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Biocatalytic nucleoside (trans-)glycosylations catalyzed by nucleoside phosphorylases have graduated to a practical and convenient approach to the preparation of modified nucleosides, which are important pharmaceuticals for the treatment of various cancers and viral infections. However, the obtained yields in these reactions are generally determined exclusively by the innate thermodynamic properties of the nucleosides involved, hampering the biocatalytic access to many sought-after target nucleosides. We herein report an orthogonal dimension for reaction engineering of these systems. We show how apparent equilibrium shifts in phosphorolysis and glycosylation reactions can be effected through entropically driven, biased esterification of nucleosides with inorganic borate. Our multifaceted analysis further describes the kinetic implications of this in situ reactant esterification for a model phosphorylase. Our results suggest an unusual pseudo-non-competitive inhibitory mechanism where reversible binding of the borate ester of the nucleoside substrate yields a non-productive inhibitor-enzyme complex. This complex exhibits constricted molecular dynamics and exists in a rapid equilibrium with the productive enzyme-substrate complex via hydrolytic interconversion. Collectively, this report presents a partial departure from the stringent thermodynamic constraints of nucleoside phosphorolysis reactions and shines light on the molecular processes regulating the activity of nucleoside-binding enzymes in the presence of borate.

Enzyme-mediated processes have proven to be a valuable and sustainable alternative to traditional chemical methods. In this regard, the use of multi-enzymatic systems enables the realization of complex synthetic schemes, while also introducing a number of additional advantages, including the conversion of reversible reactions into irreversible processes, the partial or complete elimination of product inhibition problems, and the minimization of undesirable by-products. In addition, the immobilization of biocatalysts on magnetic supports allows for easy reusability and streamlines the downstream process. Herein we have developed a cascade system for cladribine synthesis based on the sequential action of two magnetic biocatalysts. For that purpose, purine 2′-deoxyribosyltransferase from Leishmania mexicana (LmPDT) and Escherichia coli hypoxanthine phosphoribosyltransferase (EcHPRT) were immobilized onto Ni2+-prechelated magnetic microspheres (MagReSyn®NTA). Among the resulting derivatives, MLmPDT3 (activity: 11,935 IU/gsupport, 63% retained activity, operational conditions: 40 °C and pH 5–7) and MEcHPRT3 (12,840 IU/gsupport, 45% retained activity, operational conditions: pH 5–8 and 40–60 °C) emerge as optimal catalysts for further synthetic application. Moreover, the MLmPDT3/MEcHPRT3 system was biochemically characterized and successfully applied to the one-pot synthesis of cladribine under various conditions. This methodology not only displayed a 1.67-fold improvement in cladribine synthesis (compared to MLmPDT3), but it also implied a practically complete transformation of the undesired by-product into a high-added-value product (90% conversion of Hyp into IMP). Finally, MLmPDT3/MEcHPRT3 was reused for 16 cycles, which displayed a 75% retained activity.

A series of 5′-phosphorylated (dialkyl phosphates, diaryl phosphates, phosphoramidates, H-phosphonates, phosphates) 1,2,3-triazolyl nucleoside analogues in which the 1,2,3-triazole-4-yl-β-D-ribofuranose fragment is attached via a methylene group or a butylene chain to the N-1 atom of the heterocycle moiety (uracil or quinazoline-2,4-dione) was synthesized. All compounds were evaluated for antiviral activity against influenza virus A/PR/8/34/(H1N1). Antiviral assays revealed three compounds, 13b, 14b, and 17a, which showed moderate activity against influenza virus A (H1N1) with IC50 values of 17.9 μM, 51 μM, and 25 μM, respectively. In the first two compounds, the quinazoline-2,4-dione moiety is attached via a methylene or a butylene linker, respectively, to the 1,2,3-triazole-4-yl-β-D-ribofuranosyl fragment possessing a 5′-diphenyl phosphate substituent. In compound 17a, the uracil moiety is attached via the methylene unit to the 1,2,3-triazole-4-yl-β-D-ribofuranosyl fragment possessing a 5′-(phenyl methoxy-L-alaninyl)phosphate substituent. The remaining compounds appeared to be inactive against influenza virus A/PR/8/34/(H1N1). The results of molecular docking simulations indirectly confirmed the literature data that the inhibition of viral replication is carried out not by nucleoside analogues themselves, but by their 5′-triphosphate derivatives.

International audience; BACKGROUND The recommended monitoring tools for evaluating nucleot(s)ide analogue renal toxicity, such as estimated glomerular filtration rate (eGFR) and phosphatemia, are late markers of proximal tubulopathy. Multiple early markers are available, but no consensus exists on their use. AIM To determine the 24 mo prevalence of subclinical proximal tubulopathy (SPT), as defined with early biomarkers, in treated vs untreated hepatitis B virus (HBV)-monoinfected patients. METHODS A prospective, non-randomized, multicenter study of HBV-monoinfected patients with a low number of renal comorbidities was conducted. The patients were separated into three groups: Naive, starting entecavir (ETV) treatment, or starting tenofovir disoproxil (TDF) treatment. Data on the early markers of SPT, the eGFR and phosphatemia, were collected quarterly. SPT was defined as a maximal tubular reabsorption of phosphate/eGFR below 0.8 mmoL/L and/or uric acid fractional excretion above 10%. The prevalence and cumulative incidence of SPT at month 24 (M24) were calculated. Quantitative data were analyzed using analyses of variance or Kruskal-Wallis tests, whereas chi-squared or Fisher's exact tests were used to analyze qualitative data. Multivariate analyses were used to adjust for any potential confounding factors. RESULTS Of the 196 patients analyzed, 138 (84 naive, 28 starting ETV, and 26 starting TDF) had no SPT at inclusion. At M24, the prevalence of SPT was not statistically different between naive and either treated group (21.1% vs 30.7%, P < 0.42 and 50.0% vs 30.7%, P = 0.32 for ETV and TDF, respectively); no patient had an eGFR lower than 50 mL/min/1.73 m(2) or phosphatemia less than 0.48 mmoL/L. In the multivariate analysis, no explanatory variables were identified after adjustment. The cumulative incidence of SPT over 24 mo (25.5%, 13.3%, and 52.9% in the naive, ETV, and TDF groups, respectively) tended to be higher in the TDF group vs the naive group (hazard ratio: 2.283, P = 0.05). SPT-free survival at M24 was 57.6%, 68.8%, and 23.5% for the naive, ETV, and TDF groups, respectively. The median survival time without SPT, evaluated only in the TDF group, was 5.9 mo. CONCLUSION The prevalence and incidence of SPT was higher in TDF-treated patients compared to naive patients. SPT in the naive population suggests that HBV can induce renal tubular toxicity.

Based on the fact that a search for influenza antivirals among nucleoside analogues has drawn very little attention of chemists, the present study reports the synthesis of a series of 1,2,3-triazolyl nucleoside analogues in which a pyrimidine fragment is attached to the ribofuranosyl-1,2,3-triazol-4-yl moiety by a polymethylene linker of variable length. Target compounds were prepared by the Cu alkyne-azide cycloaddition (CuAAC) reaction. Derivatives of uracil, 6-methyluracil, 3,6-dimethyluracil, thymine and quinazolin-2,4-dione with ω-alkyne substituent at the N1 (or N5) atom and azido 2,3,5-tri-O-acetyl-D-β-ribofuranoside were used as components of the CuAAC reaction. All compounds synthesized were evaluated for antiviral activity against influenza virus A/PR/8/34/(H1N1) and coxsackievirus B3. The best values of IC50 (inhibiting concentration) and SI (selectivity index) were demonstrated by the lead compound 4i in which the 1,2,3-triazolylribofuranosyl fragment is attached to the N1 atom of the quinazoline-2,4-dione moiety via a butylene linker (IC50 = 30 μM, SI = 24) and compound 8n in which the 1,2,3-triazolylribofuranosyl fragment is attached directly to the N5 atom of the 6-methyluracil moiety (IC50 = 15 μM, SI = 5). According to theoretical calculations, the antiviral activity of the 1,2,3-triazolyl nucleoside analogues 4i and 8n against H1N1 (A/PR/8/34) influenza virus can be explained by their influence on the functioning of the polymerase acidic protein (PA) of RNA-dependent RNA polymerase (RdRP). Graphic abstract Electronic supplementary material The online version of this article (10.1007/s11030-020-10141-y) contains supplementary material, which is available to authorized users.

