5-(1-Substituted) alkyl pyrimidine nucleosides as antiviral (herpes) agents

The treatment of viral diseases remains one of the major challenges to modern medicine. During the past two decades there has been increased recognition of the consequences of serious viral illnesses that are not controlled by vaccination. These illnesses include human immunodeficiency virus, human herpes viruses, and viruses that cause hepatitis. There are now eight pathogens recognized in the herpes virus family that cause infections in humans. Infections by the herpes viruses are opportunistic and often life-threatening, leading to significant morbidity and mortality in the increasing number of chronically immune compromised individuals such as AIDS patients, cancer patients and transplant recipients on immunosuppressive therapy. Nearly all individuals with AIDS are infected with one or more of the herpes viruses. Antiviral therapy with guanosine nucleoside analogs acyclovir and ganciclovir has had a major impact on diseases caused by herpes simplex virus type-1 and type-2 (HSV-1, HSV-2), Varicella zoster virus (VZV), and human cytomegalovirus (HCMV) but development of resistant virus strains and the absence of any effective treatment for other members of the herpes family provide a stimulus for increased search of new agents effective against various herpes viruses. Pyrimidine nucleosides have taken up an important role in the therapy of virus infection. Significant progress in the study of anti-herpes nucleosides has been made by the advent of 5-substituted pyrimidine nucleosides such as 5-iodo-, 5-ethyl-, 5-(2-chloroethyl)-, and (E)-5-(2-bromovinyl)- derivatives of 2'-deoxyuridine. These are highly specific inhibitors of HSV-1, HSV-2, and/or VZV infections. However, Epstein Barr virus (EBV) and HCMV are much less sensitive to these agents. In 5-substituted pyrimidine nucleosides the nature of substituents, particularly at the C-5 position, has been found to be an important determinant of anti-herpes activity. Structural requirements at the C-2 carbon of the 5-substituent of pyrimidine nucleosides have been well established for anti-herpes activity. However, there is little qualitative or mechanistic knowledge of the derivatives with substitution at the C-1 carbon of the 5-substituent of pyrimidine nucleosides. During the last few years of our research, we have investigated a variety of C-1 functionalized substituents at the 5-position of the pyrimidine nucleosides to determine their usefulness as antiviral (herpes) agents. In the 5-(1-substituted) group of pyrimidine nucleosides, we demonstrated that novel substituents present at the C-1 carbon of the 5-side chain of the pyrimidine nucleosides are important determinants of potent and broad spectrum antiviral (herpes) activity including EBV and HCMV. In this article the work on design, synthesis and structure activity relationships of several 5-[(1-substituted) alkyl (or vinyl)] pyrimidine nucleoside derivatives as potential inhibitors of herpes viruses is reviewed.     

Potential prophylactic and therapeutic vaccines for HSV infections

The human herpesviruses herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) can cause severe recurrent disease in humans and establish lifelong infection in their hosts. Several antiviral therapies are available to control disease and spread, but these are not completely effective and do not affect latent virus. The need for vaccines for HSV is urgent, both for controlling initial infection and spread of disease as well as to limit recurrences. Several approaches including subunit vaccines, peptide vaccines, live virus vectors and DNA vaccine technology have been used in developing both prophylactic and therapeutic vaccines for HSV and these are reviewed here.     

Virucidal activity of polysaccharide extracts from four algal species against herpes simplex virus

