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WO2018014091A1 - Analogues de l'insuline - Google Patents

Analogues de l'insuline Download PDF

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Publication number
WO2018014091A1
WO2018014091A1 PCT/AU2017/050758 AU2017050758W WO2018014091A1 WO 2018014091 A1 WO2018014091 A1 WO 2018014091A1 AU 2017050758 W AU2017050758 W AU 2017050758W WO 2018014091 A1 WO2018014091 A1 WO 2018014091A1
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WIPO (PCT)
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absent
leu
val
glu
cys
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PCT/AU2017/050758
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English (en)
Inventor
John Gerbrandt Tasman Menting
Brian Smith
Danny Hung-Chieh Chou
Helena SAFAVI-HEMAMI
Michael Colin Lawrence
Olivera M. BALDOMERO
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La Trobe University
Walter and Eliza Hall Institute of Medical Research
University of Utah Research Foundation Inc
Original Assignee
La Trobe University
Walter and Eliza Hall Institute of Medical Research
University of Utah Research Foundation Inc
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Priority claimed from AU2016902883A external-priority patent/AU2016902883A0/en
Priority to JP2019503259A priority Critical patent/JP7143275B2/ja
Priority to BR112019000991A priority patent/BR112019000991A2/pt
Priority to CR20190096A priority patent/CR20190096A/es
Priority to IL264330A priority patent/IL264330B2/en
Priority to SG11201900181RA priority patent/SG11201900181RA/en
Priority to MX2019000829A priority patent/MX2019000829A/es
Priority to AU2017298565A priority patent/AU2017298565B2/en
Priority to EP17830123.0A priority patent/EP3487876A4/fr
Priority to CN202310855095.4A priority patent/CN116874584A/zh
Priority to RU2019100497A priority patent/RU2769476C2/ru
Priority to KR1020237015079A priority patent/KR20230070049A/ko
Application filed by La Trobe University, Walter and Eliza Hall Institute of Medical Research, University of Utah Research Foundation Inc filed Critical La Trobe University
Priority to CN201780050247.6A priority patent/CN110072884B/zh
Priority to KR1020197004760A priority patent/KR102529353B1/ko
Priority to NZ750355A priority patent/NZ750355B2/en
Priority to CA3030930A priority patent/CA3030930A1/fr
Priority to US16/319,450 priority patent/US11248034B2/en
Publication of WO2018014091A1 publication Critical patent/WO2018014091A1/fr
Priority to PH12019500090A priority patent/PH12019500090A1/en
Anticipated expiration legal-status Critical
Priority to ZA2019/01095A priority patent/ZA201901095B/en
Priority to AU2021269301A priority patent/AU2021269301B2/en
Priority to US17/568,698 priority patent/US12410228B2/en
Priority to JP2022146483A priority patent/JP2022191233A/ja
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G16B15/30—Drug targeting using structural data; Docking or binding prediction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B29/00—Single crystals or homogeneous polycrystalline material with defined structure characterised by the material or by their shape
    • C30B29/54—Organic compounds
    • C30B29/58—Macromolecular compounds
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B7/00—Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions
    • C30B7/02—Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions by evaporation of the solvent
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • the present invention relates generally to insulin analogs. More particularly, the present invention relates to rapid acting insulin analogs having shortened B chains. The present invention also relates to the crystal structure of insulin from the venom of cone snails and to methods of using the crystal and related structural information to screen for and design insulin analogs that interact with or modulate the insulin receptor.
  • Insulin is a polypeptide hormone that plays a central role in the regulation of glucose metabolism, reproduction and cognition.
  • Human insulin monomer consists of two polypeptide chains, the A- and B -chains, which are covalently linked by two disulfide bridges (CysA7-CysB7 and CysA19-B20).
  • the A-chain consists of 21 amino acids and the B chain consists of 30 amino acids.
  • a third disulfide bridge is located within the A chain (CysA6-CysAl l).
  • insulin exists as monomers, dimers and hexamers.
  • the hexamer consists of three insulin dimers held together by two central zinc ions.
  • Diabetes mellitus (referred to as diabetes) is a group of disorders that is characterized by high blood sugar levels over a prolonged period of time. Diabetes can arise if the pancreas does not produce enough insulin or if the body does not respond properly to insulin. Administration of insulin or insulin analogs remains the most effective method of treating conditions such as diabetes. Treatment of diabetes often involves administration of a combination of rapid acting, pre-prandial insulin as well as a longer-acting insulin to maintain basal levels of the hormone.
  • Rapid-acting insulin analogs have a fast onset of activity. Typically, they are either monomeric or rapidly dissociate into the monomeric form on injection into an affected individual. Structurally, these insulin analogs differ from normal human insulin by having modifications within the B-chain C-terminal region (residues B26- B30) that are deleterious to insulin multimerization. However, further C-terminal truncation of the B chain in order to abolish self-association has led to near complete loss of activity, presumably because PheB24 is critical for activity. For example, des- octapeptide[B23-B30] insulin (DOI), a monomeric analogue, preserves less than 0.1 % bioactivity (Bao at al., 1997).
  • DOI des- octapeptide[B23-B30] insulin
  • PheB24 lies immediately C-terminal to a Type 1 ⁇ -turn formed by residues GlyB20-GluB21-ArgB22-GlyB23, with both the triplet PheB24- PheB25-TyrB26 and the Type 1 ⁇ -turn being highly conserved in vertebrate insulins.
  • the inventors have characterised the newly identified insulin Con-Ins Gl from the venom of Conus geographus and show that it is monomeric, but still binds to the human insulin receptor and retains signalling activity.
  • the inventors further successfully produced crystals of Con-Ins Gl and elucidated its three-dimensional structure using X-ray crystallography.
  • the structural data presented herein have now enabled identification, for the first time, the key amino acid positions and interactions that permit Con-Ins Gl to retain its activity, despite lacking the aromatic triplet, PheB24-PheB25-TyrB26, of human insulin.
  • the present invention provides an insulin analog comprising an A chain peptide and a B chain peptide, wherein the B chain comprises an aromatic or large aliphatic residue at a position corresponding to amino acid number 20 of the B chain of human insulin and/or an aromatic or large aliphatic residue at a position corresponding to amino acid number 15 of the B chain of human insulin, wherein the analog comprises at least one amino acid found in human insulin but lacking in the corresponding position of Conus geographus venom insulin, and wherein the A chain peptide and the B chain peptide are bonded together across at least one pair of cysteine residues.
  • the aromatic residue is selected from the group consisting of tyrosine, phenylalanine, tryptophan, histidine and 4-methylphenylalanine.
  • the large aliphatic residue is selected from the group consiting of isoleucine, cyclohexylalanine, cyclopentylalanine and methionine.
  • the aromatic or large aliphatic residue at a position corresponding to amino acid number 20 of the B chain of human insulin is selected from the group consisting of tyrosine, phenylalanine, 4-methylphenylalanine, histidine, tryptophan, methionine, cyclopentylalanine and cyclohexylalanine.
  • the aromatic or large aliphatic residue at a position corresponding to amino acid number 15 of the B chain of human insulin is selected from the group consisting of tyrosine, phenylalanine, 4-methylphenylalanine, histidine, tryptophan, methionine, cyclopentylalanine and cyclohexylalanine.
  • the B chain is truncated at the C-terminal end when compared to human insulin. In some embodiments, the B chain is lacking one or more or all of the nine C-terminal amino acids of human insulin. In some embodiments, the B chain is at least lacking PheB24 of human insulin. In some embodiments, the B chain is at least lacking the human B chain aromatic triplet (amino acids PheB24-PheB25- TyrB26 of human insulin).
  • the insulin analog comprises an A chain peptide comprising the sequence Gly-XA2-XA3-XA4-XA5-Cys A 6-CysA7-XA8-XA9-XAio-Cys A ii- XA12-XA13-XA14-XA15-XA16-XA17-XA18-XA1 -CySA20-XA21-XA22-XA23-XA24-XA25-XA26-
  • the B chain peptide comprises the sequence X B1 -X B 2-
  • X B 1 Thr, Asn, Ser or is absent;
  • X B2 Phe, Ser, Asn, Thr, Gin or is absent;
  • X B 3 Asp, Gly, Pro, Leu, Phe, or His;
  • X B 4 Thr, Pro, Asp, Val or Gly;
  • XBS Asn, Pro, His, Thr, Arg, Ser or hydroxyproline;
  • X B 6 Lys, Glu, Asn
  • the B chain peptide comprises the sequence X B j-X B 2-
  • XBI Thr, Asn, Ser or is absent
  • X B 2 Phe, Ser, Asn, Thr, Gin or is absent
  • X B 3 Asp, Gly, Pro, Leu, Phe, or His
  • X B 4 Thr, Pro, Asp, Val or Gly
  • XBS Asn, Pro, His, Thr, Arg, Ser or hydroxyproline
  • X B 6 Lys, Glu, Asn
  • the B chain peptide comprises the sequence XBI-XB2- X B 3-XB4-XB5-XB6-XB 7 -XB8-CySB9-Gly-Ser-X B 12-XB13-XB14-XB15-XB16-XB17-XB18-XB19-
  • the B chain peptide comprises the sequence X B1 -X B 2- X B 3-X B 4-X B 5-X B 6-X B 7-X B 8-CyS B 9-Gly-Ser-X B 1 2-X B 13-XB14-XB15-XB16-XB17-XB18-XB19- XB20-CyS B 21-X B 22 " XB23- X B 24-XB25-XB26-XB27-XB28-XB29-XB30-XB31-XB32-XB33-XB34 "
  • the B chain peptide comprises the sequence X B1 -X B 2- X B 3-X B 4-X B 5-X B 6-X B 7-XB8-CyS B -Gly-Ser-X B i2-X B 13-XB14-XB15-XB16-XB17-XB18-XB19-
  • the B chain peptide comprises the sequence X B I-XB2-
  • X B n and X B 22 are Tyr. In some embodiments, X B 22 isTyr. In some embodiments, ⁇ isTyr.
  • the A chain peptide comprises the sequence Gly-X A 2-
  • the insulin analog comprises an A chain peptide comprising a sequence Gly-X A 2-Val-X A 4-X A 5-CysA6-CysA7-X A g-X A 9-X A1 o-CysAl l- Ser-X A13 -X A1 4-X A15 -X A16 -X A i7-XAi8-Tyr-Cys A 2o-X A 2i, wherein X A2 is Val or He, X A4 is Glu or gamma carboxyglutamate, X A s is His or Gin, X A g is His or Thr, X A 9 is Arg or Ser, X A IO is Pro or He, X A i3 is Asn or Leu, X A M is Ala or Tyr, X A is is Glu or Gin, X A I6 is Phe or Leu, ⁇ ⁇ ⁇ is Lys or Glu, X A IS is Lys or Asn
  • the insulin analog comprises an A chain peptide comprising a sequence Gly-XA2-Val-XA4-XA5-CysA6-CysA7-XA8-XA9-XAio-CysAii-Ser- X A i3-XAi4-XAi5-Phe-X A i7-XAi8-Tyr-Cys A 20-XA2i, wherein XA2 is Val or He, X A 4 is Glu or gamma carboxyglutamate, X A s is His or Gin, X A 8 is His or Thr, X A9 is Arg or Ser, X A IO is Pro or He, X A i3 is Asn or Leu, X A M is Ala or Tyr, X A is is Glu or Gin, ⁇ ⁇ ⁇ is Lys or Glu, X A IS is Lys or Asn and X A 2i is Asn or absent (S
  • the insulin analog comprises an A chain peptide comprising a sequence Gly-Val-Val-X A 4-XA5-CysA6-CysA7-XA8-XA9-XAio-Cys A ii-Ser- XAi3-XAi4-XAi5-Phe-X A i7-XAi8-Tyr-Cys A 20-XA2i, wherein X A4 is Glu or gamma carboxyglutamate, X A s is His or Gin, X A s is His or Thr, X A9 is Arg or Ser, X A IO is Pro or He, X A I3 is Asn or Leu, X A M is Ala or Tyr, X A IS is Glu or Gin, ⁇ ⁇ ⁇ is Lys or Glu, X A I8 is Lys or Asn and X A 2i is Asn or absent (SEQ ID NO: 14); and an B chain peptide compris
  • the insulin analog is identical to human insulin with the exception of a truncated B-chain at the C-terminus and an aromatic residue or large aliphatic residue at amino acid number 15 and/or 20 of the B chain. Examples are provided, but are not limited to, the three below embodiments where Xaa is an aromatic residue or large aliphatic residue.
  • the insulin analog comprises an A chain peptide comprising the sequence Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr- Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO: 16); and a B chain peptide comprising the sequence Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Xaa-Tyr- Leu-Val-Cys-Gly-Glu, where Xaa is an aromatic residue or large aliphatic residue (SEQ ID NO: 17).
  • the insulin analog comprises an A chain peptide comprising the sequence Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr- Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO: 18); and a B chain peptide comprising the sequence Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr- Leu-Val-Cys-Xaa-Glu, where Xaa is an aromatic residue or large aliphatic residue (SEQ ID NO: 19).
  • the insulin analog comprises an A chain peptide comprising the sequence Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr- Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO: 20); and a B chain peptide comprising the sequence Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Xaa-Tyr- Leu-Val-Cys-Xaa-Glu (SEQ ID NO: 21), where Xaa is an aromatic residue or large aliphatic residue.
  • the insulin analog comprises a number of modified amino acids. In some embodiments, the insulin analog comprises one or more or all of the following;
  • XA4 is gamma carboxyglutamate
  • XB5 hydroxyproline
  • XBI2 gamma carboxyglutamate.
  • Cys B 9 of the B chain peptide is bonded to Cys A 6 of the A chain peptide.
  • Cys B 2i of the B chain peptide is bonded to Cys A 20 of the A chain peptide.
  • Cys A 7 is bonded to Cys A11 .
  • the A chain peptide and the B chain peptide are linked together at one pair of their respective terminal ends. In some embodiments, the A chain peptide and the B chain peptide are linked together at both terminal ends.
  • the insulin analog has an IC50 against the human IR-B receptor of less than 10 ⁇ 6 M. In some embodiments, the insulin analog does not bind human IGF-IR or binds IGF-IR weakly. In some embodiments, the analog has an affinity (3 ⁇ 4) for human IGF-IR of weaker than 100 nM.
  • the insulin analog is predominantly monomeric. In some embodiments, at least 75% of the analog is monomeric in solution.
  • the insulin analog has increased bioavailability when administered to a human when compared human insulin. In some embodiments, the insulin analog has a peak bioavailability within 0.5 to 3 hours of administration to a human. In some embodiments, the insulin analog has an onset of activity within 10 minutes of administration.
  • the present invention provides a pharmaceutical composition, comprising the insulin analog as defined herein or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers.
  • the present invention provides a method for treating and/or preventing an insulin-related condition, comprising administering a therapeutically effective amount of the insulin analog as defined herein to a subject in need thereof.
  • the insulin related condition is hyperglycemia, insulin resistance, type-1 diabetes, gestational diabetes or type-2 diabetes.
  • the present invention provides a method for decreasing blood glucose levels, comprising administering a therapeutically effective amount of the insulin analog as defined herein to a subject in need thereof.
  • the present invention provides use of the insulin analog as defined herein in the manufacture of a medicament for treating and/or preventing an insulin-related condition in a subject.
  • the present invention provides use of the insulin analog as defined herein in the manufacture of a medicament for decreasing blood glucose levels in a subject.
  • the present invention provides an insulin analog as defined herein for use in treating and/or preventing an insulin-related condition in a subject. In still a further aspect, the present invention provides an insulin analog as defined herein for use in decreasing blood glucose levels in a subject.
  • the present invention provides peptides comprising an insulin A chain peptide and an insulin B chain peptide, wherein the B chain peptide comprises a substitution at amino acid 10 and amino acid 20.
  • the substitution at amino acid 20 is G20Y, G20F, or G20P.
  • the substitution at amino acid 10 is H10E, H10D or H10Q.
  • the peptides comprise an insulin A chain peptide and an insulin B chain peptide, wherein the B chain peptide comprises a substitution at amino acid 10 and amino acid 20, further comprising at least one substitution in the A chain peptide.
  • the at least one substitution in the A chain peptide is T8H, T8Y, T8K, or S9R.
  • the peptides comprise an insulin A chain peptide and an insulin B chain peptide, wherein the B chain peptide comprises a substitution at amino acid 10 and amino acid 20, further comprising at least two substitutions in the A chain peptide.
  • the at least two substitutions in the A chain peptide are two of the substitutions selected from: T8H, T8Y, T8K, and S9R.
  • the peptide is a des-octapeptide insulin.
  • the B chain peptide comprises the sequence of FVNQHLCGSELVEALYLVCYER (SEQ ID NO: 30).
  • the the A chain comprises the sequence of GIVEQCCHRICSLYQLENYCN (SEQ ID NO: 39).
  • the A chain peptide and B chain peptide are bonded via at least one disulfide bond.
  • the peptide is a monomer.
  • the insulin A chain peptide is at least 70% identical to wild type human insulin A chain peptide.
  • the present invention provides pharmaceutical compositions comprising an insulin analog, peptide or compound as defined herein.
  • the present invention provides pharmaceutical compositions comprising a peptide comprising an insulin A chain peptide and an insulin B chain peptide, wherein the B chain peptide comprises a substitution at amino acid 10 and amino acid 20 and a pharmaceutically acceptable carrier.
  • the present invention provides methods of increasing insulin receptor activation in a subject comprising administering a therapeutically effective amount of an insulin analog, peptide or compound as defined herein. In some embodiments, the present invention provides methods of increasing insulin receptor activation in a subject comprising administering a therapeutically effective amount of a peptide comprising an insulin A chain peptide and an insulin B chain peptide, wherein the B chain peptide comprises a substitution at amino acid 10 and amino acid 20 to a subject in need thereof.
  • the present invention provides methods of lowering the blood sugar in a subject comprising administering a therapeutically effective amount of an insulin analog, peptide or compound as defined herein.
  • the present invention provides methods of lowering the blood sugar in a subject comprising administering a therapeutically effective amount of a peptide comprising an insulin A chain peptide and an insulin B chain peptide, wherein the B chain peptide comprises a substitution at amino acid 10 and amino acid 20 to a subject in need thereof.
  • the present invention methods of treating type 1 diabetes in a subject comprising administering a therapeutically effective amount of an insulin analog, peptide or compound as defined herein.
  • the present invention provides methods of treating type 1 diabetes in a subject comprising administering a therapeutically effective amount of a peptide comprising an insulin A chain peptide and an insulin B chain peptide, wherein the B chain peptide comprises a substitution at amino acid 10 and amino acid 20 to a subject in need thereof.
  • the subject has been diagnosed with type 1 diabetes prior to administering the peptide.
  • a therapeutic protein having an A chain peptide bonded to a B chain peptide via at least one disulfide bond, wherein the A chain comprises the sequence of GIVEQCCHRICSLYQLENYCN (SEQ ID NO: 39), and wherein the B chain peptide comprises the sequence of FVNQHLCGSELVEALYLVCYER (SEQ ID NO: 30).
  • the present invention provides a method of redesigning or modifying a polypeptide which is known to bind to an insulin receptor (IR) comprising performing structure-based evaluation of a structure defined by the atomic coordinates of Appendix I or a subset thereof and redesigning or chemically modifying the polypeptide as a result of the evaluation.
