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. Author manuscript; available in PMC: 2021 Apr 1.
Published in final edited form as: Trends Biochem Sci. 2020 Jan 31;45(4):332–346. doi: 10.1016/j.tibs.2019.12.008

Table 1.

Typical characteristics and considerations for miniproteins with comparison to other modalities

Molecular properties
Category Subcategory Class Common screening methods Size (kDa) Description Specificity PPI disruption Enzymatic activity Inhibition Product homogeneity
Miniproteins Cystine reinforced Avimers Phage display 4 Loop-rich A-domains from human cell surface receptors, stabilized by cystines and a coordinated calcium ion. High Excellent Moderate as novel function, excellent as native function. Moderate to High
Kunitz Yeast display 7 Protease inhibitor domain with a hydrophobic core and cystine stabilization.
CDP Yeast display, mammalian display 3–7 Diverse structure and taxonomic lineage, folding and stability driven primarily by cystine dense core.
Hydrophobic core Affibody Phage display, bacterial display 7 Three-helix bundle from FcR. High Excellent Moderate Moderate to High
Adnectin + Centyrins mRNA display, CIS display 10 Fibronectin type 3 (10Fn3) fragment. Highly sheet rich, similar to VH.
Nanofittins + Affitins Ribosome display 7 7–kDa DNA binding protein family, such as Sulfolobus acidocaldarius Sac7d; mini β-barrel capped by an α-helix.
Fynomer Phage display, OBOC 7 SH3 domain of Fyn kinase. Some β-sheet elements with surface-exposed loops.
Chemically stabilized β-hairpins Phage display 1–2 Pair of short β-sheets joined by small turn, often cyclized. High Moderate to excellent High High
Stapled peptides Phage display, OBOC 1–2 Single α-helix with chemical staple to stabilize helical fold and impart additional function.
Bicycles Phage display, OBOC 1–2 Short peptide loop with three reactive residues cross-linked to form bicyclic compound.
Typical Large proteins Rational design, mammal immunization for antibodies > 10 Whole or modular domain of a protein, applying native function as the therapeutic with minimal structural modifications. High Excellent Poor Poor
Typical (unstructured) Peptides Phage display, OBOC, rational design 0.2–4 Short, flexible amino acid polymer. Minimal, if any, secondary structure. Moderate to High Moderate High High to Excellent
Typical Small molecules Single-well cell-based assays < 1 Synthetic organic molecules of non-protein origin. Many are from natural sources. Low to Moderate Poor Excellent Excellent
Pharmacologic Properties Manufacturing
Category Subcategory Class Tissue/cell penetration Viable compartments Serum half-life Route Production Characterization Cost of goods Typical storage PDB used in Fig. 1 Ref
Miniproteins Cystine reinforced Avimers Moderate to high Extracellular, intracellular, proteolytic Typically short, can be engineered Parenteral, some oral Synthetic or recombinant (eukaryotic or prokaryotic) Straightforward Low to moderate Refrigerated or room temperature 1AJJ [17]
Kunitz 5C67 [87]
CDP 6ATW [67]
Hydrophobic core Affibody Moderate to high Extracellular Typically short, can be engineered Parenteral Synthetic or recombinant (eukaryotic or prokaryotic) Straightforward Low to moderate Refrigerated or room temperature 3MZW [88]
Adnectin + Centyrins 3QWQ [7]
Nanofittins + Affitins 2XIW [10]
Fynomer 4AFQ [8]
Chemically stabilized β-hairpins Moderate to High Extracellular, intracellular, proteolytic Typically short, can be engineered Parenteral, some oral Synthetic or recombinant (prokaryotic) for primary screening, synthetic for manufacture Straightforward Low to moderate Refrigerated or room temperature 5V63 [89]
Stapled peptides 5AFG [90]
Bicycles 5EEL [91]
Typical large proteins Poor Extracellular Antibodies: extended.
Others: variable		 Parenteral Recombinant (eukaryotic) Complex High Refrigerated [92]
Typical (unstructured) peptides Moderate to high Extracellular, proteolytic with non-natural amino acids Typically short, can be engineered Parenteral, some intranasal, oral Synthetic or recombinant (eukaryotic or prokaryotic) Straightforward Low to moderate Refrigerated or room temperature [19]
Typical small molecules High to excellent Extracellular, intracellular, proteolytic Typically short Oral, parenteral, other Synthetic Facile Low Room temperature [80]