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J Biomol Screen. 2016 Feb;21(2):156-64. doi: 10.1177/1087057115604141. Epub 2015 Sep 3.

Direct Measurement of Intracellular Compound Concentration by RapidFire Mass Spectrometry Offers Insights into Cell Permeability.

Author information

1
Department of Biological Sciences, Molecular Discovery Research, GlaxoSmithKline, Stevenage, UK.
2
Department of Pharmacy, Uppsala University, Uppsala, Sweden Uppsala University Drug Optimization and Pharmaceutical Profiling Platform (UDOPP) at Chemical Biology Consortium, Uppsala, Sweden.
3
Department of Chemical Sciences, Molecular Discovery Research, GlaxoSmithKline, Stevenage, UK.
4
Department of Pharmacy, Uppsala University, Uppsala, Sweden.
5
Department of Target and Pathway Validation, Molecular Discovery Research, GlaxoSmithKline, Stevenage, UK.
6
Department of Chemical Sciences, Molecular Discovery Research, GlaxoSmithKline, Stevenage, UK Andy.West@gsk.com.

Abstract

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.

KEYWORDS:

RapidFire-MS; cell penetration; drug accumulation; intracellular drug concentrations

PMID:
26336900
DOI:
10.1177/1087057115604141
[Indexed for MEDLINE]

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