Engineering and Functionalization of the Disulfide‐Constrained Miniprotein Min‐23 as a Scaffold for Diagnostic Application

Abstract

Miniproteins are scaffolds for the development of alternative non-immunoglobin binding agents for medical applications. This peptide format features high tolerance to sequence mutagenesis, excellent proteolytic stability, and fast blood pool clearance. Herein we present the total chemical synthesis of the disulfide-constrained scaffold Min-23 and its functionalization for in vitro and in vivo application. Optimized solid-phase peptide chemistry and oxidative folding strategies were developed to engineer this miniprotein with native-like disulfide configuration. High levels of serum stability and proteolytic resistance, as well as a beneficial pharmacokinetic profile for diagnostic imaging, were determined by using radiolabeling techniques such as positron emission tomography. The reported achievements highlight Min-23 as a promising scaffold for the development of novel recognition molecules for medical application.

Synthetic Cystine-Knot Miniproteins – Valuable Scaffolds for Polypeptide Engineering

“…A two-disulfide scaffold of a miniprotein Min23 [56,104] derived from knottin EETI-II by its miniaturization has been applied as a template for phage display by variations in its surface-exposed loop [105]. Being radioactively labeled, it was used for molecular imaging in AR42J tumor-bearing rat, showing good serum stability [104].…”

“…Being radioactively labeled, it was used for molecular imaging in AR42J tumor-bearing rat, showing good serum stability [104].…”

Combination of Phage Display and Molecular Grafting Generates Highly Specific Tumor‐Targeting Miniproteins

“…[10] Furthermore, Min-23 has already been applied successfully as a structural template for phage display by insertion of up to ten amino acids into its hypervariable, surface-exposed GPNG loop. [10,12,13] Therefore, we combined the Min-23 based phage-display technology with a different peptide scaffold-the sunflower trypsin inhibitor (SFTI-I)-to generate a readily accessible peptide format for hit-to-lead development in vitro and in vivo. [10,12,13] Therefore, we combined the Min-23 based phage-display technology with a different peptide scaffold-the sunflower trypsin inhibitor (SFTI-I)-to generate a readily accessible peptide format for hit-to-lead development in vitro and in vivo.…”

“…[9] Min-23 offers outstanding proteolytic stability and beneficial pharmacokinetic properties for in vivo applications including molecular imaging. [10] Furthermore, Min-23 has already been applied successfully as a structural template for phage display by insertion of up to ten amino acids into its hypervariable, surface-exposed GPNG loop. [11] However, random amino acid substitutions within disulfide-constrained proteins often negatively influence the synthesis yield, because autonomous folding results in regioisomer formation owing to incorrect disulfide connectivity, and requires the appropriate oxidative folding methods.…”

“…[11] However, random amino acid substitutions within disulfide-constrained proteins often negatively influence the synthesis yield, because autonomous folding results in regioisomer formation owing to incorrect disulfide connectivity, and requires the appropriate oxidative folding methods. [10,12,13] Therefore, we combined the Min-23 based phage-display technology with a different peptide scaffold-the sunflower trypsin inhibitor (SFTI-I)-to generate a readily accessible peptide format for hit-to-lead development in vitro and in vivo.…”