  • the structure-based evaluation comprises comparison of the structure defined by the atomic coordinates of Appendix I or a subset thereof, with the atomic coordinates of insulin or a subset thereof.
  • the structure-based evaluation further comprises molecular modelling of a complex formed between the structure defined by the atomic coordinates of Appendix I or a subset thereof with the atomic coordinates of an insulin receptor or a subset thereof.
  • the method further comprises synthesising or obtaining the redesigned or chemically modified polypeptide and testing for its ability to bind IR.
  • the method further comprises synthesising or obtaining the redesigned or chemically modified polypeptide and determining the ability of the redesigned or chemically modified polypeptide to modulate IR activation.
  • the method further comprises synthesising or obtaining the redesigned or chemically modified polypeptide and determining the ability of the redesigned or chemically modified polypeptide to lower blood glucose levels.
  • the polypeptide which is known to bind to IR is insulin.
  • the insulin is human insulin.
  • the polypeptide is monomeric.
  • the present invention provides an isolated molecule which is an IR agonist, wherein the molecule is identified and/or designed based on the 3D structure of Con-Ins Gl defined by the atomic coordinates of Appendix I or a subset thereof.
  • the molecule is a peptide, polypeptide or peptidomimetic.
  • the molecule is monomeric.
  • the molecule has an IC5 0 against the human IR-B receptor of less than 10 ⁇ 6 M.
  • the present invention provides a method of identifying a compound which binds IR, the method comprising:
  • generating a three-dimensional structure model comprises generating a model of the polypeptide bound to IR or regions thereof.
  • the method further comprises synthesising the compound which potentially binds the IR.
  • the compound modulates at least one biological activity of IR.
  • the compound is monomeric.
  • the method further comprises testing the compound designed or screened for in ii) for its ability to modulate blood glucose levels. In some embodiments, steps i) and ii) are performed in silico.
  • the present invention provides a computer-based method of identifying a compound which mimics insulin activity, the method comprising
  • generating a three-dimensional structure model comprises generating a model of the polypeptide bound to IR or regions thereof.
  • the method further comprises synthesising the compound which potentially binds the IR.
  • the compound modulates at least one biological activity of IR.
  • the compound is monomeric.
  • the method further comprises testing the compound designed or screened for in ii) for its ability to modulate blood glucose levels. In some embodiments, steps i) and ii) are performed in silico.
  • the present invention provides a compound identified using a method defined herein.
  • the present invention provides the structure of Con-Ins Gl polypeptide as defined by the atomic coordinates of Appendix I.
  • the present invention provides for the use of the structure of Con-Ins Gl polypeptide as defined by the atomic coordinates of Appendix I as a structural model.
  • the structural model is used for identification of insulin analogs.
  • the present invention also provides insulin analogs identified by the use defined herein.
  • the present invention provides a pharmaceutical composition comprising the insulin analog, polypeptide molecule and/or compound as defined herein.
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • Figure 1 Sequence comparison with human insulin. The sequence of Con- Ins Gl and comparison with human insulin. The conserved cysteine residues and aromatic triplet are shaded grey. The disulphide bonds are indicated with a solid line connecting the two cysteine residues, ⁇ : ⁇ -carboxylated-glutamate; O: hydroxyproline; *: C-terminal amidation.
  • sCon-Ins Gl Con-Ins Gl with diselenide bond between SecA6 and Sec A 10.
  • Figure 4a The x-ray crystal structure of Con-Ins Gl. Superposition of Con- Ins Gl and hins (PDB entry 1MSO). The backbones of the Con-Ins Gl B- and A chains are in grey, and the backbones of the hins B- and A chains are in black and white (respectively).
  • Figure 4b The hydrophobic core of Con-Ins Gl. Core structure of Con- Ins Gl compared to that of hins. The backbones of the Con-Ins Gl B- and A chains are in grey, and the backbones of the hins B- and A chains are in black and white (respectively).
  • Figure 4c, 4d and 4e Post-translational modifications of Con-Ins Gl. Side chain interactions of GlaA4, GlaBlO and HypB3 (respectively, with that of GlaA4 being compared to that of hins GluA4).
  • the backbones of the Con-Ins Gl B- and A chains are in grey, and the backbones of the hins B- and A chains are in black and white (respectively).
  • Figure 5 The x-ray crystal structure of Con-Ins Gl. Stereo image showing the arrangement within the crystal of Con-Ins Gl monomers around the crystallographic four-fold axis.
  • Four sulphate molecules (centre) are modelled— with unrestrained coordinates and effective occupancy each of 0.25— into a relatively featureless blob of difference electron density on the four-fold axis.
  • the sulphate ion forms part of a charge-compensated cluster comprising the amino-terminal group of GlyAl and a side-chain carboxylate of GlaA4 from each Con-Ins Gl monomer.
  • FIG. 6 Con-ins Gl binding hIR.
  • Molecular model of Con-Ins Gl in the context of the primary insulin binding site of the human insulin receptor, hins residues B22-B27 (in their hIR-bound form from PDB entry 40GA) are overlaid in black.
  • the Figure illustrates how the side chain of Con-Ins Gl TyrB 15, once rotated from its receptor free conformation, may together with that of Con-Ins Gl TyrB20 act as a surrogate for that of hins PheB24 in formation of the Con-Ins Gl / hIR complex.
  • the A chain of Con-Ins Gl (foreground) is transparent for clarity.
  • Figure 7 TyrB15 and TyrB20.
  • Figure 8 GlaA4 of Con-Ins Gl. Schematic diagram showing the interaction of the side chain of Con-Ins Gl PTM residue GlaA4 with side chain of hIR ocCT residue Asn711 , as observed within the molecular model of the complex of Con-Ins Gl with the primary binding site of MR.
  • the molecular surface is that of the hIR LI domain.
  • Figure 9 Insulin signalling measured by Akt phosphorylation analysis. Akt phosphorylation analysis of hlns, hIns[DOI] and Mns[TyrB 15, TyrB20, DOI].
  • Figure 10 Characterization of Con-Ins Gl. Sedimentation equilibrium analysis of Con-Ins Gl at 30,000 rpm (black points) and 45,000 rpm (grey points) with the best fit (lines) to a single species of apparent MW 5380 ⁇ 55 g/mol.
  • FIG. 11 Con-InsGl in co-complex with Fv83-7.IR310.T and IR-A 704-719 .
  • one copy is shown as a grey Ca trace with relatively thick linkages and the other as a black Ca trace with relatively thin linkages.
  • the overlay is based on common residues within the IR310.T moiety.
  • the CR domains and their attached Fv83-7 are omitted for clarity.
  • FIG. 12 Con-InsGl in co-complex with Fv83-7.IR310.T and IR-A 704"719 .
  • the Con-InsGl complex is shown as a light grey Ca trace (with thicker lines) and the hlns complex as a black Ca trace (with thinner lines, except for residues B22-B30 which are shown in thick black).
  • the overlay is based on common residues within the IR310.T moiety.
  • the cysteine -rich domain of IR310.T and its attached antibody fragment are omitted for clarity.
  • Figure 13 TyrBIS and TyrB20. Overlay of hlns in complex with Fab83-7, IR310.T and IR-A 704"719 (PDB entry 40GA; labelled) and Con-Ins Gl in complex with Fv83-7, IR310.T and IR-A 704"719 (underlined labels) based on the common domain LI of IR310.T.
  • the LI domain of IR310.T is shown in cartoon ribbon representation, while hins, Con-Ins Gl and IR-A 704"719 are shown in Ca trace representation.
  • the CR domain of IR310.T is omitted for clarity.
  • Figure 14 Molecular modelling of hIns[DOI] bound to components that comprise the primary binding site (site 1) of the hIR.
  • site 1 the primary binding site of the hIR.
  • the IR-A 704"719 segment is shown in cartoon ribbon representation and is coloured dark grey.
  • the hins [DOI] A chain and B chain are shown in cartoon ribbon representation and are labelled.
  • the transparent molecular surface is that of the hIR LI domain.
  • the hIR LI domain is shown in cartoon ribbon representation with the side chain of Tyr67 shown.
  • Figure 15 Molecular modelling of hIns[TyrB15, DOI] bound to components that comprise the primary binding site (site 1) of the hIR.
  • site 1 the primary binding site of the hIR.
  • the IR-A 704"719 segment is shown in cartoon ribbon representation and is coloured dark grey.
  • the hIns[TyrB15, DOI] A chain and B chain are shown in cartoon ribbon representation and are labelled.
  • the molecular surface is that of the hIR LI domain.
  • the figure illustrates how the side-chain of TyrB 15 projects into the hydrophobic core of the DOI- (IR-A 704 719 )-L1 interface occupying space otherwise occupied by hins LeuB 15.
  • Figure 16 Molecular modelling of hIns[DOI, TyrB20] bound to components that comprise the primary binding site (site 1) of the hIR Molecular model of hIns[TyrB20, DOI] in complex with the IR LI domain (residues Gly5 to Cysl55) and the IR-A 704"719 .
  • the figure illustrates how the side -chain of TyrB20 remained in the hins B24 binding site, with all other interactions with the receptor appearing native-like.
  • the IR-A 704"719 segment is shown in cartoon ribbon representation and is coloured dark grey.
  • the hIns[TyrB20, DOI] A chain and B chain are shown in cartoon ribbon representation and are labelled.
  • the transparent molecular surface is that of the hIR LI domain.
  • the hIR LI domain is shown in cartoon ribbon representation with the side chain of Tyr67 shown.
  • Figure 17 Positional scan of hIns[DOI]. The resultant mutational AAG (kcal/mol) contribution at each site of hIns[DOI].
  • Figure 18 Positional scan of hIns[TyrB15, DOI].
  • the resultant mutational A AG (kcal/mol) contribution at each site of Mns[TyrB15, DOI].
  • Figure 19 Positional scan of Mns[TyrB20, DOI]. The resultant mutational AAG (kcal/mol) contribution at each site of hIns[TyrB20, DOI].
  • FIG. 20 Schematic of insulin multimer equilibrium. Figure shows that insulin monomerization slows absorption rate.
  • Figure 21 Chemical total synthesis of human DOI insulin.
  • Figure shows the chemical total synthesis of human DOI insulin.
  • Thr-Ser isopeptide (boxed in red) was used to increase the solubility of insulin A chain.
  • Figure 22 Insulin signalling activation by exemplified insulin analogs.
  • FIG. 1 Figure shows the effects of B 15 Tyr and B20 Tyr on hIR activation. The sequence for each peptide used is also shown.
  • Figure 23 Insulin signalling activation by exemplified insulin analogs.
  • Figure shows the effects of B10 Glu, B20 Tyr on hIR activation. The sequence for each peptide used is also shown.
  • Figure 24 Insulin signalling activation by exemplified insulin analogs.
  • Figure 24A and 24B show peptide sequences/modified amino acids and effects of B20 residues in activating insulin signaling, respectively.
  • Figure 25 Insulin signalling activation by exemplified insulin analogs.
  • Figure shows the effects of A8 His, A9 Arg on hIR activation. The sequence for each peptide used is also shown.
  • Figure 26 Insulin signalling activation by exemplified insulin analogs.
  • Figure shows the individual effect of A8, A9, B 10 and B20 on hIR activation.
  • Figure 27 Insulin signalling activation by venom insulins.
  • Figure shows the insulin signaling activation of several venom insulins with similar potencies to Con-Ins Gl (top panel). Sequence alignment of these venom insulins (bottom panel). Residues at position 9 and 10 in the A chain and 10 and 20 in the B chain are highlighted, ⁇ and * denote post-translational modifications (gamma-carboxyglutmate and C-terminal amidation, respectively). KEY TO THE SEQUENCE LISTING
  • SEQ ID NO: 1 - 41 Insulin analogs, peptides and/or compounds according to embodiments of the present disclosure.
  • insulin means human insulin, pig insulin, guinea pig insulin, chicken insulin, mouse insulin, beef insulin or venom insulin. In some embodiments, insulin means human insulin.
  • venom insulin means a cone snail venom insulin. Preferably, venom insulin means Con-Ins Gl .
  • insulin analog refers to any agent that is capable of mimicking the activity of insulin.
  • the insulin analog is at least an insulin receptor agonist.
  • the insulin analog binds to the insulin receptor.
  • insulin analogs may be peptides, polypeptides, proteins or peptidomimetics.
  • the insulin analog is a peptide.
  • insulin analog also include the IR agonists, molecules, compounds and the like identified by the methods disclosed herein.
  • peptide refers to a polymer of amino acids ranging from two to about fifty amino acids (e.g., 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, or 45 amino acids in length).
  • the term peptide encompasses both unmodified peptides, modified peptides, and otherwise chemically derivatized peptides (for example phosphorylated, sulphated, amidated and the like).
  • the peptide may be an unnatural peptide oligomer, such as those described in Sadowsky et al. (2005) and Sadowsky et al. (2007).
  • polypeptide refers to a polymer of amino acids generally greater than about 50 amino acids in total length and typically having stable characteristic secondary and tertiary structures.
  • polypeptide or “protein” may also include a combination of such polymers (for example two or more) associating with stable tertiary quaternary structure resulting either through their non-covalent or covalent association.
  • the peptide, protein or polypeptide comprises amino acids that occur naturally in the subject to be treated.
  • the peptide or polypeptide comprises one or more unnatural amino acids, modified amino acids or synthetic amino acid analogues.
  • Such amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, oc-amino isobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic acid, 2- amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, cyclopentylalanine, ⁇ -alanine, fluoro- amino acids, designer amino acids such as ⁇ -methyl amino acids, Coc-methyl amino acids, Noc-methyl amino acids, and amino acid analogues in general.
  • peptides, polypeptides or proteins which are differentially modified during or after synthesis, for example, by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the peptide, protein or polypeptide.
  • amino acid "modification” refers to a substitution of an amino acid, or the derivation of an amino acid by the addition and/or removal of chemical groups to/from the amino acid, and includes substitution with any of the 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids.
  • Commercial sources of atypical amino acids include Sigma-Aldrich (Milwaukee, Wis.), ChemPep Inc. (Miami, Fla.), and Genzyme Pharmaceuticals (Cambridge, Mass.).
  • Atypical amino acids may be purchased from commercial suppliers, synthesized de novo, or chemically modified or derivatized from naturally occurring amino acids.
  • substitution refers to the replacement of one amino acid residue by a different amino acid residue.
  • the substitued amino acid may be any of the 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids.
  • the terms “A chain peptide” and “B chain peptide” are interchangeable with “insulin A chain peptide” and “insulin B chain peptide.”
  • reference to a compound that is a "derivative thereof refers to a compound that is adapted or modified from an ancestral compound and has a similar but new structure and which has a similar biological activity as the ancestral compound.
  • the ancestral compound is a small molecule, a peptide, polypeptide, protein or an insulin analog as described herein.
  • the ancestral compound is a peptide, polypeptide, protein or insulin analog which may be modified to include any chemical modification, comprise single or multiple substitutions, deletions and/or additions of any molecules associated with the protein or peptide, such as carbohydrates, lipids and/or proteins or peptides.
  • derivatives of proteins, polypeptides or peptides include those modified analogues resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protective/blocking groups, proteolytic cleavage or binding to an antibody or to another cellular ligand.
  • references to a particular amino acid position by letter and number refer to the amino acid at that position of either the A chain (e.g. position A5) or the B chain (e.g. position B5) in the respective A chain or B chain of venom insulin Con-Gl Ins from Conus geographus, or the corresponding amino acid position in any analogs thereof.
  • a reference herein to "position B17" absent any further elaboration would mean the corresponding position B 15 of the B chain of human insulin as Con-Ins Gl has two additional N- terminal B chain residues.
  • the phrase "at a position corresponding to amino acid number” refers to the relative position of the amino acid compared to surrounding amino acids with reference to a defined amino acid sequence.
  • the B chain of the insulin analog of the invention may have one or two additional N-terminal amino acids, such as present in Con-Ins Gl.
  • the leucine (15 th amino acid) of the B chain of naturally occurring human insulin corresponds to the 17 th amino acid of the B chain Con-Ins Gl (see Figure 1).
  • this 15 th amino acid of the B chain of naturally occurring human insulin is an aromatic residue or a large aliphatic residue and/or 20 th amino acid of the B chain of naturally occurring human insulin is an aromatic residue or a large aliphatic residue.
  • the term "monomeric insulin” refers to insulin and insulin analogs that are less prone to forming higher order species (such as dimers, tetramers, hexamers etc) than human insulin.
  • the insulin or insulin analog is fully or substantially monomeric, e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% monomeric.
  • a therapeutic refers to a treatment, therapy, or drug that can treat a disease or condition or that can ameliorate one or more symptoms associated with a disease or condition.
  • a therapeutic can refer to a therapeutic compound, including, but not limited to proteins, peptides, nucleic acids (e.g. CpG oligonucleotides), small molecules, vaccines, allergenic extracts, antibodies, gene therapies, other biologies or small molecules.
  • the term "subject” refers to any organism susceptible to insulin related disorders.
  • the term “subject” and “patient” can be used interchangeably.
  • the subject can be a mammal, avian, arthropod, chordate, amphibian or reptile.
  • Exemplary subjects include but are not limited to human, primate, livestock (e.g. sheep, cow, chicken, horse, donkey, pig), companion animals (e.g. dogs, cats), laboratory test animals (e.g. mice, rabbits, rats, guinea pigs, hamsters), captive wild animal (e.g. fox, deer).
  • livestock e.g. sheep, cow, chicken, horse, donkey, pig
  • companion animals e.g. dogs, cats
  • laboratory test animals e.g. mice, rabbits, rats, guinea pigs, hamsters
  • captive wild animal e.g. fox, deer.
  • the subject is a mammal.
  • the subject is human.
  • treating includes prophylaxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms.
  • treating diabetes will refer in general to maintaining glucose blood levels within acceptable levels and may include increasing or decreasing blood glucose levels depending on a given situation.
  • insulin analogs will be administered in a therapeutically effective amount.
  • effective amount or “therapeutically effective amount,” as used herein, refer to an amount of an insulin analog being administered sufficient to relieve to some extent one or more of the symptoms of the disease or condition being treated.
  • an "effective amount" of an insulin analog is an amount effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects.
  • therapeutically effective amounts may be determined by routine experimentation, including but not limited to a dose escalation clinical trial.
  • therapeutically effective amount includes, for example, a prophylactically effective amount.
  • an effective amount or "a therapeutically effective amount” can vary from subject to subject, due to variation in metabolism of the compound of any of age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. Where more than one therapeutic agent is used in combination, a “therapeutically effective amount" of each therapeutic agent can refer to an amount of the therapeutic agent that would be therapeutically effective when used on its own, or may refer to a reduced amount that is therapeutically effective by virtue of its combination with one or more additional therapeutic agents.
  • onset of activity refers to the length of time before insulin reaches the blood stream and begins to lower blood glucose levels
  • peak refers to the time period when the insulin analog best lowers blood glucose levels
  • duration refers to how long the insulin continues to work, i.e. lower blood glucose levels.
