A HIGH-CAPACITY MICROBIAL SCREEN FOR INHIBITORS OF HUMAN RHINOVIRUS PROTEASE 3C

We have developed a high capacity screen for compounds that inhibit th e 3C protease of human rhinovirus-1b. The assay uses a recombinant str ain of Escherichia coli expressing both the protease and a tetracyclin e resistance-conferring protein modified to contain the minimal protea se cleavage site. Cultures growing in microtiter plates containing tet racycline are treated with potential inhibitors and simultaneously mon itored for change in growth over time using an oxygen probe. Most of t he cultures, not containing an inhibitor of the 3C protease, show redu ced growth due to cleavage of the essential gene product; normal growt h is seen only in the infrequent culture that contains an inhibitor. I n the present example, we have used the tetA gene of plasmid pACYC184 as the modified gene. The system has been validated using inhibitors o f protease 3C, and has been used to identify three new inhibitors of t he enzyme, active in the micromolar range.