Supplementary nucleosides improve the performance through enhanced enzyme activity, intestinal development and transporter genes in broiler chickens.

A biological experiment was carried out to evaluate dietary nucleoside supplementation on growth performance, digestive enzymes activities, immune response, and intestinal transporter genes expression in broiler chicken. A total of 720 newly hatched CARIBRO VISHAL broiler chicks were weighed and randomly divided into eight groups with nine replicates. The dietary treatments were as follows: Group I: diet without antibiotic supplement (control), group II: diet supplemented with antibiotic (positive control), groups III, IV and V: diet supplemented with combination of nucleosides at 0.5, 1.0 and 1.5 g/kg feed, respectively, for 14 days, groups VI, VII and VIII: diet supplemented with nucleosides at 0.5, 1.0 and 1.5 g/kg feed, respectively, for 21 days. The combination of nucleosides (equal proportion (1:1:1:1) adenosine, guanosine, cytosine, and uridine with 99% purity) were used in the study. Body weight was significantly higher in the birds fed diets containing antibiotics and 1.5 g/kg nucleosides fed groups. The supplementation had positive effect on the activity of amylase and lipase enzymes and the absorptive surface (villi length). It could be concluded that, the dietary supplementation of nucleosides improved the performance of broilers with better cellular and humoral immunity than control. The study further confirmed that nucleosides supplementation improved gut development and could be an alternative to antibiotic growth promoters in broiler production.

Pyrimidine inhibitors synergize with nucleoside analogues to block SARS-CoV-2.

The SARS-CoV-2 virus has infected more than 261 million people and has led to more than 5 million deaths in the past year and a half1 ( https://www.who.org/ ). Individuals with SARS-CoV-2 infection typically develop mild-to-severe flu-like symptoms, whereas infection of a subset of individuals leads to severe-to-fatal clinical outcomes2. Although vaccines have been rapidly developed to combat SARS-CoV-2, there has been a dearth of antiviral therapeutics. There is an urgent need for therapeutics, which has been amplified by the emerging threats of variants that may evade vaccines. Large-scale efforts are underway to identify antiviral drugs. Here we screened approximately 18,000 drugs for antiviral activity using live virus infection in human respiratory cells and validated 122 drugs with antiviral activity and selectivity against SARS-CoV-2. Among these candidates are 16 nucleoside analogues, the largest category of clinically used antivirals. This included the antivirals remdesivir and molnupiravir, which have been approved for use in COVID-19. RNA viruses rely on a high supply of nucleoside triphosphates from the host to efficiently replicate, and we identified a panel of host nucleoside biosynthesis inhibitors as antiviral. Moreover, we found that combining pyrimidine biosynthesis inhibitors with antiviral nucleoside analogues synergistically inhibits SARS-CoV-2 infection in vitro and in vivo against emerging strains of SARS-CoV-2, suggesting a clinical path forward.

Repurposing Nucleoside Analogs for Human Coronaviruses.

Coronavirus disease 2019 (COVID-19) is a serious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or CoV-2). Some reports claimed certain nucleoside analogs to be active against CoV-2 and thus needed confirmation. Here, we evaluated a panel of compounds and identified novel nucleoside analogs with antiviral activity against CoV-2 and HCoV-OC43 while ruling out others. Of significance, sofosbuvir demonstrated no antiviral effect against CoV-2, and its triphosphate did not inhibit CoV-2 RNA polymerase.

Deploying Fluorescent Nucleoside Analogues for High-Throughput Inhibitor Screening.