  • IR as used herein includes wild-type IR and variants thereof including allelic variants and naturally occurring mutations and genetically engineered variants. It will be readily apparent to the skilled person that IR may be derived from other species not specifically disclosed herein. Furthermore, the skilled person will have no difficulties identifying such other suitable IR given the known conservation of IR sequences from primitive organisms through to mammals and humans.
  • the present invention provides a crystal comprising venom insulin.
  • crystal means a structure (such as a three dimensional (3D) solid aggregate) in which the plane faces intersect at definite angles and in which there is a regular structure (such as internal structure) of the constituent chemical species.
  • crystal refers in particular to a solid physical crystal form such as an experimentally prepared crystal.
  • Crystals according to the invention may be prepared using venom insulin from organisms in the genus Conus, such as Conus geographus and Conus tulipa. Some embodiments relate to insulins from the venom of Conus geographus. However, the venom insulin may also be from other species. Typically, these insulins comprise a 20 amino acid A chain and a 23 amino acid B, however the length of the A and B chain can vary.
  • the amino acids in the A and B chain may be post-translationally modified; example post-translational modifications include but are not limited to glutamic acid may be replaced by ⁇ -carboxylated glutamic acid (also referred to as the conjugate base gamma carboxyglutamate), proline may be replaced by hydroxyproline, the C-terminus may be amidated, cysteine may be replaced by seleoncysteine.
  • glutamic acid may be replaced by ⁇ -carboxylated glutamic acid (also referred to as the conjugate base gamma carboxyglutamate)
  • proline may be replaced by hydroxyproline
  • the C-terminus may be amidated
  • cysteine may be replaced by seleoncysteine.
  • post-translational modifications include but are not limited to glutamic acid may be replaced by ⁇ -carboxylated glutamic acid (also referred to as the conjugate base gamma carboxyglutamate), proline may be replaced by hydroxyproline,
  • A-chain GVVyHCCHRPCSNAEFKKYC* (SEQ ID NO: 22)
  • B-chain TFDTOKHRCGSylTNSYMDLCYR (SEQ ID NO: 23) where y is ⁇ -carboxylated glutamic acid, O is hydroxyproline and *the C-terminus of the A-chain is amidated.
  • the insulin polypeptide may also be obtained from other species or a non-native designed sequence.
  • Crystals may be constructed with wild-type sequences or variants thereof, including naturally occurring mutations as well as genetically engineered variants. Typically, variants have at least 90, 95 or 98% sequence identity with a corresponding wild-type venom insulin.
  • a crystal comprising venom insulin has the atomic coordinates set forth in Appendix I.
  • atomic coordinates refer to a set of values which define the position of one or more atoms with reference to a system of axes. It will be understood by those skilled in the art that atomic coordinates may be varied, without affecting significantly the accuracy of models derived therefrom; thus, although the invention provides a very precise definition of a preferred atomic structure, it will be understood that minor variations are envisaged and the claims are intended to encompass such variations.
  • RMSD root mean square deviation
  • a crystal structure of a venom insulin, or a region thereof is also provided.
  • the venom insulin is Con-Ins Gl.
  • the crystal structure of a venom insulin is the structure of Con-Ins Gl as defined by the atomic coordinates of Appendix I. The atomic coordinates obtained experimentally for venom insulin are shown in Appendix I.
  • a person skilled in the art will appreciate that a set of atomic coordinates determined by X-ray crystallography is not without standard error. Accordingly, any set of structure coordinates for venom insulin that has a root mean square deviation of protein backbone atoms of less than 0.75 A when superimposed (using backbone atoms) on the atomic coordinates listed in Appendix I shall be considered identical.
  • the present invention also comprises the atomic coordinates of venom insulin that substantially conform to the atomic coordinates listed in Appendix I.
  • a structure that "substantially conforms" to a given set of atomic coordinates is a structure wherein at least about 50% of such structure has an RMSD of less than about 2.0 A for the backbone atoms in secondary structure elements in each domain, preferably less than about 1.5 A for the backbone atoms in secondary structure elements in each domain, and more preferably, less than about 1.3 A for the backbone atoms in secondary structure elements in each domain, and, in increasing preference, less than about 1.0 A, less than about 0.7 A, less than about 0.5 A, and most preferably, less than about 0.3 A for the backbone atoms in secondary structure elements in each domain.
  • a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 75% of such structure has the recited RMSD value, and more preferably, at least about 90% of such structure has the recited RMSD value, and most preferably, about 100% of such structure has the recited RMSD value.
  • the above definition of “substantially conforms” can be extended to include atoms of amino acid side chains.
  • the phrase “common amino acid side chains” refers to amino acid side chains that are common to both the structure which substantially conforms to a given set of atomic coordinates and the structure that is actually represented by such atomic coordinates.
  • a set of atomic coordinates for a polypeptide is a relative set of points that define a shape in three dimensions.
  • an entirely different set of coordinates could define a similar or identical shape.
  • slight variations in the individual coordinates will have little effect on overall shape.
  • the variations in coordinates may be generated due to mathematical manipulations of the structure coordinates.
  • the structure coordinates set forth in Appendix I could be manipulated by crystallographic permutations of the structure coordinates, fractionalisation of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates, or any combination thereof.
  • modification in the crystal structure due to mutations, additions, substitutions, and/or deletions of amino acids, or other changes in any of the components that make up the crystal could also account for variations in structure coordinates.
  • Various computational analyses are used to determine whether a molecular complex or a portion thereof is sufficiently similar to all or parts of the structure of the venom insulin described above. Such analyses may be carried out using software known to the person skilled in the art, for example PDBeFOLD (Krissinel and Henrick, 2004), DALI (Holm and Rosenstrom, 2010), LSQMAN (Kleywegt and Jones, 1994) and CHIMERA (Pettersen et al. 2004).
  • Comparisons typically involve calculation of the optimum translations and rotations required such that the root mean square difference of the fit over the specified pairs of equivalent atoms is an absolute minimum. This number is given in angstroms. Accordingly, structural coordinates of venom insulin within the scope of the present invention include structural coordinates related to the atomic coordinates listed in Appendix I by whole body translations and/or rotations. Accordingly, RMSD values listed above assume that at least the backbone atoms of the structures are optimally superimposed which may require translation and/or rotation to achieve the required optimal fit from which to calculate the RMSD value.
  • subsets of said atomic coordinates listed in Appendix I and subsets that conform substantially thereto define one or more regions of the venom insulin, for example, (i) the A chain, (ii) the B chain, (iii) the hydrophobic core (for example in Con-Ins Gl the hydrophobic core comprises the side chains of residues ValA2, CysA6, CysAl l, PheA16, TyrA19, ArgB6, IleBl l, TyrB15 and LeuB18), (iv) the PTM and residues interacting with the PTM, (v) the receptor binding surface (for example, the IR binding surface of Con-Ins Gl); (vi) TyrB 15 and residues interacting with TyrB 15; (vii) TyrB20 and residues interacting with TyrB20; (viii) PheA16 and residues interacting with PheA16. (ix) subsets of residues in the immediate vicinity the respective A-chain termin
  • a three dimensional structure of a venom insulin or region thereof which substantially conforms to a specified set of atomic coordinates can be modelled by a suitable modelling computer program such as MODELER (Sali and Blundell, 1993), as implemented in the Insight II Homology software package (Insight II (97.0), MSI, San Diego), using information, for example, derived from the following data: (1) the amino acid sequence of the venom insulin; (2) the amino acid sequence of the related portion(s) of the protein represented by the specified set of atomic coordinates having a three dimensional configuration; and, (3) the atomic coordinates of the specified three dimensional configuration.
  • a three dimensional structure of a venom insulin which substantially conforms to a specified set of atomic coordinates can also be calculated by a method such as molecular replacement, which is described in detail below.
  • Structure coordinates/atomic coordinates are typically loaded onto a machine readable-medium for subsequent computational manipulation.
  • models and/or atomic coordinates are advantageously stored on machine -readable media, such as magnetic or optical media and random-access or read-only memory, including tapes, diskettes, hard disks, CD-ROMs and DVDs, flash memory cards or chips, servers and the internet.
  • the machine is typically a computer.
  • the present invention also provides a computer readable media having recorded thereon data representing a model and/or the atomic coordinates of Con-Ins Gl.
  • the present invention also provides a set of atomic coordinates as shown in Appendix I, or a subset thereof or either, in which said coordinates define a three dimensional structure of a venom insulin.
  • the structure coordinates/atomic coordinates may be used in a computer to generate a representation, e.g. an image, of the three- dimensional structure of the cone snail insulin crystal which can be displayed by the computer and/or represented in an electronic file.
  • the structure coordinates/atomic coordinates and models derived therefrom may also be used for a variety of purposes such as drug discovery, biological reagent (binding protein) selection and X-ray crystallographic analysis of other protein crystals.
  • the use of the structure of Con-Ins Gl as a structural model is provided.
  • the structural model may be used for identification of insulin analogs.
  • the present invention also encompasses insulin analogs identified using the structure of Con-Ins Gl as a structural model.
  • Con-Ins Gl or a region thereof may be used to develop models useful for drug design and in silico screening of candidate compounds that interact with and/or modulate IR.
  • Other physicochemical characteristics may also be used in developing the model, e.g. bonding, electrostatics etc.
  • in silico refers to the creation in a computer memory, i.e., on a silicon or other like chip. Unless stated otherwise "in silico” means “virtual.” When used herein the term “in silico” is intended to refer to screening methods based on the use of computer models rather than in vitro or in vivo experiments.
  • the crystal structure of Con-Ins Gl provided herein may also be used to model/solve the structure of a new crystal using molecular replacement.
  • the present invention also provides the use of the structure of venom insulin, or a subset thereof, as a structural model.
  • the atomic coordinates of venom insulin such as those set forth in Appendix I, or a region of venom insulin can be used for determining at least a portion of the three-dimensional structure of a molecular complex which contains at least some structural features similar to the venom insulin.
  • structural information about another crystallised venom insulin or insulin analog may be obtained. This may be achieved by any of a number of well-known techniques, including molecular replacement.
  • Methods of molecular replacement are generally known by those of skill in the art, for example PHASER (McCoy et al. 2007). Methods of molecular replacement are generally described in Brunger, 1997; Navaza and Saludjian, 1997; Tong and Rossmann, 1997; Bentley, 1997; Lattman, 1985; Rossmann, 1972.
  • X-ray diffraction data are collected from the crystal of a crystallised target structure.
  • the X-ray diffraction data is transformed to calculate a Patterson function.
  • the Patterson function of the crystallised target structure is compared with a Patterson function calculated from a known structure (referred to herein as a search structure).
  • the Patterson function of the crystallised target structure is rotated on the search structure Patterson function to determine the correct orientation of the crystallised target structure in the crystal.
  • the translation function is then calculated to determine the location of the target structure with respect to the crystal axes.
  • initial phases for the experimental data can be calculated. These phases are necessary for calculation of an electron density map from which structural differences can be observed and for refinement of the structure.
  • the structural features e.g., amino acid sequence, conserved di-sulphide bonds, and beta-strands or beta-sheets
  • the structural features e.g., amino acid sequence, conserved di-sulphide bonds, and beta-strands or beta-sheets
  • the structural features
  • the electron density map can, in turn, be subjected to any well-known model building and structure refinement techniques to provide a final, accurate structure of the unknown crystallised molecular complex (e.g. see Jones et al. 1991; Brunger et al. 1998).
  • the structure of any portion of any crystallised molecule/molecular complex that is sufficiently homologous to any portion of the venom insulin may be solved by this method. This method is especially useful in determining the structure of insulin analogs that were designed using the methods described herein.
  • All of the molecules/molecular complexes referred to herein may be studied using well-known X-ray diffraction techniques and may be refined versus 1.5-3.5 A resolution X-ray data to an R value of about 0.25 or less using computer software, such as X-PLOR (Yale University, distributed by Molecular Simulations, Inc. ; see Briinger, 1996), REFMAC (Murshudov et al. 1997), PHENIX (Adams et al. 2010) and BUSTER (distributed by Global Phasing Ltd, Bricogne et al. 2011). This information may thus be used to optimize known insulin analogs, and more importantly, to design new or improved insulin analogs.
  • the present invention also provides a method for producing crystals comprising venom insulin.
  • Crystal forms of venom insulin disclosed herein can be obtained by the following crystallization methods.
  • the present invention provides a method for crystallizing venom insulin comprising the steps:
  • step (b) optionally, concentrating the solution of step (a);
  • step (c) diluting the solution of step (a) or step (b) with a precipitant solution so that the final concentration of the venom insulin is in the range of 0.5 mg/mL to 10 mg/mL.
  • the aqueous solution of venom insulin provided in step (a) may be buffered or unbuffered. Any suitable buffer known to a person skilled in the art may be used. Non- limiting examples for the venom insulin solutions include Tris-HCI, sodium phosphate, and triethanolamine buffer. In case the desired pH value is not obtained, adjusting the pH by addition of a base or acid such as ammonium chloride, sodium acetate, sodium hydroxide, potassium hydroxide, or hydrochloric acid can be performed. Preferably, the solution of step (a) contains 10 mM HC1.
  • the solution may optionally then be concentrated.
  • the solution is concentrated by centrifugal filtration.
  • the concentration of the venom insulin should be in the range of 1.0 mg/mL to 20 mg/mL.
  • the concentration of the venom insulin is approximately 4.0 mg/mL. This is the concentration of the venom insulin at the end of step (b) before dilution with the buffered precipitant solution of step (c).
  • the concentration of the venom insulin in the concentrated solution of step (b) or during the concentration in step (b) or after the concentration of step (b) could be determined, for instance, by the Bradford protein assay or by other routine methods including UV absorbance spectroscopy.
  • step (c) a precipitant solution is added to the solution of step (b).
  • the solution of step (b) is diluted with the precipitant solution at a ratio of between 1:4 to 4: 1 (solution of step (b): buffered precipitant solution), most preferably at a ratio of 1 : 1.
  • the precipitant solution may comprise at least one small organic amphiphilic molecule and/or at least one inorganic salt.
  • the small organic amphiphilic molecule may be selected from the group comprising or consisting of glycerol, ethylene glycol, butyl ether, benzamidine, dioxane, ethanol, isopropanol, butanol, pentanol, methyl pentanediol, pentanediol, hexanediol, heptanetriol, dithiothreitol, MES, Tris, Tris-HCI, Bis-Tris, imidazole, bicine, trimethylamine N- oxide, succinic acid, DL-malate, CHES, CAPS, glycine, CAPSO, ADA, MOPS, Bis- Tris propane, SPG, MIB, PCB, MMT, N-[2- hydroxyethyl]piperazine-N'-[2- ethanethan
  • the small organic amphiphilic molecule comprises DL-malate, TRIS and MES.
  • the small organic amphiphilic molecules may act as a buffer.
  • the pH of the small organic amphiphilic molecules in solution may be adjusted to between about 2.0 and about 11.0 and any pH in between, for example 2.0, 2.5, 3.0, 3.5, 4.0, 4..5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5 and 11.0.
  • the pH is between about 8.0 and about 10.0.
  • the pH is 9.0.
  • the amount of all small organic amphiphilic molecules in the precipitant solution is between 1 % weight/volume and 50% weight/volume, preferably between 5% weight/volume and 20% weight/volume , and more preferably between 8% weight/volume and 12% weight/volume of the small organic amphiphilic molecule in regard to the total weight of the buffered precipitant solution.
  • the buffered precipitant solution comprises 10% weight/volume of the small organic amphiphilic molecule.
  • the term "at least one small organic amphiphilic molecule” means that mixtures of the afore-mentioned small organic amphiphilic molecules may be used. In case mixtures of small organic amphiphilic molecules are used, the amount used refers to the total amount of all small organic amphiphilic molecules together.
  • the precipitant solution may also comprise an inorganic salt.
  • the inorganic salts is selected from the group comprising or consisting of ammonium chloride, ammonium sulphate, ammonium acetate, ammonium fluoride, ammonium bromide, ammonium iodide, ammonium nitride, cadmium chloride, cadmium sulphate, calcium chloride, calcium acetate, cesium chloride, cesium sulphate, cobalt chloride, ferric chloride, lithium acetate, lithium chloride, lithium nitrate, lithium sulphate, magnesium acetate, magnesium formate, magnesium nitrate, nickel chloride, potassium acetate, potassium bromide, potassium fluoride, potassium formate, potassium iodide, potassium nitrate, potassium thiocyanate, potassium/sodium tartrate, sodium acetate, sodium bromide, sodium fluoride, sodium iodide, sodium nitrate, sodium phosphate
  • the inorganic salt is ammonium sulphate.
  • the amount of the at least one inorganic salt in the precipitant solution is between about 50 mM and about 4000 mM, between about 1000 mM and about 3000 mM, between about 1500 mM and about 2500 mM or about 2000 mM.
  • the afore-mentioned concentration refers to the concentration of all inorganic salts together and not to the concentration of each single salt used in the mixture of inorganic salts.
  • the precipitant solution may be buffered. Any buffer known to a person may be used.
  • the buffer comprises or consists of citric acid, sodium acetate, sodium citrate, sodium cacodylate, HEPES sodium, TRIS HC1, CAPSO, CAPS, sodium malate, sodium MES and the like and combinations thereof.
  • the precipitant solution may have a pH between about 2.0 and about 11.0 and any pH in between, for example 2.0, 2.5, 3.0, 3.5, 4.0, 4..5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5 and 11.0.
  • the precipitant solution may have a pH between about 8.0 and about 10.0.
  • the precipitant solution may have a pH of 9.0.
  • the buffered precipitant solution contains more than 1 M of at least one inorganic salt and/or between 5% by weight and 20% by weight of at least one small organic amphiphilic molecule.
  • the precipitant solution comprises 2.0M ammonium sulphate and 10% DL-malate-MES- Tris (pH 9.0).
  • At least one precipitating agent in the diluted solution of step (c) competes with the protein molecules for water, thus leading to supersaturation of the protein. Crystals can normally only grow from supersaturated states, and thus they can grow from precipitates. Salts, polymers, and organic solvents are suitable precipitating agents.
  • the solution of step (c) may contain further precipitating agents.
  • the hanging drop or the sitting drop methods are used for crystallization.
  • the "hanging drop vapor diffusion" technique is the most popular method for the crystallization of macromolecules.
  • a drop composed of a mixture of sample and reagent is placed in vapor equilibration with a liquid reservoir of reagent.
  • the drop typically contains a lower reagent concentration than the reservoir.
  • water vapor leaves the drop and eventually ends up in the reservoir.
  • the sample undergoes an increase in relative supersaturation. Both the sample and reagent increase in concentration as water leaves the drop for the reservoir. Equilibration is reached when the reagent concentration in the drop is approximately the same as that in the reservoir.
  • Wild type insulin comprises an A chain peptide and a B chain peptide.
  • Wild type human insulin A chain is represented by the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 24).
  • Wild type human insulin B chain is represented by the sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 25).
  • the present inventors have determined the three-dimensional structure of Con- Gl Ins, a monomeric insulin that lacks an equivalent to the aromatic triplet PheB24- PheB25-TyrB26 of human insulin. Without wishing to be bound by theory it is thought that the side chain of TyrB 15 may compensate for the absence of the critical human insulin PheB24 in terms of IR engagement. It is also thought that the side chain of Con- Ins Gl TyrB20 may be involved in compensating for the lack of an equivalent to human insulin PheB24. The potential importance of these residues could not be predicted based on sequence analysis.
