Enhancing the Cell Permeability and Metabolic Stability of Peptidyl Drugs by Reversible Bicyclization

Abstract

Therapeutic applications of peptides are currently limited by their proteolytic instability and impermeability to the cell membrane. Here, we report a general, reversible bicyclization strategy to increase both the proteolytic stability and cell permeability of peptidyl drugs. A peptide drug is fused with a short cell-penetrating motif and converted into a conformationally constrained bicyclic structure through the formation of a pair of disulfide bonds. The resulting bicyclic peptide has greatly enhanced proteolytic stability as well as cell-permeability. Once inside the cell, the disulfide bonds are reduced to produce a linear, biologically active peptide. This strategy was Supporting information for this article is given via a link at the end of the document HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript applied to generate a cell-permeable bicyclic peptidyl inhibitor against the NEMO-IKK interaction. Drug deliveryPeptide bicyclization via a pair of disulphide bonds increases its proteolytic stability and cell permeability and yet allows for regeneration of the functional linear peptide once inside the cytosol of the cell. KeywordsCell-penetrating peptide; cyclic peptide; NEMO inhibitor; bicyclization; protein-protein interactionCompared to small-molecule drugs, peptides are highly selective and efficacious and, at the same time, relatively safe and well tolerated. A particularly exciting application of peptides is the inhibition of protein-protein interactions (PPIs), which remain challenging targets for small molecules. [1] Consequently, there is an increased interest in peptides in pharmaceutical research and development, and ~140 peptide therapeutics are currently being evaluated in clinical trials. [2] However, peptides are inherently susceptible to proteolytic degradation. Additionally, peptides are generally impermeable to the cell membrane, largely limiting their applications to extracellular targets. Although N-methylation of the peptide backbone and formation of intramolecular hydrogen bonds have been shown to improve the proteolytic stability and membrane permeability of certain cyclic peptides, [3,4] alternative strategies to increase both the metabolic stability and cell permeability of peptide drugs are clearly needed.NF-κB is a transcription factor that controls the expression of numerous gene products involved in immune, stress, inflammatory responses, cell proliferation, and apoptosis. [5] Aberrant activation of NF-κB signaling has been implicated in a number of autoimmune diseases (e.g., rheumatoid arthritis) and cancer (e.g., diffuse large B-cell lymphoma), among others. [6] Canonical NF-κB signaling is mediated by the interaction between the inhibitor of κB (IκB)-kinase (IKK) complex and regulatory protein NF-κB essential modifier (NEMO). [7] Binding to NEMO activates IKK, which in turn phosphorylates IκB, promoting the proteasomal degradation of IκB and release of active NF-κB. Modulators targeting various steps of the NF-κB signaling pa...

Overcoming Endosomal Entrapment in Drug Delivery

“…The cyclic CPPs have been used to deliver a variety of cargos including small molecules, [30][31][32] linear peptides, 31,98 cyclic peptides, 31,[99][100][101][102][103][104] proteins, 31 and nucleic acids (M.B. and D.P., unpublished results) into mammalian cells in vitro and in vivo.…”

Ionpair-π interactions favor cell penetration of arginine/tryptophan-rich cell-penetrating peptides

“…However, it later appeared that amphipathicity had no direct impact on internalization of the KLA peptide, but could promote binding to intracellular organelle membranes. Strategies exploiting switchable and/or structurally constrained amphipathic helical peptides to control or enhance uptake are still being developed [26][27][28]. CPPs are often classified according to their ability to adopt a secondary amphipathic structure [23,29,30] possibly by analogy with cationic α-helical antimicrobial peptides, another class of membrane-active peptides [31,32].…”

Designing Cell-Permeable Macrocyclic Peptides

“…Reversible bicyclization provides an alternative approach to introducing additional conformational rigidity into the macrocyclic peptide. Qian et al first demonstrated this strategy by designing a cell-permeable inhibitor against NF-κB essential modulator (NEMO) (29). They fused the NEMO-binding domain (NBD) of IκB kinase β (ALDWSWLQ) with a short CPP motif (RRRRΦF, where Φ is 2-naphthylalanine) and adding two cysteine residues, one at the Cterminus and the other in between the NBD and CPP sequences.…”