Abstract:
Protection of proteins against fibrillation may be afforded by introduction of certain histidine substitutions into the protein, such that a pair of histidines are present with sufficient spacing as to allow the histidines to coordinate with zinc. In the case of insulin, introduction of histidine residue substitutions at residues A4 and A8 together or a histidine residue substitution at residue B1, provides increased resistance to fibrillation while maintaining at least a majority of the activity of the insulin analogue. Introduction of a histidine residue substitution at residue A8 restores at least a portion of fibrillation resistance that may have been harmed by substitutions present on the B-chain such as those present in fast-acting insulins. Proteins protected by such histidine substitutions may be used to provide a pharmaceutical composition. A method of treating a patient includes administering a physiologically effective amount of the pharmaceutical composition to the patient.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims priority from U.S. Provisional Patent Application Nos. 60/756,930 and 60/757,364 filed on Jan. 6, 2006 and Jan. 9, 2006, respectively. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention was made with government support under cooperative agreements awarded by the National Institutes of Health, Contract No. NIH RO1DK069764. The U.S. government may have certain rights to the invention. 
    
    
     BACKGROUND OF THE INVENTION 
     The stability of proteins used in medical treatment is an important concern in medicine. Protein degradation can be classified as physical or chemical degradation. Chemical degradation of proteins can include deamination or transamination of the protein. Physical degradation may include a change in conformation that leads to aggregation of the protein and formation of protein fibrils. The present invention concerns the prevention of fibrillation. 
     Administration of the protein insulin has long been established as a treatment for diabetes. Insulin is a small globular protein that plays a central role in metabolism in vertebrates. Insulin contains two chains, an A chain, containing 21 residues and a B chain containing 30 residues. The hormone is stored in the pancreatic β-cell as a Zn 2+ -stabilized hexamer, but functions as a Zn 2+ -free monomer in the bloodstream. Insulin is the product of a single-chain precursor, proinsulin, in which a connecting region (35 residues) links the C-terminal residue of B chain (residue B30) to the N-terminal residue of the A chain ( FIG. 1A ). Although the structure of proinsulin has not been determined, a variety of evidence indicates that it consists of an insulin-like core and disordered connecting peptide ( FIG. 1B ). Formation of three specific disulfide bridges (A6-A11, A7-B7, and A20-B19;  FIG. 1B ) is thought to be coupled to oxidative folding of proinsulin in the rough endoplasmic reticulum (ER). Proinsulin assembles to form soluble Zn 2+ -coordinated hexamers shortly after export from ER to the Golgi apparatus. Endoproteolytic digestion and conversion to insulin occurs in immature secretory granules followed by morphological condensation. Crystalline arrays of zinc insulin hexamers within mature storage granules have been visualized by electron microscopy (EM). Assembly and disassembly of native oligomers is thus intrinsic to the pathway of insulin biosynthesis, storage, secretion, and action ( FIG. 2 ). 
     Insulin readily misfolds in vitro to form a prototypical amyloid. Unrelated to native assembly, fibrillation is believed to occur via an amyloidogenic partial fold ( FIG. 1C ). Factors that accelerate or hinder fibrillation have been extensively investigated in relation to pharmaceutical formulations. Zinc-free insulin is susceptible to fibrillation under a broad range of conditions and is promoted by factors that impair native dimerization and higher order self-assembly. It is believed that the structure of active insulin is stabilized by axial zinc ions coordinated by the side chains of His B10 . 
     Amino-acid substitutions in the A- and/or B-chains of insulin have widely been investigated for possible favorable effects on the pharmacokinetics of insulin action following subcutaneous injection. Examples are known in the art of substitutions that accelerate or delay the time course of absorption. Such substitutions (such as Asp B28  in the insulin analogue sold under the trademark NOVALOG® and [Lys B28 , Pro B29 ] in the insulin analogue sold under the trademark HUMALOG®) can be and often are associated with more rapid fibrillation and poorer physical stability. Indeed, a series of ten analogs of human insulin for susceptibility to fibrillation, including Asp B28 -insulin and Asp B10 -insulin have been tested. All ten were found to be more susceptible to fibrillation at pH 7.4 and 37° C. than is human insulin. The ten substitutions were located at diverse sites in the insulin molecule and are likely to be associated with a wide variation of changes in classical thermodynamic stability. These results suggest that substitutions that protect an insulin analogue from fibrillation under pharmaceutical conditions are rare; no structural criteria or rules are apparent for their design. 
