Production of Modified Nucleosides in a Continuous Enzyme Membrane Reactor.

Nucleoside analogues are important compounds for the treatment of viral infections or cancers. While (chemo-)enzymatic synthesis is a valuable alternative to traditional chemical methods, the feasibility of such processes is lowered by the high production cost of the biocatalyst. As continuous enzyme membrane reactors (EMR) allow the use of biocatalysts until their full inactivation, they offer a valuable alternative to batch enzymatic reactions with freely dissolved enzymes. In EMRs, the enzymes are retained in the reactor by a suitable membrane. Immobilization on carrier materials, and the associated losses in enzyme activity, can thus be avoided. Therefore, we validated the applicability of EMRs for the synthesis of natural and dihalogenated nucleosides, using one-pot transglycosylation reactions. Over a period of 55 days, 2'-deoxyadenosine was produced continuously, with a product yield >90%. The dihalogenated nucleoside analogues 2,6-dichloropurine-2'-deoxyribonucleoside and 6-chloro-2-fluoro-2'-deoxyribonucleoside were also produced, with high conversion, but for shorter operation times, of 14 and 5.5 days, respectively. The EMR performed with specific productivities comparable to batch reactions. However, in the EMR, 220, 40, and 9 times more product per enzymatic unit was produced, for 2'-deoxyadenosine, 2,6-dichloropurine-2'-deoxyribonucleoside, and 6-chloro-2-fluoro-2'-deoxyribonucleoside, respectively. The application of the EMR using freely dissolved enzymes, facilitates a continuous process with integrated biocatalyst separation, which reduces the overall cost of the biocatalyst and enhances the downstream processing of nucleoside production.

A Comprehensive Mini-review on COVID-19 Pathogenesis on Perspectives of Cytokine Storm and Recent Developments in Anti-Covid Nucleotide Analogues

The world has been rocked by the 2019 coronavirus disease (COVID-19), which has significantly changed our way of life. Despite the unusual measures taken, COVID-19 still exists and affects people all over the world. A remarkable amount of study has been done to find ways to combat the infection's unsurpassed level. No ground-breaking antiviral agent has yet been introduced to remove COVID-19 and bring about a return to normalcy, even though numerous pharmaceuticals and therapeutic technologies have been reused and discovered. The cytokine storm phenomenon is of utmost importance since fatality is strongly connected with the severity of the disease. This severe inflammatory phenomenon marked by increased amounts of inflammatory mediators can be targeted for saving patients' life. Our analysis demonstrates that SARS-CoV-2 specifically generates a lot of interleukin-6 (IL-6) and results in lymphocyte exhaustion. Tocilizumab is an IL-6 inhibitor that is currently thought to be both generally safe and effective. Additionally, corticosteroids, tumor necrosis factor (TNF)-blockers and Janus kinase (JAK) inhibitors could be effective and dependable methods to reduce cytokine-mediated storm in SARS-CoV-2 patients.Copyright © The Author(s) 2023.

Molnupiravir for the treatment of COVID-19.

Molnupiravir (MK-4482, EIDD-2801) is a promising broad-spectrum experimental antiviral developed by Merck & Co. It is a nucleoside analogue prodrug that undergoes rapid conversion into nucleoside triphosphate (NTP) by intracellular metabolic processes. NTP inhibits viral polymerase by acting as an alternative substrate. Molnupiravir was initially developed to treat influenza and Venezuelan equine encephalitis virus (VEEV) infection as it exerts its antiviral activity by inhibiting RNA-dependent RNA polymerase (RdRp). Currently, it is being developed for the treatment of SARS-CoV-2 infection. Molnupiravir has demonstrated potent in vitro antiviral activity against positive-sense RNA viruses including influenza viruses, SARS-CoV, SARS-CoV-2 and MERS-CoV with low cytotoxicity and a high resistance barrier. Molnupiravir has been evaluated in phase I, II and III trials where it has demonstrated good efficacy, dose-dependent pharmacokinetics and a sound safety profile. In an interim analysis of a phase III study, treatment with molnupiravir reduced the risk of hospitalization or death by 50% in patients with COVID-19; in the final analysis, the reduction was 30%. On the basis of positive results in clinical trials, molnupiravir has been authorized for emergency use by the U.K. Medicines and Healthcare products Regulatory Agency (MHRA) and the U.S. Food and Drug Administration (FDA) in adults with mild to moderate COVID-19.

Synthesis of N-(2,4-dinitrophenyl) derivatives of D-ribosylamines; unexpected reaction and hydrolysis products.

