Anti-picornaviral compounds
a technology of anti-picornaviral compounds and anti-viral drugs, which is applied in the field of screening viral binding compounds, can solve the problems of insufficient prophylactic effect of available drugs, high cost and time-consuming process of screening procedures, and achieve the effect of rapid screening of anti-viral compounds
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example 1
Formation of Virus-Compound Complexes
[0027] The initial phase of the assay involves the incubation of virus with libraries of compounds to allow for the specific interaction between the compounds and virus. Poliovirus (P1 / Mahoney, or P1M) is grown in HeLa cells and purified by differential centrifugation and CsCl density gradient fractionation according to standard methods (Rueckert, R. R. and Pallansch, M., Methods in Enzymol. 78:315-325 (1981)). The virus is stored frozen in phosphate buffered saline (PBS) at −80° C. until use. Stock solutions of the virus are in PBS, while mixtures of potential ligands are dissolved in DMSO. These stock solutions are diluted such that all incubations are carried out at a final DMSO concentration of 5% in PBS. Final volumes ranged from 0.5 to 1.0 mL. Once mixed, the incubations were left at room temperature for one to four hours or kept at 4° C. overnight (See FIG. 1 for schematic).
[0028] The sensitivity of the assay is largely dictated by two ...
example 2
Purification of Virus-Compound Complexes
[0030] Virus-drug mixtures are loaded onto a size exclusion column (1.4 cm diameter×8.4 cm height) having a Sephacryl S-200 or S-300 matrix (Pharmacia) to separate virus-drug complexes from unbound drug. The running buffer is 5% DMSO in PBS. One mL fractions are collected at 0.5 mL / min. Identification of fractions containing the void volume, and therefore possibly containing viruses bound with compound, is accomplished with a Pharmacia Uvicord SII UV monitor which measures the OD280 of the material as it is pumped out of the column (See FIG. 1 for schematic).
example 3
Extraction and Concentration of Virus-Bound Drug
[0031] Fractions containing virus bound with compounds are mixed with a double volume of ethyl acetate, vortexed for 30 seconds, then centrifuged for 10 minutes at 12,000×g. At this point the viruses will be completely denatured. Previously virus-bound drug is released, and will partition into the ethyl acetate phase. The upper organic phase is separated from the aqueous phases. The organic phase containing the compounds is dried in a centrivap (See FIG. 1 for schematic).
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