     Fibrillation, which is a serious concern in the manufacture, storage and use of insulin and insulin analogues for diabetes treatment, is enhanced with higher temperature, lower pH, stifling or the presence of urea, guanidine, ethanol co-solvent, or hydrophobic surfaces. Current US drug regulations demand that insulin be discarded if fibrillation occurs at a level of one percent or more. Because fibrillation is enhanced at higher temperatures, diabetic individuals optimally must keep insulin refrigerated prior to use. Fibrillation of insulin or insulin analogue can be a particular concern for diabetic patients utilizing an insulin pump, in which small amounts of insulin or insulin analogue are injected into the patient&#39;s body at regular intervals. In such a usage, the insulin or insulin analogue is not kept refrigerated within the pump apparatus and fibrillation of insulin can result in blockage of the catheter used to inject insulin or insulin analogue into the body, potentially resulting in unpredictable blood glucose level fluctuations or even dangerous hyperglycemia. At least one recent report has indicated that lispro insulin (an analogue in which residues B28 and B29 are interchanged relative to their positions in wild-type human insulin; the product sold under the trademark HUMALOG®) may be particularly susceptible to fibrillation and resulting obstruction of insulin pump catheters. 
     Insulin fibrillation is an even greater concern in implantable insulin pumps, where the insulin would be contained within the implant for 1-3 months at high concentration and at physiological temperature (i.e. 37° C.), rather than at ambient temperature as with an external pump. Additionally, the agitation caused by normal movement would also tend to accelerate fibrillation of insulin. In spite of the increased potential for insulin fibrillation, implantable insulin pumps are still the subject of research efforts, due to the potential advantages of such systems. These advantages include intraperitoneal delivery of insulin to the portal circulatory system, which mimics normal physiological delivery of insulin more closely than subcutaneous injection, which provides insulin to the patient via the systemic circulatory system. Intraperitoneal delivery provides more rapid and consistent absorption of insulin compared to subcutaneous injection, which can provide variable absorption and degradation from one injection site to another. Administration of insulin via an implantable pump also potentially provides increased patient convenience. Whereas efforts to prevent fibrillation, such as by addition of a surfactant to the reservoir, have provided some improvement, these improvements have heretofore been considered insufficient to allow reliable usage of an implanted insulin pump in diabetic patients outside of strictly monitored clinical trials. 
     Resistance to fibrillation caused by heat or other causes would be advantageous not only for insulin and insulin analogs, but for a variety of medically useful proteins, especially in tropical and sub-tropical regions of the developing world. The major barrier to the storage and practical use of presently available pharmaceutical formulations of insulin and insulin analogues at temperatures above 30° C. is due to accelerated fibrillation of the protein. The major reason for limitations to the shelf life of presently available pharmaceutical formulations of insulin and insulin analogues at temperatures above 10° C. is due to fibrillation of the protein. Fibrillation is of special concern for fast-acting or “mealtime” insulin analogues (such as the products sold under the trademarks HUMALOG® and NOVALOG®) when these formulations are diluted by the patient and stored at room temperature for more than 15 days. 
     Modifications of proteins such as insulin are known to increase resistance to fibrillation but impair biological activity. For example, “mini-proinsulin,” a proinsulin analogue containing a dipeptide linker between the A and B chains of insulin, is resistant to fibrillation but is impaired in its activity. Therefore, a need exists for insulin analogues and other protein analogues that are resistant to fibrillation and that maintain at least a majority of their biological activity. 
     SUMMARY OF THE INVENTION 
     It is, therefore, an aspect of the present invention to provide protein analogues that are more resistant to fibrillation than their wild-type counterparts, while maintaining biological activity. 
     It is another aspect of the present invention to provide insulin analogues that are more resistant to fibrillation than their counterpart insulins but maintain at least a majority of their biological activity. 
     It is still another aspect of the present invention to provide an insulin A- or B chain analogue that, as a part of an insulin analogue, provides increased resistance to fibrillation compared to the non-analogue counterpart insulin. 
     It is still another aspect of the present invention to provide amino-acid substitutions in the insulin A- or B chains such that, as a part of an unrelated insulin analogue with enhanced susceptibility to fibrillation, the substitutions described herein at least partially compensate for the instability of the original analogue to provide increased resistance to fibrillation relative to non-substituted counterpart analogue. 
     It is yet another aspect of the present invention to provide a nucleic acid that encodes for at least a portion of an insulin analogue that displays increased resistance to fibrillation compared to a non-analogue insulin. 
