skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Novel four-disulfide insulin analog with high aggregation stability and potency

Abstract

Although insulin was first purified and used therapeutically almost a century ago, there is still a need to improve therapeutic efficacy and patient convenience. A key challenge is the requirement for refrigeration to avoid inactivation of insulin by aggregation/fibrillation. Here, in an effort to mitigate this problem, we introduced a 4th disulfide bond between a C-terminal extended insulin A chain and residues near the C-terminus of the B chain. Insulin activity was retained by an analog with an additional disulfide bond between residues A22 and B22, while other linkages tested resulted in much reduced potency. Furthermore, the A22-B22 analog maintains the native insulin tertiary structure as demonstrated by X-ray crystal structure determination. We further demonstrate that this four-disulfide analog has similar in vivo potency in mice compared to native insulin and demonstrates higher aggregation stability. In conclusion, we have discovered a novel four-disulfide insulin analog with high aggregation stability and potency.

Authors:
ORCiD logo [1]; ORCiD logo [1];  [2];  [3];  [1];  [1];  [1]; ORCiD logo [3];  [2]; ORCiD logo [1]; ORCiD logo [1]
  1. Department of Biochemistry, University of Utah, Salt Lake City UT 84112, USA
  2. Department of Nutrition and Integrative Physiology, University of Utah, Salt Lake City UT 84112, USA
  3. Department of Chemical & Biomolecular Engineering, University of Notre Dame, USA
Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH), The National Institute of Diabetes and Digestive and Kidney (NIDDK); NIH National Institute of General Medical Sciences (NIGMS)
OSTI Identifier:
1574037
Alternate Identifier(s):
OSTI ID: 1624983
Grant/Contract Number:  
AC02-76SF00515; R01DK120430; 5-CDA-2018-572 AN; P41-GM103311
Resource Type:
Published Article
Journal Name:
Chemical Science
Additional Journal Information:
Journal Name: Chemical Science Journal Volume: 11 Journal Issue: 1; Journal ID: ISSN 2041-6520
Publisher:
Royal Society of Chemistry (RSC)
Country of Publication:
United Kingdom
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Chemistry

Citation Formats

Xiong, Xiaochun, Blakely, Alan, Karra, Prasoona, VandenBerg, Michael A., Ghabash, Gabrielle, Whitby, Frank, Zhang, Yi Wolf, Webber, Matthew J., Holland, William L., Hill, Christopher P., and Chou, Danny Hung-Chieh. Novel four-disulfide insulin analog with high aggregation stability and potency. United Kingdom: N. p., 2020. Web. doi:10.1039/C9SC04555D.
Xiong, Xiaochun, Blakely, Alan, Karra, Prasoona, VandenBerg, Michael A., Ghabash, Gabrielle, Whitby, Frank, Zhang, Yi Wolf, Webber, Matthew J., Holland, William L., Hill, Christopher P., & Chou, Danny Hung-Chieh. Novel four-disulfide insulin analog with high aggregation stability and potency. United Kingdom. doi:10.1039/C9SC04555D.
Xiong, Xiaochun, Blakely, Alan, Karra, Prasoona, VandenBerg, Michael A., Ghabash, Gabrielle, Whitby, Frank, Zhang, Yi Wolf, Webber, Matthew J., Holland, William L., Hill, Christopher P., and Chou, Danny Hung-Chieh. Wed . "Novel four-disulfide insulin analog with high aggregation stability and potency". United Kingdom. doi:10.1039/C9SC04555D.
@article{osti_1574037,
title = {Novel four-disulfide insulin analog with high aggregation stability and potency},
author = {Xiong, Xiaochun and Blakely, Alan and Karra, Prasoona and VandenBerg, Michael A. and Ghabash, Gabrielle and Whitby, Frank and Zhang, Yi Wolf and Webber, Matthew J. and Holland, William L. and Hill, Christopher P. and Chou, Danny Hung-Chieh},
abstractNote = {Although insulin was first purified and used therapeutically almost a century ago, there is still a need to improve therapeutic efficacy and patient convenience. A key challenge is the requirement for refrigeration to avoid inactivation of insulin by aggregation/fibrillation. Here, in an effort to mitigate this problem, we introduced a 4th disulfide bond between a C-terminal extended insulin A chain and residues near the C-terminus of the B chain. Insulin activity was retained by an analog with an additional disulfide bond between residues A22 and B22, while other linkages tested resulted in much reduced potency. Furthermore, the A22-B22 analog maintains the native insulin tertiary structure as demonstrated by X-ray crystal structure determination. We further demonstrate that this four-disulfide analog has similar in vivo potency in mice compared to native insulin and demonstrates higher aggregation stability. In conclusion, we have discovered a novel four-disulfide insulin analog with high aggregation stability and potency.},
doi = {10.1039/C9SC04555D},
journal = {Chemical Science},
number = 1,
volume = 11,
place = {United Kingdom},
year = {2020},
month = {1}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1039/C9SC04555D

Citation Metrics:
Cited by: 2 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

