Antivirals in seasonal and pandemic influenza--future perspectives.

Antiviral drugs continue to be an important option for the treatment of influenza disease and will likely be the only option during the early phases of pandemic. However, the limited number of drug classes licensed for treatment of influenza raises several issues, particularly in the face of drug resistance. Two classes of drugs are presently licensed for treatment of influenza, M2 and neuraminidase inhibitors. M2 inhibitors are currently not recommended for treatment of influenza because of widespread resistance and resistance to neuraminidase inhibitors has been observed during the past influenza seasonal outbreaks. Additional antiviral drugs with novel mechanisms of action are clearly needed for the treatment of influenza. Fortunately, the landscape of drugs in early and advanced development has dramatically increased over the last 5 years. Drugs targeting viral functions such as attachment, entry/fusion, transcription, and polymerase and drugs targeting host factors affecting viral replication are currently in clinical trials. Examples of these novel antiviral drugs and the challenges for influenza antiviral drug development are discussed in this article.

Modifications of C-2 on the pyrroloquinoline template aimed at the development of potent herpesvirus antivirals with improved aqueous solubility.

A series of C-2 pyrroloquinoline analogs designed to improve aqueous solubility were examined for herpesvirus polymerase and antiviral activity. Several analogs were identified that maintained the antiviral activity of the previous development candidate against HCMV, HSV-1 and VZV, but with significantly improved aqueous solubility.

The design and development of 2-aryl-2-hydroxy ethylamine substituted 1H,7H-pyrido[1,2,3-de]quinoxaline-6-carboxamides as inhibitors of human cytomegalovirus polymerase.

Discovery efforts were focused on identifying a non-nucleoside antiviral for treating infections caused by human cytomegalovirus (HCMV) with equal or better potency and diminished toxicity compared to current therapeutics. This Letter describes the HCMV DNA polymerase inhibition and in vitro antiviral activity of various 2-aryl-2-hydroxy ethylamine substituted 1H,7H-pyrido[1,2,3-de]quinoxaline-6-carboxamides.

Synthesis of 4-oxo-4,7-dihydrofuro[2,3-b]pyridine-5-carboxamides with broad-spectrum human herpesvirus polymerase inhibition.

A versatile synthesis of 4-oxo-4,7-dihydrofuro[2,3-b]pyridine-5-carboxylate esters has been developed which has lead to the identification of a new series of non-nucleoside inhibitors of human herpesvirus polymerases HCMV, HSV-1, EBV, and VZV with high specificity compared to human DNA polymerases.

7-Oxo-4,7-dihydrothieno[3,2-b]pyridine-6-carboxamides: synthesis and biological activity of a new class of highly potent inhibitors of human cytomegalovirus DNA polymerase.

We report a new class of non-nucleoside antivirals, the 7-oxo-4,7-dihydrothieno[3,2-b]pyridine-6-carboxamides, some of which possess remarkable potency versus a broad spectrum of herpesvirus DNA polymerases and excellent selectivity compared to human DNA polymerases. A critical factor in the level of activity is hypothesized to be conformational restriction of the key 2-aryl-2-hydroxyethylamine sidechain by an adjacent methyl group.

2-Aryl-2-hydroxyethylamine substituted 4-oxo-4,7-dihydrothieno[2,3-b]pyridines as broad-spectrum inhibitors of human herpesvirus polymerases.

A novel series of 2-aryl-2-hydroxyethylamine substituted 4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxamides have been identified as potent antivirals against human herpesviruses. These compounds demonstrate broad-spectrum inhibition of the herpesvirus polymerases HCMV, HSV-1, EBV, and VZV with high specificity compared to human DNA polymerases.

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.

Amino acid changes within conserved region III of the herpes simplex virus and human cytomegalovirus DNA polymerases confer resistance to 4-oxo-dihydroquinolines, a novel class of herpesvirus antiviral agents.

The 4-oxo-dihydroquinolines (PNU-182171 and PNU-183792) are nonnucleoside inhibitors of herpesvirus polymerases (R. J. Brideau et al., Antiviral Res. 54:19-28, 2002; N. L. Oien et al., Antimicrob. Agents Chemother. 46:724-730, 2002). In cell culture these compounds inhibit herpes simplex virus type 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), and human herpesvirus 8 (HHV-8) replication. HSV-1 and HSV-2 mutants resistant to these drugs were isolated and the resistance mutation was mapped to the DNA polymerase gene. Drug resistance correlated with a point mutation in conserved domain III that resulted in a V823A change in the HSV-1 or the equivalent amino acid in the HSV-2 DNA polymerase. Resistance of HCMV was also found to correlate with amino acid changes in conserved domain III (V823A+V824L). V823 is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, Epstein-Barr virus, and HHV-8) of the eight human herpesviruses; the HHV-6 and HHV-7 polymerases contain an alanine at this amino acid. In vitro polymerase assays demonstrated that HSV-1, HSV-2, HCMV, VZV, and HHV-8 polymerases were inhibited by PNU-183792, whereas the HHV-6 polymerase was not. Changing this amino acid from valine to alanine in the HSV-1, HCMV, and HHV-8 polymerases alters the polymerase activity so that it is less sensitive to drug inhibition. In contrast, changing the equivalent amino acid in the HHV-6 polymerase from alanine to valine alters polymerase activity so that PNU-183792 inhibits this enzyme. The HSV-1, HSV-2, and HCMV drug-resistant mutants were not altered in their susceptibilities to nucleoside analogs; in fact, some of the mutants were hypersensitive to several of the drugs. These results support a mechanism where PNU-183792 inhibits herpesviruses by interacting with a binding determinant on the viral DNA polymerase that is less important for the binding of nucleoside analogs and deoxynucleoside triphosphates.

