Structure-based design of guanosine analogue inhibitors targeting GTP cyclohydrolase IB towards a new class of antibiotics.

GTP cyclohydrolase (GCYH-I) is an enzyme in the folate biosynthesis pathway that has not been previously exploited as an antibiotic target, although several pathogens including N. gonorrhoeae use a form of the enzyme GCYH-IB that is structurally distinct from the human homologue GCYH-IA. A comparison of the crystal structures of GCYH-IA and -IB with the nM inhibitor 8-oxo-GTP bound shows that the active site of GCYH-IB is larger and differently shaped. Based on this structural information, we designed and synthesized a small set of 8-oxo-G derivatives with ether linkages at O6 and O8 expected to displace water molecules from the expanded active site of GCYH-IB. The most potent of these compounds, G3, is selective for GCYH-IB, supporting the premise that potent and selective inhibitors of GCYH-IB could constitute a new class of small molecule antibiotics.

Molecular dynamics simulation revealed binding of nucleotide inhibitors to ZIKV polymerase over 444 nanoseconds.

In the year 2015, new Zika virus (ZIKV) broke out in Brazil and spread away in more than 80 countries. Scientists directed their efforts toward viral polymerase in attempt to find inhibitors that might interfere with its function. In this study, molecular dynamics simulation (MDS) was performed over 444 ns for a ZIKV polymerase model. Molecular docking (MD) was then performed every 10 ns during the MDS course to ensure the binding of small molecules to the polymerase over the entire time of the simulation. MD revealed the binding ability of four suggested guanosine inhibitors (GIs); (Guanosine substituted with OH and SH (phenyl) oxidanyl in the 2' carbon of the ribose ring). The GIs were compared to guanosine triphosphate (GTP) and five anti-hepatitis C virus drugs (either approved or under clinical trials). The mode of binding and the binding performance of GIs to ZIKV polymerase were found to be the same as GTP. Hence, these compounds were capable of competing GTP for the active site. Moreover, GIs bound to ZIKV active site more tightly compared to ribavirin, the wide-range antiviral drug.

Synthesis of 1-beta-D-ribofuranosyl-3-ethynyl-[1,2,4]triazole and its in vitro and in vivo efficacy against Hantavirus.

There are no FDA approved drugs for the treatment of hemorrhagic fever with renal syndrome (HFRS), a serious human illnesses caused by hantaviruses. Clinical studies using ribavirin (RBV) to treat HFRS patients suggest that it provides an improved prognosis when given early in the course of disease. Given the unique antiviral activity of RBV and the lack of other lead scaffolds, we prepared a diverse series of 3-substituted 1,2,4-triazole-beta-ribosides and identified one with antiviral activity, 1-beta-d-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR). ETAR showed an EC(50) value of 10 and 4.4 microM for Hantaan virus (HTNV) and Andes virus, respectively. ETAR had weak activity against Crimean Congo hemorrhagic fever virus, but had no activity against Rift Valley fever virus. Intraperitoneally delivered ETAR offered protection to suckling mice challenged with HTNV with a approximately 25% survival at 12.5 and 25mg/kg ETAR, and a MTD of 17.1+/-0.7 days. ETAR was phosphorylated in Vero E6 cells to its 5'-triphosphate and reduced cellular GTP levels. In contrast to RBV, ETAR did not increase mutation frequency of the HTNV genome, which suggests it has a different mechanism of action than RBV. ETAR is an exciting and promising lead compound that will be elaborated in further synthetic investigations as a framework for the rational design of new antivirals for treatment of HFRS.

Suppression of NO production and 8-nitroguanosine formation by phenol-containing endocrine-disrupting chemicals in LPS-stimulated macrophages: involvement of estrogen receptor-dependent or -independent pathways.

