Cooperative glucose-binding element based glucose-responsive insulins

Abstract

An insulin analogue is provided comprising an A chain, modified by the attachment of a glucose-binding element, optionally at or near its N terminus, and a B chain, modified by the attachment of a glucose-binding element, optionally at or near its C terminus. The glucose-binding element at either or both chains may be contain a phenylboronic acid or boroxazole. Compositions comprising such insulin analogues are used in methods of treating a patient with diabetes mellitus.

Description

[0001] COOPERATIVE GLUCOSE-BINDING ELEMENT BASED GLUCOSERESPONSIVE INSULINS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 594078 filed on October 30, 2023, the disclosure of which is expressly incorporated herein.

[0004] STATEMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with government support under DK 127761 awarded by National Institutes of Health. The Government has certain rights in the invention.

[0006] BACKGROUND

[0007] 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 subop timal 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).

[0008] 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).

[0009] 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 Fig. 1 A. Fig. IB depicts a structural model of proinsulin, consisting of an insulin-like moiety and a disordered connecting peptide (C- domain); Fig. 1C is a schematic representation of the sequence of human insulin including disulfide connectivities. Specific residues in the insulin molecule are indicated by the aminoacid type (typically in standard three- letter code; e.g., Lys and Ala indicate Lysine and Alanine) and in superscript the chain (A or B) and position in that chain; or as single-letter code; e.g., A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S T, V, W, and Y indicate Alanine, Cysteine, Aspartic Acid, Glutamic Acid, Phenylalanine, Glycine, Histidine, Isoleucine, Lysine, Methionine, Asparagine, Proline, Arginine, Serine, Threonine, Valine, Tryptophan, and Tyrosine, respectively. For example, Alanine at position 14 of the B chain of human insulin is indicated by AlaB14or AB14, and likewise Lysine at position B28 of insulin lispro (the active component of Humalog®; Eli Lilly and Co.) is indicated by LysB28or KB28. Amino acids (except glycine) are chiral, and the configurations are designated L or D; L is presumed unless otherwise stated. D-Amino acids are designated “D-Cys” or “D-Ala” (and so forth) in three-letter code; the small capital “D” (d) may be used to avoid ambiguity with “D” as code for Asp. Alternatively, D-amino acids are designated “dC” or “dA” (and so forth) in one- letter code, generally within brackets in sequence string. Because of the special role played by amino-acid residues at the native N terminus of the A chain and / or by N-terminal- extended A chains, these respective positions were designated as “Aj, Ao and A-i,” where the subscript highlights potential attachment point(s) for GBEs.

[0010] Although the insulin hormone is stored in the pancreatic P-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 Fig. 2. Pertinent to the logic of the present disclosure is the proximity of the C terminus of the B chain (B30) to the N terminus of the A chain ( Ai), often engaged in a salt bridge (Fig. 3A). Covalent tethering of the B-chain C terminus (either from B30 or from neighboring residues B28, B29, B31 or B32) to A chain N terminus (or neo-N terminus at positions Ao or A-i) blocks binding of the hormone to the insulin receptor (Fig. 3B); such tethers block a conformational switch on receptor engagement. Inserting four or more residues into a foreshortened C domain restores receptor binding and agonist activity. Thus, it is herein recognized that control of conformation(s) of the C-terminal B chain in its closed (inactive) and open (active) forms by use of reversible covalent bonding, provides the opportunity for ligand-controlled glucose responsivity (Fig. 3C; Chen, Y. S., et al. Proceedings of the National Academy of Sciences, 118(30) (2021)).

[0011] An aspect of the present disclosure pertains to the chirality of amino acids. Whereas Glycine is achiral, biosynthetic proteins ordinarily are comprised of L-amino acids, where the chiral center is the alpha-carbon of the amino acid. It is a feature of the present disclosure that the elements of the glucose-regulated conformational switch — namely, the glucose-binding elements (GBEs) respectively attached to one or the other chain — may be attached to D- amino acids, such that the positions in space of the switch elements are optimized and, on binding glucose, with least perturbation to receptor-binding affinity. An example of a D- amino-acid substitution known in the art to perturb receptor binding is provided by the substitution of GlyB8by D-Ala (Nakagawa, S., et al., Biochemistry 44(13), 4984-99 (2005)). An example of a D-amino-acid substitution known in the art to enhance receptor binding is provided by the substitution of PheB24by D-Phe (Mirmira, R., and Tager, H.S. J. Biol. Chem. 264(11), 6349-54 (1989)). Pertinent to the placement of switch elements, substitution of GlyA1by D-amino acids generally preserves receptor-binding affinity whereas substitution of GlyA1by L-amino acids generally impairs receptor-binding affinity (Wan, Z.L. and Liang, D.C. Scientia Sinica 31(12), 1426-38 (1988); Wan, Z.L. and Liang, D.C. Scientia Sinica 33(7), 810-20 (1990)).

[0012] Administration of insulin has long been established as a treatment for diabetes mellitus. A major goal of conventional insulin replacement therapy in such patients 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 in 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.

[0013] 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.

[0014] 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 of 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 development of closed-loop systems in which the pump and monitor are combined with a computer-based algorithm as an “artificial pancreas.”

[0015] For more than three decades, there has been interest in the design and 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 glucoseresponsive 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.

[0016] 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 a- amino group of residue Ai or via the a-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 a-amino group of residue Bl or via the a-amino group of a Lysine substituted at positions B2, B3, B4 or Bs) 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 aptamers) 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 A 1 -linked affinity ligand and Bi -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 Bi -linked agents are typically as large as or larger than insulin itself.

[0017] The suboptimal properties of insulin analogues modified at or near residue Ai by an affinity ligand and simultaneously modified at or near residue B 1 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 without modification of the B chain’s amino terminus and without the need for large domains unrelated in structure or composition to insulin.

