Solid Phase Synthesis of an Analogue of Insulin, A0:R glargine, That Exhibits Decreased Mitogenic Activity

Numerous analogues of insulin have been prepared over the past three decades for use in diabetic therapy. However, only two long-acting insulins have been approved for clinical use. One is Levemir (Novo Nordisk) and the other is Lantus (Sanofi-Aventis). Glargine (commercial name: Lantus) is characterized by a substitution of Gly in place of Asn at the C terminus of the A-chain and addition of two Arg residues to the C terminus of the B-chain. Despite the clinical advantages of glargine, it is not without concern that its increased affinity for the IGF-1 receptor may correlate with increased mitogenic activity. Recently, a systematic study of modified analogues of glargine showed that placement of an extra Arg residue at the N terminus of the A-chain conferred improved insulin:IGF-1 receptor selectivity without significant loss of pharmacological profile. However, as it is difficult to prepare such an analogue in high yield by recombinant DNA methods, we undertook its chemical assembly by our refined solid phase synthesis method. We describe herein its chemical preparation and biological activity in both insulin receptor binding assays and DNA synthesis assays. The synthetic analogue, A0:R glargine, showed slightly reduced affinity for IR-B (twofold) compared to native insulin. In stimulating DNA synthesis, A0:R glargine was slightly less potent compared to insulin or glargine. This result ultimately confirms the previous report that A0:R glargine has a lower potency in mitogenic assays compared to glargine. This glargine analogue thus could be a potential lead compound for drug design and development for the treatment of diabetes.     

A novel high-affinity peptide antagonist to the insulin receptor

In this publication we describe a peptide insulin receptor antagonist, S661, which is a single chain peptide of 43 amino acids. The affinity of S661 for the insulin receptor is comparable to that of insulin and the selectivity for the insulin receptor versus the IGF-1 receptor is higher than that of insulin itself. S661 is also an antagonist of the insulin receptor of other species such as pig and rat, and it also has considerable affinity for hybrid insulin/IGF-1 receptors. S661 completely inhibits insulin action, both in cellular assays and in vivo in rats. A biosynthetic version called S961 which is identical to S661 except for being a C-terminal acid seems to have properties indistinguishable from those of S661. These antagonists provide a useful research tool for unraveling biochemical mechanisms involving the insulin receptor and could form the basis for treatment of hypoglycemic conditions.     

Perturbation of insulin-receptor interactions by intramolecular hormone cross-linking. Analysis of relative movement among residues A1, B1, and B29

We have evaluated, by use of isolated canine hepatocytes, the importance of intramolecular hormone cross-linking (and of concomitant changes in molecular flexibility) to the interaction of insulin with its plasma membrane receptor. Cross-linked hormone analogs were prepared by reacting porcine insulin, N alpha A1-t-butyloxycarbonyl insulin or N alpha A1-t-butyloxycarbonyl [D-LysA1]insulin with various dicarboxylic acid active esters to obtain alpha-GlyA1/epsilon-LysB29-, alpha-PheB1/epsilon-LysB29-, and epsilon-D-LysA1/epsilon-LysB29-cross-linked insulins, respectively. In the aggregate, insulin analogs cross-linked by groups containing 2-12 atoms retained 1.4-35% of the receptor binding potency of native insulin. Analysis of our results suggests that: (a) loss of chemical functionality, steric interference, and restriction of potential intramolecular movement can all play roles in determining the receptor binding potencies of cross-linked insulin analogs; (b) restriction of intramolecular movement between residues A1 and B29 affects negatively the binding of insulin to its receptor (but accounts for only a fraction of the conformational change which insulin must undergo to achieve a high affinity state of ligand-receptor interaction); and (c) introduction of a cross-link between residues B1 and B29 (residues that are in fact in proximity in one crystalline form of the hormone) decreases markedly the receptor binding potencies of the corresponding analogs. The importance of these findings is discussed in relation to the potential structure of insulin when it is bound to its plasma membrane receptor.     

