Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 1. [21-Desasparagine,20-cysteinamide-A]insulin and [21-desasparagine,20-cysteine isopropylamide-A]insulin

The C-terminal region of the A chain of insulin has been shown to play a significant role in the expression of the biological activity of the hormone. To further delineate the contribution of this segment, we have synthesized [21-desasparagine,20-cysteinamide-A]insulin and [21-desasparagine,20-cysteine isopropylamide-A]insulin, in which the C-terminal amino acid residue of the A chain of insulin, asparagine, has been removed and the resulting free carboxyl group of the A20 cysteine residue has been converted to an amide and an isopropylamide, respectively. Both insulin analogues display biological activity, 14-15% for the unsubstituted amide analogue and 20-22% for the isopropylamide analogue, both relative to bovine insulin. In contrast, a [21-desasparagine-A]insulin analogue has been reported to display less than 4% of the activity of the natural hormone [Carpenter, F. (1966) Am. J. Med. 40, 750-758]. The implications of these findings are discussed, and we conclude that the A20-A21 amide bond plays a significant role in the expression of the biological activity of insulin.     

The A14 position of insulin tolerates considerable structural alterations with modest effects on the biological behavior of the hormone

As part of our aim to investigate the contribution of the tyrosine residue found in the 14 position of the A-chain to the biological activity of insulin, we have synthesized six insulin analogues in which the A14 Tyr has been substituted by a variety of amino acid residues. We have selected three hydrophilic and charged residues—glutamic acid, histidine, and lysine—as well as three hydrophobic residues—cycloleucine, cyclohexylalanine, and naphthyl-(1)-alanine—to replace the A14 Tyr. All six analogues exhibit full agonist activity, reaching the same maximum stimulation of lipogenesis as is achieved with procine insulin. The potency for five of the six analogues, [A14 Glu]-, [A14 His]-, [A14 Lys]-, [A14 cycloleucine]-, and [A14 naphthyl-(1)-alanine]-insulins in receptor binding assays ranges from 40–71% and in stimulation of lipogenesis ranges from 35-120% relative to porcine insulin. In contrast, the potency of the sixth analogue, [A14 cyclohexylalanine]insulin, in both types of assays is less than 1% of the natural hormone. The retention time on reversed-phase high-performance liquid chromatography for the first five analogues is similar to that of bovine insulin, whereas for the sixth analogue, [A14 cyclohexylalanine]insulin, it is approximately 11 min longer than that of the natural hormone. This suggests a profound change in conformation of the latter analogue. Apparently, the A14 position of insulin can tolerate a wide latitude of structural alterations without substantial decrease in potency. This suggests that the A14 position does not participate directly in insulin receptor interaction. Only when a substitution which has the potential to disrupt the conformation of the molecule is made at this position, is the affinity for the receptor, and hence the biological potency, greatly reduced.     

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％。     

Synthesis and properties of [A19-(p-fluorophenylalanine)] insulin

The synthesis of [Phe(F)A19]insulin (porcine) is described. First the protected [Phe(F)19]A-chain was assembled by segment condensation of [1-12] and [13-21] using the dicyclohexyldiimide/1-hydroxybenzotriazole procedure. [Phe(F)19]A-chain was purified by ion exchange chromatography after removal of all the protecting groups (Boc, But, OBut and S-Trt) and its conversion into the tetra-S-sulfonated derivative. [Phe(F)A19]insulin was prepared by combination with porcine B-chain and purified by gel filtration and ion-exchange chromatography. The in vitro biological activity of this analogue was 60%. CD spectra in the near and far UV are qualitatively very similar to those of insulin.     

Specific transformations at the N-terminal region of phospholipase A2.

