Patent Application
20240218041
Incorporated by reference in its entirety is a computer-readable nucleotide/amino-acid sequence listing submitted concurrently herewith and identified as follows: 65 kilobytes ACII (text) file named “354919_ST25.txt,” created on May 3, 2022.
The engineering of non-standard proteins, including therapeutic agents and vaccines, may have broad medical and societal benefits. Naturally occurring proteins—as encoded in the genomes of human beings, other mammals, vertebrate organisms, invertebrate organisms, or eukaryotic cells in general—may have evolved to function optimally within a cellular context but may be suboptimal for therapeutic applications. Analogues of such proteins may exhibit improved biophysical, biochemical, or biological properties. A benefit of protein analogues would be to achieve enhanced activity (such as metabolic regulation of metabolism leading to reduction in blood-glucose concentration under conditions of hyperglycemia) with decreased unfavorable effects (such as induction of hypoglycemia or its exacerbation).
An example of a therapeutic protein is provided by insulin. Wild-type human insulin and insulin molecules encoded in the genomes of other mammals bind to insulin receptors in multiple organs and diverse types of cells, irrespective of the receptor isoform generated by alternative modes of RNA splicing or by alternative patterns of post-translational glycosylation. An example of a medical benefit would be the non-standard design of a soluble insulin analogue whose intrinsic affinity for insulin receptors on the surface of target cells, and hence whose biological potency, would depend on the concentration of glucose in the blood stream. Such an analogue may have a three-dimensional conformation that changes as a function of glucose concentration and/or may have a covalent bond to an inhibitory molecular entity that is detached at high glucose concentrations. Although it is not presently known in the art how to engineer such hypothetical analogues, this long-sought class of protein analogues or protein derivatives is collectively designated “glucose-responsive insulins” (GRIs).
The insulin molecule contains two chains, an A chain, containing 21 residues, and a B chain containing 30 residues. The mature hormone is derived from a longer single-chain precursor, designated proinsulin, as outlined in
Although the insulin hormone is stored in the pancreatic β-cell as a Zn2+-stabilized hexamer, it functions as a Zn2+-free monomer in the bloodstream. The three-dimensional structure of an insulin monomer is shown as a ribbon model in
An aspect of the present invention pertains to the chirality of amino acids. Whereas Glycine is achiral, biosynthetic proteins ordinarily are comprised of
Administration of insulin has long been established as a treatment for diabetes mellitus. A major goal of conventional insulin replacement therapy in patients with diabetes mellitus is tight control of the blood-glucose concentration to prevent its excursion above or below the normal range characteristic of healthy human subjects. Excursions above the normal range are associated with increased long-term risk of microvascular disease, including retinopathy, blindness, and renal failure. Hypoglycemia in patients with diabetes mellitus is a frequent complication of insulin replacement therapy and when severe can lead to significant morbidity (including altered mental status, loss of consciousness, seizures, and death). Indeed, fear of such complications poses a major barrier to efforts by patients (and physicians) to obtain rigorous control of blood-glucose concentrations (i.e., exclusions within or just above the normal range), and in patients with long-established Type 2 diabetes mellitus such efforts (“tight control”) may lead to increased mortality. In addition to the above consequences of severe hypoglycemia (designated neuroglycopenic effects), mild hypoglycemia may activate counter-regulatory mechanisms, including over-activation of the sympathetic nervous system leading to turn to anxiety and tremulousness (symptoms designated adrenergic). Patients with diabetes mellitus may not exhibit such warning signs, however, a condition known as hypoglycemic unawareness. The absence of symptoms of mild hypoglycemia increases the risk of major hypoglycemia and its associated morbidity and mortality.
Multiple and recurrent episodes of hypoglycemia are also associated with chronic cognitive decline, a proposed mechanism underlying the increased prevalence of dementia in patients with long-standing diabetes mellitus. There is therefore an urgent need for new diabetes treatment technologies that would reduce the risk of hypoglycemia while preventing upward excursions in blood-glucose concentration above the normal range.
Diverse technologies have been developed in an effort to mitigate the threat of hypoglycemia in patients treated with insulin. Foundational to all such efforts is education of the patient (and also members of his or her family) regarding the symptoms of hypoglycemia and following the recognition of such symptoms, the urgency of the need to ingest a food or liquid rich in glucose, sucrose, or other rapidly digested form of carbohydrate; an example is provided by orange juice supplemented with sucrose (cane sugar). This baseline approach has been extended by the development of specific diabetes-oriented products, such as squeezable tubes containing an emulsion containing glucose in a form that can be rapidly absorbed through the mucous membranes of the mouth, throat, stomach, and small intestine. Preparations of the counter-regulatory hormone glucagon, provided as a powder, have likewise been developed in a form amenable to rapid dissolution and subcutaneous injection as an emergency treatment of severe hypoglycemia. Insulin pumps have been linked to a continuous glucose monitor such that subcutaneous injection of insulin is halted and an alarm is sounded when hypoglycemic readings of the interstitial glucose concentration are encountered. Such a device-based approach has led to the experimental testing of closed-loop systems in which the pump and monitor are combined with a computer-based algorithm as an “artificial pancreas.”
For more than three decades, there has been interest in the development of glucose-responsive materials for co-administration with an insulin analogue or modified insulin molecule such that the rate of release of the hormone from the subcutaneous depot depends on the interstitial glucose concentration. Such systems in general contain a glucose-responsive polymer, gel or other encapsulation material; and may also require a derivative of insulin containing a modification that enables binding of the hormone to the above material. An increase in the ambient concentration of glucose in the interstitial fluid at the site of subcutaneous injection may displace the bound insulin or insulin derivative either by competitive displacement of the hormone or by physical-chemical changes in the properties of the polymer, gel or other encapsulation material. The goal of such systems is to provide an intrinsic autoregulation feature to the encapsulated or gel-coated subcutaneous depot such that the risk of hypoglycemia is mitigated through delayed release of insulin when the ambient concentration of glucose is within or below the normal range. To date, no such glucose-responsive systems are in clinical use.
A recent technology exploits the structure of a modified insulin molecule, optionally in conjunction with a carrier molecule such that the complex between the modified insulin molecule and the carrier is soluble and may enter into the bloodstream. This concept differs from glucose-responsive depots in which the polymer, gel or other encapsulation material remains in the subcutaneous depot as the free hormone enters into the bloodstream. An embodiment of this approach is known in the art wherein the A chain is modified at or near its N-terminus (utilizing the α-amino group of residue A1 or via the ε-amino group of a Lysine substituted at positions A2, A3, A4 or A5) to contain an “affinity ligand” (defined as a saccharide moiety or diol-containing moiety), the B chain is modified at its or near N-terminus (utilizing the α-amino group of residue B1 or via the ε-amino group of a Lysine substituted at positions B2, B3, B4 or B5) to contain a “monovalent glucose-binding agent.” In this description the large size of the exemplified or envisaged glucose-binding agents (monomeric lectin domains, DNA aptamers, or peptide aptomers) restricted their placement to the N-terminal segment of the B chain as defined above. In the absence of exogenous glucose or other exogenous saccharide, intramolecular interactions between the A1-linked affinity ligand and B1-linked glucose-binding agent was envisaged to “close” the structure of the hormone and thereby impair its activity. Only modest glucose-responsive properties of this class of molecular designs were reported. In this class of analogues the B1-linked agents are typically as large or larger than insulin itself.
The suboptimal properties of insulin analogues modified at or near residue A1 by an affinity ligand and simultaneously modified at or near residue B1 by a large glucose-binding agent (i.e., of size similar or greater than that of an insulin A or B chain) are likely to be intrinsic to this class of molecular designs. Overlooked in the above class of insulin analogues are the potential advantages of an alternative type of glucose-regulated switch engineered exclusively within the B chain without modification of its amino-terminus and without the need for large domains unrelated in structure or composition to insulin. The insulin analogues of the present invention thus conform to one of four design schemes sharing the properties that (a) in the absence of glucose the modified insulin exhibits marked impairment in binding to the insulin receptor whereas (b) in the presence of a high concentration of glucose breakage of a covalent bond to a diol-modified B chain either leads to an active hormone conformation or liberates an active hormone analogue (
Surprisingly, applicant has found that this fundamentally different class of molecular designs may optimally provide a glucose-dependent conformational switch between inactive and active states of the insulin molecule without the above disadvantages. Novel diol modifications of the B chain at or near its C terminus have been previously disclosed in U.S. Provisional Application 63/104,196, entitled “Molecular Designs of Glucose-Responsive and Glucose-Cleavable Insulin Analogues”; incorporated by reference herein. Displacement of the insulin diol from the A-chain-linked glucose-binding element by glucose would lead to detachment of the tethered molecular entity, which in turn enables high-affinity receptor binding; as illustrated in
The present disclosure relates to polypeptide hormone analogues that contain a glucose-regulated molecular structure or glucose-detachable molecular moiety, designed respectively to either (a) confer glucose-responsive binding to cognate cellular receptors and/or (b) enable glucose-mediated liberation of an active insulin analogue. More particularly, in one embodiment the present disclosure focuses on novel combinations of modified A chains by incorporation of bis-boron-containing glucose-binding elements (GBE) that provide increase opportunity for selective binding to glucose through interaction(s) of two boronic acid moieties binding to one glucose molecule (
In one embodiment the present disclosure is directed to the use of the insulin analogues disclosed herein for use in the treatment of patients and non-human mammals with Type 1 or Type 2 diabetes mellitus by subcutaneous, intraperitoneal or intravenous injection.