[0018] The insulin analogues of the present disclosure 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 inter-chain complex between: (i) two inter- distant PBA diol binding moieties each individually installed at the A chain N terminus (such as at positions Ao and / or Ai) and the second at or near the B chain C terminus (positions B26- B30) either modified through side chain positions or the main-chain amide nitrogen atom(s) to form a GBE across the insulin structure (as illustrated in Fig. 4A) or (ii) two or more sets of glucose-binding elements installed at the A chain N terminus (such as at positions Ao and / or Ai) and the second at or near the B-chain C terminus (positions B26-B30) either modified through side chain positions or the main-chain amide nitrogen atom(s) (as illustrated in Fig. 4B and 5) either leads to an active hormone conformation or liberates an active hormone analogue or (iii) a combination of a single PBA as in (i) and one or more GBEs as in (ii) providing opportunity for various inter-distant GBE combinations to form. Notably the presently disclosed design modifications of the insulin molecule are in each case smaller than the native A or B chains.

[0019] Surprisingly, it has been 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 sets of boron-based glucose-binding elements circumvent the need for diol modifications of the B chain at or near its C terminus as previously disclosed in US Provisional Application 63 / 104,196, entitled “Molecular Designs of Glucose-Responsive and Glucose-Cleavable Insulin Analogues”; incorporated by reference herein. Displacement of the chemical bonds between the glucose-binding elements attached at or near the N terminus of the A chain and the glucose-binding elements at or near the C terminus of the B chain would lead to detachment of the tethered molecular entity, which in turn enables high-affinity receptor binding; as illustrated in Figs. 4 and 5, and, as prior art, at the molecular level where 3 -fluoro phenylboronic acid (3fPBA) binds reversibly with a fructose molecule in Fig. 6A.

[0020] Prior art in GRI design considers there are three mutually important design components required for glucose responsive insulin switch: 1) Composition and chemical nature of the glucose-binding element (GBE; mono boronic acid, / v.v-boronic acid -symmetrical, asymmetrical, and the intervening molecular connectivity i.e., branched, backbone tandem, other methods to fuse boronic acids together). These GBEs carry out dual roles: (i) to competitively bind glucose to varying degrees based on glucose concentration while at the same time undergo competitive binding to an intermolecularly placed diol or poly-ol as advanced in component (2, below). This in essence provides the intermolecular (intrinsic) switch plays into its critical design scheme role, (ii) in constraining an intramolecular switch (inactive to active) component (3, below) through competing reaction choices of the GBE with the diol component (2) depending on available glucose concentrations.

[0021] 2) Composition and chemical nature of the diol component or multiple diol components. In such prior schemes the poly-ol component is a critical design element of the molecular switch that functions to dynamically lock and tether, within a “closed,” inactive conformation, the C terminus of the B chain (preferably through residues B27-B30) with the N terminus of the A chain (preferably through residues Ao and Ai). The poly-ol component binds to the GBE to form a dynamically stable inactive, closed (“locked”) state under hypoglycemic, and eugylcemic blood glucose levels (BGLs) that have been disclosed previously, i.e., C-terminal main-chain diols, such as via incorporation of a diol adduct at LysB29, and combinations of the same. The novelty of the GRI design schemes that place dual di-boron GBEs that have molecular complementarity with poly-ols has been previously disclosed.

[0022] 3) Composition and specific design placement of an intramolecular hinge is one element that enhances the ability of the GBE (1) and poly-ol(s) (2) glucose sensors to provide a switch between inactive and active states of the hormone. The present class of switches is broadly bio-inspired based on the native mechanism by which insulin binds to the ectodomain of the insulin receptor.

[0023] Rationale for Bio-Inspired Switch Underlying Glucose-Responsive Mechanism:

[0024] The present design approach begins with the canonical hinge-opening mechanism through which insulin interacts with its receptor, thereby intrinsically coupling IR-binding interactions with glucose sensing. In such schemes, the conformation of an insulin monomer in the blood stream will be tethered in a closed, inactive state at low glucose concentrations, but could become unconstrained — thereby allowing the open receptor-binding conformation — at high glucose concentrations. Prior art in GRI design rely on modified insulins carrying GBE components attached to one chain of the hormone and a saccharide mimic diol, poly-ol, bzs-diols) attached to the other chain. The prior art demanded that both the GBE and diol components are required for inter-chain cross-linking; their chemical bonding at low glucose concentration stabilizes the closed, inactive conformation that impairs receptor binding whereas competitive displacement of the tether at high glucose concentrations might restore sufficient protein flexibility to enable high-affinity IR binding in the open, active conformation. Design of such glucose-responsive tethers could thereby exploit the opening of the B chain’s N- or C-terminal segments relative to the critical IR- binding Ai-As a-helix, which is integral to the native hormone-receptor interface.

[0025] The illustration in Fig. 3C illustrates the compulsory interaction between a GBE and diol moiety in prior art for intrinsic GRI designs (Chen, Y. S., et al. Proceedings of the National Academy of Sciences, 118(30) (2021)). Specifically, there is a prerequisite for the GBE-diol interaction for glucose responsivity between the closed, inactive and the opened, glucose-bound active state.

[0026] Bio-inspired GRI Design Scheme without use of Diol Components:

[0027] As disclosed herein, the present embodiments extend beyond the bio-inspired switch mechanism concept and is predicated on the assertion that there is no prerequisite for a GBE- diol interaction for glucose responsivity. More particularly, mechanistically, glucose alone can drive conformational change in a dual GBE-modified insulin.

[0028] Two suitably placed GBE units are sufficient to produce a closed, inactive GRI at low BG thru a mechanism of cooperative binding of two GBEs to one glucose. Upon increasing BGL, the GRI will switch to open, active state through a mechanism that each GBE would glucose binding cooperativity would deteriorate leading to a 1:1 glucose-GBE binding. Fig. 4A depicts a simplified design for placement of two separate phenylboronic acid (PBA) diol- binding moieties that create a single GBE component that spans an inter-chain distance within the insulin structure. Fig 4B depicts a more elaborate design for placement of two separate inter-chain distant GBEs each carrying two PBA diol-binding moieties.

[0029] SUMMARY

[0030] The present disclosure provides for carbohydrate-binding elements and specifically for glucose-binding elements in peptides and proteins, such that binding of a monosaccharide can impose or relieve a conformational constraint in peptides or proteins. The design is based on pairs of boron-containing adducts and is exemplified in insulin analogues and model peptide systems of both A- and B-chain insulin sequences.

