agonist, reduces inflammatory responses that may contribute to ischemic cerebral injury. Postischemic treatment with LJ529 markedly reduced cerebral ischemic injury caused by 1.5-hour middle cerebral artery occlusion, followed by 24-hour reperfusion in rats. This effect was abolished by the simultaneous administration of the A3AR antagonist MRS1523, but not the A2AAR antagonist SCH58261. LJ529 prevented the infiltration/migration of microglia and monocytes occurring after middle cerebral artery occlusion and reperfusion, and also after injection of lipopolysaccharides into the corpus callosum. The reduced migration of microglia by LJ529 could be related with direct inhibition of chemotaxis and down-regulation of spatiotemporal expression of Rho GTPases (including Rac, Cdc42, and Rho), rather than by biologically relevant inhibition of inflammatory cytokine/chemokine release (eg, IL-1ß, TNF-α, and MCP-1) or by direct inhibition of excitotoxicity/oxidative stress (not affected by LJ529). The present findings indicate that postischemic activation of A3AR and the resultant reduction of inflammatory response should provide a promising therapeutic strategy for the treatment of ischemic stroke.

Conditioned media from lung cancer cell line A549 and PC9 inactivate pulmonary fibroblasts by regulating protein phosphorylation.

Pulmonary fibrosis is a devastating condition resulting from excess extracellular matrix deposition that leads to progressive lung destruction and scarring. In the pathogenesis of fibrotic diseases, activation of myofibroblasts by transforming growth factor-ß (TGF-ß) plays a crucial role. Since no effective therapy for pulmonary fibrosis is currently recognized, finding an effective antifibrotic agent is an important objective. One approach might be through identification of agents that inactivate myofibroblasts. In the current study we examined the potential of conditioned medium obtained from several types of cells to exhibit myofibroblast inactivating activity. Conditioned media from lung cancer cell lines A549 and PC9 were found to have this action, as shown by its ability to decrease α-smooth muscle actin expression in MRC-5 cells. Subsequently the inhibitory factor was purified from the medium and identified as 5'-deoxy-5'-methylthioadenosine (MTA), and its mechanism of action elucidated. Activation of protein kinase A and cAMP responsive element binding protein (CREB) were detected. MTA inhibited TGF-ß-induced mitogen-activated protein kinase activation. Furthermore, the gain-of-function mutant CREB caused inactivation of myofibroblasts. These results show that A549 and PC9 conditioned media have the ability to inactivate myofibroblasts, and that CREB-phosphorylation plays a central role in this process.

4-Thiofuranoid Glycal: Versatile Glycosyl Donor for the Selective Synthesis of ß-anomer of 4'-thionucleoside and its Biological Activities.

The first highly diastereoselective synthesis of ß-anomers of 4'-thionucleosides has been carried out by means of electrophilic glycosidation utilizing 3,5-O-(di-tertbutylsilylene) (DTBS)-4-thiofuranoid glycal as a glycosyl donor. The resulting glycosides were transformed into ribo-, 2'-deoxy-, and arabinofuranosyl nucleosides through a chemical transformation of the 2'-substituent. The additive Pummerer reaction of the glycal Soxide gave 1,2-di-O-acetyl-3,5-O-DTBS-4-thioribofuranose. The utility of the DTBSprotected 4-thioribofuranose has been demonstrated by the preparation of 4'-thio analogues of pyrimidine- and purine-4'-thioribonucleosides based on the Vorbrüggen glycosidation. Synthesis of 4'-thio-counterpart of C-nucleoside antibiotic tiazofurin has also been carried out. α-Face selective hydroboration of 1-C-aryl- or 1-C-heteroaryl-glycals obtained by cross-coupling of 1-tributylstannylglycal has furnished the respective ß- anomer of 4'-thio-C-ribonucleosides, including 4'-thio analogue of nucleoside antibiotic pseudouridine and 9-deazaadenosine. On the basis of lithiation chemistry, 1-C- and 2-Ccarbon- carbon-substituted 3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3- diyl) (TIPDS)- 4- thiofuranoid glycal were synthesized. These glycals enabled us to prepare 1'-C- and 2'-ß- C-carbon-substituted 2'-deoxy-4'-thionucleosides, including thio-counterpart of antitumor nucleoside antibiotic angustmycin C. Furthermore, 1'-C-methyl-4'-thiothymidine emerged as a potent inhibitor of angiogenesis. In addition, 1'-C-methyl-4'-thiothymidine exhibited more potent inhibitory activity against thymidine kinase-deficient mutant of herpes virus than that of ganciclovir. Among the 4'-substituted 4'-thiothymidines, the 4'- C-cyano- and 4'-C-ethynyl derivatives inhibited replication of HIV variant resistant to 3TC (HIVM184V) as potently as HIV-1IIIB. In terms of the value of selectivity index (SI), 4'-C-cyano-4'-thiothymidine showed a 3-fold selective index (SI) than that of the corresponding thymidine derivative. Furthermore, 4'-C-ethynyl-2'-deoxy-4'-thioguanosine has a 20-fold better value (>18,200) than that of 2'-deoxyguanosine counterpart (933). Furthermore, 4'-azido-4'-thiothymidine emerged as a selective and potent anti-EBV agent. In terms of antineoplastic activity, 4'-azido- and 4'-C-fluoromethyl-2'-deoxy-4'-thiocytidine inhibited proliferation of human B-cell (CCRF-SB) and T-cell leukemia (Molt-4) cell lines, although the parent compound 2'-deoxy-4'-thiocytidine did not exhibit any cytotoxicity up to 100 µM. These facts concerning the biological activities suggested that replacement of the furanose oxygen with a sulfur atom is a promising approach for the development of less toxic antiviral and antineoplastic nucleoside antimetabolites. 4'- Thionucleoside also acts as a monomer for oligonucleotides (ONs) therapeutics, exhibiting superior biological properties. Therefore, this review provides a wide range of potential monomers for antisense ON and siRNA.

