A high-throughput synthetic platform enables the discovery of proteomimetic cell penetrating peptides and bioportides

Collectively, cell penetrating peptide (CPP) vectors and intrinsically active bioportides possess tremendous potential for drug delivery applications and the discrete modulation of intracellular targets including the sites of protein–protein interactions (PPIs). Such sequences are usually relatively short (< 25 AA), polycationic in nature and able to access the various intracellular compartments of eukaryotic cells without detrimental influences upon cellular biology. The high-throughput platform for bioportide discovery described herein exploits the discovery that many human proteins are an abundant source of potential CPP sequences which are reliably predicted using QSAR algorithms or other methods. Subsequently, microwave-enhanced solid phase peptides synthesis provides a high-throughput source of novel proteomimetic CPPs for screening purposes. By focussing upon cationic helical domains, often located within the molecular interfaces that facilitate PPIs, bioportides which act by a dominant-negative mechanism at such sites can be reliably identified within small number libraries of CPPs. Protocols that employ fluorescent peptides, routinely prepared by N-terminal acylation with carboxytetramethylrhodamine, further enable both the quantification of cellular uptake kinetics and the identification of specific site(s) of intracellular accretion. Chemical modifications of linear peptides, including strategies to promote and stabilise helicity, are compatible with the synthesis of second-generation bioportides with improved drug-like properties to further exploit the inherent selectivity of biologics