HIV-1 integrase inhibition by modified oligonucleotides: optimization of the inhibitor structure

Integration of human immunodeficiency virus type 1 DNA into the infected cell genome is one of the key steps of the viral replication cycle. Therefore viral enzyme integrase, which realizes the integration, is of interest as a target for new antiviral drugs. Conjugates of 11-mer single stranded oligonucleotides with hydrophobic molecules are shown to be efficient integrase inhibitors since they induce dissociation of the integrase-viral DNA complex. The effect of the oligonucleotide length and structure as well as the structure of hydrophobic molecules on the conjugate inhibitory activity has been studied. Conjugates with eosin and oleic acid are shown to be the most active. Conjugates of these molecules with 2'-O-methyl-oligonucleotide inhibit integrase at 50-100 nM and have no influence on a number of other DNA-binding enzymes.     

Antisense oligonucleotides delivered to the lysosome escape and actively inhibit the hepatitis B virus

The subcellular fate and activity in inhibiting the hepatitis B virus of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-phosphorothioate oligonucleotides were studied. Their internalization and subcellular fate were monitored with confocal microscopy. A fraction of the internalized free oligonucleotides escaped into the cytoplasm and nucleus of Hep G2 cells but were not active antiviral agents. Covalently attaching the oligonucleotides to the HPMA copolymers via nondegradable dipeptide GG spacers resulted in sequestering the oligonucleotides in vesicles after internalization. Conjugation of the oligonucleotides to an HPMA copolymer via a lysosomally cleavable tetrapeptide GFLG spacer resulted in release of the oligonucleotide in the lysosome and subsequent translocation into the cytoplasm and nucleus of the cells. The HPMA copolymer-oligonucleotide conjugate possessed antiviral activity, indicating that phosphorothioate oligonucleotides released from the carrier in the lysosome were able to escape into the cytoplasm and nucleus and remain active. The Hep G2 cells appeared to actively internalize the phosphorothioate oligonucleotides as oligonucleotide-HPMA copolymer conjugates were internalized to a greater extent than unconjugated polymers.     

Approaches to antiviral drug development

At present, only a few drugs have been approved by the FDA for therapy of viral infections in humans. There is a great need for antiviral drugs with increased potency and decreased toxicity, as well as drugs to treat viral diseases for which no drug or vaccine is currently available. Two approaches for development of antiviral drugs are described--an empirical strategy and a rational strategy--with several examples of each. Although many compounds have potent antiviral activity in cell culture, only a small fraction of these will go on to become antiviral drugs for use in humans. At this time, only seven synthetic compounds and alpha interferon have been approved by the FDA for therapy of viral infections in humans. None of these approved drugs are without toxicities, however, and hence there is a great need for antiviral drugs with increased potency and decreased toxicity, as well as for drugs to treat viral diseases for which no drug or vaccine is currently available. Two approaches for the development of antiviral drugs--the empirical and the rational strategies--and their applications and future directions are discussed.     

COVID-19: Pathogenesis,cytokine storm and therapeutic potential of interferons

The outbreak of the novel SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) responsible for coronavirus disease 2019 (COVID-19) has developed into an unprecedented global pandemic. Clinical investigations in patients with COVID-19 has shown a strong upregulation of cytokine and interferon production in SARS-CoV2- induced pneumonia, with an associated cytokine storm syndrome. Thus, the identification of existing approved therapies with proven safety profiles to treat hyperinflammation is a critical unmet need in order to reduce COVI-19 associated mortality. To date, no specific therapeutic drugs or vaccines are available to treat COVID-19 patients. This review evaluates several options that have been proposed to control SARS-CoV2 hyperinflammation and cytokine storm, eincluding antiviral drugs, vaccines, small-molecules, monoclonal antibodies, oligonucleotides, peptides, and interferons (IFNs).     

Impact of delivery method on antiviral activity of phosphodiester,phosphorothioate, and phosphoryl guanidine oligonucleotides in MDCK cells infected with H5N1 bird flu virus

