WO2016172269A2 - Analogues d'insuline ayant des peptides à chaîne b raccourcie et procédés associés - Google Patents

Analogues d'insuline ayant des peptides à chaîne b raccourcie et procédés associés Download PDF

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WO2016172269A2
WO2016172269A2 PCT/US2016/028526 US2016028526W WO2016172269A2 WO 2016172269 A2 WO2016172269 A2 WO 2016172269A2 US 2016028526 W US2016028526 W US 2016028526W WO 2016172269 A2 WO2016172269 A2 WO 2016172269A2
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cys
absent
val
leu
glu
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WO2016172269A3 (fr
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Helena SAFAVI-HEMAMI
Baldomero M. Olivera
Joanna Gajewiak
Santhosh KARANTH
Amnon SCHLEGEL
Pradip K. Bandyopadhyay
Mark Yandell
Samuel D. ROBINSON
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University Of Utah Research Foundation
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • venomous cone snails More than 100 species of venomous cone snails (genus Conus) are highly effective predators of fish.
  • the vast majority of venom components identified and functionally characterized to date are neurotoxins specifically targeted to receptors, ion channels, and transporters in the nervous system of prey, predators, or
  • bioactive venom components are small disulfide-rich peptides termed conotoxins, which target specific receptors and ion channel subtypes located in the prey's nervous system.
  • mRNAs messenger RNAs
  • Conotoxin precursors exhibit a characteristic primary structure: a hydrophobic signal peptide (prepeptide) sequence, followed by a propeptide region and commonly a cysteine-rich mature peptide region.
  • prepeptide signal peptide sequence
  • the signal sequence of a precursor peptide is responsible for targeting it to the cellular secretory pathway, but is removed prior to secretion of the mature peptide.
  • Conotoxins can be classified into gene superfamilies according to this signal peptide sequence.
  • Members of a conotoxin superfamily share a high percentage of sequence identity in their signal peptide sequence but less so in their pro-peptide sequence, and can be highly variable in their mature peptide sequence, (often with the exception of the cysteine framework).
  • FIG. 1 A is a graphical representation of data according to an example embodiment
  • FIG. IB is a graphical representation of data according to an example embodiment
  • FIG. 1C is a graphical representation of data according to an example embodiment
  • FIG. ID is a graphical representation of data according to an example embodiment
  • FIG. IE is a graphical representation of data according to an example embodiment
  • FIG. IF is a graphical representation of data according to an example embodiment
  • FIG. 2 is a graphical representation of mass spec data according to an example embodiment
  • FIG. 3 A is a graphical representation of experimental data according to an example embodiment
  • FIG. 3B is a graphical representation of experimental data according to an example embodiment
  • FIG. 4A is a graphical representation of an insulin analog sequence according to an example embodiment
  • FIG. 4B is a graphical representation of HPLC data according to an example embodiment
  • FIG. 5A is a graphical representation of an insulin analog sequence according to an example embodiment
  • FIG. 5B is a graphical representation of HPLC data according to an example embodiment
  • FIG. 6A is a graphical representation of data according to an example embodiment
  • FIG. 6B is a graphical representation of data according to an example embodiment
  • FIG. 7A is a graphical representation of peptide sequences according to an example embodiment
  • FIG. 7B is a graphical representation of data according to an example embodiment
  • FIG. 7C is a graphical representation of data according to an example embodiment.
  • FIG. 7D is a graphical representation of data according to an example embodiment.
  • the term “substantially” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result.
  • an object that is “substantially” enclosed would mean that the object is either completely enclosed or nearly completely enclosed.
  • Insulin is amongst the most versatile hormones described to date, which plays central physiological roles in regulating glucose metabolism, reproduction, cognition, and the like. Vertebrate insulin is versatile hormone that is synthesized in pancreatic ⁇ cells, and is the key hormone regulator of carbohydrate and fat metabolism. In the brain, insulin functions as a neuromodulator of energy homeostasis and cognition. Insulin is initially synthesized as a precursor comprising three regions, A, B, and C, from which proteolytic cleavage of the C peptide in the Golgi releases the mature insulin heterodimer with an A and B chain connected by disulfide bonds.
