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.

Peptide-based molecules hold great potential as targeted inhibitors of intracellular protein–protein interactions (PPIs).

Discovery of false-positive target binding, due to assay interference or aggregation, presents a significant problem for drug discovery programs. These issues may often be unrealized and could lead researchers astray if not subject to independent verification of reproducibility and/or on-target mechanism of action. Although well-documented for small molecules, this issue has not been widely explored for peptide modality. As a case study, we demonstrate that two purported KRas inhibitors, stapled peptide SAH-SOS1A and macrocyclic peptide cyclorasin 9A5, exemplify false-positive moleculesboth in terms of their sub-micromolar KRas binding affinities and their on-target cellular activities. We observed that the apparent binding of fluorescein-labeled SAH-SOS1A given by a fluorescence polarization assay is sensitive to detergent. False-positive readouts can arise from peptide adsorption to the surface of microplates.Hence, we used surface plasmon resonance and isothermal titration calorimetry to unambiguously show that both SAH-SOS1A and cyclorasin 9A5 are non-binders for KRas.Thermal shift assay and hydrogen-deuterium exchange mass spectrometry further demonstrate

Peptide alcohols are clinically important compounds that are underexplored in structure–activity relationship (SAR) studies in drug discovery. One reason for this underutilization is that current syntheses are laborious and time consuming. Herein, we describe the preparation and utility of Rink, Ramage, and Sieber‐chloride resins, which enables the use of a general, easy and practical method for the attachment of fluorenylmethoxycarbonyl (Fmoc)‐amino alcohols to a solid support, in the synthesis of peptide alcohols. This method is the first straightforward Fmoc/tBu synthesis of peptide alcohols starting from a pre‐loaded resin. The synthesized peptide alcohols can be detached from the linkers through conventional methods. Treatment with trifluoroacetic acid (TFA) (95 %) and scavengers such as triisopropylsilane and water for 2 h is sufficient to obtain a fully deprotected peptide alcohol, while treatment with 20 % hexafluoroisopropanol in dichloromethane renders a fully protected peptide alcohol that can be further modified at the C‐terminus. As examples, the new resins were used in straightforward, relatively rapid syntheses of the peptide alcohols octreotide, alamethicin, and a segment of trichogin GA IV, as well as the first synthesis of stapled peptide alcohols.