Protein engineering of insulin: Two novel fast-acting insulins [B16Ala]insulin and [B26Ala]insulin

Blood glucose lowering assay proved that [B16Ala]insulin and [B26Ala]insulin exhibit potency of acute blood glucose lowering in normal pigs, which demonstrates that they are fast- acting insulin. Single-chain precursor of [B16Ala]insulin and [B26Ala]insulin is [B16Ala]PIP and [B26Ala]PIP, respectively, which are suitable for gene expression. Secretory expression level of the precursors in methylotrophic yeast Pichia pastoris was quite high, 650 mg/L and 130 mg/L, respectively. In vivo biological assay showed that the two fast-acting insulins have full or nearly full biological activity. So both [B16Ala]insulin and [B26Ala]insulin can be well developed as fast-acting insulin for clinic use.     

Four new monomeric insulins obtained by alanine scanning the dimer-forming surface of the insulin molecule

The residues A21Asn, B12Val, B16Tyr, B24Phe, B25Phe, B26Tyr and B27Thr, buried in the dimer of insulin, were identified by means of alanine-scanning mutagenesis. The receptor binding activity, in vivo biological potency and self-association properties of the seven single alanine human insulin mutants were determined. Four of the seven single alanine mutants, [B12Ala]human insulin, [B16Ala]human insulin, [B24Ala]human insulin and [B26Ala]human insulin, are monomeric insulin, which indicates that B12Val, B16Tyr, B24Phe and B26Tyr are crucial for the formation of insulin dimer. The monomeric [B16Ala]human insulin and [B26Ala]human insulin retain 27 and 54% receptor binding activity, respectively, and nearly the same in vivo biological potency compared with native insulin, so they could be developed as the fast-acting insulin.     

胰岛素蛋白质工程：[B9Glu]人胰岛素

用基因定位突变方法将胰岛素Ｂ链第９位的Ｓｅｒ改为Ｇｌｕ。获得速效胰岛素─—［Ｂ９Ｇｌｕ］人胰岛素．它的受体结合能力和体内生物活力分别为猪胰岛素的２１％和４０％。     

B17-D-leucine]insulin and [B17-norleucine]insulin: synthesis and biological properties

The chemical synthesis of two porcine insulin analogues is described. Leucine in position B17 of the native molecule was substituted by its D-enantiomer and by L-norleucine, respectively. Both B-chain derivatives were synthesized by fragment condensation and purified as di-S-sulphonates by gel filtration followed by ion exchange chromatography on SP-Sephadex at pH3. Combination with native sulphhydryl A-chain yielded [DLeuB17]insulin and [NleB17]insulin. Both insulin analogues were isolated by gel filtration followed by ion exchange chromatography on CM-cellulose at pH 4.0. Biological activities of the analogues were determined relative to native pork insulin: 1) glucose oxidation in rat epididymal adipocytes was 6% for [DLeuB17]insulin and 16% for [NleB17]insulin, 2) receptor-binding affinity tested with cultured human fibroblasts and with rat adipocytes was 3% for [DLeuB17]insulin and 26% for [NleB17]insulin, and 3) thymidine incorporation into DNA of human fibroblasts was 35% for [DLeuB17]insulin and 100% for [NleB17]insulin.     

Chemical synthesis of [des(tetrapeptide B27--30), Tyr(NH2)26-B] and [des(pentapeptide B26--30), Phe(NH2)25-B] bovine insulins.