Herpes simplex virus types 1 and 2 (HSV-1, HSV-2) infections are common, but can cause serious infections in neonates and the immunocompromised. Drugs currently used to treat cutaneous or genital HSV infections are effective in limiting disease, but the emergence of drug resistant viruses in immunocompromised individuals can be problematic. While the prophylactic oral treatment with antiviral drugs can reduce virus shedding and transmission, there is a need for topical microbicides that have the potential to limit sexual transmission of the virus. Previous reports demonstrated the antiviral activity of complex sulfated polysaccharides extracted from various species of marine algae and suggested that they interfered with the attachment of virions to host cells. Here, we evaluated the antiviral activity of extracts from Undaria pinnatifida, Splachnidium rugosum, Gigartina atropurpurea, and Plocamium cartilagineum against HSV-1 and HSV-2. These extracts exhibited good activity when added during the first hour of viral infection, but were ineffective if added later. Plaque reduction assays, when the extracts were added prior to viral inoculation, yielded EC₅₀ values that ranged from 2.5–3.6 μg/ml for HSV-1 and 0.7–6.6 μg/ml for HSV-2. None of the extracts exhibited significant toxicity in a neutral red uptake assay (IC₅₀ >100 μg/ml). Subsequent assays showed that the compounds had potent virucidal activity and were active at very low concentrations. We conclude that these extracts are nontoxic and effective virucidal agents that warrant further investigation to examine their potential role in the prevention of HSV infections of humans.     

The role of viruses in human cancer development and antiviral approaches for intervention

Viruses are estimated to be linked to at least 15% of human cancers. Hepatitis B and C viruses, human papilloma viruses, Epstein-Barr virus, human herpes virus-8 and human T-cell leukemia virus have been definitively linked to human cancer. A brief overview of the molecular mechanisms of carcinogenesis elucidated for these viruses and antiviral approaches which may provide clues to reducing the occurrence or progression of the resulting virally derived cancers are described.     

Developments in antiviral drug design, discovery and development in 2004

This article summarizes key aspects of progress made during 2004 toward the design, discovery and development of antiviral agents for clinical use. Important developments in the identification, characterization and clinical utility of inhibitors of human immunodeficiency virus; the hepatitis viruses, hepatitis B, hepatitis C; the herpes family of viruses, herpes simplex viruses 1 and 2, varicella zoster virus, Epstein-Barr virus and human cytomegalovirus; the respiratory viruses, influenza, respiratory syncytial virus, human metapneumovirus, picornaviruses, measles and the severe acute respiratory syndrome coronavirus; human papilloma virus; rotavirus; Ebola virus and West Nile virus, are reviewed.     

Novel targets for the development of anti-herpes compounds

Herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) are members of the Herpesviridae family. HSV infections have been known since ancient times and are one of the most common communicable diseases in humans. Although infections are often subclinical, HSV can cause mild to severe diseases, especially in immunocompromised patients. Herpes simplex viruses establish latency in the nuclei of neuronal cells and may reactivate, with or without symptoms, throughout the host's lifetime. Over one third of the world's population suffer from recurrent HSV infections several times a year and are thus capable of transmitting HSV by close personal contact. There are few drugs licensed for the treatment of HSV infections. Most target the viral DNA polymerase, and indeed acyclovir remains the reference treatment some thirty years after its discovery! Extensive clinical use of this drug has led to the emergence of resistant viral strains, mainly in immunocompromised patients. This highlights the crucial need for the development of new anti-herpes drugs that can inhibit infection by both wild-type viruses and drug-resistant strains. Over the last few years, significant efforts have been made to set up a range of strategies for the identification of potential new anti-viral drugs. One alternative is to develop drugs with different mechanisms of action. The present article reviews potential viral and cellular targets that are now known to be involved in HSV infection and for which specific inhibitors with anti-HSV activity, at least in cell culture, have been identified.     

Antiviral activity of Australian tea tree oil and eucalyptus oil against herpes simplex virus in cell culture