  • the structural findings provided herein provide a platform for the design of a novel class of therapeutic human insulin analogues that are intrinsically monomeric and rapid-acting.
  • the present invention provides an insulin analog comprising an A chain peptide and a B chain peptide, wherein the B chain comprises an aromatic or large aliphatic residue at a position corresponding to amino acid number 15 of the B chain of human insulin and/or an aromatic or large aliphatic residue at a position corresponding to amino acid number 20 of the B chain of human insulin, wherein the analog comprises at least one amino acid found in human insulin but lacking in the corresponding position of Conus geographus insulin, and wherein the A chain peptide and the B chain peptide are bonded together across at least one pair of cysteine residues.
  • the aromatic residue or large aliphatic residue can be a natural or a non-natural amino acid.
  • the co-crystal structure of an insulin-IR complex revealed that the side chain of PheB24 plays a unique role as an "anchor" within a nonpolar pocket (referred to as the B24 related binding pocket) defined by the IR and insulin B chain (Menting et al. 2014).
  • the present invention envisions that the large aliphatic or aromatic susbtitutions at position 15 and/or position 20 of the B chain of human insulin may compensate for the lack of PheB24 by inserting in the B24 related binding pocket.
  • the side-chains of large aliphatic or aromatic residues may be physical and chemically compatable with B24 related binding pocket.
  • the aromatic or large aliphatic residue at a position corresponding to amino acid number 15 of the B chain of human insulin is selected from the group consisting of tyrosine, phenylalanine, 4-methylphenylalanine, histidine, tryptophan, methionine, cyclopentylalanine and cyclohexylalanine.
  • the aromatic or large aliphatic residue at a position corresponding to amino acid number 20 of the B chain of human insulin is selected from the group consisting of tyrosine, phenylalanine, 4-methylphenylalanine, histidine, tryptophan, methionine, cyclcopentylalanine and cyclohexylalanine.
  • the aromatic residue or large aliphatic may be a natural or non-natural amino acid.
  • the B chain comprises an aromatic residue at a position corresponding to amino acid number 15 of the B chain of human insulin and/or an aromatic residue at a position corresponding to amino acid number 20 of the B chain of human insulin.
  • the aromatic residue may be a natural or non-natural amino acid.
  • the aromatic amino acid can be tyrosine, phenylalanine, tryptophan, histidine, 4-acetylphenylalanine and the like.
  • the insulin analog has a tyrosine at a position corresponding to amino acid number 15 of the B chain of human insulin. In some embodiments, the insulin analog has a tyrosine at a position corresponding to amino acid number 20 of the B chain of human insulin. In some embodiments, the insulin analog has a tyrosine at a position corresponding to amino acid number 15 of the B chain of human insulin and/or a tyrosine at a position corresponding to amino acid number 20 of the B chain of human insulin.
  • the insulin analog has a phenylalanine at a position corresponding to amino acid number 15 of the B chain of human insulin and/or a phenylalanine at a position corresponding to amino acid number 20 of the B chain of human insulin. In some embodiments, the insulin analog has a tryptophan at a position corresponding to amino acid number 15 of the B chain of human insulin and/or a tryptophan at a position corresponding to amino acid number 20 of the B chain of human insulin.
  • the insulin analog has a 4- acetylphenylalanine at a position corresponding to amino acid number 15 of the B chain of human insulin and/or a 4-acetylphenylalanine at a position corresponding to amino acid number 20 of the B chain of human insulin.
  • the B chain comprises a large aliphatic residue at a position corresponding to amino acid number 15 of the B chain of human insulin and/or a large aliphatic residue at a position corresponding to amino acid number 20 of the B chain of human insulin.
  • a "large aliphatic" residue has a side-chain that is larger than the leucine (naturally occurring in human insulin at position 15 and 20) side-chain.
  • the side-chain of large aliphatic residue may have the same number or more non-hydrogen atoms compared to leucine.
  • the side-chain of the large aliphatic residue has a greater side-chain volume when compared to leucine.
  • the side-chain of large aliphatic residue has a greater molecular weigth when compared to leucine. In some embodiments, the side-chain of the large aliphatic residue has more conformational flexibility when compared to leucine.
  • the large aliphatic" residue may be may be a natural or non-natural amino acid. For example, the large aliphatic residue may methionine, isoleucine, cyclopentylalanine or cyclohexylalanine and the like.
  • the insulin analog has a methionine at a position corresponding to amino acid number 15 of the B chain of human insulin. In some embodiments, the insulin analog has a methionine at a position corresponding to amino acid number 20 of the B chain of human insulin. In some embodiments, the insulin analog has a cyclohexylalanine at a position corresponding to amino acid number 15 of the B chain of human insulin and/or a cyclohexylalanine at a position corresponding to amino acid number 20 of the B chain of human insulin.
  • the insulin analog has a cyclopentylalanine at a position corresponding to amino acid number 15 of the B chain of human insulin and/or a cyclopentylalanine at a position corresponding to position 20 of the B chain of human insulin.
  • the insulin analogs have modified B chains which lack the aromatic triplet PheB24-PheB25-TyrB26 thought essential for IR binding, and have, in most cases, shorter B-chains compared to human insulin.
  • the B chain is truncated at the C-terminal end when compared to human insulin.
  • the B chain is lacking one or more of the nine C- terminal amino acids of human insulin, for example, the B chain is lacking one, two, three, four, five, six, seven, eight or nine C-terminal amino acids of human insulin.
  • the B chain is at least lacking PheB24 of human insulin.
  • the B chain is at least lacking the human B chain aromatic triplet (amino acids PheB24-PheB25-TyrB26 of human insulin). These residues may be absent or may be substituted such that the amino acids at a position corresponding to amino acid number 24, 25 and/or 26 of the B chain of human insulin are not phenylalanine, phenylalanine or tyrosine, respectively.
  • the insulin analog comprises an A chain peptide comprising the sequence Gly-X A 2-X A 3-X A 4-X A 5-Cys A 6- Cys A7 -X A 8-X A 9-X A1 o-Cys A11 - XA12-XA13-XA14-XA15-XA16-XA17-XA18-XA1 -CySA20-XA21-XA22-XA23-XA24-XA25-XA26-XA27-
  • the insulin analog comprises a the B chain peptide comprises the sequence X B I-XB2-XB3-XB4-XBS-XB6-XB7-XB8- Cys B 9-XBio-XBii-XBi2- B13- B14- B15- B16- B17- B18- B1 - B20-CyS B 21- B22- B23- B24- B25- B26- B27-
  • the insulin analog comprises a B chain peptide comprises the sequence ⁇ ⁇ 1 - ⁇ ⁇ 2- ⁇ ⁇ 3- ⁇ ⁇ 4- ⁇ ⁇ 5- ⁇ ⁇ 6- ⁇ ⁇ 7 - ⁇ ⁇ 8- ⁇ 8 ⁇ 9- ⁇ - ⁇ - ⁇ 2- ⁇ 3- ⁇ 4-
  • the insulin analog comprises a B chain peptide comprises the sequence X B I-XB2-XB3-XB4-XBS-XB6-XB 7 -XB8- Cys B 9-Gly-Ser-X B i2-XBi3-XBi4-XBi5- XB16-XB17-XB18-XB19-XB20-CySB21-XB22-XB23- XB24-XB25-XB26-XB27-XB28-XB29"XB30-
  • the insulin analog comprises a B chain peptide comprises the sequence X B i-X B2 -X B3 -X B4 -X B5 -X B6 -X B7 -X B8 -CysB9-Gly-Ser-X B i2-XBi3-XBi4-XBi5- XB16-XB17-XB18-XB19-XB20-CySB21-XB22-XB23- XB24-XB25-XB26-XB27-XB28-XB29"XB30-
  • the insulin analog comprises an A chain peptide comprising the sequence Gly-X A 2-X A 3-X A 4-X A 5-Cys A 6-Cys A 7-X A 8-X A 9-X A io-Cys A11 - XA12-XA13-XA14-XA15-Phe-X A 17-XA18-XA19-CyS A 20-XA21- XA22-XA23-XA24-XA25-XA26 "
  • the insulin analog comprises an A chain peptide comprising a sequence Gly-X A 2-Val-X A 4-X A 5-Cys A 6-Cys A7 -X A 8-X A 9-X A1 o-Cys A11 -Ser- XAi3-XAi4-XAi5-XAi6-XAi7-XAi8-Tyr-Cys A20 -X A 2i, wherein X A2 is Val or lie, X A4 is Glu or gamma carboxyglutamate, X A s is His or Gin, X A g is His or Thr, X A 9 is Arg or Ser, X A io is Pro or He, X A1 3 is Asn or Leu, AU is Ala or Tyr, X A1 s is Glu or Gin, X A 1 ⁇ 2 is Phe or Leu, ⁇ is Lys or Glu, X A is is Lys or Asn and X
  • the insulin analog comprises an A chain peptide comprising a sequence Gly-XA 2 -Val-X A 4-XA5-CysA6-CysA7-XA8-XA9-XAio-Cys A ii-Ser- X A i3-XAi4-XAi5-Phe-X A i7-XAi8-Tyr-Cys A2 o-XA2i, wherein X A2 is Val or He, X A 4 is Glu or gamma carboxyglutamate, X A S is His or Gin, X A S is His or Thr, X A is Arg or Ser, X A IO is Pro or He, X A i3 is Asn or Leu, X A M is Ala or Tyr, X A is is Glu or Gin, ⁇ ⁇ ⁇ is Lys or Glu, X A IS is Lys or Asn and X A2 i is Asn or
  • the insulin analog comprises an A chain peptide comprising a sequence Gly-Val-Val-X A 4-XA5-CysA6-CysA7-XA8-XA9-XAio-Cys A ii-Ser- XAi3-XAi4-XAi5-Phe-X A i7-XAi8-Tyr-Cys A 20-XA2i, wherein X A 4 is Glu or gamma carboxyglutamate, X A s is His or Gin, X A 8 is His or Thr, X A 9 is Arg or Ser, X A IO is Pro or He, X A i3 is Asn or Leu, X A M is Ala or Tyr, X A is is Glu or Gin, ⁇ ⁇ ⁇ is Lys or Glu, X A I8 is Lys or Asn and X A2 i is Asn or absent (SEQ ID NO: 14);; and
  • the insulin analog comprises an A chain peptide comprising a sequence Gly-X A2 -Val-X A 4-XA5-CysA6-CysA7-XA8-XA9-XAio-CysAii-Ser- X A i3-XAi4-XAi5-Phe-X A i7-XAi8-Tyr-Cys A2 o-XA2i, wherein XA2 is Val or He, X A 4 is Glu or gamma carboxyglutamate, X A s is His or Gin, X A 8 is His or Thr, X A 9 is Arg or Ser, X A IO is Pro or He, X A i3 is Asn or Leu, X A M is Ala or Tyr, X A is is is Glu or Gin, ⁇ ⁇ ⁇ is Lys or Glu, X A IS is Lys or Asn and X A 2i is Asn or
  • the insulin analog comprises an A chain peptide comprising a sequence Gly-Val-Val-X A 4-X A5 -Cys A6 -CysA7-X A8 -XA9-XAio-Cys A ii-Ser- XAi3-XAi4-XAi5-Phe-X A i7-XAi8-Tyr-Cys A 20-XA2i, wherein X A 4 is Glu or gamma carboxyglutamate, X A s is His or Gin, X A8 is His or Thr, X A9 is Arg or Ser, X A IO is Pro or He, X A i3 is Asn or Leu, X A M is Ala or Tyr, X A is is Glu or Gin, ⁇ ⁇ ⁇ is Lys or Glu, X A I 8 is Lys or Asn and X A 2i is Asn or absent (SEQ ID NO: 28); and
  • the insulin analog comprises an A chain peptide comprising the sequence Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr- Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO: 16); and a B chain peptide comprising the sequence Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Xaa-Tyr- Leu-Val-Cys-Gly-Glu, where Xaa is an aromatic residue or large aliphatic residue (SEQ ID NO: 17).
  • the insulin analog comprises an A chain peptide comprising the sequence Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr- Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO: 18); and a B chain peptide comprising the sequence Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr- Leu-Val-Cys-Xaa-Glu, where Xaa is an aromatic residue or large aliphatic residue (SEQ ID NO: 19).
  • the insulin analog comprises an A chain peptide comprising the sequence Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr- Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO: 20); and a B chain peptide comprising the sequence Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Xaa-Tyr- Leu-Val-Cys-Xaa-Glu, where Xaa is an aromatic residue or large aliphatic residue (SEQ ID NO: 21).
  • the insulin analog has a histidine residue at the position corresponding to ThrA8 of human insulin.
  • the insulin analogs may comprise one or more unnatural amino acids, modified amino acids or synthetic amino acid analogues, some of which are indicated with the sequences herein.
  • amino acids include, but are not limited to, the D- isomers of the common amino acids, 2,4-diaminobutyric acid, oc-amino isobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, gamma carboxyglutamate, hydroxyproline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, cyclopentylalanine, selenocysteine, amidated cyst
  • peptides which are differentially modified during or after synthesis, for example, by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
  • one or more Glu residues can be replaced with gamma-carboxyglutamate (Gla), for example at the X A 4 or X A s Glu positions of various A chains, or at the X B i2 Glu position of some B chains.
  • one or more Pro residues can be replaced with hydroxyproline (Hyp), for example at the X B s Pro position of certain B chains.
  • the C-terminal ends can be amidated, such as an amidated Cys (*) at the terminal end of various A chains, among others.
  • the insulin analogs have an A chain peptide and the B chain peptide that are bonded together across at least one pair of cysteine residues.
  • Cys B 9 of the B chain peptide is bonded to Cys A 6 of the A chain peptide
  • Cys B 2i of the B chain peptide is bonded to Cys A 2o of the A chain peptide
  • Cys A7 is bonded to CysAii.
  • the A chain peptide and the B chain peptide can be linked together at one or more terminal ends. In some embodiments the A chain peptide and the B chain peptide are linked together at a terminal end.
  • the N- terminus of the A chain peptide can be linked to the N- or C-terminus of the B chain peptide
  • the C-terminus of the A chain peptide can be linked to the N- or C-terminus of the B chain peptide
  • the C-terminus of the A chain peptide can be linked to the N- terminus of the A chain peptide or the C-terminus of the B chain peptide can be linked to the N- terminus of the B chain peptide.
  • the A chain peptide and the B chain peptide are linked together at both terminal ends.
  • the insulin analog is acyclic
  • the insulin analogs may be cyclic, while retaining the Cys-bonding pattern described.
  • the insulin analog has cyclized backbone such that the A and B chains have no free N- or C-terminus (for the embodiment whereby both terminal ends are linked).
  • the linkage at the one or more terminal ends can be directly between amino acids of the A and B chain peptide backbones, or there can be a linker of one or more amino acids or other linker molecules bonded therebetween.
  • chemical groups, residues or groups of residues known to the person skilled in the art to improve stability can be added to the C-terminus and/or N-terminus.
  • chemical groups, residues or groups of residues known to the person skilled in the art to improve bioavailability can be added to the C- terminus and/or N-terminus.
  • residues or groups that can be added to the N-terminus can also replace Gly within the insulin analogs.
  • fluorescent tags are may be attached to either the C- or N-terminus.
  • Insulin analog peptides as disclosed herein can be made by any technique or method known to the person skilled in the art, and any of such techniques or methods are considered to be within the present scope.
  • techniques include, but are not limited to, chemical synthesis, solid phase peptide synthesis, recombinant expression, a combination of peptide synthesis and recombinant expression, and the like.
  • Example techniques are described further in the Examples section.
  • the insulin analog may be prepared in various forms, for example native, fusions, glycosylated, lipidated, etc.
  • the insulin analogs are preferably prepared in substantially pure form (i.e. substantially free from host cell proteins or other contaminants). Typically, the insulin analog is substantially pure when it is at least 60%, by weight, of total protein present. For example, the insulin analog is at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, more preferably at least 90%, by weight, of total protein present.
  • the present disclosure also provides salts or derivatives of the insulin analogs.
  • salt denotes acidic and/or basic salts, formed with inorganic or organic acids and/or bases, preferably basic salts. While pharmaceutically acceptable salts are generally preferred, particularly when employing the insulin analogs as medicaments, other salts find utility, for example, in processing these compounds, or where non-medicament-type uses are contemplated. Salts of these compounds may be prepared by any technique know to a person skilled in the art.
  • pharmaceutically acceptable salt refers to salts of compounds that retain the biological activity of the parent compound, and which are not biologically or otherwise undesirable.
  • Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases, include by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines. Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene- sulfonic acid, salicylic acid, and the like.
  • derivative as used herein includes alpha amino acids wherein one or more side groups found in the naturally occurring alpha-amino acids have been modified.
  • the naturally-occurring amino acids may be replaced with a variety of uncoded or modified amino acids such as the corresponding D-amino acid or N-methyl amino acid.
  • Other modifications are known to the person skilled in the art and include substitution of hydroxyl, thiol, amino and carboxyl functional groups with chemically similar groups, for example substitution of -SH with -SeH in cysteine.
  • the insulin analog, salt or derivative thereof is able to bind the IR.
  • the IR is the human IR-B receptor.
  • the IC5 0 or affinity (3 ⁇ 4) against the human IR-B receptor of less than 10 ⁇ 4 M, 10 ⁇ 5 M, 10 ⁇ 6 M 10 "7 M, 10 "8 M, 10 "9 M or 10 "10 M.
  • the IC 50 against the human IR-B receptor of less than 10 ⁇ 6 M.
  • the insulin analog, salt or derivative thereof does not bind IGF-IR or binds the IGF-IR weakly.
  • weakly refers to an insulin analog that does not bind with sufficient affinity to the IGF-IR to result in activation of the IGF-IR and/or cause signal transduction via IGF-IR.
  • the insulin analog has an IC 50 or K d for IGF-IR of weaker than 10 "9 M, 10 "8 M, 10 "7 M, 10 "6 M, 10 ⁇ 5 M or 10 "4 M, preferably, the insulin analog has an affinity (3 ⁇ 4) for IGF-IR of weaker than 100 nM.
  • the insulin analog, salt or derivative thereof is predominantly monomeric in solution.
  • at least 50% of the insulin analog is a monomer
  • at least 60% of the insulin analog is a monomer
  • at least 70% of the insulin analog is a monomer
  • at least 75% of the insulin analog is a monomer
  • at least 80% of the insulin analog is a monomer
  • at least 85% of the insulin analog is a monomer
  • at least 90% of the insulin analog is a monomer
  • at least 95% of the insulin analog is a monomer
  • at least 98% of the insulin analog is a monomer
  • at least 99% of the insulin analog is a monomer or approximately 100 % of the insulin analog is a monomer.
  • the insulin analog is monomeric.
  • the insulin analog may be at least partially monomeric and dissociate into monomeric form upon administration to a subject.
  • the insulin analog is monomeric or dissociates into a monomeric form in a subjects blood stream.