Two analogs of bovine insulin, [des(tetrapeptide B27--30), Tyr(NH2)26-B] and [des(pentapeptide B26--30), Phe(NH2)25-B] insulin, which differ from the parent molecule in that the C-terminal tetrapeptide and pentapeptide sequences, respectively, from the B chain have been eliminated and the newly exposed residues are amidated, have been synthesized. The [des(tetrapeptide B27--30), Tyr(NH2)26-B] insulin shows potencies of 16.8 IU/mg by the mouse convulsion assay method and 10.8 IU/mg by the radioimmunoassay method. The [des(pentapeptide B26--30), Phe(NH2)25-B] insulin possesses a potency of 10.5 IU/mg when assayed by the mouse convulsion method and 14 IU/mg by the radioimmunoassay technique. The potencies of these analogs are higher than the potencies of the respective non-amidated derivatives (Katsoyannis et al., 1973, 1974). It is speculated that the gradual decline of biological activity observed as amino acid residues are eliminated from the C-terminal region of the B chain of insulin is due to the proximity of a hydrophilic carboxyl group to the hydrophobic core of the protein molecule.     

[B2-Lys]-胰岛素的制备与性质

本文报道了用化学半合成途径从天然猪胰岛素制备[B2-Lys]-胰岛素的过程。人胎盘细胞膜胰岛素受体结合试验表明:[B2-Lys]-胰岛素的受体结合能力只有天然胰岛素的80％,降兔血糖作用与时间关系的结果表明它没有长效作用。本文还对这些结果进行了讨论。     

Contribution of the B16 and B26 tyrosine residues to the biological activity of insulin

We report the synthesis and biological evaluation of five insulin analogues in which one or both of the B-chain tyrosine residues have been substituted. [B16 Phe]insulin and [B16 Trp]insulin display a very modest reduction in potency (c. 65%) relative to porcine insulin; [B26 Phe]insulin is less active (30–50%), and the doubly substituted [B16 Phe, B26 Phe]insulin displays still lower potency (c. 35%). The further substitution of Asp for B10 His in [B16 Phe, B26 Phe]insulin raises its activity to approximately twofold greater than natural insulin, an increase of approximately fivefold over the parent compound. We conclude that the bulk and/or aromaticity of the amino acid residue at position B16, but not its hydrogen-bonding capacity, contributes to the biological activity of the hormone. We further conclude that hydrogen-bonding capacity or special side-chain packing characteristics are required at the B26 position for insulin to display high biological activity.     

The relationship between the connecting peptide of recombined single chain insulin and its biological function

To investigate the relationship between the biological activity of recombined single chain insulin and the length of the connecting peptide, we designed and prepared three single chain insulin molecules, namely, PIP, [A]5PIP and [A]10PIP, by site-directed mutagenesis, in which B30 and A1 were linked through dipeptide A-K, heptapeptide A-A-A-A-A-A-K, and dodecapeptide A-A-A-A-A-A-A-A-A-A-A-K, respectively. Their receptor binding capacities were 0.14%, 14.3% and 11.1% of that of insulin respectively and their in vivo biological activities were in consistence with their receptor binding capacity; whereas their growth promoting activities were 17%, 116.3% and 38% of that of insulin. These results suggested the following conclusions. (i) The recombined single chain insulin could also possess the same metabolic and mitogenic function as insulin. (ii) The receptor binding capacity of recombined single chain insulin to insulin receptor was closely related to the length and amino acid composition of the connectin     

胰岛素蛋白质工程：高活性［B10Asp］人胰岛素

用基因定位突变方法将胰岛素Ｂ链第１０位的Ｈｉｓ变为Ａｓｐ,获得高活力胰岛素──［Ｂ１０Ａｓｐ］人胰岛素。其受体结合能力和离体生物活力分别为猪胰岛素的２６２％和２３５％;体内生物活力也明显高于猪胰岛素;它的促细胞生长能力为猪胰岛素的１７４％。     

The relationship between the connecting peptide of recombined single chain insulin and its biological function

