Variant Single-Chain Insulin Analogues
20220235111 · 2022-07-28
Inventors
- Michael A. WEISS (Indianapolis, IN, US)
- Michael D. GLIDDEN, II (Cleveland, OH, US)
- Faramarz ISMAIL-BEIGI (Cleveland, OH, US)
Cpc classification
International classification
Abstract
A single-chain insulin analogue containing (i) diverse amino-acid substitutions at position A14; (ii) wild-type or variant residues at positions A8 and A14; and (ii) an engineered C-domain segment of lengths 4-6 containing a specific set of Alanine substitutions and/or deletions derived from the prototype C-domain sequence Glu-Glu-Gly-Pro-Arg-Arg. The analogue may otherwise be an analogue of a mammalian insulin, such as human insulin, may optionally include standard or non-standard modifications that (i) augment the stability of insulin, (ii) cause a shift in the isoelectric point to enhance or impair the solubility of the protein at neutral pH or (iii) reduce cross-binding of the protein to the Type I IGF receptor. Formulations of the above analogues at successive strengths U-100 to U-1000 in soluble solutions under acidic or neutral pH values (e.g., pH 3.0-4.2 and 6.5-7.8, respectively) and optionally in the presence of zinc ions at a molar ratio of 2.2-10 zinc ions per six insulin analogue monomers. A method of treating a patient with diabetes mellitus comprising the administration of a physiologically effective amount of the protein or a physiologically acceptable salt thereof to a patient. Use of a single-chain insulin analogue of the present invention in an insulin delivery device (such as a pump or pen) is envisioned.
Claims
1. A single-chain insulin analogue comprising a polypeptide having the sequence of the B-chain of insulin, a polypeptide comprising the sequence of the A-chain of insulin and a C-domain connecting polypeptide between the B-chain polypeptide sequence and the A-chain polypeptide sequence, wherein the A-chain polypeptide sequence comprises a substitution at at least one position corresponding to position A8 and position A14 of insulin, and wherein the C-domain comprises residues selected from the group consisting of residues 31-36 of SEQ ID NO:5, residues 31-37 of SEQ ID NO: 8, residues 31-38 of SEQ ID NO: 9, and residues 31-39 of SEQ ID NO: 10.
2. The single-chain insulin analogue of claim 1, comprising a substitution at position A14 of any amino acid other than Tyr and Pro.
3. The single-chain insulin analogue of claim 2, wherein the substitution at position A14 is Glu.
4. A single-chain insulin analogue comprising any one of SEQ ID NOs: 6-10.
5. The single-chain insulin analogue according to claim 4, comprising SEQ ID NO: 6.
6. The single-chain insulin analogue according to claim 4, comprising SEQ ID NO: 7.
7. The single-chain insulin analogue according to claim 4, comprising SEQ ID NO: 8.
8. The single-chain insulin analogue according to claim 4, comprising SEQ ID NO: 9.
9. The single-chain insulin analogue according to claim 4, comprising SEQ ID NO: 10.
10. A method for lowering the blood sugar level of a patient in need thereof, the method comprising, subcutaneously administering a pharmaceutical composition to the patient, wherein the pharmaceutical composition comprises a single-chain insulin comprising a polypeptide having the sequence of the B-chain of insulin, a polypeptide comprising the sequence of the A-chain of insulin and a C-domain connecting polypeptide between the B-chain polypeptide sequence and the A-chain polypeptide sequence, wherein the A-chain polypeptide sequence comprises a substitution at at least one position corresponding to position A8 and position A14 of insulin, and wherein the C-domain comprises residues selected from the group consisting of residues 31-36 of SEQ ID NO:5, residues 31-37 of SEQ ID NO: 8, residues 31-38 of SEQ ID NO: 9, and residues 31-39 of SEQ ID NO: 10.
11. The method of claim 10, wherein the insulin analogue comprises a substitution at position A14 of any amino acid other than Tyr and Pro.
12. The method of claim 11, wherein the substitution at position A14 is Glu.
13. A method of treating a patient comprising administering a physiologically effective amount of a single-chain insulin analogue or a physiologically acceptable salt thereof to the patient, wherein the single-chain insulin analogue comprises the single-chain insulin analogue of claim 4.
