Nucleotide Analogues Research Articles

Aptamer nucleotide analog drug conjugates in the targeting therapy of cancers.

Aptamers are short single-strand oligonucleotides that can form secondary and tertiary structures, fitting targets with high affinity and specificity. They are so-called "chemical antibodies" and can target specific biomarkers in both diagnostic and therapeutic applications. Systematic evolution of ligands by exponential enrichment (SELEX) is usually used for the enrichment and selection of aptamers, and the targets could be metal ions, small molecules, nucleotides, proteins, cells, or even tissues or organs. Due to the high specificity and distinctive binding affinity of aptamers, aptamer-drug conjugates (ApDCs) have demonstrated their potential role in drug delivery for cancer-targeting therapies. Compared with antibodies which are produced by a cell-based bioreactor, aptamers are chemically synthesized molecules that can be easily conjugated to drugs and modified; however, the conventional ApDCs conjugate the aptamer with an active drug using a linker which may add more concerns to the stability of the ApDC, the drug-releasing efficiency, and the drug-loading capacity. The function of aptamer in conventional ApDC is just as a targeting moiety which could not fully perform the advantages of aptamers. To address these drawbacks, scientists have started using active nucleotide analogs as the cargoes of ApDCs, such as clofarabine, ara-guanosine, gemcitabine, and floxuridine, to replace all or part of the natural nucleotides in aptamer sequences. In turn, these new types of ApDCs, aptamer nucleotide analog drug conjugates, show the strength for targeting efficacy but avoid the complex drug linker designation and improve the synthetic efficiency. More importantly, these classic nucleotide analog drugs have been used for many years, and aptamer nucleotide analog drug conjugates would not increase any unknown druggability risk but improve the target tumor accumulation. In this review, we mainly summarized aptamer-conjugated nucleotide analog drugs in cancer-targeting therapies.

Treatment with nucleoside and nucleotide analogues in patients with chronic hepatitis B virus infection

This study aimed to compare the efficacy of treatment with entecavir (ETV) and tenofovir (TDF) in patients with chronic hepatitis B infection. Cross-sectional, descriptive, retrolective study. Realized in the “Hospital de especialidades Siglo XXI”. We included patients >18 years with chronic hepatitis B infection in treatment and follow-up from January 1st, 2015, to March 1st, 2021. Descriptive statistics were performed and to show differences Wilcoxon test was used. Approved by the institutional ethics committee and informed consent was obtained. We included 33 patients, male gender predominated in 51.5% (17), mean age was 59 years (+/- 11.25). Co-infected with HIV were 18% (6). Median baseline viral load was 2´500,00 (3940 – 191´500,000 copies/ml). Median baseline APRI 0.3 (0.2-1.6) and FIB-4 1.33 (1.0-2.2). Exposure to previous treatments was 45.8% (16). The mean follow-up was 9.48 years (+/-4.82). Current treatment TDF 60.6% (20), ETV 27% (9). Incidence of hepatocellular carcinoma occurred in 3% (1). At 6 and 12 months of treatment, 69% and 64% (16/23 and 16/28), respectively, with undetectable viral load. There was a difference in baseline APRI compared to current p <0.05; there was no difference in APRI throughout treatment. Treatment is effective for HBV both in chronic infection and liver cirrhosis, maintaining viral suppression with low seroconversion rates and low incidence of hepatocellular carcinoma. Treatment with nucleotide and nucleoside analogues is effective for the suppression of the hepatitis B virus. The resources used in this study were from the hospital without any additional financing The authors declare no potential conflicts of interest.