The insulin analogues of the present invention may also exhibit other enhanced pharmaceutical properties, such as increased thermodynamic stability, augmented resistance to thermal fibrillation above room temperature, decreased mitogenicity, and/or altered pharmacokinetic and pharmacodynamic properties. More particularly, this invention relates to insulin analogues that may confer either rapid action (relative to wild-type insulin in its regular soluble formulation), intermediate action (comparable to NPH insulin formulations known in the art) or protracted action (comparable to basal insulins known in the art as exemplified by insulin detemir and insulin glargine), such that the affinity of the said analogues for the insulin receptor is higher when dissolved in a solution containing glucose at a concentration above the physiological range (>140 mg/dL; hyperglycemia) than when dissolved in a solution containing glucose at a concentration below the physiological range (<80 mg/dL; hypoglycemia).
It is, therefore, an aspect of the present invention to provide insulin analogues that are inactive or exhibit reduced, prolonged activity under hypoglycemic conditions but are activated at high glucose concentrations and so may bind to the insulin receptor with high affinity. The analogues of the present invention contain two essential elements. The first element is a diol-containing side chain in the B chain C-terminal segment (residues B27-B30 or as attached to extended B-chain residues B31 or B32) and/or a C-terminal main chain diol in the B chain; the second is a glucose-binding element (GBE) attached at or near the N terminus of the A chain. This overall scheme is shown in
One embodiment of the present disclosure pertains to the design and synthesis of glucose-responsive insulins (GRIs) containing modified A chains such that paired boron-containing moieties (BCMs) are tethered at or near the N-terminal residue (position A1); the B chains are modified to comprise diol adducts of arbitrary chemical composition at or near its C terminus. Disclosed herein are specific linkage strategies and chemical approaches for synthesis of A-chain analogues that contain paired BCMs. BCMs may include phenyl-boronic acid (PBA)-based monomeric diol binders (illustrated in
The A chain of the present invention can thus be the standard 21 residues in length or contain an N-terminal extension of one residue (A0), two residues (A−1-A0), or three residues (A−2-A−1-A0) as illustrated in
In accordance with one embodiment, an insulin analogue comprising an insulin A chain and an insulin B chain is provided, wherein the insulin A chain comprises a D-amino acid at position A1 or A0 and a glucose-binding element covalently linked at or near the insulin A-chain N terminus, optionally attached to the side chain of the amino acid at position A1 or A0; and the insulin B chain comprises a diol group at or near the C terminus of the insulin B chain, optionally wherein a diol-bearing moiety is linked to the side chain of the amino acid at position B28, B29 or B30 or wherein the diol group is a modified C-terminal amino acid (a) bearing a side chain hydroxyl group and (b) having the C-terminal carboxyl group replaced with CH2OH, such that the two hydroxyl groups together may bind to an A-chain-linked GBE.
In one embodiment the glucose-binding element is covalently linked to the side chain of an amino acid of the A chain, optionally wherein the glucose-binding element-bearing amino acid is a
In another embodiment the glucose-binding element bearing insulin A chains of the present disclosure comprises a single amino acid at the N-terminus linked via a peptide bond to the alpha-amino group of the amino acid bearing the glucose-binding element disclosed in
In yet another embodiment the insulin analogue of the present disclosure comprises a B chain that is truncated, lacking residue B30, residues B29-B30, residues B28-B30, residues B27-B30 or residues B26-B30, with a diol group located at the C terminus of the truncated B chain. In another embodiment the native B chain is extended by one or two amino acids with a diol group located at the C terminus of the extended B chain, optionally in conjunction with a diol-modified side chain.
In accordance with one embodiment, an insulin analogue is provided wherein
In one embodiment the insulin B chain is a polypeptide comprising the sequence of
wherein
In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.
The term “about” as used herein means greater or lesser than the value or range of values stated by 10 percent but is not intended to limit any value or range of values to only this broader definition. Each value or range of values preceded by the term “about” is also intended to encompass the embodiment of the stated absolute value or range of values.
As used herein, the term “purified” and like terms relate to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment. As used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative definition.
The term “isolated” requires that the referenced material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.
As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.
As used herein, the term “treating” includes alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms.
As used herein an “effective” amount or a “therapeutically effective amount” of a drug refers to a nontoxic but enough of the drug to provide the desired effect. The amount that is “effective” will vary from subject to subject or even within a subject overtime, depending on the age and general condition of the individual, mode of administration, and the like. 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.
As used herein the term “patient” without further designation is intended to encompass any warm blooded vertebrate domesticated animal (including for example, but not limited to livestock, horses, cats, dogs and other pets) and humans receiving a therapeutic treatment with or without physician oversight.
The term “inhibit” defines a decrease in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
As used herein, the term “Diol-bearing amino acids” comprise natural or unnatural amino acids whose side chains are modified by linkage of a diol moiety to an attachment point located on the amino acid side chain. Examples of side chain attachment points are provided by a thiol function, amino function or carboxylate function. Amino-containing side chains, for example, may be provided by the natural amino acid Lysine or by unnatural amino acids Ornithine (Orn), Diaminobutyric Acid (Dab) and Diaminoproprionic acid (Dap), each in either the
As used herein, the term “threoninol” absent any further elaboration encompasses
As used herein the term “main-chain” defines the backbone portion of a polypeptide, and distinguishes the atoms comprising the backbone from those that comprise the amino acid side chains that project from the main-chain.
As used herein, the term “pharmaceutically acceptable salt” refers to those salts with counter ions which may be used in pharmaceuticals. See, generally, S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977, 66, 1-19. Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of subjects without undue toxicity, irritation, or allergic response. A compound described herein may possess a sufficiently acidic group, a sufficiently basic group, both types of functional groups, or more than one of each type, and accordingly react with a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt. Such salts include:
Acceptable salts are well known to those skilled in the art, and any such acceptable salt may be contemplated in connection with the embodiments described herein. Examples of acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, besylates, xylenesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates, and mandelates. Lists of other suitable acceptable salts are found in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, Pa., 1985.
As used herein the term “native insulin peptide” is intended to designate the 51 amino acid heterodimer comprising the A chain of SEQ ID NO: 2 and the B chain of SEQ ID NO: 3, as well as single-chain insulin analogues that comprise SEQ ID NOS: 2 and 3. The term “insulin peptide” as used herein, absent further descriptive language is intended to encompass the 51 amino acid heterodimer comprising the A chain of SEQ ID NO: 2 and the B chain of SEQ ID NO: 3, as well as heterodimers and that comprise modified derivatives of the native A chain and/or B chain. An “insulin A chain” is defined as the 21 amino acid sequence of SEQ ID NO: 2 as well as any modified derivatives of the native A chain, and an insulin B chain is defined as the 30 amino acid sequence of SEQ ID NO: 3 as well as any modified derivatives of the native B chain. Modified derivatives of the “insulin peptide”, “insulin A chain” and “insulin B chain” included one or more amino-acid substitutions at positions selected from A1, A5, A8, A9, A10, A12, A14, A15, A17, A18, A21, B1, B2, B3, B4, B5, B9, B10, B13, B14, B17, B20, B21, B22, B23, B26, B27, B28, B29 and B30, or deletions of any or all of positions B1-4 and B26-30, or the addition of 1-3 amino acids to the N-terminus of the A chain or at the C-terminus of the B chain. Additional amino acids linked to the insulin A chain peptide at the N-terminus are numbered starting with 0 and increasing in negative integer value as they are further removed from the native insulin A chain sequence. For example, the position of an amino acid within an N-terminal extension of the A chain is designated A−1 or A0, wherein A0 represents the position of an amino acid added directly through the use of a standard amide-bond connectivity to the native N-terminal amino acid of the insulin A chain, and A−1 represents the position of an amino acid having a single amino acid intervening between the A−1 amino acid and the native N-terminal A1 amino acid of the insulin A chain.
As used herein, an amino acid “modification” refers to a substitution, addition or deletion of an amino acid through amide bond coupling or other amide bond isosteric mimetice bond connectivity, 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 or addition of 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, WI), ChemPep Inc. (Miami, FL), and Genzyme Pharmaceuticals (Cambridge, MA). Atypical amino acids may be purchased from commercial suppliers, synthesized de novo, or chemically modified or derivatized from naturally occurring amino acids.
As used herein an amino acid “substitution” refers to the replacement of one amino acid residue by a different amino acid residue. Throughout the application, all references to a particular amino acid position by letter and number (e.g. position A5) 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 native human insulin A chain (SEQ ID NO: 2) or B chain (SEQ ID NO: 3), or the corresponding amino acid position in any analogues thereof. For example, a reference herein to “position B28” absent any further elaboration would mean the corresponding position B27 of the B chain of an insulin analogue in which the first amino acid of SEQ ID NO: 3 has been deleted.
As used herein, the term “conservative amino acid substitution” is defined herein as exchanges within one of the following five groups:
As used herein a “linker” is a bond, molecule or group of molecules that binds two separate entities to one another. In one embodiment the linker provides optimal spacing of the two entities.
As used herein a “glucose-binding elements (GBE)” is molecular structure that comprises two or more boron-containing moieties (BCMs). A bis-boronic acid GBE is a GBE comprising two boron-containing moieties wherein the two boron-containing moieties are linked to one another either directly or through a linker.
As used herein a “boron-containing moiety (BCM)” is a chemical structure comprising a boron molecule covalently linked to two oxygen atoms, wherein the boron-containing moiety is capable of interacting with a diol containing entity to form a reversible covalent link between the diol and the boron atom (see
As used herein a “a glucose responsive insulin (GRI)” is an insulin analogue that comprises a glucose-binding elements (GBEs). Such GRIs are inactive or exhibit reduced activity relative to native insulin under hypoglycemic conditions but become activated at elevated glucose concentrations, optionally at blood-glucose concentrations greater than 150 mg/dL after 8 hours of fasting, and bind the insulin receptor with high affinity.