We have previously described nanocomposites containing conjugates or complexes of native oligodeoxyribonucleotides with poly-L-lysine and TiO₂ nanoparticles. We have shown that these nanocomposites efficiently suppressed influenza A virus reproduction in MDCK cells. Here, we have synthesized previously undescribed nanocomposites that consist of TiO₂ nanoparticles and polylysine conjugates with oligonucleotides that contain phosphoryl guanidine or phosphorothioate internucleotide groups. These nanocomposites have been shown to exhibit antiviral activity in MDCK cells infected with H5N1 influenza A virus. The nanocomposites containing phosphorothioate oligonucleotides inhibited virus replication ~130-fold. More potent inhibition, i.e., ~5000-fold or ~4600-fold, has been demonstrated by nanocomposites that contain phosphoryl guanidine or phosphodiester oligonucleotides, respectively. Free oligonucleotides have been nearly inactive. The antiviral activity of oligonucleotides of all three types, when delivered by Lipofectamine, has been significantly lower compared to the oligonucleotides delivered in the nanocomposites. In the former case, the phosphoryl guanidine oligonucleotide has appeared to be the most efficient; it has inhibited the virus replication by a factor of 400. The results make it possible to consider phosphoryl guanidine oligonucleotides, along with other oligonucleotide derivatives, as potential antiviral agents against H5N1 avian flu virus.     

Pharmacological cyclin-dependent kinase inhibitors inhibit replication of wild-type and drug-resistant strains of herpes simplex virus and human immunodeficiency virus type 1 by targeting cellular,not viral,proteins

Pharmacological cyclin-dependent kinase (cdk) inhibitors (PCIs) block replication of several viruses, including herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus type 1 (HIV-1). Yet, these antiviral effects could result from inhibition of either cellular cdks or viral enzymes. For example, in addition to cellular cdks, PCIs could inhibit any of the herpesvirus-encoded kinases, DNA replication proteins, or proteins involved in nucleotide metabolism. To address this issue, we asked whether purine-derived PCIs (P-PCIs) inhibit HSV and HIV-1 replication by targeting cellular or viral proteins. P-PCIs inhibited replication of HSV-1 and -2 and HIV-1, which require cellular cdks to replicate, but not vaccinia virus or lymphocytic choriomeningitis virus, which are not known to require cdks to replicate. P-PCIs also inhibited strains of HSV-1 and HIV-1 that are resistant to conventional antiviral drugs, which target viral proteins. In addition, the anti-HSV effects of P-PCIs and a conventional antiherpesvirus drug, acyclovir, were additive, demonstrating that the two drugs act by distinct mechanisms. Lastly, the spectrum of proteins that bound to P-PCIs in extracts of mock- and HSV-infected cells was the same. Based on these observations, we conclude that P-PCIs inhibit virus replication by targeting cellular, not viral, proteins.     

Self-Interest versus Group-Interest in Antiviral Control

Antiviral agents have been hailed to hold considerable promise for the treatment and prevention of emerging viral diseases like H5N1 avian influenza and SARS. However, antiviral drugs are not completely harmless, and the conditions under which individuals are willing to participate in a large-scale antiviral drug treatment program are as yet unknown. We provide population dynamical and game theoretical analyses of large-scale prophylactic antiviral treatment programs. Throughout we compare the antiviral control strategy that is optimal from the public health perspective with the control strategy that would evolve if individuals make their own, rational decisions. To this end we investigate the conditions under which a large-scale antiviral control program can prevent an epidemic, and we analyze at what point in an unfolding epidemic the risk of infection starts to outweigh the cost of antiviral treatment. This enables investigation of how the optimal control strategy is moulded by the efficacy of antiviral drugs, the risk of mortality by antiviral prophylaxis, and the transmissibility of the pathogen. Our analyses show that there can be a strong incentive for an individual to take less antiviral drugs than is optimal from the public health perspective. In particular, when public health asks for early and aggressive control to prevent or curb an emerging pathogen, for the individual antiviral drug treatment is attractive only when the risk of infection has become non-negligible. It is even possible that from a public health perspective a situation in which everybody takes antiviral drugs is optimal, while the process of individual choice leads to a situation where nobody is willing to take antiviral drugs.     

