[Interaction of DNA Methyltransferase Dnmt3a with Phosphorus Analogs of S-Adenosylmethionine and S-Adenosylhomocysteine].

Enzymatic methyltransferase reactions are of crucial importance for cell metabolism. S-Adenosyl-L-methionine (AdoMet) is a main donor of the methyl group. DNA, RNA, proteins, and low-molecular-weight compounds are substrates of methyltransferases. In mammals, DNA methyltransferase Dnmt3a de novo methylates the C5 position of cytosine residues in CpG sequences in DNA. The methylation pattern is one of the factors that determine the epigenetic regulation of gene expression. Here, interactions with the catalytic domain of Dnmt3a was for the first time studied for phosphonous and phosphonic analogs of AdoMet and S-adenosyl-L-homocysteine (AdoHcy), in which the carboxyl group was substituted for respective phosphorus-containing group. These AdoMet analogs were shown to be substrates of Dnmt3a, and the methylation efficiency was only halved as compared with that of natural AdoMet. Both phosphorus-containing analogs of AdoHcy, which is a natural methyltransferase inhibitor, showed similar inhibitory activities toward Dnmt3a and were approximately four times less active than AdoHcy. The finding that the phosphonous and phosphonic analogs are similar in activity was quite unexpected because the geometry and charge of their phosphorus-containing groups differ substantially. The phosphorus-containing analogs of AdoMet and AdoHcy are discussed as promising tools for investigation of methyltransferases.

Synthesis of 1-[ω-(Bromophenoxy)alkyl]-3-naphthalenylmethyl Derivatives of Uracil and Their Analogues As Probable Inhibitors of Human Cytomegalovirus Replication.

The synthesis of a new series of 1-[ω-(bromophenoxy)alkyl]-uracil derivatives containing in position 3 naphthalen-1-yl-, naphthalen-2-yl-, 1-bromonaphthalen-2-ylmethyl, benzyl, and anthracene 9-methyl fragment was carried out. The antiviral properties of the synthesized compounds were studied against human cytomegalovirus. It was found that the compound that contained a bridge of five methylene groups has a high anti-cytomegalovirus activity in vitro.

[How Histone Deacetylase 3 Controls Hepcidin Expression and Hepatitis C Virus Replication].

The key role of histone deacetylases (HDAC) in the regulation of the cellular response to infection with the hepatitis C virus (HCV) was first demonstrated in 2008. When studying the metabolism of iron in the liver tissues of patients with chronic hepatitis C, the authors found that the expression of the hepcidin gene (HAMP), a hormone regulator of iron export, is markedly reduced in hepatocytes under conditions of oxidative stress caused by viral infection. HDAC were involved in the regulation of hepcidin expression through the control of acetylation level of histones and transcription factors, primarily STAT3, associated with the HAMP promoter. The purpose of this review was to summarize current data on the functioning of the HCV-HDAC3-STAT3-HAMP regulatory circuit as an example of a well-characterized interaction between the virus and the epigenetic apparatus of the host cell.

New Analogues of Uridine as Possible Anti-Viral Agents Specific to SARS-CoV-2.

The development of specific drugs against SARS-CoV-2 infection is a major challenge facing global science and healthcare. Despite numerous attempts, there are still no truly effective drugs. Currently, the main approach in the creation of drugs against COVID-19 is repurposing, i.e., re-profiling existing drugs approved for medical use, for example, the use of a drug for the treatment of Ebola-Remdesivir, and the use of a drug for the treatment of influenza-Favipiravir. However, it is already obvious that these drugs are not specific enough nor effective enough. Another promising approach is the creation of new molecules, but it should be noted immediately that implementation requires much more time and costs. However, the search for new SARS-CoV-2 specific antiviral agents continues. The aim of our work was the creation of new 5-substituted uridine derivatives as potential inhibitors of coronavirus RNA-dependent RNA polymerase. The substances were obtained in high yields by the Suzuki‒Miyaura reaction and characterized using modern physicochemical methods. However, testing of their antiviral activity against SARS-CoV-2 did not reveal a significant inhibitory effect.

[New Analogues of Uridine as Possible Anti-Viral Agents Specific to SARS-CoV-2].

