Synthesis and antiviral activity of certain carbamoylpyrrolopyrimidine and pyrazolopyrimidine nucleosides.

Following our recent discovery that 9-beta-D-ribofuranosylpurine-6-carboxamide (1) exhibits potent antiviral activity, we were prompted to synthesize certain pyrrolopyrimidine and pyrazolopyrimidine nucleosides containing a carbamoyl function (7a,b and 13). The key precursor, 7-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-4- carbonitrile (8a), required for the synthesis of 7a was prepared from the corresponding 4-chloro analogue (4a). Reaction of 4a with methanethiol, followed by oxidation, gave the 4-methylsulfonyl derivative (6a), which with NaCN in DMF gave 8a. Alkaline hydrolysis of 8a provided 7a. Similarly, 7b was prepared from 4-chloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo[3,4-d] pyrimidine (4b) via the carbonitrile intermediate 8b. Starting with thioformycin B or 7-chloro-3-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo[4,3-d]pyrimidine (10) and following the similar sequence of reactions, we obtained compound 13. The in vitro antiviral studies of these carbamoyl and certain related nucleosides indicated 7a to be a potent antiviral agent against vaccinia virus, whereas 13 was moderately active. 4-Chloro-7-beta-D-ribofuranosylpyrrolo[2,3-d]pyrimidine was found to be one of the most active compounds against RVF, PICH, YF, and SF viruses in culture.

In vitro virucidal activity of selected anthraquinones and anthraquinone derivatives.

Anthraquinones and anthraquinone derivatives were characterized for their antiviral and virucidal activities against viruses representing several taxonomic groups. One of these compounds, hypericin, had activity against vesicular stomatitis virus, herpes simplex virus types 1 and 2, parainfluenza virus, and vaccinia virus (from 0.5 to 3.8 log10 reductions in infectivity) at concentrations of less than 1 microgram/ml as determined by a direct pre-infection incubation assay. Human rhinovirus was not sensitive to hypericin at concentrations up to 10 micrograms/ml. Addition of small amounts of Tween-80 to solutions containing hypericin enhanced, by up to 2.6 log10, hypericin's virucidal activity. Anthraquinones and anthraquinone derivatives with the hydroxyl and alkyl substitution pattern of emodin (i.e. emodin, emodin anthrone, emodin bianthrone and hypericin) were active against the enveloped viruses tested. The following general pattern of activity was found: hypericin greater than emodin bianthrone greater than emodin anthrone greater than emodin. Chrysophanic acid, aloe-emodin, and sennosides A and B did not possess activity against any of the viruses tested.

Thymidine kinase: diagnostic and prognostic potential.

Thymidine kinase is a cell cycle-dependent marker that can be detected in the serum of patients diagnosed with many different types of cancer. Serum levels of thymidine kinase have also been shown to reflect the progression of cancer as well as an indication of the efficacy of chemotherapeutic intervention. A new monoclonal antibody assay for thymidine kinase has been developed, which is capable of detecting thymidine kinase in both serum and tumor tissue. Thymidine kinase assay kits should be available at low cost and could serve as an effective low cost test for the detection and progression of many types of human cancer.

The importance of IMP dehydrogenase inhibition in the broad spectrum antiviral activity of ribavirin and selenazofurin.

The nucleosides ribavirin, selenazofurin, tiazofurin and bredinin all exhibit the lowering of guanylate pools in vitro and in vivo by the inhibition of IMP dehydrogenase. However, each of these nucleosides has a separate profile of antiviral and antitumor activity. The IMP dehydrogenase inhibition in the case of ribavirin and bredinin appears to be due to the nucleoside 5'-monophosphate and in the case of selenazofurin and tiazofurin to the NAD analogs formed intracellularly. With regard to the antiviral activity of these nucleosides, although selenazofurin was the most potent antiviral agent in vitro, its antiviral activity was also most readily reversed by exogenous guanosine. The antiviral effects of ribavirin were only partially reversed under the conditions studied. These and related studies show that each of these nucleosides form nucleotide metabolites which act as enzyme inhibitors at additional sites other than IMP dehydrogenase. As in the case of ribavirin such inhibition of IMP dehydrogenase may result in an increased "self potentiation" by the lowering of guanylate pools in those instances where guanylate analogs are involved as inhibitors of viral specific or viral induced enzymes. Further studies should more clearly elucidate the importance of the simultaneous inhibition of various enzyme sites by different metabolic nucleotide forms of the same nucleoside analog.