反义寡核苷酸体外抗流感病毒活性

为了获得具有抗流感病毒活性的反义寡核苷酸,针对A型流感病毒基因组3′和5′端保守序列,设计并合成了多条硫代寡核苷酸(ODN)：3′端反义ODN(IV3#)与3′端正义ODN(IV3S);5′端反义ODN(IV4#)与5′端正义ODN(IV4S)以及由5′和3′端正义/反义保守序列组成的复合序列ODN(IV6#和IV7#)。测定了PSODN的体外细胞毒性和在MDCK细胞中对流感病毒复制的影响。结果表明：(1)PSODN浓度高达50μmol/L时对MDCK细胞末表现有毒性作用;(2)与流感病毒基因组5′端互补的ODN IV4#以及由5′和3′端保守序列构成的IV6#ODN和IV7#ODN均具有较高的抗病毒活性;如IV4#ODN浓度为1μmol/L时对流感病毒A/京防/861(H1N1)抑制率近50%,浓度为10μmol/L或更高时抑制率超过70%,且IV4#抑制病毒活性呈现明显的序列和剂量依赖性;(3)IV4#ODN不仅对A型流感病毒H1N1亚型有抑制作用,对H3N2亚型也表现较高的抑制活性;(4)病毒感染复数(MOI)对IV4#ODN抗病毒活性有一定影响,当MOI较低时,IV4#ODN表现的剂量效应关系更加明显。抗流感病毒反义寡核苷核IV4#ODN的发现为进一步研究流感新型药物奠定了实验基础。〖HTH〗关键词〖HTSS〗：流感病毒, 反义寡核苷酸, 体外细胞毒性, 抗病毒活性, 感染复数     

Oligonucleotide-based strategies to inhibit human hepatitis C virus

Hepatitis C virus (HCV) infection represents a worldwide problem, and current antiviral regimens are not satisfactory. The need to develop novel, specific, anti-HCV antiviral drugs is clear. Antisense oligonucleotides (AS-ON), ribozymes, and more recently, small interfering RNAs (siRNAs) have been widely used to control gene expression, and several clinical trials are in progress. The potential to use AS-ON as tools to control HCV infection, either by promoting an RNase H mediated cleavage of viral genomic RNA or by interfering with the assembly of a translation initiation complex on the internal ribosome entry site (IRES) is reviewed. Extensive knowledge of IRES structure and conservation among HCV genotypes have rendered the HCV IRES (and, in particular, its IIId loop) particularly attractive for antisense approaches. Encouraging data have been obtained with IRES-targeted RNase H-competent and incompetent ON analogs. We demonstrate here that very short steric blocking ONs can inhibit the formation of translation preinitiation complexes on the IRES and block IRES-mediated translation in a cell-free translation assay and in a transfected hepatoma cell line.     

Can Antiviral Drugs Contain Pandemic Influenza Transmission?

Antiviral drugs dispensed during the 2009 influenza pandemic generally failed to contain transmission. This poses the question of whether preparedness for a future pandemic should include plans to use antiviral drugs to mitigate transmission.Simulations using a standard transmission model that allows for infected arrivals and delayed vaccination show that attempts to contain transmission require relatively few antiviral doses. In contrast, persistent use of antiviral drugs when the reproduction number remains above 1 use very many doses and are unlikely to reduce the eventual attack rate appreciably unless the stockpile is very large. A second model, in which the community has a household structure, shows that the effectiveness of a strategy of dispensing antiviral drugs to infected households decreases rapidly with time delays in dispensing the antivirals. Using characteristics of past pandemics it is estimated that at least 80% of primary household cases must present upon show of symptoms to have a chance of containing transmission by dispensing antiviral drugs to households. To determine data needs, household outbreaks were simulated with 50% receiving antiviral drugs early and 50% receiving antiviral drugs late. A test to compare the size of household outbreaks indicates that at least 100-200 household outbreaks need to be monitored to find evidence that antiviral drugs can mitigate transmission of the newly emerged virus.Use of antiviral drugs in an early attempt to contain transmission should be part of preparedness plans for a future influenza pandemic. Data on the incidence of the first 350 cases and the eventual attack rates of the first 200 hundred household outbreaks should be used to estimate the initial reproduction number R and the effectiveness of antiviral drugs to mitigate transmission. Use of antiviral drugs to mitigate general transmission should cease if these estimates indicate that containment of transmission is unlikely.     

Molnupiravir,favipiravir and other antiviral drugs with proposed potentials for management of COVID-19: a concern on antioxidant aspect

COVID-19 is an important global public health problem that causes millions of infections worldwide. The specific antiviral drug for this new infection is still under research. Some new antiviral drugs, including molnupiravir and favipiravir, are proposed for usefulness in management of COVID-19. Additionally, some classic antiviral drugs used for other viral infections are also reproposed for the potentials for management of COVID-19. In the management of COVID-19, there are several pharmacological actions. An important consideration in antiviral therapy is the management of oxidative stress, which plays important roles in viral infections including to COVID-19. The analysis of antioxidative properties of alternative drugs for management of COVID-19 is interesting and can give basic data for further new antiviral drug researching. Here, the authors perform a molecular analysis on molnupiravir, favipiravir and other antiviral drugs with proposed potentials for management of COVID-19 to determine their antioxidative properties. Data from electron acceptor and donor calculation for each drug is used for further estimating overall antioxidative characteristic. Based on the present study, all studied drugs have overall antioxidative properties. Hence, the advantage of molnupiravir, favipiravir and other antiviral drugs with proposed potentials for the management of COVID-19 is their direct action on viral molecule via binding-blocking process as well as antixodiative process. For management of COVID-19 antioxidative stress, other non-antiviral drugs that are proposed for clinical advantage might also be useful.     