COVID-19 has been confirmed in millions of individuals worldwide, rendering it a global medical emergency. In the absence of vaccines and the unavailability of effective drugs for the SARS-CoV-2 infection, vaccine development is being continuously explored and several antiviral compounds and immunotherapies are currently being investigated. Given the high similarity in genetic identity between SARS-CoV and SARS-CoV-2, the present investigation identified the interaction between the physicochemical properties and the antiviral activity of different potential and clinically approved antiviral drugs against SARS-CoV using hierarchically weighted principal component analysis. Representative drugs from the classes of neuraminidase inhibitors, reverse transcriptase inhibitors, protease inhibitors, nucleoside analogues, and other compounds with potential antiviral activity were examined. The pharmacologic classification and the biological activity of the different antiviral drugs were described using indices, namely, rotatable bond count, molecular weight, heavy atom count, and molecular complexity (92.32% contribution rate). The physicochemical properties and inhibitory action against SARS-CoV-2 of lopinavir, chloroquine, ivermectin, and ciclesonide validated the adequacy of the current computational approach. The findings of the present study provide additional information, although further investigation is warranted to identify potential targets and establish exact mechanisms, in the emergent search and design of antiviral drug candidates and their subsequent synthesis as effective therapies for COVID-19.

A highly efficient and versatile synthetic approach for the synthesis of 4-(pyren-1-ylmethyl)-1-(d-glycosyloxy) phthalazine nucleosides 11a,b, 13, β-S-nucleosides 16, 18, 20, and acyclo C-nucleosides 23a,b, 24, 25 and 27a–f was described and fully characterized. Furthermore, a series of desired new nucleoside analogues containing Se of 4-(pyren-1-ylmethyl) phthalazine-1(2H)-selenone 28–33 were synthesized. The structures of all reported compounds were confirmed by IR, 1H-NMR, 13C-NMR, MS and elemental analysis. All compounds have been screened for their antibacterial and antifungal activities. Maximum activity was shown by 20 and 33a comparable to the standard drugs with lower toxicity. The cytotoxicity of the selected compound was measured and evaluated. The energy gap between the highest occupied molecular orbital and lowest unoccupied molecular orbital was calculated using theoretical computations to reflect the chemical reactivity and kinetic stability of the synthesized compounds. Using density functional theory (DFT), electronic parameters such as the highest occupied and lowest unoccupied molecular orbitals (HOMO and LUMO) and the molecular electrostatic potential (MEPS) were calculated. On the basis of different studied structures, these properties were computed in order to elucidate the chemical reactivity and the kinetic stability. Obviously, the band gap energy (Eg) of structures studied reveals that the lowest band gap obtained for the structure 16-a indicates that it has the highest chemical reactivity and lowest kinetic stability.

The spread of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged suddenly at the end of 2019 and the disease came to be known as coronavirus disease 2019 (COVID-19). To date, there is no specific therapy established to treat COVID-19. Identifying effective treatments is urgently required to treat patients and stop the transmission of SARS-CoV-2 in humans. For the present review, >100 publications on therapeutic agents for COVID-19, including in vitro and in vivo animal studies, case reports, retrospective analyses and meta-analyses were retrieved from PubMed and analyzed, and promising therapeutic agents that may be used to combat SARS-CoV-2 infection were highlighted. Since the outbreak of COVID-19, different drugs have been repurposed for its treatment. Existing drugs, including chloroquine (CQ), its derivative hydroxychloroquine (HCQ), remdesivir and nucleoside analogues, monoclonal antibodies, convalescent plasma, Chinese herbal medicine and natural compounds for treating COVID-19 evaluated in experimental and clinical studies were discussed. Although early clinical studies suggested that CQ/HCQ produces antiviral action, later research indicated certain controversy regarding their use for treating COVID-19. The molecular mechanisms of these therapeutic agents against SARS-CoV2 have been investigated, including inhibition of viral interactions with angiotensin-converting enzyme 2 receptors in human cells, viral RNA-dependent RNA polymerase, RNA replication and the packaging of viral particles. Potent therapeutic options were reviewed and future challenges to accelerate the development of novel therapeutic agents to treat and prevent COVID-19 were acknowledged.

Eight different compounds, all nucleoside analogues, could presently be considered as potential drug candidates for the treatment of Ebola virus (EBOV) and/or other hemorrhagic fever virus (HFV) infections. They can be considered as either (i) adenine analogues (3‐deazaneplanocin A, galidesivir, GS‐6620 and remdesivir) or (ii) guanine analogues containing the carboxamide entity (ribavirin, EICAR, pyrazofurin and favipiravir). All eight owe their mechanism of action to hydrogen bonded base pairing with either (i) uracil or (ii) cytosine. Four out of the eight compounds (galidesivir, GS‐6620, remdesivir and pyrazofurin) are C‐nucleosides, and two of them (GS‐6620, remdesivir) also contain a phosphoramidate part. The C‐nucleoside and phosphoramidate (and for the adenine analogues the 1′‐cyano group as well) may be considered as essential attributes for their antiviral activity., It is all about potential: Eight different compounds, four adenine analogues (3‐deazaneplanocin A, galidesivir, GS‐6620 and remdesivir) and four guanine analogues containing the carboxamide entity (ribavirin, EICAR, pyrazofurin and favipiravir) can be considered as potential drug candidates for the treatment of Ebola virus and/or other hemorrhagic fever virus infections.

Therapeutic options to treat HIV infection have widened in the past years, improving both effectiveness and tolerability, but nucleoside reverse transcriptase inhibitors (NRTIs) are still considered the standard backbone of the combination regimens. Therapeutic drug monitoring (TDM) can be useful for these drugs, due to concentration–effect relationship, with risk of ineffectiveness, toxicity or adherence concerns: in this scenario, robust and multiplexed methods are needed for an effective TDM activity. In this work, the first validated ultra-high spectrometry liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method is described for the high-sensitive simultaneous quantification of all the currently used NRTIs in human plasma, including tenofovir alafenamide (TAF), following FDA and EMA guidelines. The automated sample preparation consisted in the addition of an internal standard (IS) working solution, containing stable-isotope-linked drugs, protein precipitation and drying. Dry extracts were reconstituted with water, then, these underwent reversed phase chromatographic separation: compounds were detected through electrospray ionization and multiple reaction monitoring. Accuracy, precision, recovery and IS-normalized matrix effect fulfilled guidelines’ requirements. The application of this method on samples from people living with HIV (PLWH) showed satisfactory performance, being capable of quantifying the very low concentrations of tenofovir (TFV) in patients treated with TAF.

The de novo synthesis of piperidine nucleosides from our homologating agent 5,6-dihydro-1,4-dithiin is herein reported. The structure and conformation of nucleosides were conceived to faithfully resemble the well-known nucleoside drugs Immucillins H and A in their bioactive conformation. NMR analysis of the synthesized compounds confirmed that they adopt an iminosugar conformation bearing the nucleobases and the hydroxyl groups in the appropriate orientation.

The current pandemic caused by SARS-CoV2 and named COVID-19 urgent the need for novel lead antiviral drugs. Recently, United States Food and Drug Administration (FDA) approved the use of remdesivir as anti-SARS-CoV-2. Remdesivir is a natural product-inspired nucleoside analogue with significant broad-spectrum antiviral activity. Nucleosides analogues from marine sponge including spongouridine and spongothymidine have been used as lead for the evolutionary synthesis of various antiviral drugs such as vidarabine and cytarabine. Furthermore, the marine sponge is a rich source of compounds with unique activities. Marine sponge produces classes of compounds that can inhibit the viral cysteine protease (Mpro) such as esculetin and ilimaquinone and human serine protease (TMPRSS2) such as pseudotheonamide C and D and aeruginosin 98B. Additionally, sponge-derived compounds such as dihydrogracilin A and avarol showed immunomodulatory activity that can target the cytokines storm. Here, we reviewed the potential use of sponge-derived compounds as promising therapeutics against SARS-CoV-2. Despite the reported antiviral activity of isolated marine metabolites, structural modifications showed the importance in targeting and efficacy. On that basis, we are proposing a novel structure with bifunctional scaffolds and dual pharmacophores that can be superiorly employed in SARS-CoV-2 infection.

Background Thymidine kinase 1 (TK1) plays a key role in the synthesis of deoxythymidine triphosphate (dTTP) and is thus important for DNA replication and cell proliferation. The expression of TK1 is highest during S-phase, and it is rapidly degraded after mitosis. In cancer cells, TK1 is upregulated, resulting in leakage of excess TK1 into the blood. Consequently, serum TK1 has been used as a diagnostic and prognostic cancer biomarker, mainly in human medicine. The aims of this work were to characterize equine TK1 and to evaluate its suitability as a serum biomarker for equine lymphoma. Results Equine TK1 was cloned, expressed in E. coli and affinity purified. The purified recombinant horse TK1 showed broad substrate specificity, phosphorylating pyrimidine deoxyribo- and ribonucleosides and, to some extent, purine deoxynucleosides, including anticancer and antiviral nucleoside analogues. ATP was the preferred phosphate donor. Serum TK1 activity was measured in samples collected from horses with confirmed or suspected lymphoma and control horses with and without concurrent diseases. Serum TK1 activity levels were significantly higher in horses with lymphoma (p p p p Conclusion Equine TK1 showed high specific activity and broader substrate specificity than human TK1. Anticancer and antiviral thymidine analogues were efficiently phosphorylated by horse TK1, suggesting that these analogues might be good candidates for chemotherapy in horses. Serum TK1 activity was significantly higher in horses with lymphoma than in controls. ROC analysis indicated that serum TK1 could serve as a promising cancer biomarker in horses.