Since the endocrine and immune systems share portions of some intracellular signaling pathways, endocrine-disrupting chemicals (EDCs) are considered potential agents for influencing inflammatory responses. Here, we investigated the effect of EDCs on lipopolysaccharide (LPS)-induced NO production and NF-kappaB activation in the RAW264.7 mouse macrophage cell line. Five phenol-containing EDCs were investigated, namely bisphenol A (BPA), the alkyl phenols p-n-nonylphenol (NP) and p-n-octylphenol (OP), and the chlorinated phenols 2,4-dichlorophenol (DCP) and pentachlorophenol (PCP). Our results revealed that these chemicals dose-dependently suppressed LPS-induced NO production, as reflected by decreased NO(x) content. The suppressive effects of BPA, NP and OP, but not PCP or DCP, were blocked by the estrogen receptor (ER) inhibitor, ICI182780. ELISA-based quantification of the DNA-binding activity of free p65 NF-kappaB showed that LPS-induced NF-kappaB activation was significantly diminished by EDC treatment. Furthermore, immunocytochemical analysis of 8-nitroguanosine, a unique index of NO-mediated signaling, showed that 8-nitroguanosine formation increased in LPS-stimulated cells, but this increase was inhibited by the tested EDCs. These results demonstrate that EDCs suppress NO production and NF-kappaB activation in LPS-stimulated macrophages through ER-dependent (BPA, NP, OP) and -independent (PCP, DCP) pathways. The EDCs further inhibited 8-nitroguanosine formation, suggesting that they interfere with NO-mediated signaling. Thus, EDCs might play important roles in the inflammatory response and host defense system against foreign pathogens.

Effects of P1 and P2Y purinoceptor antagonists on endothelium-dependent and -independent relaxations of rat mesenteric artery to GTP and guanosine.

1. Guanosine 5'-triphosphate (GTP) and guanosine can relax both endothelium-intact and -denuded arterial preparations. In the present work the P1 and P2Y purinoceptor antagonists, 8-phenyltheophylline and reactive blue 2, respectively, were used to study the mechanisms of relaxation responses induced by GTP, guanosine, adenosine 5'-triphosphate (ATP) and adenosine in noradrenaline-precontracted rat mesenteric artery rings. 2. GTP (10 microM-1mM) dose-dependently relaxed endothelium-intact mesenteric artery rings and also induced moderate relaxation responses in endothelium-denuded preparations. Pretreatment of the rings with 8-phenyltheophylline (10 microM) or reactive blue 2 (10 microM) did not attenuate the relaxant effect of GTP. 3. Guanosine (10 microM-1mM) relaxed both endothelium-intact and -denuded artery rings in a dose-dependent manner. The presence of 8-phenyltheophylline or reactive blue 2 had no effects on guanosine-induced relaxations. 4. ATP-induced (0.1 microM-0.1 mM) relaxation of endothelium-intact artery rings was attenuated by reactive blue 2 while 8-phenyltheophylline was ineffective. ATP also relaxed endothelium-denuded artery rings and this relaxation was inhibited by 8-phenyltheophylline, but not by reactive blue 2. 5. Adenosine-induced (10 microM-1 mM) relaxation of endothelium-intact and -denuded artery rings was attenuated by the presence of 8-phenyltheophylline, but not of reactive blue 2. 6. In conclusion, the endothelium-dependent and -independent relaxations of rat mesenteric arteries to GTP and guanosine are not mediated via P1 and P2Y purinoceptors. Therefore, these results support our previous suggestion on the presence of a novel guanine nucleotide-specific receptor, a putative PG receptor, on both endothelial and smooth muscle cells, which may participate in the regulation of arterial tone.

Extracellular guanosine increases astrocyte cAMP: inhibition by adenosine A2 antagonists.

Both extracellular guanosine and adenosine stimulated astrocyte proliferation in vitro and increased intracellular cAMP 6-fold within 2 min. The effects of both guanosine and adenosine on proliferation and cAMP levels were inhibited by antagonists of adenosine A2 receptors but augmented by A1 receptor antagonists. The correlation between cAMP accumulation and stimulation of cell proliferation by adenosine and guanosine indicates that increased intracellular cAMP may be one of the second messengers involved in these effects. Guanosine is not an adenosine A2 receptor agonist and does not activate adenylate cyclase. It may exert its effects indirectly by increasing the endogenous extracellular adenosine concentration.