  • insulin analog formulations are available for use that differ in their onset and length of action. Rapid-acting insulins, for example, have a fast onset of activity, and are thus typically given before meals. Structurally, these insulin analogs differ from normal human insulin by having amino acid substitutions that are deleterious to insulin
  • Human insulin is a protein complex that includes two chains, an A chain (21 residues) and a B chain (30 residues), which are cross-linked by two disulfide bridges, with a third, intra-chain disulfide bridge occurring within the A chain. Residues at the C-terminus of the B chain promote insulin dimerization through an anti-parallel ⁇ - strand interaction.
  • insulin is stored as a hexamer comprising three insulin dimers held together by two central zinc ions that coordinate a histidine at position BIO of each monomer. Hexamer-to-monomer conversion is thus crucial to the bioavailability of insulin in many treatment regimes. Rapid-acting insulin analogs have reduced rates of self-association, and are more readily absorbed after
  • a lysine and proline at position B28 and B29 are reversed, creating steric hindrance and a reduced ability to self-associate.
  • the present technology provides various insulin analogs that have modified B chains lacking the aromatic triplet, and have, in most cases, shorter B chains compared to previously described insulins.
  • Portions of the presently described insulins have some similarity to insulin peptide found in the venom of fish-hunting cone snails.
  • two fish-hunting cone snails, Conus geographus and Conus tulipa have evolved specialized insulins that are expressed as components of their venoms, and that appear to be targeting prey energy metabolism.
  • these venom insulins When injected into fish, these venom insulins elicit hypoglycemic shock, a condition characterized by low blood glucose.
  • Con-Ins Gl (A chain, SEQ ID NO: 001; B chain SEQ ID NO: 002) is a specialized C. geographus insulin, having characteristic conotoxin amino acid modifications of a ⁇ -carboxyglutamate (Gla) at the A04 and B 12 positions of the A and B chains, and a hydroxyproline (Hyp) at the B05 position of the B chain, as well as an amidated cysteine (*) at the terminal end of the A chain.
  • Gla ⁇ -carboxyglutamate
  • Hyp hydroxyproline
  • insulin analogs can include an A chain peptide and a B chain peptide bonded together across at least one pair of Cys residues.
  • the A chain peptide and the B chain peptide can be bonded together across multiple pairs of Cys residues.
  • Such bonding can be a disulfide bridge, or any other known and useful bridge-type bond.
  • Such bonding can be accomplished across Cys residues, modified Cys residues, or other compatible amino acid or molecule with sufficient similarity to Cys so as to not destroy the functionality of the insulin analog.
  • Non-limiting examples of such bonds can include dicarba, lactam, diselenide, triazole, and the like, including combinations thereof.
  • an insulin analog can include an intra-chain disulfide bridge occurring within one of the peptide chains, which in some cases is the A chain.
  • the A chain peptide and the B chain peptide can be linked together at one or more terminal ends.
  • the insulin analog is acyclic
  • derivatives of the insulin analogs can be cyclic, while retaining the Cys-bridging pattern described.
  • the insulin analog has an amide 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.
  • residues or groups of residues known to the skilled artisan to improve stability can be added to the C-terminus and/or N-terminus.
  • residues or groups of residues known to the skilled artisan 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 additionally contemplated for attachment to either the C- or N- terminus.
  • the A chain peptide can include the sequence Gly-XA2-XA3-
  • XA5 Glu, His or Val; Cys A 6, Cys A 7, and CA I I are independently Cys or
  • XA 8 His, Asp, Gin, Tyr, Lys or Val
  • XA 9 Arg, Asn, His or Lys
  • X A i 8 Lys, Thr, Asn, Gin or Glu
  • XA I9 Tyr or Phe
  • XA24 Arg, Thr, Met, Gin, Leu or is absent
  • XA25 Glu, Gly or is absent
  • from XA26 Ser, Leu or is absent
  • XA27 to XA3 I are
  • X A 32 Ala, Ser or is absent
  • X A 33 Ala, Val or is absent
  • XA34 Ala or is absent.