Comparison of the functional insulin binding epitopes of the A and B isoforms of the insulin receptor

The human insulin receptor is expressed as two isoforms that are generated by alternate splicing of its mRNA; the B isoform has 12 additional amino acids (718-729) encoded by exon 11 of the gene. The isoforms have been reported to have different ligand binding properties. To further characterize their insulin binding properties, we have performed structure-directed alanine-scanning mutagenesis of a major insulin binding site of the receptor, formed from the receptor L1 domain (amino acids 1-470) and amino acids 705-715 at the C terminus of the alpha subunit. Alanine mutants of each isoform were transiently expressed as recombinant secreted extracellular domain in 293 cells, and their insulin binding properties were evaluated by competitive binding assays. Mutation of Arg(86) and Phe(96) of each isoform resulted in receptors that were not secreted. The Kds of unmutated receptors were almost identical for both isoforms. Several new mutations compromising insulin binding were identified. In L1, mutation of Leu(37) decreased affinity 20- to 40-fold and mutations of Val(94), Glu(97), Glu(120), and Lys(121) 3 to 10-fold for each isoform. A number of mutations produced differential effects on the two isoforms. Mutation of Asn(15) in the L1 domain and Phe(714) at the C terminus of the alpha subunit inactivated the A isoform but only reduced the affinity of the B isoform 40- to 60-fold. At the C terminus of the alpha subunit, mutations of Asp(707), Val(713), and Val(715) produced 7- to 16-fold reductions in affinity of the A isoform but were without effect on the B isoform. In contrast, alanine mutations of Tyr(708) and Asn(711) inactivated the B isoform but only reduced the affinities of the A isoform 11- and 6-fold, respectively. In conclusion, alanine-scanning mutagenesis of the insulin receptor A and B isoforms has identified several new side chains contributing to insulin binding and indicates that the energetic contributions of certain side chains differ in each isoform, suggesting that different molecular mechanisms are used to obtain the same affinity.     

文昌鱼胰岛素样肽的表达、纯化、鉴定及其多克隆抗体制备

为了深入研究胰岛素和胰岛素样生长因子1（IGF－1）的起源和进化以及结构与功能的关系。表达了胰岛素和IGF－1的祖先分子－－文昌鱼胰岛素样肽（ILP）。重组单链ILP的基因用化学方法合成（从cDNA推测的ILPB结构域的C端和A结构域的N端用Ala-Ala-Lys三肽连接起来,并钭B28Arg突变为Lys）,克隆到表达载体pVT102-U中,ILP在酿酒酵母中得到有效表达。发酵液经4步分离纯化,得到均一的单链ILP,经质谱测定分子量和氨基酸组成分析证明表达产物正确。通过Lys-C蛋白内切酶处理将重组单链ILP转化成双链形式。虽然双链ILP与人胰岛素受体没有结合活力。但圆二色性光谱显示它与胰岛素的结构非常相似,用表达的单链ILP免疫新西兰大白兔,获得了高滴度的多克隆抗体。     

Role of the phenylalanine B24 side chain in directing insulin interaction with its receptor. Importance of main chain conformation

We have investigated (by use of semisynthetic insulin analogs and isolated canine hepatocytes) the role of invariant residue PheB24 in determining the affinity of insulin-receptor interactions. Our results confirm that replacement of PheB24 by D-Phe is not detrimental to ligand binding to receptor, show that D-Ala is well tolerated at position B24 (whereas Ala is not), and demonstrate that [GlyB24]insulin retains as much as 78% of the receptor binding potency of native insulin. Additional findings show that replacement of PheB24 by D-Pro or by alpha-aminoisobutyric acid results in analogs with severely decreased binding potency, and that the COOH-terminal domain containing residues B26-B30 plays a positive role in determining receptor binding potency in GlyB24-substituted insulin (whereas it plays a negative role in determining the receptor binding potency of its GlyB25-substituted counterpart). We interpret our results as identifying (a) a critical role for the insulin main chain near residue B24 in determining the affinity of receptor for ligand, (b) the importance of main chain flexibility in achieving a high affinity state of receptor-bound hormone, and (c) a potential interaction of the PheB24 side chain with receptor which initiates main chain structural changes in the natural hormone, but which does not itself confer affinity to ligand-receptor interactions.     

