Version 2 2021-02-09, 13:34Version 2 2021-02-09, 13:34
Version 1 2021-02-04, 21:29Version 1 2021-02-04, 21:29
journal contribution
posted on 2021-02-09, 13:34authored byAndrea Peier, Lan Ge, Nicolas Boyer, John Frost, Ruchia Duggal, Kaustav Biswas, Scott Edmondson, Jeffrey D. Hermes, Lin Yan, Chad Zimprich, Ahmad Sadruddin, Hung Yi Kristal Kaan, Arun Chandramohan, Christopher J. Brown, Dawn Thean, Xue Er Lee, Tsz Ying Yuen, Fernando J. Ferrer-Gago, Charles W. Johannes, David P. Lane, Brad Sherborne, Cesear Corona, Matthew B. Robers, Tomi K. Sawyer, Anthony W. Partridge
Macrocyclic peptides
open new opportunities to target intracellular
protein–protein interactions (PPIs) that are often considered
nondruggable by traditional small molecules. However, engineering
sufficient membrane permeability into these molecules is a central
challenge for identifying clinical candidates. Currently, there is
a lack of high-throughput assays to assess peptide permeability, which
limits our capacity to engineer this property into macrocyclic peptides
for advancement through drug discovery pipelines. Accordingly, we
developed a high throughput and target-agnostic cell permeability
assay that measures the relative cumulative cytosolic exposure of
a peptide in a concentration-dependent manner. The assay was named
NanoClick as it combines in-cell Click chemistry with an intracellular
NanoBRET signal. We validated the approach using known cell penetrating
peptides and further demonstrated a correlation to cellular activity
using a p53/MDM2 model system. With minimal change to the peptide
sequence, NanoClick enables the ability to measure uptake of molecules
that enter the cell via different mechanisms such as endocytosis,
membrane translocation, or passive permeability. Overall, the NanoClick
assay can serve as a screening tool to uncover predictive design rules
to guide structure–activity–permeability relationships
in the optimization of functionally active molecules.