  • the A chain peptide can include the sequence Gly-Val-
  • XA 8 His, Tyr or Lys
  • XA I O Pro or Ala
  • ⁇ ⁇ Lys or Met
  • XA I8 Lys or Gin
  • XA I 9 Tyr or Phe
  • CysA2o Cys, selenocysteine, amidated Cys, or amidated selenocysteine
  • XA2 I Ser, Gly or is absent
  • XA22 Asn or is absent
  • XA23 Ser or is absent.
  • the A chain peptide can include the sequence Gly-Ile-
  • XA9 Asn or Lys
  • XA I O Tyr, Ser, Phe, -His or Thr
  • XA B Asn or Asp
  • XA I 4 A, Gin, Asp or -Glu
  • XA1 ⁇ 2 Phe or A
  • ⁇ ⁇ Arg, Met, Thr or Ser
  • XA I S Lys, Gin or - Glu
  • CysA2o Cys, selenocysteine, amidated Cys, or amidated selenocysteine
  • XA2 I Pro, His, Ser, Ala, or is absent
  • XA 2 2 Pro, Thr, Leu, Ser or is absent
  • XA23 Thr, Leu, Val, or is absent
  • X A 24 Arg, Thr, Met, Gin, Leu or is absent
  • X A 25 Glu, Gly or is absent
  • from XA26 Ser, Leu or is absent
  • XA27 to XA32 are independently Ser or is
  • the B chain peptide for example, can include the sequence X BI -X B 2-X B 3-X B 4-
  • the B chain peptide can include the sequence Xei-Ser- Phe-Gly-Ser-XB6-His-XB 8 -CySB9-XBio-Pro-XBi2-XBi 3 -XBi4-XBi5-XBi6-XBi7-XBi 8 -XBi9-
  • an A chain peptide can include a sequence selected from the following sequences. It is noted that a "_" is a space indicator that is intended to facilitated alignment of residues.
  • a B chain peptide can include a sequence selected from the following. It is noted that a "_" is a space indicator that is intended to facilitated alignment of residues.
  • the insulin analogs can include various amino acid modifications, some of which are indicated with the sequences herein.
  • one or more Glu residues can be replaced with ⁇ -carboxyglutamate (Gla), for example at the A04 or A05 Glu positions of various A chains, or at the B 12 Glu position of some B chains (the position may vary, depending on the sequence).
  • one or more Pro residues can be replaced with hydroxyproline (Hyp), for example at the B05 Pro position of certain B chains.
  • C- terminal ends can be amidated, such as an amidated Cys (*) at the terminal end of various A chains, among others.
  • an insulin analog chimera including a native human insulin or modified human insulin A chain peptide and a synthetic B chain peptide.
  • a native human A chain peptide can include the sequence of Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys- Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO: 056).
  • the synthetic B chain peptide can include any of the B chain peptides disclosed herein that compliment, and therefore provide insulin functionality with, the native human A chain peptide.
  • the synthetic B chain peptide can include the sequence of SEQ ID NO: 008.
  • Other example B chain peptides include SEQ ID NO: 006, SEQ ID NO: 007, SEQ ID NO: 009-011, and SEQ ID NO: 0034- 0055.
  • an insulin analog chimera including a human insulin analog A chain peptide and a synthetic B chain peptide.
  • the synthetic B chain peptide can include any of the B chain peptides disclosed herein that compliment, and therefore provide insulin functionality with, the human insulin analog A chain peptide.
  • the synthetic B chain peptide can include the sequence of SEQ ID NO: 008.
  • Other example B chain peptides include SEQ ID NO: 006, SEQ ID NO: 007, SEQ ID NO: 009-011, and SEQ ID NO: 0034-0055.