     In general, the present invention provides a vertebrate insulin analogue or a physiologically acceptable salt thereof, comprising at least one of the following substitutions: histidine residue substitutions at residues A4 and A8 together, and a histidine residue substitution at residue B1. In one example, the vertebrate insulin analogue is a mammalian insulin analogue, such as a human, porcine, bovine or equine insulin analogue. The histidine substitutions specified above may be used in combination with each other (A4, A8 and B1 tri-substitutions); the B1 or paired A4-A8 histidine substitutions may also be combined with unrelated substitutions elsewhere in the insulin molecule intended to introduce other favorable pharmaceutical properties unrelated to resistance to fibrillation. 
     Additionally, in some embodiments, the present invention provides an insulin analogue with an increased resistance to fibrillation over its counterpart analogues. Known insulin analogs (such as the products sold under the trademarks HUMALOG® and NOVALOG®) have an increased susceptibility to fibrillation. The present invention also specifies the use of histidine (i) singly at position B1, (ii) singly at position A8, (iii) at A8 in combination with histidine also at A4, or (iv) jointly at positions B1, A4, and A8 to compensate for the instability of insulin analogues containing amino-acid substitutions in the B-chain (such as but not restricted to substitutions at position B28 and/or position B29) for the purpose of augmenting their resistance to fibrillation. 
     The present invention likewise provides a pharmaceutical composition comprising such insulin analogues and which may additionally include zinc ions. Optimally, in the presence of phosphate-containing buffers, zinc ions may be included in such a composition at a level of a molar ratio of between 2.2 and 3.0 per hexamer of the insulin analogue; in the presence of buffers based on Tris Hydroxymethylaminoethane (Tris), or other buffers that, like Tris, do not bind well to zinc ions to form an insoluble salt, zinc ions may be included in such a composition at a level of a molar ratio of between 2.2 and 5.0 per hexamer of the insulin analogue. In such a formulation the concentration of the insulin analogue would typically be between 0.1 and 1.5 mM. Excipients may include glycerol, glycine, other buffers and salts, and anti-microbial preservatives such as phenol and meta-cresol; the latter preservatives are known to enhance the stability of the insulin hexamer. Such a pharmaceutical composition may be used to treat a patient having diabetes or other medical condition by administering a physiologically effective amount of the composition to the patient. 
     The present invention also provides a nucleic acid comprising a sequence that encodes a polypeptide selected from the group consisting of an A chain insulin analogue containing histidine residue substitutions at residues A4 and A8, a B chain insulin analogue containing a histidine residue substitution at residue B1, and combinations thereof. The nucleic acid sequence may encode either the isolated A- and B chain analogue, a modified A- or B chain containing an unrelated substitution or extension elsewhere in the polypeptide, modified proinsulin analogues, or single-chain insulin analogues containing a modified or foreshortened connecting domain. The nucleic acid may also be a portion of an expression vector, and that vector may be inserted into a host cell such as a prokaryotic host cell like an  E. coli  cell line, or a eukaryotic cell line such as as  S. cereviciae  or  Pischia Pastoris  strain or cell line. 
     The present invention likewise provides a method of increasing the resistance of polypeptides to fibrillation. The method includes introducing a histidine substitution or two paired histidine substitutions into the polypeptide such that a pair of histidine are present in the polypeptide with sufficient spacing between the pair of histidines as to allow the side chains of the histidines to coordinate with zinc. In one example, there are three intervening amino acids between the pair of histidines. In another example, the pair of histidines are present in an α-helix of the polypeptide. It is envisioned that the method would also work with a spacing of two intervening amino acids between the histidines. The method may be utilized to introduce one or more histidine substitutions into the A or B chain of a vertebrate insulin to increase the resistance of the resulting insulin to fibrillation. Irrespective of theory, the method may also utilize a histidine substitution in one chain of insulin to compensate for enhanced susceptibility to fibrillation caused by substitutions in the other chain. For example, Histidine substitutions at A8 alone or at A4 and A8 together may compensate for enhanced susceptibility to fibrillation caused by substitutions or other changes in the B-chain. 
     Chemical degradation of insulin, such as deamination, isopeptide bond formation, and disulfide interchange leading to formation of covalent polymers, is known to be reduced by formulations or modifications that enhance the thermodynamic stability of the native molecular structure of the insulin or insulin analogue. While not wishing to be bound by theory, introduction of a pair of histidine residues on the surface of a protein may confer a zinc-dependent increase in thermodynamic stability and so delay chemical degradation of the protein. 
