Improved insulin stability through amino acid substitution

Abstract

Insulin analogs designed to decrease self-association and increase absorption rates from subcutaneous tissue were found to have altered stability. Replacement of HB10 with aspartic acid increased stability while substitutions at B28 and/or B29 were either comparable to insulin or had decreased stability. The principal chemical degradation product of accelerated storage conditions was a disulfide-linked multimer that was formed through a disulfide interchange reaction which resulted from beta-elimination of the disulfides. The maintenance of the native state of insulin was shown to be important in protecting the disulfides from reduction by dithiothreitol and implicitly from the disulfide interchange reaction that occurs during storage. To understand how these amino acid changes alter chemical stability, the intramolecular conformational equilibria of each analog was assessed by equilibrium denaturation. The Gibbs free energy of unfolding was compared with the chemical stability during storage for over 20 analogs. A significant positive correlation (R2 = 0.8 and P less than 0.0005) exists between the conformational stability and chemical stability of these analogs, indicating that the chemical stability of insulin's disulfides is under the thermodynamic control of the conformational equilibria.

Mechanism of Insulin Chain Combination

“…Analysis by the two-state formalism (36) in each case indicates decreased stability (column 3 in Table II). At neutral pH (Table II, part A) 2G-, 3G-, and 4G-DKP-insulin are each ϳ2.0 kcal/mol less stable than DKP-insulin; 5G-DKP-insulin is ϳ 2.4 kcal/mol less than DKP-insulin (Table II, enhances the stability of an insulin monomer (22), the 5G A chain was also combined with the wild-type B chain. Like 5G-DKP-insulin, 5G-insulin is ϳ2.4 kcal/mol less stable than its parent structure (insulin).…”

Physicochemical basis for the rapid time‐action of LysB28ProB29‐insulin: Dissociation of a protein‐ligand complex

“…In addition, the hydrogen bonds at both ends of the P-sheet structure near the C-terminus of the B chain are weakened. Although LysPro has proven to be fast-acting upon injection, soluble zinc-free preparations require stabilization with respect to chemical degradation to be of practical use (Brems et al, 1992b).…”

Design of an Active Ultrastable Single-chain Insulin Analog

“…Synthesis of Single-chain Insulin Analog-SCI-57 was synthesized by native chemical ligation (46) of three peptides: segment I (B1-B6; polypeptide residues 1-6), segment II (B7-A6; polypeptide residues 7-42), and segment III (A7-A21; polypeptide residues [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57]. Segments III and II were first ligated; their product was then ligated to segment I to yield the reduced and unfolded 57-residue polypeptide.…”

“…These substitutions also enhance the thermodynamic stability of insulin (44,45). Such enhancement has a theoretical foundation.…”

“…2E, blue) contains a central proline (boldface) to facilitate a chain reversal (31) and retains a dibasic element (underlined) at the CA junction. The insulin moiety contains four substitutions (one in the A-domain and three in the B-domain; Thr A8 3 His, His B10 3 Asp, Pro B28 3 Asp, and Lys B29 3 Pro) collectively designed (i) to achieve electrostatic balance (40), (ii) to prevent self-assembly (41), (iii) to restore wild-type receptor-binding activity in compensation for the connecting segment (42,43), and (iv) to optimize thermodynamic stability (44,45). The corresponding two-chain insulin analog (designated 2CA) was prepared as a control to probe the effects of these substitutions in the absence of a connecting segment.…”