Assessment of the role of nucleoside transporters, P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2 in the placental transport of entecavir using in vitro, ex vivo, and in situ methods.

The nucleoside analog entecavir (ETV) is a first-line pharmacotherapy for chronic hepatitis B in adult and pediatric patients. However, due to insufficient data on placental transfer and its effects on pregnancy, ETV administration is not recommended for women after conception. To expand knowledge of safety, we focused on evaluating the contribution of nucleoside transporters (NBMPR sensitive ENTs and Na+ dependent CNTs) and efflux transporters, P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), and multidrug resistance-associated transporter 2 (ABCC2), to the placental kinetics of ETV. We observed that NBMPR and nucleosides (adenosine and/or uridine) inhibited [3H]ETV uptake into BeWo cells, microvillous membrane vesicles, and fresh villous fragments prepared from the human term placenta, while Na+ depletion had no effect. Using a dual perfusion study in an open-circuit setup, we showed that maternal-to-fetal and fetal-to-maternal clearances of [3H]ETV in the rat term placenta were decreased by NBMPR and uridine. Net efflux ratios calculated for bidirectional transport studies performed in MDCKII cells expressing human ABCB1, ABCG2, or ABCC2 were close to the value of one. Consistently, no significant decrease in fetal perfusate was observed in the closed-circuit setup of dual perfusion studies, suggesting that active efflux does not significantly reduce maternal-to-fetal transport. In conclusion, ENTs (most likely ENT1), but not CNTs, ABCB1, ABCG2, and ABCC2, contribute significantly to the placental kinetics of ETV. Future studies should investigate the placental/fetal toxicity of ETV, the impact of drug-drug interactions on ENT1, and interindividual variability in ENT1 expression on the placental uptake and fetal exposure to ETV.

Dual computational and biological assessment of some promising nucleoside analogs against the COVID-19-Omicron variant.

Nucleoside analogs/derivatives (NAs/NDs) with potent antiviral activities are now deemed very convenient choices for the treatment of coronavirus disease 2019 (COVID-19) arisen by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. At the same time, the appearance of a new strain of SARS-CoV-2, the Omicron variant, necessitates multiplied efforts in fighting COVID-19. Counteracting the crucial SARS-CoV-2 enzymes RNA-dependent RNA polymerase (RdRp) and 3'-to-5' exoribonuclease (ExoN) jointly altogether using the same inhibitor is a quite successful new plan to demultiplicate SARS-CoV-2 particles and eliminate COVID-19 whatever the SARS-CoV-2 subtype is (due to the significant conservation nature of RdRps and ExoNs in the different SARS-CoV-2 strains). Successive in silico screening of known NAs finally disclosed six different promising NAs, which are riboprine/forodesine/tecadenoson/nelarabine/vidarabine/maribavir, respectively, that predictably can act through the planned dual-action mode. Further in vitro evaluations affirmed the anti-SARS-CoV-2/anti-COVID-19 potentials of these NAs, with riboprine and forodesine being at the top. The two NAs are able to effectively antagonize the replication of the new virulent SARS-CoV-2 strains with considerably minute in vitro anti-RdRp and anti-SARS-CoV-2 EC50 values of 189 and 408 nM for riboprine and 207 and 657 nM for forodesine, respectively, surpassing both remdesivir and the new anti-COVID-19 drug molnupiravir. Furthermore, the favorable structural characteristics of the two molecules qualify them for varied types of isosteric and analogistic chemical derivatization. In one word, the present important outcomes of this comprehensive dual study revealed the anticipating repurposing potentials of some known nucleosides, led by the two NAs riboprine and forodesine, to successfully discontinue the coronaviral-2 polymerase/exoribonuclease interactions with RNA nucleotides in the SARS-CoV-2 Omicron variant (BA.5 sublineage) and accordingly alleviate COVID-19 infections, motivating us to initiate the two drugs' diverse anti-COVID-19 pharmacological evaluations to add both of them betimes in the COVID-19 therapeutic protocols.

Assessment of nucleotide/nucleoside analog intervention in primer-dependent viral RNA-dependent RNA polymerases.

