Antiviral assay

Further explorations around 4 led to identification of molecules such as tropane-based 5 and 6 (Figure 2), which were practically equipotent in both the MIP-1 (3 inhibition of binding and in the PM-1 /BaL antiviral assay [23]. However, 6 also proved to be a potent inhibitor of hERG ion channel (99% inhibition at 1 (iM concentration [23] and 80% inhibition at the rate of 300 nM [22]). 

Reports on the antiviral activity of flavanones isolated from medicinal plants have also been found in the literature. Two new antiviral flavanone derivatives were isolated from a methanol extract of leaves of Pithecellobium clypearia (Jack.) Benth (Fabaceae), guided by antiviral assays.The structures were characterized as 7-0-galloyltricetifavan (Fig. 14) and 7,4 -di-0-galloyltricetifavan. CPE reduction assay showed that both compounds possess antiviral activity against respiratory syncytial virus, influenza A HlNl virus, CVB3 and HSV-1. Examples of other... 

Amathaspiramide E (128) from the New Zealand bryozoan Amathia wilsoni exhibits strong activity in the antiviral assay against Polio virus [102],... 

Interferon solutions in MEM (30 lU/mL) were preincubated with the indicated gan-glioside concentrations at 37°C for 30 min before addition to the L-cell monolayer. Antiviral assays are described in Materials and Methods. Y EMC titer in the absence of interferon. This titer was unchanged in the presence of both ganglioside preparations up to a concentration corresponding to 100 p.M sialic acid. 

The 7-azabenzisoselenazol-3(27/)-ones (169) (Fig. 12), substituted at the 2-position with phenyl or alkyl groups, and the methiodides (170) were found in the antiviral assay to be strong inhibitors of cytopathic activity of herpes simplex type 1 virus (HSV-1) and encephalomyocarditis virus (EMCV), more potent than ebselen. The minimal inhibitory concentration (MIC) values were in the range 0.4-6.0 pg mL 1, substantially lower than those when toxicity was observed. The vesicular stomatis virus (VSV) remained resistant toward tested compounds, except moderately active methiodide (171) [51, 271],... 

Thus the infection process by enveloped virus VSV is generally more sensitive to the triterpenic glycosides than that of naked viruses like HRV. In view of the interesting antiviral activity of triterpenoids we extended the antiviral assays to triterpenes 1,8 and 9 and their esters 11-14 isolated from E. japomca (3). 

With the increase in understanding of viral replication and in particular viral protein and nucleic acid synthesis, and the development of reliable and sophisticated antiviral assays, the young science of antiviral chemotherapy has progressed tremendously over the last 50 years. Acyclovir (Fig. 5.9) was probably the first truly effective and selective... 

Advances in the screening of chemical libraries has led to the discovery of nonnucleoside inhibitors of RT (NNRTIs). Two approaches can be followed. When an appropriate antiviral assay is available compounds can be screened in whole cells. These assays are frequently labor intensive and therefore generally have a lower throughput than isolated enzyme assays. These assays have the advantage that chemical leads generated by this approach have already overcome the barriers of cell penetration and stability. 

The discovery of another NNRTI, nevirapine, illustrates the second approach (see Scheme 6). Here it is no longer necessary to have access to an antiviral assay to discover active entities. Compounds are screened against an isolated enzyme. High throughput of compounds in the screen is a key advantage of this method enabling the testing of thousands of compounds however, com-... 

To set up compounds for toxicity treatment For each compound, a total of four concentrations will be examined. This will require nine sets of four sterile, 1.1-mL minitubes (36 tubes/compound). Into the first tube of each set, aliquot sufficient compound to make up 465 pL of the highest test concentration. Leave the other tubes empty. Cover the tubes with the rack lids, and store the racks at -20°C or at an appropriate temperature. Tubes for the last day of treatment for toxicity testing contain the same amount of compound as used the previous days (not 3X as for the antiviral assays). 

Toxicity is evaluated by determining the level of stain uptake in wells treated with dilutions of test compound(s) covering the desired target range for antiviral assays. The assay should mirror the antiviral assay to be used in cell type, incubation temperature, overlay medium, and incubation time, but no virus should be used. For the evaluation of toxicity, a stain, such as crystal violet,... 

This chapter describes the procedures that can be used to determine compounds that have antiviral activity against HIV. These include maintenance of lymphoblastoid cell lines, preparation of peripheral blood mononuclear cells (PMMCs), and determination of the infectivity of the HIV stock-supernatant and antiviral assays. The assays described use both acutely and chronically infected cells. Toxicity of compounds is assessed by measuring 14C uptake. These protocols are used for the evaluation of compounds that can be carried out by a single individual in a Category 3 containment laboratory. The number of compounds analyzed would be about 10, which is a convenient number to fill a single 96-well p24 enzyme-linked immunosorbent assay (ELISA) plate. 

A sensitive method to measure toxic effects of drugs is to monitor cellular metabolism by incorporation of 3H-thymidine or 14C-protein hydrolysate. 14C-protein is used in preference to 3H-thymidine when it is necessary to avoid competition in uptake between the labeled thymidine and an unlabeled nucleoside reverse-transcriptase inhibitor. These methods are used mainly to monitor sublethal toxicity after initial screening or when comparing structure-activity relationships. The assays described here are carried out using 6-mL culture tubes (Note 1). These assays are carried out in parallel with the antiviral assay. 

Pipet 2 pL of the same concentration range of each compound as used in the antiviral assay. Add 200 pL of the uninfected cells. If an organic solvent, such as DMSO, is used then add 2 pL to the untreated controls. This gives a final concentration of the solvent of 1%. 

Steps 1-6 are the same as the previous assay (the period of contact between the cells and the virucide should be the same as that selected for the antiviral assays). 

The inhibitory effects of the pyrrolinone derivatives were evaluated using enzyme inhibition and cellular activation assays. Compound 6 (Fig. 4.3-5) showed an IC50 of 10 nM, compared to 0.6 nM for the related peptide inhibitor 5 (L682,679). However, the synthetic agent 6 showed better cell transport capacity. In a cellular antiviral assay, 5 and 6 showed CIC95 values (the concentration that inhibits 95% of virus multiplication in the cellular cultures) of 6.0 and 1.5 pM, respectively. Smith and Hirschmann proposed that the improved cellular uptake properties of polypyrrolinones are due to a reduction in the inhibitor solvation. Solvation is an impediment to transport because extraction of a molecule into a lipid bilayer from an aqueous phase is... 

Malibatol A (3) Cytotoxicity to the host cells (CEM SS) in a HIV antiviral assay 51... 

Antiviral assays of the cycHc diesters 100c—e and lOOg as PFA prodmgs were conducted on HSV-1-infected confluent human fibroblast cells. Prodrug lOOd displays a similar activity to PFA whereas 100c, lOOe, and lOOg are less active than PFA. 

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