     It is still another aspect of the present invention to provide a method treating a patient comprising administering a fibrillation-resistant insulin analogue to the patient. In one example, the fibrillation-resistant insulin analogue is a single-chain insulin analogue. In another example, the insulin is administered by an external or implantable insulin pump. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         FIG. 1A  is a schematic representation of the sequence of human proinsulin (SEQ ID NO: 11) including the A and B chains and the connecting region shown with flanking dibasic cleavage sites (filled circles) and C-peptide (open circles). The line labeled “foreshortened connecting peptide” represents the connecting region in mini-proinsulin, which is a proinsulin analogue containing a dipeptide (Ala-Lys) linker between the A chain and B chain portions of insulin. 
         FIG. 1B  provides a structural model of proinsulin, consisting of an insulin-like moiety and disordered connecting peptide (dashed line). The asterisk indicates the location of a foreshortened AK linker in mini-proinsulin. 
         FIG. 1C  provides a representation of a proposed pathway of insulin fibrillation via partial unfolding of monomer. The native state is protected by classic self-assembly (far left). Disassembly leads to equilibrium between native- and partially folded monomers (open triangle and trapezoid, respectively). This partial fold may unfold completely as an off-pathway event (open circle) or aggregate to form a nucleus en route to a protofilament (far right). 
         FIG. 2  is a representation of the pathway of insulin biosynthesis, storage, and secretion. A, nascent proinsulin folds as a monomer in ER wherein zinc-ion concentration is low; in Golgi apparatus zinc-stabilized proinsulin hexamer assembles, which is processed by cleavage of connecting peptide to yield mature insulin. Zinc-insulin crystals are observed in secretory granules. B, on metabolic stimulus, zinc-insulin crystals are released into portal circulation (pink) and disassociate in steps to liberate the functional monomer. 
         FIG. 3A  is a representation of a variant T 3 R 3   f  zinc hexamer. The side chains of His A4,A8  and His B10  in a Zn 2+ -coordination site are shown. 
         FIG. 3B  is a ribbon representation of a crystal structure of a His A4,A8  dimer. Ribbon representation of crystal structure of His A4,A8 -insulin. The A-chain of molecule 1 and 2 are shown in black and B-chain and B-chain N-terminal (B1-B8) α-helix in gray, residues of His A4,A8  side chains are shown in black stick, and a Zn 2+ -coordination site is noted. 
         FIG. 3C  is a representation of a crystal structure of His A4,A8  monomer. 
         FIG. 3D  is a space-filling representation of T-state promoter showing His A4,A8  relation to classical the surface. The side chains of His A4,A8  are shown in black with a Zn 2+ -coordination site between the two His residues. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is directed toward recombinant proteins that provide increased resistance to fibrillation. Particularly, the present invention provides insulin analogues that are resistant to fibrillation. To that end, the present invention provides insulin analogues that contain histidine (His) residue substitutions at positions A8, A4 and A8, or at B1. The insulin analogues of the present invention may include insulin analogues containing His substitutions at all three of the positions A4, A8, and B1. In one particular example, an insulin analogue of the present invention contains only a His substitution at position B1. 
     While not wishing to be bound by theory, it is believed that when the His B1  substitution is present, the side chain of the B1 His residue, in combination with the B5 histidine side chain, provides a potential B1-B5 bi-histidine Zn-binding site, which confers Zn-dependent protection from fibrillation. Similarly, while not wishing to be bound by theory, it is believed that the [His A4 , His A8 ] substitutions also provide a potential bi-histidine Zn-binding site, which confers protection from fibrillation. It is further envisioned that zinc stabilization of insulin will not affect activity in vivo because such zinc-protein structures are predicted to dissociate at protein and zinc concentrations less than about 1 μM. The protective effect of zinc binding may be mediated either though binding to the native molecular structure or to a distorted molecular structure as is thought to occur as an intermediate in the process of fibrillation. 
     Additionally, while not wishing to be bound by theory, it is believed that when the His A8  substitution is present in the presence or absence of zinc and irrespective of histidines substitutions at B1 and/or A4, insulin analogues containing otherwise fibrillation-enhancing amino acid substitutions in the B-chain will regain at least some resistance to fibrillation compared to the corresponding insulin analogue containing only the fibrillation enhancing B-chain substitution or substitutions. 