As disclosed herein analogues of native insulin have been prepared that are biological sensors that activate only under hyperglycemic conditions. In one embodiment the insulin analogue comprises an insulin A chain peptide modified by the linkage of a glucose-binding element at or near its N terminus and a B chain modified by the linkage of one or more diol adducts at or near its C terminus, wherein the glucose-binding element comprises two or more boron-containing diol-binding elements disclosed in
In addition, the modified A chain may also contain a broad molecular diversity of paired BCMs, either as branched adducts attached at one site of the modified A chain (e.g. at a single amino acid side chain) or as separate adducts attached at two or more peptide sites (e.g. at the side chain of two separate amino acids) as exemplified in
The GRI design related to the present invention considers three interlocking design elements required in concert to enable the function of a glucose-responsive conformational switch: element 1 is defined by the composition and chemical nature of the GBEs (e.g., mono-boronic acid, bis-boronic acid [with subclasses symmetrical and asymmetrical, each incorporating various BCMs as illustrated in
Design element 2 is defined by the composition and chemical nature of B-chain-tethered diol moieties. The element functions as a glucose-competable “lock” to stabilize an inactive conformation of the hormone analogue. The modified A chains of the present invention can be combined with a combination of side-chain and main-chain diol modifications of the B chain at or near its C terminus as previously disclosed (USPTO Provisional Application 63/104,196, entitled “Molecular Designs of Glucose-Responsive and Glucose-Cleavable Insulin Analogues”; incorporated by reference herein).
Design element 3 specifies the placement of the intramolecular switch that effects a glucose-dependent conformational transition between active and inactive states. Optimal placement of the A-chain modifications depends on the location of the B-chain diol(s) and vice versa. The present invention focuses on the combination of (a) A-chain analogues modified by pairs of boron-containing moieties (BCMs) at or near its N terminus and (b) B-chain analogues modified by diol moieties at or near its C terminus. Such co-modifications permit a ligand-regulated reversible conformational cycle between active and inactive states of insulin, where the ligand is preferably glucose (
Although analogous glucose-displaceable bridges may be placed at other sites in the insulin molecule, the present scheme and its reverse (i.e., diol modification of the A chain at or near its N terminus with cognate modification of the B chain by pairs of boron-containing moieties, SEQ IDs offer the unique advantage of a closed state that mirrors that ultra-stable structure of a “mini-proinsulin” in which a direct peptide bond connects a C-terminal B chain residue (B28, B29, or B30) to GlyA1 or in which 1-3 intervening residues are placed between residues B30 and A1 as foreshortened connecting domains. Such tethering of the C terminus of the B chain to the N terminus of the A chain constrains conformational fluctuation otherwise associated with chemical and physical degradation of insulin in pharmaceutical formulations. Accordingly, applicant anticipates that, in addition to conferring the desired functional properties of a glucose-regulated switch in vivo, the present class of designs confers augmented shelf life of a pharmaceutical formulation in the absence of glucose or competing diol in the solution.
In accordance with one embodiment a novel design scheme is provided that reduces to practice a glucose-cleavable tether between the C terminus of the B chain and N terminus of the A chain, thereby providing a novel class of insulin analogues that would be closed and inactive at low glucose concentration but open and active at high glucose concentration. The resulting cycle of conformational states (
In accordance with another embodiment an insulin analogue is provided comprising a first and second glucose-binding element, wherein the first and second glucose-binding element each comprises a boron-containing diol-binding moiety. The insulin analogue comprises any of the known analogues of the native insulin A chain and B chain. In one embodiment an insulin A chain peptide and an insulin B chain peptide are provided, wherein said insulin A chain and B chain peptides are linked to each other by disulfide bonds, further wherein either
In one embodiment the insulin analogue comprises a first and second glucose-binding elements linked to a first and second amino acid, respectively, of the insulin A chain peptide, wherein the first and second amino acids are located at positions i and i+1 or i and i+2 relative to each other. In one embodiment the first and second glucose-binding elements are bound to each other via a linker to form a complex, wherein the complex is covalently linked to a single amino acid of the insulin A chain peptide. In one embodiment the first and second glucose-binding elements are linked to the side chains of one or more amino acids of the insulin A chain peptide. In another embodiment the first and second glucose-binding elements are linked to the backbone amide nitrogens of one or more amino acids of the insulin A chain peptide. In one embodiment the first and second glucose-binding elements, each comprising a boron-containing diol-binding element, are covalently linked to the side chain of an amino acid selected from the group consisting of
The analogues of the present invention may optionally contain an additional saccharide-binding element attached to residue B1 as a mechanism intended to provide glucose-sensitive binding of the insulin analogue to surface lectins in the subcutaneous depot. In addition, the analogues of the present invention may optionally contain substitutions known in the art to confer rapid action (such as AspB28, a substitution found in insulin aspart (the active component of Novolog®); [LysB28, ProB29], pairwise substitutions found in insulin lispro (the active component of Humalog®); GluB29 or the combination [LysB3, GluB29] as the latter is found in insulin glulisine (the active component of Apridra®), or modifications at position B24 associated with accelerated disassembly of the insulin hexamer (e.g., substitution of PheB24 by Cyclohexanylalanine or by a derivative of Phenylalanine containing a single halogen substitution within the aromatic ring). Alternatively, the analogues of the present invention may optionally contain modifications known in the art to confer protracted action, such as modification of the ε-amino group of LysB29 by an acyl chain or acyl-glutamic acid adduct as respectively illustrated by insulin detemir (the active component of Levemir®) and insulin degludec (the active component of Tresiba®); or contain basic amino-acid substitutions or basic chain extensions designed to shift the isoelectric point (pI) to near neutrality as exemplified by the ArgB31-ArgB32 extension of insulin glargine (the active component of Lantus®). Analogues of the present invention designed to exhibit such a shifted pI may also contain a substitution of AsnA21, such as by Glycine, Alanine or Serine. Analogues of the present invention may optionally also contain non-beta-branched amino-acid substitutions of ThrA8 associated with increased affinity for the insulin receptor and/or increased thermodynamic stability as may be introduced to mitigate deleterious effects of the primary two above design elements (a phenylboronic acid derivative at or near the N-terminus of the A chain and one or more saccharide derivatives at or near the C-terminus of the B chain) on receptor-binding affinity and/or thermodynamic stability. Examples of such A8 substitutions known in the art are HisA8, LysA8, ArgA8, and GluA8.
The insulin analogues of the present invention may exhibit an isoelectric point (pI) in the range 4.0-6.0 and thereby be amenable to pharmaceutical formulation in the pH range 6.8-7.8; alternatively, the analogues of the present invention may exhibit an isoelectric point in the range 6.8-7.8 and thereby be amenable to pharmaceutical formulation in the pH range 4.0-4.2. The latter conditions are known in the art to lead to isoelectric precipitation of such a pI-shifted insulin analogue in the subcutaneous depot as a mechanism of protracted action. An example of such a pI-shifted insulin analogue is provided by insulin glargine, in which a basic two-residue extension of the B chain (ArgB31-ArgB32) shifts the pI to near-neutrality and thus enables prolonged pharmacokinetic absorption from the subcutaneous depot. In general, the pI of an insulin analogue may be modified through the addition of basic or acidic chain extensions, through the substitution of basic residues by neutral or acidic residues, and through the substitution of acidic residues by neutral or basic residues; in this context we define acidic residues as Aspartic Acid and Glutamic Acid, and we define basic residues as Arginine, Lysine, and under some circumstances, Histidine. We further define a “neutral” residue in relation to the net charge of the side chain at neutral pH.
It is an additional aspect of the present invention that absolute in vitro affinities of the insulin analogue for insulin receptor (isoforms IR-A and IR-B) are in the range 5-100% relative to wild-type human insulin and so unlikely to exhibit prolonged residence times in the hormone-receptor complex; such prolonged residence times are believed to be associated with enhanced risk of carcinogenesis in mammals or more rapid growth of cancer cell lines in culture. It is yet an additional aspect of the present invention that absolute in vitro affinities of the insulin analogue for the Type 1 insulin-like growth factor receptor (IGF-1R) are in the range 5-100% relative to wild-type human insulin and so unlikely either to exhibit prolonged residence times in the hormone/IGF-1R complex or to mediate IGF-1R-related mitogenesis in excess of that mediated by wild-type human insulin.
The insulin analogues of the present invention consist of two polypeptide chains that contain a novel paired PBA-PBA, PBA-BXB, BXB-PBA and/or BXB-BXB modifications in the A chain such that the analogue, in the absence of glucose or other exogenous saccharide, contains covalent bonds the paired boron-containing elements and diol adducts in the B chain. Although we do not wish to be restricted by theory, we envisage that these two design elements form a covalent interaction in the absence of exogenous glucose such that the structure of the hormone is stabilized in a less active conformation. Two alternative design schemes are envisioned that would follow the same principles to provide a glucose-responsive insulin. The first switches the positions of the glucose-binding elements and diol modifications such that the former are attached at or near the C terminus of the B chain whereas the latter are attached at or near the N terminus of the A chain. The second embodiment replaces diol modifications by glucose-binding elements such that both chains are modified by pairs of boron-containing moieties.