  • an insulin analog chimera including a cone snail insulin analog A chain peptide and a synthetic B chain peptide derived from a modified human insulin B chain.
  • a cone snail insulin analog A chain peptide can include the sequence SEQ ID NO: 001.
  • a cone snail insulin analog A chain peptide can include the sequence SEQ ID NO: 012.
  • the cone snail insulin analog A chain peptide can include a sequence selected from SEQ ID NO : 003-005 or SEQ ID NO: 013-033.
  • the synthetic B chain peptide derived from a modified human insulin B chain can include any modified human B chain sequence that results in an active insulin analog.
  • Insulin analog peptides can be made by any known technique or method, and any of such techniques or methods are considered to be within the present scope.
  • Example techniques can include peptide synthesis, recombinant expression, a combination of peptide synthesis and recombinant expression, and the like. Example techniques are described further in the Examples section.
  • Various insulin analog formulations are provided for the treatment of insulin- related conditions such as diabetes myelitis, hyperglycemia, and the like, as well as for diabetes drug discovery and for other research activities.
  • a formulation can generally include any independent combination of A chain peptide sequence and B chain peptide sequence described herein, including equivalents thereof.
  • the formulation can be a soluble monomeric insulin analog.
  • the formulation can be at least partially in a multimeric configuration.
  • Formulations can generally be for subcutaneous or other parenteral injection, for continuous or intermittent infusion, or any other available administration route.
  • Such formulations can comprise an insulin analog as described herein, including pharmaceutically acceptable salt of the insulin analog, in a pharmaceutical carrier.
  • the formulation can independently include various preservatives, stabilizing agents, isotonicity agents, solubilizers, metal ions, and the like, including combinations thereof.
  • Such ingredients are generally well known in the art.
  • Pharmaceutical compositions can generally be prepared according to conventional pharmaceutical techniques. See, for example, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, 2005.
  • any pharmaceutical carrier that can be used in an insulin analog formulation is considered to be within the present scope.
  • Non-limiting examples of carriers can include sodium phosphate, sodium acetate, sodium citrate, TRIS, arginine, such as L- arginine, and the like, including combinations thereof.
  • the pH of the formulations is controlled by the buffering of the pharmaceutical carrier.
  • the concentration of the buffering component of the carrier can be such as to provide adequate buffering of the pH during storage, which is well known to those skilled in the art.
  • TRIS refers to 2-amino-2-hydroxymethyl-l, 3, -propanediol, and to any pharmacologically acceptable salt thereof.
  • the free base and the hydrochloride form are two common forms of TRIS.
  • TRIS is also known in the art as trimethylol aminomethane, tromethamine, and tris(hydroxymethyl)aminomethane.
  • An optional isotonicity agent is a compound that is physiologically tolerated, and that imparts a suitable tonicity to a formulation to prevent or otherwise limit the net flow of water across cell membranes that are in contact with the formulation.
  • Compounds such as glycerin, for example, are commonly used, concentrations of which are well known.
  • Other potential isotonicity agents include salts, such as sodium chloride, dextrose, lactose, and the like.
  • salt denotes acidic and/or basic salts, formed with inorganic or organic acids and/or bases, preferably basic salts.
  • salts of these compounds 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 art-recognized techniques.
  • the insulin analog is preferably administered in a therapeutically effective amount.
  • a therapeutically effective amount or simply “effective amount” of the insulin analog is meant a sufficient amount of the compound to treat the desired condition at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or specialists, and typically takes account of the nature of the insulin disorder, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.
  • a strategy incorporating a pair of A chain selenocysteines was used to synthesize Con-Ins Gl (A chain SEQ ID NO: 012) (B chain SEQ ID NO: 034).
  • Diselenide bond-containing peptide analogs have similar biological activities to their native peptides and, in some cases, even improved potency or selectivity. Due to the lower redox potential, diselenide bond formation is favored over the disulfide bond formation under acidic conditions.