High-throughput small-molecule screening in drug discovery processes commonly rely on fluorescence-based methods including fluorescent polarization and fluorescence/Förster resonance energy transfer. These techniques use highly accessible instrumentation; however, they can suffer from high false-negative rates and background signals, or might involve complex schemes for the introduction of fluorophore pairs. Herein we present the synthesis and application of fluorescent nucleoside analogues as the foundation for directed approaches for competitive binding analyses. The general approach describes selective fluorescent environment-sensitive (ES) nucleoside analogues that are adaptable to diverse enzymes that act on nucleoside-based substrates. We demonstrate screening a set of uridine analogues and development of an assay for fragment-based lead discovery with the TcdB glycosyltransferase (GT), an enzyme associated with virulence in Clostridium difficile. The uridine-based probe used for this high-throughput screen has a KD value of 7.2 µm with the TcdB GT and shows a >30-fold increase in fluorescence intensity upon binding. The ES-based probe assay is benchmarked against two other screening approaches.

Nucleoside Analogs with Selective Antiviral Activity against Dengue Fever and Japanese Encephalitis Viruses.

Dengue virus (DENV) and Japanese encephalitis virus (JEV) are important arthropod-borne viruses from the Flaviviridae family. DENV is a global public health problem with significant social and economic impacts, especially in tropical and subtropical areas. JEV is a neurotropic arbovirus endemic to east and southeast Asia. There are no U.S. FDA-approved antiviral drugs available to treat or to prevent DENV and JEV infections, leaving nearly one-third of the world's population at risk for infection. Therefore, it is crucial to discover potent antiviral agents against these viruses. Nucleoside analogs, as a class, are widely used for the treatment of viral infections. In this study, we discovered nucleoside analogs that possess potent and selective anti-JEV and anti-DENV activities across all serotypes in cell-based assay systems. Both viruses were susceptible to sugar-substituted 2'-C-methyl analogs with either cytosine or 7-deaza-7-fluoro-adenine nucleobases. Mouse studies confirmed the anti-DENV activity of these nucleoside analogs. Molecular models were assembled for DENV serotype 2 (DENV-2) and JEV RNA-dependent RNA polymerase replication complexes bound to nucleotide inhibitors. These models show similarities between JEV and DENV-2, which recognize the same nucleotide inhibitors. Collectively, our findings provide promising compounds and a structural rationale for the development of direct-acting antiviral agents with dual activity against JEV and DENV infections.

Synthesis and Antiviral Evaluation of 3'-Fluoro-4'-modified-5'-norcarbocyclic Nucleoside Phosphonates.

The synthesis and anti-HIV evaluation of hitherto unknown 3'-fluoro-5'-norcarbocyclic nucleoside phosphonates bearing adenine with modifications at the 4' position (ethynyl, vinyl, ethyl, hydroxymethyl) is described. One of the synthesized compounds was found to be an inhibitor of HIV-1 replication, but with moderate efficiency relative to (R)-9-(2-phosphonylmethoxypropyl)adenine ((R)-PMPA, tenofovir), with no concomitant cytotoxicity.

Nucleoside supplementation modulates mitochondrial DNA copy number in the dguok -/- zebrafish.

Deoxyguanosine kinase (dGK) is an essential rate-limiting component of the mitochondrial purine nucleotide salvage pathway, encoded by the nuclear gene encoding deoxyguanosine kinase (DGUOK). Mutations in DGUOK lead to mitochondrial DNA (mtDNA) depletion typically in the liver and brain, causing a hepatocerebral phenotype. Previous work has shown that in cultured DGUOK patient cells it is possible to rescue mtDNA depletion by increasing substrate amounts for dGK. In this study we developed a mutant dguok zebrafish (Danio rerio) line using CRISPR/Cas9 mediated mutagenesis; dguok-/- fish have significantly reduced mtDNA levels compared with wild-type (wt) fish. When supplemented with only one purine nucleoside (dGuo), mtDNA copy number in both mutant and wt juvenile animals was significantly reduced, contrasting with previous cell culture studies, possibly because of nucleotide pool imbalance. However, in adult dguok-/- fish we detected a significant increase in liver mtDNA copy number when supplemented with both purine nucleosides. This study further supports the idea that nucleoside supplementation has a potential therapeutic benefit in mtDNA depletion syndromes by substrate enhancement of the purine nucleoside salvage pathway and might improve the liver pathology in patients.