Reaction of 2,3-O-isopropylidene-d-ribofuranosylamine with 2,4-dinitrofluorobenzene afforded the crystalline 2,3-O-isopropylidene-N-(2,4-dinitrophenyl)-ß-d-ribofuranosylamine (3) and a 1:1 crystalline complex of 2,3-O-isopropylidene-N-(2,4-dinitrophenyl-α-d-ribofuranosylamine and 2,3-O-isopropylidene-ß-d-ribofuranose; controlled acidic hydrolysis of 3 afforded N-(2,4-dinitrophenyl-α-d-ribopyranosylamine and not the expected ß-d-furanosylamine derivative. The structures of the new compounds were confirmed by NMR spectroscopy and X-ray crystallography.

A Nanopore Based Molnupiravir Sensor.

Nucleoside analogues are reagents that resemble the structure of natural nucleosides and are widely applied in antiviral and anticancer therapy. Molnupiravir, a recently reported nucleoside analogue drug, has shown its inhibitory effect against SARS-CoV-2. Rapid tracing of molnupiravir and its metabolites is important in the evaluation of its pharmacology effect, but direct sensing of molnupiravir as a single molecule has not been reported to date. Here, we demonstrate a nanopore-based sensor with which direct sensing of molnupiravir and its two major metabolites ß-d-N4-hydroxycytidine and its triphosphate can be achieved simultaneously. In conjunction with a custom machine learning algorithm, an accuracy of 92% was achieved. This sensing strategy may be useful in the current pandemic and is in principle suitable for other nucleoside analogue drugs.

Nucleosides and emerging viruses: A new story.

With several US Food and Drug Administration (FDA)-approved drugs and high barriers to resistance, nucleoside and nucleotide analogs remain the cornerstone of antiviral therapies for not only herpesviruses, but also HIV and hepatitis viruses (B and C); however, with the exception of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which vaccines have been developed at unprecedented speed, there are no vaccines or small antivirals yet available for (re)emerging viruses, which are primarily RNA viruses. Thus, herein, we present an overview of ribonucleoside analogs recently developed and acting as inhibitors of the viral RNA-dependent RNA polymerase (RdRp). They are new lead structures that will be exploited for the discovery of new antiviral nucleosides.

Semi-Automated High-Throughput Substrate Screening Assay for Nucleoside Kinases.

Nucleoside kinases (NKs) are key enzymes involved in the in vivo phosphorylation of nucleoside analogues used as drugs to treat cancer or viral infections. Having different specificities, the characterization of NKs is essential for drug design and nucleotide analogue production in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening method for NKs is of great importance. Here, we report on the validation of a well-known luciferase-based assay for the detection of NK activity in a 96-well plate format. The assay was semi-automated using a liquid handling robot. Good linearity was demonstrated (r² > 0.98) in the range of 0-500 µM ATP, and it was shown that alternative phosphate donors like dATP or CTP were also accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplarily used for the comparison of the substrate spectra of four NKs using 20 (8 natural, 12 modified) substrates. The screening results correlated well with literature data, and additionally, previously unknown substrates were identified for three of the NKs studied. Our results demonstrate that the developed semi-automated high-throughput assay is suitable to identify best performing NKs for a wide range of substrates.

Current treatment strategies for COVID­19 (Review).

The spread of the novel severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) emerged suddenly at the end of 2019 and the disease came to be known as coronavirus disease 2019 (COVID­19). To date, there is no specific therapy established to treat COVID­19. Identifying effective treatments is urgently required to treat patients and stop the transmission of SARS­CoV­2 in humans. For the present review, >100 publications on therapeutic agents for COVID­19, including in vitro and in vivo animal studies, case reports, retrospective analyses and meta­analyses were retrieved from PubMed and analyzed, and promising therapeutic agents that may be used to combat SARS­CoV­2 infection were highlighted. Since the outbreak of COVID­19, 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 COVID­19 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 COVID­19. The molecular mechanisms of these therapeutic agents against SARS­CoV2 have been investigated, including inhibition of viral interactions with angiotensin­converting enzyme 2 receptors in human cells, viral RNA­dependent 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 COVID­19 were acknowledged.

Remdesivir triphosphate blocks DNA synthesis and increases exonucleolysis by the replicative mitochondrial DNA polymerase, Pol γ.

The COVID-19 pandemic prompted the FDA to authorize a new nucleoside analogue, remdesivir, for emergency use in affected individuals. We examined the effects of its active metabolite, remdesivir triphosphate (RTP), on the activity of the replicative mitochondrial DNA polymerase, Pol γ. We found that while RTP is not incorporated by Pol γ into a nascent DNA strand, it remains associated with the enzyme impeding its synthetic activity and stimulating exonucleolysis. In spite of that, we found no evidence for deleterious effects of remdesivir treatment on the integrity of the mitochondrial genome in human cells in culture.

Therapeutic approaches against coronaviruses acute respiratory syndrome.

