Objective

Assessment of whether a candidate drug and its intended mechanism was tested in clinical studies requires knowledge of whether it was sufficiently exposed in the physiologically relevant tissue (1, 2). Similarly, exposure of investigated compounds at the intracellular site of action directly influence the output of functional cell assays, commonly translating to reductions in potency between biochemical and cellular assay formats. This discrepancy between assays translates to time consuming medicinal chemistry cycles aimed at understanding the root cause of functional activity loss and whether it depends on a lack of target engagement or poor compound exposure. Hence methodologies for assessment of intracellular compound availability has seen a recent growth of interest, including low throughput protocols amenable to testing in 96-deepwell format (3). These methods generally include a microdialysis step against disintegrated cells to calculate the quantity of available compound. However, cell disintegration and dilution is associated with a risk of disturbing the equilibria that control cellular compound distribution, especially since the protocols include extensive and lengthy cell washing.

We aimed to develop a protocol that allowed miniaturization to 384- and 1536-well formats, where we work with permeabilized “structurally intact” cells, incorporating fast washing schemes to minimize compound redistribution during measurement. The approach was validated by assessment of cellular compartment leakage from permeabilized cells using Western blots towards representative nuclear, membrane-associated, mitochondrial and cytoplasmic proteins. These data underpinned the implementation of a preliminary workflow for the assessment of compound distribution profiles, including the kinetics of compound uptake and its´ dependence on compound concentration. Compound measurements require highly sensitive LC/MS methods applied to pooled samples alongside quality controls to follow assay performance and reproducibility.

Miniaturization of the assay workflow was enabled through acoustic dispensing from low µL volumes, with profile assessments being made in identical cells and conditions to those applied in the functional assays. Here we present our preliminary results for two different sets of diverse compounds, assess how these compared with primary functional datasets and share our view on how such protocols can improve our interpretation of functional cell assay responses. 

1. Bunnage ME, Chekler EL, Jones LH. Target validation using chemical probes. Nat Chem Biol. 2013;9(4):195-9.

2. Morgan P, Brown DG, Lennard S, Anderton MJ, Barrett JC, Eriksson U, et al. Impact of a five-dimensional framework on R&D productivity at AstraZeneca. Nat Rev Drug Discov. 2018;17(3):167-81.

3. Gordon LJ, Allen M, Artursson P, Hann MM, Leavens BJ, Mateus A, et al. Direct Measurement of Intracellular Compound Concentration by RapidFire Mass Spectrometry Offers Insights into Cell Permeability. J Biomol Screen. 2016;21(2):156-64.