Non-nucleoside inhibitors of herpesviruses.

While the treatment of herpes simplex virus with acyclovir and similar nucleoside analogues was one of the first success stories in antiviral chemotherapy, substantial unmet medical needs remain for herpesvirus diseases. In particular, the increasing numbers of immunosuppressed people due to AIDS, transplantation, cancer and aging has driven the need for improved antivirals to treat diseases caused by human cytomegalovirus (HCMV). Currently available drugs for the treatment of HCMV diseases are less than ideal agents due to issues of toxicity, modest efficacy and poor oral bioavailability. High throughput screening of large compound collections for inhibitors of specific viral enzymes or inhibition of viral growth in cell culture have identified a number of new HCMV inhibitors at several pharmaceutical companies. These compounds act by inhibition of novel molecular targets such as the viral protein kinase, viral protease and viral proteins involved in DNA cleavage/packaging. In addition, novel non-nucleoside inhibitors of the herpesvirus DNA polymerase have recently been described. This review will summarise some of these research efforts and will focus on non-nucleoside compounds that directly inhibit a viral process.

Broad-spectrum antiviral activity of PNU-183792, a 4-oxo-dihydroquinoline, against human and animal herpesviruses.

We identified a novel class of 4-oxo-dihydroquinolines represented by PNU-183792 which specifically inhibit herpesvirus polymerases. PNU-183792 was highly active against human cytomegalovirus (HCMV, IC(50) value 0.69 microM), varicella zoster virus (VZV, IC(50) value 0.37 microM) and herpes simplex virus (HSV, IC(50) value 0.58 microM) polymerases but was inactive (IC(50) value >40 microM) against human alpha (alpha), gamma (gamma), or delta (delta) polymerases. In vitro antiviral activity against HCMV was determined using cytopathic effect, plaque reduction and virus yield reduction assays (IC(50) ranging from 0.3 to 2.4 microM). PNU-183792 antiviral activity against both VZV (IC(50) value 0.1 microM) and HSV (IC(50) ranging from 3 to 5 microM) was analyzed using plaque reduction assays. PNU-183792 was also active (IC(50) ranging 0.1-0.7 microM) in cell culture assays against simian varicella virus (SVV), murine cytomegalovirus (MCMV) and rat cytomegalovirus (RCMV). Cell culture activity was compared with the appropriate licensed drugs ganciclovir (GCV), cidofovir (CDV) and acyclovir (ACV). PNU-183792 was also active against both GCV-resistant and CDV-resistant HCMV and against ACV-resistant HSV. Toxicity assays using four different species of proliferating mammalian cells indicated PNU-183792 was not cytotoxic at relevant drug concentrations (CC(50) value >100 microM). PNU-183792 was inactive against unrelated DNA and RNA viruses indicating specificity for herpesviruses. In animals, PNU-183792 was orally bioavailable and was efficacious in a model of lethal MCMV infection.

Broad-spectrum antiherpes activities of 4-hydroxyquinoline carboxamides, a novel class of herpesvirus polymerase inhibitors.

Through broad screening of the compound library at Pharmacia, a naphthalene carboxamide was identified as a nonnucleoside inhibitor of human cytomegalovirus (HCMV) polymerase. Structure-activity relationship studies demonstrated that a quinoline ring could be substituted for naphthalene, resulting in the discovery of a 4-hydroxyquinoline-3-carboxamide (4-HQC) class of antiviral agents with unique biological properties. In vitro assays with the 4-HQCs have demonstrated potent inhibition of HCMV, herpes simplex virus type 1 (HSV-1), and varicella-zoster virus (VZV) polymerases but no inhibition of human alpha, delta, and gamma polymerases. Antiviral cell culture assays have further confirmed that these compounds are active against HCMV, HSV-1, HSV-2, VZV, and many animal herpesviruses. However, these compounds were not active against several nonherpesviruses representing different DNA and RNA virus families. A strong correlation between the viral DNA polymerase and antiviral activity for this class of compounds supports inhibition of the viral polymerase as the mechanism of antiviral activity. Northern blot analysis of immediate-early and late viral transcripts also pointed to a block in the viral life cycle consistent with inhibition of viral DNA replication. In vitro HCMV polymerase assays indicate that the 4-HQCs are competitive inhibitors of nucleoside binding. However, no cross-resistance could be detected with ganciclovir-resistant HCMV or acyclovir-resistant HSV-1 mutants. The unique, broad-spectrum activities of the 4-HQCs may offer new opportunities for treating many of the diseases caused by herpesviruses.