THE SELECTIVE INHIBITION OF VIRAL DNA SYNTHESIS BY CHEMOTHERAPEUTIC AGENTS: AN INDICATOR OF CLINICAL USEFULNESS? *

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II Letter to the editor in response to J. Suhnel's comment on the paper: A three-dimensional model to analyze drug-drug interactions : Prichard, M.N. and Shipman, C., Jr. (1990) Antiviral Res. 14, 181-206

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I Letter to the editor in response to the paper: Efficacy of ganciclovir in combination with zidovudine against cytomegalovirus in vitro and in vivo : Freitas, V.R., Fraser-Smith, E.B., Chiu, S., Michelson, S. and Schatzman, R.C. (1993) Antiviral Res. 21, 301-315

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Antiviral activity of 2,3-Dihydro-1H-imidazo[1,2-b]pyrazole in herpes simplex virus type 1-infected mammalian cells

2,3-Dihydro-1H-imidazo[1,2-b]pyrazole (IMPY), a known inhibitor of DNA synthesis, has been shown to be a useful drug for the synchronization of mammalian cells in culture. Recent studies in our laboratory indicate that IMPY may possess significant antiviral activity against herpes simplex virus (HSV) type 1. IMPY. at a concentration of 80 [mu]g/ml or 0.73 mM, reduced syncytia formation approximately 80 per cent. A 50 per cent inhibitory dose was calculated for each drug in order to compare potency in syncytia reduction of IMPY with that of arabinosyladenine (ara-A) and arabinosyl-hypoxanthine (ara-H). Our results indicated that the antiviral potencies of the three drugs were ranked in the order ara-A > ara-H > IMPY, the 50 per cent inhibitory doses being 22, 195 and 309 [mu]M respectively. Utilizing the microplate procedure of Sidwell and Huffman [Appl. Microbiol. 22, 797 (1971)], inhibition of viral cytopathic effect was rated against drug cytotoxicity and a virus rating (VR) established. A virus rating of 0.68 was calculated for IMPY. In comparison, VR values of 0.84 and 0.66 were obtained for ara-A and ara-H respectively. In contrast to the syncytia reduction studies, IMPY appeared to possess antiviral activity equivalent to that of ara-H according to the criteria of the virus rating assay. A technique was developed for evaluating the degree of selectivity ([deg]S) of a drug with respect to its differential effect on viral and cellular DNA synthesis. IMPY was found to possess a negative selectivity at all concentrations studied, reflecting the fact that it inhibited cellular DNA synthesis more than viral DNA synthesis. In contrast, ara-A and ara-H both expressed positive degrees of selectivity in that they inhibited viral DNA synthesis more extensively than cellular DNA synthesis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21651/1/0000035.pd

Selective inhibition of herpes simplex virus ribonucleoside diphosphate reductase by derivatives of 2-acetylpyridine thiosemicarbazone

The effects of thiosemicarbazone derivatives of 2-acetylpyridine on mammalian and viral ribonucleoside diphosphate reductases were investigated. The enzymes were partially purified from uninfected and herpes simplex virus type-1 (HSV-1)-infected KB cells by sequential salt fractionation with streptomycin sulfate and ammonium sulfate and by affinity chromatography on ATP-agarose. The five thiosemicarbazone derivatives investigated were all potent inhibitors of the virus-induced reductase. Fifty percent inhibitory concentrations (50 values) range from 2 to 13 [mu]M. Four of the five derivatives also were inhibitors of the host cell reductase . A semicarbazone was inactive against the cellular enzyme and relatively weak as an inhibitor of the viral enzyme . Four of the six compounds were preferential inhibitors of the viral reductase based on a comparison of 50 values (5- to > 85-fold difference). Kinetic experiments revealed that inhibition of the HSV-1 reductase by the thiosemicarbazones was noncompetitive with respect to CDP and dithiothreitol. A comparison of the inhibitory effects of 2-acetylpyridine thiosemicarbazone itself on viral reductase and on virus replication in vitro demonstrated a similarity in the dose-response relationships for the two parameters. This observation supports the hypothesis that the HSV-induced ribonucleoside diphosphate reductase is an important target for the design of antiviral drugs.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26189/1/0000268.pd