Anticonvulsant effect of GMP depends on its conversion to guanosine.

Studies on the purinergic system normally deal with adenine-based purines, namely, adenine nucleotides and adenosine. However, a guanine-based purinergic system may also have important neuromodulatory roles. Guanine-based purines exert trophic effects on neural cells, protect brain slices in a model of hypoxia and stimulate glutamate uptake. In vivo, both guanosine 5'-monophosphate (GMP) and guanosine (GUO) protected against seizures. In this study, we investigated if the anticonvulsant effect of GMP is mediated by guanosine and if guanosine or GMP treatments were able to increase adenosine levels. Intraperitoneal (i.p.) treatments with 7.5 mg/kg GMP or guanosine prevented 50% of seizures by quinolinic acid (QA) and increased guanosine cerebrospinal fluid (CSF) levels around twofold and threefold, respectively; GMP and adenosine levels remained unchanged. Intracerebroventricular treatment with 960 nmol GMP prevented 80% of seizures and the 5'-nucleotidase inhibitor alpha-beta-methyleneadenosine 5'-diphosphate (AOPCP), when injected 3 min before, reduced this anticonvulsant effect to 30% protection as well as significantly decreased the conversion of GMP into guanosine measured in the CSF. This study shows that the previously reported effect of GMP as an anticonvulsant seems to be related to its ability to generate guanosine through the action of ecto-5'-nucleotidase.

Quinolinic acid promotes seizures and decreases glutamate uptake in young rats: reversal by orally administered guanosine.

Quinolinic acid (QA) has been used as a model for experimental overstimulation of the glutamatergic system. Glutamate uptake is the main mechanism involved in the maintenance of extracellular glutamate below toxic levels. Guanosine systemically administered prevents quinolinic acid-induced seizures in adult mice and increases basal glutamate uptake by cortical astrocyte culture and slices from young rats. The immature brain differs from the adult brain in its susceptibility to seizures, seizure characteristics, and responses to antiepileptic drugs (AED). Here we investigated the effect of guanosine p.o. on QA-induced seizures in young rats (P12-14) and upon ex vivo glutamate uptake by cortical slices from these animals. I.c.v. infusion of 250 nmol QA induced seizures in all animals and decreased glutamate uptake. I.p. injection of MK-801 and phenobarbital 30 min before QA administration prevented seizures in all animals. Guanosine (7.5 mg/kg) 75 min before QA prevented seizures in 50% of animals as well as prevented the decrease of glutamate uptake in the protected animals. To investigate if the anticonvulsive effect of guanosine was specific for QA-induced seizures, the picrotoxin-induced seizures model was also performed. Pretreatment with phenobarbital i.p. (60 mg/kg-30 min) prevented picrotoxin-induced seizures in all animals, whereas guanosine p.o. (7.5 mg/kg-75 min) and MK-801 i.p. (0.5 mg/kg-30 min) had no effect. Thus, guanosine protection on the QA-induced seizures in young rats and on the decrease of glutamate uptake showed some specificity degree towards the QA-induced toxicity. This points that guanosine could be considered for treatments of epilepsy, and possibly other neurological disorders in children.

Doridosine derivatives: binding at adenosine receptors and in vivo effects.