Inhibiting pyrimidine biosynthesis impairs Ebola virus replication through depletion of nucleoside pools and activation of innate immune responses.

Specific host pathways that may be targeted therapeutically to inhibit the replication of Ebola virus (EBOV) and other emerging viruses remain incompletely defined. A screen of 200,000 compounds for inhibition of an EBOV minigenome (MG) assay that measures the function of the viral polymerase complex identified as hits several compounds with an amino-tetrahydrocarbazole scaffold. This scaffold was structurally similar to GSK983, a compound previously described as having broad-spectrum antiviral activity due to its impairing de novo pyrimidine biosynthesis through inhibition of dihydroorotate dehydrogenase (DHODH). We generated compound SW835, the racemic version of GSK983 and demonstrated that SW835 and brequinar, another DHODH inhibitor, potently inhibit the MG assay and the replication of EBOV, vesicular stomatitis virus (VSV) and Zika (ZIKV) in vitro. Nucleoside and deoxynucleoside supplementation studies demonstrated that depletion of pyrimidine pools contributes to antiviral activity of these compounds. As reported for other DHODH inhibitors, SW835 and brequinar also induced expression of interferon stimulated genes (ISGs). ISG induction was demonstrated to occur without production of IFNα/ß and independently of the IFNα receptor and was not blocked by EBOV-encoded suppressors of IFN signaling pathways. Furthermore, we demonstrated that transcription factor IRF1 is required for this ISG induction, and that IRF1 induction requires the DNA damage response kinase ATM. Therefore, de novo pyrimidine biosynthesis is critical for the replication of EBOV and other RNA viruses and inhibition of this pathway activates an ATM and IRF1-dependent innate immune response that subverts EBOV immune evasion functions.

Nucleosides for the treatment of respiratory RNA virus infections.

Influenza virus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, and rhinoviruses are among the most common viruses causing mild seasonal colds. These RNA viruses can also cause lower respiratory tract infections leading to bronchiolitis and pneumonia. Young children, the elderly, and patients with compromised cardiac, pulmonary, or immune systems are at greatest risk for serious disease associated with these RNA virus respiratory infections. In addition, swine and avian influenza viruses, together with severe acute respiratory syndrome-associated and Middle Eastern respiratory syndrome coronaviruses, represent significant pandemic threats to the general population. In this review, we describe the current medical need resulting from respiratory infections caused by RNA viruses, which justifies drug discovery efforts to identify new therapeutic agents. The RNA polymerase of respiratory viruses represents an attractive target for nucleoside and nucleotide analogs acting as inhibitors of RNA chain synthesis. Here, we present the molecular, biochemical, and structural fundamentals of the polymerase of the four major families of RNA respiratory viruses: Orthomyxoviridae, Pneumoviridae/Paramyxoviridae, Coronaviridae, and Picornaviridae. We summarize past and current efforts to develop nucleoside and nucleotide analogs as antiviral agents against respiratory virus infections. This includes molecules with very broad antiviral spectrum such as ribavirin and T-705 (favipiravir), and others targeting more specifically one or a few virus families. Recent advances in our understanding of the structure(s) and function(s) of respiratory virus polymerases will likely support the discovery and development of novel nucleoside analogs.

Flexibility-Not just for yoga anymore!

Over the past few years, nucleosides have maintained a prominent role as one of the cornerstones of antiviral and anticancer therapeutics, and many approaches to nucleoside drug design have been pursued. One such approach involves flexibility in the sugar moiety of nucleosides, for example, in the highly successful anti-HIV and HBV drug tenofovir. In contrast, introduction of flexibility to the nucleobase scaffold has only more recently gained significance with the invention of our fleximers. The history, development, and some biological relevance for this innovative class of nucleosides are detailed herein.

Current and future use of nucleo(s)tide prodrugs in the treatment of hepatitis C virus infection.