To investigate the relationship between the biological activity of recombined single chain insulin and the length of the connecting peptide, we designed and prepared three single chain insulin molecules, namely, PIP, [A]₅PIP and [A]₁₀PIP, by site-directed mutagenesis, in which B30 and A1 were linked through dipeptide A-K, heptapeptide A-A-A-A-A-A-K, and dodecapeptide A-A-A-A-A-A-A-A-A-A-A-K, respectively. Their receptor binding capacities were 0.14%, 14.3% and 11.1% of that of insulin respectively and theirin vivo biological activities were in consistence with their receptor binding capacity; whereas their growth promoting activities were 17%, 116.3% and 38% of that of insulin. These results suggested the following conclusions. (i) The recombined single chain insulin could also possess the same metabolic and mitogenic function as insulin. (ii) The receptor binding capacity of recombined single chain insulin to insulin receptor was closely related to the length and amino acid composition of the connecting peptide and could change from 0 to 100% of insulin depending on the different connecting peptides. This result further illustrated the necessity of B chain C-terminus swaying away from A chain N-terminus when insulin binds to its receptor. (iii) The mitogenic activity of recombined single chain insulin also depended on the length and the amino acid composition of the connecting peptide and was higher than its metabolic activity.     

Contribution of the absolutely conserved B8Gly to the foldability of insulin

B8Gly is absolutely conserved in insulin from different species, and in other members of the insulin superfamily the corresponding position is always occupied by a Gly residue. However, the reasons for its conservation are still unclear; probably many factors contribute to this phenomenon. In our previous work, B8Gly was replaced by an Ala residue, which suggested that biological activity is one of the factors contributing to its conservation. In order to identify more factors contributing to this positional conservation, the secretion efficiency, structural stability, disulfide stability, and in vitro refolding of single-chain insulin (PIP) and a mutant with B8Gly replaced by Ala, were investigated. Compared with wild-type PIP, the B8Ala replacement decreased the secretion efficiency, structural stability, disulfide stability, and in vitro refolding efficiency of the PIP sequence. These results suggest that B8Gly is important to the secretion, folding, and stability of the insulin sequence.     

B22-D-arginine]insulin: synthesis and biological properties

An analogue of porcine insulin which differs from the native molecule in that the amino-acid residue B22-L-arginine is replaced by its D-enantiomer has been synthesized. The [D ArgB22]B-chain was synthesized by the segment condensation method and purified as the di-S-sulfonate by ion exchange chromatoggraphy on SP-Sephadex at pH 3.5. Combination with native porcine sulfhydryl A-chain gave [DArgB22]insulin which was purified by ion exchange chromatography on SP-Sephadex at pH 4.5 with a linear NaCl gradient. The biological activity of this analogue as measured by glucose oxidation in rat epididymal adipocytes was 2%. Thymidine incorporation into DNA of human fibroblast was 16%. The immunoreactivity using antipork insulin antibody in a double antibody immunoassay was 4%. The receptor-binding affinity as measured by radioreceptor assays was 2% with cultured human fibroblasts and 1% with rat adipocytes. These results suggest that the L-configuration at B22-arginine is essential for retaining the biological, immunological and receptor-binding properties of the hormone.     

Effect of N-methylation of selected peptide bonds on the biological activity of insulin. [2-N-methylisoleucine-A]insulin and [3-N-methylvaline-A]insulin

Hydrogen bonding involving peptide bonds of the backbone of the insulin molecule may play an important role in insulin-receptor interaction. Our previous work suggested that the A2-A8 helical segment of the hormone molecule participates in this interaction. To investigate the possible involvement of peptide bonds of this segment in insulin-receptor interaction the [2-N-methylisoleucine-A]insulin and [3-N-methylvaline-A]insulin ([MeIle2-A]- and [MeVal3-A]insulins) were synthesized. The circular dichroic spectra of the analogues were obtained and their properties were examined in several biological assays. The circular dichroic spectra suggested that the analogues remained monomeric at concentrations at which insulin is predominantly dimeric, and that their A2-A8 helical segments are distorted. The in vitro biological activity and the receptor binding affinity of these analogues were compared with that of natural insulin. Both analogues are weak full agonists. [MeIle2-A]insulin displayed a potency of 5.4 +/- 0.3% in stimulating lipogenesis and 4.6 +/- 2.3% in receptor binding affinity in rat fat cells and rat liver plasma membranes respectively. [MeVal3-A]insulin displayed a potency of 2.1 +/- 0.2% in lipogenesis and 1.0 +/- 0.3% in receptor binding assays. In radioimmunoassays [MeIle2-A]- and [MeVal3-A]insulins exhibited potencies of 13% and 11% respectively relative to the natural hormone. The substantially decreased biological activity and receptor binding affinity of these analogues may be attributed partly to the change of conformation and partly to the loss of hydrogen bonding capacity of the A2-A8 segment brought about by N-methylation of the A1-A2 or A2-A3 peptide bonds.     