14. The method of claim 13, wherein the single-chain insulin analogue comprises SEQ ID NO: 6.
15. The method of claim 13, wherein the single-chain insulin analogue comprises SEQ ID NO: 7.
16. The method of claim 13, wherein the single-chain insulin analogue comprises SEQ ID NO: 8.
17. The method of claim 13, wherein the single-chain insulin analogue comprises SEQ ID NO: 9.
18. The method of claim 13, wherein the single-chain insulin analogue comprises SEQ ID NO: 10.
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
Description
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0030] The present invention is directed toward a single-chain insulin analogue that provides (i) enhanced stability, solubility and resistance to fibrillation due to the presence of a foreshortened C domain (length 4-11 residues) and (ii) ready and convenient co-optimization of biological, biophysical and pharmacodynamics properties. The single-chain insulin analogues of the present invention may have an isoelectric point between 4.0 and 6.0 (and so be suitable for formulation under neutral pH conditions as a rapid-acting insulin analogue formulation) or may have an isoelectric point between 6.5 and 8.0 (and so be suitable for formulation under acidic pH conditions as a basal insulin analogue formulation). Molecular embodiments of this strategy were prepared by biosynthetic expression in the yeast Pichia pastoris. The parent SCI has previously been disclosed in U.S. Pat. No. 9,499,600, issued Nov. 22, 2016, which is incorporated by reference herein. The variant SCIs of the present invention differ from this prototype in containing amino-acid substitutions at positions A8 and/or A14 (Table 1A, B). The foreshortened C domains of the present invention are given in Table 1C. The N-terminal residue of the C domain is acidic (Aspartic Acid or Glutamic Acid) in order to impair binding of the analogues to the mitogenic Type 1 IGF receptor (IGF-1R) relative to insulin receptor isoforms (IR-A and IR-B).
TABLE-US-00001 TABLE 1 Sequence features of SCI variant and control insulin analogs A. Control Analogs Analog Mutations Relative to Name WT Insulin KP (lispro) Lys.sup.B28, Pro.sup.B29; no C domain EA14 + HA8.sup.a Asp.sup.B28, Pro.sup.B29, His.sup.A8, Glu.sup.A14; EEGPRR C domain .sup.aEA14 + HA8 has an identical sequence to previously characterized SCI-b (1, 2). B. A Domain SCI Variants Analog A8 and A14 Name Residues Present.sup.b EA14 +TA8 Glu.sup.A14 and Thr.sup.A8 YA14 +HA8 Tyr.sup.A14 and His.sup.A8 YA14 +TA8 Tyr.sup.A14 and Thr.sup.A8 .sup.bAll A8 and A14 variants have the template C domain sequence EEGPRR (1, 2). C. C Domain SCI Variants Analog Mutations relative to Name template SCI-b.sup.c EAGPRR Ala.sup.C2 AEGPRR Ala.sup.C1 EEGARR Ala.sup.C4 EAGARR Ala.sup.C2 and Ala.sup.C4 EFGPRR Phe.sup.C2 EDGPRR Asp.sup.C2 EAAAAA Ala.sup.C2, Ala.sup.C3, Ala.sup.C4, Ala.sup.C5, Ala.sup.C6 EGPRR del(Glu.sup.C2) .sup.cAll C domain variants possess Glu.sup.A14 and His.sup.A8 as in the EA14 + HA8 (SCI-b) template.
[0031] In
[0032] In
[0033] In
[0034] While not intending to be constrained by theory, the A8 side chain is positioned to interact through electrostatic interactions with the helical dipole axis (white horizontal arrow in
[0035] The SCIs of the present invention may also contain substitutions at position A14 as exemplified in Table 2. Although not constrained by theory, these data suggest that substitution of the hyper-exposed Tyr.sup.A14 on the surface of wild-type insulin by Glu or other less hydrophobic side chains may mitigate an unfavorable “reverse-hydrophobic effect”—thereby augmenting thermodynamic stability—and simultaneously remove a potential aromatic site of chemical degradation. It is another aspect of the present invention that substitutions at position A14—and a combination of substitutions at positions A8 and A14—can alter both the thermodynamic stability and the PD profile of an SCI. In the data provided in Table 2, thermodynamic stabilities were measured by CD-monitored chemical denaturation and in selected cases also by .sup.1H-NMR amide proton exchange as described in the case of the prototype SCIs.