This review describes the current state of discovery of past most important nucleoside and nucleotide prodrugs in the treatment of hepatitis C virus infection as well as future potential drugs currently in discovery or clinical evaluation. I highlight first generation landmark prodrug compounds which have been the foundations of incremental improvements toward the discovery and approval milestone of Sofosbuvir. Sofosbuvir is the first nucleotide prodrug marketed for hepatitis C virus treatment and the backbone of current combination therapies. Since this approval, new nucleotide prodrugs using the same design of Sofosbuvir McGuigan prodrug have emerged, some of them progressing through advanced clinical trials and may become available as new incremental alternative hepatitis C virus treatments in the future. Although since Sofosbuvir success, only minimal design efforts have been invested in finding better liver targeted prodrugs, a few novel prodrugs are being studied and their different modes of activation may prove beneficial over the heart/liver targeting ratio to reduce potential drug-drug interaction in combination therapies and yield safer treatment to patients. Prodrugs have long been avoided as much as possible in the past by development teams due to their metabolism and kinetic characterization complexity, but with their current success in hepatitis C virus treatment, and the knowledge gained in this endeavor, should become a first choice in future tissue targeting drug discovery programs beyond the particular case of nucleos(t)ide analogs.

Discovery of novel purine nucleoside derivatives as phosphodiesterase 2 (PDE2) inhibitors: Structure-based virtual screening, optimization and biological evaluation.

Phosphodiesterase 2 (PDE2) has received much attention for the potential treatment of the central nervous system (CNS) disorders and pulmonary hypertension. Herein, we identified that clofarabine (4), an FDA-approved drug, displayed potential PDE2 inhibitory activity (IC50 = 3.12 ±â€¯0.67 µM) by structure-based virtual screening and bioassay. Considering the potential therapeutic benefit of PDE2, a series of purine nucleoside derivatives based on the structure and binding mode of 4 were designed, synthesized and evaluated, which led to the discovery of the best compound 14e with a significant improvement of inhibitory potency (IC50 = 0.32 ±â€¯0.04 µM). Further molecular docking and molecular dynamic (MD) simulations studies revealed that 5'-benzyl group of 14e could interact with the unique hydrophobic pocket of PDE2 by forming extra van der Waals interactions with hydrophobic residues such as Leu770, Thr768, Thr805 and Leu809, which might contribute to its enhancement of PDE2 inhibition. These potential compounds reported in this article and the valuable structure-activity relationships (SARs) might bring significant instruction for further development of potent PDE2 inhibitors.

Flex-nucleoside analogues - Novel therapeutics against filoviruses.

Fleximers, a novel type of flexible nucleoside that have garnered attention due to their unprecedented activity against human coronaviruses, have now exhibited highly promising levels of activity against filoviruses. The Flex-nucleoside was the most potent against recombinant Ebola virus in Huh7 cells with an EC50=2µM, while the McGuigan prodrug was most active against Sudan virus-infected HeLa cells with an EC50 of 7µM.

Effects of 4-Pyridone-3-carboxamide-1ß-D-ribonucleoside on adenine nucleotide catabolism in the aortic wall; Implications for atherosclerosis in ApoE-/-LDLR-/- mice.

