Direct Measurement of Intracellular Compound Concentration by RapidFire Mass Spectrometry Offers Insights into Cell Permeability
Direct Measurement of Intracellular Compound Concentration by RapidFire Mass Spectrometry Offers Insights into Cell Permeability
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DOI：
10.1177/1087057115604141
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发表时间：
2016-02-01
影响因子：
--
通讯作者：
West, Andy
中科院分区：
文献类型：
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作者：
Gordon, Laurie J.;Allen, Morven;West, Andy
早期药物发现面临的关键挑战之一是了解生化测定和细胞测定中化合物活性之间常见的差异。传统上，使用间接或估计的细胞渗透性测量（例如 logP 或人工膜渗透性的估计）来解释差异。缺失的环节是直接测量整个细胞中的细胞内化合物浓度。在某些情况下，这可以根据细胞活动来估计，但如果细胞活动弱或不存在，这也可能是有问题的。分析技术的灵敏度和通量的进步使我们能够开发出一种高通量测定法，用于测量细胞内化合物浓度，以供常规使用以支持先导化合物优化。该测定使用基于 RapidFire-MS 的 HeLa 细胞中化合物浓度读数，将细胞与测试化合物一起孵育后。通过超高效液相色谱串联质谱法进行初始测定验证，随后将测定转移至RapidFire串联质谱法。进行了进一步的小型化和优化，以简化流程、提高样品通量并缩短周期时间。这种优化提供了一个半自动化平台，具有每天最多可生产 100 种化合物的生产规模化合物分析潜力。
One of the key challenges facing early stage drug discovery is understanding the commonly observed difference between the activity of compounds in biochemical assays and cellular assays. Traditionally, indirect or estimated cell permeability measurements such as estimations from logP or artificial membrane permeability are used to explain the differences. The missing link is a direct measurement of intracellular compound concentration in whole cells. This can, in some circumstances, be estimated from the cellular activity, but this may also be problematic if cellular activity is weak or absent. Advances in sensitivity and throughput of analytical techniques have enabled us to develop a high-throughput assay for the measurement of intracellular compound concentration for routine use to support lead optimization. The assay uses a RapidFire-MS based readout of compound concentration in HeLa cells following incubation of cells with test compound. The initial assay validation was performed by ultra-high performance liquid chromatography tandem mass spectrometry, and the assay was subsequently transferred to RapidFire tandem mass spectrometry. Further miniaturization and optimization were performed to streamline the process, increase sample throughput, and reduce cycle time. This optimization has delivered a semi-automated platform with the potential of production scale compound profiling up to 100 compounds per day.