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Cell Penetration Profiling Using the Chloroalkane Penetration Assay.

Leila Peraro1, Kirsten L Deprey1, Matthew K Moser1

  • 1Department of Chemistry , Tufts University , Medford , Massachusetts 02155 , United States.

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|August 18, 2018
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Summary

A new assay, chloroalkane penetration assay (CAPA), enables high-throughput measurement of biotherapeutic cell penetration. This method aids in optimizing drug delivery for enhanced cytosolic and nuclear penetration.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Drug Discovery

Background:

  • Biotherapeutics offer therapeutic potential but face limitations in reaching intracellular targets due to poor cell penetration.
  • Development of high-throughput assays is crucial for optimizing biotherapeutic cell penetration and efficacy.

Purpose of the Study:

  • To develop and validate a quantitative, high-throughput, and compartment-specific assay for measuring biotherapeutic cell penetration.
  • To investigate the factors influencing cytosolic penetration and explore structure-penetration relationships of peptides.

Main Methods:

  • Development of a HaloTag-based assay termed the chloroalkane penetration assay (CAPA).
  • Utilizing CAPA to quantitatively profile cytosolic penetration under various conditions (concentration, serum, temperature, time).
  • Assessing nuclear penetration using a cell line with HaloTag localized to the nucleus.

Main Results:

  • CAPA successfully quantified cytosolic penetration and its dependence on experimental conditions.
  • Structure-penetration relationships were investigated for stapled peptides and cell-penetrating peptide fusions.
  • Quantitative measurement of nuclear penetration was demonstrated, expanding the assay's utility.

Conclusions:

  • The chloroalkane penetration assay (CAPA) is a versatile and effective tool for measuring and optimizing biotherapeutic cell penetration.
  • CAPA facilitates the study of factors affecting penetration and structure-activity relationships.
  • This assay holds significant value for advancing the development of cell-penetrating biotherapeutics.