TABLE-US-00002 TABLE 2 Thermodynamic Stabilities of SCI Analogs. ΔG.sub.CD ΔG.sub.NMR A14 Residue (kcal/mol).sup.a m-value C.sub.mid (M).sup.b (kcal/mol).sup.c Non-polar Y 3.9 ± 0.1 0.74 ± 0.02 5.3 ± 0.1 3.98 ± 0.05.sup.d F 4.2 ± 0.1 0.81 ± 0.02 5.2 ± 0.1 — W 4.2 ± 0.1 0.76 ± 0.02 5.5 ± 0.1 — V 3.9 ± 0.1 0.77 ± 0.01 5.1 ± 0.1 — G 4.2 ± 0.1 0.83 ± 0.01 5.0 ± 0.1 4.20 ± 0.10 A 4.6 ± 0.1 0.79 ± 0.02 5.8 ± 0.1 — Polar N 4.2 ± 0.1 0.79 ± 0.02 5.4 ± 0.1 4.20 ± 0.06 Q 4.6 ± 0.1 0.83 ± 0.01 5.6 ± 0.1 — S 4.5 ± 0.1 0.82 ± 0.02 5.5 ± 0.1 — Charged D 4.4 ± 0.1 0.81 ± 0.01 5.4 ± 0.1 — E 4.7 ± 0.1 0.82 ± 0.01 5.7 ± 0.1 4.67 ± 0.01 R 4.5 ± 0.1 0.79 ± 0.01 5.7 ± 0.1 — .sup.a From two-state transition fits of (D-guanidine titration performed at 25° C. and pH 7.4 .sup.bC.sub.mid is guanidine denaturant concentration at which 50% of the protein is in the unfolded state. .sup.cFrom pH 7.4 amide exchange and fitting the B18 global amide resonance, unless otherwise noted. Hyphen = ΔG.sub.NMR not measured by amide exchange. .sup.dFrom amide exchange performed at pH 2.8 and fitting the B18 global amide reaonance.
[0036] The single-chain insulin analogues of the present invention may also contain substitutions within their respective A- and B domains. B-domain substitutions can include variants known in the art to weaken self-association and thus confer rapid absorption on subcutaneous injection; examples include AspB28 (as in Novolog®; insulin aspart), Lys.sup.B28-Pro.sup.B29 (as in Humalog®; insulin lispro) or Asp.sup.B28-Pro.sup.B29 (as described in Hua, Q.-X., et al. 2008). The analogues of the present invention exclude the substitution His.sup.B10.fwdarw.Asp, which has been associated with enhanced mitogenicity in cell culture and carcinogenesis in rat testing (Hansen, B. F., et al. (2011)).
[0037] In view of the similarity between human and animal insulins, and use in the past of animal insulins in human patients with diabetes mellitus, it is also envisioned that other minor modifications in the sequence of insulin may be introduced, especially those substitutions considered “conservative.” For example, additional substitutions of amino acids may be made within groups of amino acids with similar side chains, without departing from the present invention. These include the neutral hydrophobic amino acids: Alanine (Ala or A), Valine (Val or V), Leucine (Leu or L), Isoleucine (Ile or I), Proline (Pro or P), Tryptophan (Trp or W), Phenylalanine (Phe or F) and Methionine (Met or M). Likewise, the neutral polar amino acids may be substituted for each other within their group of Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T), Tyrosine (Tyr or Y), Cysteine (Cys or C), Glutamine (Glu or Q), and Asparagine (Asn or N). Basic amino acids are considered to include Lysine (Lys or K), Arginine (Arg or R) and Histidine (His or H). Acidic amino acids are Aspartic acid (Asp or D) and Glutamic acid (Glu or E). Unless noted otherwise or wherever obvious from the context, the amino acids noted herein should be considered to be L-amino acids. Standard amino acids may also be substituted by non-standard amino acids belong to the same chemical class. By way of non-limiting example, the basic side chain Lys may be replaced by basic amino acids of shorter side-chain length (Ornithine, Di-aminobutyric acid, or Di-aminopropionic acid). Lys may also be replaced by the neutral aliphatic isostere Norleucine (Nle), which may in turn be substituted by analogues containing shorter aliphatic side chains (Aminobutyric acid or Aminopropionic acid).
[0038] In the sequences described herein, nucleotides should be understood to be represented by their standard one letter abbreviations: A for adenine, G for guanine, C for cytosine, T for thymine and U for uracil. The presence of a mixture of nucleotides may also be indicated with an abbreviation as recognized in the art: N for any base (A,C,G or T/U), R for purine (G or A), Y for pyrimidine (T/U or C), M for amino (A or C), K for keto (G or T/U), S for G or C, W for A or T/U, V for nucleotides other than T (A, C, or G), D for nucleotides other than C (A, G, or T), B for nucleotides other than A (C, G, or T), and H for nucleotides other than G (A, C, or T).