Zoonotic spillover, i.e. pathogen transmission from animal to human, has repeatedly introduced RNA viruses into the human population. In some cases, where these viruses were then efficiently transmitted between humans, they caused large disease outbreaks such as the 1918 flu pandemic or, more recently, outbreaks of Ebola and Coronavirus disease. These examples demonstrate that RNA viruses pose an immense burden on individual and public health with outbreaks threatening the economy and social cohesion within and across borders. And while emerging RNA viruses are introduced more frequently as human activities increasingly disrupt wild-life eco-systems, therapeutic or preventative medicines satisfying the “one drug-multiple bugs”-aim are unavailable. As one central aspect of preparedness efforts, this review digs into the development of broadly acting antivirals via targeting viral genome synthesis with host- or virus-directed drugs centering around nucleotides, the genomes’ universal building blocks. Following the first strategy, selected examples of host de novo nucleotide synthesis inhibitors are presented that ultimately interfere with viral nucleic acid synthesis, with ribavirin being the most prominent and widely used example. For directly targeting the viral polymerase, nucleoside and nucleotide analogues (NNAs) have long been at the core of antiviral drug development and this review illustrates different molecular strategies by which NNAs inhibit viral infection. Highlighting well-known as well as recent, clinically promising compounds, structural features and mechanistic details that may confer broad-spectrum activity are discussed. The final part addresses limitations of NNAs for clinical development such as low efficacy or mitochondrial toxicity and illustrates strategies to overcome these.

Nucleos(t)ide analogues are commonly used in the treatment of infectious disease and cancer. SAMHD1 is a deoxyribonucleotide (dNTP) triphosphohydrolase which is involved in the regulation of the intracellular dNTP pool, whose function has been linked to viral restriction, cancer development, and autoimmune disorders. Here, we evaluate SAMHD1 function on the antiviral and antiproliferative efficacy of a wide range of nucleos(t)ide analogues which are currently used to treat infections and cancer. The anti-HIV-1 and cytotoxic activity of compounds was assessed in primary and established cell lines in the presence or absence of SAMHD1. SAMHD1 effectively modified the anti-HIV-1 activity of all the nucleos(t)ide analogues tested, whereas sensitivity to a non-nucleoside inhibitor (nevirapine) or nucleoside phosphonates (cidofovir and tenofovir) was not affected. Interestingly, SAMHD1 could either enhance (gemcitabine, capecitabine, fluorouracil, and floxuridine) or inhibit (Ara-C, fludarabine, cladribine, clofarabine, and nelarabine) the antiviral potency of anticancer analogues, an effect that was not dependent on the specific nucleotide targeted. When cytotoxicity was evaluated, SAMHD1-dependent changes were less evident and were restricted to the increased efficacy of fluorouracil and floxuridine and reduced efficacy of nelarabine and ara-C in the presence of SAMHD1. In summary, our results demonstrate that SAMHD1 modifies the efficacy of a wide variety of nucleoside analogues which are used to treat infections, cancer, and other diseases. In addition, the anti-HIV activity of nucleos(t)ide analogues may represent a more sensitive measure of SAMHD1’s impact on drug efficacy. Thus, modulation of SAMHD1’s function may constitute a promising target for the improvement of multiple therapies.