Doridosine, an adenosine analogue, causes, in vivo, hypotension, reduction of heart rates, muscle relaxation and anti-inflammatory effects through adenosine A1 and A2 receptors. A series of doridosine derivatives was synthesized in a search for compounds with more selective adenosine A1 receptor activity. These derivatives were characterized for binding to the respective adenosine receptors and for their cardiovascular effects. We used competition binding studies with highly selective radioligands: [3H]cyclohexyladenosine for adenosine A1 and [3H]CGS 21680 for adenosine A2 binding assays. The results for eight doridosine derivatives revealed that 1-cyclopropylisoguanosine (BN-063) and 1-allylisoguanosine (AZ-108-1) were more selective for the adenosine A1 receptor. In vivo, both BN-063 and AZ-108-1 caused significant bradycardia but no obvious effect on blood pressure. The bradycardia was almost completely blocked by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, a specific adenosine A1 receptor antagonist).

Inhibition of O6-alkylguanine-DNA alkyltransferase by O6-benzyl-N2-acetylguanosine increases chloroethylnitrosourea-induced apoptosis in Mer+ human melanoma cells.

The exposure of cells to -benzyl- 2-acetylguanosine (BNAG) and several guanine derivatives is known to reduce -alkylguanine-DNA alkyltransferase (AGAT) activity and to decrease the resistance of methyl enzyme repair positive (Mer ) cells to chloroethylnitrosoureas (CENUs) and. We evaluated the influence of AGAT activity inhibition by BNAG on the ability of two CENUs, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 3-(2-chloroethyl)-1-(2-methylsulphonyl)ethyl-3-nitrosourea (cystemustine), to induce an apoptotic response in two melanoma cell lines, M3 Dau (Mer ) and IPC 227F (Mer ). The apoptotic morphology of cells was assessed by microscopy after Wright-Giemsa or Hoechst 33342 staining of cells, and DNA internucleosomal cleavage was demonstrated by the ladder-like pattern of DNA separated by agarose gel electrophoresis. The concentration-dependent number of apoptotic cells assessed using a terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labelling (TUNEL) assay 72 h after BCNU or cystemustine treatment (0-400 microM for 2 h) was increased by prior AGAT depletion with BNAG pretreatment (300 microM for 4 h) in Mer cells. These results suggest that the DNA lesions on the position of guanine are a key event in inducing an apoptotic response in melanoma cells. We also observed that cystemustine was a more potent inducer of apoptosis than BCNU, and that the synergism with BNAG was more potent with cystemustine than with BCNU. These results suggest that the nature of the CENUs associated with an AGAT inhibitor is a determinant factor in forecasting the clinical efficacy of the association, especially in melanoma.

Millimolar Mn2+ influences agonist binding to 5-HT1A receptors by inhibiting guanosine nucleotide binding to receptor-coupled G-proteins.

Manganese is an essential trace element but its overexposure causes poisoning (called manganism) that shares several symptoms with Parkinson's disease, but with a mechanism that is still not well understood: in addition to involvement of the dopaminergic system, both serotonergic and peptiergic systems have been implicated. In the present report we have studied the influence of Mn(2+) on 5-HT(1A) receptor signaling complexes in rat brain and found that Mn(2+) in millimolar concentration caused an increase of high-affinity agonist binding to rat hippocampal membranes in comparison with experiments in the presence of Mg(2+), but not in rat cortical membranes and in Sf9 cell membranes expressing 5-HT(1A) receptors and G(i1) heterotrimers. Activation of G proteins with 30µM GTPγS turned all 5-HT(1A) receptors in these preparations into a low-affinity state for agonist binding in the presence of 1mM Mg(2+), but not in the presence of 1mM Mn(2+) in rat hippocampal membranes. However, if 1µM GTPγS was used for G protein activation, a substantial amount of high affinity agonist binding was detected in the presence of Mn(2+) also in cortical membranes and Sf9 cells, but not with Mg(2+) or EDTA. Comparison of the abilities of GDP and GTPγS to modulate high affinity agonist binding to 5-HT(1A) receptors indicated that both nucleotides were almost 10-fold less potent in the presence of MnCl(2) compared to MgCl(2). This means that by inhibiting guanosine nucleotide binding to G proteins in complex with 5-HT(1A) receptors, Mn(2+) acts as an enhancer for agonist binding and signal transduction. As the influence of Mn(2+) resembles the hypersensitivity of dopaminergic system in Parkinsonial models, it can be proposed that at least some symptoms of manganism are connected with a change of signal transduction complex caused by manganese-nucleotide complexes.