The antiviral effect of Australian tea tree oil (TTO) and eucalyptus oil (EUO) against herpes simplex virus was examined. Cytotoxicity of TTO and EUO was evaluated in a standard neutral red dye uptake assay. Toxicity of TTO and EUO was moderate for RC-37 cells and approached 50% (TC50) at concentrations of 0.006% and 0.03%, respectively. Antiviral activity of TTO and EUO against herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells using a plaque reduction assay. The 50% inhibitory concentration (IC50) of TTO for herpes simplex virus plaque formation was 0.0009% and 0.0008% and the IC50 of EUO was determined at 0.009% and 0.008% for HSV-1 and HSV-2, respectively. Australian tea tree oil exhibited high levels of virucidal activity against HSV-1 and HSV-2 in viral suspension tests. At noncytotoxic concentrations of TTO plaque formation was reduced by 98.2% and 93.0% for HSV-1 and HSV-2, respectively. Noncytotoxic concentrations of EUO reduced virus titers by 57.9% for HSV-1 and 75.4% for HSV-2. Virus titers were reduced significantly with TTO, whereas EUO exhibited distinct but less antiviral activity. In order to determine the mode of antiviral action of both essential oils, either cells were pretreated before viral infection or viruses were incubated with TTO or EUO before infection, during adsorption or after penetration into the host cells. Plaque formation was clearly reduced, when herpes simplex virus was pretreated with the essential oils prior to adsorption. These results indicate that TTO and EUO affect the virus before or during adsorption, but not after penetration into the host cell. Thus TTO and EUO are capable to exert a direct antiviral effect on HSV. Although the active antiherpes components of Australian tea tree and eucalyptus oil are not yet known, their possible application as antiviral agents in recurrent herpes infection is promising.     

4-Oxo-4,7-dihydrothieno[2,3-b]pyridines as non-nucleoside inhibitors of human cytomegalovirus and related herpesvirus polymerases

A novel series of 4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxamides have been identified as potential antivirals against human herpesvirus infections resulting from human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), and varicella-zoster virus (VZV). Compounds 10c and 14 demonstrated broad-spectrum inhibition of the herpesvirus polymerases HCMV, HSV-1, and VZV. High specificity for the viral polymerases was observed compared to human alpha polymerase. The antiviral activity of 10c and 14, as determined by plaque reduction assay, was comparable or superior to that of existing antiherpes drugs, ganciclovir (for HCMV) and acyclovir (for HSV-1 and VZV). Drug resistance to compound 14 correlated to point mutations in conserved domain III of the herpesvirus DNA polymerase, but these mutations do not confer resistance to existing nucleoside therapy. In addition, compound 14 maintained potent antiviral activity against acyclovir-resistant HSV-1 strains. Substitution to the pyridone nitrogen (N7) was found to be critical for enhanced in vitro antiviral activity.     

Suppression of proliferation of poliovirus and porcine parvovirus by novel phenoxazines, 2-amino-4,4 alpha-dihydro-4 alpha-7-dimethyl-3H-phenoxazine-3-one and 3-amino-1,4 alpha-dihydro-4 alpha-8-dimethyl-2H-phenoxazine-2-one

The present study aimed at investigating the antiviral effects of 2-amino-4,4alpha-dihydro-4alpha-7-dimethyl-3H-phenoxazine-3-one (Phx-1) and 3-amino-1,4alpha-dihydro-4alpha-8-dimethyl-2H-phenoxazine-2-one (Phx-2) on 6 representative viruses: poliovirus, porcine parvovirus, simian virus 40 (SV-40), herpes simplex virus-1 (HSV-1), Sindbis virus, and vesicular stomatitis virus (VSV). Phx-1 and Phx-2 suppressed the proliferation of poliovirus in Vero cells and that of porcine parvovirus in ESK cells at concentrations between 0.25 microg/ml and 2 microg/ml, when the cells were treated with Phx-1 and Phx-2 for 1 h and then inoculated with these viruses. The proliferation of the other viruses, SV-40, HSV-1, Sindbis virus, and VSV, in the host cells was not influenced by Phx-1 or Phx-2 at concentrations less than 20 microg/ml. The results suggest that Phx-1 and Phx-2 may be useful to prevent the proliferation of poliovirus and porcine parvovirus infection and may contribute to developing new antiviral drugs in future.     