The role of Hepatitis B Core Antibody: Significance and Clinical practice

Hepatitis B infection remains a global health problem, with progression to acute-chronic hepatitis, severe liver failure, and death making hepatitis B one of the most serious infections worldwide. The disease is most widely transmitted from an infected mother to her baby, after exposure to infected blood or body fluids or unsafe sexual contact. Pregnant women, adolescents, and all adults at high risk for chronic infection are recommended to be screened for hepatitis B. Serological tests allow the distinction between acute and chronic hepatitis. Meanwhile, the molecular tests performed provide detection and quantification of viral DNA, genotyping, drug resistance, and pre-core/core mutation analysis to confirm infection and follow monitoring disease progression in patients with chronic hepatitis B. Because, the current treatment is only based on nucleotide analogs and pegylated interferons that save lives by decreasing liver cancer death, liver transplant, slowing or reversing the progression of liver disease as well as the virus infectivity. In this review, we clearly light the role of Hepatitis B Core Antibody, therefore clinicians understand the need to screen for hepatitis B core antigen (Anti-HBc), proper interpretation of HBV biomarkers, and that “anti-HBc only” indicates HBV exposure, lifelong persistence of cccDNA with incomplete infection control, and potential risk for reactivation. Key words: Hepatitis B virus (HBV), Hepatitis B Core Antibody (Anti-HBc/HbcAb).

Multifaceted Defects in Monocytes in Different Phases of Chronic Hepatitis B Virus Infection: Lack of Restoration after Antiviral Therapy.

Monocytes play an important role in the control of microbial infection, but monocyte biology during chronic hepatitis B virus (HBV) infection (CHI) remains inadequately studied. We investigated the frequency, phenotype, and functions of monocyte subsets in different phases of CHI, namely, immune tolerance (IT), hepatitis B early antigen (HBeAg)-positive/HBeAg-negative chronic hepatitis B (EP-/EN-CHB, respectively), and inactive carrier (IC), identified factors responsible for their functional alterations, and determined the impact of antiviral therapy on these cells. Flow cytometric analysis indicated that HLA-DR+ CD14++ CD16- classical monocytes were significantly reduced while HLA-DR+ CD14++ CD16+ intermediate and HLA-DR+ CD14+ CD16++ nonclassical monocytes were expanded in IT and EP-/EN-CHB compared with those in IC and healthy controls (HC). In comparison to IC/HC, monocytes in IT and CHB exhibited diminished expression of Toll-like receptor 2 (TLR-2)/TLR-4/TLR-9 and cytokines interleukin-12 (IL-12)/tumor necrosis factor alpha (TNF-α)/IL-6 but produced higher levels of IL-10/transforming growth factor β (TGF-β). Further, monocytes in CHB/IT showed impaired phagocytosis and oxidative response relative to those in IC/HC. In vitro assays indicated that high titers of hepatitis B surface antigen (HBsAg) present in IT/CHB and of IL-4 in CHB triggered the functional defects in monocytes via induction of β-catenin. Additionally, monocyte-derived M1 macrophages of CHB/IT produced fewer proinflammatory and more anti-inflammatory cytokines than those of IC/HC, while in CHB/IT, the monocytes skewed the differentiation of CD4+ T cells more toward regulatory T cells and a Th2 phenotype. Moreover, monocytes in CHB and IT overexpressed chemokine receptor CCR2, which coincided with increased intrahepatic accumulation of β-catenin+ CD14+ cells. One year of tenofovir therapy failed to normalize monocyte functions or reduce serum HBsAg/IL-4 levels. Taken together, monocytes are functionally perturbed mostly in IT and EP-/EN-CHB phases. Targeting intramonocytic β-catenin or reducing HBsAg/IL-4 levels might restore monocyte function and facilitate viral clearance. IMPORTANCE Chronic HBV infection (CHI) is a major cause of end-stage liver disease for which pharmacological treatments currently available are inadequate. Chronically HBV-infected patients fail to mount an efficient immune response to the virus, impeding viral clearance and recovery from hepatitis. Monocytes represent a central part of innate immunity, but a comprehensive understanding on monocyte involvement in CHI is still lacking. We here report a multitude of defects in monocytes in chronically HBV-infected patients that include alteration in subset distribution, Toll-like receptor expression, cytokine production, phagocytic activity, oxidative response, migratory ability, polarization of monocyte-derived macrophages, and monocyte-T-cell interaction. We demonstrated that high levels of hepatitis B virus surface antigen and IL-4 potentiate these defects in monocytes via β-catenin induction while therapy with the nucleotide analog tenofovir fails to restore monocyte function. Our findings add to the continuing effort to devise new immunotherapeutic strategies that could reverse the immune defects in CHI.

Cell and Tissue Specific Metabolism of Nucleoside and Nucleotide Drugs: Case Studies and Implications for Precision Medicine.