Insulin and insulin mutants stimulate glucose uptake in rat adipocytes

A simple method to determine the in vitro biological activity of insulin by measuring glucose uptake in the rat adipocytes is presented here. In the presence of insulin, the glucose uptake is 5-6 times more than the basal control. And the uptake of D-[3-3H]-glucose is linear as the logarithm of insulin concentration from 0.2 μg/L to 1.0 μg/L. Glucose and 3-O-methyl-glucose inhibit D-[3-3H]-glucose uptake into adipocytes. By this method, the in vitro biological activity of [B2-Lys]-insulin and [B3-Lys]-insulin was measured to be 61.6% and 154% respectively, relative to that of insulin.     

Degradation, receptor binding, insulin secreting and antihyperglycaemic actions of palmitate-derivatised native and Ala8-substituted GLP-1 analogues

The hormone glucagon-like peptide-1(7-36)amide (GLP-1) is released in response to ingested nutrients and acts to promote glucose-dependent insulin secretion ensuring efficient postprandial glucose homeostasis. Unfortunately, the beneficial actions of GLP-1 which give this hormone many of the desirable properties of an antidiabetic drug are short lived due to degradation by dipeptidyl-peptidase IV (DPP IV) and rapid clearance by renal filtration. In this study we have attempted to extend GLP-1 action through the attachment of palmitoyl moieties to the epsilon-amino group in the side chain of the Lys26 residue and to combine this modification with substitutions of the Ala8 residue, namely Val or amino-butyric acid (Abu). In contrast to native GLP-1, which was rapidly degraded, [Lys(pal)26]GLP-1, [Abu8, Lys(pal)26]GLP-1 and [Val8 Lys(pal)26]GLP-1 all exhibited profound stability during 12 h incubations with DPP IV and human plasma. Receptor binding affinity and the ability to increase cyclic AMP in the clonal beta-cell line BRIN-BD11 were decreased by 86- to 167-fold and 15- to 62-fold, respectively compared with native GLP-1. However, insulin secretory potency tested using BRIN-BD11 cells was similar, or in the case of [Val8,Lys(pal)26]GLP-1 enhanced. Furthermore, when administered in vivo together with glucose to diabetic (ob/ob) mice, [Lys(pal)26]GLP-1, [Abu8,Lys(pal)26]GLP-1 and [Val8,Lys(pal)26]GLP-1 did not demonstrate acute glucose-lowering or insulinotropic activity as observed with native GLP-1. These studies support the potential usefulness of fatty acid linked analogues of GLP-1 but indicate the importance of chain length for peptide kinetics and bioavailability.     

B22 Glu Des-B30 Insulin： A Novel Monomeric Insulin

Studies on monomeric insulin with reduced self-association are important in the development of insulin pharmaceutical preparations with rapid hypoglycemic action on patients with diabetes. Here we report a novel monomeric insulin, B22 Glu des-B30 insulin, prepared from a single chain insulin precursor with B22 Arg mutated to Glu, which was expressed in Pichia pastoris and converted to B22 Glu des-B30 insulin by tryptic digestion. It still retains 50% of the in vivo biological activity of porcine insulin and does not form a dimer even at a concentration of 10 mg/ml, showing that B22 Glu plays a key role in reducing the self- association of the insulin molecule without greatly reducing its biological activity. This novel monomeric insulin might have potential applications in the clinic.     