The development of specific drugs against SARS-CoV-2 infection is a major challenge facing global science and healthcare. Despite numerous attempts, there are still no truly effective drugs. Currently, the main approach in the creation of drugs against COVID-19 is repurposing, i.e., re-profiling existing drugs approved for medical use, for example, the use of a drug for the treatment of Ebola-Remdesivir, and the use of a drug for the treatment of influenza-Favipiravir. However, it is already obvious that these drugs are not specific enough nor effective enough. Another promising approach is the creation of new molecules, but it should be noted immediately that implementation requires much more time and costs. However, the search for new SARS-CoV-2 specific antiviral agents continues. The aim of our work was the creation of new 5-substituted uridine derivatives as potential inhibitors of coronavirus RNA-dependent RNA polymerase. The substances were obtained in high yields by the Suzuki-Miyaura reaction and characterized using modern physicochemical methods. However, testing of their antiviral activity against SARS-CoV-2 did not reveal a significant inhibitory effect.

Synthesis of 1-[ω-(Bromophenoxy)alkyl]-3-Naphthalenylmethyl Uracil Derivatives and Their Analogues as Probable Inhibitors of Human Cytomegalovirus Replication.

A new series of 1-[ω-(bromophenoxy)alkyl]-uracil derivatives containing naphthalen-1-yl, naphthalen-2-yl, 1-bromonaphthalen-2-ylmethyl, benzyl, and anthracene-9-ylmethyl fragments in position 3 of uracil residue was synthesized. The antiviral properties of the synthesized compounds against human cytomegalovirus were studied. It was found that the compound containing a bridge consisting of five methylene groups exhibits a high anti-cytomegalovirus activity in vitro.

[Glycol and Phosphate Depot Forms of 4- and/or 5-Modified Nucleosides Exhibiting Antibacterial Activity].

Resistance developed to the majority of drugs used to treat infectious diseases warrants the design of new compounds effective against drug-resistant strains of pathogens. Recently, several groups of modified nucleosides have been synthesized and showed significant antibacterial activity in vitro, but their further studies were difficult to undertake because of their low solubility in aqueous solutions. Nevertheless, new compounds, well soluble in water-organic solutions, were synthesized and found to be more effective in inhibiting the growth of Gram-positive bacteria and mycobacteria. The water-soluble forms of modified nucleosides under study were assumed to be their depot forms. To check the assumption, the compounds were tested for hydrolysis in various media and their molecular docking was performed into the active center of the putative target, Mycobacterium tuberculosis flavin-dependent thymidylate synthase ThyX. Computer modelling showed that the water-soluble analogs do not act as ThyX inhibitors, supporting the assumption of their depot nature. The compounds were resistant to chemical hydrolysis but were hydrolyzed when incubated with porcine liver carboxylesterase, human serum, or Staphylococcus aureus 209P. The results demonstrate that the compounds are most likely depot forms of modified nucleosides.

Evaluation of the Antiviral Potential of Modified Heterocyclic Base and 5'-Norcarbocyclic Nucleoside Analogs Against SARS-CoV-2.

The pandemic caused by the novel betacoronavirus SARS-CoV-2 has already claimed more than 3.5 million lives. Despite the development and use of anti-COVID-19 vaccines, the disease remains a major public health challenge throughout the world. Large-scale screening of the drugs already approved for the treatment of other viral, bacterial, and parasitic infections, as well as autoimmune, oncological, and other diseases is currently underway as part of their repurposing for development of effective therapeutic agents against SARS-CoV-2. In this work, we present the results of a phenotypic screening of libraries of modified heterocyclic bases and 5'-norcarbocyclic nucleoside analogs previously synthesized by us. We identified two leading compounds with apparent potential to inhibit SARS-CoV-2 replication and EC50 values in a range of 20-70 µM. The structures of these compounds can be further optimized to develop an antiviral drug.

Synthesis and Antiviral Properties of 1-Substituted 3-[ω-(4-Oxoquinazolin-4(3H)-yl)alkyl]uracil Derivatives.

A series of uracil derivatives containing a 4-oxoquinazoline fragment bound to the nitrogen atom N3 of the pyrimidine ring by a short methylene bridge was synthesized to search for new antiviral agents. Some compounds in this series are shown to exhibit high inhibitory activity against human cytomegalovirus and the varicella zoster virus in a HEL cell culture.

Identification of a Novel Substrate-Derived Spermine Oxidase Inhibitor.

