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We have developed a high capacity screen for compounds that inhibit the 3C protease of human rhinovirus-1b. The assay uses a recombinant strain of Escherichia coli expressing both the protease and a tetracycline resistance-conferring protein modified to contain the minimal protease cleavage site. Cultures growing in microtiter plates containing tetracycline are treated with potential inhibitors and simultaneously monitored for change in growth over time using an oxygen probe. Most of the cultures, not containing an inhibitor of the 3C protease, show reduced growth due to cleavage of the essential gene product; normal growth is seen only in the infrequent culture that contains an inhibitor. In the present example, we have used the tetA gene of plasmid pACYC184 as the modified gene. The system has been validated using inhibitors of protease 3C, and has been used to identify three new inhibitors of the enzyme, active in the micromolar range.

A High Capacity Microbial Screen for Inhibitors of Human Rhinovirus Protease 3C