Small Scaffolds, Big Potential: Developing Miniature Proteins as Therapeutic Agents

Abstract

Preclinical Research Miniature proteins are a class of oligopeptide characterized by their short sequence lengths and ability to adopt well-folded, three-dimensional structures. Because of their biomimetic nature and synthetic tractability, miniature proteins have been used to study a range of biochemical processes including fast protein folding, signal transduction, catalysis and molecular transport. Recently, miniature proteins have been gaining traction as potential therapeutic agents because their small size and ability to fold into defined tertiary structures facilitates their development as protein-based drugs. This research overview discusses emerging developments involving the use of miniature proteins as scaffolds to design novel therapeutics for the treatment and study of human disease. Specifically, this review will explore strategies to: (i) stabilize miniature protein tertiary structure; (ii) optimize biomolecular recognition by grafting functional epitopes onto miniature protein scaffolds; and (iii) enhance cytosolic delivery of miniature proteins through the use of cationic motifs that facilitate endosomal escape. These objectives are discussed not only to address challenges in developing effective miniature protein-based drugs, but also to highlight the tremendous potential miniature proteins hold for combating and understanding human disease. Drug Dev Res 78 : 268-282, 2017. © 2017 Wiley Periodicals, Inc.

Overcoming Endosomal Entrapment in Drug Delivery

“…Patients with genetic diseases caused by missing or defective genes may be treated with recombinant proteins chemically modified with a cyclic CPP 31 or genetically fused to a miniature protein. 105 Monoclonal antibodies (and their fragments/surrogates) may be delivered into cells to specifically block the function of a target protein, as therapeutic agents or mechanistic probes. As discussed above, cyclic CPPs may be integrated into macrocyclic peptide design to generate cell-permeable, metabolically stable peptide therapeutics to target challenging proteins such as those involved in intra-cellular protein−protein interactions.…”

“…An exciting application is the delivery of proteins into the cytosol of mammalian cells. Patients with genetic diseases caused by missing or defective genes may be treated with recombinant proteins chemically modified with a cyclic CPP or genetically fused to a miniature protein . Monoclonal antibodies (and their fragments/surrogates) may be delivered into cells to specifically block the function of a target protein, as therapeutic agents or mechanistic probes.…”

Synthesis of Scyllatoxin‐Based BH3 Domain Mimetics with Diverse Patterns of Native Disulfide Bonds

“…Such approaches have been especially successful for inhibiting PPIs involving extracellular targets (Laraia, McKenzie, Spring, Venkitaraman, & Huggins, 2015). At the same time, many labs have expanded the utility of miniature proteins through “protein grafting”, a re‐engineering technique that replaces sequences of structured peptides or small proteins with recognition elements that are complementary to the targeted protein surface (Holub, 2017; Sia & Kim, 2003; Tsomaia, 2015). This strategy has gained considerable traction among chemical biologists over the last 20 years and has been used to successfully target PPIs previously considered to be “undruggable.” Since being introduced in the early 2000s, the protein grafting strategy has become a well‐established approach to develop molecules that can be used for therapeutic intervention or as tools to help researchers better understand the molecular nature of PPIs.…”

“…Despite their small size, the globular folds of miniature proteins are often stabilized by the same forces (e.g., electrostatic interactions, salt bridges, metal‐ion complexation, hydrophobic effects, and disulfide linkages) found in much larger proteins. Owing to their synthetic tractability and well‐defined architectures, miniature proteins have emerged as attractive alternatives to small‐molecule‐ and protein‐based drugs for targeting therapeutically‐relevant PPIs (Holub, 2017; Li et al., 2008; Vita et al., 1999). Indeed, miniature proteins occupy a unique ‘middle‐space’, combining compact size with elements of well‐folded secondary/tertiary structures that can be used to target protein interaction surfaces.…”

Peptide Mimetics: Fast‐Forward Look

“…Readers are referred to the comprehensive review by Marshall and Ballante in this issue, discussing novel strategies for designing type III peptide mimetics, that is, peptide mimetics having unique templates that position the necessary functional epitopes for serving as topographical mimetics [Marshall and Ballante, ]. In another research overview in this issue, Justin Holub discusses general aspects encountered during the development of miniature proteins as therapeutics, in particular, including epitopes that facilitate endosomal escape for improving bioavailability [Holub, ]. Further reviews in this issue discuss specific examples for peptide mimetic drug development.…”