[0039] Representative analogues of the present invention were purified from an engineered strain of the yeast Pichia pastoris as described (Glidden, M. D., et al., 2018a and 2018b). The prototype SCI contained four substitutions in the insulin moiety: Thr.sup.A8.fwdarw.His and Tyr.sup.A14—Glu and with B-domain substitutions Pro.sup.B28.fwdarw.Asp and Lys.sup.B29.fwdarw.Glu. The respective rationales for these substitutions were as follows. His.sup.A8 was introduced to augment receptor-binding affinity and thermodynamic stability; Glu.sup.A14 was introduced to enhance stability and reduce the isoelectric point (otherwise increased by the partial charge of His.sup.A8); and the Asp.sup.B28.fwdarw.Pro.sup.B29 element was introduced to weaken dimerization and further reduce the isoelectric point. The pharmacodynamics features of this analogue were tested following subcutaneous injection in a rat model of diabetes mellitus as described (Menting, J. G., et al., 2014). Its biological activity, onset of action, and duration of action were defined relative insulin lispro and two single-chain analogues containing the more complex C domain previously known in the art (Glu-Glu-Gly-Pro-Arg-Arg; EEGPRR). The PD profile of the prototype SCI is biphasic as described in Glidden et al. (2018a). This biphasic property was retained on substitution by a variety of C-domain sequences (Table 1C). A variant was constructed in which the A8 residue was reverted to Threonine and in which the A14 residue was reverted to Glutamic Acid. Remarkably, its pharmacodynamic profile is essentially identical to that of insulin lispro with respect to onset of action, offset of action, and integrated potency (area over the curve). The absence of a prolonged tail despite the presence of His.sup.A8 and Glu.sup.A14 demonstrates an interplay between C-domain sequence and modifications in the insulin moiety.
[0040] We note that the presence or absence of a “tail” of insulin action following the immediate peak of activity (as observed following intravenous bolus injection in a mammal) may be advantageous or disadvantageous depending on the clinical context and administration device. On the one hand, the absence of a tail could enable more precise control of prandial dosing. For use in an insulin pump, for example, a “tail-less” form of an insulin analogue would be desirable to reduce the risk of late hypoglycemia and to enhance the robustness of control algorithms in closed-loop systems (i.e., in which an algorithm controls the pump delivery rate in response to the output of a continuous glucose monitor). Other features of the insulin analogue, such as enhanced stability or resistance to aggregation at high protein concentration, may be desirable in such a device. On the other hand, the presence of a tail could enhance overall glycemic control in a biphasic insulin formation, especially in patients with diabetes mellitus in underprivileged environments not suitable for advanced closed-loop technologies or in patients with long-standing Type 2 diabetes mellitus for whom approximate glucose control may be safer than tight control. In challenged regions of the developing world, for example, the absence of refrigeration and limited educational backgrounds of many patients with Type 2 diabetes mellitus create conditions in which ultra-stable biphasic single-chain insulin analogue formulations would be of particular clinical value. The present invention provides a method for the dual tuning of the magnitude of the tail of insulin action and the thermodynamic stability of the SCI as a globular protein structure. The nature of the amino-acid substitutions at A8 and A14 and the specific C-domain sequence may be chosen to adjust the isoelectric point (pI) of the protein as a whole, such that the pI is in the range 4.0-5.5 (as suitable for a neutral-pH rapid-acting or biphasic formulation) or in the range 6.5-8.0 (as suitable for an acidic basal or biphasic formulation).
[0041] The biological activity of insulin analogues of the claimed invention was investigated in tissue culture cells using MCF-7 human breast cancer cell, rat hepatoma-devived HepG2 cells and rat myoblast L6 cells. The insulin analogues tested had the following sequences:
[0042] 3303—SEQ ID NO: 4
[0043] 3401—SEQ ID NO: 8
[0044] 3402—SEQ ID NO: 9
[0045] 3403—SEQ ID NO: 10.