Mycophenolic acid inhibits hepatitis C virus replication and acts in synergy with cyclosporin A and interferon-alpha.

BACKGROUND & AIMS: Chronic hepatitis C virus (HCV) infection is the leading indication for liver transplantation. Clinical evidence suggests that particular immunosuppressive agents can have an influence on HCV recurrence. Cyclosporine A (CsA) specifically inhibits HCV replication through blocking the viral RNA polymerase enzyme NS5B. In this study, we investigated the effect of mycophenolic acid (MPA) and other immunosuppressants on HCV replication. METHODS: MPA and other compounds were tested in vitro using an HCV-replication model containing a luciferase reporter gene. RESULTS: At clinically relevant concentrations (1.0-6.0 microg/mL), MPA inhibited HCV replication to approximately 75%. CsA and interferon (IFN)-alpha also showed inhibition in a dose-dependent manner. In these short-term (18 hours) experiments, MPA did not inhibit cell proliferation or induce cell death, which could have accounted for the antiviral effect. In contrast to the antiviral activity of MPA against West Nile virus, the effect of MPA on HCV replication was guanosine independent. When combined, MPA and CsA showed significant synergistic inhibition of replication, reaching maximum inhibition of approximately 90% at the highest doses. Synergistic effects were observed with suboptimal concentrations of IFN-alpha with MPA or CsA. The kinetics of HCV inhibition by MPA, CsA, and IFN-alpha were clearly distinct, with earliest effects seen with IFN-alpha. No specific inhibitory effects were observed with tacrolimus or rapamycin. CONCLUSIONS: The immunosuppressive drug MPA is as potent as CsA as an inhibitor of HCV replication. MPA was shown to have a distinct anti-HCV mechanism of action, independent of cell proliferation and guanosine depletion.

Purine nucleosides and nucleotides stimulate proliferation of a wide range of cell types.

Presumptive astrocytes isolated from 10-day white Leghorn chick embryos, Factor VIII-positive human brain capillary endothelial cells, meningeal fibroblasts from 10-day chick embryos, Swiss mouse 3T3 cells, and human astrocytoma cell lines, SKMG-1 and U373, were rendered quiescent when placed in culture medium that contained 0 or 0.2% serum for 48 h; their proliferation was markedly reduced and they incorporated [3H]thymidine at a low rate. [3H]Thymidine incorporation and cell proliferation were induced in all types of cells by addition of guanosine, GMP, GDP, GTP, and to a lesser extent, adenosine, AMP, ADP or ATP to the culture medium. The stimulation of proliferation by adenosine and guanosine was abolished by 1,3-dipropyl-7-methylxanthine (DPMX), an adenosine A2 receptor antagonist, but not by 1,3-dipropyl-8-(2-amino-4-chorophenyl)xanthine (PACPX), an A1 antagonist. Stimulation of proliferation by the nucleotides was not abolished by either DPMX or PACPX. The P2 receptor agonists, alpha, beta-methyleneATP and 2-methylthioATP, also stimulated [3H]thymidine incorporation into the cells with peak activity at approximately 3.5 and 0.03 nM, respectively. These data imply that adenosine and guanosine stimulate proliferation of these cell types through activation of an adenosine A2 receptor, and the stimulation of cell proliferation by the nucleotides may be due to the activation of purinergic P2y receptors. As the primary cultures grew older their growth rate slowed. The capacity of the purine nucleosides and nucleotides to stimulate their growth diminished concomitantly. The 3T3 cells showed neither decreased growth with increased passages nor reduced response to the purines. In contrast, although the doubling time of the immortalized human astrocytoma cell lines SKMG-1 and U373 remained constant, the responsiveness to purinergic stimulation of the U373 cells decreased but that of the SKMG-1 did not. These data are compatible with a decrease in the number, or the ligand-binding affinity of the purinergic receptors, or a decreased coupling of purinergic receptors to intracellular mediators in primary cells aged in tissue culture.