Many clinically used antiviral drugs are nucleoside or nucleotide analog drugs, which have a unique mechanism of action that requires intracellular phosphorylation. This dependence on intracellular activation presents novel challenges for the discovery and development of nucleoside/nucleotide analog drugs. Contrary to many small molecule drug development programs that rely on plasma pharmacokinetics and systemic exposures, the precise mechanisms that result in efficacious intracellular nucleoside triphosphate concentrations must be understood in the process of nucleoside/nucleotide drug development. The importance is highlighted here, using the following as case studies: the herpes treatment acyclovir, the cytomegalovirus therapy ganciclovir, and human immunodeficiency virus (HIV) treatments based on tenofovir, which are also in use for HIV prophylaxis. For each drug, the specificity of metabolism that results in its activation in different cells or tissues is discussed, and the implications explored. Acyclovir's dependence on a viral enzyme for activation provides selective pressure for resistance mutations. Ganciclovir is also dependent on a viral enzyme for activation, and suicide gene therapy capitalizes on that for a novel oncology treatment. The tissue of most relevance for tenofovir activation depends on its use as treatment or as prophylaxis, and the pharmacogenomics and drug-drug interactions in those tissues must be considered. Finally, differential metabolism of different tenofovir prodrugs and its effects on toxicity risk are explored. Taken together, these examples highlight the importance of understanding tissue specific metabolism for optimal use of nucleoside/nucleotide drugs in the clinic. SIGNIFICANCE STATEMENT: Nucleoside and nucleotide analogue drugs are cornerstones in current antiviral therapy and prevention efforts that require intracellular phosphorylation for activity. Understanding their cell and tissue specific metabolism enables their rational, precision use for maximum efficacy.

Efficacy and Safety of Bepirovirsen in Chronic Hepatitis B Infection

BackgroundBepirovirsen is an antisense oligonucleotide that targets all hepatitis B virus (HBV) messenger RNAs and acts to decrease levels of viral proteins.MethodsWe conducted a phase 2b, randomized, investigator-unblinded trial involving participants with chronic HBV infection who were receiving or not receiving nucleoside or nucleotide analogue (NA) therapy. Participants were randomly assigned (in a 3:3:3:1 ratio) to receive weekly subcutaneous injections of bepirovirsen at a dose of 300 mg for 24 weeks (group 1), bepirovirsen at a dose of 300 mg for 12 weeks then 150 mg for 12 weeks (group 2), bepirovirsen at a dose of 300 mg for 12 weeks then placebo for 12 weeks (group 3), or placebo for 12 weeks then bepirovirsen at a dose of 300 mg for 12 weeks (group 4). Groups 1, 2, and 3 received loading doses of bepirovirsen. The composite primary outcome was a hepatitis B surface antigen (HBsAg) level below the limit of detection and an HBV DNA level below the limit of quantification maintained for 24 weeks after the planned end of bepirovirsen treatment, without newly initiated antiviral medication.ResultsThe intention-to-treat population comprised 457 participants (227 receiving NA therapy and 230 not receiving NA therapy). Among those receiving NA therapy, a primary-outcome event occurred in 6 participants (9%; 95% credible interval, 0 to 31) in group 1, in 6 (9%; 95% credible interval, 0 to 43) in group 2, in 2 (3%; 95% credible interval, 0 to 16) in group 3, and 0 (0%; post hoc credible interval, 0 to 8) in group 4. Among participants not receiving NA therapy, a primary-outcome event occurred in 7 participants (10%; 95% credible interval, 0 to 38), 4 (6%; 95% credible interval, 0 to 25), 1 (1%; post hoc credible interval, 0 to 6), and 0 (0%; post hoc credible interval, 0 to 8), respectively. During weeks 1 through 12, adverse events, including injection-site reactions, pyrexia, fatigue, and increased alanine aminotransferase levels, were more common with bepirovirsen (groups 1, 2, and 3) than with placebo (group 4).ConclusionsIn this phase 2b trial, bepirovirsen at a dose of 300 mg per week for 24 weeks resulted in sustained HBsAg and HBV DNA loss in 9 to 10% of participants with chronic HBV infection. Larger and longer trials are required to assess the efficacy and safety of bepirovirsen. (Funded by GSK; B-Clear ClinicalTrials.gov number, NCT04449029.)