Specific binding of insulin-like growth factors 1 and 2 to the type 1 and type 2 receptors respectively.

1. Competitive binding and receptor cross-linking experiments have been used to examine the receptor-ligand interactions between three bovine insulin-like growth factors (IGF) and monolayer cultures of myoblasts and fibroblasts. 2. Labelled IGF-2 bound predominantly to the type 2 receptor with negligible label cross-linked to the type 1 receptor, notwithstanding the ability of IGF-2 to compete effectively for the binding of IGF-1 to the type 1 receptor. Approx. 100-fold higher concentrations of IGF-1 or the N-terminal truncated (des-Gly-Pro-Glu) IGF-1 (-3N:IGF-1) were required to produce competition equivalent to IGF-2. 3. All IGF peptides, but especially IGF-1, enhanced the binding of labelled IGF-2 to the type 2 receptor of lung fibroblasts. This unusual effect was probably a consequence of the displacement of labelled IGF-2 otherwise bound to a medium protein, a conclusion supported by the demonstration of a 38 kDa membrane protein cross-linked to labelled IGF-2. 4. Both IGF-1 and -3N:IGF-1 bound only to the type 1 IGF receptor in L6 myoblasts, rat vascular smooth-muscle cells and human lung fibroblasts. The peptides competed for labelled IGF-1 binding with potencies in the order -3N:IGF-1 greater than IGF-1 greater than IGF-2 much greater than insulin. Since the IGF peptides were equipotent in skin fibroblasts, it was proposed that the apparently higher affinity of -3N:IGF-1 for receptors in the other cell types was instead a consequence of a low affinity of this peptide for the competing 38 kDa binding protein.     

Residues within the transmembrane domain of the glucagon-like peptide-1 receptor involved in ligand binding and receptor activation: modelling the ligand-bound receptor

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9-39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9-39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.     

Engineering of insulin receptor isoform-selective insulin analogues

Background

The insulin receptor (IR) exists in two isoforms, A and B, and the isoform expression pattern is tissue-specific. The C-terminus of the insulin B chain is important for receptor binding and has been shown to contact the IR just adjacent to the region where the A and B isoforms differ. The aim of this study was to investigate the importance of the C-terminus of the B chain in IR isoform binding in order to explore the possibility of engineering tissue-specific/liver-specific insulin analogues.

Methodology/Principal Findings

Insulin analogue libraries were constructed by total amino acid scanning mutagenesis. The relative binding affinities for the A and B isoform of the IR were determined by competition assays using scintillation proximity assay technology. Structural information was obtained by X-ray crystallography. Introduction of B25A or B25N mutations resulted in analogues with a 2-fold preference for the B compared to the A isoform, whereas the opposite was observed with a B25Y substitution. An acidic amino acid residue at position B27 caused an additional 2-fold selective increase in affinity for the receptor B isoform for analogues bearing a B25N mutation. Furthermore, the combination of B25H with either B27D or B27E also resulted in B isoform-preferential analogues (2-fold preference) even though the corresponding single mutation analogues displayed no differences in relative isoform binding affinity.

Conclusions/Significance

We have discovered a new class of IR isoform-selective insulin analogues with 2–4-fold differences in relative binding affinities for either the A or the B isoform of the IR compared to human insulin. Our results demonstrate that a mutation at position B25 alone or in combination with a mutation at position B27 in the insulin molecule confers IR isoform selectivity. Isoform-preferential analogues may provide new opportunities for developing insulin analogues with improved clinical benefits.     