While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
We envisage that such an insulin analogue formulation would be compatible with multiple devices (such as insulin vials, insulin pens, and insulin pumps) and could be integrated with modifications to the insulin molecule known in the art to confer rapid-, intermediate-, or prolonged insulin action. In addition, the present glucose-regulated conformational switch in the insulin molecule, engineered between the C-terminus of the B chain and N-terminus of the A chain, could be combined with other glucose-responsive technologies (such as closed-loop systems or glucose-responsive polymers) to optimize their integrated properties. We thus envisage that the products of the present invention will benefit patients with either Type 1 or Type 2 diabetes mellitus both in Western societies and in the developing world.
In one embodiment a method of treating a diabetic patient while decreasing the risk of hypoglycemia is provided. In accordance with the present disclosure the method comprising administering a physiologically effective amount of any of the insulin analogues disclosed herein that comprise a glucose-binding elements, or a physiologically acceptable salt thereof, to the patient.
In one embodiment a glucose responsive insulin analogue is provided wherein the insulin analogue comprises
In one embodiment the boron-containing diol-binding moiety of said first and second glucose-binding elements of the insulin analogue are independently selected from the group consisting of BCM1-BCM12 as shown in
linked to the side chain or backbone amide of the insulin B chain peptide. In one embodiment the first and second glucose-binding elements are bound to each other via a linker to form a complex having the general structure
wherein BCM1 and BCM2 are boron-containing moieties; n and m are independently an integer selected from the range of 1 to 3;
In one embodiment a glucose-sensing insulin analogue is provided wherein the insulin A chain peptide
In one embodiment the insulin analog comprises an insulin A chain peptide comprising a sequence of GX0GIVEQX6CX8SIX11SLYQLENYCX21 (SEQ ID NO: 13);
In one embodiment the glucose sensing insulin analog comprises an insulin A chain peptide comprising a sequence of GIVEQCCX8SICSLYQLENYCX21 (SEQ ID NO: 27); and said B chain comprises the sequence the sequence FVKQX25LCGSHLVEALYLVCGERGFFYTEKT (SEQ ID NO: 28), FVNQX25LCGSHLVEALYLVCGERGFFYTDKT (SEQ ID NO: 29), FVNQX25LCGSHLVEALYLVCGERGFFYTKPT (SEQ ID NO: 30), FVNQHLCGSHLVEALYLVCGERGFFYTKKP (SEQ ID NO: 31) or FVNQX25LCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 32) wherein X8 is selected from the group consisting of threonine and histidine; X21 is selected from the group consisting of asparagine, glycine and alanine; X25 is selected from the group consisting of histidine and threonine, and a lysine at position 28 or 29 of said B chain has been modified to comprise a diol adduct.
In accordance with one embodiment any of the glucose-sensing insulin analogues disclosed herein can be provided with two or more glucose-binding moieties that comprise the same boron-containing diol-binding motif. In one embodiment the boron-containing diol-binding moiety is phenylboronic acid, a halogen-modified fluorophenylboronic acid or benzoboroxole. The glucose-binding elements of the present invention contain two or more boron atoms, and so this encompasses pairs, triplets or high-order combinations of single boron-containing moieties (such as PBA and Bxb); these can be respectively attached to different amino acids selected from positions A−1, A0 and/or A1; or they can be moieties within a single complex adduct attached to only one of these peptide sites (see
Representative examples of the glucose-responsive insulin analogues of the present invention were prepared for functional testing in mammalian cell culture in the context of insulin lispro such that the epsilon-amino group of LysB28 was modified by 2, 3-dihydroxybenzoic acid (DHBA), thereby providing a diol at or near the C terminus of the B chain. The A chain was modified by a
We note in passing that strengths of clinical insulin formulations are measured not in molar concentrations, but instead in relation to biological potency in a standard test animal (usually rabbits). For example, Levemir contains four times the hormone concentration in molar units as other clinical insulin products, but is nonetheless labeled as “U-100” in strength. This is because the per molecule activity of insulin detemir (the active component of Levemir) is fourfold reduced relative to human insulin. By the same token, the per-molecule activity of the present insulin analogues are typically reduced by the A-chain modifications or by the B-chain modifications. U-100 strength can nonetheless be achieved by increasing the analogue's protein concentration by analogy to Levemir. The example of GRI-1 highlighted its Levemir-like reduction in per-molecule potency, whose precise value was observed to depend on the blood-glucose concentration. Thus, experimental design exploited prior baseline studies of intrinsic potency to equalize activities rather than to equalize molar concentrations (see
Control studies of “partial analogues” (i.e., containing only an A-chain-linked GBE or only a B-chain-linked diol moiety) were undertaken to test whether both components of the designed switch would be required for (a) a change in protein conformation or (b) glucose-dependent activation of biological activity. These controls were analogous to those described by Chen, Y.-S., et al. (2021) in the case of a fructose-responsive insulin (FRI). Protein conformation and glucose binding were monitored by 1H-13C HSQC NMR spectra. Glucose-dependent activities were assessed in normal rats versus streptozotocin (STZ)-induced diabetic rats. These control data validated that, as in the FRI, GRI-1 requires both components of the switch in the same protein molecule as envisioned in our design scheme. Neither “partial analogue” exhibited GRI-like functional properties.
The closed-open transition of the present GRI analogues was further validated by cryo-EM single-particle image reconstruction employing the isolated ectodomain of the human insulin receptor. Complexes were made either in the absence of glucose or in the presence of 50 mM glucose. Whereas addition of glucose led to an ectodomain-hormone structure essentially identical to that of the wild-type complex in its signaling conformation (Weis, F., et al. The signalling conformation of the insulin receptor ectodomain. Nature Communications 9(1), 4420 (2018)), in the absence of glucose the structure was of lower resolution and without binding of the insulin analogue to “Site 1” of the receptor; the overall ectodomain conformation was not in the active, signaling state. Thus, the predicted mechanism underlying the present class of GRI analogues was explicitly visualized in these studies.
Another, related prototype GRI (designated GRI-2) was prepared that differed from GRI-1 in one respect: the same dual-boron-containing GBE (GBE1-C) was attached to
Yet another related prototype GRI (designated GRI-3) was prepared that differed from GRI-1 in only by substitution of cystine A6-A11 by a diselenide bridge (SecA6 and SecA11). This modification stabilizes insulin as described by Weil-Ktorza, O., et al. (Substitution of an Internal Disulfide Bridge with a Diselenide Enhances both Foldability and Stability of Human Insulin. Chemistry: a European Journal 25(36), 8513-21 (2019)). GRI-3 was tested in HepG2 cells in culture (as described in Chen, Y.-S., et al. (2021) and observed to exhibit glucose-dependent activity.
Yet another related prototype GRI (designated GRI-4) was prepared that differed from GRI-2 in only by substitution of cystine A6-A11 by a diselenide bridge (SecA6 and SecA11). This modification stabilizes insulin as described by Weil-Ktorza, O., et al. (Substitution of an Internal Disulfide Bridge with a Diselenide Enhances both Foldability and Stability of Human Insulin. Chemistry: a European Journal 25(36), 8513-21 (2019)). GRI-4 was tested in HepG2 cells in culture (as described in Chen, Y.-S., et al. (2021) and observed to exhibit glucose-dependent activity.
A related prototype GRI (designated GRI-5) was prepared that differed from GRI-1 in two respects: (i) the aromatic diol moiety (DHBA) was attached to an Ornithine side chain at position B28, as substituted for LysB28 in insulin lispro; and (ii) the same dual-boron-containing moiety (GBE1-C) was attached to
A fructose-responsive insulin (FRI) was prepared to demonstrate proof of principle as described in Chen, Y.-S., et al. (2021) and incorporated by reference herein. This provided a model for Examples 1-5 above.
A switchable insulin analogue (designated FRI; fructose-responsive insulin) contains meta-fluoro-PBA* (meta-fPBA or m-fPBA) as a diol sensor linked to the α-amino group of GlyA1 and an aromatic diol (3,4-dihydroxybenzoic acid; DHBA) attached to the e-amino group of LysB28 of insulin lispro. Although fructose and glucose each contain diols, the sensor preferentially binds to aligned 1,2-diol groups as found in β-
Western Blot Assays Demonstrated Fructose-Dependent Signaling. Structural studies suggest that insulin's hinge-opening at a dimer-related αCT/L1 interface is coupled to closure of IR ectodomain legs, leading to TK-mediated trans-phosphorylation and receptor activation. Signal propagation was probed via a cytoplasmic kinase cascade and changes in metabolic gene expression in HepG2 cells. Control studies indicated that addition of 0 to 100 mM fructose or glucose did not trigger changes in signaling outputs. An overview of IR autophosphorylation (probed by anti-pTyr IR antibodies) and downstream phosphorylation of Ser-Thr protein kinase AKT (protein kinase B; ratio p-AKT/AKT), forkhead transcription factor 1 (p-FOXO1/FOXO1), and glycogen synthase kinase-3 (p-GSK-3/GSK-3) at a single hormone dose (50 nM) was provided by Western blot (WB). In each case, WBs demonstrated fructose-dependent signaling by FRI and fructose-independent signaling by KP and DFC. The activity of FRI in the absence of fructose is low.
Plate Assays Demonstrated Ligand-Selective Signaling. Quantitative dose-dependent and ligand-selective IR autophosphorylation were evaluated in a 96-well plate assay. FRI triggered a robust signal on addition of 50 mM fructose whereas baseline activity in the absence of fructose was low. As expected, KP and DFC exhibited high signaling activity in the presence or absence of fructose, respectively). Ligand-dependent activation of FRI is specific to fructose as addition of 50 mM glucose did not influence its activity (nor the activities of KP and DFC). These data indicate that in 50 mM fructose FRI is almost as active as KP.
