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.