Nucleotide/nucleoside analogs (NAs) are important compounds used in antiviral drug development. To understand the action mode of NA drugs, we present an enzymology protocol to initially evaluate the intervention mechanism of the NTP forms of NAs on a coronaviral RNA-dependent RNA polymerase (RdRP). We describe the preparation of SARS-CoV-2 RdRP proteins and RNA constructs, followed by a primer-dependent RdRP assay to assess NTP forms of NAs. Two representative NA drugs, sofosbuvir and remdesivir, are used for demonstration of this protocol. For complete details on the use and execution of this protocol, please refer to Wu et al. (2021).

An efficient synthesis tetrazole and oxadiazole analogues of novel 2'-deoxy-C-nucleosides and their antitumor activity.

Various tetrazole and oxadiazole C-nucleoside analogues were synthesized starting from pure α- or ß-glycosyl-cyanide. The synthesis of glycosyl-cyanide as key precursor was optimized on gram-scale to furnish crystalline starting material for the assembly of C-nucleosides. Oxadizole C-nucleosides were synthesized via two independent routes. First,  the glycosyl-cyanide was converted into an amidoxime which upon ring closure offered an alternative pathway for the assembly of 1,2,4-oxadizoles in an efficient manner. Second, both anomers of glycosyl-cyanide were transformed into tetrazole nucleosides followed by acylative rearrangement to furnish 1,3,4-oxadiazoles in high yields. These protocols offer an easy access to otherwise difficult to synthesize C-nucleosides in good yield and protecting group compatibility. These C-nucleosides were evaluated for their antitumor activity. This work paves a path for facile assembly of library of new chemical entities useful for drug discovery.

Facile Solid-Phase Synthesis and Assessment of Nucleoside Analogs as Inhibitors of Bacterial UDP-Sugar Processing Enzymes.

The privileged uptake of nucleosides into cells has generated interest in the development of nucleoside-analog libraries for mining new inhibitors. Of particular interest are applications in the discovery of substrate mimetic inhibitors for the growing number of identified glycan-processing enzymes in bacterial pathogens. However, the high polarity and the need for appropriate protecting group strategies for nucleosides challenges the development of synthetic approaches. Here, we report an accessible, user-friendly synthesis that branches from a common solid phase-immobilized uridinyl-amine intermediate, which can be used as a starting point for diversity-oriented synthesis. We demonstrate the generation of five series of uridinyl nucleoside analogs for investigating inhibitor structure-activity relationships. This library was screened for inhibition of representative enzymes from three functional families including a phosphoglycosyl transferase, a UDP-aminosugar acetyltransferase, and a glycosyltransferase. These candidates were taken from the Gram-negative bacteria Campylobacter concisus and Campylobacter jejuni and the Gram-positive bacterium Clostridium difficile, respectively. Inhibition studies show that specific compound series preferentially inhibit selected enzymes, with IC50 values ranging from 35 ± 7 µM to 174 ± 21 µM. Insights from the screen provide a strong foundation for further structural elaboration, to improve potency, which will be enabled by the same synthetic strategy. The solid-phase strategy was also used to synthesize pseudouridine analogs of lead compounds. Finally, the compounds were found to be nontoxic to mammalian cells, further supporting the opportunities for future development.

Drugs in development for herpes simplex and varicella zoster virus.

The alpha-herpesviruses varicella zoster virus (VZV) and herpes simplex virus (HSV) share common features including lifelong persistence in sensory ganglia and the risk of recurrences. For both HSV and VZV, standard-of-care (SoC) is based on nucleoside analogs (NAs), which require specific activation in infected cells. These existing drugs exhibit substantial limitations, warranting the development of new and more effective drugs.

An initial assessment of correlations between host- and virus-related factors affecting analogues antiviral therapy in HBV chronically infected patients.

