Potent inhibition of HIV-1 replication by novel non-peptidyl small molecule inhibitors of protease dimerization
Potent inhibition of HIV-1 replication by novel non-peptidyl small molecule inhibitors of protease dimerization
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DOI：
10.1074/jbc.m703938200
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发表时间：
2007-09-28
影响因子：
4.8
通讯作者：
Mitsuya, Hiroaki
中科院分区：
文献类型：
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作者：
Koh, Yasuhiro;Matsumi, Shintaro;Mitsuya, Hiroaki
HIV-1蛋白酶亚基的二聚化是其蛋白水解活性所必需的，蛋白水解活性在HIV-1复制中起着关键作用。因此，蛋白酶二聚化的抑制代表了HIV-1潜在干预的独特靶标。我们开发了一种基于分子间荧光共振能量转移的HIV-1表达检测方法，采用青色和黄色荧光蛋白标记的蛋白酶单体。使用该测定，我们鉴定了蛋白酶二聚化的非肽基小分子抑制剂。这些抑制剂，包括地瑞那韦和两种实验性蛋白酶抑制剂，在低至0.01 μ M的浓度下阻断蛋白酶二聚化，并阻断HIV-1复制，IC 50值为0.0002-0.48 μ M。这些试剂还抑制成熟蛋白酶的蛋白水解活性。除了替拉那韦（一种CCR 5抑制剂）和可溶性CD 4外，其他已批准的抗HIV-1药物均未能阻断二聚化事件。一旦蛋白酶单体二聚化成为成熟蛋白酶，成熟蛋白酶不会通过这种二聚化抑制机制解离，表明这些试剂在蛋白酶成熟的新生阶段阻断二聚化。尽管存在这些试剂，但仍设法经历二聚化的成熟蛋白酶的蛋白水解活性可能被用作常规蛋白酶抑制剂的相同试剂抑制。这种双重抑制机制应导致对HIV-1的高度有效抑制。
Dimerization of HIV-1 protease subunits is essential for its proteolytic activity, which plays a critical role in HIV-1 replication. Hence, the inhibition of protease dimerization represents a unique target for potential intervention of HIV-1. We developed an intermolecular fluorescence resonance energy transfer-based HIV-1-expression assay employing cyan and yellow fluorescent protein-tagged protease monomers. Using this assay, we identified non-peptidyl small molecule inhibitors of protease dimerization. These inhibitors, including darunavir and two experimental protease inhibitors, blocked protease dimerization at concentrations of as low as 0.01 mu M and blocked HIV-1 replication with IC50 values of 0.0002-0.48 mu M. These agents also inhibited the proteolytic activity of mature protease. Other approved anti-HIV-1 agents examined except tipranavir, a CCR5 inhibitor, and soluble CD4 failed to block the dimerization event. Once protease monomers dimerize to become mature protease, mature protease is not dissociated by this dimerization inhibition mechanism, suggesting that these agents block dimerization at the nascent stage of protease maturation. The proteolytic activity of mature protease that managed to undergo dimerization despite the presence of these agents is likely to be inhibited by the same agents acting as conventional protease inhibitors. Such a dual inhibition mechanism should lead to highly potent inhibition of HIV-1.