title: The road to the first, fully active and more stable human insulin variant with an additional disulfide bond.
abstract: Insulin, a small peptide hormone, is crucial in maintaining blood glucose homeostasis. The stability and activity of the protein is directed by an intricate system involving disulfide bonds to stabilize the active monomeric species and by their non-covalent oligomerization. All known insulin variants in vertebrates consist of two peptide chains and have six cysteine residues, which form three disulfide bonds, two of them link the two chains and a third is an intra-chain bond in the A-chain. This classical insulin fold appears to have been conserved over half a billion years of evolution. We addressed the question whether a human insulin variant with four disulfide bonds could exist and be fully functional. In this review, we give an overview of the road to engineering four-disulfide bonded insulin analogs. During our journey, we discovered several active four disulfide bonded insulin analogs with markedly improved stability and gained insights into the instability of analogs with seven cysteine residues, importance of dimerization for stability, insulin fibril formation process, and the conformation of insulin binding to its receptor. Our results also open the way for new strategies in the development of insulin biopharmaceuticals.
authors.affiliation: Global Research, Novo Nordisk A/S, DK-2730, Måløv, Denmark.
authors.forename: Tine N
authors.initials: TN
authors.lastname: Vinther
authors.affiliation: Global Research, Novo Nordisk A/S, DK-2730, Måløv, Denmark.
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Kjeldsen
authors.affiliation: Faculty of Science, Department of Chemistry, University of Copenhagen, DK-1871, Frederiksberg, Denmark.
authors.forename: Knud J
authors.initials: KJ
authors.lastname: Jensen
authors.affiliation: Global Research, Novo Nordisk A/S, DK-2730, Måløv, Denmark.
authors.forename: František
authors.initials: F
authors.lastname: Hubálek
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism; D003553:Cystine / Q000737:chemistry*; D003922:Diabetes Mellitus, Type 1 / Q000188:drug therapy* / Q000378:metabolism; D019281:Dimerization; D015195:Drug Design; D004355:Drug Stability; D006801:Humans; D007004:Hypoglycemic Agents / Q000737:chemistry / Q000627:therapeutic use*; D061386:Insulin, Regular, Human / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D055550:Protein Stability; D011972:Receptor, Insulin / Q000819:agonists* / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000627:therapeutic use
chemical_list: D015703:Antigens, CD; D007004:Hypoglycemic Agents; D061386:Insulin, Regular, Human; D011994:Recombinant Proteins; D003553:Cystine; C435941:INSR protein, human; D011972:Receptor, Insulin
keywords: cysteine chemistry; disulfide bonds; insulin; protein engineering