Binding and autophosphorylating activity of human insulin analogs

Insulin receptor binding and autophosphorylating activities of a number of synthetic analogs of human insulin have been examined using highly purified insulin receptor from human placenta. In general, autophosphorylation correlates well with the ability of the analogs to stimulate glucose oxidation and to inhibit lipolysis in adipocytes although their biological activities varied over a wide range. These findings support the hypothesis that autophosphorylation is an obligatory step in the pathways leading to glucose oxidation and inhibition of lipolysis. The relative biological potencies of the analogs in the autophosphorylation assay also correlated well with their receptor-binding affinities except for the peptides [endo-TyrB16a]insulin, in which an additional Tyr has been inserted between TyrB16 and LeuB17 and [ProA2]insulin. The relative receptor binding affinity of [endo-TyrB16a]insulin is significantly greater than its biological activity in the adipocyte or receptor autophosphorylation assays. The converse is true for [ProA2]insulin. These results demonstrate that the amino-acid residues involved in binding and receptor activation may not be identical.     

The importance of the B10 amino acid residue to the biological activity of insulin. [Lys10-B] human insulin

An analog of human insulin, which differs from the parent molecule in that the histidine residue at position 10 of the B chain (B¹⁰) is replaced by lysine, has been synthesized and isolated in purified form. This analog, [10-lysine-B] insulin ([Lys¹⁰-B] insulin), in stimulating lipogenesis and in radioimmunoassays, exhibited potencies of 14.2% and 14.7%, respectively, as compared to the natural hormone. In insulin receptor binding in rat liver membranes, [Lys¹⁰-B] insulin was found to possess a potency of 17% compared to insulin. We have shown previously that substitution of the B¹⁰ polar residue histidine with the nonpolar leucine results in an analog exhibiting inin vivo assays 50% of the activity of the parent molecule. It is speculated that in insulin the relative size of the amino acid residue at B¹⁰, rather than its polarity, is the most important factor in maintaining a structure commensurate with high biological activity.For the previous paper in this series see Schwartzet al. (1981).     

Impaired negative cooperativity of the semisynthetic analogues human [LeuB24]- and [LeuB25]-insulins

Several semisynthetic analogues of human insulin were prepared by enzyme-assisted coupling of synthetic octapeptides to the C-terminal of porcine desoctapeptide insulin. We report the receptor-binding and biological properties of [Leu^B24]- and [Leu^B25]-insulins, one of which has the same sequence as a “mutant” insulin recently found in a diabetic patient (Tager, H. et al.(1979) Nature $\text{28}$:121–125). [Leu^B24]- and [Leu^B25]-insulins had, respectively, 8–12% and 0.9–1.1% of the binding affinity of human insulin, and 11% and 2.7% of its potency in stimulating lipogenesis in isolated rat fat cells. Neither one was an antagonist of the biological effects of native insulin. While the ability of [Leu^B24]-insulin to induce negative cooperativity was clearly impaired, that of [Leu^B25]-insulin was almost abolished. [Leu^B25]-insulin was also a potent antagonist of the negative cooperativity induced by native insulin.     

The thermodynamic stability of insulin disulfides is not affected by the C-domain of insulin-like growth factor 1

Both Insulin and insulin-like growth factor 1 are members of insulin superfamily. They share homologous primary and tertiary structure as well as weakly overlapping biological activity. However, their folding behavior is different: insulin and its recombinant precursor (PIP) fold into one unique tertiary structure, while IGF-1 folds into two disulfides isomers with similar thermodynamic stability. To elucidate the molecular mechanism of their different folding behavior, we prepared a singlechain hybrid of insulin and IGF-1, [B10Glu]Ins/IGF-1(C), and studied its folding behavior compared with that of PIP and IGF-1. We also separated a major non-native disulfides isomer of the hybrid and studied its refolding. The data showed that the C-domain of IGF-1 did not affect the folding thermodynamics of insulin, that is, the primary structure of the hybrid encoded only one thermodynamically stable disulfides linkage. However, the folding kinetics of insulin was affected by the C-domain of IGF-1.