[0031] An alternative design for an intrinsic (or unimolecular) glucose-responsive insulin (GRI) analogue is presented herein. One distinctive feature of the present embodiment is the absence of an internal diol that was utilized in previous design schemes to confer a glucose- displaceable tether between the A- and B chains of insulin. In accordance with one embodiment of the present disclosure, an alternative GRI design scheme is provided wherein an insulin is modified by two internal glucose-binding elements (GBE; such as phenyl- boronic acids [PBAs] or boroxazoles) such that (a) inter-chain PBA-PBA interactions in the free hormone inhibit activity and (b) a 2:1 bridged state (GBE-glucose-GBE) at low glucose concentration inhibits activity but a 2:2 glucose-saturated state is conformationally unrestricted and active. In the active state the two GBE-glucose moieties are non-interacting and so do not provide an inactivating tether. It is further envisaged that this “2-1 cooperative GBE lock” approach can be extended to other non-canonical switch mechanisms.

[0032] In accordance with one embodiment, the present disclosure thus relates to polypeptide hormone analogues that contain a glucose-regulated molecular structure, 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 / As-boron-containing glucose-binding elements (GBE) that provide increased opportunity for selective binding to glucose through interaction(s) of two boronic acid moieties binding to one glucose molecule (Fig. 6B), thereby allowing for the design and discovery of novel glucose responsive insulins (GRIs).

[0033] In another 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.

[0034] The insulin analogues of the present disclosure 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).

[0035] BRIEF DESCRIPTION OF DRAWINGS

[0036] FIG. 1 A is a schematic representation of the sequence of human proinsulin including the A- and B-chains and the connecting region shown with flanking dibasic cleavage sites (filled circles) and C-peptide (open circles).

[0037] FIG. IB is a structural model of proinsulin, consisting of an insulin-like moiety and a disordered connecting peptide (dashed line).

[0038] FIG. 1C is a schematic representation of the sequence of human insulin including the A chain with a sequence of GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1) and B chain with a sequence of FVNQHLCGSHLVEALYLVCGERGFFYTPLT (SEQ ID NO: 2) and indicating the position of residues B27 and B30 in the B-chain.

[0039] FIG. 2 is cylinder model of insulin in which the side chains of TyrB16, PheB25and TyrB26are shown. The A- and B chain ribbons are shown in light gray and dark gray, respectively.

[0040] FIG. 3A provides a ribbon / cylinder diagram highlighting a potential salt bridge between the C-terminal carboxylate of the B chain (its negative charge is depicted as a minus - within circle at B30) and a-amino group of the A chain (its positive charge is depicted as a plus + within circle) as observed in a subset of wild-type insulin crystallographic protomers.

[0041] FIG. 3B provides a ribbon / cylinder diagram highlighting a peptide amide bond (box) between the C-terminal carboxylate of the B chain and a-amino group of the A chain as observed in inactive single-chain insulin analogues.

[0042] FIG. 3C provides a generic scheme of prior art in which a diol-modified B chain containing a main-chain hydroxyl group (boxed) in combination with a neighboring hydroxyl group (which may be on a side chain or attached via one or more intervening atoms to the main- chain nitrogen) binds to a glucose-binding element at or near the N terminus of the A chain (horseshoe).

[0043] FIG. 4A provides a design scheme of monosaccharide-responsive insulin and depicts a simplified design for placement of two separate inter-distant Phenylboronic Acid (PBA) diol- binding moieties that create a single GBE component that span an inter-distance within the insulin structure. The ribbon model of closed inactive insulin (T-state monomer, left) is shown with bound glucose molecule adjacent; the horseshoe shape indicates glucose-binding elements inter-distant dual di-boron GBE). The envisioned glucose-regulated conformational cycle in which a monosaccharide acts as a competitive ligand to regulate a conformational switch between the closed state (inactive in absence of ligand) and the open state (active in presence of ligand).

[0044] Fig. 4B depicts a more elaborate design for placement of two separate inter-distant GBEs each carrying two PBA diol-binding moieties.

[0045] FIG. 5 illustrates sequence of insulin showing the A-chain (light) and B-chain (dark) with sites of chemical modification (underlined) including A chain N-terminal residues A-i, Ao, and GlyA1and B-chain residues TyrB26-ThrB30to affect monosaccharide responsivity. Amino-acid residues are labelled using their standard single letter codes. Glucose-binding element (GBE) (horseshoe) is installed at the A chain N terminus (such as at positions Ao and / or Ai). The second inter-distant dual di-boron GBE (horseshoe) is installed at or near the B-chain C terminus (positions B26-B30), modified either through side chain positions or the main-chain amide nitrogen atom(s).

[0046] FIG. 6A depicts, as prior art, the reversible interaction between 3-fluoro phenylboronic acid (3fPBA) placed at or near the A chain N terminus with dihydroxybenzoic acid (DHBA) placed in the B-chain C terminus. Such a reversible interaction between a boronic acid and a diol moiety contributes to the conformation switching between closed ‘inactive’ and open ‘active’ insulin conformations.

[0047] FIG. 6B depicts basis for preferential glucose-binding of / ? / .v-boronic acids when attached through appropriately placed linker(s) (curved line) (Norrlid, 1995).

[0048] FIG. 6C provides a molecular representation of the embodied GRI framework and its conformational switch between the closed state (left) and open state (right). The GBE elements (light gray) are attached at or near the C terminus of the B chain (medium gray) and the inter- distant dual di-boron GBE moiety is attached at or near the N terminus of the A chain (dark). FIG. 7 provides listing of the eight (8) current diol-less GRIs in hand; two (2) generic structures of future design analogues.

[0049] FIG. 8 provides listing of all the GBE linked model peptides from A chain sequences (A-i through A?) and B-chain sequences (B23-B30) each was synthesized, HPLC-purified, and LC-MS characterized. These were studied for their glucose-binding abilities (See FIG. 9 and 10).

[0050] FIG. 9 presents circular dichroism (CD) spectra monitoring the binding of D-glucose to model peptides GFFYT[Xxx(GBE)]PT (8P). Such spectra represent average ensemble of productive and nonproductive conformation(s) in the presence and absence of D-glucose (25uM). Model peptide, K(GBE1-C-Ac)-8P exhibited greatest CD changes in glucose-free versus bound conformation(s). Dap(GBE3)-8P exhibited second largest CD change in glucose free versus bound conformation(s). Om(GBE5)-8P exhibited the least CD change in glucose free versus bound conformation(s). Average structure of GBE5 may include the binding conformation.