  • the insulin analog, salt or derivative thereof is a rapid acting insulin analog. In some embodiments, has increased bioavailability when administered to a human when compared human insulin. In some embodiments, the insulin analog, salt or derivative thereof has a peak bioavailability within 10 minutes to 6 hours of administration to a human. In some embodiments, the maximum plasma concentration of the insulin analog after administration occurs earlier than the maximum plasma concentration of human insulin after administration. For example, the peak availability of the insulin analog, salt or derivative thereof occurs within 10 minutes to 4 hours of administration, within 15 minutes to 3 hours of administration, within 30 minutes to 1 hour of administration or within 40 to 55 minutes of administration.
  • the insulin analog, salt or derivative thereof has an onset of activity within 2 min, 5 min, 10 minute, 15 minute, 20 minute or 30 minutes of administration. Preferably, the onset of activity is within 10 to 30 minutes of administration.
  • Insulin analogs of the present invention also include those designed or identified using a method of the invention and those which are capable of recognising and binding to a target binding site.
  • Target binding sites include physiological binding partners of insulin, such as the IR, as well as regions of physiological binding partners.
  • a target binding site may be a short polypeptide defining an epitope (e.g. corresponding to a loop structure identified below as a target binding site) or a mimetic, e.g. a peptidomimetic, mimicking a loop structure.
  • the target binding site is the insulin receptor, preferably human insulin receptor. In some embodiments, the target binding site is a region of IR involved in insulin docking to the receptor. In some embodiments, the region of the IR includes low affinity target binding sites comprising one or more of the following: the LI domain, the CT peptide and the CR domain of IR ectodomain.
  • the target binding site preferably comprises portions of the molecular surface of the central ⁇ -sheet of LI and portions of the molecular surface of the second LRR which contain Phe39 or the loop in the fourth LRR rung of LI, or preferably both.
  • the target binding site preferably comprises module 6 of the CR domain.
  • the low affinity target binding site may comprise one or more amino acids from one or more of the following amino acid sequences: (i) amino acids 1-156; (ii) amino acids 704-719; and (iii) amino acids 157-310.
  • the target binding site preferably comprises at least one amino acid from the amino acid sequence 1-68, preferably 1-55, and more preferably amino acid sequence 27-55.
  • the target binding site preferably comprises at least one amino acid selected from Argl4, Asnl5, Gln34, Leu36, Leu37, Phe39, Pro43- Phe46, Phe64, Leu87, Phe88, Asn90 and Phe89, more preferably at least one amino acid selected from Argl4, Asnl5, Gln34, Leu37, Phe39, Pro43-Phe46, Phe64, yet more preferably at least one amino acid selected from Phe39 and Pro43-Phe46, and most preferably at least Phe39.
  • the target binding site preferably comprises at least one amino acid from the amino acid sequence 192-310, more preferably at least one amino acid from the sequence 227-303, yet more preferably least one amino acid selected from the sequence 259-284.
  • the present invention also provides peptides comprising an insulin A chain peptide and an insulin B chain peptide, wherein the B chain peptide comprises a substitution at amino acid 10 and amino acid 20.
  • the B chain peptide comprises a substitution at amino acid 10 and amino acid 20 compared to wild type human insulin.
  • any conservative amino acid substitution can be present at positions 10, 20, or both positions.
  • another hydrophilic amino acid, polar amino acid, or aliphatic amino acid could be substituted at one or both positions.
  • the substitution at amino acid 20 of the B chain peptide can be G20Y, G20F, or G20P. In some instances, the substitution at amino acid 20 is G20Y. In some instances, the substitution at amino acid 20 can be G20P and the peptide further comprises a substitution at amino acid 21, wherein the substitution at amino acid 21 can be G21H. In some instances, the amino acid substitution can be any conservative substitution from glycine.
  • the substitution at amino acid 10 of the B chain peptide can be H10E, H10D or H10Q. In some instances, the substitution at amino acid 10 is H10E. In some instances, the amino acid substitution can be any conservative substitution from histidine.
  • both the insulin A chain peptide and the B chain peptide can contain substitutions compared to wild type insulin.
  • peptides comprising an insulin A chain peptide and an insulin B chain peptide, wherein the B chain peptide comprises a substitution at amino acid 10 and amino acid 20 and further comprising at least one substitution in the A chain peptide.
  • the at least one substitution can be found at position 8 or 9.
  • the at least one substitution in the A chain peptide can be T8H, T8Y, T8K, or S9R.
  • any conservative amino acid substitution can be present at position 8 or 9 or both positions.
  • another hydrophilic amino acid could be substituted or other polar amino acids could be substituted.
  • peptides comprising an insulin A chain peptide and an insulin B chain peptide, wherein the B chain peptide comprises a substitution at amino acid 10 and amino acid 20 and further comprising at least two substitutions in the A chain peptide.
  • the at least two substitutions can be found at positions 8 and 9.
  • the at least two substitutions in the A chain peptide can be selected from: T8H, T8Y, T8K, and S9R.
  • any conservative amino acid substitution can be present at position 8 or 9 or both positions.
  • another hydrophilic amino acid could be substituted or other polar amino acids could be substituted at one or both positions.
  • the B chain peptide is lacking one or more, up to eight, of the
  • the disclosed peptides can be des- octapeptide insulin peptides (missing the last 8 amino acids of the C-terminus of the human insulin B chain).
  • the disclosed peptides can have a B chain peptide that comprises the sequence of:
  • FVNQHLCGS QL VE ALYL VCPER (SEQ ID NO: 38).
  • the disclosed peptides can have an A chain comprising the sequence of GIVEQCCHRICSLYQLENYCN (SEQ ID NO: 39),
  • the A chain peptide and B chain peptide can be bonded via at least one disulfide bond. In some instances, the A chain peptide and B chain peptide can be bonded via at least two disulfide bonds. In some instances, the disclosed peptides are monomers. In other words, in some instances, the disclosed peptides are less likely to form dimers, tetramers, hexamers, etc.
  • the insulin A chain peptide can be at least 70% identical to wild type human insulin A chain peptide. In some instances, the insulin A chain peptide can be at least 60, 65, 70, 75, 80, 85, 90, 95, 99% identical to wild type human insulin A chain peptide. In some instances, the percent identity can be reached by the deletion of one or more amino acids from the N-terminus or C-terminus end of the disclosed peptides.
  • the insulin B chain peptide can be at least 70% identical to wild type human insulin B chain peptide. In some instances, the insulin B chain peptide can be at least 60, 65, 70, 75, 80, 85, 90, 95, 99% identical to wild type human insulin B chain peptide. In some instances, the percent identity can be reached by the deletion of one or more amino acids from the N-terminus or C-terminus end of the disclosed peptides.
  • the disclosed peptides can comprise one or more unnatural amino acids, modified amino acids or synthetic amino acid analogues.
  • amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4- diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, cyclopentylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ - methyl amino acids, Ca-methyl amino acids, Na-methyl amino
  • peptides which are differentially modified during or after synthesis, for example, by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the peptide.
  • therapeutic proteins having an A chain peptide bonded to a B chain peptide via at least one disulfide bond, wherein the A chain comprises the sequence of GIVEQCCHRICSLYQLENYCN (SEQ ID NO: 39), and wherein the B chain peptide comprises the sequence of FVNQHLCGSELVEALYLVCYER (SEQ ID NO: 30).
  • therapeutic proteins can be employed in pharmaceutical compositions and used in connection with treatment of disorders including diabetes.
  • the three-dimensional structure of venom insulin provided by the present invention may be used to design insulin analogs (also referred to herein as IR agonists, molecules and compounds), particularly rapid acting insulin analogs.
  • insulin analogs also referred to herein as IR agonists, molecules and compounds
  • rapid acting insulin analogs there is provided the use of the structure of Con-Ins Gl as defined by the atomic coordinates of Appendix I as a structural model.
  • the structural model is used for identification of insulin analogs.
  • a method of identifying, designing or screening for a compound that can potentially interact with IR comprises performing structure-based identification, design or screening of a compound based on the compound's interactions with an IR structure defined by the three-dimensional structure of Con-Ins Gl, or a subset thereof.
  • known IR binding molecules can be screened against the 3D structure of Con-Ins Gl defined by the atomic coordinates of Appendix I or a portion thereof, and an assessment made of the ability to self-associate and the potential to interact with IR.
  • the known IR binding molecule could be redesigned (i.e. chemically modified) so as to impart one or more of the following properties: (i) reduce self-association, (ii) improve its affinity for the low affinity binding site of IR (i.e. the binding site governing selectivity), (iii) improve its affinity for the high affinity binding site for IR (i.e.
  • a method of redesigning or modifying a polypeptide which is known to bind to IR, or a region of the IR comprises performing structure-based evaluation of a structure defined by the atomic coordinates of Appendix I or a subset thereof, and redesigning or chemically modifying the polypeptide as a result of the evaluation.
  • structure-based evaluation comprises comparison of the structure defined by the atomic coordinates of Appendix I or a subset thereof, with the atomic coordinates of insulin or a subset thereof.
  • structure-based evaluation further comprises molecular modelling of a complex formed between the structure defined by the atomic coordinates of Appendix I or a subset thereof with the atomic coordinates of an insulin receptor or a subset thereof.
  • the model is defined by the atomic coordinates of Appendix II or a subset thereof.
  • the method further comprises synthesising or obtaining the redesigned or chemically modified polypeptide and testing for its ability to bind IR. In some embodiments, the ability of the redesigned or chemically modified polypeptide to modulate IR activation is determined. In some embodiments, the ability of the redesigned or chemically modified polypeptide to lower blood glucose levels may be determined.
  • the polypeptide which is known to bind to IR is an insulin-like growth factor (IGF).
  • IGFs include those from humans, pigs, cattle, birds, mice and the like.
  • the IGF is human IGF-I or IGF-II.
  • the polypeptide which is known to bind to IR is insulin.
  • Suitable insulin includes human insulin, porcine insulin bovine insulin, ovine insulin, murine insulin, guinea pig insulin and the like. In some embodiments, the insulin is human insulin.
  • modeling includes the quantitative and qualitative analysis of molecular structure and/or function based on atomic structural information and interaction models.
  • the term “modelling” includes conventional numeric-based molecular dynamic and energy minimisation models, interactive computer graphic models, modified molecular mechanics models, distance geometry and other structure- based constraint models.
  • Molecular modelling techniques can be applied to the atomic coordinates of Con-Ins Gl or a region thereof to derive a range of 3D models and to investigate the structure of binding sites, such as the binding site of IR and other protein targets.
  • a region of Con-Ins Gl as referred to herein may be defined by a single amino acid (or side-chain thereof), by a continuous amino acid sequence or by two or more separate amino acids and/or stretches of amino acids. Such separate amino acids and/or stretches of amino acids may exist in close spatial proximity to one another in the three dimensional structure or may have the potential to be brought into close spatial proximity, for example, upon the binding of a suitable ligand.
  • regions of Con-Ins Gl comprise amino acid sequences involved in the binding of IR, both the initial selective low affinity binding and the subsequent high affinity binding to the other monomer in the IR dimer.
  • the screen may employ a solid 3D screening system or a computational screening system.
  • such modelling methods are to design or select chemical entities (for example a polypeptide, peptide, peptidomimetic, compound and the like) that possess stereochemical complementary to particular regions of IR.
  • stereochemical complementarity we mean that the compound or a portion thereof makes a sufficient number of energetically favourable contacts with the receptor as to have a net reduction of free energy on binding to the receptor.
  • a number of methods may be used to identify chemical entities possessing stereo-complementarity to a region of the IR.
  • the process may begin by visual inspection of Con-Ins Gl structure on a computer screen based on the atomic coordinates of Con-Ins Gl, or region thereof, in Appendix I generated from the machine -readable storage medium.
  • the process may begin by molecular modelling of a complex formed between the structure defined by the atomic coordinates of Appendix I or a subset thereof with the atomic coordinates of an insulin receptor or a subset thereof. Modelling software that is well known and available in the art may be used, for example MODELLER (v9.15) (Webb and Sali, (2014).
  • This modelling step may be followed by energy minimization with standard molecular mechanics force fields such as CHARMM (Guvench et al. 2011 ; Best et al. 2012). Modelling and energy minimization may be followed by molecular dynamics simulations using software known in the art, for example NAMD (Phillips et al. 2005).
  • NAMD Phillips et al. 2005.
  • polypeptide or salt or analog thereof which has been redesigned or modified by the method of redesigning or modifying a polypeptide defined herein.
  • the redesigned or modified polypeptide is monomeric, or dissociates to a monomer when administered to a subject.
  • Preferred regions of the IR are those governing specificity, for example those described as target binding sites above.
  • an isolated molecule which is an IR agonist wherein the molecule is identified and/or designed based on the 3D structure of Con- Ins Gl defined by the atomic coordinates of Appendix I or a subset thereof.
  • Suitable molecules include peptides, polypeptides or peptidomimetics.
  • peptidomimetic is a molecule that mimics the biological activity of a peptide but is no longer completely peptidic in chemical nature.
  • a peptidomimetic is a molecule that no longer contains any peptide bonds (that is, amide bonds between amino acids).
  • peptide mimetic can be used to describe molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi-peptides and peptoids.
  • peptidomimetics of the invention provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of active groups in the peptide on which the peptidomimetic is based.
  • peptidomimetic has effects on biological systems which are similar to the biological activity of the peptide.
  • Suitable peptidomimetics based on venom insulin can be developed using readily available techniques and/or the methods described herein.
  • peptide bonds can be replaced by non-peptide bonds that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original peptide.
  • Further modifications can also be made by replacing chemical groups of the amino acids with other chemical groups of similar structure.
  • the development of peptidomimetics derived from venom insulin can be aided by reference to the three dimensional structure of these residues as provided in Appendix I or a subset thereof.
  • This structural information can be used to search three-dimensional databases to identify molecules having a similar structure, using programs such as MACCS-3D and ISIS/3D (Molecular Design Ltd., San Leandro, CA), ChemDBS-3D (Chemical Design Ltd., Oxford, U.K.), and Sybyl/3DB Unity (Tripos Associates, St. Louis, MO).
  • programs such as MACCS-3D and ISIS/3D (Molecular Design Ltd., San Leandro, CA), ChemDBS-3D (Chemical Design Ltd., Oxford, U.K.), and Sybyl/3DB Unity (Tripos Associates, St. Louis, MO).
  • peptidomimetic may require slight structural alteration or adjustment of a chemical structure designed or identified using the methods of the invention.
  • mimetics identified or designed using the methods of the invention can be synthesized chemically and then tested for ability to modulate insulin receptor activity using any of the methods described herein.
  • the methods of the invention are particularly useful because they can be used to greatly decrease the number of potential mimetics which must be screened for their ability to modulate insulin receptor activity.
  • the isolated molecule is able to bind the IR.
  • the IR is the human IR-B receptor.
  • the IC5 0 or 3 ⁇ 4 against the human IR-B receptor is stronger than 10 "4 M, 10 "5 M, 10 "6 M 10 "7 M, 10 "8 M, 10 "9 M or 10 ⁇ 10 M.
  • the IC5 0 or K d against the human IR-B receptor is stronger than 10 " 6 M.
  • the isolated molecule does not bind IGF-IR or binds IGF- IR weakly.
  • the isolated molecule has an affinity (3 ⁇ 4) for IGF-IR of weaker than 100 nM.
  • the isolated molecule is predominantly monomeric in solution. In some embodiments, at least 50% of the isolated molecule is a monomer, at least 60% of the isolated molecule is a monomer, at least 70% of the isolated molecule is a monomer, at least 75% of the isolated molecule is a monomer, at least 80% of the isolated molecule is a monomer, at least 85% of the isolated molecule is a monomer, at least 90% of the isolated molecule is a monomer, at least 95% of the isolated molecule is a monomer, at least 98% of the isolated molecule is a monomer, at least 99% of the isolated molecule is a monomer or approximately 100 % of the isolated molecule is a monomer. In some embodiments, the isolated molecule may be at least partially monomeric and dissociate into monomeric form upon administration to a subject. In some embodiments, the isolated molecule is monomeric or dissociates into a monomeric form in a subjects blood stream.
  • the present invention is also useful in the identification and/or design of insulin analogs which do not bind or only bind weakly to IGF-IR.
  • insulin analogs identified using the methods of this invention can be screened in silico, in vitro and/or in vivo for their ability to bind the IGF-IR. Any insulin analogs found or suspected to bind to IGF-IR can be redesigned so as to be more selective for IR.
  • an insulin analog (which includes an isolated molecule, compound or IR agonist) identified by the methods herein does not bind IGF-IR or binds the IGF-IR weakly.
  • the insulin analog has an IC5 0 or 3 ⁇ 4 for IGF-IR of weaker than 10 "9 M, 10 "8 M, 10 "7 M, 10 "6 M, 10 "5 M or 10 "4 M, preferably, the insulin analog has an affinity (K d ) for IGF-IR of weaker than 100 nM.
  • the present disclosure also provides a method of identifying a compound which binds IR, the method comprising:
  • generating a three-dimensional structure model comprises generating a model of the polypeptide bound to IR or regions thereof.
  • Preferred regions of the IR are those governing specificity, for example those described as target binding sites above.
  • the model is defined by the atomic coordinates of Appendix II or a subset thereof.
  • the model may be adaptive in a sense that it allows for slight surface changes to improve the fit between the candidate compound and the protein, e.g. by small movements in side chains or main chain.
  • the methods further comprise synthesising the compound which potentially binds the IR.
  • compound modulates at least one biological activity of IR.
  • the method may further comprise testing the compound designed or screened for in ii) for its ability to modulate at least one biological activity of IR.
  • the method may further comprise testing the compound designed or screened for in ii) for its ability to modulate blood glucose levels.
  • steps i) and ii) are performed in silico.
  • the present invention also provides a computer-based method of identifying a compound which mimics insulin activity, the method comprising
  • generating a three-dimensional structure model comprises generating a model of the polypeptide bound to IR or regions thereof.
  • the model is defined by the atomic coordinates of Appendix II or a subset thereof.
  • Preferred regions of the IR are those governing specificity, for example those described as target binding sites above.
  • the methods further comprise synthesising the compound which potentially binds the IR.
  • compound modulates at least one biological activity of IR.
  • the method may further comprise testing the compound designed or screened for in ii) for its ability to modulate at least one biological activity of IR.
  • the method may further comprise testing the compound designed or screened for in ii) for its ability to modulate blood glucose levels.
  • steps i) and ii) are performed in silico.
  • the compounds identified by the methods of the present invention are able to bind the IR.
  • the IR is the human IR-B receptor.
  • the IC5 0 against the human IR-B receptor of less than 10 ⁇ 4 M, 10 ⁇ 5 M, 10 "6 M 10 "7 M, 10 "8 M, 10 "9 M or 10 "10 M.
  • the IC 50 against the human IR- B receptor of less than 10 ⁇ 6 M.
  • the compounds identified by the methods of the present invention do not bind IGF-IR or binds the IGF-IR weakly.
  • the insulin analog has an IC 50 or K d for IGF-IR of weaker than 10 "9 M, 10 "8 M, 10 "7 M, 10 “6 M, 10 ⁇ 5 M or 10 "4 M, preferably, the insulin analog has an affinity (3 ⁇ 4) for IGF-IR of weaker than 100 nM.
  • the compounds identified by the methods of the present invention are predominantly monomeric in solution.
