Evaluation of a system to screen for stimulators of non-specific DNA nicking by HIV-1 integrase: application to a library of 50,000 compounds.
Evaluation of a system to screen for stimulators of non-specific DNA nicking by HIV-1 integrase: application to a library of 50,000 compounds.
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DOI：
10.3851/imp1857
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发表时间：
2011-10-07
影响因子：
--
通讯作者：
Katzman M
中科院分区：
文献类型：
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作者：
Sudol M;Fritz JL;Tran M;Robertson GP;Ealy JB;Katzman M
除了催化整合所需的活性外，逆转录病毒整合酶还表现出非特异性核酸内切酶活性，这种活性可以通过某些小化合物增强，这表明整合酶可以在整合发生之前被刺激以破坏病毒DNA。使用非放射性、基于板的溶液相荧光测定来筛选 50,080 种药物样化学物质的库，以刺激人类免疫缺陷病毒 1 型 (HIV-1) 整合酶产生非特异性 DNA 切口。建立了半自动化工作流程，并且可以从图形输出中轻松识别主要命中。总体而言，0.6% 的化学物质导致荧光（主要命中率）大幅增加，但同时也没有可能人为导致此结果的可见颜色。然而，来自这个中等大小文库的潜在刺激物均未通过包括失活整合酶突变体的二次测试，该测试评估荧光的增加是否取决于整合酶的核酸内切酶活性。这是鉴定整合酶刺激剂 (IS) 化合物的首次尝试，建立了必要的物流和工作流程，这将鼓励更大规模的高通量筛选，以推进刺激整合酶破坏逆转录病毒 DNA 的新型抗病毒策略。
In addition to activities needed to catalyze integration, retroviral integrases exhibit nonspecific endonuclease activity that is enhanced by certain small compounds, suggesting that integrase could be stimulated to damage viral DNA before integration occurs. A nonradioactive, plate-based, solution-phase, fluorescence assay was used to screen a library of 50,080 drug-like chemicals for stimulation of nonspecific DNA nicking by human immunodeficiency virus type 1 (HIV-1) integrase. A semi-automated workflow was established, and primary hits were readily identified from a graphic output. Overall, 0.6% of the chemicals caused a large increase in fluorescence (the primary hit rate) without also having visible color that could have artifactually caused this result. None of the potential stimulators from this moderate-size library, however, passed a secondary test that included an inactive integrase mutant that assessed whether the increased fluorescence depended on the endonuclease activity of integrase. This first attempt at identifying integrase stimulator (IS) compounds establishes the necessary logistics and workflow, which should encourage larger scale high-throughput screening to advance the novel antiviral strategy of stimulating integrase to damage retroviral DNA.