Remdesivir inhibits the progression of glioblastoma by enhancing endoplasmic reticulum stress

Glioblastoma (GBM) is one of the most aggressive primary malignant brain tumors. The major challenge is the lack of effective therapeutic drugs due to the blood–brain barrier (BBB) and tumor heterogeneity. Remdesivir (RDV), a new member of the nucleotide analog family, has previously been shown to have excellent antiviral effects and BBB penetration, and was predicted here to have anti-GBM effects. In vitro experiments, RDV significantly inhibited the growth of GBM cells, with IC50 values markedly lower than those of normal cell lines or the same cell lines treated with temozolomide. Moreover, in multiple mouse models, RDV not only distinctly inhibited the progression and improved the prognosis of GBM but also exhibited a promising biosafety profile, as manifested by the lack of significant body weight loss, liver or kidney dysfunction or organ structural damage after administration. Furthermore, we investigated the anti-GBM mechanism by RNA-seq and identified that RDV might induce apoptosis of GBM cells by enhancing endoplasmic reticulum (ER) stress and activating the PERK-mediated unfolded protein response. In conclusion, our results indicated that RDV might serve as a novel agent for GBM treatment by increasing ER stress and inducing apoptosis in GBM cells.

Two-in-one combination therapy of fluoronucleoside analogues and triplex forming oligonucleotides

Antigene technology based on triplex-forming oligonucleotide (TFO) has been widely used in gene-specific regulation, serving as a potential gene therapy method. The optimization of TFO backbone and base can significantly improve the formation and stability of the triplex DNA. Fluorination is widely used in small molecule drug development, but its application in TFO optimization has not been well characterized. Herein, we developed a ‘in silico-in vitro-in vivo’ screening strategy to optimize fluorine atom (F) substitution of TFO. Molecular dynamics simulations are applied to screen single-site F substitutions in the TFO bases that retained stable triplex formation ability with the Ets2-targeted duplex DNA. Combining existing nucleotide analogue floxuridine, the synergistic effect is realized via a two-in-one chemo-TFO including gene regulation and chemotherapeutic functions, which is verified through in vitro and in vivo anti-tumor experiments.

Fragile sites, chromosomal lesions, tandem repeats, and disease.

Expanded tandem repeat DNAs are associated with various unusual chromosomal lesions, despiralizations, multi-branched inter-chromosomal associations, and fragile sites. Fragile sites cytogenetically manifest as localized gaps or discontinuities in chromosome structure and are an important genetic, biological, and health-related phenomena. Common fragile sites (∼230), present in most individuals, are induced by aphidicolin and can be associated with cancer; of the 27 molecularly-mapped common sites, none are associated with a particular DNA sequence motif. Rare fragile sites ( 40 known), 5% of the population (may be as few as a single individual), can be associated with neurodevelopmental disease. All 10 molecularly-mapped folate-sensitive fragile sites, the largest category of rare fragile sites, are caused by gene-specific CGG/CCG tandem repeat expansions that are aberrantly CpG methylated and include FRAXA, FRAXE, FRAXF, FRA2A, FRA7A, FRA10A, FRA11A, FRA11B, FRA12A, and FRA16A. The minisatellite-associated rare fragile sites, FRA10B, FRA16B, can be induced by AT-rich DNA-ligands or nucleotide analogs. Despiralized lesions and multi-branched inter-chromosomal associations at the heterochromatic satellite repeats of chromosomes 1, 9, 16 are inducible by de-methylating agents like 5-azadeoxycytidine and can spontaneously arise in patients with ICF syndrome (Immunodeficiency Centromeric instability and Facial anomalies) with mutations in genes regulating DNA methylation. ICF individuals have hypomethylated satellites I-III, alpha-satellites, and subtelomeric repeats. Ribosomal repeats and subtelomeric D4Z4 megasatellites/macrosatellites, are associated with chromosome location, fragility, and disease. Telomere repeats can also assume fragile sites. Dietary deficiencies of folate or vitamin B12, or drug insults are associated with megaloblastic and/or pernicious anemia, that display chromosomes with fragile sites. The recent discovery of many new tandem repeat expansion loci, with varied repeat motifs, where motif lengths can range from mono-nucleotides to megabase units, could be the molecular cause of new fragile sites, or other chromosomal lesions. This review focuses on repeat-associated fragility, covering their induction, cytogenetics, epigenetics, cell type specificity, genetic instability (repeat instability, micronuclei, deletions/rearrangements, and sister chromatid exchange), unusual heritability, disease association, and penetrance. Understanding tandem repeat-associated chromosomal fragile sites provides insight to chromosome structure, genome packaging, genetic instability, and disease.

