The application of the phosphoramidate ProTide approach confers micromolar potency against Hepatitis C virus on inactive agent 4′-azidoinosine: Kinase bypass on a dual base/sugar modified nucleoside

Novel phosphoramidate ProTides derived from 4′-azidoinosine have been prepared and evaluated in the replicon assay against hepatitis C Virus (HCV). The parent nucleoside analogue is inactive in this assay, while the ProTides are active at low μM levels in some cases. This is a rare example of an inosine nucleoside analogue with potent antiviral activity and further supports the notion of ProTides as a drug discovery motif.     

Synthesis and biological evaluation of 6-substituted-5-fluorouridine ProTides

A new family of thirteen phosphoramidate prodrugs (ProTides) of different 6-substituted-5-fluorouridine nucleoside analogues were synthesized and evaluated as potential anticancer agents. In addition, antiviral activity against Chikungunya (CHIKV) virus was evaluated using a cytopathic effect inhibition assay. Although a carboxypeptidase Y assay supported a putative mechanism of activation of ProTides built on 5-fluorouridine with such C6-modifications, the Hint docking studies revealed a compromised substrate-activity for the Hint phosphoramidase-type enzyme that is likely responsible for phosphoramidate bioactivation through P–N bond cleavage and free nucleoside 5′-monophosphate delivery. Our observations may support and explain to some extent the poor in vitro biological activity generally demonstrated by the series of 6-substituted-5-fluorouridine phosphoramidates (ProTides) and will be of guidance for the design of novel phosphoramidate prodrugs.     

Human uridine-cytidine kinase phosphorylation of ribavirin: a convenient method for activation of ribavirin for conjugation to proteins

Ribavirin is a synthetic nucleoside analog that is used for the treatment of hepatitis C virus (HCV) infection. Its primary toxicity is hemolytic anemia, which sometimes necessitates dose reduction or discontinuation of therapy. Selective delivery of ribavirin into liver cells would be desirable to enhance its antiviral activity and avoid systemic side effects. One approach to liver-specific targeting is conjugation of the ribavirin with asialoglycoprotein that is taken up specifically by liver cells. Human uridine-cytidine kinase-1 (UCK-1) was used for ribavirin phosphorylation to its monophosphate form. 1-Ethyl-3-diisopropylaminocarbodiimide (EDC) was used as a coupling agent. The best results were obtained using direct conjugation protocol with a molar ratio of 6.5 ribavirin monophosphate (RMP) molecules per one asialoorosomucoid (AsOR) molecule. Our findings show that ribavirin is a potential substrate of UCK-1, and RMP formed could be chemically coupled to AsOR to form a conjugate for liver specific targeting.     

Could 5′-N and S ProTide analogues work as prodrugs of antiviral agents?

The nucleoside/nucleotide derived antiviral agents have been the most important components of antiviral therapy used in clinics. Recently, the focus of the medicinal chemists within this exciting research field has been affected mainly by the lack of effective therapies for the Hepatitis C virus (HCV) infection and several other “neglected” diseases caused by viruses such as Zika or Dengue. 2′-Methyl modified nucleosides and their monophosphate prodrugs (ProTides) have revolutionized the therapies for HCV in the last few years and, according to the latest research efforts, have also brought a promise for treatment of diseases caused by other members of Flaviviridae family. Here, we report on the design and synthesis of 5’-N and S modified ProTides derived from 2′-methyladenosine. We studied potential applicability of these derivatives as prodrugs of this archetypal antiviral compound.     

Ribavirin can be mutagenic for arenaviruses

Arenaviruses include several important human pathogens, and there are very limited options of preventive or therapeutic interventions to combat these viruses. An off-label use of the purine nucleoside analogue ribavirin (1-β-d-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is the only antiviral treatment currently available for arenavirus infections. However, the ribavirin antiviral mechanism action against arenaviruses remains unknown. Here we document that ribavirin is mutagenic for the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) in cell culture. The mutagenic activity of ribavirin on LCMV was observed under single- and multiple-passage regimes and could not be accounted for by a decrease of the intracellular GTP pool promoted by ribavirin-mediated inhibition of inosine monophosphate dehydrogenase (IMPDH). Our findings suggest that the antiviral activity of ribavirin on arenaviruses might be exerted, at least partially, by lethal mutagenesis. Implications for antiarenavirus therapy are discussed.     