  • This Cys-to-Sec replacement strategy was combined with orthogonal protection of the remaining two pairs of cysteines to sequentially form intra- and intermolecular disulfide bridges, as shown in FIGs. 1 A-F. CysA6 and CysAl 1 were replaced with Sec residues, which formed an intradiselenide bridge after peptide cleavage and reduction (FIG. 1 A).
  • CysA20 and CysB21 of the respective chains were used to form the first intermolecular disulfide bridge on DMSO treatment (FIG. 1C).
  • sCon-Ins Gl Gly-Val-Val-Glu-His-Sec-Cys-His-Arg- Pro-Sec-Ser-Asn-Ala-Glu-Phe-Lys-Lys-Tyr-Cys (A chain SEQ ID NO: 062) (B chain SEQ ID NO: 034), in a total yield of 10.5%, based on the starting amount of purified chain A.
  • RP-HPLC confirmed the purity (95%), and electrospray ionization MS sequencing confirmed the correct identity of the product (FIG. 2).
  • FIGs. 1A-F show synthesis of sCon-Ins Gl .
  • A Purified chain A with the intramolecular Sec-Sec bridge formed and Cys-7 protected with the Acm group.
  • B Purified chain B with Cys-9 protected with the Acm group.
  • C sCon-Ins Gl heterodimer formation by treatment with 20% DMSO for 30 h in 0.1 M Tris HCl containing 1 mM EDTA, pH 7.5.
  • D Purified sCon-Ins Gl heterodimer.
  • E Purified sCon-Ins Gl heterodimer.
  • FIG. 2 shows MS analysis of synthetic sCon-Ins Gl .
  • the integrity of synthetic sCon-Ins Gl was determined by electrospray ionization MS at the Salk Institute for Biological Studies, La Jolla, CA.
  • the monoisotopic MH+1 ion was 5238.1004.
  • the inset shows the isotopic distribution.
  • sCon-Ins Gl Lowers Blood Glucose and Alters swimming Activity in Fish.
  • the streptozotocin (STZ)-induced model of hyperglycemia was used to assess whether sCon-Ins Gl could effectively lower blood glucose levels in a model prey: adult zebrafish.
  • Animals were first rendered hyperglycemic through i.p. injection of the ⁇ -cell poison STZ (1.5 g/kg), and the effect of subsequent injection of sCon-Ins Gl was examined.
  • FIGs. 3 A-B show that sCon-Ins Gl lowers blood glucose and disrupts swimming behavior in zebrafish.
  • sCon-Ins Gl (A chain SEQ ID NO: 062) (B chain SEQ ID NO: 034) containing Cys A6 U to Sec A6 U modifications in the A chain was chemically synthesized, purified and oxidized as described above with the exception that corrected extinction coefficients were used for the quantification of the B chain (2,980 M _1 -cm _1 ) and fully oxidized sCon-Ins Gl (4,470 M ⁇ crn -1 ). Synthesis of sCon-Ins Gl (A chain SEQ ID NO: 062) (B chain SEQ ID NO: 034) containing Cys A6 U to Sec A6 U modifications in the A chain was chemically synthesized, purified and oxidized as described above with the exception that corrected extinction coefficients were used for the quantification of the B chain (2,980 M _1 -cm _1 ) and fully oxidized sCon-Ins Gl (4,470 M ⁇ crn -1 ). Synthesis of sCon-Ins
  • sCon-Ins G1[ A4 E] 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.
  • Reagent K was prepared using 2 mL TFA (Fisher Scientific, Fair Lawn, NJ), 66 H 2 0, 12 mg 2,2-dithiobis(5-nitropyridine) (DTNP; Aldrich; Saint Louis, MO), and 150 mg phenol, followed by addition of 25 [iL thioanisole.
  • the cleavage mixture was filtered and precipitated with 10 mL of cold methyl -tert-butyl ether (MTBE; Fisher Scientific, Fair Lawn, NJ).
  • 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% acetonitrile (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 (Malinckrodt, St.