PCR Assays Demonstrated Ligand-Selective Metabolic Gene Regulation. Insulin-signaling in hepatocytes extends to metabolic transcriptional regulation as recapitulated in HepG2 cells. At hypoglycemic conditions, the cells exhibited stronger gluconeogenesis-related responses following insulin stimulation than at hyperglycemic conditions. In this protocol, FRI, when activated by fructose, regulated downstream expression of the gene encoding phosphoenolpyruvate carboxykinase (PEPCK; a marker for hormonal control of gluconeogenesis). Under normoglycemic conditions, FRI, when activated by fructose, regulated the genes encoding carbohydrate-response-element and sterol response-element binding proteins (ChREBP and SREBP; markers for hormonal control of lipid biosynthesis). No fructose dependence was observed in control studies of KP and DFC; no effects were observed on addition of glucose instead of fructose. Control studies were undertaken in the absence of insulin analogues to assess potential confounding changes in metabolic gene expression on addition of 0 to 100 mM fructose or 0 to 100 mM glucose. No significant effects were observed in either case, indicating that the present short-term fructose exposure (to activate FRI) is unassociated with the transcriptional signature of longer-term exposure.
Ligand-Binding to FRI Affects Protein Structure. Far-UV circular dichroism (CD) spectra of FRI and DFC are indistinguishable from parent analogue insulin lispro (KP), indicating that secondary structure is not affected by the modifications at A1 and B28. Difference CD spectra calculated on addition of 100 mM fructose or glucose were in each case featureless. High-resolution NMR spectroscopy [as enabled by the monomeric KP template] corroborate essential elements of the intended fructose-selective switch
19F-NMR spectra monitored fructose sensor. The fluorine atom in meta-fPBA provided an NMR-active nucleus. Addition of 0 to 100 mM fructose led to an upfield change in 19F-NMR chemical shift in slow exchange on the NMR time scale. This upfield shift presumably reflects displacement of an aromatic diol by a nonaromatic ligand. No change in FRI 19F chemical shift was observed on addition of glucose. Although an analogous 19F resonance was observed in the NMR spectrum of DFC, its chemical shift did not change on addition of glucose or fructose. Interestingly, a broadened 19F signal was observed in ligand-free DFC, probably due to conformational exchange or self-association; this signal sharpened on addition of ligand (fructose or glucose).
Dual 19F- and 1H NMR-monitored titration and natural-abundance 1H-13C heteronuclear single quantum coherence (HSQC) spectra provided further evidence of a specific interaction between FRI and fructose.
1H-13C 2D HSQC spectra monitored “closed” conformation of ligand-free FRI. One-dimensional (1D) 1H and 1H-13C HSQC spectra of DFC were similar to those of parent analogue insulin lispro (KP), excepting methyl resonances of IleA2 and ValA3 (adjacent to the GlyA1-attached meta-fPBA). Patterns of 1H-13C chemical shifts of FRI and DFC were also similar. Those NMR features provided evidence that FRI and DFC retain a native-like structure. However, in FRI, the resonances of IleA2, ValA3, LeuB11, ValB12, and LeuB15 exhibited larger chemical shift differences (relative to KP) than in DFC. These findings suggest that FRI exhibits a local change in conformation and/or dynamics, presumably due to the intended DHBA/meta-fPBA tether. We envision that constraining the C-terminal B-chain segment alters aromatic ring currents affecting the central B-chain α-helix (via TyrB26-LeuB11, TyrB26-ValB12, and PheB24-LeuB15 packing) and N-terminal A-chain helix (via native-like TyrB26-IleA2 and TyrB26-ValA3 packing).
Aromatic 1H-13C two-dimensional (2D) HSQC spectra monitor hinge-opening. 1H-13C HSQC spectra provide probes of aromatic resonances in FRI's DHBA/meta-fPBA adducts in the absence of fructose and in the presence of 100 mM fructose. Significant chemical shift changes in both 1H/13C dimensions were observed. Resonance assignments were corroborated by model studies of meta-fPBA- and DHBA-modified peptides. DHBA chemical shifts in fructose-free FRI are similar to those in the complex of model peptides, whereas such chemical shifts in fructose bound FRI are similar to that of free DHBA-modified octapeptide. In addition, methyl resonances sensitive to addition of fructose exhibited a trend toward corresponding chemical shifts observed in spectra of insulin lispro and ligand-free DFC. Together, these NMR features provide evidence that in FRI the LysB28-attached DHBA binds GlyA1-linked meta-fPBA in absence of fructose, but this tether is displaceable by fructose.
Methyl 1H-13C 2D HSQC spectra monitor protein core. Aliphatic 1H-13C spectra reflect tertiary structure as probed by upfield-shifted methyl resonances. Changes in cross-peak chemical shifts were observed in FRI on overlay of spectra acquired in the absence of an added monosaccharide or on addition of 100 mM fructose. Fructose-binding accentuated upfield 1H secondary shifts with smaller changes in 13C chemical shifts. These changes presumably reflect altered aromatic ring currents within insulin's core. Control studies of DFC suggested that such chemical shift changes require the interchain DHBA/meta-fPBA tether; in these spectra, changes were restricted to IleA2 immediately adjoining the sensor. Addition of 50 mM glucose caused essentially no changes in 1H-13C fingerprints of FRI or DFC in accordance with the fructose selectivity of meta-fPBA.
Methods pertaining to these assays, in addition to those described above, are as follows.
Chemical Synthesis of Single-Chain Insulin Precursors (SCIs) and Analogues. We employed solid-phase peptide synthesis to prepare an extensive collection of single-chain insulin precursors (Table 1). The peptides were synthesized starting with Pre-loaded Fmoc-Asn(Trt)-HMBA-CM resins using traditional Fmoc/tBu chemistry with repetitive DIC/6-Cl-HOBt activation/coupling cycles using DIC/6-Cl-HOBt activation (10 Equivalents) and IR or induction heating at 60° C. for 10 min per cycle and 50° C. for Fmoc deprotection (20% piperidine/DMF, 2×5 min). Tribute or Chorus automated peptide synthesizers (Gyros Protein Technology, Tucson, AZ) were used. All amino acids, DIC and 6-Cl-HOBt were purchased from Gyros Protein Technology (Tucson, AZ) or ChemImpex (Chicago, Ill). Peptides were cleaved from resin and deprotected by treatment with trifluoroacetic acid (TFA) containing 2.5% triisopropylsilane (TIS), 2.5% water, 2.5% DODT (ethylenedioxy)-diethenethiol), and 2.5% of Anisole for 3-4 hr.
SCIs were chemically synthesized using Fmoc/OtBu solid-phase chemistry on a Pre-loaded H-Asn(Trt)-HMBP-Chemmatrix resin, with repetitive coupling cycles using DIC/6-Cl-HOBt or DIC/Oxyma Pure (Ethyl cyano(hydroxyimino)acetate) activation (10 Equivalents) and IR or induction heating at 60° C. for 10 min per cycle and 50° C. for Fmoc deprotection (20% piperidine/DMF, 2×5 min). Tribute or Chorus automated peptide synthesizers (Gyros Protein Technology, Tucson, AZ) were used. Pre-loaded Fmoc-Asn(Trt)-Chemmatrix resin was used. All amino acids, DIC and 6-Cl-HOBt and Oxyma Pure were purchased from Gyros Protein Technology (Tucson, AZ). The peptide was cleaved and deprotected by treatment with trifluoroacetic acid (TFA) containing 2.5% triisopropylsilane (TIS), 2.5% water, 2.5% DODT (ethylenedioxy)-diethenethiol), and 2.5% of anisole. Peptides containing Sec(Mob) were cleaved in presence of 2,2′-dithio-bis-(5-nitropyridine) (DTNP, 2 equivalents per Sec; see K. M. Harris, S. Flemer Jr, R. J. Hondal, J. Pept. Sci. 2007, 13, 81-93). Cleavage mixture was precipitated with ether (5-10 fold with respect to TFA) and solid was isolated by centrifugation. Precipitate was further washed with twice with ether and dried in vacuo.
Oxidative folding of Single-chain Insulins. Crude linear reduced insulin precipitated and dried (in vacuo) were suspended and air oxidized at 0.1 mM in a folding buffer (Cys, 2.0 mM, Gly 20 mM, pH adjusted to 10.5 with NaOH (10 M)) with vigorous stirring for 17-48 h at 10° C. The reaction was monitor by RP-HPLC and LC-MS for reaction completion. Preparative HPLC was carried out using a C8 column was used for purification. Crude reaction was acidified (HCl, 5M to pH 3) filtered (0.2 μM) and then purified identity of the SCI was confirmed by LC-MS (Finnigan LCQ Advantage, Thermo) on a TARGA C8 (4.6×250 mm, 5 μm, Higgins Analytical) with 0.1% TFA/H2O (A) and 0.1% TFA/CH3CN as eluents.
To generate the des-octapeptide insulin (DOI), the dried single-chain DesDI precursor was treated with Trypsin-TPCK (10% w/w) in 1 M urea and 0.1 M ammonium bicarbonate for 24 h at room temperature. After completion of the cleavage as indicated by HPLC, the DOI was purified by preparative RP-HPLC on a C4 or C8 (20×250 mm, 5 μm, Higgins Analytical) column with 0.1% TFA/H2O (A) and 0.1% TFA/CH3CN (B) as elution buffers.