Ribavirin and inosiplex: a review of their present status in viral diseases

A considerable amount of information has accumulated during the past 10 years in the search for antiviral agents. Ribavirin and inosiplex are 2 interesting developments to come out of this search. Ribavirin, a synthetic nucleoside, has an unusually wide spectrum of antiviral activity, especially when tested in vitro. A large number of RNA and DNA viruses are sensitive, especially herpes viruses, poxvirus, influenza, parainfluenza, reovirus, togavirus, and RNA tumour viruses. The in vivo antiviral spectrum of activity is much narrower, with activity against herpes virus, influenza, parainfluenza, measles and adenoviruses. However, controlled clinical trials have not been uniformly successful in treating influenza, hepatitis, herpes simplex and herpes zoster. Inosiplex has been shown to have antiviral activity in vivo against influenza, herpes simplex, rhinovirus and vaccinia virus infections. However, antiviral activity has not been consistently demonstrated, and this observation led to further studies which revealed its immunomodulating effects. The accumulated evidence has indicated that inosiplex is more a prohost agent rather than an antiviral drug. Immune functions which are depressed during viral infection can be restored to normal by inosiplex therapy. At present, neither ribavirin nor inosiplex alone has been shown to be uniformly successful in the treatment of human viral diseases. Nevertheless, their potential place in chemotherapy should not be neglected, although further data are needed to determine what this place will be. Whether combining them with other antiviral agents such as interferon, acyclovir, Ara-A, and so on, would produce a potentiation of action and improved antiviral chemotherapy, will be an interesting area for further study.     

Isolation and characterization of herpes simplex virus type 1 resistant to aminothiazolylphenyl-based inhibitors of the viral helicase-primase

The aminothiazolylphenyl-containing compounds BILS 179 BS and BILS 45 BS are novel inhibitors of the herpes simplex virus helicase-primase with antiviral activity in vitro and in animal models of HSV disease. To verify the mechanism of antiviral action, resistant viruses were selected by serial passage or by single-step plaque selection of HSV-1 KOS in the presence of inhibitors. Three resistant isolates K138r3, K22r5, and K22r1 were found to be 38-, 316-, and 2500-fold resistant to BILS 22 BS, a potent analog of BILS 45 BS. All three viruses had growth properties in vitro similar to wild-type HSV-1 KOS but they were sensitive to acyclovir. Cutaneous and intra-cerebral inoculation of mice with K22r1 or K22r5 resulted in pathogenicity equivalent to that of HSV-1 KOS. Both isolates were fully competent for reactivation from latency following corneal inoculation. Helicase-primase purified from cells infected with resistant viruses showed decreased inhibition in an in vitro DNA-dependent ATPase assay that correlated well with antiviral resistance. Marker transfer experiments and DNA sequence analysis identified single base pair mutations clustered in the N-terminus of the UL5 gene that resulted in single amino acid changes in the UL5 protein. Taken together, the results indicate that helicase-primase inhibitors prevent HSV growth by inhibiting HSV helicase-primase through specific interaction with the UL5 protein.     

Pharmacotherapy of post-transplant viral infections

Background: Management of a number of significant viral pathogens in transplant recipients remains challenging. Objectives: To define an optimal antiviral approach to the management of cytomegalovirus (CMV), human herpes virus-6 (HHV-6), Epstein–Barr (EBV)-associated post-transplant lymphoproliferative disorder (PTLD), and polyoma virus-associated nephropathy in transplant recipients. Methods: Clinical trials and existing data regarding use of antiviral agents for these viruses were reviewed to develop evidence-based recommendations for their management. Conclusions: Weighing the current evidence regarding the use of valganciclovir as pre-emptive therapy or prophylaxis, the former approach offers a greater benefit for the overall prevention of CMV disease. Limited data show that prophylaxis with antiviral agents is associated with a reduction in the risk of EBV-associated PTLD. Treatment options for HHV-6 and polyoma virus-associated nephropathy are still limited.     

