The endosomal escape vehicle platform enhances delivery of oligonucleotides in preclinical models of neuromuscular disorders.

Biological therapeutic agents are highly targeted and potent but limited in their ability to reach intracellular targets. These limitations often necessitate high therapeutic doses and can be associated with less-than-optimal therapeutic activity. One promising solution for therapeutic agent delivery is use of cell-penetrating peptides. Canonical cell-penetrating peptides, however, are limited by low efficiencies of cellular uptake and endosomal escape, minimal proteolytic stability, and toxicity. To overcome these limitations, we designed a family of proprietary cyclic cell-penetrating peptides that form the core of our endosomal escape vehicle technology capable of delivering therapeutic agent-conjugated cargo intracellularly. We demonstrated the therapeutic potential of this endosomal escape vehicle platform in preclinical models of muscular dystrophy with distinct disease etiology. An endosomal escape vehicle-conjugated, splice-modulating oligonucleotide restored dystrophin protein expression in striated muscles in the mdx mouse, a model for Duchenne muscular dystrophy. Furthermore, another endosomal escape vehicle-conjugated, sterically blocking oligonucleotide led to knockdown of aberrant transcript expression levels in facioscapulohumeral muscular dystrophy patient-derived skeletal muscle cells. These findings suggest a significant therapeutic potential of our endosomal escape vehicle conjugated oligonucleotides for targeted upregulation and downregulation of gene expression in neuromuscular diseases, with possible broader application of this platform for delivery of intracellular biological agents.

Cyclic Peptidyl Inhibitors against CAL/CFTR Interaction for Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, encoding for a chloride ion channel. Membrane expression of CFTR is negatively regulated by CFTR-associated ligand (CAL). We previously showed that inhibition of the CFTR/CAL interaction with a cell-permeable peptide improves the function of rescued F508del-CFTR. In this study, optimization of the peptidyl inhibitor yielded PGD97, which exhibits a KD value of 6 nM for the CAL PDZ domain, ≥ 130-fold selectivity over closely related PDZ domains, and a serum t1/2 of >24 h. In patient-derived F508del homozygous cells, PGD97 (100 nM) increased short-circuit currents by ∼3-fold and further potentiated the therapeutic effects of small-molecule correctors (e.g., VX-661) by ∼2-fold (with an EC50 of ∼10 nM). Our results suggest that PGD97 may be used as a novel treatment for CF, either as a single agent or in combination with small-molecule correctors/potentiators.

A Peptidyl Inhibitor that Blocks Calcineurin-NFAT Interaction and Prevents Acute Lung Injury.

Acute respiratory distress syndrome (ARDS) is an inflammatory lung disease with a high morbidity and mortality rate, for which no pharmacologic treatment is currently available. Our previous studies discovered that a pivotal step in the disease process is the activation of the nuclear factor of activated T cells (NFAT) c3 in lung macrophages, suggesting that inhibitors against the upstream protein phosphatase calcineurin should be effective for prevention/treatment of ARDS. Herein, we report the development of a highly potent, cell-permeable, and metabolically stable peptidyl inhibitor, CNI103, which selectively blocks the interaction between calcineurin and NFATc3, through computational and medicinal chemistry. CNI103 specifically inhibited calcineurin signaling in vitro and in vivo and exhibited a favorable pharmacokinetic profile, broad tissue distribution following different routes of administration, and minimal toxicity. Our data indicate that CNI103 is a promising novel treatment for ARDS and other inflammatory diseases.

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.

Understanding Cell Penetration of Cyclic Peptides.

Approximately 75% of all disease-relevant human proteins, including those involved in intracellular protein-protein interactions (PPIs), are undruggable with the current drug modalities (i.e., small molecules and biologics). Macrocyclic peptides provide a potential solution to these undruggable targets because their larger sizes (relative to conventional small molecules) endow them the capability of binding to flat PPI interfaces with antibody-like affinity and specificity. Powerful combinatorial library technologies have been developed to routinely identify cyclic peptides as potent, specific inhibitors against proteins including PPI targets. However, with the exception of a very small set of sequences, the vast majority of cyclic peptides are impermeable to the cell membrane, preventing their application against intracellular targets. This Review examines common structural features that render most cyclic peptides membrane impermeable, as well as the unique features that allow the minority of sequences to enter the cell interior by passive diffusion, endocytosis/endosomal escape, or other mechanisms. We also present the current state of knowledge about the molecular mechanisms of cell penetration, the various strategies for designing cell-permeable, biologically active cyclic peptides against intracellular targets, and the assay methods available to quantify their cell-permeability.