En les darreres dècades, l’ús d’anàlegs de nucleòsids (ANs) ha despertat un gran interès de recerca per a la teràpia antiviral i anticancerigen. La conformació i el plegament de la unitat de sucre dels nucleòsids tenen un paper crític en la modulació de la seva activitat biològica. En aquest context, s’han dissenyat i sintetitzat nous anàlegs de carbanucleòsids (ACNs) restringits conformacionalment per imitar el comportament conformacional de l’anell de furanosa natural. Els nucleòsids ciclohexenílics són un tipus prometedor de compostos antivirals, en què la substitució de l’àtom d’oxigen de l’anell de furanosa per un doble enllaç indueix una flexibilitat anular, similar a la dels nucleòsids naturals. Recentment, s’ha descrit que ambdós enantiòmers del ciclohexenil G (DCG i LCG) mostren una potent i selectiva activitat antivirus. Tenint en compte la interessant activitat anti-HSV del ciclohexenil G, es va proposar una nova sèrie d’ANs biciclo[4.1.0]heptà al nostre grup de recerca, en què el doble enllaç va ser substituït per un ciclopropà fusionat. La present tesi doctoral està enfocada a la síntesi enantioselectiva d’una nova sèrie d’ANs carbocíclis de sis membres basats en l’esquelet del ciclohexenil G que conté la unitat de biciclo[4.1.0]heptà. Concretament, es descriu l’assoliment dels objectius següents: - En primer lloc, s’han optimitzat dues estratègies sintètiques desenvolupades en el nostre grup de recerca per preparar la cetona clau 36, que és l’intermedi comú per a la preparació dels ANs biciclo[4.1.0]heptà. La cetona 36 s’ha obtingut en una seqüencia de 6 passos robusta, reproduïble i eficaç a escala de multi-gram amb un rendiment total del 49% a partir de l’1-ciclohexadiona. -En segon lloc, ja que els ANs 5-hidroximetilbiciclo[4.1.0]heptà, BCH-(A, G, T i U), prèviament obtinguts al grup de recerca, no mostraven activitat antiviral significativa, es va decidir de completar la família de 5-hidroximetilbiciclo[4.1.0]heptà, on es va sintetitzar l’anàleg de citosina que es va obtenir amb un rendiment total del 4% en 17 etapes. Tot i això, no va presentar activitat antiviral ni citotoxicitat. Tota aquesta família de ANs han estat avaluades en assajos enzimàtics per determinar l’afinitat amb la timidina quinasa (TK) vírica. El compost basat en timidina BCH-T té una interessantíssima afinitat cap a HSV-TK (EC50 = 1.6 ± 0.1 µg / ml), que coincideix amb el nostre model computacional realitzat per a la primera etapa de fosforilació per aquests productes. -En tercer lloc, s’ha preparat una nova família d’ANs 5-hidroximetil-4-hidroxibiciclo[4.1.0]heptan-2-il. En particular, els ANs de timina i guanina s’han obtingut en 17 i 18 passos i amb un rendiment global del 18% i 10%, respectivament. L’epímer-C4 de la timina i l’alquè de la timina 4,5-é s’han sintetitzat en 19 etapes i un rendiment total del 6% i del 3%, respectivament. Aquests quatre ANs han estat avaluats per determinar la seva activitat antiviral, encara que cap d’ells mostra cap activitat significativa. -Finalment, també s’ha obtingut una nova família d’ACNs 1,2,3-triazole-biciclo[4.1.0]heptà. Aquest enfocament ha permès la preparació de quatre compostos 1,2,3-triazol-ACNs (4-substituït) via CuAAc amb un rendiment global del 19-16% en 15 etapes. A més, es va obtenir un compost 4,5-substituït mitjançant una reacció de cicloaddició i un 12% de rendiment en 14 etapes. S’ha avaluat l’activitat biològica d’aquests anàlegs contra diversos virus, tot i que només el propilbenzen-1,2,3-triazolo-ACNs 151 mostra una activitat considerable contra el virus Coxsackie B4 (EC50 = 13.5-9.4 µg / mL). En las últimas décadas, el uso de análogos de nucleósidos (ANs) ha generado un amplio interés de investigación para la terapia antiviral y anticancerígena. La conformación y el plegamiento de la unidad de azúcar de los nucleósidos juegan un papel crítico en la modulación de su actividad biológica. En este contexto, se han diseñado y sintetizado nuevos análogos de carbanucleósidos (ACNs) restringidos conformacionalmente para imitar el comportamiento conformacional del anillo de furanosa. Los nucleósidos de ciclohexenilo son un tipo prometedor de compuestos antivirales, donde el reemplazo del átomo de oxígeno del anillo de furanosa por un doble enlace induce flexibilidad anular, similar a la de los nucleósidos regulares. Recientemente, se ha descrito que ambos enantiómeros de ciclohexenilo G (DCG y LCG) muestran una actividad potente y selectiva contra el virus del herpes (HSV). Teniendo en cuenta la interesante actividad anti-HSV del ciclohexenilo G, en nuestro grupo de investigación se propuso una nueva serie de ANs biciclo[4.1.0]heptano en el que el doble enlace fue reemplazado por un ciclopropano fusionado. La presente Tesis doctoral se centra en la síntesis enantioselectiva de una nueva serie de ANs carbocíclicos de seis miembros basados en el esqueleto del ciclohexenilo G conteniendo la estructura de biciclo[4.1.0]heptano a partir de un intermedio clave común. Específicamente, se describe el logro de los siguientes objetivos: - En primer lugar, se han optimizado dos estrategias sintéticas desarrolladas en nuestro grupo de investigación para preparar la cetona clave 36, que es el intermedio común para la preparación de los ANs biciclo[4.1.0]heptano. La cetona 36 se ha obtenido en un secuencia robusta, reproducible y eficiente de 6 pasos en una escala de multi-gramo con un rendimiento general del 49% a partir de 1,4-ciclohexadiona. -En segundo lugar, dado que los análogos 5’-hidroximetilbiciclo[4.1.0]heptano BCH-(A, G, T y U), obtenidos previamente en el grupo de investigación, no mostraron actividad antiviral significativa, se decidió completar la familia 5’-hidroximetilbiciclo [4.1.0]heptanilo sintetizando el análogo de citosina que ha sido obtenido con un rendimiento global del 4% en 17 pasos. Sin embargo, no mostró actividad antiviral o citotoxicidad. Toda esta familia de AN se ha evaluado en ensayos enzimáticos para determinar la afinidad de la timidina quinasa (TK) vírica. El compuesto a base de timidina BCH-T tiene una gran afinidad interesante hacia HSV-TK (EC50 = 1.6 ± 0.1 µg/mL) que es consistente con nuestro modelo computacional realizado para la primera etapa de fosforilación para estos compuestos. -En tercer lugar, se ha preparado una nueva familia de ANs 5’-hidroximetil-4’-hidroxibiciclo[4.1.0]heptan-2’-ilo. En particular, se obtuvieron los ANs de timina y guanina en 17 y 18 pasos y un rendimiento general de 18% y 10%, respectivamente. El epimero-C4 de timina y el alqueno de timina 4’,5’-eno se han sintetizado en 19 pasos y con un rendimiento general de 6% y 3%, respectivamente. Estos cuatro AN han sido seleccionados para evaluar su actividad antiviral, aunque ninguno de ellos muestra actividad significativa. -Finalmente, también se ha obtenido una nueva familia de ACNs 1,2,3-triazol-biciclo[4.1.0]heptanilo. Este enfoque ha permitido la preparación de cuatro 1,2,3-triazolo-ACNs (4-sustituido) a través de CuAAc con un rendimiento global del 19-16% en 15 pasos. Además, también se ha sintetizado un compuesto de triazol (4,5-sustituido) mediante una reacción de cicloadición y 12% de rendimiento en 14 etapas. Se ha evaluado la actividad biológica de estos análogos contra varios virus, aunque solo el propilbenceno-1,2,3-triazolo-ACN 151 muestra una actividad considerable contra el virus Coxsackie B4 (EC50 = 13.5-9.4 µg/mL). In the last decades, the use of nucleoside analogues (NAs) has garnered extensive research interest for antiviral and anticancer therapy. The conformation and puckering of the sugar moiety of nucleosides play a critical role in modulating their biological activity. In this context, new conformationally restricted carbanucleosides analogues (CNAs) have been designed and synthesised in order to mimic the conformational behaviour of the natural furanose ring. Cyclohexenyl nucleosides are a promising type of antiviral compounds, wherein replacement of the oxygen atom of the furanose ring by a double bond induces annular flexibility, similar to that of regular nucleosides. Recently, it has been described that both enantiomers of cyclohexenyl G (DCG and LCG) display potent and selective anti-herpes virus activity. Considering the interesting anti-HSV activity of cyclohexenyl G, new series of enantiopure bicyclo[4.1.0]heptanyl NAs were proposed in our research group, in which the double bond was replaced by a fused cyclopropane. The present Ph.D Thesis is focused on the enantioselective synthesis of a novel series of six-membered carbocyclic NAs based on the skeleton of cyclohexenyl G containing bicyclo[4.1.0]heptane structure from a common key intermediate. Specifically, it is described the achievement of the following objectives: - Firstly, two synthetic strategies developed in our research group have been optimized in order to prepare the key ketone 36, which is the common intermediate for the preparation of bicyclo[4.1.0]heptane NAs. Ketone 36 has been obtained in a robust, reproducible, and efficient 6-steps approach on a multigram scale in 49% overall yield from commercially available 1,4-cyclohexadione. -Secondly, since the 5’-hydroxymethylbicyclo[4.1.0]heptane analogues BCH-(A, G, T and U) NAs, previously obtained in the research group, did not show significant antiviral activity, in order to complete the 5’-hydroxymethylbicyclo[4.1.0]heptanyl family, the cytosine analogue has been synthesised in 4% overall yield in 17 steps. However, it does not show antiviral activity or cytotoxicity. All of this family of NAs have been evaluated in enzymatic assays in order to determine the virus-TK affinity. The thymidine-based compound BCH-T has an interesting great affinity towards HSV-TK (EC50 = 1.6 ± 0.1 µg/mL) which is consistent with our computational model performed for the first stage of phosphorylation. -Thirdly, a novel family of 5’-hydroxymethyl-4’-hydroxybicyclo[4.1.0]heptan-2’-yl NAs has been prepared. In particular, thymine and guanine NAs have been obtained in 17 and 18 steps and 18% and 10% overall yield, respectively. The thymine C4-epimer and the thymine alk-4,5-ene have been synthesised in 19 steps and 6% and 3% overall yield, respectively. These four NAs have been screened for antiviral activity, although none of them show significant activity. -Finally, a new family of 1,2,3-triazolo-bicyclo[4.1.0]heptanyl CNAs has also been obtained. This approach has allowed the preparation of the four 4-substituted-1,2,3-triazolo-CNAS via CuAAc in 19-16% overall yield in 15 steps. Furthermore, the 4,5-substituted-1,2,3-triazolo-CNAS has been synthesised via cycloaddition and 12% yield in 14 steps. The biological activity of these analogues against several viruses has been evaluated, although only the propylbenzene-1,2,3-triazolo-CNAs 151 shows a considerable activity against Coxsackie B4 virus (EC50 = 13.5-9.4 µg/mL). Universitat Autònoma de Barcelona. Programa de Doctorat en Química

Homonucleoside analogues cis-16 and trans-17 having a (5-methoxycarbonyl)isoxazolidine framework were synthesized via the 1,3-dipolar cycloaddition of nucleobase-derived nitrones with methyl acrylate. Hydrogenolysis of the isoxazolidines containing thymine, dihydrouracil, theophylline and adenine moieties efficiently led to the formation of the respective &gamma, lactam analogues. &gamma, Lactam analogues having 5-bromouracil and 5-chlorouracil fragments were synthesized by treatment of uracil-containing &gamma, lactams with NBS and NCS. Isoxazolidine and &gamma, lactam analogues of homonucleosides obtained herein were evaluated for activity against a broad range of DNA and RNA viruses. None of the compounds that were tested exhibited antiviral or cytotoxic activity at concentrations up to 100 &micro, M. The cytostatic activities of all compounds toward nine cancerous cell lines was tested. &gamma, Lactams trans-15e (Cl-Ura) and cis-15h (Theo) appeared the most active toward pancreatic adenocarcinoma cells (Capan-1), showing IC50 values 21.5 and 18.2 &micro, M, respectively. Isoxazolidine cis-15e (Cl-Ura) inhibited the proliferation of colorectal carcinoma (HCT-116).

The rational design of broad‐spectrum antivirals requires data on antiviral activity of compounds against multiple viruses, which are often not available. We have developed a panel of (+)ssRNA viruses composed of Enterovirus and Flavivirus genera members allowing to study these activity spectra. Antiviral activity profiling of a set of nucleoside analogues revealed N 4‐hydroxycytidine as an efficient inhibitor of replication of coxsackieviruses and other enteroviruses, but ineffective against tick‐borne encephalitis virus. Furano[2, 3‐d]pyrimidine nucleosides with n‐pentyl or n‐hexyl tails showed selective inhibition of Enterovirus A representatives. 5‐(Tetradec‐1‐yn‐1‐yl)‐uridine showed selective inhibition of tick‐borne encephalitis virus at the micromolar level., Antiviral activity spectrum assessment showed that broad‐spectrum antiviral N4‐hydroxycytidine inhibits reproduction of enteroviruses, but not tick‐borne encephalitis virus (TBEV), a flavivirus. Furano[2, 3‐d]pyrimidine nucleosides suppressed only Enterovirus A species members, Enterovirus A71 and Coxsackievirus A16, but not other enteroviruses or TBEV. 5‐(Tetradec‐1‐yn‐1‐yl)‐uridine, on the other hand, inhibited TBEV only.

