The chiral synthesis and biochemical properties of electron rich phenolic sulfoxide analogs of sparsomycin.

A novel route to activated phenolic sulfoxide analogs of sparsomycin has been developed. These analogs display an enhanced "preincubation effect" as inhibitors of peptide-bond formation. This time-dependent component of inhibition, which is postulated to result from an enzyme-mediated Pummerer rearrangement, is the dominant route to inhibition in these activated analogs.

Inhibition of herpes simplex virus replication by anthracycline compounds.

The replication of type 1 and type 2 strains of herpes simplex virus (HSV) was inhibited greater than 99.9% by low concentrations (0.1-0.2 microM) of anthracycline compounds. The degree of viral inhibition was dependent upon the host cell. N,N-dimethyl daunomycin (NDMD), a non-mutagenic compound, was more potent as an inhibitor of HSV synthesis than either daunomycin (DM) or adriamycin (AD). The depression of viral yield by DM or AD was attributable, in part, to a temperature-dependent direct effect on infectious virions. Tritium-labeled DM bound tightly to HSV particles. NDMD did not directly inactivate virions in spite of superior potency in reducing viral yields. All three anthracyclines could be added late in the infectious cycle (6-8 h p.i.) and retain effectiveness. Cesium chloride density gradient analysis verified that viral DNA synthesis was blocked by addition of all three anthracyclines early in the infectious cycle. The inhibition of HSV replication was not a simple consequence of the suppression of host DNA synthesis since treatment of cells with compounds for 24 h before infection did not reduce virus yields even though host DNA synthesis was inhibited by 90%. Further, the kinetics of inhibition of cellular DNA synthesis by anthracyclines was similar in HFF or Vero cells but the degree of inhibition of virus replication was markedly different. The data suggest that anthracyclines with substitutions on the sugar moiety may be useful anti-herpes agents.

Butyrate-induced reversal of herpes simplex virus restriction in neuroblastoma cells.

The synthesis of herpes simplex virus (HSV) in mouse neuroblastoma cells (NB, clone 41A3) is restricted. There was a disappearance of infectious virus upon serial passage of infected cells. NB cells treated with sodium-n-butyrate for 24 hr before infection synthesized 200-2000 times more HSV than untreated cells. Infectious center assays demonstrated that the number of cells capable of producing HSV was increased as a result of butyrate pretreatment. Although host protein synthesis was inhibited by HSV infection, viral-induced protein and DNA syntheses were not detected in the absence of butyrate. Cycloheximide blocked the induction of permissiveness by butyrate suggesting that a protein(s) was responsible for allowing HSV synthesis in NB cells. Regulatable host factors involved in HSV replication in neural cells can be studied in the system described.

Sulfoxide configuration in sparsomycin determines time-dependent and competitive inhibition of peptidyl transferase.

Sparsomycin, ScRs configuration, was the most potent of the four possible stereoisomers as a competitive inhibitor of peptide bond formation. In addition, the configuration of the two chiral centers dictated whether the compound exhibited time- and temperature-dependent inhibition of peptidyl transferase when incubated with polysomes prior to enzyme assay. The data corroborate the thesis that a peptidyl transferase-mediated acylation of the pivotal sulfoxide moiety and subsequent Pummerer rearrangement play a significant role in the inhibitory properties of sparsomycin.

Importance of the hydrophobic sulfoxide substituent on nontoxic analogs of sparsomycin.

Nontoxic analogs of sparsomycin were competitive inhibitors of puromycin in the peptidyl transferase assay with Escherichia coli polysomes. The sensitivity of HeLa cells in vitro to the analogs was used as a preliminary index of cellular toxicity. In vitro killing of HeLa cells by this class of compounds correlated well with in vivo 50% lethal doses. The data indicate that modification of the hydrophobic sulfoxide substituent on sparsomycin decreases the toxicity of the molecule for mammalian cells by several hundredfold. Such modifications have less of an effect on the inhibitory activity of the compounds for peptidyl transferase. The differential effects of an analog active against bacterial but not mammalian cells was due to a decreased uptake of the compound by HeLa cells.

Necessity of the sulfoxide moiety for the biochemical and biological properties of an analog of sparsomycin.

An analog of the peptidyl transferase inhibitor sparsomycin was a competitive inhibitor (Ki = 1.8 microM) of peptidyl-puromycin synthesis on E. coli polysomes. Preincubation of polysomes with the compound enhanced the degree of inhibition of peptide bond formation. A model for the involvement of a histidine residue in peptidyl transferase activity is presented as a result of our observations which include direct association of [3H] labelled analog with 70S ribosomes. The correct oxidation state of sulfur in the compound was necessary for the "preincubation effect" and entry of the compound into bacterial cells.