A Synthetic Route to Human Insulin Using Isoacyl Peptides
journal, March 2014

  • Liu, Fa; Luo, Ethan Y.; Flora, David B.
  • Angewandte Chemie International Edition, Vol. 53, Issue 15
  • DOI: 10.1002/anie.201310735

The signalling conformation of the insulin receptor ectodomain
journal, October 2018


Mechanisms of Stabilization of the Insulin Hexamer through Allosteric Ligand Interactions
journal, May 1997

  • Rahuel-Clermont, Sophie; French, Cherie A.; Kaarsholm, Niels C.
  • Biochemistry, Vol. 36, Issue 19
  • DOI: 10.1021/bi963038q

Fish-hunting cone snail venoms are a rich source of minimized ligands of the vertebrate insulin receptor
journal, February 2019

  • Ahorukomeye, Peter; Disotuar, Maria M.; Gajewiak, Joanna
  • eLife, Vol. 8
  • DOI: 10.7554/eLife.41574

Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis
journal, February 2018

  • Scapin, Giovanna; Dandey, Venkata P.; Zhang, Zhening
  • Nature, Vol. 556, Issue 7699
  • DOI: 10.1038/nature26153

An ultra-stable single-chain insulin analog resists thermal inactivation and exhibits biological signaling duration equivalent to the native protein
journal, November 2017

  • Glidden, Michael D.; Aldabbagh, Khadijah; Phillips, Nelson B.
  • Journal of Biological Chemistry, Vol. 293, Issue 1
  • DOI: 10.1074/jbc.M117.808626

Design of an Active Ultrastable Single-chain Insulin Analog: SYNTHESIS, STRUCTURE, AND THERAPEUTIC IMPLICATIONS
journal, March 2008

  • Hua, Qing-xin; Nakagawa, Satoe H.; Jia, Wenhua
  • Journal of Biological Chemistry, Vol. 283, Issue 21
  • DOI: 10.1074/jbc.M800313200

Synthesis of Four-Disulfide Insulin Analogs via Sequential Disulfide Bond Formation
journal, March 2017

  • Wu, Fangzhou; Mayer, John P.; Gelfanov, Vasily M.
  • The Journal of Organic Chemistry, Vol. 82, Issue 7
  • DOI: 10.1021/acs.joc.6b03078

Specialized insulin is used for chemical warfare by fish-hunting cone snails
journal, January 2015

  • Safavi-Hemami, Helena; Gajewiak, Joanna; Karanth, Santhosh
  • Proceedings of the National Academy of Sciences, Vol. 112, Issue 6
  • DOI: 10.1073/pnas.1423857112

Pursuit of a perfect insulin
journal, March 2016

  • Zaykov, Alexander N.; Mayer, John P.; DiMarchi, Richard D.
  • Nature Reviews Drug Discovery, Vol. 15, Issue 6
  • DOI: 10.1038/nrd.2015.36

Extreme Stabilization of a Thermolysin-like Protease by an Engineered Disulfide Bond
journal, April 1997

  • Mansfeld, Johanna; Vriend, Gert; Dijkstra, Bauke W.
  • Journal of Biological Chemistry, Vol. 272, Issue 17
  • DOI: 10.1074/jbc.272.17.11152

Structural Origins of the Functional Divergence of Human Insulin-Like Growth Factor-I and Insulin ,
journal, July 2002

  • Brzozowski, Andrzej M.; Dodson, Eleanor J.; Dodson, Guy G.
  • Biochemistry, Vol. 41, Issue 30
  • DOI: 10.1021/bi020084j

Display of Single-Chain Insulin-like Peptides on a Yeast Surface
journal, September 2018


A Disulfide Scan of Insulin by [3 + 1] Methodology Exhibits Site-Specific Influence on Bioactivity
journal, June 2019


Supramolecular PEGylation of biopharmaceuticals
journal, November 2016

  • Webber, Matthew J.; Appel, Eric A.; Vinciguerra, Brittany
  • Proceedings of the National Academy of Sciences, Vol. 113, Issue 50
  • DOI: 10.1073/pnas.1616639113

R 6 hexameric insulin complexed with m -cresol or resorcinol
journal, December 2000

  • Smith, G. David; Ciszak, Ewa; Magrum, Lucy A.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 56, Issue 12
  • DOI: 10.1107/S0907444900012749

Insulin analog with additional disulfide bond has increased stability and preserved activity: Insulin Analog with Additional Disulfide Bond
journal, January 2013

  • Vinther, Tine N.; Norrman, Mathias; Ribel, Ulla
  • Protein Science, Vol. 22, Issue 3
  • DOI: 10.1002/pro.2211

Protective hinge in insulin opens to enable its receptor engagement
journal, August 2014

  • Menting, J. G.; Yang, Y.; Chan, S. J.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 33
  • DOI: 10.1073/pnas.1412897111

Binding of phenol to R6 insulin hexamers*
journal, January 1999


A Synthetic Route to Human Insulin Using Isoacyl Peptides
journal, March 2014