McCall J O, Kadam S, Katz L
Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064.
Biotechnology (N Y). 1994 Oct;12(10):1012-6. doi: 10.1038/nbt1094-1012.
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.
我们开发了一种用于筛选抑制人鼻病毒1b型3C蛋白酶的化合物的高通量筛选方法。该检测方法使用一种重组大肠杆菌菌株，该菌株同时表达蛋白酶和一种经修饰含有最小蛋白酶切割位点的赋予四环素抗性的蛋白质。在含有四环素的微量滴定板中生长的培养物用潜在抑制剂处理，并同时使用氧探头监测其随时间的生长变化。大多数不含3C蛋白酶抑制剂的培养物由于必需基因产物的切割而生长减少；只有在极少数含有抑制剂的培养物中才能观察到正常生长。在本实例中，我们使用质粒pACYC184的tetA基因作为修饰基因。该系统已用3C蛋白酶抑制剂进行了验证，并已用于鉴定三种新的该酶抑制剂，其活性在微摩尔范围内。