Herpesvirus type 1 serum antibodies and brain tumors in humans.

Sixty-five patients underwent craniotomy for brain tumors; of these 35 had glioblastoma multiforme (GM). The GM cases, as a group, showed significantly higher serum titers for herpesvirus type 1 (HSV-1) neutralizing antibodies (NT) than the non-glioblastoma cases. Both the GM group and the pituitary adenoma group had high levels of HSV-1 serum antibodies with the enzyme-linked immunosorbent assay (ELISA). These data, combined with other evidence, lead us to speculate that HSV-1 infections may be associated with certain brain tumors in humans. However, coincidental causes for these elevated HSV-1 titers must be ruled out.

A monoclonal antibody specific for human thymidine kinase 1.

Previous research has shown that thymidine kinase 1 (TK1), a nucleotide salvage pathway enzyme, is an accurate prognostic and diagnostic tumor marker. However, the current radioisotope assay for TK1 is cumbersome and has hampered the clinical application of this diagnostic technique in cancer management. To overcome the problems of the current radioisotope assay, we have produced monoclonal antibodies (MAbs) using purified TK1 from Raji cell extract. Production and confirmation of their specificity was confirmed using Western blot, immunohistochemical staining, TK1 activity inhibition assays, and enzyme-linked immunoadsorbent assay (ELISA) techniques. Thus, in the future, these antibodies may aid in the early detection of cancer and more accurate prognosis, as well as allowing for an increased ability to study the function of TK1 in basic cellular processes.

Conversion of herpetic lesions to malignancy by ultraviolet exposure and promoter application.

Many lines of evidence exist associating herpes simplex virus (HSV) with the development of carcinoma, but much of this evidence is anecdotal or associative in nature and does not prove a cause and effect. The purpose of this research was to investigate the oncogenic potential of HSV type 2 (HSV-2) in vivo. A mouse model for lip carcinogenesis was designed to combine HSV-2 infection, u.v. exposure and tetradecanoyl-phorbol-acetate (TPA) application. HSV-2 inoculation on to abraded mouse lips was capable of causing vesicular ulcerative lesions which healed completely after 10 to 14 days. Ultraviolet irradiation of the HSV lesion site daily for 6 min at 42 ergs/mm2/s on days 3 to 6 post-infection caused hyperkeratosis, acanthosis and dysplasia to develop in several lips; while the same u.v. exposure by itself failed to alter the histologic appearance. The addition to repeated TPA application to the HSV+ u.v. regimen promoted tumour emergence. Thirty-two of 156 Balb/c mice developed tumours. Although the majority were papillomas, six were squamous cell carcinomas. These tumour-bearing mice had increased HSV-specific antibody titres. HSV antigens were shown to be present in outgrowths from explanted tumours as well as in tumour biopsies by immunoperoxidase staining with HSV-specific antisera. It is suggested that the in vivo u.v.-irradiated HSV acted as the inducer and TPA as the promoter, analogous to the classical two-stage carcinogenesis model.

Protection from oral herpes simplex virus infection by a nucleic acid-free virus vaccine.

The effect of immunization with inactivated herpes virus vaccines, including a vaccine free of all nucleic acid, was investigated in a mouse model system. Protection against oral lesions induced by herpes simplex virus type 1 was demonstrated by several criteria: (i) reduction in the incidence and severity of primary oral lesions; (ii) decrease in acute and latent infection of the regional sensory ganglia; and (iii) protection from viral encephalitis and death. The immune response of mice to the vaccine and to subsequent virus challenge was measured by following serum-neutralizing antibody titers.