  • the HPLC solvents were 0.1% (vol/vol) TFA in water (solvent A) and 0.1% TFA (vol/vol) in 90% aqueous ACN (vol/vol) (solvent B).
  • the eluent was monitored by measuring absorbance at 220 and 280 nm.
  • Purity of the peptide was assessed by analytical C18 Vydac RP-HPLC (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 .
  • the peptide was cleaved from 94 mg resin by a 3-h treatment with 1 mL of Reagent K (TF A/water/ phenol/ thioanisole/l,2-ethanedithiol 82.5/5/5/2.5 by volume) 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 Da, determined monoisotopic MH +1 :
  • a total of 100 nmol of each chain was combined and dried using a SpeedVac.
  • the peptide mixture was dissolved in 100 [iL 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 ⁇ .
  • 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 C18 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.
  • 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.
  • Iodine (I 2) assisted formation of fully folded sCon-Ins Gl[ E; P; E]
  • a solution of I 2 (Acros Organics, Geel, Belgium) was prepared as follows: 10 mg of I 2 was added to 5 mL of ACN. After 20 min of stirring, the I 2 was completely dissolved, and 15 mL of water and 600 ⁇ of TFA were added. A total of 300 ⁇ of the I 2 mixture was added to 149 nmol (90% purity) and 106 nmol (72% purity) of sCon-Ins G1 [ A4 E; B5 P; B12 E] 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.
  • FIG. 4 shows sequence and HPLC profile of fully oxidized sCon-Ins
  • Arg was always double coupled at room temperature for 25 minutes then at 15 W with a maximum temperature of 50 °C for 12 min. 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 minutes at 35 W followed by 5 min deprotection at 35 W with a maximum temperature of 60 °C.
  • Con-Ins Gl chain A cleavage, intramolecular disulfide bond formation and purification
  • the intramolecular disulfide bridge between A6 Cys and A11 Cys was formed on the resin using a non-oxidative method.
  • S-t-Bu of A6 Cys was removed by reduction to liberate free thiol by treating the resin (760 mg) with 20%
  • ME mercaptoethanol
  • NMM N-Methylmorpholine
  • the resin was treated with 1% trifluoroacetic acid (TFA) in dichloromethane (DCM) 8 mL in the presence of 2 iL triisopropylsilane (TIS) as a scavenger for 20 minutes to deprotect A11 Cys(Mmt) and to form the disulfide bridge between A6 Cys and A11 Cys at the same time.
  • TIS triisopropylsilane
  • Chain A and chain B (7 ⁇ each) were dissolved together in 0.1%>
  • the purity of the peptide was determined to be 89% (FIG. 5).
  • Capillary electrophoresis (CE) 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%.
  • FIG. 5 shows sequence and HPLC profile of fully oxidized Con-Ins Gl .
  • HPLC conditions C18 Vydac RP-HPLC column, linear gradient ranging from 15% to 45% of solvent B in 30 min with I niL / min flow rate monitored at 220 nm.
  • pAkt Ser473 levels were measured in a mouse fibroblast cell line, NIH 3T3, overexpressed with human IR-B.
  • the cell line was cultured in DMEM with 10% FBS, pen/strep and 2ug/mL puromycin.
  • 40,000 cells per well were plated in a 96-well plates with culture media containing 1% FBS. 24 hours later, 50uL of insulin solution was pipetted into each well after the removal of the original media. After a 30-min treatment, the insulin solution was aspirated and the HTRF pAkt Ser473 kit (Cisbio, Massachusetts, USA) was used to measure the intracellular level of pAkt Ser473.