Immobilized insulin for high capacity affinity chromatography of insulin receptors

Insulin receptors can be purified by affinity chromatography on immobilized insulin, but published methods all suffer from a rather low capacity of the affinity columns. By using insulin that has been protected in positions A1 and B29, we have been able to couple the insulin selectively through the B1 amino group to divinyl sulfone-activated agarose. The N terminus of the B-chain is the most innocuous site as far as receptor-insulin interaction is concerned, and this strategy allowed us to make affinity columns with capacities of several milligrams of receptor/ml of resin. The receptor used was the soluble ectodomain of the human insulin receptor, produced in transfected baby hamster kidney cells. The column preparation and the elution conditions are described in detail, as the efficacy of the purification depends strongly on both. The purity of the eluted receptors was so high that quantitative amino acid analysis fitted with theory. The molar absorption coefficient at 278.5 nm was 296,000 M-1 cm-1. Finally, it could be unequivocally established that the soluble receptor binds two molecules of insulin with equal affinity.     

Amino substituted analogs of 1-phenyl-3-phenylimino-2-indolones with potent galanin Gal3 receptor binding affinity and improved solubility

A series of amino analogs of 1,3-dihydro-1-phenyl-3-[[3-(trifluoromethyl)phenyl]imino]-2H-indol-2-one (1) were synthesized to improve aqueous solubility, while retaining high affinity for the human galanin Gal3 receptor. A very potent analog (9e, 1,3-dihydro-1-[3-(2-pyrrolidinylethoxy)phenyl]-3-[[3-(trifluoromethyl)phenyl]imino]-2H-indol-2-one, Ki=5 nM) shows good selectivity and solubility of 48 microg/mL at pH 7.4.     

The differential effects of pp120 (Ceacam 1) on the mitogenic action of insulin and insulin-like growth factor 1 are regulated by the nonconserved tyrosine 1316 in the insulin receptor

pp120 (Ceacam 1) undergoes ligand-stimulated phosphorylation by the insulin receptor, but not by the insulin-like growth factor 1 receptor (IGF-1R). This differential phosphorylation is regulated by the C terminus of the beta-subunit of the insulin receptor, the least conserved domain of the two receptors. In the present studies, deletion and site-directed mutagenesis in stably transfected hepatocytes derived from insulin receptor knockout mice (IR(-/-)) revealed that Tyr(1316), which is replaced by the nonphosphorylatable phenylalanine in IGF-1R, regulated the differential phosphorylation of pp120 by the insulin receptor. Similarly, the nonconserved Tyr(1316) residue also regulated the differential effect of pp120 on IGF-1 and insulin mitogenesis, with pp120 downregulating the growth-promoting action of insulin, but not that of IGF-1. Thus, it appears that pp120 phosphorylation by the insulin receptor is required and sufficient to mediate its downregulatory effect on the mitogenic action of insulin. Furthermore, the current studies revealed that the C terminus of the beta-subunit of the insulin receptor contains elements that suppress the mitogenic action of insulin. Because IR(-/-) hepatocytes are derived from liver, an insulin-targeted tissue, our observations have finally resolved the controversy about the role of the least-conserved domain of insulin and IGF-1Rs in mediating the difference in the mitogenic action of their ligands, with IGF-1 being more mitogenic than insulin.     

Insulin and insulin-like growth factor-1 binding specificity is determined by distinct regions of their cognate receptors.

Chimeric insulin/insulin-like growth factor-1 receptors and insulin receptor alpha-subunit point mutants were characterized with respect to their binding properties for insulin and insulin-like growth factor-1 (IGF-1) and their ability to translate ligand interaction into tyrosine kinase activation in intact cells. We found that replacement of the amino-terminal 137 amino acids of the insulin receptor (IR) with the corresponding 131 amino acids of the IGF-1 receptor (IGF-1R) resulted in loss of affinity for both ligands. Further replacement of the adjacent cysteine region with IGF-1R sequences fully reconstituted affinity for IGF-1, but only marginally for insulin. Unexpectedly, replacement of the IR cysteine-rich domain alone by IGF-1R sequences created a high affinity receptor for both insulin and IGF-1. The binding characteristics of all receptor chimeras reflected the potential of both ligands to regulate the receptor tyrosine kinase activity in intact cells. Our chimeric receptor data, in conjunction with IR amino-terminal domain point mutants, strongly suggest major contributions of structural determinants in both amino- and carboxyl-terminal IR alpha-subunit regions for the formation of the insulin-binding pocket, whereas, surprisingly, the residues defining IGF-1 binding are present predominantly in the cysteine-rich domain of the IGF-1R.     