Hematopoietic Flt3+ Multipotent Progenitors Are Organized in Discrete Functionally and Hierarchically Distinct Populations

Blood production in the bone marrow (BM) is classically viewed as a constant hematopoietic flux originating from a small pool of self-renewing hematopoietic stem cells (HSCs). In this model, quiescent HSCs regularly differentiate and give rise to a complex set of transient multipotent progenitors (MPPs) that serve as compartments of amplification through division, while undergoing progressive lineage specification Recent studies exploiting sophisticated genetic marking for in vivo cell-fate tracking have challenged this paradigm and suggested that HSCs have a limited contribution to steady state blood production (Sun, Nature 2014, Busch, Nature 2015; Rodriguez-Fraticelli, Nature 2018). The interpretation of these results and the extent of the HSC contribution to native hematopoiesis remains a matter of debate (Pucella, Annu Rev Cell Dev Biol 2020; Rodriguez-Fraticelli, Curr. Opin. Hematol 2021). Beyond this debate, these studies have highlighted the underestimated contribution of the MPP compartment to the daily hematopoietic output in unperturbed conditions. This new understanding raises questions about the functional properties of the MPP compartment in steady state condition and its ability to independently sustain prolonged hematopoietic cell production. Here we revisited the early hematopoietic hierarchy present in the Lin- Kit+ Sca1+ (LSK) fraction that includes all HSC and MPP (MPP1-5) compartments. Highly-multiplexed CITE-Seq single cell analyses with over 60 antibodies identified a novel LSK CD62L- Flt3+ population (CD62L- MPP4) that clusters within the MPP compartments but displays a cell cycle gene signature similar to the one found in quiescent HSCs. Analysis of the Mki67 reporter mouse model and nucleotide analogue incorporation assays directly confirmed the existence of this unexpected cell cycle diversity in MPP4 during unperturbed hematopoiesis. Consistent with the molecular data, these analyses characterized a resting CD62L- and a proliferative CD62L+ MPP4 compartments. These two populations showed similar cloning efficiency in methylcellulose assays indicating that both populations behave as intermediary MPP compartments. When assessed in short-term culture, both populations were responsive to Flt3L stimulation but displayed specific kinetics of division. They also showed distinct short-term reconstitution ability upon transplantation in correlation with their cell cycle activity. Analysis of these populations in native condition using the H2B-GFP label-retaining system further confirmed the specific properties of the resting CD62L- MPP4 cells, distinct from the other proliferative MPP2, MPP3 and CD62L+ MPP4 compartments. Finally, these studies uncovered a novel early path of differentiation that involves the MPP5 cells (LSK Flt3+ CD48- CD150-) sequentially acquiring Flk2 and CD48 expression to generate the resting CD62L- MPP4 compartment, which in turn gives rise to cycling CD62L+ MPP4 cells. Altogether, these results challenge the accepted dichotomy between quiescent HSCs and dividing progenitors. While difference in lineage potential has been historically a major criterium to classify MPP populations, we established a new dimension at the MPP diversity, based on their proliferative activity at steady state. These results particularly highlight a new path of differentiation directly linking MPP5, a quiescent compartment with residual self-renewal capacity, to the resting CD62L- Flt3+ MPP cells. Based on these results, we proposed that slow-dividing Flt3+ MPPs act as a key transitional compartment that can buffer the hematopoietic cell production and therefore regulate hematopoietic homeostasis at steady state. We envision that this novel compartment could be a key intermediary for hematopoietic malignancies driven by the acquisition of Flt3 mutations and a cellular target for intervention.