Treatment of porcine pancreatic prophospholipase A2 with methyl acetimidate converted all lysine residues into epsilon-acetimidolysine residues. Enzymatically active epsilon-amidinated phospholipase A2 (AMPA) was obtained from the epsilon-amidinated zymogen by limited tryptic proteolysis cleaving the Arg7-Ala8 bond. AMPA was used to prepare des-Ala8-, des-(Ala8,Leu9)- and des-(ALa8),Leu9,Trp10)-AMP by successive Edman degradations, and des-(A la 8-Arg13)-AMPA by selective splitting of the Arg13-Ser14 bond by trypsin. Structural analogues of AMPA with different N-terminal amino acid residues, viz., D-Ala, beta-Ala, and Gly, have been prepared by reacting des-Ala8-AMPA with the corresponding N-t-Boc-N-hydroxysuccinimide esters of these amino acids. Similarly, the only Trp10 residue has been substituted for Phe by coupling of des-(Ala8-,Leu9,Trp10)-AMPA with N-t-Boc-L-Ala-L-Leu-L-Phe-N-hydroxysuccinimide ester. The feasibility of these substitutions has been proven unambiguously by the retroconversion of des-Ala8-AMPA and of [Ala7]AMPA into AMPA having identical enzymatic activity as the starting AMPA. The single Trp10 residue in native phospholipase A2 and its zymogen was specifically sulfenylated using 0-nitrophenyl-sulfenyl chloride. The homogenous proteins were kinetically analyzed using short-chain lecithins in the monomeric and micellar region. All modified AMPA analogues, except those in which two or more of the N-terminal amino acid residues are removed, show enzymatic activities toward monermic substrate comparable to that of AMPA, indicating that the active site region is still intact. Only [Gly8]-, [beta-Ala8]-, and [Ala8,Leu9,Phe10]AMPA exhibit a dramatic increase in enzymatic activity similar to that of AMPA upon passing the critical micellar concentration (cmc) of the substrate. From these results it can be concluded that the N-terminal region of the enzyme requires a very precise architecture in order to interact with lipid-water interfaces and consequently to display its full enzymatic activity.     

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％。     

Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 2. [21-Asparagine diethylamide-A]insulin

We have synthesized [21-asparagine diethylamide-A]insulin, which differs from the parent molecule in that the free carboxyl group of the C-terminal amino acid residue, asparagine, of the A chain moiety has been converted to a diethylamide group. The analogue displays equivalent potency in receptor binding and biological activity, 48% and 56%, respectively, relative to bovine insulin. In contrast, we have reported previously [Burke, G. T., Chanley, J. D., Okada, Y., Cosmatos, A., Ferderigos, N., & Katsoyannis, P. G. (1980) Biochemistry 19, 4547-4556] that [21-asparaginamide-A]insulin exhibits a divergence in these properties, ca. 60% in receptor binding and ca. 13% in biological activity. The disparity in the biological behavior of these analogues is discussed, and we ascribe the modulation of biological activity independent of receptor binding activity observed between these analogues to the difference in the negativity of the carbonyl oxygen of the A chain moiety C-terminal amino acid residue.     

Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 3. [21-Desasparagine,20-cysteine ethylamide-A]insulin and [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin

We have synthesized [21-desasparagine,20-cysteine ethylamide-A]insulin and [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin, which differ from natural insulin in that the C-terminal amino residue of the A chain, asparagine, has been removed and the resulting free carboxyl group of the A20 cysteine residue has been converted to an ethylamide and a trifluoroethylamide group, respectively. [21-Desasparagine,20-cysteine ethylamide-A]insulin displayed equivalent potency in receptor binding and biological activity, ca. 12% and ca. 14%, respectively, relative to bovine insulin. In contrast, [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin displayed a divergence in these properties, ca. 13% in receptor binding and ca. 6% in biological activity. This disparity is ascribed to a difference in the electronic state of the A20-A21 amide bond in these two analogues. A model is proposed to account for the observation of divergence between receptor binding and biological activity in a number of synthetic insulin analogues and naturally occurring insulins. In this model, changes in the electronic state and/or the orientation of the A20-A21 amide bond can modulate biological activity independently of receptor binding affinity. The A20-A21 amide bond is thus considered as an important element in the "message region" of insulin.     

