Gordon Laurie J, Allen Morven, Artursson Per, Hann Michael M, Leavens Bill J, Mateus André, Readshaw Simon, Valko Klara, Wayne Gareth J, West Andy
Department of Biological Sciences, Molecular Discovery Research, GlaxoSmithKline, Stevenage, UK.
Department of Pharmacy, Uppsala University, Uppsala, Sweden Uppsala University Drug Optimization and Pharmaceutical Profiling Platform (UDOPP) at Chemical Biology Consortium, Uppsala, Sweden.
J Biomol Screen. 2016 Feb;21(2):156-64. doi: 10.1177/1087057115604141. Epub 2015 Sep 3.
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.
早期药物发现面临的关键挑战之一是理解在生化分析和细胞分析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通透性测量方法，如根据logP估计或人工膜通透性来解释这些差异。缺失的环节是对全细胞内化合物浓度的直接测量。在某些情况下，这可以从细胞活性中估计出来，但如果细胞活性较弱或不存在，这也可能存在问题。分析技术在灵敏度和通量方面的进步使我们能够开发一种高通量分析方法，用于测量细胞内化合物浓度，以便常规使用以支持先导化合物优化。该分析方法在将细胞与测试化合物孵育后，基于RapidFire-MS读取HeLa细胞中的化合物浓度。最初的分析验证是通过超高效液相色谱串联质谱法进行 的，随后该分析方法被转移到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。这种优化带来了一个半自动平台，并具有每天对多达100种化合物进行生产规模化合物分析的潜力
