Discovery of High Potency, Single-Chain Insulin Analogs with a Shortened B-Chain and Nonpeptide Linker

2013 ◽  
Vol 8 (8) ◽  
pp. 1822-1829 ◽  
Author(s):  
Zachary P. Kaur ◽  
Alexander R. Ochman ◽  
John P. Mayer ◽  
Vasily M. Gelfanov ◽  
Richard D. DiMarchi
Keyword(s):  
Insulin Analogs ◽  
Single Chain ◽  
High Potency ◽  
Single Chain Insulin

scholarly journals Age of diabetes onset in the mutant proinsulin syndrome correlates with mutational impairment of protein foldability and stability

2021 ◽  
Author(s):  
Balamurugan Dhayalan ◽  
Yen-Shan Chen ◽  
Yanwu Yang ◽  
Mark Jarosinski ◽  
Deepak Chatterjee ◽  
...  
Keyword(s):  
Molten Globule ◽  
Disease Onset ◽  
Single Chain ◽  
Human Insulin Gene ◽  
Disulfide Pairing ◽  
Single Chain Insulin ◽  
Hormone Analogs ◽  
Α Helix ◽  
Core Packing

Diverse heterozygous mutations in the human insulin gene cause a monogenic diabetes mellitus (DM) syndrome due to toxic misfolding of the variant proinsulin. Whereas mutations that add or remove cysteines (thereby leading to an odd number of thiol groups) generally lead to neonatal-onset DM, non-Cys-related mutations can be associated with a broad range of ages of onset. Here, we compare two mutations at a conserved position in the central B-chain α-helix: one neonatal in DM onset (ValB18→Gly) and the other with onset delayed until adolescence (AlaB18). The substitutions were introduced within a 49-residue single-chain insulin precursor optimized for folding efficiency (Zaykov, A., et al. ACS Chem. Biol. 9, 683-91 (2014)). Although mutations are each unfavorable, GlyB18 (a) more markedly perturbs DesDi folding efficiency in vitro than does AlaB18 and (b) more severely induces endoplasmic reticulum (ER) stress in cell-based studies of the respective proinsulin variants. In corresponding two-chain hormone analogs, GlyB18 more markedly perturbs structure, function and thermodynamic stability than does AlaB18. Indeed, the GlyB18-insulin analog forms a molten globule with attenuated α-helix content whereas the AlaA18 analog retains a nativelike cooperative structure with reduced free energy of unfolding (ΔΔGu 1.2(±0.2) kcal/mole relative to ValB18 parent). We propose that mutations at B18 variably impede nascent pairing of CysB19 and CysA20 to an extent correlated with perturbed core packing once native disulfide pairing is achieved. Differences in age of disease onset (neonatal or adolescent) reflect relative biophysical perturbations (severe or mild) of an obligatory on-pathway protein folding intermediate.

scholarly journals Combinations of Single Chain Variable Fragments From HIV Broadly Neutralizing Antibodies Demonstrate High Potency and Breadth

2021 ◽  
Vol 12 ◽  
Author(s):  
Rebecca T. van Dorsten ◽  
Kshitij Wagh ◽  
Penny L. Moore ◽  
Lynn Morris
Keyword(s):  
Neutralizing Antibodies ◽  
Passive Immunization ◽  
Neutralization Assay ◽  
Single Chain ◽  
Broadly Neutralizing Antibodies ◽  
High Potency ◽  
Hill Model ◽  
Mucosal Tissues ◽  
Action Model ◽  
Single Chain Variable Fragments

