Targeting the Expanded TCF4/Fuchs' Endothelial Corneal Dystrophy CUG Repeat with Morpholino Peptide Conjugates.

Fuchs' corneal endothelial dystrophy (FECD) is a major cause of vision loss. Corneal transplantation is the only effective curative treatment, but this surgery has limitations. A pharmacological intervention would complement surgery and be beneficial for many patients. FECD is caused by an expanded CUG repeat within intron 2 of the TCF4 RNA. Agents that recognize the expanded repeat can reverse the splicing defects associated with the disease. Successful drug development will require diverse strategies for optimizing the efficacy of anti-CUG oligomers. In this study, we evaluate anti-CUG morpholinos conjugated to cyclic cell penetrating peptides. The morpholino domain of the conjugate is complementary to the repeat, while the peptide has been optimized for import across cell membranes. We show that morpholino conjugates can enter corneal endothelial cells and block the CUG RNA foci associated with the disease. These experiments support morpholino peptide conjugates as an approach for developing anti-CUG therapies for FECD.

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.

Leveraging a "Catch-Release" Logic Gate Process for the Synthesis and Nonchromatographic Purification of Thioether- or Amine-Bridged Macrocyclic Peptides.

Macrocyclic peptides containing N-alkylated amino acids have emerged as a promising therapeutic modality, capable of modulating protein-protein interactions and an intracellular delivery of hydrophilic payloads. While multichannel automated solid-phase peptide synthesis (SPPS) is a practical approach for peptide synthesis, the requirement for slow and inefficient chromatographic purification of the product peptides is a significant limitation to exploring these novel compounds. Herein, we invent a "catch-release" strategy for the nonchromatographic purification of macrocyclic peptides. A traceless catch process is enabled by the invention of a dual-functionalized N-terminal acetate analogue, which serves as a handle for capture onto a purification resin and as a leaving group for macrocyclization. Displacement by a C-terminal nucleophilic side chain thus releases the desired macrocycle from the purification resin. By design, this catch/release process is a logic test for the presence of the key components required for cyclization, thus removing impurities which lack the required functionality, such as common classes of peptide impurities, including hydrolysis fragments and truncated sequences. The method was shown to be highly effective with three libraries of macrocyclic peptides, containing macrocycles of 5-20 amino acids, with either thioether- or amine-based macrocyclic linkages; in this latter class, the reported method represents an enabling technology. In all cases, the catch-release protocol afforded significant enrichment of the target peptides purity, in many cases completely obviating the need for chromatography. Importantly, we have adapted this process for automation on a standard multichannel peptide synthesizer, achieving an efficient and completely integrated synthesis and purification platform for the preparation of these important molecules.

Spiroligozymes for transesterifications: design and relationship of structure to activity.

Transesterification catalysts based on stereochemically defined, modular, functionalized ladder-molecules (named spiroligozymes) were designed, using the "inside-out" design strategy, and mutated synthetically to improve catalysis. A series of stereochemically and regiochemically diverse bifunctional spiroligozymes were first synthesized to identify the best arrangement of a pyridine as a general base catalyst and an alcohol nucleophile to accelerate attack on vinyl trifluoroacetate as an electrophile. The best bifunctional spiroligozyme reacted with vinyl trifluoroacetate to form an acyl-spiroligozyme conjugate 2.7 × 10(3)-fold faster than the background reaction with a benzyl alcohol. Two trifunctional spiroligozymes were then synthesized that combined a urea with the pyridine and alcohol to act as an oxyanion hole and activate the bound acyl-spiroligozyme intermediate to enable acyl-transfer to methanol. The best trifunctional spiroligozyme carries out multiple turnovers and acts as a transesterification catalyst with k(1)/k(uncat) of 2.2 × 10(3) and k(2)/k(uncat) of 1.3 × 10(2). Quantum mechanical calculations identified the four transition states of the catalytic cycle and provided a detailed view of every stage of the transesterification reaction.

Hydrophobic substituent effects on proline catalysis of aldol reactions in water.

Derivatives of 4-hydroxyproline with a series of hydrophobic groups in well-defined orientations have been tested as catalysts for the aldol reactions. All of the modified proline catalysts carry out the intermolecular aldol reaction in water and provide high diastereoselectivity and enantioselectivity. Modified prolines with aromatic groups syn to the carboxylic acid are better catalysts than those with small hydrophobic groups (1a is 43.5 times faster than 1f). Quantum mechanical calculations provide transition structures, TS-1a(water) and TS-1f(water), that support the hypothesis that a stabilizing hydrophobic interaction occurs with 1a.