  • at least 50% of the compound is a monomer
  • at least 60% of the compound is a monomer
  • at least 70% of the compound is a monomer
  • at least 75% of the compound is a monomer
  • at least 80% of the compound is a monomer
  • at least 85% of the compound is a monomer
  • at least 90% of the compound is a monomer
  • at least 95% of the compound is a monomer
  • at least 98% of the compound is a monomer
  • at least 99% of the compound is a monomer or approximately 100 % of the compound is a monomer.
  • the compound may be at least partially monomeric and dissociate into monomeric form upon administration to a subject.
  • the compound is monomeric or dissociates into a monomeric form in a subjects blood stream.
  • the methods of the present invention provide a rational method for designing and selecting insulin analog proteins which interact with the insulin receptor. In the some cases these proteins may require further development in order to increase activity. Such further development is routine in this field and will be assisted by the structural information provided in this application. It is intended that in particular embodiments the methods of the present invention includes such further developmental steps.
  • an insulin analog that has been designed or selected to bind the IR must also preferably traverse a volume not overlapping that occupied by the binding site when it is bound to the native IR.
  • An insulin analog designed or selected as binding to IR may be further computationally optimised so that in its bound state it would preferably lack repulsive electrostatic interaction with the target protein.
  • Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions.
  • the sum of all electrostatic interactions between the compound and the insulin analog when the insulin analog is bound to IR preferably make a neutral or favourable contribution to the enthalpy of binding.
  • substitutions may then be made in some of its atoms or side groups to improve or modify its binding properties.
  • initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided.
  • substituted insulin analogs may then be analysed for efficiency of fit to IR by the same computer methods described in detail above.
  • the present invention encompasses insulin analogs (including IR agonists, molecules, compounds and the like) identified using a method described herein. Some embodiments, also relate pharmaceutical compositions comprising the insulin analogs (including IR agonists, molecules, compounds and the like) identified using a method described herein. Screening Assays and Confirmation of Binding and Biological Activity
  • Insulin analogs which includes compounds and molecules identified using the methods of the present disclosure
  • Insulin analogs of the present invention are preferably assessed by a number of in vitro and in vivo assays of IR and/or IGF-1R function to confirm their ability to interact with and modulate IR and/or IGF-1R activity.
  • compounds may be tested for their ability to bind to IR and/or IGF-1R and/or for their ability to modulate e.g. activate or disrupt IR and/or IGF-1R signal transduction.
  • IR or IGF-1R may be joined to a label, where the label can directly or indirectly provide a detectable signal.
  • labels include radioisotopes, fluorescent molecules, chemiluminescent molecules, enzymes, specific binding molecules, particles, e.g., magnetic particles, and the like.
  • Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin, etc.
  • the complementary member would normally be labelled with a molecule that provides for detection, in accordance with known procedures.
  • reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc., which are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., may be used. The components are added in any order that produces the requisite binding. Incubations are performed at any temperature that facilitates optimal activity, typically between 4 and 40 °C.
  • Direct binding of compounds to IR or IGF-1R can also be done by Surface Plasmon Resonance (BIAcore) (reviewed in Morton and Myszka, 1998).
  • the receptor is immobilized on a CM5 or other sensor chip by either direct chemical coupling using amine or thiol-disulphide exchange coupling (Nice and Catimel, 1999) or by capturing the receptor ectodomain as an Fc fusion protein to an appropriately derivatised sensor surface (Morten and Myszka, 1998).
  • the potential insulin analog (called an analyte) is passed over the sensor surface at an appropriate flow rate and a range of concentrations.
  • the classical method of analysis is to collect responses for a wide range of analyte concentrations.
  • a range of concentrations provides sufficient information about the reaction, and by using a fitting algorithm such as CLAMP (see Morton and Myszka, 1998), rate constants can be determined (Morton and Myszka, 1998; Nice and Catimel, 1999).
  • CLAMP a fitting algorithm
  • rate constants can be determined (Morton and Myszka, 1998; Nice and Catimel, 1999).
  • the ligand surface is regenerated at the end of each analyte binding cycle. Surface regeneration ensures that the same number of ligand binding sites is accessible to the analyte at the beginning of each cycle.
  • Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Normally, between 0.1 and 1 hour will be sufficient.
  • a plurality of assay mixtures is run in parallel with different test agent concentrations to obtain a differential response to these concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.
  • the basic format of an in vitro competitive receptor binding assay as the basis of a heterogeneous screen for insulin analog replacements for native insulin may be as follows: occupation of the active site of IR or IGF-IR is quantified by time-resolved fluorometric detection (TRFD) as described by Denley et al. (2004). RTR-A, RTR-B and P6 cells are used as sources of IR-A, IR-B and IGF-IR respectively.
  • TRFD time-resolved fluorometric detection
  • lysis buffer (20 mM HEPES, 150 mM NaCl, 1.5 mM MgCl 2 , 10% (v/v) glycerol, 1% (v/v) Triton X-100, 1 mM EGTA pH 7.5) for 1 hour at 4°C. Lysates are centrifuged for 10 minutes at 3500 rpm and then 100 ⁇ is added per well to a white Greiner Lumitrac 600 plate previously coated with anti-insulin receptor antibody 83-7 or anti-IGF-lR antibody 24-31. Neither capture antibody interferes with receptor binding by insulin, IGF-I or IGF-II.
  • suitable assays which may be employed to assess the binding and biological activity of compounds to and on IR are well known in the art.
  • suitable assays may be found in PCT International Publication Number WO 03/027246.
  • suitable assays include the following:
  • R IR-B cells or P6 cells are plated in a Falcon 96 well flat bottom plate at 2.5 x 10 4 cells/well and grown overnight at 37°C, 5% C(3 ⁇ 4. Cells are washed for 4 hours in serum-free medium before treating with one of either insulin, IGF-I or IGF-II in ⁇ DMEM with 1% BSA for 10 minutes at 37°C, 5% C0 2 . Lysis buffer containing 2mM Na 3 V0 4 and 1 mg/ml NaF is added to cells and receptors from lysates are captured on 96 well plates precoated with antibody 83-7 or 24-31 and blocked with lx TBST/0.5 BSA.
  • Glucose uptake using 2-deoxy-[U- 14 C] glucose (as described by Olefsky, 1978).
  • Adipocytes between days 8-12 post-differentation in 24-well plates are washed twice in Krebs-Ringer Bicarbonate Buffer (25mM Hepes, pH 7.4 containing 130 mM NaCl, 5 mM KC1, KH 2 P0 4 , 1.3 mM MgS0 4 .7H 2 0, 25 mM NaHC0 3 and 1.15 mM CaCl 2 ) supplemented with 1% (w/v) RIA-grade BSA and 2 mM sodium pyruvate.
  • Krebs-Ringer Bicarbonate Buffer 25mM Hepes, pH 7.4 containing 130 mM NaCl, 5 mM KC1, KH 2 P0 4 , 1.3 mM MgS0 4 .7H 2 0, 25 mM NaHC0 3 and 1.15 mM CaCl 2
  • Adipocytes are equilibrated for 90 min at 37°C prior to insulin addition, or for 30 min prior to agonist or antagonist addition.
  • Insulin Actrapid, Novogen
  • Agonist or antagonist (0 to 500 mM) is added to adipocytes for 90 min followed by the addition of native insulin in the case of antagonists.
  • Uptake of 50 mM 2-deoxy glucose and 0.5 mCi 2-deoxy-[U- 14 C] glucose (NEN, PerkinElmer Life Sciences) per well is measured over the final 10 min of agonist stimulation by scintillation counting.
  • Glucose transporter GLUT4 translocation using plasma membrane lawns (as described by Robinson and James (1992) and Marsh et al. (1995)).
  • GLUT4 translocation using plasma membrane lawns (as described by Marsh et al. 1995).
  • 3T3-L1 fibroblasts are grown on glass coverslips in 6-well plates and differentiated into adipocytes. After 8-12 days post-differentiation, adipocytes are serum-starved for 18 hrs in DMEM containing 0.5% FBS.
  • adipocytes are washed twice in Krebs-Ringer Bicarbonate Buffer, pH 7.4 and equilibrated for 90 min at 37°C prior to insulin ( ⁇ ) addition, or for 30 min prior to compound ( ⁇ ) addition.
  • adipocytes are washed in 0.5 mg/ml poly-L-lysine in PBS, shocked hypotonically by three washes in 1 :3 (v/v) membrane buffer (30 mM Hepes, pH 7.2 containing 70 mM KC1, 5 mM MgCl 2 , 3 mM EGTA and freshly added 1 mM DTT and 2 mM PMSF) on ice.
  • the washed cells are then sonicated using a probe sonicator (Microson) at setting 0 in 1: 1 (v/v) membrane buffer on ice, to generate a lawn of plasma membrane fragments that remain attached to the coverslip.
  • the fragments are fixed in 2% (w/v) paraformaldehyde in membrane buffer for 20 min at 22°C and the fixative quenched by 100 mM glycine in PBS.
  • the plasma membrane fragments are then blocked in 1% (w/v) Blotto in membrane buffer for 60 min at 22°C and immunolabelled with an in-house rabbit affinity purified anti-GLUT4 polyclonal antibody (clone R10, generated against a peptide encompassing the C-terminal 19 amino acids of GLUT4) and Alexa 488 goat anti-rabbit secondary antibody (Molecular Probes; 1:200).
  • Coverslips are mounted onto slides using FluoroSave reagent (Calbiochem), and imaged using an OptiScan confocal laser scanning immunofluoroscence microscope (Optiscan, VIC, Australia). Data are analysed using ImageJ (NIH) imaging software. At least six fields are examined within each experiment for each condition, and the confocal microscope gain settings over the period of experiments are maintained to minimise between-experiment variability.
  • Insulin analog activity may be determined using an adipocyte assay. Insulin increases uptake of H glucose into adipocytes and its conversion into lipid. Incorporation of H into a lipid phase is determined by partitioning of lipid phase into a scintillant mixture, which excludes water-soluble H products. The effect of insulin analogs on the incorporation of H glucose at a sub-maximal insulin dose is determined. The method is adapted from Moody et al. (1974).
  • Mouse epididymal fat pads are dissected out, minced into digestion buffer (Krebs-Ringer 25 mM HEPES, 4% HSA, 1.1 mM glucose, 0.4 mg/ml Collagenase Type 1, pH 7.4), and digested for up to 1.5 hours at 36.5 C. After filtration, washing (Krebs-Ringer HEPES, 1% HSA) and resuspension in assay buffer (Krebs-Ringer HEPES, 1% HSA), free fat cells are pipetted into 96-well Picoplates containing test solution.
  • the assay is started by addition of H glucose (e.g. ex. Amersham TRK 239), in a final concentration of 0.45 mM glucose.
  • H glucose e.g. ex. Amersham TRK 239
  • the assay is incubated for 2 hours at 36.5 °C, in a Labshaker incubation tower, 400 rpm, then terminated by the addition of Permablend/Toluene scintillant (or equivalent), and the plates sealed before standing for at least 1 hour and detection in a Packard Top Counter or equivalent.
  • a full native insulin standard curve (8 dose) is run as control on each plate.
  • the assay can also be run at basal or maximal insulin concentration.
  • Male Mol:Wistar rats weighing about 300 g, are divided into two groups. A 10 ⁇ sample of blood is taken from the tail vein for determination of blood glucose concentration. The rats are then anaesthe
  • Insulin analogs, compounds and/or molecules designed or selected according to the methods of the present invention may also be assessed by a number of biophysical methods. Suitable methods include x-ray crystallography, analytical ultracentrifugation, size exclusion chromatography, isothermal calorimetry and the like.
  • insulin analogs (which includes compounds and molecules identified using the methods of the present disclosure) may be subjected to further confirmation by crystallization of the analog and structural determination, as described herein.
  • the multimerisation state in solution may be determined by analytical ultracentrifugation. Analytical ultracentrifugation is carried out at 20°C using a Beckman XLI analytical centrifuge in 12 mm path-length cells.
  • sample containing the compound is diluted in 10 mM HC1 into 10 mM Tris, 50 mM NaCl, pH 7.4 to a final concentration of 100 ⁇ g/mL.
  • samples may also be prepared that contain 0.2 mM ⁇ (3 ⁇ 4, 2 mM CaCh, 1 mM sodium phosphate (pH 7.4) or 0.1 M ammonium sulfate.
  • An equal volume of 10 mM NaOH is added to neutralize any pH change. A total sample volume of 100 ⁇ . is used.
  • the insulin analogs, compounds and molecules with which the present disclosure is concerned are of value in the treatment of conditions which are responsive to activation and/or modulation of IR. These conditions include those for which regulation of glucose metabolism and/or blood glucose levels is indicated.
  • Conditions which are responsive to activation and/or modulation of IR include, but are not limited to, diabetes myelitis (e.g. type 1 diabetes, type 2 diabetes, gestational diabetes), hyperglycemia, insulin resistance, impaired glucose tolerance and the like.
  • the condition is an insulin-related condition.
  • Insulin related conditions include but are not limited to, hyperglycemia, insulin resistance, type-1 diabetes, gestational diabetes or type-2 diabetes.
  • the insulin analogs of the present invention are suitable for use in a subject, such as a mammal including a human, in order to regulate glucose metabolism. Accordingly, there is provided methods for regulating glucose metabolism. Some embodiments relate to a method for regulating glucose metabolism by administering to a subject in need thereof a therapeutically effective amount of such an insulin analog. Some embodiments relate to a method for treating an insulin-related condition, comprising administering a therapeutically effective amount of the insulin analog as defined herein to a subject in need thereof. Some embodiments relate to a method for treating diabetes by administering to a subject in need thereof a therapeutically effective amount of an insulin analog. Diabetes, includes but is not limited to, type 1 diabetes, type 2 diabetes or gestational diabetes.
  • Some embodiments relate to a method for treating hyperglycemia by administering to a subject in need thereof a therapeutically effective amount of such an insulin analog. Some embodiments relate to a method for treating insulin resistance by administering to a subject in need thereof a therapeutically effective amount of such an insulin analog. Some embodiments relate to a method for treating impaired glucose tolerance by administering to a subject in need thereof a therapeutically effective amount of such an insulin analog. Some embodiments relate to a method for decreasing blood glucose levels in a subject by administering to a subject in need thereof a therapeutically effective amount of such an insulin analog.
  • a method of treating type 1 diabetes in a subject comprising administering a therapeutically effective amount of a peptide comprising an insulin A chain peptide and an insulin B chain peptide, wherein the B chain peptide comprises a substitution at amino acid 10 and amino acid 20 to a subject in need thereof.
  • the substitution at amino acid 20 of the B chain peptide can be G20Y, G20F, or G20P.
  • the substitution at amino acid 10 of the B chain peptide can be H10E, H10D or H10Q.
  • any combination of the B chain substitutions at amino acid 10 and 20 can be present.
  • the A chain of the administered peptide can also comprise at least one substitution.
  • the at least one substitution in the A chain peptide can be T8H, T8Y, T8K, or S9R.
  • the amino acid substitution can be present at position 8 or 9 or both positions.
  • any combination of the disclosed B chain peptide substitutions and A chain peptide substitutions can be present. Also disclosed herein are methods of treating type 1 diabetes in a subject comprising administering a therapeutically effective amount of an insulin analog as defined herein. In some instances, the subject has been diagnosed with type 1 diabetes prior to administering the peptide. In some instances, the subject has been diagnosed with being at risk for developing type 1 diabetes prior to administering the peptide.
  • an insulin analog, compound or molecule as defined herein in the manufacture of a medicament is also provided. Some embodiments relate to the use of the insulin analog as defined herein in the manufacture of a medicament for regulating glucose metabolism in a subject. Some embodiments relate to the use of the insulin analog as defined herein in the manufacture of a medicament for treating and/or preventing an insulin-related condition in a subject. Some embodiments relate to the use of the insulin analog as defined herein in the manufacture of a medicament for treating and/or preventing an diabetes in a subject. Some embodiments relate to the use of the insulin analog as defined herein in the manufacture of a medicament for treating and/or preventing an hyperglycemia in a subject.
  • Some embodiments relate to the use of the insulin analog as defined herein in the manufacture of a medicament for treating and/or preventing insulin resistance in a subject. Some embodiments relate to the use of the insulin analog as defined herein in the manufacture of a medicament for treating and/or preventing impaired glucose tolerance in a subject. Some embodiments relate to the use of the insulin analog as defined herein in the manufacture of a medicament for decreasing blood glucose levels in a subject.
  • Some embodiments also relate to an insulin analog as defined herein for use in regulating glucose metabolism in a subject. Some embodiments relate to an insulin analog as defined herein for use in treating and/or preventing an insulin-related condition in a subject. Some embodiments relate to an insulin analog as defined herein for use in treating and/or preventing diabetes in a subject. Some embodiments relate to an insulin analog as defined herein for use in treating and/or preventing hyperglycemia in a subject. Some embodiments relate to an insulin analog as defined herein for use in treating and/or preventing insulin resistance in a subject. Some embodiments relate to an insulin analog as defined herein for use in treating and/or preventing impaired glucose tolerance in a subject. Some embodiments relate to an insulin analog as defined herein for use in decreasing blood glucose levels in a subject.
  • administration of the insulin analog results in a decrease in blood glucose levels.
  • the insulin analog is a rapid acting insulin analog such that administration of the insulin analog results in a decrease in blood glucose levels within 60 minutes of administration.
  • administration of the insulin analog results in a decrease in blood glucose levels within minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes or 5 minutes of administration.
  • administration of the insulin analog results in a decrease in blood glucose levels for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.
  • the preferred compounds that have been discussed in details herein could be administered. Blood glucose levels can be measured by any means known to one of skill in the art.
  • a subject in need thereof can be a subject known to have decreased insulin receptor activation compared to a standard activation level.
  • a standard activation level of insulin receptor activation can be based on established levels in healthy individuals.
  • a standard activation level of insulin receptor activation can be based on established levels in the subject being treated prior to the determination of a need for increased insulin receptor activation.
  • a method of increasing insulin receptor activation in a subject comprising administering a therapeutically effective amount of a peptide comprising an insulin A chain peptide and an insulin B chain peptide, wherein the B chain peptide comprises a substitution at amino acid 10 and amino acid 20 to a subject in need thereof.
  • the substitution at amino acid 20 of the B chain peptide can be G20Y, G20F, or G20P.
  • the substitution at amino acid 10 of the B chain peptide can be H10E, H10D or H10Q.
  • any combination of the B chain substitutions at amino acid 10 and 20 can be present.
  • the A chain of the administered peptide can also comprise at least one substitution.
  • the at least one substitution in the A chain peptide can be T8H, T8Y, T8K, or S9R.
  • the amino acid substitution can be present at position 8 or 9 or both positions.
  • any combination of the disclosed B chain peptide substitutions and A chain peptide substitutions can be present. Also disclosed are methods of increasing insulin receptor activation in a subject comprising administering a therapeutically effective amount of an insulin analog as defined herein.
  • Disclosed are methods of lowering the blood sugar in a subject comprising administering a therapeutically effective amount of any one of the disclosed insulin analogs, peptides, compounds or pharmaceutical compositions to a subject in need thereof.
  • a subject in need thereof can be a subject known to have increased blood sugar compared to a standard blood sugar level.
  • a standard activation level of insulin receptor activation can be based on established levels in healthy individuals.