2'-5' Oligoadenylate synthetase plays a critical role in interferon-gamma inhibition of respiratory syncytial virus infection of human epithelial cells

Respiratory syncytial virus (RSV), associated with bronchiolitis and asthma, is resistant to the antiviral effects of type-I interferons (IFN), but not IFN-gamma. However, the antiviral mechanism of IFN-gamma action against RSV infection is unknown. The molecular mechanism of IFN-gamma-induced antiviral activity was examined in this study using human epithelial cell lines HEp-2 and A549. Exposure of these cells to 100-1000 units/ml of IFN-gamma, either before or after RSV infection, results in a significant decrease in RSV infection. After 1 h of exposure, IFN-gamma induces protein expression of IFN regulatory factor-1 (IRF-1) but not IRF-2, double-stranded RNA-activated protein kinase, and inducible nitric-oxide synthase in these cells. The mRNA for IRF-1, p40, and p69 isoforms of 2'-5' oligoadenylate synthetase (2-5 AS) are detectable, respectively, at 1 and 4 h of IFN-gamma exposure. Studies using cycloheximide and antisense oligonucleotides to IRF-1 indicate a direct role of IRF-1 in activating 2-5 AS. Cells transfected with 2-5 AS antisense oligonucleotides inhibit the antiviral effect of IFN-gamma. A stable cell line of HEp-2 overexpressing RNase L inhibitor, RLI-14, which exhibits an IFN-gamma-induced gene expression pattern similar to that of the parent cell line, shows a significant reduction in RNase L activity and IFN-gamma-mediated antiviral effect, compared with HEp-2 cells. These results provide direct evidence of the involvement of 2-5 AS in IFN-gamma-mediated antiviral activity in these cells.     

Synthesis of nucleoside-based antiviral drugs in ionic liquids

Nucleoside-based antiviral drugs have been synthesized using imidazolium-based ionic liquids as reaction medium. The ionic liquids were proved to be better solvents for all the nucleoside in terms of solubility and reaction medium as compared to conventional molecular solvents.     

Finding a chink in the armor: Update,limitations, and challenges toward successful antivirals against flaviviruses

Flaviviruses have caused large epidemics and ongoing outbreaks for centuries. They are now distributed in every continent infecting up to millions of people annually and may emerge to cause future epidemics. Some of the viruses from this group cause severe illnesses ranging from hemorrhagic to neurological manifestations. Despite decades of research, there are currently no approved antiviral drugs against flaviviruses, urging for new strategies and antiviral targets. In recent years, integrated omics data-based drug repurposing paired with novel drug validation methodologies and appropriate animal models has substantially aided in the discovery of new antiviral medicines. Here, we aim to review the latest progress in the development of both new and repurposed (i) direct-acting antivirals; (ii) host-targeting antivirals; and (iii) multitarget antivirals against flaviviruses, which have been evaluated both in vitro and in vivo, with an emphasis on their targets and mechanisms. The search yielded 37 compounds that have been evaluated for their efficacy against flaviviruses in animal models; 20 of them are repurposed drugs, and the majority of them exhibit broad-spectrum antiviral activity. The review also highlighted the major limitations and challenges faced in the current in vitro and in vivo evaluations that hamper the development of successful antiviral drugs for flaviviruses. We provided an analysis of what can be learned from some of the approved antiviral drugs as well as drugs that failed clinical trials. Potent in vitro and in vivo antiviral efficacy alone does not warrant successful antiviral drugs; current gaps in studies need to be addressed to improve efficacy and safety in clinical trials.     

Mathematical modeling of multi-drugs therapy: a challenge for determining the optimal combinations of antiviral drugs

In the current era of antiviral drug therapy, combining multiple drugs is a primary approach for improving antiviral effects, reducing the doses of individual drugs, relieving the side effects of strong antiviral drugs, and preventing the emergence of drug-resistant viruses. Although a variety of new drugs have been developed for HIV, HCV and influenza virus, the optimal combinations of multiple drugs are incompletely understood. To optimize the benefits of multi-drugs combinations, we must investigate the interactions between the combined drugs and their target viruses. Mathematical models of viral infection dynamics provide an ideal tool for this purpose. Additionally, whether drug combinations computed by these models are synergistic can be assessed by two prominent drug combination theories, Loewe additivity and Bliss independence. By combining the mathematical modeling of virus dynamics with drug combination theories, we could show the principles by which drug combinations yield a synergistic effect. Here, we describe the theoretical aspects of multi-drugs therapy and discuss their application to antiviral research.     