Coronaviruses represent global health threat. In this century, they have already caused two epidemics and one serious pandemic. Although, at present, there are no approved drugs and therapies for the treatment and prevention of human coronaviruses, several agents, FDA-approved, and preclinical, have shown in vitro and/or in vivo antiviral activity. An in-depth analysis of the current situation leads to the identification of several potential drugs that could have an impact on the fight against coronaviruses infections. In this review, we discuss the virology of human coronaviruses highlighting the main biological targets and summarize the current state-of-the-art of possible therapeutic options to inhibit coronaviruses infections. We mostly focus on FDA-approved and preclinical drugs targeting viral conserved elements.

Marine Sponge is a Promising Natural Source of Anti-SARS-CoV-2 Scaffold.

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.

Broad-Spectrum Antiviral Strategies and Nucleoside Analogues.

The emergence or re-emergence of viruses with epidemic and/or pandemic potential, such as Ebola, Zika, Middle East Respiratory Syndrome (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus 1 and 2 (SARS and SARS-CoV-2) viruses, or new strains of influenza represents significant human health threats due to the absence of available treatments. Vaccines represent a key answer to control these viruses. However, in the case of a public health emergency, vaccine development, safety, and partial efficacy concerns may hinder their prompt deployment. Thus, developing broad-spectrum antiviral molecules for a fast response is essential to face an outbreak crisis as well as for bioweapon countermeasures. So far, broad-spectrum antivirals include two main categories: the family of drugs targeting the host-cell machinery essential for virus infection and replication, and the family of drugs directly targeting viruses. Among the molecules directly targeting viruses, nucleoside analogues form an essential class of broad-spectrum antiviral drugs. In this review, we will discuss the interest for broad-spectrum antiviral strategies and their limitations, with an emphasis on virus-targeted, broad-spectrum, antiviral nucleoside analogues and their mechanisms of action.

Identification of FDA approved drugs and nucleoside analogues as potential SARS-CoV-2 A1pp domain inhibitor: An in silico study.

Coronaviruses are known to infect respiratory tract and intestine. These viruses possess highly conserved viral macro domain A1pp having adenosine diphosphate (ADP)-ribose binding and phosphatase activity sites. A1pp inhibits adenosine diphosphate (ADP)-ribosylation in the host and promotes viral infection and pathogenesis. We performed in silico screening of FDA approved drugs and nucleoside analogue library against the recently reported crystal structure of SARS-CoV-2 A1pp domain. Docking scores and interaction profile analyses exhibited strong binding affinity of eleven FDA approved drugs and five nucleoside analogues NA1 (-13.84), nadide (-13.65), citicholine (-13.54), NA2 (-12.42), and NA3 (-12.27). The lead compound NA1 exhibited significant hydrogen bonding and hydrophobic interaction at the natural substrate binding site. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface (SASA), hydrogen bond formation, principle component analysis, and free energy landscape calculations for NA1 bound protein displayed stable complex formation in 100 ns molecular dynamics simulation, compared to unbound macro domain and natural substrate adenosine-5-diphosphoribose bound macro domain that served as a positive control. The molecular mechanics Poisson-Boltzmann surface area analysis of NA1 demonstrated binding free energy of -175.978 ± 0.401 kJ/mol in comparison to natural substrate which had binding free energy of -133.403 ± 14.103 kJ/mol. In silico analysis by modelling tool ADMET and prediction of biological activity of these compounds further validated them as putative therapeutic molecules against SARS-CoV-2. Taken together, this study offers NA1 as a lead SARS-CoV-2 A1pp domain inhibitor for future testing and development as therapeutics against human coronavirus.

Tandem mass spectrometry of small-molecule antiviral drugs: 3. antiviral agents against herpes, influenza and other viral infections.

For the treatment of various viral infections, antiviral drugs may be used. Liquid chromatography-mass spectrometry (LC-MS) with tandem mass spectrometry (MS-MS) operated in selected-reaction monitoring (SRM) mode is the method of choice in quantitative bioanalysis of drugs, e.g., to establish bioavailability, to study pharmacokinetics, and later on possibly for therapeutic drug monitoring. In this study, the fragmentation in MS-MS of small-molecule antiviral drugs against herpes and influenza viruses is reviewed. In this way, insight is gained on the identity of the product ions used in SRM. Fragmentation schemes of antiviral agents are also relevant in the identification of drug metabolites or (forced) degradation products. As information of the fragmentation of antiviral drugs in MS-MS and the identity of the product ions is very much scattered in the scientific literature, it was decided to collect this information and to review it. In this third study, attention is paid to small-molecule antiviral agents used against herpes and influenza virus infections. In addition, some attention is paid to broad-spectrum antiviral agents, that are investigated with respect to their efficacy in challenging virus infections of this century, e.g., involving Ebola, Zika and corona viruses, like SARS-CoV-2, which is causing a world-wide pandemic at this very moment. The review provides fragmentation schemes of ca. 35 antiviral agents. The identity of the product ions used in SRM, i.e., elemental composition and exact-m/z, is tabulated, and more detailed fragmentation schemes are provided.