     As used in this specification and the claims, various analogues may be noted by the amino acid being substituted, followed by the position of the amino acid, optionally in superscript. The position of the amino acid in question includes the A or B chain of insulin where the substitution is located. Thus, His B1  denotes a histidine substitution at the first amino acid of the B chain of insulin. His A4  denotes a histidine substitution at the fourth amino acid of the A chain of insulin, and His A8  denotes a histidine substitution at the eighth amino acid of the A chain of insulin. Unless noted otherwise or wherever obvious from the context, the amino acids noted herein should be considered to be L-amino acids. 
     It is envisioned that the substitutions of the present invention may be made in any of a number of existing insulin analogues. For example, the His A8 , [His A4 , His A8 ], or His B1  substitutions, or combinations thereof, may be made in insulin analogues such as Lispro insulin (sold under the trademark HUMALOG®), insulin Aspart (sold under the trademark NOVALOG®), other modified insulins or insulin analogues, or within various pharmaceutical formulations, such as regular insulin, NPH insulin, lente insulin or ultralente insulin, in addition to human insulin. These substitutions may also be introduced into analogues of human insulin that, while not clinically used, are still useful experimentally, such as DKP-insulin, which contains the substitutions Asp B10  (D), Lys B28  (K) and Pro B29  (P), or miniproinsulin, a proinsulin analogue containing a dipeptide (Ala-Lys) linker between the A chain and B chain portions of insulin in place of the normal 35 amino acid connecting region between the C-terminal residue of the B chain and the N-terminal residue of the A chain. 
     The present invention is not limited to human insulin and its analogues however. It is also envisioned that these substitutions may also be made in animal insulins such as porcine, bovine, equine, and canine insulins, by way of non-limiting examples. 
     Furthermore, in view of the similarity between human and animal insulins, and use in the past of animal insulins in human diabetic patients, it is also envisioned that other minor modifications in the sequence of insulin may be introduced, especially those substitutions considered “conservative” substitutions. For example, additional substitutions of amino acids may be made within groups of amino acids with similar side chains, without departing from the present invention. These include the neutral hydrophobic amino acids: Alanine (Ala or A), Valine (Val or V), Leucine (Leu or L), Isoleucine (Ile or I), Proline (Pro or P), Tryptophan (Trp or W), Phenylalanine (Phe or F) and Methionine (Met or M) Likewise, the neutral polar amino acids may be substituted for each other within their group of Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T), Tyrosine (Tyr or Y), Cysteine (Cys or C), Glutamine (Glu or Q), and Asparagine (Asn or N). Basic amino acids are considered to include Lysine (Lys or K), Arginine (Arg or R) and Histidine (His or H). Acidic amino acids are Aspartic acid (Asp or D) and Glutamic acid (Glu or E). In one example, the insulin analogue of the present invention contains three or fewer conservative substitutions other than the [His A4 , His A8 ], or His substitutions of the present invention. In another example, the insulin analogue of the present invention contains one or fewer conservative substitutions other than the His A8 , [His A4 , His A8 ], or His B1  substitutions of the present invention. 
     The amino-acid sequence of the A chain of human insulin is provided, for comparative purposes, as SEQ ID NO: 1. The amino acid sequence of an analogue of the A chain of human insulin containing the His A4  and His A8  substitutions is provided as SEQ ID NO: 2. The amino acid sequence of the B chain of human insulin is provided, for comparative purposes, as SEQ ID NO: 3. The amino acid sequence of an analogue of the B chain of human insulin containing the His substitution is provided as SEQ ID NO: 4. Further combinations of other substitutions are also within the scope of the present invention. It is also envisioned that the substitutions of the present invention may also be combined with substitutions of prior known insulin analogues. For example, the amino acid sequence of an analogue of the B chain of human insulin containing the Lys B28  Pro B29  substitutions of lispro insulin combined with the His B1  substitution is provided as SEQ ID NO: 5. Likewise, the amino acid sequence of an analogue of the B chain of human insulin containing the Asp B28  substitution of aspart insulin, combined with the His B1  substitutions, is provided as SEQ ID NO: 6. 
     By way of example and not of limitation, it is also envisioned that the [His A4 , His A8 ], and/or His B1  substitutions of the present invention may also be made in an insulin analogue or proinsulin analogue having the amino acid sequence represented by formula I (SEQ ID NO: 11), 
                     (I)       R1-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-               Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-               Phe-Tyr-Thr-R2-R3-Thr-Xaa 0-35 -Gly-Ile-Val-R4-Gln-               Cys-Cys-R5-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-               Asn-Tyr-Cys-Asn;            
wherein R1 is His or Phe; wherein R2 is Pro, Lys, or Asp; wherein R3 is Lys or Pro; wherein R4 is His or Glu; wherein R5 is His or Thr; and wherein Xaa 0-35  is 0-35 of any amino acid or a break in the amino acid chain;
 