Cytotoxicity and Antiviral Activity of Kyllinga nemoralis Methanolic Extract

Herpes Simplex Virus (HSV) is a double-stranded DNA virus in the Herpesviridae family. The most prevalent in humans is Human Simplex Virus Type 1 (HSV-1). It can reach the brain without causing the symptoms [1]. Currently, the most antiviral drug used is Acyclovir (ACV); nucleotide analogues target DNA polymerase inhibiting viral replication. However, ACV drug-resistant occurs, especially in immunocompromised patients [2]. Therefore, alternate therapy for patients with established resistance was required. Plant extract is commonly used as antiviral agent. Kyllinga nemoralis is known as White Spike Head had antiviral, antibacterial, antibleeding and anti-poisoning properties [3]. It contains antiviral sources such as the highest flavonoid and flavonol concentrations [4].&#x0D; &#x0D; The objective of this experimental study was to investigate the potential of K. nemoralis methanolic extract as antiviral agent against HSV-1. The root of K. nemoralis methanolic plant extract, Vero Cells and HSV-1 were used in this study. The Cytotoxic Concentration (CC50) of the K. nemoralis methanolic extract against Vero Cells and the antiviral activity of K. nemoralis methanolic extract against HSV-1 by using post-treatment, pre-treatment and virucidal assay were performed in this study.&#x0D; &#x0D; Results showed that the percentage of cell population versus concentration of root of K. nemoralis methanolic extract (figure 1). The CC50 of this extract was determined at 0.91 mg/mL. This showed that the root of K. nemoralis methanolic extract was considered not toxic to the cells as the CC50 of extract showed more than 4 µg/mL [5].&#x0D; &#x0D; The results indicate that the root of K. nemoralis methanolic extract was more effective in protecting the Vero cells. In pre-treatment, the SI value of the extract was 8.27. This finding shows that the root of K. nemoralis methanolic extract had a moderate ability to bind to the Vero cells and inhibited the binding of HSV-1 to the cell surface of the Vero cells. In a virucidal test, the root extract towards HSV-1 showed the SI value was 8.125. This data suggests that the root of the extract was moderately inactivates the extracellular component of HSV-1.&#x0D; In conclusion, the findings of this study show that K. nemoralis methanolic extract was non-toxic to the Vero cells and has the potential to be an antiviral agent against HSV-1.&#x0D;

The hepatitis delta virus and chronic hepatitis D.

The hepatitis delta virus and chronic hepatitis D.

Effect of GS-441524 in combination with the 3C-like protease inhibitor GC376 on the treatment of naturally transmitted feline infectious peritonitis.

The main objectives of this study were to investigate the efficacy of the nucleotide analog GS-441524 in combination with the 3C-like protease inhibitor GC376 for the treatment of naturally aquired feline infectious peritonitis (FIP) and to test whether their combination shortens the dosing period and improves the cure rate. In total, 46 FIP-affected cats were enrolled in this experiment, including 36 with wet FIP (29 with abdominal effusion, six with thoracic effusion, and one with thoracic+abdominal effusion), and 10 with dry FIP. The cats were aged from 3 to 96 months. Thoracic+abdominal effusion, lymph-node puncture fluid and perirenal puncture fluid was collected from the affected cats for qPCR testing, and all 46 cats were positive for feline coronavirus (FCoV). The cats divided into different dose groups, all treated for 4 weeks: group 1 (GS-441524, 5 mg/kg.sc.q.24 h; GC376, 20 mg/kg.sc.q.12 h), group 2 (GS-441524, 2.5 mg/kg.sc.q.24 h; GC376, 20 mg/kg.sc.q.12 h), group 3 (GS-441524, 2.5 mg/kg.sc.q.24 h; GC376, 10 mg/kg.sc.q.12 h), and group 4 (GS-441524, 5 mg/kg.sc.q.24 h; GC376, 10 mg/kg.sc.q.12 h). After the 4-week combination treatment, 45 of the 46 (97.8%) cats survived, and 43 of those became clinically normal. Two cats required longer (7 to 12 weeks) treatment to achieve full recovery. As of writing (10 months after completion of the trial), all 45 cats were alive and no relapse was observed. GS-441524 combined with GC376 can be safely and effectively used to treat FIP and reduces the treatment period to 4 weeks, with an excellent cure rate.

Extrahepatic Manifestations of Chronic HBV Infection and the Role of Antiviral Therapy.