[0051] FIG. 10 describes an ARS-based assay to determine GBE-glucose association constants. The ARS assay is a two-part competition assay between a fluorescent indicator (Alizarin Red S), boronic-acid-based sensor, and diol (Brooks, W. L., Deng, C. C., & Sumerlin, B. S. (2018) Structure-reactivity relationships in boronic acid-diol complexation. ACS omega, 3(12), 17863-17870; Springsteen, G., & Wang, B. (2002). A detailed examination of boronic acid- diol complexation. Tetrahedron, 58(26), 5291-5300).

[0052] FIG. 11 A provides explicit structures of the glucose-binding elements (GBEs) that are defined as GBE1, GBE2, GBE3, GBE4, GBE5, and GBE6 containing paired BCM2 groups attached to the branch-mers (Dap and Dab). In GBEs 1 and 4, the BCMs are directly linked to both amino groups of the Dap or Dab amino groups to as exemplified in the left panel. GBEs 2 and 5 (center panel) differ from GBE 1 and 4 in that the alpha amino group of Dap (n = 1 ) or Dab (n = 2) is modified with the amino acid glycine prior to paired BCM2 coupling, denoted as BCM2-Xxx-Dap(BCM2)-OH (GBE2) and BCM2-Dab(BCM2-Xxx)-OH (GBE5) respectively, where Xxx = Gly. The GBEs 3 and 6 (left panel) differ from GBE 1 and 4 in that the sidechain amino group of Dap (n = 1 ) or Dab (n = 2) is modified with the amino acid, denoted as BCM2-Xxx-Dab(BCM2)-OH (GBE3) and BCM2-Dab(BCM2-Xxx)-OH (GBE6) respectively, where Xxx = glycine, prior to paired BCM2 coupling. The use of Xxx recognizes the substitution by any other L-amino acids, D-amino acids, or non-natural amino acids would create unique GBEs, thus allowing for modulation of binding properties of each GBE with respect to affinity and selectivities toward glucose, other monosaccharides, or polysaccharides.

[0053] FTG. 1 IB provides six specific examples of symmetrical placement of paired BCM2 on the amino groups of either Dap (n = 1) or Dab (n = 2) to produce sidechain amide linked GBE1 thru GBE6 onto the insulin A chain at positions Ao (replaced by Dap) or Ai (replaced by D- Dap) the insulin sequence

[0054] FIG. 12 depicts reaction schemes used for solid-phase synthesis of glucose-binding element (GBE): GBEs 1-6 herein explicitly built out to connect (1) GBEs 4, 5, 6, onto the sidechain amino group of Dab Ao, and D-Dab at the Ao and Ai positions on insulin sequences (2) on the [B23-B30] octapeptide sequence, GFFYTX(GBE1-6)PT that represents the octapeptide (8P) sequence of insulin lispro (KP) residues B23-B30, where X = Lys, Orn, Dab, and Dap, and (3) any ‘acid’ (i.e., Wang, Cl-Trtyl) or ‘amide’ (i.e., Rink, RAM, aminomethyl) functionalized solid supports. A similar approach using Dap or D-Dap was used to install GBEs 1, 2, and 3 onto at the Ao and Ai positions of the insulin analogues.

[0055] FIG. 13 depicts the generalized solid-phase synthetic scheme to prepare and install amide bond linked glucose-binding elements (GBEs 1-6). Illustrated here for GlyAo, [D- Dap(GBE4)]A1and GlyAo, [D-Dap(GBE5)]A1-DesDI single-chain intermediates. The same scheme was also used to prepare additional DesDi single-chain analogues carrying GBEs 1-6.

[0056] FIG. 14 depicts the generalized solid-phase synthetic scheme to prepare and install amide-bond-linked glucose-binding elements (GBEs 1-6) as a GlyA 1, [Dap(GBEl)]A1, HisA8and GA-i, [Dap(GBE3)]A1, HisASDesDi single-chain insulin intermediates. The same scheme was also used to prepare additional DesDi single-chain analogues carrying GBEs 1-6.

[0057] DETAILED DESCRIPTION

[0058] DEFINITIONS

[0059] 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.

[0060] 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 "purified polypeptide" is used herein to describe a polypeptide that has been separated from other compounds including, but not limited to nucleic acid molecules, lipids and carbohydrates.

[0061] 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.

[0062] "Subject" refers to any mammal for whom diagnosis, treatment, or therapy is desired including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits and guinea pigs), livestock (e.g., cows, sheep, goats, and pigs), household pets (e.g., dogs, cats, and rodents), and horses.

[0063] “Treat," "treating" or "treatment" refer to an action to obtain a beneficial or desired clinical result including, but not limited to, alleviation or amelioration of one or more signs or symptoms of a disease (e.g., regression, partial or complete), diminishing the extent of disease, stability (i.e., not worsening, achieving stable disease) of the state of disease, amelioration or palliation of the disease state, diminishing rate of or time to progression, and remission (whether partial or total). For example, treating an intracellular pathogen includes decreasing the ability of the pathogen to infect, replicate or maintain viability in a host cell.

[0064] 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.

[0065] The term "inhibit" refers to 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.

[0066] 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: 1 and the B chain of SEQ ID NO: 2, as well as single-chain insulin analogues that comprise SEQ ID NOS: 1 and 2. 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: 2, as well as heterodimers and that comprise modified derivatives of the native A chain and / or B chain, including one or more amino-acid substitutions at positions selected from A5, A8, A9, A10, A12, A14, A15, A17, A18, A21, Bl, 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 Bl-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 (AA) within an N- terminal extension of the A chain is designated AA-i or AAo wherein A Ao represents the position of an amino acid added directly to the native N-terminal amino acid of the insulin A chain, and AA-i represents the position of an amino acid having a single amino acid intervening between the AA-i amino acid and the native N-terminal amino acid of the insulin A chain.