The effect of placement of tryptophan residues in selected A-chain positions on the biological profile of insulin

In continuation of our efforts to study the solution structure and conformational dynamics of insulin by time-resolved fluorescence spectroscopy, we have synthesized and examined the biological activity of five insulin analogues in which selected naturally occurring residues in the A-chain have been replaced with the strongly fluorescent tryptophan residue. The potency of these analogues was evaluated in lipogenesis assays in isolated rat adipocytes, in receptor binding assays using rat liver plasma membranes, and in two cases, in receptor binding assays using adipocytes. [A3 Trp]insulin displays a potency of 3% in receptor binding assays in both liver membranes and in adipocytes, but only 0.06% in lipogenesis assays as compared to porcine insulin. [A10 Trp] insulin displays a potency ofca. 40% andca. 25% in rat liver receptor binding and lipogenesis assays, respectively. [A13 Trp]insulin displays a potency ofca. 39% in rat liver receptor binding assays, but onlyca. 9% in receptor binding in adipocytes; in lipogenesis assays, [A13 Trp] insulin displays a potency ofca. 12%, comparable to its potency in adipocyte receptor binding assays. [A15 Trp]insulin exhibits a potency of 18% and 9% in rat liver receptor binding and lipogenesis assays, respectively. The doubly substituted analogue, [A14 Trp, A19 Trp] insulin, displays a potency ofca. 0.7% in both rat liver receptor binding assays and lipogenesis assays. These data suggest two major conclusions: (1) the A3 and A15 residues lie in sensitive regions in the insulin molecule, and structural modifications at these positions have deleterious effects on biological activity of the hormone; and (2) [A13 Trp]insulin appears to be a unique case in which an insulin analogue exhibits a higher potency when assayed in liver tissue than when assayed in fat cells.     

(A1-D-alanine) insulin (author's transl)]

Starting from porcine insulin, A1-glycine was substituted by D-alanine and by L-alanine for comparison. Replacement of A1-glycine by L-alanine revealed the known decrease in the biological activity. [A1-D-Alanine]insulin, however, has the same blood sugar lowering activity as insulin and is slightly more active in its influence on the glucose uptake into the rat diaphragm. The specific binding to insulin receptors of rat liver is decreased as, compared to insulin, but increased as compared to the L-alanine analogue.     

The synthesis of [A 19-3-iodotyrosine] and [A 19-3,5-diiodotyrosine]insulin (porcine)

The chemical synthesis of [Tyr(I)A19] and [Tyr(I2)A19]insulin (porcine), using the amino-acid derivatives 3-iodotyrosine and 3,5-diiodotyrosine is described. The synthesis of the iodinated A-chains were performed by segment condensation in solution using acid labile protecting groups. The hydroxyl groups of Tyr(I) and Tyr(I2) were unprotected. For the temporary protection of the alpha-amino groups of the A-chain segments containing iodinated tyrosines, the 1-(4-biphenylyl)-1-methylethoxycarbonyl group was selected. After deprotection and sulphitolysis the iodinated A-chain tetra-S-sulphonates were purified by ion exchange chromatography on DEAE cellulose at pH 5.6. Reduction to the sulphhydryl form and the combination with native porcine B-chain yielded [Tyr(I)A19] and [Tyr(I2)A19]insulin (porcine), respectively. Purification of the first product was achieved by gel filtration and of the later by ion exchange chromatography on CM-cellulose at pH 4.5 and gel filtration. The monoiodinated insulin had a biological activity of 24 +/- 2% and the diiodinated analogue 2.6 +/- 0.2% as determined in an in vitro lipogenesis assay with epididymal adipocytes.     