High resolution separation and quantitation of ribonucleotides using capillary electrophoresis.

A method for the analysis of ribonucleotides using capillary electrophoresis has been developed. A cross-linked polyacrylamide coated column, Tris-HCl, and phosphate-mixed buffer were used, which produced reproducible separations (solute migration time average RSD% of 1.2) of 14 ribonucleotides within 50 min. Linear relationships between peak areas and sample concentrations, an average minimum detectable concentration of 5.4 microM, and an average minimum detectable quantity of 0.08 pmol were obtained. The described method allowed reproducible and reliable qualitative and quantitative analysis of intracellular ribonucleotide pools in HeLa cells.

Immune responses to labial infection of BALB/c mice with herpes simplex virus type 1.

The kinetics of appearance of five humoral antibody responses (micro-neutralization assay [NT], complement fixation [CF], enzyme-linked immunosorbent assay [ELISA], radioimmunoassay [RIA], antibody-dependent cell-mediated cytotoxicity [ADCC]), were compared during labial infection of BALB/c mice with herpes simplex virus type 1 strain Patton. The ELISA/RIA antibody responses were present in most mice by day 5 after infection, at the beginning of the herpetic lip lesions; antibody effective in ADCC showed identical early kinetics. In contrast, NT/CF antibodies were not detected in most mice until day 10, at the time of resolution of the herpetic lip lesions. The humoral immune responses persisted for at least 6 months after infection. The NT and CF responses were closely correlated in time of appearance and titers (r = 0.9), as were the ELISA and RIA responses (r = 0.99). However, there was little correlation between NT/CF and ELISA/RIA responses (r = 0.02). The kinetics of the delayed type hypersensitivity response showed similar kinetics of appearance to the ELISA/RIA/ADCC humoral responses, and peaked similarly, but waned gradually over 2 months. The importance of antibody in protection against labial herpes simplex virus type 1 infection was demonstrated by the ability of passively transferred convalescent serum (that produced a minimum NT titer of 10 in recipient mice) to protect against development of herpetic lesions and death.

Effect of pyran on latency after herpes simplex virus infections.

The immunomodulator pyran protected mice against both herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections. In infections of the lip with HSV-1, prophylactic administration of pyran reduced the severity of the herpetic lesions and enhanced their resolution, but did not decrease the high incidence of development of latent HSV-1 infection of the trigeminal ganglia. In vaginal infections with HSV-2, prophylactic administration of pyran either systemically or locally reduced mortality, reduced the incidence of mice with vaginal HSV-2 infection, and did not alter the low incidence of latent infection of the spinal dorsal root ganglia. Pyran treatment before systemic herpetic infection after intravenous inoculation of HSV-2 also reduced mortality and virus replication, as evidenced by a decreased antibody response in the survivors, and it either reduced latent infection in the spinal dorsal root ganglia or did not predispose mice to latent infection. Treatment with the immunomodulator appeared to inhibit or reduce HSV infection early in viral pathogenesis in all three model systems, producing protection from clinical disease and resulting in less virus to induce a systemic antibody response, with either a reduction in latent virus infection or no enhancement of development of latency. In all of the HSV models, the development of latent herpetic infection was closely correlated with sufficient virus replication early in the infection to induce a systemic neutralizing-antibody response.

Broad-spectrum synergistic antiviral activity of selenazofurin and ribavirin.