  • the cells were first treated with cell lysis buffer (50uL per well) for 1 hour under mild shaking. 16uL of cell lysate was then added to 4uL of detecting reagent in a white 384-well plate. After 4-hour incubation, the plate was read in a Synergy Neo plate reader (Biotek, Vermont, USA). The data was processed according to the manufacturer's protocol. Cone snail insulin vs. mammalian insulin receptor
  • Insulin signaling activation assay To determine the extent of insulin signaling induced by conus insulin molecules, pAkt Ser473 levels were measured in a mouse fibroblast cell line, NIH 3T3, overexpressed with human IR-B. The cell line was cultured in DMEM with 10% FBS, pen/strep and 2ug/mL puromycin. For the assay, 40,000 cells per well were plated in a 96-well plates with culture media containing 1% FBS. 24 hours later, 50uL of insulin solution was pipetted into each well after the removal of the original media.
  • the insulin solution was aspirated 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 (50uL per well) for 1 hour under mild shaking. 16uL of cell lysate was then added to 4uL of detecting reagent in a white 384-well plate. After 4-hour incubation, the plate was read in a Synergy Neo plate reader (Biotek, Vermont, USA). The data was processed according to the manufacturer's protocol. Results are shown in FIG. 6A. It is clear from the data that the cone snail insulin activates the human insulin signaling pathway.
  • Con-InsGl The association state of Con-InsGl in solution was assessed at 100 ⁇ g/ml using sedimentation equilibrium analysis at 30,000 and 45,000 rpm. The data are well described by a Con-InsGl being a single sedimenting species of apparent MW 5380 ⁇ 55 g/mol (FIG. 7C). Based on a calculated theoretical mass of 5143, it is concluded that Con-InsGl is overwhelmingly monomeric in solution, with at most 5% possibly being dimeric.
  • FIG. 7 Characterization of Con-Ins Gl .
  • A Sequence comparison with human insulin, ⁇ -: ⁇ — carboxylated-glutamate, O: hydroxyproline, *: C-terminal amidation.
  • B Competition binding analysis of Con-Ins Gl against human insulin receptor (isoform B) compared to hins.

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Abstract

La présente invention concerne des analogues d'insuline ayant des peptides à chaîne B raccourcie et des formulations d'analogues de l'insuline et des procédés de traitement correspondants.
PCT/US2016/028526 2015-04-20 2016-04-20 Analogues d'insuline ayant des peptides à chaîne b raccourcie et procédés associés WO2016172269A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3506945A4 (fr) * 2016-08-30 2020-07-15 Board of Regents, The University of Texas System Production de séléno-biologiques dans des organismes génétiquement recodés
WO2020214955A1 (fr) * 2019-04-19 2020-10-22 The Trustees Of Indiana University Stabilisation d'analogues d'insuline prandiale ou basale par un pont diséléniure interne
US10919949B2 (en) 2017-08-17 2021-02-16 Novo Nordisk A/S Acylated insulin analogues and uses thereof
US11098102B2 (en) 2018-12-11 2021-08-24 Sanofi Insulin conjugates
US11155804B2 (en) 2016-07-11 2021-10-26 Board Of Regents, The University Of Texas System Recombinant polypeptides comprising selenocysteine and method for producing the same

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11155804B2 (en) 2016-07-11 2021-10-26 Board Of Regents, The University Of Texas System Recombinant polypeptides comprising selenocysteine and method for producing the same
EP3506945A4 (fr) * 2016-08-30 2020-07-15 Board of Regents, The University of Texas System Production de séléno-biologiques dans des organismes génétiquement recodés
US11492650B2 (en) 2016-08-30 2022-11-08 Board Of Regents, The University Of Texas System Production of seleno-biologics in genomically recoded organisms
US10919949B2 (en) 2017-08-17 2021-02-16 Novo Nordisk A/S Acylated insulin analogues and uses thereof
US11098102B2 (en) 2018-12-11 2021-08-24 Sanofi Insulin conjugates
WO2020214955A1 (fr) * 2019-04-19 2020-10-22 The Trustees Of Indiana University Stabilisation d'analogues d'insuline prandiale ou basale par un pont diséléniure interne
CN114144426A (zh) * 2019-04-19 2022-03-04 印地安纳大学理事会 餐时或基础胰岛素类似物通过内部二硒桥的稳定化

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