Insulin analog with additional disulfide bond has increased stability and preserved activity

Tine N. Vinther; Mathias Norrman; Ulla Ribel; Kasper Huus; Morten Schlein; Dorte B. Steensgaard; Thomas Å. Pedersen; Ingrid Pettersson; Svend Ludvigsen; Thomas Kjeldsen; Knud Jørgen Jensen; František Hubálek

doi:10.1002/pro.2211

Insulin analog with additional disulfide bond has increased stability and preserved activity

Tine N. Vinther, Mathias Norrman, Ulla Ribel, Kasper Huus, Morten Schlein, Dorte B. Steensgaard, Thomas Å. Pedersen, Ingrid Pettersson, Svend Ludvigsen, Thomas Kjeldsen, Knud Jørgen Jensen, František Hubálek

Department of Chemistry

45 Citations (Scopus)

Abstract

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα-Cα distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the B-chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.

Original language	English
Journal	Protein Science
Volume	22
Issue number	3
Pages (from-to)	296-305
Number of pages	10
ISSN	0961-8368
DOIs	https://doi.org/10.1002/pro.2211
Publication status	Published - Mar 2013

Keywords

Adipocytes
Amino Acid Substitution
Animals
Antigens, CD
Biological Transport
Blood Glucose
Cells, Cultured
Cystine
Dose-Response Relationship, Drug
Drug Stability
Glucose
Humans
Hypoglycemic Agents
Insulin, Regular, Human
Mutant Proteins
Protein Conformation
Protein Stability
Rats
Rats, Mutant Strains
Rats, Wistar
Receptor, Insulin
Recombinant Proteins
Zinc

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10.1002/pro.2211

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title = "Insulin analog with additional disulfide bond has increased stability and preserved activity",

abstract = "Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα-Cα distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the B-chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.",

keywords = "Adipocytes, Amino Acid Substitution, Animals, Antigens, CD, Biological Transport, Blood Glucose, Cells, Cultured, Cystine, Dose-Response Relationship, Drug, Drug Stability, Glucose, Humans, Hypoglycemic Agents, Insulin, Regular, Human, Mutant Proteins, Protein Conformation, Protein Stability, Rats, Rats, Mutant Strains, Rats, Wistar, Receptor, Insulin, Recombinant Proteins, Zinc",

author = "Vinther, {Tine N.} and Mathias Norrman and Ulla Ribel and Kasper Huus and Morten Schlein and Steensgaard, {Dorte B.} and Pedersen, {Thomas {\AA}.} and Ingrid Pettersson and Svend Ludvigsen and Thomas Kjeldsen and Jensen, {Knud J{\o}rgen} and Franti{\v s}ek Hub{\'a}lek",

note = "Copyright {\textcopyright} 2012 The Protein Society.",

year = "2013",

month = mar,

doi = "10.1002/pro.2211",

language = "English",

volume = "22",

pages = "296--305",

journal = "Protein Science",

issn = "0961-8368",

publisher = "Wiley-Blackwell",

number = "3",

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TY - JOUR

T1 - Insulin analog with additional disulfide bond has increased stability and preserved activity

AU - Vinther, Tine N.

AU - Norrman, Mathias

AU - Ribel, Ulla

AU - Huus, Kasper

AU - Schlein, Morten

AU - Steensgaard, Dorte B.

AU - Pedersen, Thomas Å.

AU - Pettersson, Ingrid

AU - Ludvigsen, Svend

AU - Kjeldsen, Thomas

AU - Jensen, Knud Jørgen

AU - Hubálek, František

PY - 2013/3

Y1 - 2013/3

N2 - Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα-Cα distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the B-chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.

AB - Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα-Cα distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the B-chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.

KW - Adipocytes

KW - Amino Acid Substitution

KW - Animals

KW - Antigens, CD

KW - Biological Transport

KW - Blood Glucose

KW - Cells, Cultured

KW - Cystine

KW - Dose-Response Relationship, Drug

KW - Drug Stability

KW - Glucose

KW - Humans

KW - Hypoglycemic Agents

KW - Insulin, Regular, Human

KW - Mutant Proteins

KW - Protein Conformation

KW - Protein Stability

KW - Rats

KW - Rats, Mutant Strains

KW - Rats, Wistar

KW - Receptor, Insulin

KW - Recombinant Proteins

KW - Zinc

U2 - 10.1002/pro.2211

DO - 10.1002/pro.2211

M3 - Journal article

C2 - 23281053

SN - 0961-8368

VL - 22

SP - 296

EP - 305

JO - Protein Science

JF - Protein Science

IS - 3

ER -

Insulin analog with additional disulfide bond has increased stability and preserved activity

Abstract

Keywords

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