Semi-synthesis with Diol containing octapeptides. The DOI from the above step was combined with de protected octapeptide Gly-Phe-Phe-Tyr-Thr-Lys(DHBA)-Pro-Thr (reacted as a crude or previously HPLC-purified) in presence of 10% w/w Trypsin-TPCK (K. Inouye, K. Watanabe, K. Morihara, Y. Tochino, T. Kanaya, J. Emura, S. Sakakibara, J. Am. Chem. Soc. 1979, 101, 751-752). Generally, a 1:5 molecular ratio was used for trypsin-mediated ligation, typically 3-9 mg of DOI were dissolved along with similar 3-9 mg of octapeptide in 200 μl of a mixed solvent system containing Tris-acetate (pH 8.5), 1,4-butanediol and dimethylacetamide. pH was adjusted to 7.5 with 2 μl of 4-Methylmorpholine and the reaction was carried out for 24-48 h. Full-length insulin product proteins was purified by preparative RP-HPLC on a C8 column with 0.1% TFA/H2O (A) and 0.1% TFA/CH3CN (B) as elution buffers. Identity was confirmed by LC-MS. The DOI precursor and resultant GRI candidate compounds are given in the SEQ ID section. After semi-synthesis, reaction mixtures were purified by RP-HPLC as described above. The general disulfide conjugation method is as follows: GBE-1-C(NPYS)-OH, 1.5 equivalents were reacted with an SCI containing a free thiol group (2-4 mg/ml). The reaction are performed in a buffer consisting of ammonium bicarbonate (0.1 M), urea (1M) at pH 8.5 and at room temperature for 15-60 min. The reaction mixture was re-purified by RP-HPLC.
Cell culture. Human hepatocellular carcinoma cell line HepG2 was cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin as recommended by the American Type Culture Collection. A protocol employing 24-h serum starvation wherein FBS was applied at 70-75% confluence. After starvation, cells were treated in parallel with a set of insulin analogues in serum-free medium.
Real-time qPCR assays. Following serum starvation, HepG2 cells were treated with medium containing an insulin analogue (50 nM) for 8 h. In studies related to possible glucose responsiveness and lipid metabolism, the cells were treated with analogues for 3 or 4 h in media containing either low or normal glucose concentrations. Readouts were provided by downregulation of PEPCK and G6P and upregulation of ChREBP and SREBP. mRNA (messenger ribonucleic acid) abundances were measured in triplicate by quantitative polymerase chain reaction (qPCR). Samples were prepared as described by the vendor (One-Step rt-PCR reagent kits; Bio-Rad). The following sets of primers were used: (PEPCK), GTTCAATGCCAGGTTCCCAG (SEQ ID NO: 33) and TTGCAGGCCAGTTGTTGAC (SEQ ID NO: 34); (ChREBP), AGAGACAAGATCCGCCTGAA (SEQ ID NO: 35) and CTTCCAGTAGTTCCCTCCA (SEQ ID NO: 36); (SREBP), CGACATCGAAGACATGCTTCAG (SEQ ID NO: 37) and GGAAGGCTTCAAGAGAGGAGC (SEQ ID NO: 38); and (GAPDH), ATGGTTTACATGTTCCAATAT (SEQ ID NO: 39) and ATGAGGTCCACCACCCTGGTTG (SEQ ID NO: 40).
In-cell pIR immunoblotting. This cell-based assay probed insulin-dependent IR activation via fluorescent readouts. HepG2 cells were seeded (˜8000 cells/well) into a 96-well black plate with clear bottom and cultured (Fisher). After serum starvation in 100 μl plain Hanks' Balanced Salt Solution (HBSS) for 2 h at 37° C., serial analogue dilutions (100 μl) were applied to each well; cells were then incubated for 20 min at 37° C. After removing medium, 150 μl of 3.7% formaldehyde (Fisher) was added to each well, and plates incubated for 20 min at 37° C. 200 μl of 0.1% Triton-X-100 (Sigma) was then added to permeabilize the cells, followed by their fixation by 100-μl Odyssey Blocking Buffer (LI-COR). A blocking procedure was applied for 1 h at room temperature on an orbital shaker. Fixed cells were then exposed to the primary antibody (10 μL anti-pTyr 4G10 (Sigma) into 20 ml Blocking Buffer) overnight at 4° C. The secondary antibody (anti-mouse-IgG-800-CW antibody (Sigma) in 25 ml Blocking Buffer) was added after a wash. pTyr was detected via 800 nm emission. DRAQ5 (Fisher) was also applied to enable measurement of cell number via 700 nm emission. The fluorescence signals were detected on a LI-COR Infrared Imaging system (Odyssey) under settings as follows: offset 4 mm with setting “Intensity-Auto.”
Signaling assays in a mammalian cell line. HepG2 cell line (ATCC) were cultured in DMEM with 10% FBS, 1% penicillin/streptomycin per vendor's instruction. After 70-75% cell confluence, cells were serum-starved for 24 h and then exposed to medium containing an insulin analogue (each at a protein concentration of 50 nM). After lysis in RIPA buffer (containing protease- and phosphatase-inhibitor cocktails; Roche), the total protein concentrations in the lysate was determined by BCA assay (Thermo). Blotting protocols were modified from previous publication. Briefly, for p-IR/IR blotting, samples were probed by insulin receptor β (4B8) antibody (CST antibodies unless otherwise stated) or an equal mixture of anti-phospho-insulin receptor β (Tyr1150/1151); phospho-insulin receptor (Tyr1158) antibody (Thermo); phospho-insulin receptor (Tyr1334) antibody (Thermo); phospho-insulin receptor β (Tyr1345) monoclonal antiserum; and anti-phospho-insulin receptor (phospho-Tyr972) antibody (Abcam). Dilutions for these antibodies were 1:5000 in 5% bovine serum albumin. Antibodies for AKT blotting were p-AKT antibody (Ser473) (1:400) and AKT1/2/3 antiserum (H-136) (1:1000). For p-FOXO/FOXO and p-GSK-3/GSK-3 blotting, samples were probed by phospho-Fox01 (Thr24)/FoxO3a (Thr32) antibody, FOXO1 antibody, phospho-GSK-3α/β (Ser21/9), and GSK-3α/β antibody (purchased from CST; dilution 1:1000).
NAMES AND SEQUENCES are summarized in Table 1 as follows. GRI-1, 2, 3, 4 and 5 are labeled in appropriate rows. “Single-chain sequences” and des-octapeptide[B23-B30]-insulin analogue fragments (“DOI”s) pertain to synthetic intermediates and not to GRIs of the present invention.
C)GIVEQCCTSICSLYQLENYCN (SEQ ID NO:
BA)PT (SEQ ID NO: 89)_G[dC(GBE1-
Dap(Fmoc))]IVEQCCHSICSLYQLENYCN (SEQ
Dap(NH2))]IVEQCCHSICSLYQLENYCN (SEQ
Dap(Mtt)]IVEQCCHSICSLYQLENYCN (SEQ
Dap(NH2)]IVEQCCHSICSLYQLENYCN (SEQ
Gly)]IVEQCCHSICSLYQLENYCN (SEQ ID NO:
Dap(BCM2))]IVEQCCHSICSLYQLENYCN
Dap(BCM2))]IVEQCCHSICSLYQLENYCN
BA)PT(SEQ ID NO: 89)_GC(GBE1-
C)IVEQCCTSICSLYQLENYCN (SEQ ID NO:
C)GIVEQUCTSIUSLYQLENYCN
C)IVEQUCTSIUSLYQLENYCN (SEQ ID NO:
C)]IVEQCCTSICSLYQLENYCN (SEQ ID NO:
C(Acm)GIVEQCCHSICSLYQLENYCN (SEQ
G[APD] (SEQ ID NO: 126) GdC(GBE1-
G[APD] (SEQ ID NO: 126)
BE5)PT (SEQ ID NO: 124)
GBE5)]PT (SEQ ID NO: 124)
E1)]PT (SEQ ID NO: 128)
E1)]PT (SEQ ID NO: 128)
BE2)PT (SEQ ID NO: 124)
BE2)PT (SEQ ID NO: 124)
E1)]PT (SEQ ID NO: 128)
E1)]PT (SEQ ID NO: 128)
N(APD)[DOPA]GIVEQCCTSICSLYQLENYCN
E1)]PT (SEQ ID NO: 128)
The present disclosure relates to polypeptide hormone analogues that contain a glucose-regulated molecular structure or glucose-detachable molecular moiety, designed respectively either (a) to confer glucose-responsive binding to cognate cellular receptors and/or (b) to enable glucose-mediated liberation of an active insulin analogue. More particularly, the present disclosure is directed to insulin analogues that are responsive to blood-glucose concentrations and their use in the treatment of patients and non-human mammals with Type 1 or Type 2 diabetes mellitus by subcutaneous, intraperitoneal or intravenous injection of the insulin analogues disclosed herein.
The insulin analogues of the present invention may also exhibit other enhanced pharmaceutical properties, such as increased thermodynamic stability, augmented resistance to thermal fibrillation above room temperature, decreased mitogenicity, and/or altered pharmacokinetic and pharmacodynamic properties. More particularly, this disclosure relates to insulin analogues that may confer either rapid action (relative to wild-type insulin in its regular soluble formulation), intermediate action (comparable to NPH insulin formulations known in the art) or protracted action (comparable to basal insulins known in the art as exemplified by insulin detemir and insulin glargine) such that the affinity of the said analogues for the insulin receptor is higher when dissolved in a solution containing glucose at a concentration above the physiological range (>140 mg/dL; hyperglycemia) than when dissolved in a solution containing glucose at a concentration below the physiological range (<80 mg/dL; hypoglycemia).
