A fission yeast cell-based system for multidrug resistant HIV-1 proteases
A fission yeast cell-based system for multidrug resistant HIV-1 proteases
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DOI：
10.1186/s13578-016-0131-5
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发表时间：
2017-01-11
影响因子：
7.5
通讯作者：
Zhao, Richard Y.
中科院分区：
文献类型：
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作者：
Benko, Zsigmond;Liang, Dong;Zhao, Richard Y.
背景：HIV-1蛋白水解酶(PR)是病毒产生的必需酶。因此，PR抑制剂(PI)是最有效的抗HIV药物。然而，PI类药物在患者治疗中成功应用的主要挑战是出现多药耐药PR((MDR)PR)。结果：从HIV感染者中分离到3株mdrPR，分别携带7个(M7PR)、10个(M10PR)和11个(M11PR)PR基因突变。它们被克隆并在可诱导启动子下在分裂酵母中表达，以允许测量PR特异的蛋白分解和耐药性。结果表明，这三种mdrPR都保持了其对HIV病毒底物(MA、下箭头、CA和p6)的蛋白分解能力和赋予耐药性的能力。在分裂酵母中产生这些蛋白质会导致细胞生长抑制、氧化应激和线粒体形态改变，从而导致细胞死亡。五个调查性PI被用来测试所建立的酵母系统的效用，并以FDA批准的PI药物达鲁那韦(DRV)为对照。所有6个化合物均抑制野生型PR(WtPR)和M7PR介导的活性。但对M10PR和M11PR均无抑制作用。结论：临床分离的3株mdrPR在裂殖酵母中保持了病毒的蛋白分解活性和耐药性。此外，这些病毒mdrPR活性与诱导生长抑制和细胞死亡相结合，可用于检测PI活性。事实上，这五个研究的PI和DRV抑制了分裂酵母中的wtPR，就像它们在哺乳动物细胞中所做的那样。值得注意的是，两个高水平(MDR)PR(M10PR和M11PR)对包括DRV在内的所有现有PI药物都具有耐药性。这一观察结果强调了继续寻找新的针对(MDR)PR的PI的重要性。
Background: HIV-1 protease (PR) is an essential enzyme for viral production. Thus, PR inhibitors (PIs) are the most effective class of anti-HIV drugs. However, the main challenge to the successful use of PI drugs in patient treatment is the emergence of multidrug resistant PRs ((mdr)PRs). This study aimed to develop a fission yeast cell-based system for rapid testing of new PIs that combat (mdr)PRs.Results: Three mdrPRs were isolated from HIV-infected patients that carried seven (M7PR), ten (M10PR) and eleven (M11PR) PR gene mutations, respectively. They were cloned and expressed in fission yeast under an inducible promoter to allow the measurement of PR-specific proteolysis and drug resistance. The results showed that all three mdrPRs maintained their abilities to proteolyze HIV viral substrates (MA down arrow CA and p6) and to confer drug resistance. Production of these proteins in the fission yeast caused cell growth inhibition, oxidative stress and altered mitochondrial morphologies that led to cell death. Five investigational PIs were used to test the utility of the established yeast system with an FDA-approved PI drug Darunavir (DRV) as control. All six compounds suppressed the wildtype PR (wtPR) and the M7PR-mediated activities. However, none of them were able to suppress the M10PR or the M11PR.Conclusions: The three clinically isolated mdrPRs maintained their viral proteolytic activities and drug resistance in the fission yeast. Furthermore, those viral mdrPR activities were coupled with the induction of growth inhibition and cell death, which could be used to test the PI activities. Indeed, the five investigational PIs and DRV suppressed the wtPR in fission yeast as they did in mammalian cells. Significantly, two of the high level (mdr)PRs (M10PR and M11PR) were resistant to all of the existing PI drugs including DRV. This observation underscores the importance of continued searching for new PIs against (mdr)PRs.