[0067] As used herein an amino-acid “modification” refers to a substitution, addition or deletion of an amino acid, or the derivation of an amino acid by the addition and / or removal of chemical groups to / from the amino acid, and includes substitution with 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.

[0068] 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 As) refer to the amino acid at that position of either the A chain (e.g., position As) or the B chain (e.g., position Bs) in the respective native human insulin A chain (SEQ ID NO: 1) or B chain (SEQ ID NO: 2), or the corresponding amino-acid position in any analogues thereof. For example, a reference herein to “position Bis” 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: 2 has been deleted.

[0069] As used herein, the term “conservative amino-acid substitution” is defined herein as exchanges within one of the following five groups:

[0070] I. Small aliphatic, nonpolar or slightly polar residues:

[0071] Ala, Ser, Thr, Pro, Gly;

[0072] II. Polar, negatively charged residues and their amides:

[0073] Asp, Asn, Glu, Gin;

[0074] III. Polar, positively charged residues:

[0075] His, Arg, Lys; Ornithine (Om)

[0076] IV. Large, aliphatic, nonpolar residues:

[0077] Met, Leu, He, Vai, Cys, Norleucine (Nle), homocysteine

[0078] V. Large, aromatic residues:

[0079] Phe, Tyr, Trp, acetyl phenylalanine

[0080] 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.

[0081] As used herein, a “glucose-binding elements (GBE)” is molecular structure that comprises one or more boron-containing moieties (BCMs). A / ? / .v-boronic acid GBE is a GBE comprising two boron-containing moieties wherein the two boron-containing moieties are linked to one another.

[0082] As used herein, a “boron-containing moiety” is a chemical structure comprising a boron molecule covalently linked to two oxygen atoms, wherein the boron-containing moiety is capable of interacting with diol containing entity to form a reversible covalent link between the diol and the boron atom.

[0083] As used herein, a “glucose responsive insulin (GRI)” is an insulin analogue that comprises a glucose-binding elements. 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 levels greater than 150 mg / dL after 8 hours of fasting, and bind the insulin receptor with high affinity.

[0084] EMBODIMENTS

[0085] In accordance with one embodiment of the present disclosure, insulin analogues are provided that are inactive or exhibit reduced, prolonged activity under hypoglycemic conditions. The insulin analogues are activated at high glucose concentrations, restoring their ability to bind with high affinity, and to activate, the insulin receptor. The analogues comprise an insulin polypeptide comprising an A chain and a B chain wherein both the insulin A chain and B chain have been modified by the attachment of a combination of either (i) two inter-distant place boron-containing moieties (BCM), (ii) a single BCM and a glucose- binding element, or (iii) a glucose-binding element. The insulin polypeptide may be either a two-chain insulin, wherein the A chain and B chain are linked only by disulfide bonds, or the insulin polypeptide may be an active single-chain analogue wherein the C terminus is covalently linked to the N terminus of the A chain, optionally via a peptide linker.

[0086] In accordance with one embodiment, the insulin analogue is a two-chain insulin comprising a first glucose-binding element that is linked at or near (within the first 5 or 10 amino acids) of the native N terminus of the A chain and a second glucose-binding element that is linked at or near (within the last 5 or 10 amino acids) of the native C terminus of the B chain. In one embodiment, the glucose-binding element is selected from phenylboronic acid and / or benzoboroxole.

[0087] In accordance with embodiment 1 , an insulin analogue comprising an insulin A chain and an insulin B chain is provided. The insulin A chain comprises a glucose-binding element covalently linked at or within the first five amino acids of the N terminus of the A chain, and the insulin B chain comprises a glucose-binding element covalently linked at or within the last five amino acids of the C terminus. In some embodiments, at least one of positions Ao and Ai may be present and may be a D-amino acid.

[0088] In accordance with embodiment 2, the insulin analogue of embodiment 1 is provided wherein said A and B chains comprise glucose-binding elements that comprise two or more boron atoms. In some embodiments, one of said glucose-binding elements may be covalently linked to (a) the side chain of an amino acid at positions A-i, Ao or Ai and / or (b) to the a- amino group of residues A.i, Ao or Ai. In accordance with embodiment 3, the insulin analogue of embodiment 1 or 2 is provided wherein said glucose-binding elements comprise a phenyl-boronic acid [PBAs] or boroxazole.

[0089] In accordance with embodiment 4, the insulin analogue of any one of embodiments 1-

[0090] 3 is provided wherein 1 or 2 amino acids are added to the N terminus of the native insulin A chain. In some embodiments, the glucose-binding elements may be covalently linked to one of the amino acids of the N-terminal extension.

[0091] In accordance with embodiment 5, the insulin analogue of any one of embodiments 1-

[0092] 4 is provided wherein the N-terminal amino acid of said insulin A chain is glycine.

[0093] In accordance with embodiment 6, the insulin analogue of any one of embodiments 1 -

[0094] 5 is provided wherein said glucose-binding element is linked to a side chain of residues B26, B27, B28, B29, B30 or to a C-terminal extension at B31 or B31-B32.

[0095] In accordance with embodiment 7, the insulin analogue of any one of embodiments 1-

[0096] 6 is provided wherein the A chain comprises a substitution at position A8 that enhances the affinity of the insulin analogue for the insulin receptor. In some embodiments, the A8 substitution may be a nitrogen-containing amino acid selected from the group Lysine, Histidine or Glutamine.

[0097] In accordance with embodiment 8, the insulin analogue of any one of embodiments 1-

[0098] 7 is provided wherein the A chain comprises a substitution at position A8 or position Al 4 that enhances thermodynamic stability of the insulin analogue for the insulin receptor. In some embodiments, the substitution at position A8 or A14 may be independently selected from the group consisting of His, Lys, Arg, and Glu.

[0099] In accordance with embodiment 9, the insulin analogue of any one of embodiments 1 -

[0100] 8 is provided wherein the A chain comprises a substitution at position A21 that protects the insulin analogue from chemical degradation.

[0101] In accordance with embodiment 10, the insulin analogue of any one of 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 glucose-binding element linked to the C terminus of the truncated B chain.