Renal secretion of the antiviral nucleoside analog AM188 is inhibited by probenecid, p-aminohippuric acid, and cimetidine in the isolated perfused rat kidney.

PURPOSE: To investigate the effects of potential inhibitors of membrane transport on the tubular secretion of AM188, an antiviral guanosine analog, in the isolated perfused rat kidney (IPK). METHODS: AM188 was administered to the IPK perfusate as a bolus/infusion regimen. In inhibitor groups, probenecid, p-aminohippuric acid (PAH), cimetidine, or nitrobenzylthioinosine was added to the perfusing medium. RESULTS: In control IPKs, the ratio of renal clearance of AM188 (CLR) to GFR was 7.7 +/- 0.51 (mean +/- SD). The CL(R)/GFR ratio for AM188 was 6.20 +/- 0.41*, 2.85 +/- 0.20*, 1.45 +/- 0.07*, and 0.80 +/- 0.01* when the concentration of probenecid in perfusate was 10, 50, 100, and 1000 microM, respectively (*p < 0.05 compared to control group); the ratio was 7.71 +/- 0.38, 6.02 +/- 0.42*, 1.71 +/- 0.15*, and 0.91 +/- 0.07* for the PAH group and 6.42 +/- 1.70*, 5.33 +/- 1.53*, 3.16 +/- 0.81*, and 1.21 +/- 0.20* for the cimetidine group when the concentrations were 10, 100, 1000 and 10,000 microM, respectively; and the ratio was 5.33 +/- 0.21* when the concentration of nitrobenzylthioinosine was 5 microM. CONCLUSIONS: These results suggest that renal tubular secretion of AM188 involves organic anion and cation transport systems.

Purinergic stimulation of astroblast proliferation: guanosine and its nucleotides stimulate cell division in chick astroblasts.

A highly active fraction that was mitogenic for astroblasts but which contained no amino acids was identified during the purification of peptides from chick embryo brains. This material was purified by ultracentrifugation, ultrafiltration through Diaflo PM-30 and YM-2 membranes and retention on Diaflo YC-05, followed by ion exchange chromatography and reversed phase high performance liquid chromatography (HPLC) on a C18 Deltapak column. On thin layer chromatography and HPLC the material co-chromatographed with authentic commercially-obtained GMP. Its ultraviolet absorption spectrum was also identical with that of GMP. 1H and 31P nuclear magnetic resonance spectra of the isolated material were identical with those of GMP. The close match between the fast atom bombardment (FAB) mass spectra of the unknown material and authentic GMP indicated that the unknown material was GMP of molecular weight 363 Da. Authentic, commercial GMP stimulated the growth of cultured chick astroblasts in the same dose-dependent manner as the material from chick embryo brains; maximal stimulation was at 50 microM. Guanosine, GDP, and GTP also stimulated cell proliferation. The nucleotides were equally as effective as guanosine. 5'-Guanylyl imidodiphosphate, guanosine 5'-O-(2-thiodiphosphate), and guanosine 5'-N-(3-thiotriphosphate), guanine nucleotides which are relatively resistant to enzymatic hydrolysis, were also mitogenic, indicating that the nucleotides do not need to be degraded to nucleosides to be active and that they probably act extracellularly. Guanine nucleosides and nucleotides promoted astroblast growth when other growth factors were removed from the culture medium. The mitogenic effects of guanosine and its nucleotides were inhibited in a dose-dependent fashion by micromolar concentrations of theophylline, a characteristic of phenomena mediated by purinergic receptors. Guanosine and its nucleotides are released in micromolar concentrations by hypoxic or dying cells. Under these circumstances these compounds may stimulate division of adjacent cells in vivo.