RMI 15,731 (1-[5-tetradecyloxy-2-furanyl]-ethanone), a new antirhinovirus compound.

RMI 15,731 (1-[5-tetradecyloxy-2-furanyl]-ethanone) is a new antiviral compound with activity specific for rhinoviruses. Virus synthesis in R-HeLa cells was susceptible to low concentrations (0.25 micrograms/ml) of the compound. RMI 15,731 was effective if added for 1 h before infection and removed or if added as late as 6 h postinfection. Low concentrations of the compound directly inactivated infectious rhinovirus in a time- and temperature-dependent fashion. RMI 15,731 appears to be a promising new antirhinovirus compound for clinical evaluation since it (i) blocks intracellular virus synthesis (without cellular toxicity), and (ii) directly inactivates virions. The last property may be important in limiting virus shedding from an infected host.

Salivary neutralizing activity against herpes simplex virus type 1.

Samples of saliva collected from 40 patients, of whom 16 had a previous clinical history of herpes simplex virus type 1 (HSV-1) infection, were concentrated and sterilized. Salivary neutralizing activity, as measured by a plaque-reduction method, was found in 62% of those patients with a history of oral lesions. Studies with antisera to human immunoglobulin indicated that the IgG class of immunoglobulin was a major HSV-1-neutralizing component of both saliva and serum.

Effect of actinomycin D on plaque formation by mengovirus.

Treatment of L cell monolayers with actinomycin D resulted in an increase in both the size and number of mengovirus plaques when compared to untreated cells. Addition of the drug to the agar overlay was as effective as pretreatment provided the addition was immediately after virus adsorption. The increased plaque number observed was apparently not due to rescue of defective particles or inhibition of interferon production. The data indicate that more mengovirus particles can successfully complete the infectious cycle in L cells in the presence of actinomycin. The data also suggest that indigenous C-type particles in L cells may interfere with picornavirus replication.

Metabolic characteristics of cells infected with a herpesvirus of turkeys.

Chicken embryo fibroblasts infected with the nononcogenic herpesvirus of turkeys (HVT) displayed an increased rate of glucose uptake, a pronounced alteration of the pH of the medium, and an increased production of lactic acid when compared to mock-infected cultures. Objective estimates of cytopathology (quantifiable neutral red uptake and cell protein determination) showed that cell deterioration was a slow process in HVT-infected cells when compared to infection by herpes simplex virus. Experiments with irradiated host cells demonstrated that HVT required functional cell DNA for replication. The inactivation of the necessary host cell function displayed multihit kinetics. In agreement with data on other herpesviruses, HVT damaged by UV light could be photoreactivated in chick cells. The results indicate that HVT shares biologic properties in common with other herpes and transforming viruses.

Optimal cooling and warming rates in the preservation of herpes simplex virus (type 2).

Fast rates of cooling and warming were optimal for survival of herpes simplex virus, type 2. Under these conditions cryoprotectants were not necessary for virus preservation.

Influence of viral envelope on Newcastle disease virus infection.

The adsorption characteristics of Newcastle disease virus (NDV) propagated in chicken cells (NDV-C) and in human cells (NDV-H) were examined. Adsorption experiments performed at different temperatures indicated that virus propagated in a particular cell infected that cell type more readily than did virus propagated in a different host. For example, NDV-C was more efficient in initiating infection of chicken cells at 22 C than was NDV-H; the reverse was true when human cells were employed. The results indicate that infection of susceptible cells by NDV is influenced by the host cell in which the virus was propagated. The data also suggest that NDV may be useful in studies on homologous and heterologous membrane-membrane interactions.

Growth characteristics of a herpesvirus of turkeys.

The herpesvirus of turkeys remains associated with cells attached to the culture vessel throughout its growth cycle. The level of infectious centers is essentially constant between 24 and 48 hr when the culture medium is unchanged. Thereafter, the number of infectious centers decreases. The cessation of virus synthesis is not due to insufficient numbers of uninfected cells or interferon production. Herpesvirus of turkeys does not replicate at 22 C and is similar to other herpesviruses in its sensitivity to interferon. The results define conditions to be considered in the preparation of infected cell suspensions commonly used as a vaccine against Marek's disease virus.