[0102] In accordance with embodiment 11 , the insulin analogue of any one of embodiments 1-10 is provided wherein said B chain is extended by one or two amino acids with a glucose- binding element linked to the C terminus of the extended B chain. In accordance with embodiment 12, the insulin analogue of any one of embodiments 1-3 is provided wherein the insulin A chain is a polypeptide selected from the group consisting of

[0103] X-iXoXiIVEQXeCXsSIXiiSLYQLENYCN (SEQ ID NO: 3), XoXiIVEQXeCXsSIXnSLYQLENYCN (SEQ ID NO: 4), and X oIVEQXeCXgSIXiiSLYQLENYCN (SEQ ID NO: 5) wherein X-i is any amino acid, optionally wherein X-i is Gly;

[0104] Xo is any amino acid or a modified amino acid comprising a BCM or glucose- binding element linked to its side chain;

[0105] Xi is any amino acid or a modified amino acid comprising a BCM or glucose- binding element linked to its side chain, optionally wherein X-i is Gly;

[0106] Xe and Xu are each Cys or selenocysteine; and

[0107] Xs is Thr, His, Lys, Arg, or Glu;

[0108] X70 is a D-amino acid comprising a BCM or glucose-binding element linked to its side chain; further wherein at least one of X-i , Xo and Xi is in the D conformation and the insulin B chain is a polypeptide selected from the group consisting of FVX24QHLCGSHLVEALYLVCGERGFFYTX49X50X51 (SEQ ID NO: 6), and FVX24QHLCGSHLVEALYLVCGERGFFYTX51 (SEQ ID NO: 7), wherein X24 is Lys or Asn;

[0109] X49 is Orn, Glu Asp, Lys, Pro, or a modified amino acid comprising a BCM or glucose-binding element linked to the side chain of the amino acid;

[0110] X50 is Orn, Lys, Pro, or a modified amino acid comprising a BCM or glucose- binding element linked to the side chain of the amino acid; and

[0111] X51 is Thr or an amino acid linked to a BCM or glucose-binding element.

[0112] In accordance with embodiment 13, the insulin analogue of any one of embodiments

[0113] 1 - 12 is provided wherein the insulin B chain is a polypeptide comprising the sequence of FVX24QHLCGSHLVEALYLVCGERGFFYTX49X50X51 (SEQ ID NO: 6), and FVX24QHLCGSHLVEALYLVCGERGFFYTX51 (SEQ ID NO: 7), wherein X24 is Lys or Asn; X49 is Orn, Glu Asp, Lys, Pro, or a modified amino acid comprising a BCM or glucose-binding element linked to the side chain of the amino acid;

[0114] X50 is Orn, Lys, Pro, or a modified amino acid comprising a BCM or glucose- binding element linked to the side chain of the amino acid; and

[0115] X51 is Thr or an amino acid linked to a BCM or glucose-binding element.

[0116] In accordance with embodiment 14, the insulin analogue of any one of embodiments 1-13 is provided wherein

[0117] X24 is Lys or Asn;

[0118] X49 is a modified amino acid comprising a glucose-binding element linked to the side chain of the amino acid;

[0119] X50 is Orn, Lys or Pro; and

[0120] X51 is an amino acid linked to a BCM or glucose-binding element, optionally wherein the BCM or glucose-binding element is linked to the side chain of the amino acid.

[0121] In accordance with embodiment 15, the insulin analogue of any one of embodiments 1-14 is provided wherein

[0122] X24 is Lys or Asn;

[0123] X49 is a modified amino acid comprising a BCM or glucose-binding element linked to the side chain of the amino acid;

[0124] X50 is Pro; and

[0125] X51 is Thr.

[0126] In accordance with embodiment 16, the insulin analogue of any one of embodiments 1-15 is provided wherein the insulin A chain is a polypeptide comprising the sequence of

[0127] XoXiVEQXeCXsSIXnSLYQLENYCN (SEQ ID NO: 4), wherein

[0128] Xo is any amino acid, optional Gly; and

[0129] Xi is a modified D-amino acid comprising a BCM or glucose-binding element linked to its side chain;

[0130] Xe and Xu are each Cys or each selenocysteine; and Xs is Thr, His, Lys, Arg, or Glu.

[0131] In accordance with embodiment 17, the insulin analogue of any one of embodiments 1 - 16 is provided wherein the glucose-binding element comprises two or more boron atoms. In some embodiments, the insulin analogue of any one of embodiments 1-16 may comprise phenyl-boronic acid or a halogen-modified phenyl-boronic acid, such that one or more moieties is selected from the following:

[0132] BCM2-Dab(BCM2)-OH BCM2-Gly-Dab(BCM2) BCM2-Dab(BCM2-G!y)-OH

[0133] GBE4 GBE5 = Xxx = Gly GBE6 = Xxx = Gfy

[0134] In accordance with one embodiment, a method of treating a diabetic patient is provided comprising administering a physiologically effective amount of an insulin analogue of any one of embodiments 1-17, or a physiologically acceptable salt thereof to the patient.

[0135] In one aspect, 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 Al); the B chains are modified to comprise paired boron-containing moieties (BCMs) are tethered at or near the 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 Fig. 6C) containing carboxylate group amino group, and aldehyde group handles to be used as monomeric library inputs. BCMs may also include boronate esters such as benzoxaborole (Bxb), or a combination of PBA and Bxb elements. Such inputs will be reacted with bifunctional scaffolds either built out separately, or prepared by solid-phase peptide synthesis using linker-based chemistries; to produce paired BCMs thus provide Z s-boronic acid GBEs. In one embodiment GBEs are specifically positioned at or near the N terminus of the A chain, optionally including modifications of D configuration of amino-acid side chains at position Al and / or modifications of side chains at one-residue, two-residue or three-residue N-terminal extensions of the A chain (respectively designated positions Ao, A.j and A-2).

[0136] The A chain of the present disclosure may be the standard 21 residues in length or contain an N-terminal extension of one residue (Ao), two residues (A-1-A0), or three residues (A-2-A-1-A0); or contain (as in insulin-like growth factors; IGFs) a C-terminal extension of 1- 5 residues as might enhance the biophysical or biochemical properties of the insulin analogue. The B chain of the present disclosure may be the standard 30 residues in length or may contain deletion of residues B30, B29-B30, B28-B30 or B27-B30; or may contain an extension to contain additional residue B31 or additional residues B31-B32 as is known in the art. The C-termini of these B chains may carry a carboxylate (as in a conventional peptide) or may be modified to contain a second inter-distant dual di-boron GBE.