4-Pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR) is an endogenously produced nucleoside that had been identified as a substrate for intracellular phosphorylation to form intracellular nucleotides. Previous studies demonstrated that 4PYR adversely affects metabolism of endothelial cells that is known risk factor for atherosclerosis. The purpose of this study was to evaluate effects of 4PYR on the progression of atherosclerosis and changes in extracellular nucleotides degradation on the surface of the vessel wall in the murine model. METHODS: Two month old ApoE-/-LDLR-/- mice were subcutaneously injected with 4PYR (4P) twice per day for one month or with saline in controls (C). Then, at the age of eight month hydrolysis rates of ATP, AMP and adenosine were evaluated in the intact aorta sections by HPLC based assays. Oil Red O (ORO) staining that indicates lipid deposition was quantified spectrophotometrically after extraction from the vessel. Serum amyloid A (SAA) content was analyzed with ELISA. RESULTS: Adenosine deamination rate (activity of eADA) increased from 8.7±1.4 nmol/min/cm2 in C to 16.0±2.6 nmol/min/cm2 in 4P (p<0.05). AMP dephosphorylation rate (activity of e5NT) and ATP hydrolysis rate (activity of eNTPD) were not different between C and 4P. ORO staining in the aorta of 4P mice increased by 75% as compared to C (p<0.01) while SAA content was similar in both groups. CONCLUSIONS: This data demonstrated that prolonged exposure to 4PYR of ApoE-/-LDLR-/- mice results in sustained elevation of vascular eADA activity and increased ORO staining indicating endothelial impairment and accelerated atherosclerosis.

Development of a reliable automated screening system to identify small molecules and biologics that promote human ß-cell regeneration.

Numerous compounds stimulate rodent ß-cell proliferation; however, translating these findings to human ß-cells remains a challenge. To examine human ß-cell proliferation in response to such compounds, we developed a medium-throughput in vitro method of quantifying adult human ß-cell proliferation markers. This method is based on high-content imaging of dispersed islet cells seeded in 384-well plates and automated cell counting that identifies fluorescently labeled ß-cells with high specificity using both nuclear and cytoplasmic markers. ß-Cells from each donor were assessed for their function and ability to enter the cell cycle by cotransduction with adenoviruses encoding cell cycle regulators cdk6 and cyclin D3. Using this approach, we tested 12 previously identified mitogens, including neurotransmitters, hormones, growth factors, and molecules, involved in adenosine and Tgf-1ß signaling. Each compound was tested in a wide concentration range either in the presence of basal (5 mM) or high (11 mM) glucose. Treatment with the control compound harmine, a Dyrk1a inhibitor, led to a significant increase in Ki-67+ ß-cells, whereas treatment with other compounds had limited to no effect on human ß-cell proliferation. This new scalable approach reduces the time and effort required for sensitive and specific evaluation of human ß-cell proliferation, thus allowing for increased testing of candidate human ß-cell mitogens.

Synthesis and Evaluation of 2'-Deoxy-2'-Spirodiflurocyclopropyl Nucleoside Analogs.

The preparation of 2'-deoxy-2'-siprodifluorocyclopropany-lnucleoside analogs has been achieved from α-d-glucose in several steps. The key step in the synthesis was the introduction of the difluorocyclopropane through a difluorocarbene type reaction at the 2'-position. Then, a series of novel 2'-deoxy-2'-spirodifluorocyclopropanyl nucleoside analogs were synthesized using the Vorbrüggen method. All the synthesized nucleosides were characterized and subsequently evaluated against hepatitis C and influenza A virus strains in vitro.

Inhibitory effect of extracellular purine nucleotide and nucleoside concentrations on T cell proliferation.

The release of nucleic acids and derivatives after tissue-injury may affect cellular immune-response. We studied the impact of extracellular ribo-, desoxyribonucleotides and nucleosides on T-cell immunity. Peripheral-blood-mononuclear-cells (PBMCs) or isolated CD3+T-cells obtained from 6 healthy donors were stimulated via CD3/CD28 Dynabeads or dendritic cells (DCs) in the presence or absence of pyrimidine-, purine-nucleotides and -nucleosides (range 2-200µM). Addition of deoxy-, guanosine-triphosphate (dGTP, GTP) and guanosine resulted concentration dependent in a complete, adenosine-triphosphate (ATP) in a partial inhibition of the induced T-cell-proliferation. Deoxyadenosine-triphosphate (dATP), adenosine and the pyrimidine-ribo- and -deoxyribonucleotides displayed no inhibitory capacity. Inhibitory effects of dGTP and GTP, but not of guanosine and ATP were culture-media-dependent and could be almost abrogated by use of the serum-free lymphocyte-culture-media X-Vivo15 instead of RPMI1640 with standard-supplementation. In contrast to RPMI1640, X-Vivo15 resulted in a significant down-regulation of the cell-surface-located ectonucleotidases CD39 (Ecto-Apyrase) and CD73 (Ecto-5'-Nucleotidase), critical for the extracellular nucleotides-hydrolysis to nucleosides, explaining the loss of inhibition mediated by dGTP and GTP, but not Guanosine. In line with previous findings ATP was found to exert immunosuppressive effects on T-cell-proliferation. Purine-nucleotides, dGTP and GTP displayed a higher inhibitory capacity, but seem to be strictly dependent on the microenvironmental conditions modulating the responsiveness of the respective T-lymphocytes. Further evaluation of experimental and respective clinical settings should anticipate these findings.