The role of the N-terminal domain of the complement fragment receptor C5L2 in ligand binding

C5L2 is a new cellular receptor found to interact with the human anaphylatoxins complement factor C5a and its C-terminal cleavage product C5a des Arg. The classical human C5a receptor (C5aR) preferentially binds C5a, with a 10-100-fold lower affinity for C5a des Arg. In contrast, C5L2 binds both ligands with nearly equal affinity. C5aR presents acidic and tyrosine residues in its N terminus that interact with the core of C5a while a hydrophobic pocket formed by the transmembrane helices interacts with residues in the C terminus of C5a. Here, we have investigated the molecular basis for the increased affinity of C5L2 for C5a des Arg. Rat and mouse C5L2 preferentially bound C5a des Arg, whereas rodent C5aR showed much higher affinity for intact C5a. Effective peptidic and non-peptidic ligands for the transmembrane hydrophobic pocket of C5aR were poor inhibitors of ligand binding to C5L2. An antibody raised against the N terminus of human C5L2 did not affect the binding of C5a to C5L2 but did inhibit C5a des Arg binding. A chimeric C5L2, containing the N terminus of C5aR, had little effect on the affinity for C5a des Arg. Mutation of acidic and tyrosine residues in the N terminus of human C5L2 revealed that 3 residues were critical for C5a des Arg binding but had little involvement in C5a binding. C5L2 thus appears to bind C5a and C5a des Arg by different mechanisms, and, unlike C5aR, C5L2 uses critical residues in its N-terminal domain for binding only to C5a des Arg.     

Synthesis and biological activity of novel small peptides with aminophosphonates moiety as NOP receptor ligands

The aim of the present study was the synthesis and the biological screening of new analogs of Ac-RYYRWK-NH₂, modified at the N-terminal with 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids. The four newly synthesized ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) have been prepared by solid-phase peptide synthesis—Fmoc-strategy. These compounds were tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of Wistar rats. Our data showed that substitution of Arg at position 1 with aminophosphonates moiety decreased significantly the affinity of ligands to the NOP receptor. Furthermore, the enlargement of the cycle (with 5–8 carbon atoms) additionally diminished both the activity and the selectivity for NOP-receptor.     

Topography of the neurotensin (NT)(8-9) binding site of human NT receptor-1 probed with NT(8-13) analogs.

A series of neurotensin (NT)(8-13) analogs featuring substitution of the Arg8 and/or Arg9 residues with non-natural cationic amino acids was synthesized and evaluated for binding to the human NT receptor-1 (hNTR-1). The modifications were designed to probe specific steric and electrostatic requirements in the N-terminal cationic region of NT(8-13) for receptor binding as a general evaluation of the feasibility of incorporating minor structural changes into a peptide at a crucial polar receptor binding site. Many of the non-natural amino acids are more or less isosteric to Arg but more lipophilic as a result of addition of alkyl groups or through removal or replacement of NH character with methylene or methyl substituents, whereas others vary the distance between the cation and the alpha-amino acid carbon. Substitution of Arg8 with N(G)-alkylated Arg derivatives or homolysine (Hlys) maintained the subnanomolar affinity of NT(8-13) to the hNTR-1. Position 8 incorporation of Hlys produced the most favorable primary amine side-chain substitution to date. Moderate losses in affinity observed with position 9 substitutions were attributed to adverse steric effects. Doubly substituted [Hlys8, DAB9]NT(8-13), in which DAB is 2,4-diaminobutyric acid, was also prepared and tested as the shorter side-chain of DAB is known to be favored in position 9 of NT(8-13). This analog maintained 60% of NT(8-13) binding affinity making it the most favored des-guanidinium-containing analog known. These results demonstrate that adequate receptor binding affinity can be maintained over a structural range of Arg analogs, thus providing a range of peptides expected to exhibit altered pharmacokinetic properties. From the standpoint of the hNTR-1 cationic binding sites, these results help to map out the structural stringency inherent in the formation of a tight binding complex with NT(8-13) and related analogs.     