Phosphoramidates of 2′-β-d-arabinouridine (AraU) as phosphate prodrugs; design,synthesis, in vitro activity and metabolism

2′-β-d-Arabinouridine (AraU), the uridine analogue of the anticancer agent AraC, was synthesized and evaluated for antiviral activity and cytotoxicity. In addition, a series of AraU monophosphate prodrugs in the form of triester phosphoramidates (ProTides) were also synthesized and tested against a range of viruses, leukaemia and solid tumour cell lines. Unfortunately, neither the parent compound (AraU) nor any of its ProTides showed antiviral activity, nor potent inhibitory activity against any of the cancer cell lines. Therefore, the metabolism of AraU phosphoramidates to release AraU monophosphate was investigated. The results showed carboxypeptidase Y, hog liver esterase and crude CEM tumor cell extracts to hydrolyse the ester motif of phosphoramidates with subsequent loss of the aryl group, while molecular modelling studies suggested that the AraU l-alanine aminoacyl phosphate derivative might not be a good substrate for the phosphoramidase enzyme Hint-1. These findings are in agreement with the observed disappearance of intact prodrug and concomitant appearance of the corresponding phosphoramidate intermediate derivative in CEM cell extracts without measurable formation of araU monophosphate. These findings may explain the poor antiviral/cytostatic potential of the prodrugs.     

Expedient synthesis and biological evaluation of alkenyl acyclic nucleoside phosphonate prodrugs

The importance of phosphonoamidate prodrugs (ProTides) of acyclic nucleoside phosphonate (ANPs) is highlighted by the approval of Tenofovir Alafenamide Fumarate for the treatment of HIV and HBV infections. In the present paper we are reporting an expedient, one-pot, two-steps synthesis of allyl phosphonoamidates and diamidates that offers a time saving strategy when compared to literature methods. The use of these substrates in the cross metathesis reactions with alkenyl functionalised thymine and uracil nucleobases is reported. ANPs prodrugs synthesized via this methodology were evaluated for their antiviral activities against DNA and RNA viruses. It is anticipated that the use of 5,6,7,8-tetrahydro-1-napthyl as aryloxy moiety is capable to confer antiviral activity among a series of otherwise inactive uracil ProTides.     

The phosphoramidate ProTide approach greatly enhances the activity of β-2′-C-methylguanosine against hepatitis C virus

β-2′-C-Methyl purines (1, 2) are known inhibitors of hepatitis C virus (HCV). We herein report the synthesis, biological and enzymatic evaluation of their 5′-phosphoramidate ProTides. Described herein are seven l-alanine phosphoramidate derivatives with variations to the amino acid ester. The 1-naphthyl phosphoramidate of β-2′-methylguanosine containing the benzyl ester (20) was the most active at 0.12 μM, an 84-fold of increase in activity compared to the parent nucleoside (2) with no increase of cytotoxicity. The carboxypeptidase mediated hydrolysis of several ProTides showed a predictive correlation with their activity versus HCV in replicon.     

Nucleoside mono- and nucleoside diphosphate kinase activities in rat liver and brain in radiation sickness

Activity of nucleoside di- and nucleoside triphosphates metabolism enzymes in tissues of rats gamma-irradiated by a dose of 30 Gy was studied 0.5, 1, 3, 6 and 24 hours after the radiation effect. It is shown that the nucleoside monophosphate kinase activity of the liver and brain is enhanced almost at all stages of the studies and the nucleoside diphosphate kinase activity is inhibited. A significant but reversible decrease of the nucleoside monophosphate kinase activity is observed in the liver 3 h later. By an end of the first day after irradiation the nucleoside mono- and nucleoside diphosphate kinase activities increase significantly both in the liver and brain.     

Synthesis of sugar-modified nucleoside 5'-triphosphates with partially purified nucleotide kinases from calf thymus.

The partial purification of some nucleoside monophosphate kinases (ATP:nucleosidemonophosphate phosphotransferases, EC 2.7.4.4) from calf thymus by chromatography on Blue Sepharose to remove interfering phosphatase activity is described. Their specificities towards nucleoside monophosphates modified in the sugar are investigated. Pyrimidine nucleoside monophosphate kinase is not very much affected by such modifications, whereas GMP kinase does not tolerate such alteration. The effect on AMP kinase is intermediate.     