BACKGROUND: Success in treating hepatitis B virus (HBV) infection with nucleoside analogues drugs is limited by the emergence of drug-resistant viral strains upon prolonged therapy. In addition to mutation patterns in the viral polymerase gene, host factors are assumed to contribute to failure of treatment in chronic HBV infections. The aim of this study was to analyze the correlation between efficacy of antiviral therapy and the prevalence of HBV pretreatment drug-resistant variants. We also analyzed the role of heterogeneity in the promoter region of the IL-10 on the HBV pol/s gene polymorphisms and efficacy of analogues-driven therapy. MATERIAL AND METHODS: HBV DNA was extracted from 54 serum samples from chronic hepatitis B (CHB) patients. Drug-resistance mutations were analyzed using MALDI-TOF mass spectrometry technology (MALDI-TOF MS) and Multi-temperature single-strand conformation polymorphism (MSSCP). IL-10 gene promoter region polymorphisms at positions -1082, -819, and -592 were determined in allele-specific PCR reactions (AS-PCR). RESULTS: Drug-resistance mutations were detected in 74% of naïve and 93% of experienced patients, but the effect of pre-existence of drug-resistant HBV variants on antiviral therapy was not statistically significant (p=0.86). The role of polymorphisms at positions -1082 (p=0.88), -819 (p=0.26), and -592 (p=0.26) of IL-10 promoter region polymorphisms was excluded from the response-predicting factors. The main host factors predicting successful response to antiviral therapy were female sex (p=0.007) and young age (p=0.013). CONCLUSIONS: The presence of drug-resistant HBV variants in baseline is not a viral predictor of good response to nucleoside/nucleotide analogues therapy. Only low HBV viral load predicted positive response to antiviral therapy. The ideal candidate for antiviral therapy is an immunocompetent, young female with low HBV viral load and elevated ALT activity.

Recent developments in the total synthesis of fungicidal natural products--a crop protection perspective.

Natural products play an important role as lead structures for the identification of novel active ingredients in crop protection. As it is the case in the pharmaceutical industry, however, many crop protection companies have substantially decreased their in-house efforts in natural product exploration. Therefore, joint projects with academic research groups become more and more important in the field. This review describes several examples for successful collaborations in the field of fungicidal natural products.

Resistance of herpes simplex viruses to nucleoside analogues: mechanisms, prevalence, and management.

Herpes simplex viruses (HSV) type 1 and type 2 are responsible for recurrent orolabial and genital infections. The standard therapy for the management of HSV infections includes acyclovir (ACV) and penciclovir (PCV) with their respective prodrugs valacyclovir and famciclovir. These compounds are phosphorylated by the viral thymidine kinase (TK) and then by cellular kinases. The triphosphate forms selectively inhibit the viral DNA polymerase (DNA pol) activity. Drug-resistant HSV isolates are frequently recovered from immunocompromised patients but rarely found in immunocompetent subjects. The gold standard phenotypic method for evaluating the susceptibility of HSV isolates to antiviral drugs is the plaque reduction assay. Plaque autoradiography allows the associated phenotype to be distinguished (TK-wild-type, TK-negative, TK-low-producer, or TK-altered viruses or mixtures of wild-type and mutant viruses). Genotypic characterization of drug-resistant isolates can reveal mutations located in the viral TK and/or in the DNA pol genes. Recombinant HSV mutants can be generated to analyze the contribution of each specific mutation with regard to the drug resistance phenotype. Most ACV-resistant mutants exhibit some reduction in their capacity to establish latency and to reactivate, as well as in their degree of neurovirulence in animal models of HSV infection. For instance, TK-negative HSV mutants establish latency with a lower efficiency than wild-type strains and reactivate poorly. DNA pol HSV mutants exhibit different degrees of attenuation of neurovirulence. The management of ACV- or PCV-resistant HSV infections includes the use of the pyrophosphate analogue foscarnet and the nucleotide analogue cidofovir. There is a need to develop new antiherpetic compounds with different mechanisms of action.

Nucleoside analogues in the treatment of haematological malignancies.