In accordance with one embodiment an insulin analogue is provided comprising an A chain modified by a glucose-binding element at or near its N terminus and a variant B chain comprising a diol group at the C terminus of the B chain such that the polypeptide chain ends with a hydroxyl group rather than with a carboxylate group. Reduced or absent activity is associated with formation of a covalent bond between the unique diol moiety in the B chain and a second molecular entity located at the N-terminus of the A chain and that contains a glucose-binding element. Displacement of the B chain diol from the A-chain-linked glucose-binding element by glucose would lead to detachment of the tethered molecular entity, which in turn enables high-affinity receptor binding. In the absence of glucose the C-terminus diols remain bound to the A-chain-linked glucose-binding element and the insulin analogue remains inactive.
In accordance with one embodiment the modified B chain may contain a broad molecular diversity of diol-containing moieties (or adducts containing an α-hydroxycarboxylate group as an alternative binding motif that might bind to a glucose-binding element), whether a saccharide or a non-saccharide reagent. Possibilities include an N-linked or O-linked saccharide or any organic moiety of similar molecular mass that contains a diol function that mimics the diol function of a monosaccharide and hence confers reversible PBA-binding activity (or adducts containing an α-hydroxycarboxylate group as an alternative PBA-binding function; PBA in the present invention may equivalently be substituted by other boron-containing diol-binding elements as known in the art to bind glucose). Such non-saccharide diol-containing organic compounds span a broad range of chemical classes, including acids, alcohols, thiol reagents containing aromatic and non-aromatic scaffolds; adducts containing an α-hydroxycarboxylate group may provide an alternative function able to bind PBA or other boron-containing diol-binding elements able to bind glucose. Convenient modes of attachment to the B chain also span a broad range of linkages in addition to the above N-linked and O-linked saccharide derivatives described above; these additional modes of attachment include (i) the side-chain amino function of Lysine, ornithine, diamino-butyric acid, diaminopropionic acid (with main-chain chirality L or D) and (ii) the side-chain thiol function of Cysteine or homocysteine (with main-chain chirality L or D). A preferred embodiment at sites of native aromatic acids (positions B16, B25 and B26) is provided by
The molecular purpose of the diol-modified B chain is to form an intramolecular bond or bonds with the A-chain-attached glucose-binding element such that the conformation of insulin is “closed” and so impaired in binding to the insulin receptor. Use of a main-chain-directed diol recapitulates the inactive structure of a single-chain insulin analogue. We envision that at high glucose concentrations, the diol-glucose-binding element bond or bonds will be broken due to competitive binding of the glucose to the glucose-binding element. Preferred embodiments contain two or more diol groups in an effort to introduce cooperativity. The main-chain element can be via substitution of the C-terminal carboxylate by a hydroxyl group together with an appropriately positioned side-chain hydroxyl group and/or via a moiety attached to the main-chain nitrogen atom. The analogues of the present invention may optionally contain an additional saccharide-binding element attached to residue B1 as a mechanism intended to provide glucose-sensitive binding of the insulin analogue to surface lectins in the subcutaneous depot. In addition, the analogues of the present invention may optionally contain substitutions known in the art to confer rapid action (such as AspB28, a substitution found in insulin aspart (the active component of Novolog®); [LysB28, ProB29], pairwise substitutions found in insulin lispro (the active component of Humalog®); GluB29 or the combination [LysB3, GluB29] as the latter is found in insulin glulisine (the active component of Apridra®), or modifications at position B24 associated with accelerated disassembly of the insulin hexamer (e.g., substitution of PheB24 by Cyclohexanylalanine or by a derivative of Phenylalanine containing a single halogen substitution within the aromatic ring). Alternatively, the analogues of the present invention may optionally contain modifications known in the art to confer protracted action, such as modification of the ε-amino group of LysB29 by an acyl chain or acyl-glutamic acid adduct as respectively illustrated by insulin detemir (the active component of Levemir®) and insulin degludec (the active component of Tresiba®); or contain basic amino-acid substitutions or basic chain extensions designed to shift the isoelectric point (pI) to near neutrality as exemplified by the ArgB31-ArgB32 extension of insulin glargine (the active component of Lantus®). Analogues of the present invention designed to exhibit such a shifted pI may also contain a substitution of AsnA21, such as by Glycine, Alanine or Serine. Analogues of the present invention may optionally also contain non-beta-branched amino-acid substitutions of ThrA8 associated with increased affinity for the insulin receptor and/or increased thermodynamic stability as may be introduced to mitigate deleterious effects of the primary two above design elements (a phenylboronic acid derivative at or near the N-terminus of the A chain and one or more saccharide derivatives at or near the C-terminus of the B chain) on receptor-binding affinity and/or thermodynamic stability. Examples of such A8 substitutions known in the art are HisA8, LysA8, ArgA8, and GluA8.
The insulin analogues of the present invention may exhibit an isoelectric point (pI) in the range 4.0-6.0 and thereby be amenable to pharmaceutical formulation in the pH range 6.8-7.8; alternatively, the analogues of the present invention may exhibit an isoelectric point in the range 6.8-7.8 and thereby be amenable to pharmaceutical formulation in the pH range 4.0-4.2. The latter conditions are known in the art to lead to isoelectric precipitation of such a pI-shifted insulin analogue in the subcutaneous depot as a mechanism of protracted action. An example of such a pI-shifted insulin analogue is provided by insulin glargine, in which a basic two-residue extension of the B chain (ArgB31-ArgB32) shifts the pI to near-neutrality and thus enables prolonged pharmacokinetic absorption from the subcutaneous depot. In general the pI of an insulin analogue may be modified through the addition of basic or acidic chain extensions, through the substitution of basic residues by neutral or acidic residues, and through the substitution of acidic residues by neutral or basic residues; in this context we define acidic residues as Aspartic Acid and Glutamic Acid, and we define basic residues as Arginine, Lysine, and under some circumstances, Histidine. We further define a “neutral” residue in relation to the net charge of the side chain at neutral pH.
It is an additional aspect of the present invention that absolute in vitro affinities of the insulin analogue for insulin receptor (isoforms IR-A and IR-B) are in the range 5-100% relative to wild-type human insulin and so unlikely to exhibit prolonged residence times in the hormone-receptor complex; such prolonged residence times are believed to be associated with enhanced risk of carcinogenesis in mammals or more rapid growth of cancer cell lines in culture. It is yet an additional aspect of the present invention that absolute in vitro affinities of the insulin analogue for the Type 1 insulin-like growth factor receptor (IGF-1R) are in the range 5-100% relative to wild-type human insulin and so unlikely either to exhibit prolonged residence times in the hormone/IGF-1R complex or to mediate IGF-1R-related mitogenesis in excess of that mediated by wild-type human insulin.
The insulin analogues of the present invention consist of two polypeptide chains that contain a novel modifications in the B chain such that the analogue, in the absence of glucose or other exogenous saccharide, contains covalent bonds between the side-chain diol in the B chain and a molecular entity containing PBA, a halogen-derivative of PBA, or any boron-containing diol-binding element able to bind glucose. The latter entity may be a C-terminal extension of the B chain or be a separate molecule prior to formation of the diol-PBA bonds.
Table 2 presents diol- or α-hydroxycarboxylate-containing precursors.
Although we do not wish to be restricted by theory, we envisage that these two design elements form a covalent interaction in the absence of exogenous glucose such that the structure of the hormone is stabilized in a less active conformation.
In accordance with first embodiment 1 an insulin analogue is provided comprising an A chain modified by a glucose-binding element at or near its N terminus and a variant B chain comprising a diol group at the C terminus of the B chain such that the polypeptide chain ends with a hydroxyl group rather than with a carboxylate group.
In accordance with first embodiment 2 an insulin analogue of first embodiment 1 is provided wherein the A chain contains a substitution at position A8 that enhances affinity of the insulin analogue for the insulin receptor, optionally wherein the substitution at position A8 is histidine.
In accordance with first embodiment 3 an insulin analogue of first embodiment 1 or 2 is provided wherein the A chain contains a substitution at position A8 or position A14 that enhances thermodynamic stability of the insulin analogue for the insulin receptor, optionally wherein the substitution at position A8 or A14 is independently selected from the group consisting of HisA8, LysA8, ArgA8, and GluA8.
In accordance with first embodiment 4 an insulin analogue of any one of first embodiments 1-3 is provided wherein the A chain contains a substitution at position A21 that protects the insulin analogue from chemical degradation.
In accordance with first embodiment 5 an insulin analogue of any one of first embodiments 1-4 is provided wherein said diol group at the C terminus of the B chain is an aliphatic (1, 2) diol.
In accordance with first embodiment 6 an insulin analogue of any one of first embodiments 1-5 is provided wherein said diol group at the C terminus of the B chain is an aliphatic (1, 3) diol.
In accordance with first embodiment 7 an insulin analogue of any one of first embodiments 1-6 is provided further comprising a modified amino acid at a position 1, 2, 3, or 4 residues N-terminal to the C-terminal amino acid, wherein said modified amino acid is an L or D amino acid comprising a side-chain diol.
In accordance with first embodiment 8 an insulin analogue of any one of first embodiments 1-7 is provided wherein the modified amino acid is
In accordance with first embodiment 9 an insulin analogue of any one of first embodiments 1-8 is provided further comprising an L Dopa at position B26 or an L or D Dopa located at 1-3 residues N-terminal to the C-terminal amino acid.
In accordance with first embodiment 10 an insulin analogue of any one of first embodiments 1-9 is provided wherein said B chain is a truncated B chain lacking residue B30, residues B29-B30, residues B28-B30, residues B27-B30 or residues B26-B30, with a diol group located at the C terminus of the truncated B chain.