     and further wherein at least one substitution selected from the group of the following amino acid substitutions is present:
         R1 is His; and   R5 is His; and   R4 and R5 together are His.       

     Analogues of insulin containing the His B1 , His A8 , or the [His A4 , His A8 ] substitutions were made in human insulin and DKP insulin as follows. The tetra-S-sulfonate derivative of the A chain was obtained by oxidative sulfitolysis of human insulin (provided by Eli Lilly and Co., Indianapolis, Ind.) followed by separation of the S-sulphonated A and B chains as known in the art. A and B-chain analogues were prepared by solid-phase chemical synthesis. 4-methylbenzhydrylamine resin (0.6 mmol of amine/g; Bachem, Inc.) was used as solid support for synthesis of A-chain analogues; (N-tert-butoxycarbonyl, O-benzyl)-threonine-PAM resin (0.56 mmol/g; Bachem, Inc.) was used as solid support for synthesis of B-chain analogues. A manual double-coupling protocol was followed. Insulin analogues were prepared by chain combination in 0.1M glycine buffer (pH 10.6) in the presence of 4.5 mM dithiothreitol (DTT). Analogues were purified by size exclusion chromatography (Bio-Gel P-4 in 3M acetic acid) and preparative reverse phase HPLC Predicted molecular masses were confirmed by matrix-assisted laser desorption ionization (MALDI-TOF) mass spectrometry (MS). 
     The following analogues were synthesized and tested for activity and resistance to fibrillation as provided herein in comparison to human insulin and otherwise unmodified DKP-insulin. An A-chain analogue having the sequence of SEQ ID NO: 2 was combined with a B chain peptide having the sequence of SEQ ID NO: 3 to provide a [His A4 , His A8 ]-insulin analogue. A [His A4 , His A8 ]-DKP-insulin analogue was prepared with the peptides identified by SEQ ID NOS: 2 and 7. A His B1 -insulin analogue was prepared with peptides having the sequences of SEQ ID NOS: 1 and 4. A His B1 -DKP-insulin analogue was prepared with the peptides identified by SEQ ID NOS: 1 and 8. A His A8 -DKP-insulin analogue was prepared with the peptides identified by SEQ ID NOS: 9 and 7. The rationale of testing of substitutions in the context of a monomeric analog is that the insulin monomer is thought to be an obligatory intermediate in the molecular pathway of fibrillation; protective effects in the context of a zinc insulin hexamer would be expected to be similar or greater. In addition, the crystal structure of an insulin analogue containing the paired substitutions [His A4 , His A8 ] was determined in the presence of zinc ions to verify the formation of a novel zinc-ion-binding site involving the introduced histidines side chains at these positions. 
     Receptor-Binding Assays. 
     Relative activity is defined as the ratio of analogue to wild-type human insulin required to displace 50 percent of specifically bound  125 I-human insulin. A human placental membrane preparation containing the insulin receptor (IR) was employed as known in the art. Membrane fragments (0.025 mg protein/tube) were incubated with  125 I-labeled insulin (ca. 30,000 cpm) in the presence of selected concentrations of unlabelled analogue for 18 hours at 4° C. in a final volume of 0.25 ml of 0.05 M Tris-HCl and 0.25 percent (w/v) bovine serum albumin at pH 8. Subsequent to incubation, mixtures are diluted with 1 ml of ice-cold buffer and centrifuged (10,000 g) for 5 min at 4° C. The supernatant will then be removed by aspiration, and the membrane pellet counted for adioactivity. Data is corrected for nonspecific binding (amount of radioactivity remaining membrane associated in the presence of 1 μM human insulin. In all assays the percentage of tracer bound in the absence of competing ligand was less than 15% to avoid ligand-depletion artifacts. A corresponding microtiter plate antibody assay using the IGF Type I receptor was employed to assess cross-binding to this homologous receptor. 