Homeostasis of the biogenic polyamines spermine (Spm) and spermidine (Spd), present in µM-mM concentrations in all eukaryotic cells, is precisely regulated by coordinated activities of the enzymes of polyamine synthesis, degradation, and transport, in order to sustain normal cell growth and viability. Spermine oxidase (SMOX) is the key and most recently discovered enzyme of polyamine metabolism that plays an essential role in regulating polyamine homeostasis by catalyzing the back-conversion of Spm to Spd. The development of many types of epithelial cancer is associated with inflammation, and disease-related inflammatory stimuli induce SMOX. MDL72527 is widely used in vitro and in vivo as an irreversible inhibitor of SMOX, but it is also potent towards N1-acetylpolyamine oxidase. Although SMOX has high substrate specificity, Spm analogues have not been systematically studied as enzyme inhibitors. Here we demonstrate that 1,12-diamino-2,11-bis(methylidene)-4,9-diazadodecane (2,11-Met2-Spm) has, under standard assay conditions, an IC50 value of 169 µM towards SMOX and is an interesting instrument and lead compound for studying polyamine catabolism.

[Low-molecular-weight regulators of biogenic polyamine metabolism affect cytokine production and expression of hepatitis С virus proteins in Huh7.5 human hepatocarcinoma cells].

Hepatitis C virus (HCV) induces the expression of the genes of proinflammatory cytokines, the excessive production of which may cause cell death, and contribute to development of liver fibrosis and hepatocarcinoma. The relationship between cytokine production and metabolic disorders in HCV-infected cells remains obscure. The levels of biogenic polyamines, spermine, spermidine, and their precursor putrescine, may be a potential regulator of these processes. The purpose of the present work was to study the effects of the compounds which modulate biogenic polyamines metabolism on cytokine production and HCV proteins expression. Human hepatocarcinoma Huh7.5 cells have been transfected with the plasmids that encode HCV proteins and further incubated with the following low-molecular compounds that affect different stages of polyamine metabolism: (1) difluoromethylornithine (DFMO), the inhibitor of ornithine decarboxylase, the enzyme that catalyzes the biosynthesis of polyamines; (2) N,N'-bis(2,3-butane dienyl)-1,4-diaminobutane (MDL72.527), the inhibitor of proteins involved in polyamine degradation; and (3) synthetic polyamine analog N^(I),N^(II)-diethylnorspermine (DENSpm), an inducer of polyamine degradation enzyme. The intracellular accumulation and secretion of cytokines (IL-6, IL-1ß, TNF-α, and TGF-ß) was assessed by immunocytochemistry and in the immunoenzyme assay, while the cytokine gene expression was studied using reverse transcription and PCR. The effects of the compounds under analysis on the expression of HCV proteins were analyzed using the indirect immunofluorescence with anti-HCV monoclonal antibodies. It has been demonstrated that, in cells transfected with HCV genes, DFMO reduces the production of three out of four tested cytokines, namely, TNF-α and TGF-ß in cells that express HCV core, Е1Е2, NS3, NS5A, and NS5B proteins, and IL-1ß in the cells that express HCV core, Е1Е2, and NS3 proteins. MDL72527 and DENSpm decreased cytokine production to a lesser extent. Incubation with DFMO led to a 28-32% decrease in the number of cells expressing NS5B or NS5A, both of which are key components of the HCV replication complex. The results obtained in the work indicate that a further detailed study of the antiviral activity of DFMO is required in order to assess its potential as an anti-hepatitis C therapeutic agent.

Novel HIV-1 Non-nucleoside Reverse Transcriptase Inhibitors: A Combinatorial Approach.

Highly active antiretroviral therapy (HAART) is one of the most effective means for fighting against HIV-infection. HAART primarily targets HIV-1 reverse transcriptase (RT), and 14 of 28 compounds approved by the FDA as anti-HIV drugs act on this enzyme. HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) hold a special place among HIV RT inhibitors owing to their high specificity and unique mode of action. Nonetheless, these drugs show a tendency to decrease their efficacy due to high HIV-1 variability and formation of resistant virus strains tolerant to clinically applied HIV NNRTIs. A combinatorial approach based on varying substituents within various fragments of the parent molecule that results in development of highly potent compounds is one of the approaches aimed at designing novel HIV NNRTIs. Generation of HIV NNRTIs based on pyrimidine derivatives explicitly exemplifies this approach, which is discussed in this review.