The nucleoside analogues are a group of antimetabolite cytotoxics which generally have to be metabolised to the equivalent nucleotide before incorporation into DNA. Cytarabine is a well established component of the treatment of acute leukaemias and has its principal action on dividing cells. New formulations include a liposome encapsulated product for intrathecal use and oral cytarabine ocfosfate which may be suitable for long-term outpatient use. Pentostatin acts by causing accumulation of deoxynucleotides and, although active against hairy cell leukaemia, is associated with a poor tolerance profile. Cladribine and fludarabine have substantial activity in the treatment of chronic lymphocytic leukaemia (CLL) and low-grade non-Hodgkin's lymphoma (NHL). Fludarabine is the more thoroughly investigated of the two and is currently being developed in combination therapies for CLL and NHL and also in a combination with cytarabine for acute myeloid leukaemia. Fludarabine's immunosuppressive activity is being exploited in the conditioning of patients for non-myeloablative stem cell transplantation. Gemcitabine is an established agent in the treatment of a number of solid tumours but also has activity in haematological malignancies which might be exploited by the use of extended infusion schedules. Newer agents including nelarabine, clofarabine and troxacitabine are undergoing clinical evaluation and show promising activity.

Comprehensive mutant enzyme and viral variant assessment of human immunodeficiency virus type 1 reverse transcriptase resistance to nonnucleoside inhibitors.

The nonnucleoside reverse transcriptase (RT) inhibitors comprise a class of structurally diverse compounds that are functionally related and specific for the human immunodeficiency virus type 1 RT. Viral variants resistant to these compounds arise readily in cell culture and in treated, infected human. Therefore, the eventual clinical usefulness of the nonnucleoside inhibitors will rely on a thorough understanding of the genetic and biochemical bases for resistance. A study was performed to assess the effects of substitutions at each RT amino acid residue that influences the enzyme's susceptibility to the various nonnucleoside compounds. Single substitutions were introduced into both purified enzyme and virus. The resulting patterns of resistance were markedly distinct for each of the tested inhibitors. For instance, a > 50-fold loss of enzyme susceptibility to BI-RG-587 was engendered by any of four individual substitutions, while the same level of relative resistance to the pyridinone derivatives was mediated only by substitution at residue 181. Similarly, substitution at residue 181. Similarly, substitution at residue 106 had a noted effect on virus resistance to BI-RG-587 but not to the pyridinones. The opposite effect was mediated by a substitution at residue 179. Such knowledge of nonucleoside inhibitor resistance profiles may help in understanding the basis for resistant virus selection during clinical studies of these compounds.

Radiosensitization efficacy of KU-2285, RP-170 and etanidazole at low radiation doses: assessment by in vitro cytokinesis-block micronucleus assay.

Since the cytokinesis-block micronucleus assay is very sensitive at low radiation doses, we used it to investigate the in vitro sensitizing effects of two new hypoxic cell sensitizers (KU-2285, a fluorinated 2-nitroimidazole and RP-170, a 2-nitroimidazole nucleoside analogue) at 1-3 Gy in comparison with etanidazole. Exponentially growing EMT6 cells were treated with the drugs under aerobic or hypoxic conditions for 40 min prior to and during irradiation, after which the drugs were removed and cytochalasin B (2 micrograms/ml) was added to the medium. The number of micronuclei in binucleate cells was counted after 42 h of culture. Under aerobic conditions the three compounds at 5 mM had no sensitizing effect. Under hypoxic conditions the sensitizer enhancement ratio (SER) at 5 mM was 3.8 for KU-2285, 3.2 for RP-170, and 2.3 for etanidazole, while the oxygen enhancement ratio was 2.9. When the cells were pretreated under hypoxic conditions with drugs at 5 mM but then irradiated under aerobic conditions, KU-2285 and RP-170 had a sensitizing effect whereas etanidazole did not. The sensitizers were also tested at 0.5 and 1 mM, and the SER values were compared with those obtained at high doses (15-30 Gy) using a colony assay. The SER at low doses was higher than that at high doses for 1 and 5 mM KU-2285 and 5 mM RP-170, while the SERs were similar for all concentrations of etanidazole and the lower concentrations of KU-2285 and RP-170. These results might suggest the potential usefulness of KU-2285 and RP-170 in clinical radiotherapy.