The hepatitis B virus (HBV) infection leads to chronic hepatitis, cirrhosis, and hepatocarcinoma. However, about 20% of patients experience extrahepatic manifestations such as polyarteritis nodosa, non-rheumatoid arthritis, non-Hodgkin lymphoma, cryoglobulinemic vasculitis, and glomerulonephritis. These influence the patient's morbidity, quality of life and mortality. The treatment of an HBV infection is based on nucleotide analogues (NAs) which are safe and effective for the suppression of HBV-DNA in almost 100% of cases. A few studies have shown that NAs induce a viral response and an improvement of extrahepatic diseases. There is a lack of a thorough analysis of the available treatments for extrahepatic HBV manifestations. In 90% to 100% of cases, the NAs stop the HBV replication, and they produce a clinical response in the majority of patients with mild to moderate extrahepatic signs/symptoms. Arthritis can definitely disappear after the HBV elimination and, in some cases, the HBV eradication following NAs therapy appears to improve the renal function in HBV-related nephropathies. Plasma exchange can be used in subjects who are suffering from the most aggressive forms of cryoglobulinemic vasculitis and glomerulonephritis, progressive peripheral neuropathy, and life-threatening cases, and this can be combined with glucocorticosteroids and antiviral agents. In selected refractory patients, the use of rituximab in conjunction with NAs therapy can be considered. The review provides an update on extrahepatic conditions that are linked to HBV and the impact of treating HBV with NAs.

A comprehensive in silico analysis of pathogenic nsSNPs in the NT5C2 gene involved in relapsed ALL

Background: About 10-20% of children suffering from acute lymphoblastic leukemia (ALL), experience a relapse, which is a major cause of their death. Purine nucleotide analogs are frequently prescribed to maintain the treatment of ALL. Cytosolic 5´-nucleotidase (NT5C2) catalyzes the 5´ dephosphorylation of purine analogs. Gain-of-function mutations in the NT5C2 gene result in resistance to the treatment with purine analogs and subsequently in the relapse of the disease.&#x0D; Materials and Methods: In this descriptive study, bioinformatics tools were used to assess the effect of single nucleotide polymorphisms (SNPs) in the NT5C2 gene on the function and structure of the protein. So, 352 missense variants were retrieved from the NCBI database and analyzed by SIFT, PROVEAN, PMut, PANTHER, PolyPhen2, SNPs &amp; Go, and PhD-SNP servers. Then, structural evaluations were performed using HOPE, NetSurp-2.0, and PyMOL. Moreover, stability and evolutionary preservation were assessed by I-Mutant2.0 and ConSurf, respectively.&#x0D; Results: As many as 31 nsSNPs were predicted to be affecting the protein function and stability. Also, the native residues were found to be evolutionarily preserved. The structural evaluation demonstrated that a change of hydrophobicity, flexibility, size, charge, or surface accessibility due to 24 nsSNPs would lead to the change of noncovalent interactions and then the conformation of the protein.&#x0D; Conclusion: Identification of biomarkers is significant in the prediction of relapses in ALL children. In this study, bioinformatics tools served to identify 24 high-risk deleterious nsSNPs in the NT5C2 gene. These mutations can be used to predict resistance to chemotherapy and relapse in ALL patients.

Synthesis of Nucleoside and Nucleotide Analogues by Cyclization of the Guanine Base with 1,1,3,3-Tetramethoxypropane.

3-(2-Deoxy-β-d-erythropentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one (M1dG) is an endogenous DNA adduct in bacterial and mammalian cells that could be explored as a biomarker for oxidative stress. Nonetheless, the lack of an efficient methodology for the preparation of M1dG hampers the deep investigation of its biosynthesis and biorelevant processes. In this project, we aimed to address this issue by developing a highly efficient method to synthesize M1dG and its analogues. This method has wide functional group tolerance, as various guanine-based nucleosides and nucleotides are suitable for the reaction. Furthermore, large-scale and derivatization reactions were carried out to showcase the possibility for biochemists to study DNA damage and repair processes in the future.

Small Interfering RNAs Containing Dioxane- and Morpholino-Derived Nucleotide Analogues Show Improved Off-Target Profiles and Chirality-Dependent In Vivo Knock-Down.

