Screening of HIV-1 Protease Using a Combination of an Ultra-High-Throughput Fluorescent-Based Assay and RapidFire Mass Spectrometry
Screening of HIV-1 Protease Using a Combination of an Ultra-High-Throughput Fluorescent-Based Assay and RapidFire Mass Spectrometry
复制标题
DOI：
10.1177/1087057115570838
复制
发表时间：
2015-06-01
影响因子：
--
通讯作者：
Adam, Gregory C.
中科院分区：
文献类型：
--
作者：
Meng, Juncai;Lai, Ming-Tain;Adam, Gregory C.
HIV-1蛋白水解酶(PR)是开发抗病毒药物治疗HIV感染者的主要靶点之一。为了识别新的PR抑制剂，利用RapidFire平台开发了一种无标记的高通量质谱分析(HTMS)，并作为正交分析应用于确认在基于荧光共振能量转移(FRET)的100万种化合物的初级筛选中确定的命中。在底物选择方面，在RapidFire平台上评估了一组来自PR天然加工场所的多肽底物。结果，KVSLNFPIL，一种新的底物，被测量在k(CAT)/K-m上分别比频繁使用的序列SQNYPIVQ和SQNYPIV提高了20倍和60倍，用于HTMS筛选。来自基于FRET的初级筛查的大约17%的命中在HTMS确证试验中得到确认，包括集合中的所有304种已知PR抑制剂，这表明HTMS分析在检测假阳性的同时捕获真实的命中是有效的。因此，RapidFire HTMS分析的采样率类似于每孔7 S，能够高通量评估多肽底物，并在发现新型PR抑制剂时作为HITS分类的有效工具。
HIV-1 protease (PR) represents one of the primary targets for developing antiviral agents for the treatment of HIV-infected patients. To identify novel PR inhibitors, a label-free, high-throughput mass spectrometry (HTMS) assay was developed using the RapidFire platform and applied as an orthogonal assay to confirm hits identified in a fluorescence resonance energy transfer (FRET)-based primary screen of > 1 million compounds. For substrate selection, a panel of peptide substrates derived from natural processing sites for PR was evaluated on the RapidFire platform. As a result, KVSLNFPIL, a new substrate measured to have a similar to 20- and 60-fold improvement in k(cat)/K-m over the frequently used sequences SQNYPIVQ and SQNYPIV, respectively, was identified for the HTMS screen. About 17% of hits from the FRET-based primary screen were confirmed in the HTMS confirmatory assay including all 304 known PR inhibitors in the set, demonstrating that the HTMS assay is effective at triaging false-positives while capturing true hits. Hence, with a sampling rate of similar to 7 s per well, the RapidFire HTMS assay enables the high-throughput evaluation of peptide substrates and functions as an efficient tool for hits triage in the discovery of novel PR inhibitors.