Ast O, Jentsch K D, Schramm H J, Hunsmann G, Lüke W, Petry H
German Primate Centre, Department of Virology and Immunology, Göttingen.
J Virol Methods. 1998 Mar;71(1):77-85. doi: 10.1016/s0166-0934(97)00202-4.
The HIV-1 protease is essential for maturation of virus particles and is, therefore, an attractive target for antiviral drugs. The function of this protease depends on the dimerization of two identical subunits. Commonly used protease inhibitors are directed mainly against the active site of the enzyme which often leads to viral resistance. To determine the inhibitory effect of peptides interfering with the dimerization site of the HIV-1 protease, a recombinant bacterial screening assay was established. Escherichia coli was co-transformed with two different plasmids, expressing the 'interface' peptide and an active HIV-1 protease toxic for the bacteria. Co-expression of inhibitory peptides overcomes the incomplete membrane transmission of supplemented inhibitors and leads to a direct interaction of the inhibitory peptide and the HIV-1 protease. The inhibitory effect of co-expressed peptides was measured by an increased growth of co-transformed bacteria, compared with a slowly growing E. coli control culture only expressing the HIV-1 protease. Using this assay several penta- and hexa-peptides were screened for their ability to inhibit HIV-1 protease activity. One of these peptides showed a significant inhibitory effect on co-expressed recombinant HIV-1 protease.
HIV-1蛋白酶对于病毒颗粒的成熟至关重要，因此是抗病毒药物的一个有吸引力的靶点。这种蛋白酶的功能依赖于两个相同亚基的二聚化。常用的蛋白酶抑制剂主要针对酶的活性位点，这常常导致病毒产生抗性。为了确定干扰HIV-1蛋白酶二聚化位点的肽的抑制作用，建立了一种重组细菌筛选试验。将大肠杆菌用两种不同的质粒共转化，这两种质粒分别表达“界面”肽和对细菌有毒性的活性HIV-1蛋白酶。抑制肽的共表达克服了补充抑制剂不完全的膜转运，并导致抑制肽与HIV-1蛋白酶直接相互作用。与仅表达HIV-1蛋白酶的生长缓慢的大肠杆菌对照培养物相比，通过共转化细菌生长的增加来测量共表达肽的抑制作用。利用该试验筛选了几种五肽和六肽抑制HIV-1蛋白酶活性的能力。其中一种肽对共表达的重组HIV-1蛋白酶显示出显著的抑制作用。
