Director, Cell Sciences
Screening & Compound Profiling
NanoClick Assay: A High Throughput, Target-Agnostic Permeability Assay that Combines NanoBRET Technology with Intracellular Click Chemistry
Macrocyclic peptides open new opportunities to target intracellular protein–protein interactions, PPIs, that are often considered non-druggable by traditional small molecules. Specifically, peptides have the potential to bind to shallow and highly expansive binding surfaces, orthosteric blocking, of such PPIs and/or other unique allosteric binding sites. However, their clinical development may be limited by their ability to efficiently penetrate into cells to modulate their cognate PPI targets. The ability to have a predictive, high-throughput assay to assess cell permeability is a critical tool to enable peptide drug discovery programs.
We developed a high throughput, target-agnostic cell permeability assay that quantitates the cumulative cytosolic exposure of a peptide in a concentration- and time-dependent manner. The assay has been named ‘NanoClick’ as it combines in-cell copper-free Click chemistry and monitoring of a NanoBRET signal in cells. The assay is based on cellular expression of the NanoLuc-HaloTag system and relies on the Click reaction of azide-tagged peptides with DiBac-chloroalkane, CA, anchored to the HaloTag. Subsequent introduction of an azido-dye followed by the NanoLuc substrate allows the detection of a BRET signal that is reduced by the presence of azide-functionalized peptides in the cytosol. The readout can be expressed as a permeability ratio of EC50s when compared to the response of a low permeability control.
We validated the assay using known cell penetrating peptides, CPPs, and were further able to demonstrate correlations to cellular activity using a p53/MDM2 model system. The assay has been applied across several MRL drug discovery programs and has been instrumental to guide and establish Structure – Permeability Relationships in the optimization of macrocyclic peptides for cellular potency across intracellular PPI target programs.
AuthorsAndrea Peier, Lan Ge, Nicolas Boyer, Kaustav Biswas, Chunhui Huang, Michael Garrigou, David Tellers, Sookhee Ha, Sir David Lane $, Chris Brown $, Charles Johannes $, Tsz Ying Yuen $, Ahmad Sadruddin, Brad Sherborne, Tomi Sawyer, and Anthony Partridge
AffiliationsMerck & Co., Inc., Kenilworth, NJ, USA. $ A*STAR, Agency for Science, Technology and Research, Singapore