[0046] Relevant tissue culture cells were challenged with 50 nM insulin analogue (or lispro control) and harvested. Analysis of Insulin Receptor (IR) activation and downstream metabolic effects were determined by Western blot analysis of expressed proteins extracted from MCF-7 human breast cancer cells (which express Insulin Receptor-A (IR-A), Insulin Receptor-B (IR-B) and Insulin-like Growth Factor 1 Receptor (IGF-1R)), rat hepatoma HepG2 cells, and rat L6 cells expressing Insulin Receptor A (IRA). Equal amounts of total protein extracts (12-30 for MCF-7, HepG2, and L6 cells) were loaded in Bio-Rad Mini-Protean TGX gels (4-20%) for separation. Briefly, supernatants collected following centrifugation of total cell lysates and after BCA protein assay (Thermo Scientific) were added with Laemmli sample buffer (Bio-Rad) containing 10% βME, heated at 95° C. for 6 min, and centrifuged quickly before loading in gels as mentioned above. Gels were usually run (100V constant) with the 20 kD protein marker (Bio-Rad) at the very bottom. Separated proteins were then transferred onto 0.2 μm PVDF membrane (Bio-Rad) for overnight (˜22 h) in cold-room (4° C.) using 25-30V (constant). Following transfer, membranes depending upon the proteins to be probed by individual primary antibodies were blocked either in 5% BSA (Cell Signaling Technology) (for example, pIR) or 5-10% nonfat dry milk (Bio-Rad) (for example, pAkt) in 1× TBS-T (Tris-buffered saline with 0.1% Tween-20) for 2 h at room temp on an orbital shaker. Membranes were then incubated for overnight (˜22 h) in cold room (at 4° C.) on an orbital shaker with the following individual antibodies—anti-insulin receptor β (1:1,000), anti-phospho insulin receptor (Y1334) (Invitrogen) (1:3,000), anti-pAkt (Ser 473) (1:400), anti-Akt (pan) (1:1,000), anti-GAPDH (1:8,000), anti-FoxO (1:1,000), anti-phospho FoxO (1:1,000), anti-GSK-3 α/β (1:1,000), anti-phospho GSK-3 α/β (1:1,000), anti-p27(kip1) (1:4,000), anti-phospho p27(S10) (1:1,000), anti-phospho p27(T157) (0.5 μg/ml), and anti-phospho glycogen synthase (1:1,000). All these antibodies were purchased from Cell Signaling Technology except stated otherwise. After the primary antibody incubation, membranes were washed thoroughly at room temp with 1×TBS-T (3×10 min) and then incubated with goat anti-rabbit IgG-HRP (1:5,000) in either 5% BSA or 5-10% non-fat dry milk in 1×TBS-T for 1-2 h at room temp on an orbital shaker. Following secondary antibody incubation, membranes were thoroughly washed as mentioned above (3×10 min) and then the protein bands were detected employing chemiluminescence reagents (Signal Fire ECL—Cell Signaling Technology) and finally developed in a dark room using autoradiography films.
[0047] As shown in
[0048] The insulin analogue of the present invention may be used as a medicament, and/or for the treatment of disease, such as diabetes mellitus, or other conditions where a reduction in blood sugar levels is advisable or necessary. In some examples, the insulin analogue of the present invention may be used for the manufacture of a medicament for the treatment of diabetes mellitus.
[0049] A method for treating a patient with diabetes mellitus comprises administering a single-chain insulin analogue as described herein. It is another aspect of the present invention that the single-chain insulin analogues may be prepared either in yeast (Pichia pastoris) or subject to total chemical synthesis by native fragment ligation. The synthetic route of preparation is preferred in the case of non-standard modifications, such as D-amino-acid substitutions, halogen substitutions within the aromatic rings of Phe or Tyr, or O-linked modifications of Serine or Threonine by carbohydrates; however, it would be feasible to manufacture a subset of the single-chain analogues containing non-standard modifications by means of extended genetic-code technology or four-base codon technology (for review, see Hohsaka, T., & Sisido, M., 2012). It is yet another aspect of the present invention that use of non-standard amino-acid substitutions can augment the resistance of the single-chain insulin analogue to chemical degradation or to physical degradation. We further envision the analogues of the present invention providing a method for the treatment of diabetes mellitus or the metabolic syndrome. The route of delivery of the insulin analogue is by subcutaneous injection through the use of a syringe or pen device.
[0050] A single-chain insulin analogue of the present invention may also contain other modifications, such as a halogen atom at positions B24, B25, or B26 as described more fully in U.S. Pat. No. 8,921,313, the disclosure of which is incorporated by reference herein. An insulin analogue of the present invention may also contain a foreshortened B-chain due to deletion of residues B1-B3 as described more fully in U.S. Pat. No. 9,725,493, the disclosure of which is incorporated by reference herein.