Analysis of the Domains of Hepatitis C Virus Core and NS5A Proteins that Activate the Nrf2/ARE Cascade.

The hepatitis C virus (HCV) triggers a chronic disease that is often accompanied by a spectrum of liver pathologies and metabolic alterations. The oxidative stress that occurs in the infected cells is considered as one of the mechanisms of HCV pathogenesis. It is induced by the viral core and NS5A proteins. It is already known that both of these proteins activate the antioxidant defense system controlled by the Nrf2 transcription factor. Here, we show that this activation is mediated by domain 1 of the NS5A protein and two fragments of the core protein. In both cases, this activation is achieved through two mechanisms. One of them is mediated by reactive oxygen species (ROS) and protein kinase C, whereas the other is triggered through ROS-independent activation of casein kinase 2 and phosphoinositide 3-kinase. In the case of the HCV core, the ROS-dependent mechanism was assigned to the 37-191 a.a. fragment, while the ROS-independent mechanism was assigned to the 1-36 a.a. fragment. Such assignment of the mechanisms to different domains is the first evidence of their independence. In addition, our data revealed that intracellular localization of HCV proteins has no impact on the regulation of the antioxidant defense system.

Data on synthesis of methylene bisphosphonates and screening of their inhibitory activity towards HIV reverse transcriptase.

Inorganic pyrophosphate (PPi) mimetics designed on a basis of methylenediphosphonic acid backbone are promising inhibitors of two key HIV replication enzymes, IN [1] and RT [2]. Herein, we present chemical synthesis of eleven methylenebisphosphonates (BPs) with their NMR and HRMS analysis synthesized via five different ways. Also, we present data on inhibition of HIV RT catalyzed phosphorolysis and polymerization by synthesized BPs using two methods based on denaturing urea PAGE. Tests were also performed for thymidine analogue mutations reverse transcriptase (TAM RT), which was expressed and purified for that. Structure-activity relationships and inhibitory activity data of synthesized BPs are presented in "Methylene bisphosphonates as the inhibitors of HIV RT phosphorolytic activity" [2].

[Effect of Hepatitis C virus proteins on the production of proinflammatory and profibrotic cytokines in Huh7.5 human hepatoma cells].

Hepatitis C virus (HCV) is a widespread dangerous human pathogen. Up to 80% of HCV-infected individuals develop chronic infection, which is often accompanied by liver inflammation and fibrosis and, at terminal stages, liver cirrhosis and cancer. Treatment of patients with end-stage liver disease is often ineffective, and even patients with suppressed HCV replication have higher risk of death as compared with noninfected subjects. Therefore, investigating the mechanisms that underlie HCV pathogenesis and developing treatments for virus-associated liver dysfunction remain an important goal. The effect of individual HCV proteins on the production of proinflammatory and profibrotic cytokines in hepatocellular carcinoma Huh7.5 cells was analyzed in a systematic manner. Cells were transfected with plasmids encoding HCV proteins. Cytokine production and secretion was accessed by immunocytochemistry and ELISA of the culture medium, and transcription of the cytokine genes was assessed using reverse transcription and PCR. HCV proteins proved to differ in effect on cytokine production. Downregulation of interleukin 6 (IL-6) production was observed in cells expressing the HCV core, NS3, and NS5A proteins. Production of transforming growth factor ß1 (TGF-ß1) was lower in cells expressing the core proteins, NS3, or E1/E2 glycoproteins. A pronounced increase in production and secretion of tumor necrosis factor α (TNF-α) was observed in response to expression of the HCV E1/E2 glycoproteins. A higher biosynthesis, but a lower level in the cell culture medium, was detected for interleukin 1ß (IL-1ß) in cells harboring NS4 and IL-6 in cells expressing NS5В. The finding was possibly explained by protein-specific retention and consequent accumulation of the respective cytokines in the cell.

Methylene bisphosphonates as the inhibitors of HIV RT phosphorolytic activity.

The structure-function analysis of 36 methylenebisphosphonates (BPs) as inhibitors of the phosphorolytic activity of native and drug-resistant forms of HIV-1 reverse transcriptase (RT) was performed. It was shown that with the increase of the inhibitory potential of BPs towards the phosphorolytic activity raises their ability to inhibit the RT-catalyzed DNA elongation. Herein, we report the impact of the thymidine analog mutations (TAM) on the activity of bisphosphonates, as well as some structural features of the BPs, allowing them to maintain the inhibitory activity on the enzyme resistant to nucleoside analog therapy. We estimated the Mg(2+)-coordinating group structure, the linker and the aromatic pharmacophore influence on the inhibitory potential of the BPs. Based on the 31 BPs SAR, several BPs with improved inhibitory properties were designed and synthesized.

