A Streamlined High Performance Liquid Chromatography with Tandem Mass Spectrometry Based Workflow for Rapid Screening of Cellular Accumulation of Small Molecules

Assessing if compounds with intracellular targets reach their site of action is crucial for success in drug development. Cell type-specific uptake goes beyond permeability studies, typically mimicking crossing the gut, the lung, or the blood-brain barrier. A medium- to high-throughput cellular accumulation protocol in 96-well format is presented using six compounds, evaluating optimal conditions varying several parameters, such as incubation time, compound concentration, and extraction protocol. An optimized assay protocol for cellular accumulation of distinct chemical classes is a compromise: No one-extraction-protocol-fits-all exists; equally, some compounds need longer incubation periods to reach maximal intracellular concentration. Reliable high performance liquid chromatography with tandem mass spectrometry based quantification of cellular accumulation for all six compounds to the nM range is achieved with a short 1 h incubation. Intracellular concentrations per cell count are determined in A549^ACE+TMPRSS2 cells, taking nonspecific binding into account. Hence, this approach adds valuable information during the pre-screening of compounds with intracellular targets. Finally, optimal assay conditions are emphasized as essential for predicting activity in vitro and in vivo, based on biochemical information and intracellular concentrations. In summary, the workflow for cellular accumulation determination can serve two scenarios: 1) Pre-selection of compounds for screening purposes or 2) systematic optimization of conditions to advance compounds with intracellular targets.