[0051] A pharmaceutical composition may comprise such insulin analogues and which may optionally include zinc. Zinc ions may be included at varying zinc ion:protein ratios, ranging from 2.2 zinc atoms per insulin analogue hexamer to 10 zinc atoms per insulin analogue hexamer. The pH of the formulation may either be in the range pH 3.0-4.5 (as a basal formulation of a pI-shifted single-chain insulin analogue) or be in the range pH 6.5-8.0 (as a prandial insulin formulation of a single-chain insulin analogue whose pI is similar to that of wild-type insulin). In either such formulation, the concentration of the insulin analogue would typically be between about 0.6-5.0 mM; concentrations up to 5 mM may be used in vial or pen; the more concentrated formulations (U-200 or higher, including in the range U-500 through U-1000) may be of particular benefit in patients with marked insulin resistance. Excipients may include glycerol, glycine, arginine, Tris, other buffers and salts, and anti-microbial preservatives such as phenol and meta-cresol; the latter preservatives are known to enhance the stability of the insulin hexamer. Such a pharmaceutical composition may be used to treat a patient having diabetes mellitus or other medical condition by administering a physiologically effective amount of the composition to the patient.
[0052] The amino-acid sequence of human proinsulin is provided, for comparative purposes, as SEQ ID NO: 1.
TABLE-US-00003 SEQ ID NO: 1 (human proinsulin) Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Lys-Thr-Arg-Arg-Glu-Ala-Glu-Asp- Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro- Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly- Ser-Leu-Gln-Lys-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys- Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr- Cys-Asn
[0053] The amino-acid sequence of the A chain of human insulin is provided as SEQ ID NO: 2.
TABLE-US-00004 SEQ ID NO: 2 (human A chain) Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser- Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn
[0054] The amino-acid sequence of the B chain of human insulin is provided as SEQ ID NO: 3.
TABLE-US-00005 SEQ ID NO: 3 (human B chain) Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Lys-Thr
[0055] The amino-acid sequence of the prototype SCI is provided as SEQ ID NO: 4.
TABLE-US-00006 SEQ ID NO: 4 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Asp-Pro-Thr-Glu-Glu-Gly-Pro-Arg-Arg- Gly-Ile-Val-Glu-Gln-Cys-Cys-His-Ser-Ile-Cys-Ser- Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn
[0056] The amino-acid sequence of the SCIs of the present invention conform to the following as provided in SEQ ID NO: 5
[0057] The amino-acid sequence of variants of an SCI containing diverse substitutions at position A14 and/or position A8 is provided as SEQ ID NO: 5 to SEQ ID NO: 10.
TABLE-US-00007 SEQ ID NO: 5 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-XXa.sub.1-XXa.sub.2-Pro-Thr-Glu-Glu-Gly-Pro-Arg- Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-Xaa.sub.4-Ser-Ile-Cys- Ser-Leu-XXa.sub.3-Gln-Leu-Glu-Asn-Tyr-Cys-Asn
[0058] Where the XXa.sub.1-XXa.sub.2 element at B-domain positions B28 and B29 either (a) contains a Proline at position B28 and a non-proline residue at position B29 (as exemplified by the wild-type sequence Pro.sup.B28-Lys.sup.B29 and its variants Pro-Arg, Pro-Glu, or Pro-Ala), (b) contains a Proline at position B29 and a non-proline at position B28 (as exemplified by Asp-Pro, Glu-Pro, Ala-Pro, Asp-Lys, Glu-Lys, and Ala-Arg) and (c) does not contain Proline at either position (as exemplified by Asp-Lys, Glu-Arg, Ala-Lys, and Ala-Arg); and where Xaa.sub.3 may be any amino acid, and Xaa.sub.4 is Thr, His, Ser, Glu, or Ala.