Molecular modelling studies on the binding of some protides to the putative human phosphoramidase Hint1

The aim of the present work is to investigate through molecular modelling the possible role of the human enzyme Hint1 in the final P-N bond cleavage of phosphoramidate ProTides, which would lead to the intracellular delivery of unmasked nucleoside analogue monophosphates. Herein, we report our preliminary analysis based on docking studies of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) related aminoacyl phosphates with Hint1 and the effect of the amino acid moiety on the enzyme-substrate binding affinity.     

Adenosine triphosphatase activity in the carotid body of the cat

Summary Three kinds of nucleoside phosphatases were demonstrated histochemically in the cat carotid body with nucleoside triphosphate, nucleoside disphosphate and nucleoside monophosphate as substrates. Each of these enzyme activities exhibited the substrate specificity respectively. The nucleoside triphosphatase activity showed specific localization in association with the parenchymal cells of the carotid body.The electronmicroscopy revealed that the reaction product was located on and between the two apposing plasma membranes of type I and type II cells, of a type II cell and its wrapping axons and of the intricate basal infolding of a type II cell itself.Some possible functions of the adenosine triphosphatase in the carotid body are discussed.     

Purine metabolism in Acholeplasma laidlawii B: novel PPi-dependent nucleoside kinase activity

Acholeplasma laidlawii B-PG9 was examined for 16 cytoplasmic enzymes with activity for purine salvage and interconversion. Phosphoribosyltransferase activities for adenine, guanine, xanthine, and hypoxanthine were shown. Adenine, guanine, xanthine, and hypoxanthine were ribosylated to their nucleoside. Adenosine, inosine, xanthosine, and guanosine were converted to their base. No ATP-dependent phosphorylation of nucleosides to mononucleotides was found. However, PPi-dependent phosphorylation of adenosine, inosine, and guanosine to AMP, inosine monophosphate, and GMP, respectively, was detected. Nucleotidase activity for AMP, inosine monophosphate, xanthosine monophosphate, and GMP was also found. Interconversion of GMP to AMP was detected. Enzyme activities for the interconversion of AMP to GMP were not detected. Therefore, A. laidlawii B-PG9 cannot synthesize guanylates from adenylates or inosinates. De novo synthesis of purines was not detected. This study demonstrates that A. laidlawii B-PG9 has the enzyme activities for the salvage and limited interconversion of purines and, except for purine nucleoside kinase activity, is similar to Mycoplasma mycoides subsp. mycoides. This is the first report of a PPi-dependent nucleoside kinase activity in any organism.     

Ribavirin and interferon-beta synergistically inhibit SARS-associated coronavirus replication in animal and human cell lines

Initial in vitro investigations demonstrated type I interferons (IFNs: IFN-alpha, IFN-beta) to inhibit replication of SARS coronavirus (SARS-CoV), but found the nucleoside analogue ribavirin ineffective in Vero cells. In this report, ribavirin was shown to inhibit SARS-CoV replication in five different cell types of animal or human origin at therapeutically achievable concentrations. Since clinical anti-SARS-CoV activity of type I interferons or ribavirin is limited, we investigated the combination of IFN-beta and ribavirin. Determination of the virus yield indicated highly synergistic anti-SARS-CoV action of the combination suggesting the consideration of ribavirin plus IFN-beta for the treatment of SARS.     

A structural basis for the inhibition of the NS5 dengue virus mRNA 2'-O-methyltransferase domain by ribavirin 5'-triphosphate

Ribavirin is one of the few nucleoside analogues currently used in the clinic to treat RNA virus infections, but its mechanism of action remains poorly understood at the molecular level. Here, we show that ribavirin 5'-triphosphate inhibits the activity of the dengue virus 2'-O-methyltransferase NS5 domain (NS5MTase(DV)). Along with several other guanosine 5'-triphosphate analogues such as acyclovir, 5-ethynyl-1-beta-d-ribofuranosylimidazole-4-carboxamide (EICAR), and a series of ribose-modified ribavirin analogues, ribavirin 5'-triphosphate competes with GTP to bind to NS5MTase(DV). A structural view of the binding of ribavirin 5'-triphosphate to this enzyme was obtained by determining the crystal structure of a ternary complex consisting of NS5MTase(DV), ribavirin 5'-triphosphate, and S-adenosyl-l-homocysteine at a resolution of 2.6 A. These detailed atomic interactions provide the first structural insights into the inhibition of a viral enzyme by ribavirin 5'-triphosphate, as well as the basis for rational drug design of antiviral agents with improved specificity against the emerging flaviviruses.     