Interaction between the A2 and A19 amino acid residues is of critical importance for high biological activity in insulin: [19-leucine-A]insulin

The replacement of tyrosine at position A19 by leucine in the insulin molecule led to an analogue, [19-leucine-A]insulin [( Leu19-A]insulin), displaying insignificant receptor binding affinity and in vitro biological activity less than 0.1 and 0.05%, respectively, compared to the natural hormone. This analogue along with the previously reported [2-glycine-A]-, [2-alanine-A]-, and [2-norleucine-A]insulins is the least potent insulin analogue we have examined. Circular dichroic studies showed that all these analogues are monomeric at concentrations at which insulin is primarily dimeric. We conclude that an aromatic ring at position A19 and the presence of the side chain of isoleucine at position A2 are each of critical importance for high biological activity in insulin. It appears that the van der Waals interaction between the side chain of isoleucine A2 and tyrosine A19, present in crystalline insulin, is among the most important determinants for high biological activity in insulin.     

[A_(15)Asn,A_(17)Pro,A_(21)Ala]-胰岛素的体内外生物活性

对胰岛素的研究使得许多生命科学中的重要问题被认识．然而对于胰岛素结构和功能关系,仍有许多不清楚的地方．在胰岛素Ａ链的Ｎ端和Ｃ端各有一个螺旋区：Ａ２－８和Ａ１２－１８．杨士珍［１］的研究表明：缺失Ａ１２－１８肽段的Ａ链类似物虽然能和天然Ｂ链重组,但其重组产物只保留了天然胰岛素的约６％的体内外生物活性．这说明Ａ１２－１８对胰岛素的生物活性十分重要．但是这一结果还不能说明是Ａ１２－１８螺旋还是Ａ１２－１８肽段对胰岛素的生物活性十分重要．根据Ｃｈｕｏ－Ｆａｓｍａｎ法,在含有Ａｓｎ和Ｐｒｏ的肽段中不能形成…     

高受体结合和低免疫原性的［B1—Ala，B2—Ala］－胰岛素

通过化学半合成从天然猪胰岛素得到［Ｂ１－Ａｌａ,Ｂ２－Ａｌａ］胰岛素。这一胰岛素类似物经聚丙烯酰胺凝胶电泳和ＨＰＬＣ鉴定证明是均一的,氨基酸组成与理论值相符生物活性测定结果表明：［Ｂ１－Ａｌａ,Ｂ２－Ａｌａ］－胰岛素的体内活力与天然猪胰岛素相同,而与人胎盘细胞膜胰岛素受体的结合能力为天然猪胰岛素的１３２％。这一结果进一步说明胰岛素Ｂ链Ｎ端肽段参子与受体相互作用。此外,［Ｂ１－Ａｌａ,Ｂ２－Ａｌａ］－胰岛素的免疫活性很低,远小于天然猪胰岛素的４％。     

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.     

Structural analogues of ZAPA as GABAA agonists

Structural analogues of ZAPA, Z-3-[(aminoiminomethyl)thio]prop-2-enoic acid, an isothiouronium analogue of GABA, are potent GABA_A agonists as seen in the isolated guinea-pig ileum and in the facilitation of [³H]diazepam binding to rat brain membranes. Compounds with guanidino or amidine groups replacing the amino functionality of GABA were also found to be active. The highest activity was displayed by the isothiouronium salts in which the conformational flexibility of the molecule is restricted by a Z-substituted carbon–carbon double bond. A series of bis-isothiouronium compounds was prepared from aliphatic ,ω-bis-thioureas as mixtures of E and Z adducts. Maximum GABA_A agonist activity for this series was found with a C₆–C₈ carbon chain, and the results were consistent with an interaction at the GABA_A receptor with only one end of the molecule, rather than the more potent effect expected of a molecule bridging two active sites. GABA_A antagonist/partial agonist activity was observed on the guinea-pig isolated ileum for a number of different analogue types, with the most potent being bis-isothiouronium derivatives. None of the substituted derivatives of ZAPA was as active as ZAPA itself, and maximum GABA_A activity was found in the n-pentyl and n-hexyl analogues.     