The antiviral effects of selenazofurin (2-beta-D-ribofuranosylselenazole-4-carboxamide, selenazole), ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), and 3-deazaguanosine (6-amino-1-beta-D-ribofuranosylimidazo-[4.5-C]pyridin-4(5H)-one) were investigated separately and in various combinations in an in vitro study. The combination interactions were evaluated at seven drug concentrations, graphically (isobolograms) or by using fractional inhibitory concentration indices against mumps, measles, parainfluenza virus type 3, vaccinia and herpes simplex virus type 2 viruses in Vero and HeLa cells. Selenazofurin in combination with ribavirin produced the greatest synergistic antiviral activity. However, the degree of synergy depended on the virus and cell line used. In contrast, selenazofurin combined with 3-deazaguanosine consistently yielded an indifferent or an antagonistic response, or both, whereas the ribavirin-3-deazaguanosine interaction was additive against the same viruses. Single-drug cytotoxicity was minimal for the cytostatic agents selenazofurin and ribavirin but was markedly higher for cytocidal 3-deazaguanosine, as determined by relative plating efficiency after drug exposure. The drug combinations did not significantly increase cytotoxicity (they were only additive) when used on uninfected cells. Therefore, the enhanced antiviral activities of the drug combinations (shown to be synergistic) were due to specific effects against viral replication. These results indicated that in Vero and HeLa cells (i) the combination of selenazofurin and ribavirin produced an enhanced antiviral effect, thus requiring smaller amounts of drug to cause the same antiviral effect relative to a single compound; (ii) selenazofurin when compared with ribavirin and 3-deazaguanosine appeared to have a somewhat different mode of antiviral action; (iii) 3-deazaguanosine combined with selenazofurin was an unsuitable antiviral combination; and (iv) the antiviral activity of 3-deazaguanosine appeared to be due largely to its general overall cytotoxic effect.

Cell cycle arrest and differential gene expression in HT-29 cells exposed to an aqueous garlic extract.

Epidemiological data show an inverse correlation between garlic consumption and the risk for colon cancer. To examine this relationship, HT-29 human adenocarcinoma cells were cultured in the presence and absence of an aqueous garlic extract. Garlic treatment resulted in a fraction of cells detaching from the culture flasks. These cells remained viable. Flow cell cytometry showed that untreated cells exhibited a normal distribution among phases of the cell cycle, with 12% of cells at the G2/M boundary. Of the garlic-treated cells remaining attached to the flask, 27% were present at the G2/M boundary. Treated cells that detached from the flask were found almost exclusively (89%) at the G2/M boundary. RNA fingerprinting and microarray analysis showed that expression of the gene for menin was twice as high in control cells as in detached treated cells. In contrast, expression of genes for epidermal growth factor receptor and integrin-alpha6 was nearly twice as high in detached treated cells as in control cells. These changes in gene expression were consistent with an arrest of the cell cycle at the G2/M boundary. Garlic's arrest of the cell cycle in human adenocarcinoma cells may explain in part its anticarcinogenic properties.

Cyclic appearance of defective interfering particles of herpes simplex virus and the concomitant accumulation of early polypeptide VP175.

Serial passage of undiluted herpes simplex virus types 1 and 2 resulted in cyclic production of infectious and defective virions. Defective virus production was characterized by the appearance of a new species of viral DNA with a higher bouyant density in CsCl than standard viral DNA. Measurement of the infectivity titer and DNA synthesis revealed that the defective particles interfered with the replication of standard virions and stimulated the overproduction of a large molecular weight (175,000 daltons) polypeptide.

In vitro virucidal effects of Allium sativum (garlic) extract and compounds.