New strategies against drug resistance to herpes simplex virus.

Herpes simplex virus (HSV), a member of the Herpesviridae family, is a significant human pathogen that results in mucocutaneous lesions in the oral cavity or genital infections. Acyclovir (ACV) and related nucleoside analogues can successfully treat HSV infections, but the emergence of drug resistance to ACV has created a barrier for the treatment of HSV infections, especially in immunocompromised patients. There is an urgent need to explore new and effective tactics to circumvent drug resistance to HSV. This review summarises the current strategies in the development of new targets (the DNA helicase/primase (H/P) complex), new types of molecules (nature products) and new antiviral mechanisms (lethal mutagenesis of Janus-type nucleosides) to fight the drug resistance of HSV.

N-Branched acyclic nucleoside phosphonates as monomers for the synthesis of modified oligonucleotides.

Protected N-branched nucleoside phosphonates containing adenine and thymine bases were prepared as the monomers for the introduction of aza-acyclic nucleotide units into modified oligonucleotides. The phosphotriester and phosphoramidite methods were used for the incorporation of modified and natural units, respectively. The solid phase synthesis of a series of nonamers containing one central modified unit was successfully performed in both 3'→5' and 5'→3' directions. Hybridization properties of the prepared oligoribonucleotides and oligodeoxyribonucleotides were evaluated. The measurement of thermal characteristics of the complexes of modified nonamers with the complementary strand revealed a considerable destabilizing effect of the introduced units. We also examined the substrate/inhibitory properties of aza-acyclic nucleoside phosphono-diphosphate derivatives (analogues of nucleoside triphosphates) but neither inhibition of human and bacterial DNA polymerases nor polymerase-mediated incorporation of these triphosphate analogues into short DNA was observed.

Identification of Nucleoside Analogs as Inducers of Neuronal Differentiation in a Human Reporter Cell Line and Adult Stem Cells.

Nucleoside analogs (NSAs) were among the first chemotherapeutic agents and could also be useful for the manipulation of cell fate. To investigate the potential of NSAs for the induction of neuronal differentiation, we developed a novel phenotypic assay based on a human neuron-committed teratocarcinoma cell line (NT2) as a model for neuronal progenitors and constructed a NT2-based reporter cell line that expressed eGFP under the control of a neuron-specific promoter. We tested 38 structurally related NSAs and determined their activity to induce neuronal differentiation by immunocytochemistry of neuronal marker proteins, live cell imaging, fluorometric detection and immunoblot analysis. We identified twelve NSAs, which induced neuronal differentiation to different extents. NSAs with highest activity carried a halogen substituent at their pyrimidine nucleobase and an unmodified or 2'-O-methyl substituted 2-deoxy-ß-D-ribofuranosyl residue as glyconic moiety. Cladribine, a purine nucleoside with similar structural features and in use to treat leukemia and multiple sclerosis, induced also differentiation of adult human neural crest-derived stem cells. Our results suggest that NSAs could be useful for the manipulation of neuronal cell fate in cell replacement therapy or treatment of neurodegenerative disorders. The data on the structure and function relationship will help to design compounds with increased activity and low toxicity.