Direct Measurement of Intracellular Compound Concentration by RapidFire Mass Spectrometry Offers Insights into Cell Permeability.
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.
Department of Biological Sciences, Molecular Discovery Research, GlaxoSmithKline, Stevenage, UK.
Laurie J
LJ
Gordon
Department of Biological Sciences, Molecular Discovery Research, GlaxoSmithKline, Stevenage, UK.
Morven
M
Allen
Department of Pharmacy, Uppsala University, Uppsala, Sweden Uppsala University Drug Optimization and Pharmaceutical Profiling Platform (UDOPP) at Chemical Biology Consortium, Uppsala, Sweden.
Per
P
Artursson
Department of Chemical Sciences, Molecular Discovery Research, GlaxoSmithKline, Stevenage, UK.
Michael M
MM
Hann
Department of Chemical Sciences, Molecular Discovery Research, GlaxoSmithKline, Stevenage, UK.
Bill J
BJ
Leavens
Department of Pharmacy, Uppsala University, Uppsala, Sweden.
André
A
Mateus
Department of Chemical Sciences, Molecular Discovery Research, GlaxoSmithKline, Stevenage, UK.
Simon
S
Readshaw
Department of Chemical Sciences, Molecular Discovery Research, GlaxoSmithKline, Stevenage, UK.
Klara
K
Valko
Department of Target and Pathway Validation, Molecular Discovery Research, GlaxoSmithKline, Stevenage, UK.
Gareth J
GJ
Wayne
Department of Chemical Sciences, Molecular Discovery Research, GlaxoSmithKline, Stevenage, UK Andy.West@gsk.com.
Andy
A
West
D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D055808:Drug Discovery / Q000379:methods; D006367:HeLa Cells; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D010539:Permeability; D004364:Pharmaceutical Preparations / Q000737:chemistry* / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods
D004364:Pharmaceutical Preparations
RapidFire-MS; cell penetration; drug accumulation; intracellular drug concentrations