Synthetic analogues of bovine bactenecin dodecapeptide reduce herpes simplex virus type 2 infectivity in mice

We have evaluated the potential of four synthetic peptides (denoted HH-2, 1002, 1006, 1018) with a distant relationship to the host defense peptide bovine bactenecin dodecapeptide for their ability to prevent genital infections with herpes simplex virus type 2 (HSV-2) in mice. All four peptides showed antiviral properties in vitro and reduced HSV-2 infection of Vero cells in a dose-dependent manner. Detailed analysis showed that the peptides were able to interfere with both viral attachment and entry, but not with replication post-entry, and were effective antivirals also when HSV-2 was introduced in human semen. Two of the peptides proved especially effective in reducing HSV-2 infection also in vivo. When admixed with virus prior to inoculation, both HH-2 and 1018 reduced viral replication and disease development in a genital model of HSV-2 infection in mice, and also when using very high infectious doses of HSV-2. These data show that peptides HH-2 and 1018 have antiviral properties and can be used to prevent genital herpes infection in mice.     

The 17th International Conference on Antiviral Research

The focus of the 17th International Conference on Antiviral Research was the discovery and development of antiviral agents (chemistry, biology, animal models and clinical trial results) against a variety of human infectious agents including HIV, herpes viruses, hepatitis viruses, respiratory viruses and emerging/re-emerging pathogens. The meeting included the symposium 'Clinical Update on Antiviral Drugs', plenary sessions dedicated to each of the individual classes of infectious agents, a symposium on new developments surrounding emerging pathogens, and three special award lectures, which discussed the history of nucleotide antiviral agents, mechanisms of viral persistence and drug resistance, and the therapy of herpes virus infections. Within each infectious agent session the presentations included those describing the development of new and novel anti-infectives, including research based on the preclinical development of new molecules, and the results of animal modelling and clinical studies on advanced-stage antiviral agents. A summary of the meeting highlights, segregated by infectious agent, will be presented in this review.     

The past, present and future of antiviral drug discovery

A review of the history, development and current status of antiviral chemotherapy is presented from its origins in the 1960s until the present day. Key issues in the development of novel antivirals are the emergence of resistant virus, safety and side effects. This review describes the current therapeutic status of the herpes viruses, HIV, hepatitis viruses and respiratory viruses, and outlines the current limitations in the field together with the future compounds likely to emerge to address these needs. The future of antiviral research is assessed in relation to the impact of potential 'emerging viruses' and biological weapons, and the potential of combination therapies involving antivirals and disease modification.     

Phenoxazine derivatives inactivate human cytomegalovirus, herpes simplex virus-1, and herpes simplex virus-2 in vitro.

We examined whether phenoxazine derivatives, 2-amino-4,4alpha-dihydro-4alpha-7-dimethyl-3H-phenoxazine-3-one (Phx-1), 3-amino-1,4alpha-dihydro-4alpha-8-dimethyl-2H-phenoxazine-2-one (Phx-2), and 2-amino-phenoxazine-3-one (Phx-3) may have antiviral activity against herpes family viruses: human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), and herpes simplex virus type 2 (HSV-2). The antiviral activity was evaluated by the selectivity index (SI), which is the ratio of 50% cytotoxic concentration (CC(50)) and 50% antiviral concentration (IC(50)). Among these phenoxazines, Phx-2 exerted strong antiviral activity to HCMV with the SI of 200, while Phx-1 and Phx-3 exerted no marked anti-HCMV activity. Phx-2 also showed moderate inhibition of HSV-1 and HSV-2, with the SI of 6.7 and 17, respectively. In the time-of-addition experiments, inhibitory effect of Phx-2 against HCMV was active even when applied to cells at 100 h after HCMV infection, while ganciclovir (GCV) showed potent inhibition when applied to cells before 42-h post-infection, but its inhibitory effects disappeared thereafter. Attachment and penetration of HCMV was not affected by the presence of Phx-2. When HCMV was pretreated with Phx-2, concentration-dependent virucidal action was observed, suggesting that Phx-2 inactivates HCMV directly. From these data, it was found that Phx-2 might have a different anti-HCMV target from GCV.     