Garlic (Allium sativum) has been shown to have antiviral activity, but the compounds responsible have not been identified. Using direct pre-infection incubation assays, we determined the in vitro virucidal effects of fresh garlic extract, its polar fraction, and the following garlic associated compounds: diallyl thiosulfinate (allicin), allyl methyl thiosulfinate, methyl allyl thiosulfinate, ajoene, alliin, deoxyalliin, diallyl disulfide, and diallyl trisulfide. Activity was determined against selected viruses including, herpes simplex virus type 1, herpes simplex virus type 2, parainfluenza virus type 3, vaccinia virus, vesicular stomatitis virus, and human rhinovirus type 2. The order for virucidal activity generally was: ajoene > allicin > allyl methyl thiosulfinate > methyl allyl thiosulfinate. Ajoene was found in oil-macerates of garlic but not in fresh garlic extracts. No activity was found for the garlic polar fraction, alliin, deoxyalliin, diallyl disulfide, or diallyl trisulfide. Fresh garlic extract, in which thiosulfinates appeared to be the active components, was virucidal to each virus tested. The predominant thiosulfinate in fresh garlic extract was allicin. Lack of reduction in yields of infectious virus indicated undetectable levels of intracellular antiviral activity for either allicin or fresh garlic extract. Furthermore, concentrations that were virucidal were also toxic to HeLa and Vero cells. Virucidal assay results were not influenced by cytotoxicity since the compounds were diluted below toxic levels prior to assaying for infectious virus. These results indicate that virucidal activity and cytotoxicity may have depended upon the viral envelope and cell membrane, respectively. However, activity against non-enveloped virus may have been due to inhibition of viral adsorption or penetration.(ABSTRACT TRUNCATED AT 250 WORDS)

Herpes simplex virus vaccine: protection from stomatitis, ganglionitis, encephalitis and latency.

A mouse model system was developed for studying the pathogenesis of oral infection with herpes simplex virus type 1 and the protection offered by prior immunization with a nucleic acid-free vaccine. Of non-immunized mice, 95-100% developed ulcerative lesions 3-5 days following application of virus to abraded oral epithelial surfaces. Infection of the ipsilateral sensory (trigeminal) ganglion and the cerebellum occurred by day 2 and sequentially progressed to the contralateral ganglion by day 4 and to the cerebrum by day 5. Prior immunization of mice with an inactivated virus vaccine, and most importantly, with a vaccine free of nucleic acid, protected mice from subsequent oral virus infection. Protection was demonstrated by: (i) reduction in the incidence and severity of primary oral lesions; (ii) a decrease in the number of mice with acute ganglionic infection or dying of encephalitis; and (iii) a reduction in the incidence of latent trigeminal ganglionic infection.

Broad-spectrum antiviral activity of 2-beta-D-ribofuranosylselenazole-4-carboxamide, a new antiviral agent.

The relative in vitro antiviral activities of three related nucleoside carboxamides, ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide), and selenazole (2-beta-D-ribofuranosylselenazole-4-carboxamide), were studied against selected DNA and RNA viruses. Although the activity of selenazole against different viruses varied, it was significantly more potent than ribavirin and tiazofurin against all tested representatives of the families Paramyxoviridae (parainfluenza virus type 3, mumps virus, measles virus), Reoviridae (reovirus type 3), Poxviridae (vaccinia virus), Herpes-viridae (herpes simplex virus types 1 and 2), Togaviridae (Venezuelan equine encephalomyelitis virus, yellow fever virus, Japanese encephalitis virus), Bunyaviridae (Rift Valley fever virus, sandfly fever virus [strain Sicilian], Korean hemorrhagic fever virus), Arenaviridae (Pichinde virus), Picornaviridae (coxsackieviruses B1 and B4, echovirus type 6, encephalomyocarditis virus), Adenoviridae (adenovirus type 2), and Rhabdoviridae (vesicular stomatitis virus). The antiviral activity of selenazole was also cell line dependent, being greatest in HeLa, Vero-76, and Vero E6 cells. Selenazole was relatively nontoxic for Vero, Vero-76, Vero E6, and HeLa cells at concentrations of up to 1,000 micrograms/ml. The relative plating efficiency at that concentration was over 90%. The effects of selenazole on viral replication were greatest when this agent was present at the time of viral infection. The removal of selenazole from the medium of infected cells did not reverse the antiviral effect against vaccinia virus, but there was a gradual resumption of viral replication in cells infected with parainfluenza type 3 or herpes simplex virus type 1 (strain KOS). However, the antiviral activity of ribavirin against the same viruses was reversible when the drug was removed.