Broadly neutralizing antibodies (bNAbs) are currently being assessed in clinical trials for their ability to prevent HIV infection. Single chain variable fragments (scFv) of bNAbs have advantages over full antibodies as their smaller size permits improved diffusion into mucosal tissues and facilitates vector-driven gene expression. We have previously shown that scFv of bNAbs individually retain significant breadth and potency. Here we tested combinations of five scFv derived from bNAbs CAP256-VRC26.25 (V2-apex), PGT121 (N332-supersite), 3BNC117 (CD4bs), 8ANC195 (gp120-gp41 interface) and 10E8v4 (MPER). Either two or three scFv were combined in equimolar amounts and tested in the TZM-bl neutralization assay against a multiclade panel of 17 viruses. Experimental IC50 and IC80 data were compared to predicted neutralization titers based on single scFv titers using the Loewe additive and the Bliss-Hill model. Like full-sized antibodies, combinations of scFv showed significantly improved potency and breadth compared to single scFv. Combinations of two or three scFv generally followed an independent action model for breadth and potency with no significant synergy or antagonism observed overall although some exceptions were noted. The Loewe model underestimated potency for some dual and triple combinations while the Bliss-Hill model was better at predicting IC80 titers of triple combinations. Given this, we used the Bliss-Hill model to predict the coverage of scFv against a 45-virus panel at concentrations that correlated with protection in the AMP trials. Using IC80 titers and concentrations of 1μg/mL, there was 93% coverage for one dual scFv combination (3BNC117+10E8v4), and 96% coverage for two of the triple combinations (CAP256.25+3BNC117+10E8v4 and PGT121+3BNC117+10E8v4). Combinations of scFv, therefore, show significantly improved breadth and potency over individual scFv and given their size advantage, have potential for use in passive immunization.

In Vitro Folding/Unfolding of Insulin/Single-Chain Insulin

2006 ◽  
Vol 13 (5) ◽  
pp. 423-429 ◽  
Author(s):  
Zhi-Song Qiao ◽  
Zhan-Yun Guo ◽  
You-Min Feng
Keyword(s):  
Single Chain ◽  
Single Chain Insulin

Single chain insulin with high bioactivity

Biofutur ◽  
1997 ◽  
Vol 1997 (163) ◽  
pp. 44
Author(s):  
RE Chance
Keyword(s):  
Single Chain ◽  
Single Chain Insulin

scholarly journals A single-chain insulin-like growth factor I/insulin hybrid binds with high affinity to the insulin receptor

1995 ◽  
Vol 305 (3) ◽  
pp. 981-986 ◽  
Author(s):  
C Kristensen ◽  
A S Andersen ◽  
M Hach ◽  
F C Wiberg ◽  
L Schäffer ◽  
...  
Keyword(s):  
Growth Factor ◽  
Insulin Receptor ◽  
Insulin Receptors ◽  
Insulin Like Growth Factor ◽  
Single Chain ◽  
Factor I ◽  
High Affinity ◽  
Igf I ◽  
Membrane Bound ◽  
Single Chain Insulin

1. To investigate the structure/function relationship of the interaction between ligand and receptor in the insulin-like growth factor I (IGF-I) and insulin receptor systems we have prepared and characterized a single-chain insulin/IGF-I hybrid. The single-chain hybrid consists of the insulin molecule combined with the C domain of IGF-I. The single-chain hybrid was found to bind with high affinity to both truncated soluble insulin receptors and membrane-bound holoreceptors. The affinity for interacting with the soluble truncated insulin receptors was 55-94% relative to insulin, and affinity for membrane-bound insulin receptors was 113% of that of insulin. Furthermore we found that the affinity of the single-chain hybrid molecule for IGF-I receptors was 19-28% relative to IGF-I. 2. The affinity of the single-chain hybrid for chimeric insulin/IGF-I receptors exceeded that of either natural ligand. This indicates that coordinately changing domains of the receptors and the ligands can induce higher affinity of ligand for receptor, supporting the idea that these receptors have a common ligand-binding site [Kjeldsen, Andersen, Wiberg, Rasmussen, Schäffer, Balschmidt, Møller and Møller (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4404-4408]. 3. In contrast with what was generally assumed about the ligand structure required for binding to the insulin receptor we demonstrate the first single-chain insulin analogue that can bind with high affinity to the insulin receptor.

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