Study of Antiherpetic Efficiency of Phosphite of Acycloguanosine Ableto Over come the Barrier of Resistance to Acyclovir.

As has been shown previously, phosphite of acycloguanosine (Hp-ACG) exhibits equal efficacy against ACV-sensitive and ACV-resistant HSV-1 strains in cell culture. Intraperitoneal administration of Hp-ACG to model mice with herpetic encephalitis caused by HSV-1 infection was shown to be effective in protecting against death. In the present work, we continue the study of the antiviral efficiency of Hp-ACG against HSV administered non-invasively; namely in vivo, orally and in the form of ointment formulations. It has been first shown that oral administration of Hp-ACG twice daily for five days prevents systemic infection in mice caused by HSV-1. Mortality in the control group of animals was 57%. Administration of Hp-ACG at doses of 600, 800 and 1,000 mg/kg per day significantly increased the survival and median day of death of the animals compared to the placebo-treated control group. A comparative evaluation of the therapeutic efficacy parameters of polyethylene glycol-based ACV ointment and Hp-ACG ointment was carried out after a 5-day course in the model of an experimental cutaneous infection of HSV-1 in guinea pigs. It was found that Hp-ACG has a significant therapeutic effect resulting in a statistically significant reduction in the lesion's surface area and the amount of vesicular structures. The exhibited therapeutic effect of 10% Hp-ACG in ointment form compares well with that of 5% ACG ointment.

Therapy of HIV Infection: Current Approaches and Prospects.

The human immunodeficiency virus type 1 (HIV-1) is the causative agent of one of the most dangerous human diseases - the acquired immune deficiency syndrome (AIDS). Over the past 30 years since the discovery of HIV-1, a number of antiviral drugs have been developed to suppress various stages of the HIV-1 life cycle. This approach has enables the suppression of virus replication in the body, which significantly prolongs the life of HIV patients. The main downside of the method is the development of viral resistance to many anti-HIV drugs, which requires the creation of new drugs effective against drug-resistant viral forms. Currently, several fundamentally new approaches to HIV-1 treatment are under development, including the use of neutralizing antibodies, genome editing, and blocking an integrated latent provirus. This review describes a traditional approach involving HIV-1 inhibitors as well as the prospects of other treatment options.

Hepatitis C virus: The role of N-glycosylation sites of viral genotype 1b proteins for formation of viral particles in insect and mammalian cells.

Hepatitis C virus (HCV) is characterized by considerable genetic variability and, as a consequence, it has 6 genotypes and multitude of subtypes. HCV envelope glycoproteins are involved in the virion formation; the correct folding of these proteins plays the key role in virus infectivity. Glycosylation at certain sites of different genotypes HCV glycoproteins shows substantial differences in functions of the individual glycans (Goffard et al., 2005; Helle et al., 2010) [1], [2]. In this study, differential glycosylation sites of HCV genotype 1b envelope proteins in insect and mammalian cells was demonstrated. We showed that part of glycosylation sites was important for folding of the proteins involved in the formation of viral particles. Point mutations were introduced in the protein N-glycosylation sites of HCV (genotype 1b) and the mutant proteins were analyzed using baculovirus expression system in mammalian and insect cells. Our data showed that, in contrast to HCV 1a and 2a, the folding of HCV 1b envelope proteins E2 (sites N1, N2, N10) and E1 (sites N1, N5) was disrupted, however that did not prevent the formation of virus-like particles (VLP) with misfolded glycoproteins having densities typical for HCV particles containing RNA fragments. Experimental data are supported by mathematical modeling of the structure of E1 mutant variants.

5-Arylaminouracil Derivatives as Potential Dual-Action Agents.

Several 5-aminouracil derivatives that have previously been shown to inhibit Mycobacterium tuberculosis growth at concentrations of 5-40 µg/mL are demonstrated to act also as noncompetitive non-nucleoside inhibitors of HIV-1 reverse transcriptase without causing toxicity in vitro (MT-4 cells) and ex vivo (human tonsillar tissue).