[0137] 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 Ai or Ao 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 Ai or Ao; and the insulin B chain comprises an inter-distant dual di-boron GBE group at or near the C terminus of the insulin B chain, optionally wherein an inter-distant dual di-boron GBE-bearing moiety is linked to the side chain of the amino acid at position B28, B29 or B30 or wherein the inter-distant dual di-boron GBE group is a modified C-terminal amino acid (a) bearing a side chain BCM and (b) having the C-terminal carboxyl group replaced with BCM, such that the BCM together with an A chain-linked GBE may bind a single molecule of D- glucose.

[0138] 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 D-amino acid. In one embodiment, the glucose-binding element comprises two or more boron-containing moieties. In one embodiment the glucose-binding element comprises two boron-containing moieties wherein the two boron-containing moieties are linked to one another (extrinsic to the polypeptide), wherein the paired boron-containing moieties are the same or different. In accordance with one embodiment, the boron-containing binding element is selected from any of those disclosed in Fig. 7, and in one embodiment the boron-containing moiety is selected from phenylboronic acid (BCM1) elements and benzoboroxole (Bxb) as in BCMs 2-4. While not wishing to be constrained by theory, it is envisaged that pairwise and higher-order combinations of boron-containing diol-binding moieties, each containing one or more boron atoms, would enhance (a) avidity and the affinity of this combination of boron-containing moieties and the glucose molecule and (b) the cooperativity of glucose-dependent activation of hormonal activity as a function of ambient glucose concentration. Enhanced cooperativity would in turn confer more effective switch- like regulation of glycemia as a function of blood-glucose concentration, a favorable property to mitigate the risk of hypoglycemia in patients with diabetes mellitus. Individual boron-containing diol-binding moieties, as isolated small molecules, are known in the art (for review, see Williams, G.T., Kedge, J.L., and Fossey, J.S. Molecular Boronic Acid- Based Saccharide Sensors. ACS Sens. 6(4), 1508-28 (2021)).

[0139] 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 Figs. 5 and 6A. In one embodiment the amino acid positioned at the N terminus of the glucose-binding-element-bearing A chain is any of the standard 20 amino acids, in another embodiment the N-terminal amino acid is an aliphatic amino acid selected from alanine, glycine, isoleucine, leucine, proline, and valine, and in yet another embodiment the N- terminal amino acid of the glucose-binding element bearing A chain is glycine. In still a further embodiment the N-terminal amino acid of the glucose-binding-element-bearing A chain is in the D-configuration, as depicted in Figs. 10B, 11A, 11C, and 12C.

[0140] 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 an inter-distant dual di-boron GBE 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 an inter-distant dual di-boron GBE group located at the C terminus of the extended B chain, optionally in conjunction with an inter-distant dual diboron GBE-modified side chain.

[0141] In accordance with one embodiment, an insulin analogue is provided wherein the insulin A chain is a polypeptide selected from the group consisting of X.iXoXiIVEQXfiCXsSIXi iSLYQLENYCN (SEQ ID NO: 3) and XoXdVEQXeCXsSIXiiSLYQLENYCN (SEQ ID NO: 4), wherein X-i is any amino acid, optionally wherein X-i is Gly;

[0142] Xo is any amino acid or a modified amino acid comprising a BCM or glucose- binding element linked to its side chain;

[0143] Xi is any amino acid or a modified amino acid comprising a BCM or glucose- binding element linked to its side chain;

[0144] Xe and Xu are each Cys or selenocysteine; and

[0145] Xg is His, Lys, Arg, or Glu; further wherein at least one of X-i , Xo and Xi is in the D conformation; and the insulin B chain is a polypeptide selected from the group consisting of FVX24QHLCGSHLVEALYLVCGERGFFYTX49X50X51 (SEQ ID NO: 6), and FVX24QHLCGSHLVEALYLVCGERGFFYTX51 (SEQ ID NO: 7), wherein X24 is Lys or Asn;

[0146] X49 is Glu Asp, Lys, Pro, or a modified amino acid comprising an inter-distant dual di-boron GBE bearing moiety linked to the side chain of the amino acid;

[0147] X50 is Orn, Lys, Pro, or a modified amino acid comprising an inter-distant dual diboron GBE bearing moiety linked to the side chain of the amino acid; and

[0148] X51 is Thr. or an inter-distant dual di-boron GBE bearing amino acid.

[0149] In one embodiment, the insulin B chain is a polypeptide comprising the sequence of FVX24QHLCGSHLVEALYLVCGERGFFYTX49X50X51 (SEQ ID NO: 6), wherein X24 is Lys or Asn;

[0150] X49 is a modified amino acid comprising an inter-distant dual di-boron GBE bearing moiety linked to the side chain of the amino acid;

[0151] X50 is Orn, Lys or Pro; and

[0152] X51 is Thr. or an inter-distant dual di-boron GBE bearing amino acid. In a further embodiment both X49 and X51 are modified amino acids comprising an inter-distant dual diboron GBE.

Claims

CLAIMS1. An insulin analogue comprising an insulin A chain and an insulin B chain, said insulin A chain comprising a first glucose-binding element covalently linked at or within the first five amino acids of the N terminus of the A chain, and an insulin B chain comprising a second glucose-binding element covalently linked at or within the last five amino acids of the C terminus.

2. An insulin analogue comprising an insulin A chain and an insulin B chain, said insulin A chain comprising a phenylboronic-acid moiety (or halogen-modified phenylboronic-acid moiety) covalently linked at or within the first five amino acids of the N terminus of the A chain, and an insulin B chain comprising a glucose-binding element covalently linked at or within the last five amino acids of the C terminus.

3. An insulin analogue comprising an insulin A chain and an insulin B chain, said insulin A chain comprising a phenylboronic-acid moiety (or halogen- modified phenylboronic-acid moiety) covalently linked at or within the first five amino acids of the N terminus of the A chain, and an insulin B chain comprising a phenylboronic-acid moiety (or halogen-modified phenylboronic-acid moiety) covalently linked at or within the last five amino acids of the C terminus.