Potent virucidal effect of pheophorbide a and pyropheophorbide a on enveloped viruses

In this study, we evaluated the inhibitory effect of ethanol and aqueous extracts from a stem of Opuntia ficus indica on replication of three kinds of viruses: two enveloped viruses [herpes simplex virus type 2 (HSV-2), influenza A virus (IFV-A)], and one non-enveloped virus [poliovirus type 1 (PV-1)]. Only ethanol extract from the cactus stem showed significant antiviral activity in vitro. Two chlorophyll derivatives, pheophorbide a and pyropheophorbide a, were isolated as active substances exhibiting potent virucidal effects on HSV-2 and IFV-A, but no activity against PV-1 was observed. These findings suggest that these active compounds might recognize specific glycoproteins of enveloped viruses, precluding their binding to host cell receptors and inhibiting viral infections.     

Antiviral activity of hop constituents against a series of DNA and RNA viruses

We investigated whether crude hop extracts and purified hop components representing every major chemical class of hop compound have antiviral activity. These hop constituents were tested for antiviral activity against bovine viral diarrhea virus (BVDV) as a surrogate model of hepatitis C virus (HCV), human immunodeficiency virus (HIV), influenza A virus (FLU-A), influenza B virus (FLU-B), rhinovirus (Rhino), respiratory syncytial virus (RSV), yellow fever virus (YFV), cytomegalovirus (CMV), hepatitis B virus (HBV), and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The extracts all failed to prevent the replication of HIV, FLU-A, FLU-B, RSV and YFV. A xanthohumol-enriched hop extract displayed a weak to moderate antiviral activity against BVDV (therapeutic index (TI)=6.0), HSV-2 (TI=>5.3), Rhino (TI=4.0) and HSV-1 (TI=>1.9) with IC(50) values in the low microg/ml range. Pure iso-alpha-acids demonstrated low to moderate antiviral activity against both BVDV (TI=9.1) and CMV (TI=4.2) with IC(50) values in the low microg/ml range. No antiviral activity was detected using beta-acids or a hop oil extract. Ultra-pure preparations (>99% pure) were used to show that xanthohumol accounted for the antiviral activity observed in the xanthohumol-enriched hop extract against BVDV, HSV-1 and HSV-2. Xanthohumol was found to be a more potent antiviral agent against these viruses than the isomer iso-xanthohumol. With Rhino, the opposite trend was observed with iso-xanthohumol showing superior antiviral activity to that observed with xanthohumol. Xanthohumol also showed antiviral activity against CMV, suggesting that it might have a generalized anti-herpesvirus antiviral activity. Again, superior antiviral activity was observed with the xanthohumol isomer against CMV. In summary, iso-alpha-acids and xanthohumol were shown to have a low-to-moderate antiviral activity against several viruses. These hop constituents might serve as interesting lead compounds from which more active anti-HCV, anti-Rhino and anti-herpesvirus antiviral agents could be synthesized.     

抗耐药疱疹病毒药物的研究进展

摘 要疱疹病毒是人类最常见的感染性疾病,根据人类感染程度可分为不同种类,其中单纯疱疹病毒通常引起人类口腔或生殖器黏膜感染,巨细胞病毒是器官移植和肿瘤患者等免疫功能不全患者最常见机会致病菌,可导致严重的疾病甚至死亡。核苷类似物是治疗疱疹病毒疗效较好的药物,但耐药病毒株的出现,为疱疹病毒感染患者的治疗设置了新的障碍,尤其对于免疫功能不全的患者。因此临床亟需能够规避疱疹病毒耐药性的有效的抗病毒药物。本文综述了耐药疱疹病毒的治疗药物的研究进展,包括非核苷类抗病毒药物、新型作用靶点、新型作用机制的抗病毒药物的研究进展。