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.

Cell-Permeable Bicyclic Peptidyl Inhibitors against NEMO-IκB Kinase Interaction Directly from a Combinatorial Library.

Macrocyclic peptides are capable of binding to flat protein surfaces such as the interfaces of protein-protein interactions with antibody-like affinity and specificity, but generally lack cell permeability in order to access intracellular targets. In this work, we designed and synthesized a large combinatorial library of cell-permeable bicyclic peptides, in which the first ring consisted of randomized peptide sequences for potential binding to a target of interest, while the second ring featured a family of different cell-penetrating motifs, for both cell penetration and target binding. The library was screened against the IκB kinase α/ß (IKKα/ß)-binding domain of NF-κB essential modulator (NEMO), resulting in the discovery of several cell-permeable bicyclic peptides, which inhibited the NEMO-IKKß interaction with low µM IC50 values. Further optimization of one of the hits led to a relatively potent and cell-permeable NEMO inhibitor (IC50 = 1.0 µM), which selectively inhibited canonical NF-κB signaling in mammalian cells and the proliferation of cisplatin-resistant ovarian cancer cells. The inhibitor provides a useful tool for investigating the biological functions of NEMO/NF-κB and a potential lead for further development of a novel class of anti-inflammatory and anticancer drugs.

Targeting intracellular protein-protein interactions with cell-permeable cyclic peptides.

Intracellular protein-protein interactions (PPIs) are challenging targets for conventional drug modalities, because small molecules generally do not bind to their large, flat binding sites with high affinity, whereas monoclonal antibodies cannot cross the cell membrane to reach the targets. Cyclic peptides in the 700-2000 molecular-weight range have the sufficient size and a balanced conformational flexibility/rigidity for binding to flat PPI interfaces with antibody-like affinity and specificity. Several powerful cyclic peptide library technologies were developed over the past decade to rapidly discover potent, specific cyclic peptide ligands against proteins of interest including those involved in PPIs. Methods are also being developed to enhance the membrane permeability of cyclic peptides through both passive diffusion and active transport mechanisms. Integration of the permeability-enhancing elements into cyclic peptide design has led to an increasing number of cell-permeable and biologically active cyclic peptides against intracellular PPIs. In this account, we review the recent developments in the design and synthesis of cell-permeable cyclic peptides.

Macrocycles as protein-protein interaction inhibitors.

Macrocyclic compounds such as cyclic peptides have emerged as a new and exciting class of drug candidates for inhibition of intracellular protein-protein interactions, which are challenging targets for conventional drug modalities (i.e. small molecules and proteins). Over the past decade, several complementary technologies have been developed to synthesize macrocycle libraries and screen them for binding to therapeutically relevant targets. Two different approaches have also been explored to increase the membrane permeability of cyclic peptides. In this review, we discuss these methods and their applications in the discovery of macrocyclic compounds against protein-protein interactions.

Monitoring the cytosolic entry of cell-penetrating peptides using a pH-sensitive fluorophore.

We report a simple, effective method to assess the cytosolic delivery efficiency and kinetics of cell-penetrating peptides using a pH-sensitive fluorescent probe, naphthofluorescein.

Structure-based optimization of a peptidyl inhibitor against calcineurin-nuclear factor of activated T cell (NFAT) interaction.

Calcineurin inhibitors such as cyclosporine A and FK506 are effective immunosuppressants but produce severe side effects. Rational modification of a previously reported peptide inhibitor, GPHPVIVITGPHEE (KD ∼ 500 nM), by replacing the two valine residues with tert-leucine and the C-terminal proline with a cis-proline analogue, gave an improved inhibitor ZIZIT-cisPro, which binds to calcineurin with a KD value of 2.6 nM and is more resistant to proteolysis.