Targeting the HIV-1 RNA leader sequence with synthetic oligonucleotides and siRNA: Chemistry and cell delivery

New candidates for development as potential drugs or virucides against HIV-1 infection and AIDS continue to be needed. The HIV-1 RNA leader sequence has many essential functional sites for virus replication and regulation that includes several highly conserved sequences. The review describes the historical context of targeting the HIV-1 RNA leader sequence with antisense phosphorothioate oligonucleotides, such as GEM 91, and goes on to describe modern approaches to targeting this region with steric blocking oligonucleotide analogues having newer and more advantageous chemistries, as well as recent studies on siRNA, towards the attainment of antiviral activity. Recent attempts to obtain improved cell delivery are highlighted, including exciting new developments in the use of peptide conjugates of peptide nucleic acid (PNA) as potential virucides.     

Targeting the HIV-1 RNA leader sequence with synthetic oligonucleotides and siRNA: chemistry and cell delivery

New candidates for development as potential drugs or virucides against HIV-1 infection and AIDS continue to be needed. The HIV-1 RNA leader sequence has many essential functional sites for virus replication and regulation that includes several highly conserved sequences. The review describes the historical context of targeting the HIV-1 RNA leader sequence with antisense phosphorothioate oligonucleotides, such as GEM 91, and goes on to describe modern approaches to targeting this region with steric blocking oligonucleotide analogues having newer and more advantageous chemistries, as well as recent studies on siRNA, towards the attainment of antiviral activity. Recent attempts to obtain improved cell delivery are highlighted, including exciting new developments in the use of peptide conjugates of peptide nucleic acid (PNA) as potential virucides.     

Targeting of antiviral drugs by coupling with protein carriers

Side effects of antiviral drugs might be circumvented by their selective delivery into infected cells. This targeting can be obtained by conjugation of the drugs to macromolecules which are taken up specifically by the infected cells. The experiments reviewed, on this approach to antiviral chemotherapy, are mainly directed at improving the chemotherapeutic index of adenine arabinoside (ara-A) in the treatment of chronic hepatitis B by its coupling to galactosyl terminating glycoproteins.     

Inhibition of HIV-1 integrase by modified oligonucleotides: Optimization of the inhibitor structure

Integration of human immunodeficiency virus type 1 DNA into the infected cell genome is one of the key steps of the viral replication cycle. Therefore, viral integrase is of interest as a target for new antiviral drugs. Conjugates of 11-mer single-stranded oligonucleotides with hydrophobic molecules were shown to be efficient integrase inhibitors, inducing dissociation of the integrase-viral DNA complex. The dependence of the conjugate inhibitory activity on the oligonucleotide length and structure as well as on the structure of hydrophobic molecules was studied. Conjugates with eosin and oleic acid proved to be the most active. Conjugates of these molecules with 2′-O-methyl-oligonucleotide inhibited integrase at concentrations 50–100 nM but did not influence some other DNA-binding enzymes.     

HIV-1 integrase complexes with DNA dissociate in the presence of short oligonucleotides conjugated to acridine

The human immunodeficiency virus type 1 (HIV-1) integrase is an essential enzyme in the life cycle of the virus and is therefore an attractive target for the development of new antiviral drugs. Among them, inhibitors which are capable of targeting the preassembled integrase/DNA complex are of particular interest, because they could suppress integrase activity in the context of the HIV-1 preintegration complex. Here, we study the mechanism of action of 11-mer oligonucleotides, which are efficient inhibitors of the catalytic activity of integrase, provided that they are conjugated to a hydrophobic compound, acridine. To understand the mechanism of the conjugate inhibitory action, we used a steady-state fluorescence anisotropy assay, which allowed us to study the stability of the integrase/DNA complex in various conditions. We found that oligonucleotide-acridine conjugates induced the efficient dissociation of preassembled integrase/DNA complexes. The simultaneous presence of both acridine and an oligonucleotidic moiety is required for the inhibitory activity of conjugates. However, the dissociation effect is not dependent on the oligonucleotide sequence. Finally, our results suggest that the conjugates bind directly to integrase within its complex with DNA at a site different from the viral DNA binding site.