Prevalence of type R virus-like particles in clones of BHK-21 cells

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Viral DNA synthesis is required for the efficient expression of specific herpes simplex virus type 1 mRNA species

Inhibition of HSV-1 DNA synthesis with either arabinosyladenine plus the adenosine deaminase inhibitor pentostatin, or with arabinosylthymine, showed a viral mRNA population identical to that seen prior to viral DNA replication (early) by the criteria of quantitative hybridization, specific mRNA species identifiable by Southern blot hybridization of size-fractionated RNA, and migration of polypeptides resolved by in vitro translation of purified viral mRNA. The amount of viral mRNA associated with infected cell polyribosomes was determined by quantitative DNA excess solution hybridization. At 2 hr postinfection (p.i.) (before viral DNA synthesis) and in drug-treated cells at 6 hr p.i., the majority of the polyadenylated RNA was cell specific with some virus-specific RNA detectable. In contrast, at 6 hr p.i., in the absence of drugs (during maximum viral DNA synthesis), nearly all the polyadenylated polyribosomal RNA was viral. Blot hybridization of size-fractionated viral RNA confirmed several specific differences between the viral mRNA species occurring before and after HSV-1 DNA synthesis, which have been reported previously from this laboratory. These differences also were reflected in the in vitro translation products encoded by the viral mRNAs. The mRNA species and the encoded polypeptides that were present in the absence of viral DNA synthesis are a subset of those viral mRNA species and polypeptides expressed in the presence of viral DNA synthesis. The viral mRNA species fall into several groups based on their relative abundance at various times of infection. These data suggest that, in the normal virus infection cycle, the onset of viral DNA synthesis is necessary for normal expression of later viral genes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23320/1/0000259.pd

A microtiter virus yield reduction assay for the evaluation of antiviral compounds against human cytomegalovirus and herpes simplex virus

Although the virus yield reduction assay is a powerful technique for evaluating the efficacy of antiviral compounds, it is not routinely utilized due to its labor-intensive nature. This procedure was modified, developed, thereby reducing greatly the time and effort required to perform yield reduction assays. Monolayer cultures of mammalian cells were grown in 96-well microtiter tissue culture plates and infected with virus. Test compounds were added and serially diluted directly with the plates. Following a cycle of virus replication, culture lysates were made and serially diluted in a separate set of uninfected cultures grown in microtiter plates. The cultures were incubated, plaques were enumerated in wells containing 5 to 20 plaques, and virus titers were calculated. To illustrate the use of the assay the known antiviral drugs acyclovir and ganciclovir were evaluated using this procedure. Ninety percent inhibitory concentrations for the respective drugs were 3 [mu]M and 0.7 [mu]M against herpes simplex virus type 1 and 60 [mu]M and 1 [mu]M against human cytomegalovirus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28636/1/0000450.pd

Molecular characterization and clinical relevance of metabolic expression subtypes in human cancers.

Metabolic reprogramming provides critical information for clinical oncology. Using molecular data of 9,125 patient samples from The Cancer Genome Atlas, we identified tumor subtypes in 33 cancer types based on mRNA expression patterns of seven major metabolic processes and assessed their clinical relevance. Our metabolic expression subtypes correlated extensively with clinical outcome: subtypes with upregulated carbohydrate, nucleotide, and vitamin/cofactor metabolism most consistently correlated with worse prognosis, whereas subtypes with upregulated lipid metabolism showed the opposite. Metabolic subtypes correlated with diverse somatic drivers but exhibited effects convergent on cancer hallmark pathways and were modulated by highly recurrent master regulators across cancer types. As a proof-of-concept example, we demonstrated that knockdown of SNAI1 or RUNX1—master regulators of carbohydrate metabolic subtypes-modulates metabolic activity and drug sensitivity. Our study provides a system-level view of metabolic heterogeneity within and across cancer types and identifies pathway cross-talk, suggesting related prognostic, therapeutic, and predictive utility