Pyrrolotriazines and related fused azaheterocycles have high potential for the synthesis of bioactive compounds, especially as a purine base isoster in carbon linked nucleosides. Although many structurally related compounds have already been synthesized and used in medicinal chemistry, pyrrolo[1,3,5]triazines have barely been described. The present work describes the synthesis of such heterocycles via condensation of 2-amino-3-ethoxycarbonylpyrrole with ethoxycarbonyliso(thio)cyanate. In a brief reactivity study of the obtained fused pyrroles, O- and S-alkylation, ester hydrolysis as well as regioselective bromination at the 6-position was demonstrated.

Hepatitis B virus infection affects over 250 million chronic carriers, causing more than 800,000 deaths annually, although a safe and effective vaccine is available. Currently used antiviral agents, pegylated interferon and nucleos(t)ide analogues, have major drawbacks and fail to completely eradicate the virus from infected cells. Thus, achieving a “functional cure” of the infection remains a real challenge. Recent findings concerning the viral replication cycle have led to development of novel therapeutic approaches including viral entry inhibitors, epigenetic control of cccDNA, immune modulators, RNA interference techniques, ribonuclease H inhibitors, and capsid assembly modulators. Promising preclinical results have been obtained, and the leading molecules under development have entered clinical evaluation. This review summarizes the key steps of the HBV life cycle, examines the currently approved anti-HBV drugs, and analyzes novel HBV treatment regimens.

Modern approaches to the prevention and treatment of chronic hepatitis B are based on the use of vaccination of the population, on the one hand, and rational choice of antiviral therapy schemes in patients - on the other. This allows the physician to predict results of treatment and preventive measures. Modern analogues of nucleosides, in particular, Entecavir, has high efficacy, good tolerance and high safety profile when used in long term conditions.

This article presents modern aspects of clinical manifestations, laboratory diagnostics and treatment of herpetic keratitis (HK). A total of 68 patients participated in the study. In 75 percent of patients high relapse rates of Herpes Simplex skin infection were revealed in the period before HK onset (one-six times per year). Trigger factors for keratitis were thoroughly analyzed. Among them we discovered a number of new ones: the instillation of prostaglandin analogues, paranasal sinus surgery, septoplasty and blepharoplasty. 51 out of 68 patients had active herpetic keratitis, whereas 17 people were in the remission phase. Polymerase chain reaction (PCR) test results for the herpes viruses (HSV, VZV, CMV, EBV, HHV-6) in various body fluids (tears, saliva, blood and urine) from patients with HK are discussed. Herpesvirus DNA was determined in 96 percent of cases (49 out of 51 patients with active HK). Among these 49 patients a monoinfection was detected in 47 percent of cases and mixed infection - in 53 percent. According to the obtained results, we proposed some optimized treatment solutions // Russian Ophthalmological Journal, 2017; 1: 90-7. doi: 10.21516/2072-0076-2017-10-1-90-97.

Introduction and objective: The aim of the present study was to investigate the significance of serum HBsAg levels in treatment cessation of nucleoside analogues (NAs) in patients with chronic hepatitis B (CHB) infection. Methods: In 158 CHB patients with long-term NAs treatment, 74 patients were in HBeAg negative and had a HBsAg level 1000 IU/mL relapsed. HBsAg of patients with a sustained response decreased slowly. In contrast, HBsAg levels increased gradually in relapsed patients, and the increase of HBsAg was precedent to relapses of HBV DNA and ALT. Multivariate analysis suggested that only HBsAg level showed a close correlation with HBV DNA relapses. ROC curve analysis suggested that the increase of HBsAg level in the 3rd and 6th month after NAs cessation had a great predictive value for relapses. Conclusion: Monitoring of base line HBsAg level can predict outcomes of NAs cessation in HBeAg-negative chronic hepatitis B. HBsAg

Here, we report a convenient synthetic procedure for the preparation of four novel indanyl carbanucleoside derivatives in the racemic form. The action of these compounds against hepatitis C virus was evaluated in vitro using the replicon cell line, Huh7.5 SG. Contrary to our expectations, all these compounds did not inhibit, but rather promoted HCV genotype 1b (HCVg1b) replication. Similar effects have been reported for morphine in the replicon cell lines, Huh7 and Huh8. Several biological experiments and computational studies were performed to elucidate the effect of these compounds on HCVg1b replication. Based on all the experiments performed, we propose that the increase in HCVg1b replication could be mediated, at least in part, by a similar mechanism to that of morphine on the enhancement of this replication. The presence of opioid receptors in Huh7.5 SG cells was indirectly determined for the first time in this work. Fil: Gómez Elías, Matías Daniel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gentile, Emiliano Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Martini, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; Argentina Fil: Cuestas, María Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Mathet, Veronica Lidia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Moltrasio, Graciela Yolanda. Universidad Nacional de Córdoba. Facultad de Cs.agropecuarias. Departamento de Fundamentación Biológica. Cátedra de Química Organica; Argentina Fil: Moglioni, Albertina Gladys. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; Argentina

Macromolecular prodrugs (MP) built on the natural phosphodiester and deoxyribose backbone are developed using marketed antiviral nucleoside analogues. These MP are synthesized using automated synthesis, have defined molecular composition, and have a natural mechanism for drug release. These unique attributes, coupled to the efficient cell entry and potent antiviral effects, position the prodrugs scaffolded on nucleic acids favorably for translational studies.

Human African trypanosomiasis is a neglected parasitic disease for which the current treatment options are quite limited. Trypanosomes are not able to synthesize purines de novo and thus solely depend on purine salvage from the host environment. This characteristic makes players of the purine salvage pathway putative drug targets. The activity of known nucleoside analogues such as tubercidin and cordycepin led to the development of a series of C7-substituted nucleoside analogues. Here, we use RNA interference (RNAi) libraries to gain insight into the mode-of-action of these novel nucleoside analogues. Whole-genome RNAi screening revealed the involvement of adenosine kinase and 4E interacting protein into the mode-of-action of certain antitrypanosomal nucleoside analogues. Using RNAi lines and gene-deficient parasites, 4E interacting protein was found to be essential for parasite growth and infectivity in the vertebrate host. The essential nature of this gene product and involvement in the activity of certain nucleoside analogues indicates that it represents a potential novel drug target.

Hepatitis B reactivation is a common complication in lymphoma patients under immunosuppressive treatment with potentially serious and life-threating consequences. In this review, we discuss the basis of chronic Hepatitis B virus (HBV) infection, the definition and risk factors for HBV reactivation. We overview the management of HBV reactivation based on virological status and immunosuppressive regimen risk stratification. We also highlight and update information about the HBV reactivation in lymphoma patients under novel agent treatment, including newer monoclonal antibodies, small molecule inhibitors, and even chimeric antigen receptor T-cell immunotherapy.

Seek, and ye shall find: After years of focusing research on synthetic antibiotics out of fear that all the useful natural ones had already been found, a novel antibacterial compound has been discovered through conventional microbial extract screening. The broad-spectrum nucleoside-analogue inhibitor pseudouridimycin is selective for bacterial RNA polymerase and elicits very low resistance rates.

Aim: We aim to explore the effects of nucleos(t)ide analogues (NUCs) on the changes of HBsAg in chronic hepatitis B (CHB) patients. Methods: A total of 264 CHB patients were enrolled in our study. All of them were treated with NUCs for at least three years. Quantification of HBsAg levels were measured by Elecsys HBsAg II. Results: Although HBsAg levels were significantly higher in HBeAg seropositive CHB patients at baseline than in HBeAg seronegative CHB patients (3.84 ± 0.82 vs 3.21 ± 0.59 IU/mL), HBsAg levels declined more rapidly in the HBeAg seropositive group (P

Apoptosis is a series of molecular signalling regulating normal cellular growth and development. Cells resistance to apoptosis, however, leads to uncontrolled proliferation. Research involving cancer cell death is one of the most important targeted areas in the discovery of novel anticancer therapy. There are several biochemical pathways that are liked towards cancer cell death of which, uridine-cytidine kinase 2 (UCK2) was recently linked to cell apoptosis induction. UCK2 is responsible for the phosphorylation of uridine and cytidine to their corresponding monophosphate in a salvage pathway of pyrimidine nucleotides biosynthesis. Cytotoxic ribonucleoside analogues that target UCK2 enzyme activity are currently being investigated in clinical trials useful for cancer treatment. Whilst findings have clearly shown that these antimetabolites inhibit cancer development in clinical settings, they have yet to establish linking cytotoxic nucleoside analogues to cancer cell death. In this present review, we propose the probable molecular crosstalk involving UCK2 protein and cancer cell death through cell cycle arrest and triggering of apoptosis involving proteins, MDM2 and the subsequent activation of p53.