4. An insulin analogue comprising an insulin A chain and an insulin B chain, said insulin A chain comprising a phenylboronic-acid moiety (or halogen- modified phenylboronic-acid moiety) covalently linked at or within the first five amino acids of the N terminus of the A chain, and an insulin B chain comprising a phenylboronic-acid moiety (or halogen-modified phenylboronic-acid moiety) covalently linked at or within the last five amino acids of the C terminus.

5. The insulin analogue of claims 1, 2, 3, or 4 wherein the covalent linkages to the A chain or B chain pertain to an attachment point in an N-terminal one- or two-residue extension of the A chain (residues Ao and A-i) or to an attachment point in a C-terminal one- or two-residue extension of the B chain (residues B31 and B32).

6. The insulin analogue of claims 1, 2, 3, 4 or 5 wherein said A and B chains comprise glucose-binding elements that comprise two or more boron atoms, optionally wherein said first glucose-binding elements is covalently linked to (a) the side chain of an amino acid at positions A-i, Ao or Ai and / or (b) to the a-amino group of residues A-i, Ao or Ai.

7. The insulin analogue of claim 6 wherein said first and second glucose-binding elements independently comprise phenyl-boronic acid [PBAs] or boroxazole.

8. The insulin analogue of claims 1-4 comprising 1 or 2 amino acids added to the N terminus of the native insulin A chain.

9. The insulin analogue of claim 1-8 wherein the N-terminal amino acid of said insulin A chain is glycine.

10. The insulin analogue of claim 1-4 wherein said glucose-binding element is linked to a side chain of residues B26, B27, B28, B29, B30 or to a C-terminal extension at B31 or B31-B32.

11. The insulin analogue of claims 1 -4 wherein the A chain contains a substitution at position A8 that enhances affinity of the insulin analogue for the insulin receptor, optionally wherein the A8 substitution is a nitrogen-containing amino acid selected from the group Lysine, Histidine or Glutamine.

12. The insulin analogue of claims 1-4 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 A 14 is independently selected from the group consisting of His, Lys, Arg, and Glu.

13. The insulin analogue of claims 1-4 wherein the A chain contains a substitution at position A21 that protects the insulin analogue from chemical degradation.

14. The insulin analogue of claims 1-4 where 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 glucose-binding element linked to the C terminus of the truncated B chain.

15. The insulin analogue of claims 1-4 where said B chain is extended by one or two amino acids with a glucose-binding element linked to the C terminus of the extended B chain.

16. The insulin analogue of claims 1 -4 wherein the insulin A chain is a polypeptide selected from the group consisting ofX.IXOXIIVEQX6CXSSIXI ISLYQLENYCN (SEQ ID NO: 3), XoXiIVEQXeCXsSIXiiSLYQLENYCN (SEQ ID NO: 4), and X7oIVEQX6CX8SIXiiSLYQLENYCN (SEQ ID NO: 5) wherein X-i is any amino acid, optionally wherein X.| is Gly;Xo is any amino acid or a modified amino acid comprising a glucose-binding element linked to its side chain;Xi is any amino acid or a modified amino acid comprising a glucose-binding element linked to its side chain;Xi, and Xu are each Cys or selenocysteine; and X8is Thr, His, Lys, Arg, or Glu;X70 is a D- amino acid comprising a glucose-binding element linked to its side chain; further wherein at least one of X-i , Xo and Xi is in the D conformation, and the insulin B chain is a polypeptide selected from the group consisting of FVX24QHLCGSHLVEALYLVCGERGFFYTX49X50X51 (SEQ ID NO: 6), and FVX24QHLCGSHLVEALYLVCGERGFFYTX51 (SEQ ID NO: 7), wherein X24 is Lys or Asn;X49 is Orn, Glu Asp, Lys, Pro, or a modified amino acid comprising a glucose- binding element linked to the side chain of the amino acid;X50 is Orn, Lys, Pro, or a modified amino acid comprising a glucose-binding element linked to the side chain of the amino acid; andX51 is Thr or an amino acid linked to glucose-binding element.

17. The insulin analogue of claim 16 wherein the insulin B chain is a polypeptide comprising the sequence ofFVX24QHLCGSHLVEALYLVCGERGFFYTX49X50X51 (SEQ ID NO: 6), whereinX24 is Lys or Asn;X49 is a modified amino acid comprising a glucose-binding element linked to the side chain of the amino acid;X50 is Orn, Lys or Pro; andX51 is Thr or an amino acid linked to glucose-binding element.

18. The insulin analogue of claim 17 whereinX24 is Lys or Asn;X49 is a modified amino acid comprising a glucose-binding element linked to the side chain of the amino acid;X50 is Orn, Lys or Pro; andX51 is an amino acid linked to a glucose-binding element, optionally wherein the glucose-binding element is linked to the side chain of the amino acid.

19. The insulin analogue of claim 17 whereinX24 is Lys or Asn;X49 is a modified amino acid comprising a glucose-binding element linked to the side chain of the amino acid;X50 is Pro; andX51 is Thr.

20. The insulin analogue of claim 17 wherein the insulin A chain is a polypeptide comprising the sequence ofX0XIIVEQX6CX8SIXI ISLYQLENYCN (SEQ ID NO: 4), whereinXo is any amino acid, optionally Gly; andXi is a modified D amino acid comprising a glucose-binding element linked to its side chain;Xe and Xu are each Cys or each selenocysteine; andX8is Thr, His, Lys, Arg, or Glu.

21. An insulin analogue of any one of claims 1-20 wherein the glucose-binding element contains two or more boron atoms, optionally containing phenyl-boronic acid or a halogen-modified phenyl boronic acid, such that one or more moieties is selected from the following:8CM2-Dab(BCM2)-OH BCM2-Gly-Dab(BCM2) BCM2-Dab(BCM2-Gly)-OHGBE4 GBE5 = Xxx - Gly GBE6 - Xxx - Gly22. A method of treating a diabetic patient comprising administering a physiologically effective amount of an insulin analogue of any one of claims 1-21, or a physiologically acceptable salt thereof to the patient.

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