  • a standard activation level of insulin receptor activation can be based on established levels in the subject being treated prior to the determination of a need for increased insulin receptor activation.
  • a method of lowering the blood sugar in a subject comprising administering a therapeutically effective amount of a peptide comprising an insulin A chain peptide and an insulin B chain peptide, wherein the B chain peptide comprises a substitution at amino acid 10 and amino acid 20 to a subject in need thereof.
  • the substitution at amino acid 20 of the B chain peptide can be G20Y, G20F, or G20P.
  • the substitution at amino acid 10 of the B chain peptide can be H10E, H10D or H10Q.
  • any combination of the B chain substitutions at amino acid 10 and 20 can be present.
  • the A chain of the administered peptide can also comprise at least one substitution.
  • the at least one substitution in the A chain peptide can be T8H, T8Y, T8K, or S9R.
  • the amino acid substitution can be present at position 8 or 9 or both positions.
  • any combination of the disclosed B chain peptide substitutions and A chain peptide substitutions can be present. Also disclosed are methods of lowering the blood sugar in a subject comprising administering a therapeutically effective amount of an insulin analog as defined herein.
  • a subject may receive a therapeutically effective amount of an insulin analog in one or more doses.
  • the actual amount administered, and the rate and time-course of administration, will vary with the route of administration, the nature of the benefit required, the nature and severity of the condition being treated, the condition of the subject being treated and will ultimately be at the discretion of the attendant veterinarian or medical professional.
  • Prescription of treatment e.g. decisions on dosage, timing, etc., is within the responsibility of the attendant veterinarian or medical professional and typically takes account of the nature of the disorder, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.
  • one or more analog(s) together or separately can be administered on any appropriate schedule, e.g., from one or more times per day to one or more times per week; including once every other day, for any number of days or weeks, or any variation thereon.
  • the insulin analog is administered one, two, three, four or more times daily.
  • the insulin analog can also be administered on an as needs basis, for example when blood glucose levels are above the normal range or when blood glucose levels need to be reduced.
  • the compound or composition of the present invention is administered with, before or after ingesting food.
  • the insulin analog is administered prior to every meal, for example breakfast, lunch and dinner.
  • the compound or composition is administered 5, 10, 20, 30, 40, 50 or 60 minutes before ingesting food.
  • the compound or composition is administered immediately before ingesting food.
  • the insulin analog, as well as pharmaceutical compositions as described herein, can be administered by any route known to one of skill in the art, the parenteral being of most interest. Accordingly, in one embodiment of the invention the insulin analogs are administered by the parenteral route, such as by injection or infusion. Other suitable administration routes, for example enteral (e.g. oral administration), are within the scope of the present invention.
  • the parenteral route is preferred and includes intravenous, intraarticular, intraperitoneal, subcutaneous, intramuscular, intrastemal injection and infusion as well as administration by the sublingual, transdermal, topical, transmucosal including nasal route, or by inhalation such as, e.g., pulmonary inhalation.
  • the insulin analogs can be administered in a suitable vehicle or they can be administered in the form of a suitable pharmaceutical composition. Such compositions are also within the scope of the invention. In the following are described suitable pharmaceutical compositions.
  • Pharmaceutical compositions are also within the scope of the invention. In the following are described suitable pharmaceutical compositions.
  • compositions may include the insulin analog and one or more pharmaceutically acceptable carriers.
  • the term "pharmaceutically acceptable carrier” includes any and all solids or solvents (such as phosphate buffered saline buffers, water, saline) dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the pharmaceutically acceptable carriers must be 'acceptable' in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin (Mack Publishing Co., Easton, Pa.); Gennaro, A.
  • compositions are described in a number of sources that are well known and readily available to those skilled in the art, for example, Remington's Pharmaceutical Sciences (Martin E. W., Easton Pa., Mack Publishing Company, 19th ed., 1995).
  • the amount of pharmaceutically acceptable carrier will depend upon the level of the compound and any other optional ingredients that a person skilled in the art would classify as distinct from the carrier (e.g., other active agents).
  • the formulations of the present invention may comprise, for example, from about 5% to 99.99%, or 25% to about 99.9 % or from 30% to 90% by weight of the composition, of a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can, in the absence of other adjuncts, form the balance of the composition.
  • the pharmaceutical composition of the present disclosure further comprises other additional components, for example therapeutic and/or prophylactic ingredients.
  • the invention thus relates in a further aspect to pharmaceutical composition comprising the compound of the present invention, one or more pharmaceutically acceptable carriers together with one or more other active agents.
  • the amount of other active agent present in the pharmaceutical composition is sufficient to provide an additional benefit either alone or in combination with the other ingredients in the composition.
  • these optional components may be categorized by their therapeutic or aesthetic benefit or their postulated mode of action. However, it is also understood that these optional components may, in some instances, provide more than one therapeutic or aesthetic benefit or operate via more than one mode of action. Therefore, classifications herein are made for the sake of convenience and are not intended to limit the component to that particular application or applications listed. Also, when applicable, the pharmaceutically-acceptable salts of the components are useful herein.
  • the dose of the compound may either be the same as or differ from that employed when the other additional components are not present. Appropriate doses will be readily appreciated by those skilled in the art.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration, e.g., local or systemic.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral, nasal, topical, transdermal, transmucosal, and rectal administration.
  • Oral and nasal administration include administration via inhalation.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, non-aqueous solutions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • Isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride can also be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, such as aluminum monostearate or gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the polynucleotide into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • suitable methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, methyl salicylate, or orange flavouring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch
  • a lubricant such as magnesium stearate or sterotes
  • a glidant such as colloidal silicon dioxide
  • Formulations suitable for administration by nasal inhalation include where the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns, which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Suitable formulations wherein the carrier is a liquid for administration by nebulizer include aqueous or oily solutions of the agent.
  • the agent(s) can also be delivered in the form of drops or an aerosol spray from a pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays, drops, or suppositories.
  • the active compound e.g., polynucleotides of the invention
  • the active compound are formulated into ointments, salves, gels, or creams, as generally known in the art.
  • compositions may be prepared by any of the method well known to a person skilled in pharmaceutical formulation. Generally, the compositions are prepared by contacting the insulin analog, molecule or compound uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation.
  • the insulin analogs, compounds or molecules of the invention can be formulated by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises a therapeutically effective amount of an insulin analog, peptide, compound or molecule according to the invention.
  • the content of the insulin analog, peptide, compound or molecule of the invention in a pharmaceutical composition of the invention is e.g. from about 0.1 to about 100% w/w of the pharmaceutical composition.
  • kits comprising one or more of the disclosed insulin analogs.
  • kits comprising one or more of the disclosed insulin analogs, peptides, compounds or pharmaceutical compositions.
  • the side chain protecting group of CysA7 and CysB7 was Acm
  • the side chain protecting group of CysA20 and CysB 19 was Trt
  • the side chain of CysAl l was Mmt protected
  • the side chain of CysA6 was S-i-Bu protected. Both chains were synthesized on a 0.1 mmol scale.
  • Cys, His, and Gla were coupled at 40 W with a maximum temperature of 50 C for 6 min.
  • Deprotection of the Fmoc group was performed with 20% piperidine containing 0.1 M HOBt in DMF in two stages (using a fresh reagent each time): with an initial deprotection of 2 min at 35 W followed by 5 min deprotection at 35 W with a maximum temperature of 60 C.
  • Con-Ins Gl chain A intramolecular disulfide bond formation, cleavage and purification.
  • the intramolecular disulfide bridge between CysA6 and CysAl l was formed on the resin using a non-oxidative method (Gaieri et al. 2005).
  • S-i-Bu of CysA6 was removed by reduction to liberate free thiol by treating the resin (760 mg) with 20% mercaptoethanol (ME) (Fluka) and 1% N-Methylmorpholine ( ⁇ ) in dimethylformamide (DMF) 8 mL overnight at room temperature.
  • ME mercaptoethanol
  • ⁇ N-Methylmorpholine
  • DMF dimethylformamide
  • the resin was treated with 1 % trifluoroacetic acid (TFA) in dichloromethane (DCM) 8 mL in the presence of 2 ⁇ ⁇ triisopropylsilane (TIS) as a scavenger for 20 min to deprotect CysAl l (Mmt) and to form the disulfide bridge between CysA6 and CysAl l at the same time.
  • TFS triisopropylsilane
  • the peptide was purified by preparative Waters HPLC (Milford, MA) on Waters PrepPak cartridge (2.5 x 10 cm) packed with Bondapak Qs (15-20 ⁇ particle size, 300 A) in solvent system A: 0.1% TFA/water, B: 0.1% TF A/40% water/60% ACN with a linear gradient ranging from 5% to 65% solvent B in 60 min at a flow rate 20 mL/min. 18.7 mg (7.7 ⁇ ) of chain A was obtained.
  • Chain A and chain B (7 ⁇ each) were dissolved together in 0.1 % TF A/water solution (7.1 mL) and added to a mixture of 14.5 mL DMSO, 14.25 mL water, 35.6 mL 0.2 M Tris containing 2 mM EDTA, pH 7.5.
  • the oxidation was monitored by analytical HPLC. After 25 h at room temperature, the reaction was quenched with 8% formic acid (1 mL), diluted with 0.1%TFA to a total volume of 225 mL and purified by preparative HPLC with a gradient ranged from 15 to 75%B in 60 min. 4.5 mg (0.85 ⁇ , 12.1% yield based on the starting amount of 7 ⁇ ) of heterodimer was obtained.
  • RP-HPLC quantitative RP-HPLC
  • capillary electrophoresis quantitative RP-HPLC
  • Quantitative RP-HPLC was performed using a GE Healthcare AKTApurifier 10 (Pittsburgh, PA) and a Phenomenex (Torrance, CA) Kinetex XB-C18 column (4.6 x 100 mm, 5.0 ⁇ particle size, 100 A pore size).
  • a gradient was performed from 20 to 80%B in 30 min at a flow rate of 1.0 mL/min. Detection was at 214 and 280 nm. The purity of the peptide was determined to be 89%.
  • Capillary electrophoresis was performed using a Groton Biosystems GPA 100 instrument. (Boxborough, MA) The electrophoresis buffer was 0.1 M sodium phosphate (15% acetonitrile), pH 2.5. Separation was accomplished by application of 20 kV to the capillary (0.75 ⁇ x 100 cm). Detection was at 214 nm. The assessed purity of the peptide was 80%.
  • Synthesis of sCon-Ins Gl [GluA4, ProB3, GluBlO] was performed as described for sCon-Ins Gl (Safavi- Hemami et al. (2015)).
  • sCon-Ins Gl [GluA4, ProB3, GluBlO]
  • sCon-Ins Gl[ GluA4 ] chain A cleavage, DTT reduction and purification.
  • the peptide was cleaved from 125 mg of resin for 1.5 h using 1 mL of enriched Reagent K (TFA/water/phenol/thioanisole/l,2-ethanedithiol, 82.5/5.0/5.0/2.5 by volume), which was prepared using 2 mL TFA (Fisher Scientific, Fair Lawn, NJ), 66 ⁇ L ⁇ H2O, 12 mg 2,2-dithiobis(5-nitropyridine) (DTNP; Aldrich; Saint Louis, MO), and 150 mg phenol, followed by addition of 25 ⁇ L ⁇ thioanisole.
  • enriched Reagent K TFA/water/phenol/thioanisole/l,2-ethanedithiol, 82.5/5.0/5.0/2.5 by volume
  • the cleavage mixture was filtered and precipitated with 10 mL of cold methyl-teri-butyl ether (MTBE; Fisher Scientific, Fair Lawn, NJ).
  • MTBE cold methyl-teri-butyl ether
  • the crude peptide was precipitated by centrifugation at 7,000 x g for 6 min and washed once with 10 mL cold MTBE.
  • the washed peptide pellet was dissolved in 50% ACN (Fisher Scientific; Fair Lawn, NJ) (vol/vol) in water and 2 mL of 100 mM dithiotreitol (DTT, EMD Chemicals, Gibbstown, NJ) in 1 mL 0.2 M Tris-HCl (Sigma, St Louis, MO) containing 2 mM EDTA (Mallinckrodt, St. Louis, MO), pH 7.5, 1 mL of water was added and vortexed gently, and the reaction was allowed to proceed for 2 h.
  • UV absorbance was measured at 220 and 280 nm to monitor the eluent. Purity of the peptide was assessed by analytical RP-HPLC on a CI 8 Vydac column (218TP54, 250 x 4.6 mm, 5 ⁇ particle size, Grace, Columbia, MD) using a linear gradient ranging from 10 to 40% of solvent B in 30 min with a flow rate 1 mL/min. The peptide was quantified by UV absorbance at 280 nm using an extinction coefficient ( ⁇ ) of 1,490 M _1 -cm _1 . From 135 mg of the resin, 3.8 mg of chain A was obtained.
  • the peptide was cleaved from 94 mg resin by a 3 h treatment with 1 mL of Reagent K and subsequently filtered, precipitated, and washed as described above.
  • the washed peptide pellet was purified as described above with the exception that the gradient ranged from 15 to 45% solvent B. The same gradient was used to assess the purity of the linear peptide as described above, and peptide quantitation was carried out using ⁇ value of 2,980 M _1 -cm _1 . From 94 mg of the cleaved resin, 2.37 mg of chain B was obtained.
  • the mass of the peptide was confirmed by ESI-MS (calculated monoisotopic MH +1 : 2,808.24, determined monoisotopic MH +1 : 2,808.25).
  • a total of 100 nmol of each chain was combined and dried using a SpeedVac.
  • the peptide mixture was dissolved in 100 of 0.1% TFA (vol/vol) and added to a mixture of 800 CuCl 2 H 2 0 (J.T. Baker, Phillipsburg, NJ) 100 1M Tris-HCl containing 10 nM EDTA, pH 7.5. The final peptide concentration was 100 ⁇ .
  • the reaction was left for 24 h at room temperature and then quenched with 8% formic acid (vol/vol), diluted with 0.1% TFA and purified by RP-HPLC using a preparative CI 8 Vydac column eluted with a linear gradient ranging from 15 to 45% of solvent B in 30 min at a flow rate 4 mL/min.
  • sCon-Ins Gl The purity of sCon-Ins Gl was assessed by analytical RP- HPLC using the same gradient as for the semi-preparative purification, at a flow rate 1 mL/min.
  • sCon-Ins Gl[GluA4, ProB3, GluBlO] was quantified at 280 nm using an ⁇ value of 4,470 M _1 -cm _1 .
  • the yield of the reaction was 28%. From 900 nmol of the 1 : 1 mixture of chain A and B, 1.36 mg of the desired product was obtained.
  • the identity of the peptide was confirmed by ESI-MS (calculated monoisotopic MH +1 : 5,278.15; determined monoisotopic MH +1 : 5278.15).
  • a solution of I 2 (Acros Organics, Geel, Belgium) was prepared as follows: 10 mg of 1 ⁇ 2 was added to 5 mL of ACN. After 20 min of stirring, the I2 was completely dissolved, and 15 mL of water and 600 uL of TFA were added. A total of 300 of the I2 mixture was added to 149 nmol (90% purity) and 106 nmol (72% purity) of partially folded sCon-Ins Gl[GluA4, Cys(Acm)7, ProB3, C(Acm)B7, GluB lO] dissolved in 300 ⁇ of 0.1% TFA each. Reactions were incubated for 5 min, quenched with 10 of 1 M L-ascorbic acid (Sigma, St.
  • sCon-Ins Gl [GluA4, ProB3, GluBlO]
  • the purity of the final product was assessed by analytical RP-HPLC on C18 Vydac column (218TP54, 250 x 4.6 mm, 5 ⁇ particle size) using the same gradient as for the semi- preparative purification, at a flow rate 1 mL/min, and was determined to be 97%.
  • sCon- Ins Gl [GluA4, ProB3, GluB lO] was quantified as described for the partially folded product.
  • hlns [DOI] is a monomeric analogue lacking residues 23 to 30 of the B chain of human insulin.
  • hIns[DOI] and Mns[TyrB15, TyrB20, DOI] were chemically synthesized, purified and oxidized following standard procedures.
  • Example 2 Con-Ins Gl Human Insulin Receptor Binding and Signalling Activation Insulin receptor binding .
  • Con-Ins Gl to bind to the human insulin receptor (hIR) was measured by a binding competition assay.
  • Competition binding assays were performed using solubilised immuno-captured human IR (isoform B) with europium-labelled human insulin and increasing concentrations of venom insulin as previously described (Denley et al. 2004).
  • Time -resolved fluorescence was measured using 340-nm excitation and 612-nm emission filters with a Polarstar Fluorimeter (BMGLab Technologies, Mornington, Australia).
  • IC5 0 values were calculated, using Prism 6, by curve-fitting with a non-linear regression (one-site) analysis. At least three assays were performed with three replicates per data point.
  • Akt phosphorylation analysis The ability of Con-Ins Gl to induce insulin signalling was assessed by Akt phosphorylation analysis. Briefly, pAkt Ser473 levels were measured in a mouse fibroblast cell line, NIH 3T3, overexpressing human IR-B. The cell line was cultured in DMEM with 10% fetal bovine serum (FBS), 100 U/mL penicillin- streptomycin and 2 ⁇ g/mL puromycin. For the assay, 40,000 cells per well were plated in a 96-well plates with culture media containing 1 % FBS. 24 h later, 50 ⁇ L ⁇ of insulin solution was pipetted into each well after the removal of the original media.
  • FBS fetal bovine serum
  • the insulin solution was removed and the HTRF pAkt Ser473 kit (Cisbio, Massachusetts, USA) was used to measure the intracellular level of pAkt Ser473. Briefly, the cells were first treated with cell lysis buffer (50 ⁇ L ⁇ per well) for 1 h under mild shaking. 16 ⁇ L ⁇ of cell lysate was then added to 4 ⁇ L ⁇ of detecting reagent in a white 384-well plate. After 4-h incubation, the plate was read in a Synergy Neo plate reader (BioTek, Vermont, USA) and the data processed according to the manufacturer's protocol. The assays were repeated for a total of four times. EC5 0 values were calculated (using Prism 6) by curve-fitting with a non-linear regression (one-site) analysis.
  • Our results highlight the existence within Con-Ins Gl of structural motifs that enable potent activity despite the venom protein's lack of an equivalent to either the canonical aromatic triplet or the B -chain C-terminal segment as a whole.
  • Example 3 Crystal Structure of Con-Ins Gl
  • Con-Ins Gl was synthesised as described above. Con-Ins Gl was prepared for crystallization in 10 mM HC1 at a concentration of 4 mg/mL.
  • a Numbers in parentheses refer to the outer resolution shell.
  • All monomer-monomer interfaces within the crystal are sparse, bar those formed between Con-Ins Gl monomers packed around the four-fold axis, each of which buries -440 A of molecular surface.
  • the four monomers coordinate an apparent sulphate ion lying close to the four-fold axis, which forms part of a charged- compensated cluster with the amides of GlyAl and a single side-chain carboxylate group of each GlaA4 ( Figure 5). Based on sedimentation equilibrium data below the inventors conclude that this association is an artefact of crystallization.