TABLE-US-00008 SEQ ID NO: 6 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Asp-Pro-Thr-Z-Gly-Ile-Val-Glu-Gln-Cys- Cys-Xaa.sub.1-Ser-Ile-Cys-Ser-Leu- Xaa.sub.2-Gln-Leu-Glu- Asn-Tyr-Cys-Asn
[0059] Where Xaa.sub.1 is Thr, His, Ser, Glu or Ala; where Xaa.sub.2 is any amino acid other than Proline; and where Z indicates a peptide of length 6 residues containing derivable from the parent sequence Glu-Glu-Gly-Pro-Arg-Arg by one, two or three Alanine substitutions (e.g., Ala-Glu-Gly-Pro-Arg-Arg, Glu-Ala-Gly-Pro-Arg-Arg, Glu-Glu-Ala-Pro-Arg-Arg, Glu-Glu-Gly-Ala-Arg-Arg, Glu-Glu-Gly-Pro-Ala-Arg, or Glu-Glu-Gly-Pro-Arg-Ala; or by two-Ala substitutions such as Glu-Ala-Gly-Pro-Arg-Ala or Glu-Glu-Ala-Ala-Arg-Arg; or by three Ala substitutions such as Glu-Ala-Gly-Ala-Arg-Ala).
TABLE-US-00009 SEQ ID NO: 7 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Asp-Pro-Thr-Z′-Gly-Ile-Val-Glu-Gln- Cys-Cys-Xaa.sub.1-Ser-Ile-Cys-Ser-Leu- Xaa.sub.2-Gln-Leu- Glu-Asn-Tyr-Cys-Asn
[0060] Where Xaa.sub.1 is Thr, His, Ser, Glu or Ala; where Xaa.sub.2 is any amino acid other than Proline; and where Z′ indicates a peptide of length 4-5 residues containing derivable from the C-domain sequences in SEQ ID NO:6 by deletion of one or two residues (e.g., Glu-Gly-Pro-Arg, Glu-Ala-Gly-Pro-Arg, Glu-Glu-Ala-Arg-Arg or Glu-Gly-Ala-Arg-Ala).
TABLE-US-00010 SEQ ID NO: 8 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Pro Thr Glu Glu Gly Ala Pro Arg Arg Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser Leu Glu Gln Leu Glu Asn Tyr Cys Asn SEQ ID NO: 9 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Pro Thr Glu Glu Gly Ala Ala Pro Arg Arg Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser Leu Glu Gln Leu Glu Asn Tyr Cys Asn SEQ ID NO: 10 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Pro Thr Glu Glu Gly Ala Ala Ala Pro Arg Arg Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser Leu Glu Gln Leu Glu Asn Tyr Cys Asn SEQ ID NO: 11 (lispro B chain) Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr- Lys- Pro-Thr
[0061] The word “comprising” and forms of the word “comprising” as used in this description and in the claims does not limit the invention claimed to exclude any variants or additions.
[0062] Based upon the foregoing disclosure, it should now be apparent that an ultra-stable single-chain insulin analogue may be made compatible with either an unperturbed duration of insulin signaling or a “tail” of insulin action tuneable through the co-engineering of amino-acid substitutions at positions A8 and A8 in the context of a foreshortened and simplified C domain. The resulting single-chain insulin analogues provided will carry out the objects set forth hereinabove. Namely, these modified proteins exhibit enhanced resistance to fibrillation while retaining desirable pharmacokinetic and pharmacodynamic features (conferring rapid or prolonged rates of absorption from a subcutaneous depot with or without a tail of insulin action as may be therapeutically desired) and maintaining at least a fraction of the biological activity of wild-type insulin. It is, therefore, to be understood that any variations evident fall within the scope of the claimed invention and thus, the selection of specific component elements can be determined without departing from the spirit of the invention herein disclosed and described.
