Liposomal Permeation Assay for Droplet-Scale Pharmacokinetic Screening

Combinatorial library screening increasingly explores chemical space beyond the Ro5 (bRo5), which is useful for investigating undruggable targets but suffers compromised cellular permeability and therefore bioavailability. Moreover, structure-permeation relationships for bRo5 molecules are unclear partially because high-throughput permeation measurement technology for encoded combinatorial libraries is still nascent. Here, we present a permeation assay that is scalable to combinatorial library screening. A liposomal fluorogenic azide probe transduces permeation of alkyne-labeled molecules into small unilamellar vesicles via copper-catalyzed azide-alkyne cycloaddition. Control alkynes (e.g., propargylamine, various alkyne-labeled PEGs) benchmarked the assay. Cell-permeable macrocyclic peptides, exemplary bRo5 molecules, were alkyne labeled and shown to retain permeability. The assay was miniaturized to microfluidic droplets with high assay quality (Z ' >= 0.5), demonstrating excellent discrimination of photocleaved known membrane-permeable and-impermeable model library beads. Droplet-scale permeation screening will enable pharmacokinetic mapping of bRo5 libraries to build predictive models.

Automated summary

A scalable permeation assay was developed for combinatorial library screening, which can determine the cellular permeability of molecules. This method is particularly useful for investigating compounds that target undruggable targets, providing important insights into their drug-like properties.