In accordance with first embodiment 11 an insulin analogue of any one of first embodiments 1-9 is provided wherein said B chain is extended by one or two amino acids with a diol group located at the C terminus of the extended B chain.
In accordance with first embodiment 12 an insulin analogue of any one of first embodiments 1-9 is provided wherein the B chain is a polypeptide selected from the group consisting of
In accordance with first embodiment 14 an insulin analogue of any one of first embodiments 1-9 is provided wherein the B chain is a polypeptide selected from the group consisting of
wherein
In accordance with first embodiment 15 an insulin analogue of any one of first embodiments 1-14 is provided wherein the A chain is a polypeptide selected from the group consisting of
In accordance with first embodiment 16 a method of preparing an analogue of any one of First embodiments 1-15 is provided by means of trypsin-mediated semi-synthesis wherein (a) any optional A-chain modification (i.e., by a monomeric glucose-binding moiety) is introduced within a des-octapeptide[B23-B30] fragment of insulin or insulin analogue and (b) the diol-containing B-chain modification is introduced within a synthetic peptide of length 5-10 amino-acid residues whose N-terminal residue is Glycine and which upon modification contains no tryptic cleavage site.
In accordance with first embodiment 17 the method of first embodiment 16 is provided wherein the des-octapeptide[B23-B30] fragment of insulin or an insulin analogue is obtained by trypsin digestion of a parent insulin or insulin analogue.
In accordance with first embodiment 18 the method of first embodiment 16 is provided wherein the des-octapeptide[B23-B30] fragment of insulin or insulin analogue is obtained by trypsin digestion of a single-chain polypeptide (such as proinsulin, a proinsulin analogue or a corresponding mini-proinsulin containing a foreshortened or absent C domain) as expressed in Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris or other microbial system for the recombinant expression of proteins.
In accordance with first embodiment 19 the method of first embodiment 16 is provided wherein the des-octapeptide[B23-B30] fragment of insulin or insulin analogue is obtained by trypsin digestion of single-chain polypeptide (such as proinsulin, a proinsulin analogue or a corresponding mini-proinsulin containing a foreshortened or absent C domain) as prepared by solid-phase chemical peptide synthesis, optionally including native fragment-ligation steps.
In accordance with first embodiment 20, a method of treating a diabetic patient is provided wherein the patient is administered a physiologically effective amount of an insulin analogue of any one of first embodiments 1-15, or a physiologically acceptable salt thereof via any standard route of administration.
In accordance with second embodiment 1, an insulin analogue comprising an insulin A chain and an insulin B chain is provided wherein,
In accordance with second embodiment 2 an insulin analogue of second embodiment 1 is provided wherein said glucose-binding element is covalently linked to the side chain of an amino acid in the
In accordance with second embodiment 3 an insulin analogue of second embodiment 1 or 2 is provided further comprising an amino acid added to the N-terminus of the insulin A chain, wherein said N-terminal amino acid is located at position A0 or A−1.
In accordance with second embodiment 4 an insulin analogue of any one of second embodiments 1-3 is provided wherein said N-terminal amino acid is glycine.
In accordance with second embodiment 5 an insulin analogue of any one of second embodiments 1-4 is provided wherein said diol group is linked to the side chain of one of the three most C-terminal amino acids of the insulin B chain, optionally at any of positions B26, B27, B30 or B31, B32 or B33 of a C-terminally extended B chain, optionally at B28, B29 or B30, optionally at B28.
In accordance with second embodiment 6 an insulin analogue of any one of second embodiments 1-5 is provided wherein said diol group is a main chain diol, having the —COOH group of the C-terminal amino acid replaced with —CH2OH and a side chain bearing an hydroxyl group.
In accordance with second embodiment 7 an insulin analogue of any one of second embodiments 1-6 is provided wherein the A chain contains a substitution at position A8 that enhances affinity of the insulin analogue for the insulin receptor, optionally wherein the substitution at position A8 is histidine.
In accordance with second embodiment 8 an insulin analogue of any one of second embodiments 1-7 is provided wherein the A chain contains a substitution at position A8 or position A14 that enhances thermodynamic stability of the insulin analogue for the insulin receptor, optionally wherein the substitution at position A8 or A14 is independently selected from the group consisting of His, Lys, Arg, and Glu.
In accordance with second embodiment 9 an insulin analogue of any one of second embodiments 1-6 is provided wherein the A chain contains a substitution at position A21 that protects the insulin analogue from chemical degradation.
In accordance with second embodiment 10 an insulin analogue of any one of second embodiments 1-9 is provided wherein said diol group at the C terminus of the B chain is an aliphatic (1, 2) diol.
In accordance with second embodiment 11 an insulin analogue of any one of second embodiments 1-10 is provided wherein said diol group at the C terminus of the B chain is an aliphatic (1, 3) diol.
In accordance with second embodiment 12 an insulin analogue of any one of second embodiments 1-11 is provided further comprising a modified amino acid at a position 1, 2, 3, or 4 residues N-terminal to the C-terminal amino acid of the B chain, wherein said modified amino acid is an L or D amino acid comprising a side-chain diol.
In accordance with second embodiment 13 an insulin analogue of any one of second embodiments 1-12 is provided wherein the modified amino acid is a thiol-containing L or D amino acid.
In accordance with second embodiment 14 an insulin analogue of any one of second embodiments 1-13 is provided further comprising an L Dopa at position B26 or an L or D Dopa located at 1-3 residues N-terminal to the C-terminal amino acid of the B chain.
In accordance with second embodiment 15 an insulin analogue of any one of second embodiments 1-14 is provided wherein said B chain is a truncated B chain lacking residue B30, residues B29-B30, residues B28-B30, residues B27-B30 or residues B26-B30, with a diol group located at the C terminus of the truncated B chain.
In accordance with second embodiment 16 an insulin analogue of any one of second embodiments 1-15 is provided wherein said B chain is extended by one or two amino acids with a diol group located at the C terminus of the extended B chain.
In accordance with second embodiment 17 an insulin analogue of any one of second embodiments 1-16 is provided wherein
In accordance with second embodiment 18 an insulin analogue of any one of second embodiments 1-17 is provided wherein
wherein
In accordance with second embodiment 19 an insulin analogue of any one of second embodiments 1-18 is provided wherein
In accordance with second embodiment 20 an insulin analogue of any one of second embodiments 1-19 is provided wherein
In accordance with second embodiment 21 an insulin analogue of any one of second embodiments 1-20 is provided wherein
In accordance with second embodiment 22 an insulin analogue of any one of second embodiments 1-21 is provided wherein the diol bearing amino acid is selected from
In accordance with second embodiment 23 an insulin analog of any one of second embodiments 1-22 is provided wherein diol bearing amino acid comprises a diol moiety having the structure of
linked to the side chain or backbone amide of the insulin B chain peptide.
In accordance with second embodiment 24 an insulin analog of any one of second embodiments 1-23 is provided wherein the glucose-binding elements are linked to the A chain and comprise a complex having the general structure
wherein BCM1 and BCM2 are boron-containing moieties; n and m are independently an integer selected from the range of 1 to 3; and R is any amino acid side chain of the L or D configurations standard 20 essential amino acids. In one embodiment R is selected from the group consisting of H, C1-C18 alkyl, C2-C18 alkenyl, (C1-C18 alkyl)OH, (C1-C18 alkyl)SH, (C2-C3 alkyl)SCH3, (C1-C4 alkyl)CONH2, (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH2, (C1-C4 alkyl)NHC(NH2+)NH2, (C0-C4 alkyl)(C3-C6 cycloalkyl), (C0-C4 alkyl)(C2-C5 heterocyclic), (C0-C4 alkyl)(C6-C10 aryl)R7, (C1-C4 alkyl)(C3-C9 heteroaryl), and C1-C12 alkyl(W1)C1-C12 alkyl, wherein W1 is a heteroatom selected from the group consisting of N, S and O. In one embodiment R is H, C1-C6 alkyl, (C1-C4 alkyl)C(O)NH2, (C1-C4 alkyl)OH, (C1-C4 alkyl)CH3OH, (C1-C4 alkyl)S, (C1-C4 alkyl)SCH3, (C1-C4 alkyl)COOH and (C1-C4 alkyl)NH2.
In accordance with second embodiment 25 an insulin analogue of any one of second embodiments 1-22 is provided wherein the insulin B chain is a polypeptide selected from the group consisting of
In accordance with second embodiment 24 an insulin analogue of any one of second embodiments 1-23 is provided wherein the B chain is a polypeptide selected from the group consisting of
In accordance with second embodiment 25 and insulin analogue of any one of second embodiments 1-24 is provided wherein the diol-bearing amino acids comprises a natural or unnatural amino acids whose side chains are modified by linkage of a diol moiety to an attachment point located on the amino acid side chain, optionally wherein the amino acid is selected from the group consisting of Cysteine Homocysteine, Lysine, Ornithine (Orn), Diaminobutyric Acid (Dab) and Diaminoproprionic acid (Dap), each in either the L- or D configuration with the covalent linkage of a diol moiety, exemplified (but not restricted to) those listed in Table 2, to the amino acid side chain; and the glucose binding element is a compound selected from those listed in
This application is a U.S. national counterpart application of international application serial No. PCT/US2022/027504 filed May 3, 2022, which claims priority to U.S. Provisional Patent Application No. 63/183,325 filed on May 3, 2021, the disclosures of which are hereby expressly incorporated herein by reference in their entireties.
This invention was made with government support under DK127761 and DK040949 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/027504 | 5/3/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63185325 | May 2021 | US |