  • the hydrophobic core of Con-Ins Gl involves the side chains of residues ValA2, CysA6, CysAl l, PheA16, TyrA19, ArgB6, IleB l l, TyrB15 and LeuB 18 (Figure 4b). Of these, three are identical in human insulin (CysA6, CysAl l and TyrA19), three differ conservatively (ValA2 ⁇ Ile, IleB l l ⁇ Leu and LeuB 18 ⁇ Val) and three are markedly different (ArgB6— >Leu, PheA16— >Leu and TyrB 15— >Leu).
  • the inventors created a model of Con-Ins Gl bound to the elements of the human insulin receptor (hIR) that form the primary binding site for the hormone.
  • hIR human insulin receptor
  • Models of Con-Ins Gl in complex with the IR Ll-CR module (residues Gly5 to Lys310) and the IR aCT segment (residues Phe705 to Ser719 of the IR-A isoform) were created using MODELLER (v9.15) (Webb and Sali, (2014)) with the templates being the above crystal structure of Con-Ins Gl, the crystal structure of the IR site 1 components in complex with hins (PDB entry 40GA; Menting et al. 2014), and the NMR structure of the A-chain of insulin (PDB entry 2HIU; Hua et al. 1995).
  • Each system was solvated using the TIP3P water model in a cubic box extending 10 A beyond all atoms.
  • Sodium and chloride ions were added to neutralize the system and provide a final ionic strength of 0.1 M.
  • the protein and solvent (including ions) were coupled separately with velocity rescaling to a thermal bath at 300 K applied with a coupling time of 0.1 ps. All simulations were performed with a single non-bonded cut-off of 10 A and applying the Verlet neighbour searching cut-off scheme with a neighbour-list update frequency of 25 steps (50 fs); the time step used in all the simulations was 2 fs.
  • Periodic boundary conditions were used with the particle-mesh Ewald method used to account for long-range electrostatics, applying a grid width of 1.2 A and a sixth-order spline interpolation. All bond lengths were constrained using the P-LINCS algorithm. Simulations consisted of an initial minimization, followed by 50 ps of MD with all protein atoms restrained. Following positionally-restrained MD, MD simulations were continued for a further 10 ns applying positional restraints on the Ca atoms of the IR excluding the C-terminal residues of aCT (residues Val715 to Ser719). Following the Ca atom-restrained MD, the simulations were continued without restraints for a further 50 ns. The coordinates of the final model are provided in Appendix II.
  • Gl TyrB15 is rotated with respect to its conformation in our crystal structure in order to avoid steric clash with the hIR aCT residue Phe714.
  • the rotation directs the side chain of Con- Ins Gl TyrB 15 into the pocket occupied by hins PheB24 in the receptor complex, suggesting that Con-Ins Gl TyrB 15 is thus a surrogate for hins PheB24 in terms of receptor engagement (Figure 6).
  • Such rotation of the TyrB 15 side chain also permits the key hIR aCT residue Phe714 to engage the venom protein core ( Figure 6).
  • vertebrate insulins have leucine at position B 15, which is strictly conserved.
  • Con-Ins Gl TyrB20 The side chain of Con-Ins Gl TyrB20 is adjacent to that of Con-Ins Gl TyrB 15 and may also be involved in compensating for the lack of an equivalent to hInsPheB24.
  • crystallographic difference electron density associated with the TyrB15 side chain is somewhat poorly defined, compatible with such mobility ( Figure 7).
  • vertebrate insulins have a glycine at position B20, which is strictly conserved.
  • the PTM The PTM
  • Con-Ins Gl contains the following four post-translational modifications (PTMs): residues A4 and BIO are ⁇ -carboxyglutamates (Gla) as opposed to Glu and His (respectively) in hlns, residue B3 is hydroxyproline (Hyp) as opposed to Asn in hlns, and the A-chain C-terminal residue CysA20 is amidated ( Figure 1); note that the Con- Ins Gl B -chain numbering begins at -1 to allow comparison with hlns). Such modifications are commonly observed in conotoxins but have not been detected previously in insulins (Safavi-Hemami et al. 2015).
  • One side-chain carboxylate group of GlaB lO forms a hydrogen bond to the backbone amide of CysB7 and may play a role in stabilizing the B-chain N-terminal region; there is no equivalent interaction within hlns, hlns HisB lO being involved in hexamer formation ( Figure 4d).
  • the side-chain hydroxyl group of HypB3 is equivalently located to the side-chain amide oxygen of hlns AsnB3 and may be able to form a (long) H-bond to the backbone amide of SerA12 ( Figure 4e).
  • the C-terminal amide of CysA20 makes no interaction with the remainder of the venom protein.
  • Akt phosphorylation analysis was assessed by Akt phosphorylation analysis as described above. Briefly, pAkt Ser473 levels were measured in a mouse fibroblast cell line, NIH 3T3, overexpressing human IR-B. The cell line was cultured in DMEM with 10% fetal bovine serum (FBS), 100 U/mL penicillin-streptomycin and 2 ⁇ g/mL puromycin. For the assay, 40,000 cells per well were plated in a 96-well plates with culture media containing 1% FBS. 24 h later, 50 ⁇ L ⁇ of insulin solution was pipetted into each well after the removal of the original media.
  • FBS fetal bovine serum
  • the insulin solution was removed and the HTRF pAkt Ser473 kit (Cisbio, Massachusetts, USA) was used to measure the intracellular level of pAkt Ser473. Briefly, the cells were first treated with cell lysis buffer (50 ⁇ L ⁇ per well) for 1 h under mild shaking. 16 ⁇ of cell lysate was then added to 4 ⁇ L ⁇ of detecting reagent in a white 384-well plate. After 4-h incubation, the plate was read in a Synergy Neo plate reader (BioTek, Vermont, USA) and the data processed according to the manufacturer's protocol. The assays were repeated for a total of four times. EC5 0 values were calculated (using Prism 6) by curve-fitting with a non-linear regression (one-site) analysis.
  • Con-Ins Gl was diluted from a 10 mg/mL stock in 10 mM HC1 into 10 mM Tris, 50 mM NaCl, pH 7.4 to a final concentration of 100 ⁇ g/mL. An equal volume of 10 mM NaOH was added to neutralize any pH change. A total sample volume of 100 ⁇ L ⁇ was used.
  • the inventors also tested whether Zn , Ca , SO 4 " or PO 4 " altered the aggregation state of Con-Ins Gl ; in particular, in the case of Zn 2+ to test whether the ion might mediate Con-Ins Glmultimerization as it does for hins, and in the case of
  • Example 8 Crystal Structure of Con-Ins Gl in complex with human insulin receptor fragments that reconstitute the primary hormone binding site of the receptor
  • Site 1 The primary insulin binding site of the human insulin receptor (hIR) can be re-created in a domain minimized and suitable for crystallographic analysis of the interaction of insulin or insulin analogues with Site 1 (Menting et al. 2013) (Lawrence et al. 2016). Integral to this process is the further attachment of fragments of the monoclonal antibody 83-7 (Soos et al. 1986) to the CR domain of the receptor fragment to assist crystallization. This technique was used to generate crystals of Con- Ins Gl in co-complex with the elements that re-create hIR Site 1.
  • Con-Ins Gl was synthesised as described in Example 1. Con-Ins Gl was resuspended in 10 mM HC1.
  • MR construct IR310.T ⁇ i.e., residues 1-310 of MR followed by the N-terminal remnant Leu-Val-Pro-Arg of a thrombin cleavage site and inclusive of the population variant Tyrl44His
  • Fv83-7 the variable domain module of the monoclonal antibody 83-7 (Soos et al. 1986), was produced and purified as described in (Lawrence et al. 2016).
  • IR310.T was then complexed with Fv83-7 and the complex purified as described in Lawrence et al. 2016.
  • the Fv83-7.IR310.T complex was then subject to endoglycosidase H treatment as described in Lawrence et al. 2016. Peptide IR-A " of the A isoform of hIR was synthesized under contract by Genscript (USA).
  • Con-Ins Gl in complex with human insulin receptor fragments that reconstitute the primary hormone binding site of the receptor was prepared by combining EndoH- treated Fv83.7.IR310.T, IR-A 704"719 and Con-Ins Gl as shown in Table 2.
  • Crystallisation conditions were optimised and single crystals of the same protein:peptide:Con-Ins Gl mixture were grown using hanging drop format in Linbro 24 plates and a reservoir buffer consisting of 1.8 to 2.0 M ammonium sulfate or 1.8 to 2.0 M ammonium sulfate, 0.1 M Tris-HCl, pH 7.5.
  • Other buffers e.g. MOPS-NaOH pH 7.0 and MES-NaOH, pH 6.5
  • MOPS-NaOH pH 7.0 and MES-NaOH, pH 6.5 were also trialled and produced similarly diffracting crystals.
  • a Numbers in parentheses refer to the outer resolution shell.
  • Figure 13 shows an overlay of the structure of the Con-Ins Gl complex determined here with that of human insulin complexed with IR310.T, IR-A 704"719 and Fab 83-7, focussing on residue TyrB15 of Con-Ins Gl.
  • the side-chain of TyrB15 is rotated from its receptor-free position to be positioned in the same location that is occupied by that of hins PheB24 in the human insulin complex.
  • the complex structure supports the conclusion from Example 5 that TyrB 15 helps compensate for the lack of PheB24. No interpretable electron density is present for Con-Ins Gl TyrB20.
  • Example 10 - Molecular modelling of hlnsrPOH, hIns
  • Temperature coupling was conducted in 2 groups with the protein and solvent coupled independently to a velocity rescaling (Bussi et al. 2007) , thermostat at 300 K, both groups utilising a time constant of 0.1 ps.
  • Isotropic pressure coupling was implemented with the Berendsen (Berendsen et al. 1984) technique using a reference pressure of 1 bar and a time constant of 0.5 ps. All simulations were performed with a universal 12 A non-bonded interactions cut-off, with long-range electrostatics accounted for using the particle-mesh Ewald method (Essmann et al. 1995) with a grid width of 1.0 A and a sixth-order spline interpolation.
  • Verlet neighbour searching cut-off scheme was applied with a neighbour-list update frequency of 25 steps (50 fs); the time step used in all the simulations was 2 fs. All bond lengths were constrained with the P-LINCS algorithm (Hess, 2008). Simulations underwent an initial steepest decent minimization followed by 50 ps of MD with all protein atoms restrained. Following positionally restrained MD, MD simulations were continued for a further 100 ns
  • the initial comparative model included a restraint to ensure TyrB20 occupied the hInsB24 binding site. Following 100 ns of MD TyrB20 remained in the hlns B24 binding site, with all other interactions with the receptor appearing native-like. Unlike hIns[DOI], the native ⁇ - ⁇ parallel displaced stacking between TyrB 16 and IR LI Phe39 was maintained, however, the salt bridge caused by the lack of B-chain C-terminal residues between ArgB22 and GluA17 was the same as that observed with hIns[DOI]. The final model is shown in Figure 16. FoldX mutational position scan analysis
  • the FoldX position scan utility provides a qualitative interpretation as to the positions within hIns[DOI] that can accommodate mutations, and those that cannot ( Figures 17, 18 and 19).
  • the solvent- exposed residues within the A chain particularly residues GluA4, GlnA5 and ThrA8 indicate a little net positive or negative effect to most mutations. This is dissimilar to that of the hIns[TyrB15, DOI] analogue which instead proposed a positive impact on mutation of these residues.
  • the MD simulations of the hlns analogues, hIns[DOI], Mns[TyrB 15, DOI] and hIns[TyrB20, DOI], in complex with the IR LI domain and IR-A 705- " 71 and subsequent mutational analysis provide insight as to the method hlns [DOI] binds despite the lack of key receptor engagement residues, particularly PheB24.
  • the simulations indicate that the tyrosine substitution at position B20 can act as a substitute for PheB24 in hlns, occupying approximately the same location, and is stable over the simulated time.
  • Con-Ins-Gl a synthetic analogue
  • sCon-Ins-Gl induces hypoglycemic shock when it is injected into fish, and it slows fish motility when it is present in the water.
  • the most special feature of Con-Ins-Gl is that it is the shortest insulin molecule reported to date with a "shortened" B chain. Because a shortened human insulin (des- octapeptide insulin, DOI) is monomeric, it indicated that Con-Ins-Gl is monomeric and can be used as an UFI (ultra-fast acting insulin).
  • Con-Ins-Gl lacks two segments that in human insulin are involved in binding to with the human insulin receptor (MR): First, A21 Asn of human insulin contacts hIR binding site 1 and its removal causes a 100- fold reduction in binding affinity. Second, the aromatic triplet (B24-B26) is one element for human insulin to bind hIR binding through contacts at hIR binding site 1. Removal of these residues leads to a 1 ,000-fold reduction in affinity.
  • MR human insulin receptor
  • Con-Ins-Gl (instead of the selenium analogue) was chemically synthesized and it was found that it binds to hIR with only 30-fold less affinity than human insulin. This surprising result raised a key question: how does Con- Ins-Gl bind to hIR without the key aromatic residues used by human insulin?
  • the structure of Con-Ins-Gl was found to display a nearly identical backbone as human insulin.
  • Con-Ins-Gl B15 Tyr and B20 Tyr (Leu and Gly in human insulin) interact with human IR to substitute for the role played by human B24 Phe.
  • UFI ultra-fast acting insulin
  • the fundamental challenge in redesigning human insulin is that the same residues involved in receptor binding also mediate dimer formation.
  • Con-Ins-Gl represents an important step forward in the creation of a monomeric, ultrafast-acting insulin because it lacks these residues (and thus does not dimerize) but retains the ability to bind and activate the insulin receptor.
  • the low sequence identity between Con-Ins-Gl and human insulin could give rise to an immune response, especially given that diabetes is a chronic disease that requires daily insulin injections.
  • DOI Des-octapeptide (B23-30) human insulin
  • B23-30 human insulin
  • DOI Des-octapeptide (B23-30) human insulin
  • Con- Ins-Gl uses the B15 Tyr and/or B20 Tyr to compensate for the loss of B24 Phe, and further indicate additional modifications that enhance the affinity of Con-Ins-Gl. Leveraging these insights, DOI can be developed into an active UFI analogue as a therapeutic lead for diabetes treatment.
  • DOI was synthesized enzymatically by trypsin cleavage of human insulin, which is not suitable for analogue synthesis. Therefore, a modular synthetic route to access DOI has been developed.
  • the primary challenge for the synthesis of human insuin is the hydrophobic character of the A chain.
  • an isoacyl peptide pair on the A8-A9 Thr-Ser an extra charged residue (amine) was introduced to the A chain to increase its solubility (Figure 21).
  • the isoacyl peptide underwent an O-to-N acyl shift at pH 8 to yield the DOI sequence.
  • This synthetic DOI has the same molecular weight (from MALDI) and hIR activation activity as the enzymatically synthetic DOI, which proves the reliability of the developed method.
  • the biphenyl analogue is 10% of the potency of human insulin (3 -fold higher than N10E, B20Y DOI). This demonstrates the power of the interdisciplinary approach using both protein engineering and structural biology.
  • the potency of DOI has been increased by 100-fold by mutating two positions.
  • Halogen-substituted naphthyl and biphenyl groups on B20 can be used to further optimize DOI analogue potency. Because the A8 position is important for interacting with hIR binding site 2, the A8 His mutation can be introduced into the current lead analogue and assay for hIR activation.
  • UFI analogue serum levels will be measured using HPLC coupled with mass spectrometry (LC/MS/MS) in diabetic mice after subcutaneous injections to measure its absorption rate (using insulin lispro as a control). For monomeric insulins, a faster absorption rate can be seen compared to the dimeric insulin lispro.
  • glycemic clamp experiments can be used to quantify the onset and duration of UFI analogues in vivo by determining the amount of glucose infusion required to maintain a targeted glucose level.
  • the glucose clamp study can show that UFI analogues have a shorter onset and duration of action due to their reduced depot effects in subcutaneous tissue. The combination of these properties can greatly reduce the risk of hypoglycemia.
  • HG12:VAL:A:3:15.144:46.176:34.323:28.02 CA:CYS:A:7:16.186:47.837:29.839:21.74
  • CD2:CGU:A:4:18.45:40.98:36.25:48.38 O:HIS:A:8:21.558:48.179:30.604:34.16
  • CB:HIS:A:8:19.847:46.811:32.972:27.96 SG:CYS:A:11:15.186:43.358:24.401:23.25
  • HZ2 LYS:A:17:10.081:39.061:15.511:32.34 HN11:CY3:A:20:4.569:36.191:27.039:51.9
  • CD1:PHE:B:0:20.187:43.786:12.753:26.31 CA:LYS:B:4:16.899:49.058:21.865:26.28

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Abstract

La présente invention concerne des analogues de l'insuline, en particulier des analogues de l'insuline présentant des chaînes B raccourcies. La présente invention concerne également la structure cristalline de l'insuline provenant du venin d'escargots marins, ainsi que des procédés d'utilisation du cristal et des informations structurales associées pour cribler et concevoir des analogues d'insuline qui interagissent avec ou modulent le récepteur de l'insuline. La présente invention concerne également des procédés thérapeutiques et prophylactiques utilisant les analogues de l'insuline.
PCT/AU2017/050758 2016-04-07 2017-07-21 Analogues de l'insuline Ceased WO2018014091A1 (fr)

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CN202310855095.4A CN116874584A (zh) 2016-07-22 2017-07-21 胰岛素类似物
BR112019000991A BR112019000991A2 (pt) 2016-07-22 2017-07-21 análogo da insulina, composição farmacêutica, método para tratar e/ou prevenir uma afeção relacionada com insulina, método para decrescer níveis de glicose no sangue, uso do análogo da insulina, método de replanejamento ou modificação de um polipeptídeo que é conhecido por se ligar a um receptor da insulina, polipeptídeo, molécula isolada, método para identificar um composto que se liga ao receptor de insulina, método de base computacional para identificar um composto que imita a atividade da insulina, composto identificado usando um método, cristal do polipeptídeo, estrutura do polipeptídeo, uso da estrutura, uso do modelo estrutural, peptídeo, método para aumentar a ativação do receptor da insulina em um sujeito, método para diminuir o açúcar no sangue em um sujeito, método para tratar diabetes tipo 1 em um sujeito e proteína terapêutica
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JP2021530210A (ja) * 2018-06-29 2021-11-11 アクストン バイオサイエンシズ コーポレーションAkston Biosciences Corporation 超長期作用性インスリン−ｆｃ融合タンパク質および使用方法
JP7417277B2 (ja) 2018-06-29 2024-01-18 アクストン バイオサイエンシズ コーポレーション 超長期作用性インスリン－ｆｃ融合タンパク質および使用方法
JP2024045117A (ja) * 2018-06-29 2024-04-02 アクストン バイオサイエンシズ コーポレーション 超長期作用性インスリン－ｆｃ融合タンパク質および使用方法
JP7510728B2 (ja) 2018-06-29 2024-07-04 アクストン バイオサイエンシズ コーポレーション 超長期作用性インスリン－ｆｃ融合タンパク質および使用方法
CN114675019A (zh) * 2022-02-10 2022-06-28 江苏省人民医院（南京医科大学第一附属医院） 一种检测胰岛素受体胞外段抗体的试剂盒
CN114675019B (zh) * 2022-02-10 2022-12-16 江苏省人民医院（南京医科大学第一附属医院） 一种检测胰岛素受体胞外段抗体的试剂盒

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