     X-Ray Crystallography. 
     Crystals of an analogue of human insulin containing histidines at positions A4 and A8 were grown by hanging-drop vapor diffusion in the presence of a 1:1.7 ratio of Zn 2+  to protein monomer and a 3.5:1 ratio of phenol to protein monomer in Tris-HCl buffer as known in the art. Drops consisted of 1 μl of protein solution (8 mg/ml in 0.02 M HCl) mixed with 1 of reservoir solution (0.38 M Tris-HCl, 0.1 M sodium citrate, 9% acetone, 4.83 mM phenol, and 0.8 mM zinc acetate at pH 8.4). Each drop was suspended over 1 ml of reservoir solution. Crystals (space group R3) were obtained at room temperature after two weeks. Data were collected from single crystals mounted in a rayon loop and flash frozen to 100° K. Reflections from 32.05-1.90 Å were measured with a CCD detector system on synchrotron radiation in Berkeley National Laboratory. Data were processed with programs DTREK. The crystal belongs to space group R3 with unit cell parameters: a=b=78.09 Å, c=36.40 Å, α=β=90°, γ=120°. The structure was determined by molecular replacement using the computer program CNS. Accordingly, a model was obtained using the native TR dimer (Protein Databank (PDB) identifier 1RWE following removal of all water molecules, zinc- and chloride ions). A translation-function search was performed using coordinates from the best solution for the rotation function following analysis of data between 15.0 and 4.0 Åresolutions. Rigid-body refinement using CNS, employing overall anisotropic temperature factors and bulk-solvent correction, yielded values of 0.325 and 0.344 for R and R free , respectively, for data between 19.2 and 3.0 Åresolution. Between refinement cycles, 2F o -F c  and F o -F c  maps were calculated using data to 3.0 Åresolution; zinc and chloride ions and phenol molecules were built into the structure using the program O. Water molecules were calculated and checked using DDQ program. The geometry was continually monitored with PROCHECK; zinc ions and water molecules were built into the difference map as the refinement proceeded. Calculation of omit maps (especially in the first eight residues of B chain N terminus of each monomer) and further refinement were carried out using CNS, which implement maximum-likelihood torsion-angle dynamics and conjugate-gradient refinement. 
     The crystal structure of [His A4 , His A8 ]-insulin as a T 3 R 3   f  zinc hexamer ( FIG. 3A ), determined to a resolution of 1.9 Å, contains five bound zinc ions instead of the two bound zinc ions that are ordinarily observed in wild-type crystal structures obtained under these conditions. Three novel zinc ions are observed coordinated by the side chains of histidines side chains at positions A4 and A8 in the three T-state protomers in addition to the two classical axial zinc ions coordinated by the wild-type histidine side chain at position B10. 
     Activities of the insulin analogues [His A4 , His A8 ]-insulin, [His A4 , His A8 ]-DKP-insulin, His B1 -insulin, His B1 -DKP-insulin, and His A8 -DKP-insulin relative to human insulin, whose affinity is 0.05 nM under assay conditions are provided in Table 1. For comparison purposes, the activity of DKP-insulin is also provided. Histidine substitutions at positions B1, A4, A8, or jointly at positions A4 and A8, do not significantly enhance cross-binding of these analogues to the IGF-I Type I receptor; any such increase is less than twofold (i.e., the level of increased cross-binding exhibited by the insulin analogue sold under the trademark HUMALOG®). 
     
