In recent years, analogs of human insulin have been engineered with the aim of improving therapy for people with diabetes. To ensure that the safety profile of the human hormone is not compromised by the molecular modifications, the toxico-pharmacological properties of insulin analogs should be carefully monitored. In this study, we compared the insulin and IGF-I receptor binding properties and metabolic and mitogenic potencies of insulin aspart (B28Asp human insulin), insulin lispro (B28Lys,B29Pro human insulin), insulin glargine (A21Gly,B31Arg,B32Arg human insulin), insulin detemir (NN304) [B29Lys(-tetradecanoyl),desB30 human insulin], and reference insulin analogs. Receptor affinities were measured using purified human receptors, insulin receptor dissociation rates were determined using Chinese hamster ovary cells overexpressing the human insulin receptor, metabolic potencies were evaluated using primary mouse adipocytes, and mitogenic potencies were determined in human osteosarcoma cells. Metabolic potencies correlated well with insulin receptor affinities. Mitogenic potencies in general correlated better with IGF-I receptor affinities than with insulin receptor off-rates. The 2 rapid-acting insulin analogs aspart and lispro resembled human insulin on all parameters, except for a slightly elevated IGF-I receptor affinity of lispro. In contrast, the 2 long-acting insulin analogs, glargine and detemir, differed significantly from human insulin. The combination of the B31B32diArg and A21Gly substitutions provided insulin glargine with a 6-to 8-fold increased IGF-I receptor affinity and mitogenic potency compared with human insulin. The attachment of a fatty acid chain to LysB29 provided insulin detemir with reduced receptor affinities and metabolic and mitogenic potencies but did not change the balance between mitogenic and metabolic potencies. The safety implications of the increased growth-stimulating potential of insulin glargine are unclear. The reduced in vitro potency of insulin detemir might explain why this analog is not as effective on a molar basis as human insulin in humans.

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Liraglutide is an acylated glucagon-like peptide-1 (GLP-1) analogue that binds to serum albumin in vivo and is approved for once-daily treatment of diabetes as well as obesity. The aim of the present studies was to design a once weekly GLP-1 analogue by increasing albumin affinity and secure full stability against metabolic degradation. The fatty acid moiety and the linking chemistry to GLP-1 were the key features to secure high albumin affinity and GLP-1 receptor (GLP-1R) potency and in obtaining a prolonged exposure and action of the GLP-1 analogue. Semaglutide was selected as the optimal once weekly candidate. Semaglutide has two amino acid substitutions compared to human GLP-1 (Aib(8), Arg(34)) and is derivatized at lysine 26. The GLP-1R affinity of semaglutide (0.38 ± 0.06 nM) was three-fold decreased compared to liraglutide, whereas the albumin affinity was increased. The plasma half-life was 46.1 h in mini-pigs following i.v. administration, and semaglutide has an MRT of 63.6 h after s.c. dosing to mini-pigs. Semaglutide is currently in phase 3 clinical testing.

Alanine scanning mutagenesis has been used to identify specific side chains of insulin which strongly influence binding to the insulin receptor. A total of 21 new insulin analog constructs were made, and in addition 7 high pressure liquid chromatography-purified analogs were tested, covering alanine substitutions in positions B1, B2, B3, B4, B8, B9, B10, B11, B12, B13, B16, B17, B18, B20, B21, B22, B26, A4, A8, A9, A12, A13, A14, A15, A16, A17, A19, and A21. Binding data on the analogs revealed that the alanine mutations that were most disruptive for binding were at positions TyrA19, GlyB8, LeuB11, and GluB13, resulting in decreases in affinity of 1,000-, 33-, 14-, and 8-fold, respectively, relative to wild-type insulin. In contrast, alanine substitutions at positions GlyB20, ArgB22, and SerA9 resulted in an increase in affinity for the insulin receptor. The most striking finding is that B20Ala insulin retains high affinity binding to the receptor. GlyB20 is conserved in insulins from different species, and in the structure of the B-chain it appears to be essential for the shift from the ␣-helix B8 -B19 to the ␤-turn B20 -B22. Thus, replacing GlyB20 with alanine most likely modifies the structure of the B-chain in this region, but this structural change appears to enhance binding to the insulin receptor.Insulin mediates its effects by binding to the insulin receptor in the plasma membrane of target cells. The molecular mechanisms for insulin interaction with the receptor are not fully understood. The crystal structure of the insulin molecule has been known for more than 25 years (1), but it remains an open question whether the structure of insulin that binds to the receptor is similar to the crystal structure. Until the structure of bound insulin and the side chains that are actually involved in binding is identified by co-crystallization of the receptor and ligand, more information about the binding domain on insulin can be obtained using mutational approaches.The binding domain of the insulin molecule has been studied by investigating receptor binding of a number of insulins from different animal species as well as chemically modified and more recently genetically engineered insulins (2-4). These studies have provided experimental support for a model in which invariant residues from both A and B chains form a surface that binds to the insulin receptor. The putative binding domain comprises a number of residues overlapping the dimerforming surface (ValB12, TyrB16, GlyB23, PheB24, PheB25, TyrB26, GlyA1, GlnA5, TyrA19, and AsnA21) and some of the residues buried beneath the COOH terminus of the B-chain (IleA2, ValA3, GluA4) (2). Cross-linking studies with an azidophenylalanine-substituted analog have shown that one of these residues, PheB25, comes into close proximity to the insulin receptor upon binding (5).Recently, a second binding site has been proposed, involving residues LeuA13 and LeuB17 (6, 7). A biphasic binding reaction involving this second binding site could explain the negative cooperativity phenomen...

