Crystal structure of the herpes simplex virus 1 DNA polymerase.

Herpesviruses are the second leading cause of human viral diseases. Herpes Simplex Virus types 1 and 2 and Varicella-zoster virus produce neurotropic infections such as cutaneous and genital herpes, chickenpox, and shingles. Infections of a lymphotropic nature are caused by cytomegalovirus, HSV-6, HSV-7, and Epstein-Barr virus producing lymphoma, carcinoma, and congenital abnormalities. Yet another series of serious health problems are posed by infections in immunocompromised individuals. Common therapies for herpes viral infections employ nucleoside analogs, such as Acyclovir, and target the viral DNA polymerase, essential for viral DNA replication. Although clinically useful, this class of drugs exhibits a narrow antiviral spectrum, and resistance to these agents is an emerging problem for disease management. A better understanding of herpes virus replication will help the development of new safe and effective broad spectrum anti-herpetic drugs that fill an unmet need. Here, we present the first crystal structure of a herpesvirus polymerase, the Herpes Simplex Virus type 1 DNA polymerase, at 2.7 A resolution. The structural similarity of this polymerase to other alpha polymerases has allowed us to construct high confidence models of a replication complex of the polymerase and of Acyclovir as a DNA chain terminator. We propose a novel inhibition mechanism in which a representative of a series of non-nucleosidic viral polymerase inhibitors, the 4-oxo-dihydroquinolines, binds at the polymerase active site interacting non-covalently with both the polymerase and the DNA duplex.

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.

Spontaneous aggregation and cytotoxicity of the beta-amyloid Abeta1-40: a kinetic model.

The time dependency of the spontaneous aggregation of the fibrillogenic beta-amyloid peptide, Abeta1-40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-Abeta antibody 6E10, raised against residues 1-17, at concentrations of 200-300 nM delayed significantly the aggregation of 50 microM amyloid peptide. The anti-Abeta antibody 4G8, raised against residues 17-24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39-43 of the full-length Abeta was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the Abeta1-40-induced toxicity toward SH-EPI cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of Abeta1-40 is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of Abeta1-40 in vitro and possibly in vivo.

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.