N6-ethyl-2-alkynyl NECAs, selective human A3 adenosine receptor agonists

A series of N6-ethyl-2-alkynyl NECA (5'-N-ethylcarboxamidoadenosine) analogs were synthesized and their binding affinity with the four human adenosine receptors was evaluated. One of the compounds ZR1121 shows high affinity with hA3 receptor and its selectivity over hA1 receptor is 1-2 log orders greater than IB-MECA or Cl-IB-MECA, the currently employed selective A3 agonists.     

The different folding behavior of insulin and insulin-like growth factor 1 is mainly controlled by their B-chain/domain

Although insulin and insulin-like growth factor 1 (IGF-1) share homologous sequence, similar tertiary structure, weakly overlapped biological activity, and a common ancestor, the two highly homologous sequences encode different folding behavior: insulin folds into one unique stable tertiary structure while IGF-1 folds into two disulfide isomers with similar thermodynamic stability. To further elucidate the molecular mechanism of their different folding behavior, we prepared two single-chain hybrids of insulin and IGF-1, Ins(A)/IGF-1(B) and Ins(B)/IGF-1(A), as well as a mini-IGF-1 by means of protein engineering and studied their structure as well as folding behavior. Both mini-IGF-1 and Ins(A)/IGF-1(B) fold into two thermodynamically stable disulfide isomers in vivo and in vitro just like that of IGF-1, while Ins(B)/IGF-1(A) folds into one unique thermodynamically stable tertiary structure in vivo and in vitro just like that of insulin. So we deduce that the different folding behavior of insulin and IGF-1 is mainly controlled by their B-chain/domain. By V8 endoproteinase digestion and circular dichroism analysis, as well as insulin receptor binding assay, we deduce that Ins(B)/IGF-1(A), isomer 2 of mini-IGF-1, and isomer 2 of Ins(A)/IGF-1(B) adopt native IGF-1/insulin-like three-dimensional structure with native disulfides, while isomer 1 of mini-IGF-1 and isomer 1 of Ins(A)/IGF-1(B) adopt the swap IGF-1-like three-dimensional structure with swap disulfides.     

Insulin-like Growth Factor-II (IGF-II) and IGF-II Analogs with Enhanced Insulin Receptor-a Binding Affinity Promote Neural Stem Cell Expansion

The objective of this study was to employ genetically engineered IGF-II analogs to establish which receptor(s) mediate the stemness promoting actions of IGF-II on mouse subventricular zone neural precursors. Neural precursors from the subventricular zone were propagated in vitro in culture medium supplemented with IGF-II analogs. Cell growth and identity were analyzed using sphere generation and further analyzed by flow cytometry. F19A, an analog of IGF-II that does not bind the IGF-2R, stimulated an increase in the proportion of neural stem cells (NSCs) while decreasing the proportion of the later stage progenitors at a lower concentration than IGF-II. V43M, which binds to the IGF-2R with high affinity but which has low binding affinity to the IGF-1R and to the A isoform of the insulin receptor (IR-A) failed to promote NSC growth. The positive effects of F19A on NSC growth were unaltered by the addition of a functional blocking antibody to the IGF-1R. Altogether, these data lead to the conclusion that IGF-II promotes stemness of NSCs via the IR-A and not through activation of either the IGF-1R or the IGF-2R.     

Insulin analog with additional disulfide bond has increased stability and preserved activity

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα‐Cα distances, solvent exposure, and side‐chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C‐terminus of the B‐chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild‐type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R‐state conformation and thus showing that the R‐state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.