21-arginine-A] insulin: a biologically active analog.

An analog of sheep insulin which differs from the parent molecule in that the C-terminal amino acid residue of the A chain, asparagine, is replaced by arginine, has been synthesized and isolated in highly purified form. The [Arg21] A chain of sheep insulin was synthesized by the fragment condensation approach and isolated as the S-sulfonated derivative. Conversion of the latter into the sulfhydryl form and interaction with the S-sulfonated B chain of bovine (sheep) insulin yielded [Arg21-A] sheep insulin, which was purified by chromatography on a carboxymethylcellulose column with an exponential sodium chloride gradient. The [Arg21-A] sheep insulin shows potencies of 10.5--12.5 IU/mg when assayed by the mouse convulsion method and 8.6 IU/mg by the radioimmunoassay method (cf. 23--25 IU/mg for the natural hormone). It has been suggested that in the insulin molecule the A21 asparagine participates in salt bridge- and hydrogen bond-forming interactions which are critical in the biological activity of the hormone. Although the [Arg21-A] analog still retains these interactions, it is only ca. 50% as active as the natural hormone. It is speculated that other factors than the above mentioned interactions come into play, which involve the side chain of the A21 amino acid residue and affect the biological activity of the hormone.     

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.     

Studies on the total synthesis of an A7,B7-dicarbainsulin. III. Assembly of segments and generation of biological activity

As a further contribution to the synthesis of an insulin analogue with a stable A7-B7 interchain bond, the synthesis of A(8-21) by solution methods, and of B(9-25) as well as [7-(2,7-diaminosuberic acid)]B(1-8) by solid phase methods is described. In the latter compound, the amino group of the diaminosuberic acid residue was acylated with A(1-6), and the resulting "U-peptide" sequentially elongated with the C-terminal A- and finally B-chain sequences. The conversion of the product into the disulfide moiety gave a mixture which could not be resolved by currently available methods. However, the low biological activity of the crude product indicates that the A7-B7 disulfide bond is not crucially important for the activity of insulin.     

Insulin-releasing properties of the frog skin peptide pseudin-2 and its [Lys18]-substituted analogue

Abstract Pseudin-2 is a cationic alpha-helical peptide that was first isolated from the skin of the paradoxical frog Pseudis paradoxa on the basis of its antimicrobial activity. We have investigated the insulin-releasing properties and cytotoxicity of the peptide, together with selected analogues with increased cationicity and hydrophobicity. At concentrations in the range 10(-9)-10(-6) m, pseudin-2, and its [Lys18], [Phe8], and [d-Lys3,d-Lys10,d-Lys14] derivatives, stimulated insulin release from the BRIN-BD11 clonal beta-cell line without increasing release of lactate dehydrogenase. The [Lys18] analogue was the most potent (46% increase in insulin release at 10(-9) m) and the most effective (215% increase in insulin release at 10(-6) m). The more cationic [Lys3,Lys10,Lys14] and [Lys3,Lys10,Lys14,Lys21] analogues lacked insulinotropic action and the more hydrophobic [Phe16] analogue was cytotoxic at concentrations > or =10(-7) m. Pseudin-2 and [Lys18]-pseudin-2 had no effect on intracellular calcium concentrations and stimulated insulin release in the absence of external calcium. [Lys18]-pseudin-2 (10(-8) m) stimulated insulin release in the presence of diazoxide and verapamil. Our results demonstrate that pseudin-2 stimulates insulin secretion from BRIN-BD11 cells by a mechanism involving Ca2+-independent pathways and identify [Lys18]-pseudin-2 as a peptide that may have potential for development as a therapeutically valuable insulinotropic agent for the treatment of type 2 diabetes.