       
         
               
             
               
               
             
           
               
                 TABLE I 
               
             
             
               
                   
               
               
                 Affinity of Insulin and Insulin Analogues to Insulin Receptor 
               
             
          
           
               
                 Sample: 
                 Affinity (relative to human insulin): 
               
               
                   
               
               
                 Human insulin 
                 100% 
               
               
                 DKP-insulin 
                 161 ± 19 
               
               
                 His A8 -DKP-insulin 
                 167 ± 21 
               
               
                 [His A4 , His A8 ]-insulin 
                 108 ± 4  
               
               
                 [His A4 , His A8 ]-DKP-insulin 
                 201 ± 14 
               
               
                 His B1 -insulin 
                 &gt;180 
               
               
                 His B1 -DKP-insulin 
                 311 ± 48 
               
               
                   
               
             
          
         
       
     
     These data indicate that the affinity of the analogues to the human insulin receptor is as great or greater than that of wild-type human insulin. 
     Circular Dichroism—Samples were dissolved in either 10 mM phosphate and 100 mM KCl (pH 7.4) or 0.01 N HCl (pH 2.0) at a protein concentration of 25 μM. To remove particulate matter and protein aggregates, samples were filtered (0.22 μM; Satorius, Goetlingen, Germany). Spectra, acquired with an Aviv spectropolarimeter (Aviv Biomedical, Inc., Lakewood, N.J.), were normalized. Data were obtained at 4° C. and fitted by non-linear least squares to a two-state model. CD Spectra for analogues were similar to native and DKP insulins (data not shown). 
     Fibrillation Assay—DKP-insulin and analogues were made 100 μM in a deoxygenated buffer consisting of 10 mM Tris-HCl (pH 7.4), 140 mM NaCl, with or without 1 mM ZnCl 2 . Samples (in triplicate) were placed in sealed glass vials and placed on an automated tilting table at 37° C. At successive times aliquots were withdrawn and analyzed by a thioflavin T (ThT) fluorescence assay to determine the onset of fibrillation. 
     Fluorescence Spectroscopy—Thioflavin T (ThT) was made 1 mM in double-distilled water and stored at 4° C. in the dark. To monitor fibrillation, 10-μl aliquots obtained at indicated time points were mixed with 3 ml of ThT assay buffer (5 μM ThT in 50 mM Tris-HCl (pH 7.5) and 100 mM NaCl). Fluorescence measurements were performed using an Aviv spectrofluorometer in 1-cm quartz cuvettes. Emission spectra were collected from 470 to 500 nm following excitation at 450 nm; the integration time was 1 s. ThT in buffer without protein was used as baseline. The fibrillation lag time is defined as the time required to observe 2-fold enhancement in ThT emission. The threshold of 2-fold-enhanced ThT fluorescence is followed a rapid increase in turbidity associated with elongation of mature fibrils and a further increase in ThT fluorescence. Under these conditions, human insulin undergoes fibrillation in 3-4 days in the absence of zinc. Lag times prior to fibrillation of the analogues are provided in Table II. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE II 
               
             
             
               
                   
               
               
                 Fibrillation Lag Times of DKP-insulin and analogues ± Zinc 
               
             
          
           
               
                   
                 Lag time 
                   
                 Lag time 
               
               
                 Sample (without Zn) 
                 (days) 
                 Sample (with Zn) 
                 (days) 
               
               
                   
               
               
                 DKP-insulin 
                 7.7 ± 0.6 
                 DKP-insulin 
                 11.7 ± 2.5 
               
               
                 [His A4 , His A8 ]-DKP- 
                 22.7 ± 1.5  
                 [His A4 , His A8 ]-DKP- 
                 &gt;92 
               
               
                 insulin 
                   
                 insulin 
               
               
                 His B1 -DKP-insulin 
                 6.8 ± 0.8 
                 His B1 -DKP-insulin 
                 20.3 ± 2.1 
               
               
                 His A8 -DKP-insulin 
                 21.3 ± 1.2  
                 His A8 -DKP-insulin 
                   34 ± 1.7 
               
               
                   
               
             
          
         
       
     
     In the presence of zinc, each of the DKP analogues provides a greater fibrillation lag time than DKP insulin. The protective effect against fibrillation was especially pronounced for the DKP-insulin analogue carrying the His A4 , His A8  substitutions in zinc. In the absence of zinc ions in a buffer consisting of 10 mM potassium phosphate and 140 mM KCl at pH 7.4 a single histidines substitution at position A8 increased the lag time prior to fibrillation from 3.5±0.9 days to 13±2.6 days at 37° C. 
     Based upon the foregoing disclosure, it should now be apparent that the polypeptide analogues provided herein will provide increased resistance to fibrillation while maintaining at least the majority of the activity of the polypeptide. The histidine substitutions specified herein may be employed to enhance the resistance of wild-type human insulin or to compensate for otherwise destabilizing effects of unrelated substitutions in the A- or B-chains. It is, therefore, to be understood that any variations evident fall within the scope of the claimed invention and thus, the selection of specific component elements can be determined without departing from the spirit of the invention herein disclosed and described.