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).

Identification of a 2-cell stage specific inhibitor of the cleavage of preimplantation mouse embryos synthesized by rat hepatoma cells as 5'-deoxy-5'-methylthioadenosine.

Rat hepatoma Reuber H-35 cells produce a unique compound designated as Fr.B-25, a 2-cell stage-specific inhibitor of the cleavage of preimplantation mouse embryos cultured in vitro. Here, we identified Fr.B-25 as a purine nucleoside, 5'-deoxy-5'-methylthioadenosine (MTA), by mass spectroscopic analysis. All of the biological activities examined of authentic MTA on the development of mouse zygotes were indistinguishable from those of Fr.B-25. The mechanism of MTA action in the development of preimplantation mouse embryos was probably different from those of hypoxanthine and adenosine, which are well-characterized purine nucleosides that act as inhibitors of the cleavage of mouse 2-cell embryos. From the shared molecular and biological properties of Fr.B-25 and MTA, we concluded that Fr.B-25 is MTA. To the best of our knowledge, this is the first delineation of the effect of MTA on the development of preimplantation mammalian embryos cultured in vitro.

LJ529 attenuates mast cell-related inflammation via A3R-PKCε-ALDH2 pathway after subarachnoid hemorrhage in rats.

BACKGROUND AND PURPOSE: Mast cells (MCs) has been recognized as an effector of inflammation or a trigger of inflammatory factors during stroke. LJ529 was reported to attenuate inflammation through a Gi protein-coupled Adenosine A3 receptor (A3R) after ischemia. Here, we aim to study the protective effect and its mechanism of LJ529 in subarachnoid hemorrhage (SAH) rat model for mast cell-related inflammation. METHODS: 155 Sprague-Dawley adult male rats were used in experiments. Endovascular perforation was used for SAH model. Intraperitoneal LJ529 was performed 1 h after SAH. Neurological scores were measured 24 h after SAH. Rotarod and morris water maze tests were evaluated for 21 days after SAH. Mast cell degranulation was assessed with Toluidine blue staining and Chymase/Typtase protein expressions. Mast cell-related inflammation was evaluated using IL-6, TNF-α and MCP-1 protein expressions. MRS1523, inhibitor of GPR18 and ε-V1-2, inhibitor of PKCε were respectively given intraperitoneally (i.p.) 1 h and 30 min before SAH for mechanism studies. Pathway related proteins were investigated with western blot and immunofluorescence staining. RESULTS: Expression of A3R, PKCε increased after SAH. LJ529 treatment attenuated mast cell degranulation and inflammation. Meanwhile, both short-term and long-term neurological functions were improved after LJ529 treatment. Administration of LJ529 resulted in increased expressions of A3R, PKCε, ALDH2 proteins and decreased expressions of Chymase, Typtase, IL-6, TNF-α and MCP-1 proteins. MRS1523 abolished the treatment effects of LJ529 on neurobehavior and protein levels. ε-V1-2 also reversed the outcomes of LJ529 administration through reduction in protein expressions downstream of PKCε. CONCLUSIONS: LJ529 attenuated mast cell-related inflammation through inhibiting degranulation via A3R-PKCε-ALDH2 pathway after SAH. LJ529 may serve as a potential treatment strategy to relieve post-SAH brain injury.