ABSTRACT
Pin1 regulates the levels and functions of phosphoproteins by catalyzing phosphorylation-dependent cis/trans isomerization of peptidyl-prolyl bonds. Previous Pin1 inhibitors contained phosphoamino acids, which are metabolically unstable and have poor membrane permeability. In this work, we report a cell-permeable and metabolically stable nonphosphorylated bicyclic peptide as a potent and selective Pin1 inhibitor, which inhibited the intracellular Pin1 activity in cultured mammalian cells but had little effect on other isomerases such as Pin4, FKBP12, or cyclophilin A.
Subject(s)
Bridged Bicyclo Compounds/pharmacology , Cell Membrane Permeability , Enzyme Inhibitors/pharmacology , Peptidylprolyl Isomerase/antagonists & inhibitors , HeLa Cells , Humans , NIMA-Interacting Peptidylprolyl Isomerase , PhosphorylationABSTRACT
Cyclic peptides have great potential as therapeutic agents and research tools but are generally impermeable to the cell membrane. Fusion of cyclic peptides with a cyclic cell-penetrating peptide produces bicyclic peptides that are cell-permeable and retain the ability to recognize specific intracellular targets. Application of this strategy to protein tyrosine phosphatase 1B and a peptidyl-prolyl cis-trans isomerase (Pin1) isomerase resulted in potent, selective, proteolytically stable, and biologically active inhibitors against the enzymes.
Subject(s)
Bridged Bicyclo Compounds/chemical synthesis , Bridged Bicyclo Compounds/pharmacology , Cell-Penetrating Peptides/chemical synthesis , Cell-Penetrating Peptides/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/pharmacology , Cell Line, Tumor , Cell Membrane Permeability , Humans , Peptidylprolyl Isomerase/antagonists & inhibitors , Peptidylprolyl Isomerase/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1/antagonists & inhibitors , Structure-Activity RelationshipABSTRACT
Cyclic heptapeptide cyclo(FΦRRRRQ) (cFΦR4, where Φ is l-2-naphthylalanine) was recently found to be efficiently internalized by mammalian cells. In this study, its mechanism of internalization was investigated by perturbing various endocytic events through the introduction of pharmacologic agents and genetic mutations. The results show that cFΦR4 binds directly to membrane phospholipids, is internalized into human cancer cells through endocytosis, and escapes from early endosomes into the cytoplasm. Its cargo capacity was examined with a wide variety of molecules, including small-molecule dyes, linear and cyclic peptides of various charged states, and proteins. Depending on the nature of the cargos, they may be delivered by endocyclic (insertion of cargo into the cFΦR4 ring), exocyclic (attachment of cargo to the Gln side chain), or bicyclic approaches (fusion of cFΦR4 and cyclic cargo rings). The overall delivery efficiency (i.e., delivery of cargo into the cytoplasm and nucleus) of cFΦR4 was 4-12-fold higher than those of nonaarginine, HIV Tat-derived peptide, or penetratin. The higher delivery efficiency, coupled with superior serum stability, minimal toxicity, and synthetic accessibility, renders cFΦR4 a useful transporter for intracellular cargo delivery and a suitable system for investigating the mechanism of endosomal escape.
