Cell Penetration Profiling Using the Chloroalkane Penetration Assay

Leila Peraro, Kirsten L. Deprey, Matthew K. Moser, Zhongju Zou, Haydn L. Ball, Beth Levine, Joshua A. Kritzer

Research output: Contribution to journalArticlepeer-review

86 Scopus citations


Biotherapeutics are a promising class of molecules in drug discovery, but they are often limited to extracellular targets due to their poor cell penetration. High-throughput cell penetration assays are required for the optimization of biotherapeutics for enhanced cell penetration. We developed a HaloTag-based assay called the chloroalkane penetration assay (CAPA), which is quantitative, high-throughput, and compartment-specific. We demonstrate the ability of CAPA to profile extent of cytosolic penetration with respect to concentration, presence of serum, temperature, and time. We also used CAPA to investigate structure-penetration relationships for bioactive stapled peptides and peptides fused to cell-penetrating sequences. CAPA is not only limited to measuring cytosolic penetration. Using a cell line where HaloTag is localized to the nucleus, we show quantitative measurement of nuclear penetration. Going forward, CAPA will be a valuable method for measuring and optimizing the cell penetration of biotherapeutics.

Original languageEnglish (US)
Pages (from-to)11360-11369
Number of pages10
JournalJournal of the American Chemical Society
Issue number36
StatePublished - Sep 12 2018

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry


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