Assay Development and Screening
Optimization of Assays for Challenging Targets for Lead Discovery and Screening
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 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 support peptide drug discovery programs.
We developed a high throughput, quantitative, target-agnostic cell permeability assay that essentially measures the cumulative cytosolic exposure of a peptide in a concentration-dependent manner. The assay has been named NanoClick as it combines in-cell 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-containing 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 Click-reactive 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 and were further able to demonstrate correlations to cellular activity using a p53/MDM2 model system. The assay has been applied across multiple programs and has been used to guide and establish structure-permeability relationships in the optimization of macrocyclic peptides for cellular potency across intracellular PPI target programs.