Cell-penetrating and mitochondrion-targeting molecules.

The mitochondrion performs critical roles in eukaryotic cells including ATP production, cell growth, survival, apoptosis, and differentiation. Many human diseases can be traced to dysfunction within the mitochondria, but selective delivery of therapeutics into the mitochondria has been challenging. This chapter describes the detailed protocols for the synthesis of a new family of mitochondrion-targeting, cell-penetrating molecules (CPMs) and their application for the delivery of small-molecule and peptidyl cargos into the mitochondrial matrix. Live-cell confocal microscopic imaging of HeLa cells treated with a variety of CPM-cargo conjugates revealed that the CPMs efficiently and specifically deliver membrane-impermeable linear and cyclic peptidyl cargos into the mitochondrial matrix, as long as the cargo carries no more than two negative charges.

Enhancing the Cell Permeability of Stapled Peptides with a Cyclic Cell-Penetrating Peptide.

Stapled peptides recapitulate the binding affinity and specificity of α-helices in proteins, resist proteolytic degradation, and may provide a novel modality against challenging drug targets such as protein-protein interactions. However, most of the stapled peptides have limited cell permeability or are impermeable to the cell membrane. We show herein that stapled peptides can be rendered highly cell-permeable by conjugating a cyclic cell-penetrating peptide to their N-terminus, C-terminus, or stapling unit. Application of this strategy to two previously reported membrane-impermeable peptidyl inhibitors against the MDM2/p53 and ß-catenin/TCF interactions resulted in the generation of potent proof-of-concept antiproliferative agents against key therapeutic targets.

Developments with bead-based screening for novel drug discovery.

Introduction: Combinatorial chemistry provides a cost-effective method for rapid discovery of drug hits/leads. The one-bead-one-compound (OBOC) library method is in principle ideally suited for this application, because it permits a large number of structurally diverse compounds to be rapidly synthesized and simultaneously screened for binding to a target of interest. However, application of OBOC libraries in drug discovery has encountered significant technical challenges. Areas covered: This Special Report covers the challenges associated with first-generation OBOC libraries (difficulty in structural identification of non-peptidic hits, screening biases and high false positive rates, and poor scalability). It also covers the many strategies developed over the past two decades to overcome these challenges. Expert opinion: With most of the technical challenges now overcome and the advent of powerful intracellular delivery technologies, OBOC libraries of metabolically stable and conformationally rigidified molecules (macrocyclic peptides and peptidomimetics, rigidified acyclic oligomers, and D-peptides) can be routinely synthesized and screened to discover initial hits against previously undruggable targets such as intracellular protein-protein interactions. On the other hand, further developments are still needed to expand the utility of the OBOC method to non-peptidic chemical scaffolds.

Designing Cell-Permeable Macrocyclic Peptides.

Peptides provide an attractive modality for targeting challenging drug targets such as intracellular protein-protein interactions. Unfortunately, peptides are generally impermeable to the cell membrane and inherently susceptible to proteolytic degradation in vivo. Macrocyclization of peptides greatly increases their proteolytic stability and in some cases the cell-penetrating activity. Conjugation of peptidyl cargoes to cyclic cell-penetrating peptides has resulted in potent, cell-permeable, and metabolically stable macrocyclic peptides against intracellular protein targets. Proper conjugation/integration of a peptidyl cargo with a cyclic cell-penetrating peptide is critical to retain the activity of each component and generate a biologically active macrocyclic peptide. This chapter describes the different conjugation strategies that have been developed (including endocyclic, bicyclic, and reversible cyclization methods) and the detailed protocols for their preparation.

Non-Peptidic Cell-Penetrating Motifs for Mitochondrion-Specific Cargo Delivery.

Mitochondrial dysfunction is linked to a variety of human illnesses, but selective delivery of therapeutics into the mitochondrion is challenging. Now a family of amphipathic cell-penetrating motifs (CPMs) is presented, consisting of four guanidinium groups and one or two aromatic hydrophobic groups (naphthalene) assembled through a central scaffold (a benzene ring). The CPMs and CPM-cargo conjugates efficiently enter the interior of cultured mammalian cells and are specifically localized into the mitochondrial matrix, as revealed by high-resolution confocal microscopy. With a membrane-impermeable peptide as cargo, the CPMs exhibited ≥170-fold higher delivery efficiency than previous mitochondrial delivery vehicles. Conjugation of a small-molecule inhibitor of heat shock protein 90 to a CPM resulted in accumulation of the inhibitor inside the mitochondrial matrix with greatly enhanced anticancer activity. The CPMs showed minimal effect on the viability or the mitochondrial membrane potential of mammalian cells.

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

Therapeutic applications of peptides are currently limited by their proteolytic instability and impermeability to the cell membrane. A general, reversible bicyclization strategy is now reported 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 applied to generate a cell-permeable bicyclic peptidyl inhibitor against the NEMO-IKK interaction.

Discovery and Mechanism of Highly Efficient Cyclic Cell-Penetrating Peptides.

Previous cell-penetrating peptides (CPPs) generally have low cytosolic delivery efficiencies, because of inefficient endosomal escape. In this study, a family of small, amphipathic cyclic peptides was found to be highly efficient CPPs, with cytosolic delivery efficiencies of up to 120% (compared to 2.0% for Tat). These cyclic CPPs bind directly to the plasma membrane phospholipids and enter mammalian cells via endocytosis, followed by efficient release from the endosome. Their total cellular uptake efficiency correlates positively with the binding affinity for the plasma membrane, whereas their endosomal escape efficiency increases with the endosomal membrane-binding affinity. The cyclic CPPs induce membrane curvature on giant unilamellar vesicles and budding of small vesicles, which subsequently collapse into amorphous lipid/peptide aggregates. These data suggest that cyclic CPPs exit the endosome by binding to the endosomal membrane and inducing CPP-enriched lipid domains to bud off as small vesicles. Together with their high proteolytic stability, low cytotoxicity, and oral bioavailability, these cyclic CPPs should provide a powerful system for intracellular delivery of therapeutic agents and chemical probes.