[0063] The following literature is cited to demonstrate that the testing and assay methods described herein would be understood by one of ordinary skill in the art. [0064] Derewenda, U., Derewenda, Z., Dodson, E. J., Dodson, G. G., Bing, X., and Markussen, J. 1991. X-ray analysis of the single chain B29-A1 peptide-linked insulin molecule. A completely inactive analogue. J. Mol. Biol. 220: 425-433. [0065] Doig, A. J, & Baldwin, R. L. N- and C-capping preferences for all 20 amino acids in alpha-helical peptides. 1995. Protein Sci. 4: 1325-36. [0066] Hansen, B. F., Kurtzhals, P., Jensen, A. B., Dejgaard, A., Russell-Jones, D. 2011. Insulin X10 revisited: a super-mitogenic insulin analogue. Diabetologia. 54: 2226-31. [0067] Glidden, M. D., Aldabbagh, K., Phillips., N. B., Carr., K., Chen, Y. S., Whittaker, J., Phillips, M., Wickramasinghe, N. P., Rege, N., Swain, M., Peng, Y., Yang, Y., Lawrence, M. C., Yee, V. C., Ismail-Beigi, F., & Weiss, M. A. 2017. An ultra-stable single-chain insulin analog resists thermal inactivation and exhibits biological signaling duration equivalent to the native protein. J. Biol. Chem. pii: jbc.M117.808626. doi: 10.1074/jbc.M117.808626 [Epub ahead of print]; in print: 293: 69-8 [0068] Glidden M D, Yang Y, Smith N A, Phillips N B, Carr K, Wickramasinghe N P, Ismail-Beigi F, Lawrence M C, Smith B J, Weiss M A. 2017. Solution structure of an ultra-stable single-chain insulin analog connects protein dynamics to a novel mechanism of receptor binding. J. Biol. Chem. pii: jbc.M117.808667. doi: 10.1074/jbc.M117.808667 [Epub ahead of print]; in print 293: 47-68. [0069] Hohsaka, T., & Sisido, M. 2012. Incorporation of non-natural amino acids into proteins. Curr. Opin. Chem. Biol. 6: 809-15. [0070] Hua, Q. X., Nakagawa, S. H., Jia, W., Huang, K., Phillips, N. B., Hu, S. & Weiss, M. A. (2008) Design of an active ultrastable single-chain insulin analog: synthesis, structure, and therapeutic implications. J. Biol. Chem. 283: 14703-14716. [0071] Kristensen, C., Andersen, A. S., Hach, M., Wiberg, F. C., Schïffer, L., & Kjeldsen, T. 1995. A single-chain insulin-like growth factor I/insulin hybrid binds with high affinity to the insulin receptor. Biochem. J. 305: 981-6. [0072] Lee, H. C., Kim, S. J., Kim, K. S., Shin, H. C., & Yoon, J. W. 2000. Remission in models of type 1 diabetes by gene therapy using a single-chain insulin analogue. Nature 408, 483-8. Retraction in: Lee H C, Kim K S, Shin H C. 2009. Nature 458: 600. [0073] Menting, J. G., Yang, Y., Chan, S. J., Phillips, N. B., Smith, B. J., Whittaker, J., Wickramasinghe, N. P., Whittaker, L. J., Pandyarajan, V., Wan, Z. L., Yadav, S. P., Carroll, J. M., Strokes, N., Roberts, C. T., Jr, Ismail-Beigi, F., Milewski, W., Steiner, D. F., Chauhan, V. S., Ward, C. W., Weiss, M. A., & Lawrence, M. C. 2014. Protective hinge in insulin opens to enable its receptor engagement. Proc. Natl. Acad. Sci. USA 111(33):E3395-404. [0074] Nakagawa S. H., & Tager H. S. 1989. Perturbation of insulin-receptor interactions by intramolecular hormone cross-linking. Analysis of relative movement among residues A1, B1, and B29. J. Biol. Chem. 264(1):272-9. [0075] Phillips, N. B., Whittaker, J., Ismail-Beigi, F., & Weiss, M. A. (2012) Insulin fibrillation and protein design: topological resistance of single-chain analogues to thermal degradation with application to a pump reservoir. J. Diabetes Sci. Technol. 6, 277-288. [0076] Rohl, C. A., Chakrabartty, A., & Baldwin, R. L. 1997. Protein Soc. 5:2623-2637. [0077] Vinther, T. N., Pettersson, I., Huus, K., Schlein, M., Steensgaard, D. B., Sorensen, A., Jensen, K. J., Kjeldsen T., and Hubalek, F. 2015. Additional disulfide bonds in insulin: Prediction, recombinant expression, receptor binding affinity, and stability. Protein Sci. 24:779-88.
[0078] Vinther, T. N., Norrman, M., Ribel, U., Huus, K., Schlein, M., Steensgaard, D. B., Pedersen, T. A., Pettersson, I., Ludvigsen, S., Kjeldsen, T., Jensen, K. J., and Hubalek, F. 2013. Insulin analog with additional disulfide bond has increased stability and preserved activity. Protein Sci. 22:296-305.
[0079] Wang, Z. X. 1995. An exact mathematical expression for describing competitive biding of two different ligands to a protein molecule FEBS Lett. 360: 111-114.
[0080] Whittaker, J., and Whittaker, L. 2005. Characterization of the functional insulin binding epitopes of the full-length insulin receptor. J. Biol. Chem. 280: 20932-20936. [0081] Yutani, K., Ogasahara, K., Sugino, Y. 1985. Effect of amino-acid substitutions on conformational stability of a protein. Adv Biophys. 20:13-29.