11 pags, 5 figs, 7 tabs, The use of enzymes for the synthesis of nucleoside analogues offers several advantages over multistep chemical methods, including chemo-, regio- and stereoselectivity as well as milder reaction conditions. Herein, the production, characterization and utilization of a purine nucleoside 2’-deoxyribosyltransferase (PDT) from Trypanosoma brucei are reported. TbPDT is a dimer which displays not only excellent activity and stability over a broad range of temperatures (50–70 °C), pH (4–7) and ionic strength (0–500 mM NaCl) but also an unusual high stability under alkaline conditions (pH 8–10). TbPDT is shown to be proficient in the biosynthesis of numerous therapeutic nucleosides, including didanosine, vidarabine, cladribine, fludarabine and nelarabine. The structure-guided replacement of Val11 with either Ala or Ser resulted in variants with 2.8-fold greater activity. TbPDT was also covalently immobilized on glutaraldehyde-activated magnetic microspheres. MTbPDT3 was selected as the best derivative (4200 IU/g, activity recovery of 22 %), and could be easily recaptured and recycled for >25 reactions with negligible loss of activity. Finally, MTbPDT3 was successfully employed in the expedient synthesis of several nucleoside analogues. Taken together, our results support the notion that TbPDT has good potential as an industrial biocatalyst for the synthesis of a wide range of therapeutic nucleosides through an efficient and environmentally friendly methodology., This work was supported by grants SAN151610 from the Santander Foundation and 2016/UEM08 from the European University of Madrid to JFL and SAF2015-64629-C2-2-R from the Spanish Ministerio de Economía y Competitividad to FG

Rigid amphipathic fusion inhibitors (RAFIs) are a family of nucleoside derivatives that inhibit the infectivity of several enveloped viruses by interacting with virion envelope lipids and inhibiting fusion between viral and cellular membranes. Here we tested the antiviral activity of two RAFIs, 5-(Perylen-3-ylethynyl)-arabino-uridine (aUY11) and 5-(Perylen-3-ylethynyl)uracil-1-acetic acid (cm1UY11) against African swine fever virus (ASFV), for which no effective vaccine is available. Both compounds displayed a potent, dose-dependent inhibitory effect on ASFV infection in Vero cells. The major antiviral effect was observed when aUY11 and cm1UY11 were added at early stages of infection and maintained during the complete viral cycle. Furthermore, virucidal assay revealed a significant extracellular anti-ASFV activity for both compounds. We also found decrease in the synthesis of early and late viral proteins in Vero cells treated with cm1UY11. Finally, the inhibitory effect of aUY11 and cm1UY11 on ASFV infection in porcine alveolar macrophages was confirmed. Overall, our study has identified novel anti-ASFV compounds with potential for future therapeutic developments.

Structure-function relationships of a novel 2 -deoxyribosyltransferase from the psychrotolerant bacterium Bacillus psychrosaccharolyticus (BpNDT) have been exhaustively studied by biochemical and high resolution crystallographic analyses. Despite BpNDT exhibiting some structural features characteristic of cold-adapted enzymes such as localized flexibility in critical loops, its biochemical properties are typical of mesophilic enzymes. BpNDT is a highly symmetrical homohexamer with tightly associated subunits that possesses flexible and short loops bordering the active sites. The catalytic center is essentially identical to that of other mesophilic homologues. Moreover, BpNDT shows that it is a mesophilic-like enzyme since it is not heat-labile and exhibits an apparent unfolding temperature (T) of 49C, being active during 96 h at 40 and 50C. Finally, BpNDT synthesizes natural and modified nucleosides, with preference for purines as acceptors and pyrimidine nucleosides as donors. Remarkably, the synthesis of several therapeutic nucleosides has been efficiently carried out. In this sense, 5-hydroxymethyl-2 -deoxyuridine (5-HMdUrd), 7-deaza-6-hydroxypurine-2-deoxyriboside (7-DHPdRib) and theophylline-2 -deoxyriboside were synthesized for the first time by an NDT enzyme, showing the biotechnological interest of BpNDT., This work was supported by grant CTQ2009-11543 from the Spanish Ministry of Science and Innovation and grant S2009/PPQ-1752 (CAPOTE) from Comunidad de Madrid. José Miguel Mancheño acknowledges the ESRF for provision of synchrotron radiation facilities.

Hepatitis B virus (HBV) poses a significant challenge for both dialysis patients and kidney transplant recipients despite its decreasing rates, especially in developed countries. The best preventive method is vaccination. Patients with chronic renal disease should ideally be vaccinated prior to dialysis, otherwise, reinforced vaccination practices and close antibody titer monitoring should be applied while on dialysis. HBV infected dialysis patients who are renal transplant candidates must be thoroughly examined by HBV-DNA, and liver enzyme testing and by liver biopsy. When needed, one must consider treating patients with tenofovir or entecavir rather than lamivudine. Depending on the cirrhosis stage, dialysis patients are eligible transplant recipients for either a combined kidney-liver procedure in the case of decompensated cirrhosis or a lone kidney transplantation since even compensated cirrhosis after sustained viral responders is no longer considered an absolute contraindication. Nucleoside analogues have led to improved transplantation outcomes with both long-term patient and graft survival rates nearing those of HBsAg(-) recipients. Moreover, in the cases of immunized HBsAg(-) potential recipients with concurrent prophylaxis, we are enabled today to safely use renal grafts from both HBsAg(+) and HBsAg(-)/anti-HBc(+) donors. In so doing, we avoid unnecessary organ discarding. Universal prophylaxis with entecavir is recommended in HBV kidney recipients and should start perioperatively. One of the most important issues in HBV(+) kidney transplantation is the duration of antiviral prophylaxis. In the absence of robust data, it seems that prophylactic treatment may be discontinued in selected stable, low-risk recipients during maintenance immunosuppression and should be reintroduced when the immune status is altered. All immunosuppressive agents in kidney transplantation can be used in HBV(+) recipients. Immunosuppression is intimately associated with increased viral replication; thus it is important to minimize the total immunosuppression burden long term.

Вирусный гепатит В остается важной медико-социальной проблемой для здравоохранения как Российской Федерации, так и всего мира. В Российской Федерации в 2013 г. экономический ущерб от острого вирусного гепатита В составил 349 049,8 тыс. руб., от хронического гепатита В 694 220,9 тыс. руб. В Республике Саха (Якутия) отмечается высокий уровень регистрации хронического гепатита В. В работе представлены результаты фармакоэкономического обоснования применения аналогов нуклеозидов у пациентов с хронической HBV-инфекцией методом моделирования определения затратной эффективности и экономии средств государственного бюджета. Эффективность и безопасность применения аналогов нуклеозидов (энтекавира, тенофовира) доказаны в рандомизированных контролируемых исследованиях и подтверждены результатами длительных наблюдений. В исследовании использованы материалы официальной статистики Территориального управления Роспотребнадзора Республики Саха (Якутия). В результате исследования было установлено, что применение противовирусной терапии позволило бы снизить число больных с декомпенсированным циррозом в 6 раз (с 3,7 до 0,6%), гепатоцеллюлярной карциномой -в 3 раза (с 2,3 до 0,8%) и в 2 раза снизить число умерших от хронического гепатита В (с 22 до 10%). Лечение аналогами нуклеозидов позволило бы предотвратить производственные потери, связанные с недопроизводством валового внутреннего продукта из-за выбытия человека из трудовой деятельности в связи с инвалидизацией или смертью по причине хронического гепатита В, в размере 908 млн руб. в год в масштабе Республики Саха (Якутия)., Viral hepatitis B is important problem for healthcare systems around the world. In 2013, economic damage for Russian Federation from acute HBV-infection is 349 049.8 thousand RUB, chronic HBV-infection 694 220.9 thousand RUB. In Republic of Sakha (Yakutia) we can notice high prevalence of HBV-infection. Authors presenting results of pharmacoeconomic validation to chronic HBV-infection ethiotropic therapy by nucleoside analogues with method of cost-effective efficiency and the state budget economy modelling. Safety and efficiency nucleoside analogues using (entecavir, tenofovir) confirmed in randomized controlled trials. In current article Territoral department of Rospotrebnadzor official statistics had been used. Resulting this article, antiviral therapy using would be decrease number of patients with decompensated liver cirrhosis at 6 times (from 3.7 to 0.6%), hepatocellular carcinoma at 3 times (from 2.3 to 0.8%), and decrease mortality at 2 times (from 22 to 10%). In addition, nucleoside analogues treatment would prevent manufactural losses related with GDP underproduction caused by retirement due to disability or death total 908 mln. RUB per year in Republic of Sakha (Yakutia).