To expand the applicability of recently developed dioxane- and morpholino-based nucleotide analogues, their seed region destabilizing properties in small interfering RNAs (siRNAs) were investigated in order to improve potential off-target profiles. For this purpose, the corresponding adenosine analogues were synthesized in two diastereomeric series as building blocks for the automated oligonucleotide synthesis. The obtained nucleotide precursors were integrated at position 7 of an siRNA antisense strand, targeting transthyretin messenger RNA. Evaluation of the melting temperatures revealed significant differences in the obtained duplex stabilities between the two diastereomeric series, while the influence of the central scaffold was small. All siRNAs containing these novel nucleotide structures showed improved off-target profiles in vitro compared to their parent sequence with the common 2'-OMe-modified adenosine at the same position. In contrast, in vivo potencies were highly dependent on the chirality within the six-membered nucleotide scaffolds and showed high mRNA downregulations for the (2R,6R)-configured diastereomers.

On the Recognition of Natural Substrate CTP and Endogenous Inhibitor ddhCTP of SARS-CoV-2 RNA-Dependent RNA Polymerase: A Molecular Dynamics Study.

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 outbreak that is affecting the entire planet. As the pandemic is still spreading worldwide, with multiple mutations of the virus, it is of interest and of help to employ computational methods for identifying potential inhibitors of the enzymes responsible for viral replication. Attractive antiviral nucleotide analogue RNA-dependent RNA polymerase (RdRp) chain terminator inhibitors are investigated with this purpose. This study, based on molecular dynamics (MD) simulations, addresses the important aspects of the incorporation of an endogenously synthesized nucleoside triphosphate, ddhCTP, in comparison with the natural nucleobase cytidine triphosphate (CTP) in RdRp. The ddhCTP species is the product of the viperin antiviral protein as part of the innate immune response. The absence of the ribose 3′-OH in ddhCTP could have important implications in its inhibitory mechanism of RdRp. We built an in silico model of the RNA strand embedded in RdRp using experimental methods, starting from the cryo-electron microscopy structure and exploiting the information obtained by spectrometry on the RNA sequence. We determined that the model was stable during the MD simulation time. The obtained results provide deeper insights into the incorporation of nucleoside triphosphates, whose molecular mechanism by the RdRp active site still remains elusive.

Remdesivir—the medicine that has proven to be a successful new health technology in the battle against COVID-19

The global COVID-19 pandemic caused by the viral pathogen SARS-CoV-2 has surged the biomedical community into action to discover and develop effective antiviral drugs. One potential therapeutic approach currently being evaluated in multiple clinical trials is the medicinal product—remdesivir, which has had a tortuous development path over the past eight years. In essence, it is a nucleotide analog prodrug that inhibits viral replication. It was initially evaluated in clinical trials to prevent the Ebola outbreak in 2014. Subsequent evaluation by multiple virology laboratories around the world demonstrated remdesivir's ability to inhibit coronavirus replication, including the SARS-CoV-2 virus which is the cause of the current pandemic. In the presented paper, the authors provide an overview of the discovery of remdesivir, its mechanism of action, and current studies investigating its clinical effectiveness.

A mechanism for SARS-CoV-2 RNA capping and its inhibition by nucleotide analog inhibitors

Decoration of cap on viral RNA plays essential roles in SARS-CoV-2 proliferation. Here we report a mechanism for SARS-CoV-2 RNA capping and document structural details at atomic resolution. The NiRAN domain in polymerase catalyzes the covalent link of RNA 5’ end to the first residue of nsp9 (termed as RNAylation), thus being an intermediate to form cap core (GpppA) with GTP catalyzed again by NiRAN. We also reveal that triphosphorylated nucleotide analogue inhibitors can be bonded to nsp9 and fit into a previously unknown ‘Nuc-pocket’ in NiRAN, thus inhibiting nsp9 RNAylation and formation of GpppA. S-loop (residues 50-KTN-52) in NiRAN presents a remarkable conformational shift observed in RTC bound with sofosbuvir monophosphate, reasoning an ‘induce-and-lock’ mechanism to design inhibitors. These findings not only improve the understanding of SARS-CoV-2 RNA capping and the mode of action of NAIs, but also provide a strategy to design antiviral drugs.