# A generalizable assay for intracellular accumulation to profile cytosolic drug delivery in mammalian cells

## TL;DR

This paper introduces a new high-throughput assay to accurately measure how well different molecules enter the cytosol of mammalian cells, improving drug development for intracellular targets.

## Contribution

The CHAMP assay uses azide tags and click chemistry to specifically measure cytosolic accumulation, overcoming limitations of existing methods.

## Key findings

- Longer polyarginine sequences in cell-penetrating peptides show enhanced cytosolic accumulation.
- C-terminal amidation and D-amino acid substitutions significantly affect cellular penetration.
- CHAMP successfully distinguishes accumulation capabilities of supercharged proteins and antibiotics.

## Abstract

The ability of biologically active molecules to access intracellular targets remains a critical barrier in drug development. While assays for measuring cellular uptake exist, they often fail to distinguish between membrane-associated or endosomal trapped compounds and those that successfully reach the cytosol. Here, we present the Chloroalkane HaloTag Azide-based Membrane Penetration (CHAMP) Assay, a high-throughput method that employs a minimally disruptive azide tag to report the cytosolic accumulation of diverse molecules in mammalian cells. The CHAMP assay utilizes HaloTag-expressing cells and strain-promoted azide-alkyne cycloaddition (SPAAC) chemistry to quantify the presence of azide-tagged test compounds in the cytosol. We demonstrate the versatility of this approach by evaluating the accumulation profiles of small molecules, peptides, and proteins, revealing how structural variations and stereochemical differences influence cytosolic penetration. Our findings with cell-penetrating peptides confirm established structure-activity relationships, with longer polyarginine sequences showing enhanced accumulation. Additionally, we observed that C-terminal amidation and D-amino acid substitutions significantly impact cellular penetration. When applied to supercharged proteins and antibiotics, CHAMP successfully discriminates between compounds with varying accumulation capabilities. This method provides a robust platform for screening cytosolic accumulation while minimizing the confounding effects of large tags on molecular permeability, potentially accelerating the development of therapeutics targeting intracellular pathways.

Cellular penetration is a crucial property of bioactive molecules to reach intracellular targets, however, current penetration assays are limited when it comes to measuring the exact cytosolic accumulation. Here, the authors introduce a chloroalkane HaloTag azide-based membrane penetration (CHAMP) assay, a high-throughput method using azide tags and click chemistry to accurately measure cytosolic accumulation, offering a powerful tool for the development of drugs targeting intracellular pathways.