Objectives This study evaluated the relationship between intracellular tenofovir diphosphate concentrations in peripheral blood mononuclear cells and prophylactic efficacy in a macaque model for HIV pre-exposure prophylaxis (PrEP). Methods Macaques were challenged with simian HIV (SHIV) via rectal inoculation once weekly for up to 14 weeks. A control group (n=34) received no drug, a second group (n=6) received oral tenofovir disoproxil fumarate/emtricitabine 3 days before each virus challenge and a third group (n=6) received the same dosing plus another dose 2 h after virus challenge. PBMCs were collected just before each weekly virus challenge. The relationship between tenofovir diphosphate in PBMCs and prophylactic efficacy was assessed with a Cox proportional hazards model. Results The percentages of animals infected in the control, one-dose and two-dose groups were 97, 83 and 17, respectively. The mean (SD) steady-state tenofovir diphosphate concentration (fmol/10(6) cells) was 15.8 (7.6) in the one-dose group and 30.7 (10.1) in the two-dose group. Each 5 fmol tenofovir diphosphate/10(6) cells was associated with a 40% (95% CI 17%-56%) reduction in risk of SHIV acquisition, P=0.002. The tenofovir diphosphate concentration associated with a 90% reduction in risk (EC90) was 22.6 fmol/10(6) cells (95% CI 13.8-60.8). Conclusions The prophylactic EC90 for tenofovir diphosphate identified in macaques exposed rectally compares well with the EC90 previously identified in men who have sex with men (MSM; 16 fmol/10(6) cells, 95% CI 3-28). These results highlight the relevance of this model to inform human PrEP studies of oral tenofovir disoproxil fumarate/emtricitabine for MSM.

Nucleoside analogues have found widespread application as antiviral and antitumor agents, but not yet as antibacterials. Naturally occurring uridine-derived &lsquo, nucleoside antibiotics&rsquo, target the bacterial membrane protein MraY, an enzyme involved in peptidoglycan biosynthesis and a promising target for the development of novel antibacterial agents. Muraymycins represent a nucleoside-peptide subgroup of such MraY-inhibiting natural products. As part of detailed structure-activity relationship (SAR) studies on muraymycins and their analogues, we now report novel insights into the effects of stereochemical variations in the nucleoside core structure. Using a simplified version of the muraymycin scaffold, it was shown that some formal inversions of stereochemistry led to about one order of magnitude loss in inhibitory potency towards the target enzyme MraY. In contrast, epimers of the core motif with retained inhibitory activity were also identified. These 5&prime, 6&prime, anti-configured analogues might serve as novel chemically tractable variations of the muraymycin scaffold for the future development of uridine-derived drug candidates.

The substitution of a hydroxyl group by a fluorine atom in a potential drug is an efficient reaction that can, in principle, improve its pharmacological properties. Herein, the synthesis of the novel compound 5′-fluoro- 5′-deoxyacadesine (5′-F-AICAR), a strict analogue of AICAR that cannot be 5′-phosphorylated to ZMP by cellular kinases, is reported. © 2012 by the authors.

Despite the introduction of vaccination, chronic hepatitis B remains a major cause of liver-related morbidity and mortality including cirrhosis, decompensated cirrhosis and hepatocellular carcinoma. Maintenance antiviral therapy is required for most people, as low rates of cure occur. The stated aim of therapy presently is HBV DNA suppression; effective suppression of viral replication is associated with significant reductions in morbidity from end-stage liver failure and to an extent, hepatocellular carcinoma. Unfortunately, major barriers to cure, such as a reservoir of episomal covalently closed circular DNA (cccDNA) (the HBV minichromosome), and a dysfunctional immune response, pose challenges. These barriers will need to be overcome to ensure higher rates of cure than can be achieved presently. Quantitative and diagnostic testing for HBV DNA is not generally available, hampering effective monitoring and treatment in low-income countries. The majority of patients in resource-constrained countries are not identified before the onset of cirrhosis. Without coordinated action, and transfer of new diagnostic technologies and treatments to low-income countries, recent therapeutic advances will have little effect on the global burden of disease. A shift to curative treatment for the majority would be a major advance in the elimination of hepatitis B. New and improved molecular therapeutics and immunological strategies for the treatment of chronic hepatitis are emerging, however. A number of promising lines of development are in progress. A curative regimen may require a combination of viral suppression via nucleoside analogue therapy to prevent cccDNA amplification and viral propagation, safe selective cccDNA inhibitors to deplete, silence or degrade cccDNA, agents to block the entry of HBV into the hepatocyte plus compounds to prevent capsid assembly and cccDNA interactions. Targeted immune activation could restore the exhausted immune cell repertoire.

Feline herpesvirus type 1 (FHV-1) is a common and important cause of ocular surface disease, dermatitis, respiratory disease, and potentially intraocular disease in cats. Many antiviral drugs developed for the treatment of humans infected with herpesviruses have been used to treat cats infected with FHV-1. Translational use of drugs in this manner ideally requires methodical investigation of their in vitro efficacy against FHV-1 followed by pharmacokinetic and safety trials in normal cats. Subsequently, placebo-controlled efficacy studies in experimentally inoculated animals should be performed followed, finally, by carefully designed and monitored clinical trials in client-owned animals. This review is intended to provide a concise overview of the available literature regarding the efficacy of antiviral drugs and other compounds with proven or putative activity against FHV-1, as well as a discussion of their safety in cats.

Backgrounds/Aims Nucleos(t)ide analogues (NUCs) effectively suppress hepatitis B virus (HBV) replication, but hepatocellular carcinoma (HCC) recurrence often leads to HBV replication despite NUC therapy. The aim of this study was to determine whether high-dose tenofovir (TNF) therapy can suppresses HCC recurrence-associated HBV replication. Methods We performed a single-arm prospective study to assess the clinical feasibility of high-dose TNF (hdTNF). We recruited 10 patients during September 2015 and followed up for 3 months or early drop-out. Results All 10 patients had HCC of advanced stages due to HCC recurrence and gradual progression. The average age of patients was 51.2±4.7 years and 9 were male. Three patients did not tolerate the increased TNF dosage and were dropped out early. The other 7 patients were relatively tolerable to the increased dosage of TNF 5 tablets per day. One patient had mild gastrointestinal symptoms and another patient complained of insomnia. Increased HBV replication and HCC progression was observed despite hdTNF for 4-8 weeks. All 7 patients showed tumor progression during the 3 month follow-up. In these patients, blood HBV DNA before hdTNF was 50-200 copies/ml; and 4-8 weeks after hdTNF, the HBV replication status was not improved with blood HBV DNA of 50-300 copies/ml. This clinical study was terminated early after these negative results were confirmed. Conclusions The results of this study indicated that high dose of TNF up to 5-fold the recommended dosage is not tolerated by a considerable proportion of patients and also ineffective in suppressing HCC progression-associated HBV replication.

Thymidine kinase 2 (TK2), encoded on chromosome 16q22 of the human genome, is a deoxynucleoside kinase (dNK) that catalyzes the phosphorylation of the pyrimidine deoxynucleosides 2’-deoxythymidine (dThd), 2’-deoxyuridine (dUrd) and 2’- deoxycytidine (dCyd) to the corresponding deoxynucleoside 5’-monophosphate derivatives. In contrast to the S-phase-specific thymidine kinase 1 (TK1), TK2 is constitutively expressed in the mitochondria and plays an important role in providing dNTPs for the replication and maintenance of mitochondrial DNA (mtDNA). The severe mitochondrial DNA depletion syndrome (MDS) has been associated with mutations in TK2, resulting in mtDNA depletion, isolated skeletal myopathy, and death of the individual at an early stage of life. Some antiviral nucleoside analogs, such as 3’-azido-dThd (AZT) that is targeting the human immunodeficiency virus (HIV)-encoded reverse transcriptase, are substrates for TK2 and it has been proposed that the mitochondrial toxicity observed after prolonged treatment with such drugs could be due to their interaction with TK2. Therefore, the design of specific TK2 inhibitors may be useful to investigate the role of TK2 in the maintenance and homeostasis of mitochondrial dNTP pools and its contribution to the mitochondrial toxicity of several antiviral and anticancer drugs. Since 2000, several potent and selective TK2 inhibitors have been described. Besides bringing together previously reported inhibitors, special attention will be paid in this review to the new families of TK2 inhibitors more recently described, together with modeling studies and biological assays. Moreover, the last section will be focused on several recent investigations that suggest that depletion of mtDNA can take place both in tumorigenesis and during cancer treatment with certain nucleoside analogues.

Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Analogues of the pyrimidine deoxynucleoside thymidine may be inserted into replicating DNA, effectively tagging dividing cells allowing their characterisation. Tritiated thymidine, targeted using autoradiography was technically demanding and superseded by 5-bromo-2-deoxyuridine (BrdU) and related halogenated analogues, detected using antibodies. Their detection required the denaturation of DNA, often constraining the outcome of investigations. Despite these limitations BrdU alone has been used to target newly synthesised DNA in over 20,000 reviewed biomedical studies. A recent breakthrough in "tagging DNA synthesis" is the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). The alkyne group in EdU is readily detected using a fluorescent azide probe and copper catalysis using 'Huisgen's reaction' (1,3-dipolar cycloaddition or 'click chemistry'). This rapid, two-step biolabelling approach allows the tagging and imaging of DNA within cells whilst preserving the structural and molecular integrity of the cells. The bio-orthogonal detection of EdU allows its application in more experimental assays than previously possible with other "unnatural bases". These include physiological, anatomical and molecular biological experimentation in multiple fields including, stem cell research, cancer biology, and parasitology. The full potential of EdU and related molecules in biomedical research remains to be explored.

Background: Several published studies indicate that the acyclic guanine nucleoside analogues possessing bis(1,2-hydroxymethyl) substituted cyclopropane rings mimicking the sugar moiety are potent inhibitors of replication of several herpes viruses. Methods: Established synthetic methods and antiviral and cytostatic activity assays were used for the evaluation of new 1,2,4-triazole and purine acyclic nucleoside analogues. Results: The synthesis of new types of acyclic nucleoside analogues which incorporate 1,2,4-triazole or purine moiety bound via flexible methylenic spacer to the bis(1,2-hydroxymethyl) cyclopropane ring. None of the new compounds showed pronounced antiviral activities at subtoxic concentrations on a broad panel of DNA and RNA viruses. Evaluation of their affinity for herpes simplex type 1 (HSV-1) and varicella-zoster virus-encoded thymidine kinases (VZV TK) also showed that none of the compounds was able to significantly inhibit 1 μM deoxythymidine phosphorylation by HSV-1 and VZV TK at 500 μM concentrations. The in vitro cytostatic activity evaluation results indicated a weak antiproliferative activity for all tested compounds. Only 6-pyrrolylpurine derivative bearing a carboxylic group substituted cyclopropane ring produced a rather slight inhibitory effect at higher micromolar concentrations on a breast carcinoma cell line (MCF-7) and no cytotoxic effect on human normal fibroblasts (WI 38). Conclusions: The lack of antiherpetic activity may be due to poor, if any, recognition of the compounds by virus-induced nucleoside kinases as an alternative substrate to become metabolically activated.

Background & Aims: Chronic hepatitis B patients are at increased risk for hepatocellular carcinoma (HCC). The effect of medium-term nucleos(t)ide analogue therapy on HCC incidence is unclear; therefore, we systematically reviewed all the data on HCC incidence from studies in chronic hepatitis B patients treated with nucleos(t)ide analogues. Methods: We performed a literature search to identify studies with chronic hepatitis B patients treated with nucleos(t)ide analogues for ⩾24months. Results: Twenty-one studies including 3881 treated and 534 untreated patients met our inclusion criteria. HCC was diagnosed in 2.8% and 6.4% of treated and untreated patients, respectively, during a 46 (32–108) month period (p=0.003), in 10.8% and 0.5% of nucleos(t)ide naive patients with and without cirrhosis (p

Antimetabolites, in particular nucleobase and nucleoside analogues, are cytotoxic drugs that, starting from the small field of paediatric oncology, in combination with other chemotherapeutics, have revolutionised clinical oncology and transformed cancer into a curable disease. However, even though combination chemotherapy, together with radiation, surgery and immunotherapy, can nowadays cure almost all types of cancer, we still fail to achieve this for a substantial proportion of patients. The understanding of differences in metabolism, pharmacokinetics, pharmacodynamics, and tumour biology between patients that can be cured and patients that cannot, builds the scientific basis for rational therapy improvements. Here, we summarise current knowledge of how tumour-specific and patient-specific factors can dictate resistance to nucleobase/nucleoside analogues, and which strategies of re-sensitisation exist. We revisit well-established hurdles to treatment efficacy, like the blood-brain barrier and reduced deoxycytidine kinase activity, but will also discuss the role of novel resistance factors, such as SAMHD1. A comprehensive appreciation of the complex mechanisms that underpin the failure of chemotherapy will hopefully inform future strategies of personalised medicine.

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.

BACKGROUND: There are currently five approved nucleos(t)ide analogues (NUCs) for the management of chronic hepatitis B (CHB): lamivudine, adefovir dipivoxil, telbivudine, entecavir, and tenofovir disoproxil fumarate. OBJECTIVE: To determine the persistence rates among patients receiving NUCs for CHB at weeks 48, 96 and 144, compare them in these periods, and analyse the evolution of treatment persistence. METHODS: We conducted a retrospective study that included patients with CHB who initiated antiviral therapy and were attended to by the pharmaceutical care office between January 2002 and December 2011. Patients included in a clinical trial or patients who did not collect their medication personally were excluded. There were two different analyses: a comparative analysis of the persistence rates in three periods (weeks 1–48, weeks 48–96, and weeks 96–144); and a Kaplan-Meier analysis to evaluate the evolution of persistence. RESULTS: A total of 102 patients were included. Persistence rates were different in the three periods. They decreased during the course of the different periods, and the decline was more rapid between the first and second period. There were statistically significant differences in the non-persistence of the five drugs (p

Introduction : There have been several important developments in antiretroviral treatment in the past two years. Randomized clinical trials have been conducted to evaluate a lower dose of efavirenz (400 mg once daily). Integrase inhibitors such as dolutegravir have been approved for first-line treatment. A new formulation of tenofovir (alafenamide) has been developed and has shown equivalent efficacy to tenofovir in randomized trials. Two-drug combination treatments have been evaluated in treatment-naive and -experienced patients. The novel pharmacokinetic booster cobicistat has been compared to ritonavir in terms of pharmacokinetics, efficacy and safety. The objective of this commentary is to assess recent developments in antiretroviral drug treatment to determine whether new treatments should be included in new international guidelines. Discussion : The use of first-line treatment with tenofovir and efavirenz at the standard 600 mg once-daily dose should remain the first-choice standard of care treatment. Evidence supporting a switch to efavirenz 400 mg once daily or integrase inhibitors is sufficient to consider these drugs as alternative first-line options, but more data are needed on their use in pregnant women and people with TB co-infection. The use of new formulations of tenofovir is currently too preliminary to justify immediate adoption and scale-up across HIV programmes in low- and middle-income countries. The evidence supporting use of two-drug combinations is not considered strong enough to justify changed recommendations from use of standard triple drug combinations. Cobicistat does not offer significant safety advantages over ritonavir as a pharmacokinetic booster. Conclusions : For continued scale-up of antiretroviral treatment in low- and middle-income countries, use of first-line triple combinations including efavirenz 600 mg once daily is supported by the largest evidence base. Additional studies are underway to evaluate new treatments in key populations, and these results may justify changes to these recommendations. Keywords: antiretroviral treatment; Universal Access; nucleoside analogues; integrase inhibitors; protease inhibitors; non-nucleosides; pregnancy; tuberculosis. (Published: 2 February 2016) Vitoria M et al. Journal of the International AIDS Society 2016, 19 :20504 http://www.jiasociety.org/index.php/jias/article/view/20504 | http://dx.doi.org/10.7448/IAS.19.1.20504

Article available at http://dx.doi.org/10.2174/138161206776873608, Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is an important target of drugs fighting HIV infection. The introduction of potent antiretroviral therapies based on the use of RT inhibitors and/or protease inhibitors has been an important achievement towards the control of AIDS. However, the development of drug resistance constitutes a major hurdle towards long-term efficacy of those therapies. With the increasing complexity of the antiretroviral regimens, novel mutational patterns conferring high-level resistance to nucleoside and nonnucleoside RT inhibitors have been identified in viral isolates. Among them, insertions and deletions in the β3- β4 hairpin-loop-coding region of HIV-1 RT have been identified in heavily-treated patients. Insertions of one, two or several residues appear to have a significant impact on nucleoside analogue resistance. The frequently found combination of a dipeptide insertion and thymidine analogue resistance mutations (i.e. T215Y) in the viral RT confers an ATP-dependent phosphorolytic activity that facilitates the removal of the inhibitor from primers terminated with zidovudine or stavudine. Furthermore, this mechanism appears to be relevant for resistance mediated by one amino acid-deletions appearing in combination with thymidine analogue resistance mutations. However, in other sequence contexts (i.e. in the presence of Q151M), the effects of the deletion are not fully understood. Drugs targeting the excision repair mechanism could be an important aid in the fight against multinucleoside-resistant HIV isolates bearing complex mutational patterns in their RT-coding region, Work in the authors’ laboratory is supported in part by grants of the Ministerio de Educación y Ciencia (BIO2003/01175), Fundación para la Investigación y Prevención del SIDA en España (FIPSE) (grant 36200/01), Fondo de Investigación Sanitaria (through “Red Temática Cooperativa de Investigación en SIDA” G03/173), and an institutional grant of Fundación Ramón Areces.