Design and evaluation of a potential mutagen for Hepatitis C virus

Pyrrolopyrimidine nucleoside 1 was designed and synthesized as a potential mutagen for HCV. An in vitro HCV NS5B enzymatic assay indicated that pyrrolopyrimidine triphosphate acts as a CTP analog rather than a UTP analog. The SATE-prodrug of pyrrolopyrimidine monophosphate showed a weak inhibitory activity in an HCV replicon system (EC(50)=60 microM) and did not exhibit cytotoxicity (CC(50)>100 microM). Investigation of phosphorylation events using nucleoside kinases and LC-MS analysis revealed that the second phosphorylation step, from monophosphate ester to diphosphate ester, is unfavorable.     

Potential application of thymidylate kinase in nucleoside analogue activation in Plasmodium falciparum

Plasmodium falciparum thymidylate kinase (PfTMPK) shows a broad range of substrate tolerance when compared to the corresponding human enzyme. Besides 2′-deoxythymidine monophosphate (dTMP), PfTMPK can phosphorylate 3′-azido-2′,3′-dideoxythymidine monophosphate (AZTMP) very efficiently. In contrast, human thymidylate kinase (hTMPK) is 200 times less active towards AZTMP. We were interested to see if we could use PfTMPK to activate 3′-azido-2′,3′-deoxythymidine (AZT) derivatives as a strategy to treat malaria. P. falciparum lacks a pyrimidine nucleoside kinase which usually activates nucleoside and nucleoside analogues to the corresponding monophosphates. Therefore, several prodrug analogues of AZT and related nucleoside monophosphates were prepared and analysed for antiparasitic activity. The prodrugs showed an increase in activity over the parent nucleoside analogues, which showed no inhibition of parasite growth at the concentration tested. The evidence here reported provides a strategy that could be exploited for further anti-malarial design.     

A conformational basis for the antiviral inactivity of tetrazole ribonucleosides

The antiviral activity of ribavirin has been associated with its inhibition of the enzyme, IMP dehydrogenase. The ability of ribavirin to inhibit this enzyme has previously been shown to be related to its stability in the high anti glycosidic conformation. The antiviral effectiveness of several analogs of ribavirin have been investigated recently. The evidence indicates their antiviral effectiveness is related to their stability in the high anti conformation. Recently the disposition of purine analogs that pass through the inosine monophosphate branch point has been investigated. The results of these studies are consistent with the concept that the conversion of IMP to XMP requires the high anti conformation and that the conversion of IMP to adenylosuccinate requires some other conformation, possibly the anti conformation.     

Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: purification to high specific activity by a two-step affinity chromatography procedure

This report describes a two-column scheme for purifying a pyrimidine nucleoside monophosphate kinase from rat bone marrow cells. Purification was achieved by affinity chromatography on Blue Sepharose and cellulose phosphate, with selective elution of the enzyme by substrates (UMP, ATP). The enzyme preparation appeared to be about 90% pure upon polyacrylamide gel electrophoresis, exhibited an exceptionally high specific activity (greater than 600 mumol/min/mg protein), and was obtained with 30-36% recovery of enzyme activity. It was concluded that UMP, dUMP, and CMP serve as phosphate acceptors for the enzyme, based on the parallel behavior displayed by enzyme activity with these substrates both during the purification process and during other procedures. The purified enzyme preparation did not display dTMP kinase activity. This report also describes a simplified radiotracer assay for pyrimidine nucleoside monophosphate kinases. Thin-layer chromatography on polyethyleneimine-cellulose is used to resolve residual substrates and products. Because both nucleoside di- and triphosphates remain at the origin, the assay is insensitive to the action of nucleoside diphosphate kinases and does not require the use of marker compounds. A variety of radiolabeled substrates can be used with this assay, including UMP, dUMP, CMP, and dTMP.