The affinities of a number of insulin analogues for the human insulin receptor, a truncated soluble form of the insulin receptor, and the human insulin-like growth factor 1 receptor were determined. Insulin analogues with substitutions in the A13 or B17 positions were shown to have anomalous binding properties. This suggests that these positions, which are located in the hexamerforming surface on the opposite side of the molecule from the classical binding site, constitute a second domain of the molecule important for receptor binding. In the present work, a model is proposed where each of the two a subunits of the insulin receptor contributes with a different binding region to the formation of the high-affinity binding site. Subsequently, a second molecule of insulin is able to bind to a low-affinity site involving only one of the a subunits, thus accounting for the curvilinear Scatchard plot. The affinity of the low-affinity site could be estimated using a high-affinity insulin analogue as the tracer. The model also provides the framework for a molecular explanation of the negative cooperativity phenomenon.Insulin exerts its effects by binding to a membrane receptor which is present in almost all mammalian cell types. The insulin receptor is a heavily glycosylated heterotetramer a& structure where the binding of insulin occurs on the extracellular a subunits [l, 21. The binding of insulin leads to activation of the tyrosine kinase function of the intracellular part of the / 3 subunits [3,4]. The molecular mechanism of insulin binding and signal transduction through the transmembrane domain has not been elucidated. The insulin receptor exhibits a curvilinear Scatchard plot which indicates the presence of multiple classes of binding sites or negative cooperativity between sites. Acceleration of dissociation of bound tracer insulin from the insulin receptor in the presence of unlabeled insulin suggests the presence of at least two interacting binding sites [5]. The binding of a number of insulin analogues to various receptor constructs has been measured, and based on these data a model for insulin binding is proposed. The proposed model is also consistent with most of the available literature data. MATERIALS AND METHODSThe soluble form of the insulin receptor (SIR), truncated just before the transmembrane domain, was expressed in baby hamster kidney cells and harvested from the supernatant by affinity chromatography as previously described [6]. The human insulin receptor (hIR) and the human insulinlike-growth-factor-1 (IGF1) receptor (hICF1 R) were expressed in baby hamster kidney cells and partially purified by solubilization and affinity chromatography or gel-permeation Abbreviations. IGFI, insulin-like growth factor I; hIR, human insulin receptor; SIR, soluble insulin receptor; hIGFlR, human IGFI receptor; hGH, human growth hormone. chromatography as described elsewhere [7, 81. In all cases, the receptor isoform without exon I1 was used. Human insulin and insulin analogues were labeled with ' *' I by the lacto...

We have synthesized insulins acylated by fatty acids in the epsilon-amino group of LysB29. Soluble preparations can be made in the usual concentration of 600 nmol/ml (100 IU/ml) at neutral pH. The time for 50% disappearance after subcutaneous injection of the corresponding TyrA14(125I)-labelled insulins in pigs correlated with the affinity for binding to albumin (r = 0.97), suggesting that the mechanism of prolonged disappearance is binding to albumin in subcutis. Most protracted was LysB29-tetradecanoyl des-(B30) insulin. The time for 50% disappearance was 14.3 +/- 2.2 h, significantly longer than that of Neutral Protamine Hagedorn (NPH) insulin, 10.5 +/- 4.3 h (p < 0.001), and with less inter-pig variation (p < 0.001). Intravenous bolus injections of LysB29-tetradecanoyl des-(B30) human insulin showed a protracted blood glucose lowering effect compared to that of human insulin. The relative affinity of LysB29-tetradecanoyl des-(B30) insulin to the insulin receptor is 46%. In a 24-h glucose clamp study in pigs the total glucose consumptions for LysB29-tetradecanoyl des-(B30) insulin and NPH were not significantly different (p = 0.88), whereas the times when 50% of the total glucose had been infused were significantly different, 7.9 +/- 1.0 h and 6.2 +/- 1.3 h, respectively (p < 0.04). The glucose disposal curve caused by LysB29-tetradecanoyl des-(B30) insulin was more steady than that caused by NPH, without the pronounced peak at 3 h. Unlike the crystalline insulins, the soluble LysB29-tetradecanoyl des-(B30) insulin does not elicit invasion of macrophages at the site of injection. Thus, LysB29-tetradecanoyl des-(B30) insulin might be suitable for providing basal insulin in the treatment of diabetes mellitus.

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The glucagon-like peptide-1 incretin receptor (GLP-1R) of family B G protein-coupled receptors (GPCRs) is a major drug target in type-2-diabetes due to its regulatory effect on post-prandial blood-glucose levels. The mechanism(s) controlling GLP-1R mediated signaling are far from fully understood. A fundamental mechanism controlling the signaling capacity of GPCRs is the post-endocytic trafficking of receptors between recycling and degradative fates. Here, we combined microscopy with novel real-time assays to monitor both receptor trafficking and signaling in living cells. We find that the human GLP-1R internalizes rapidly and with similar kinetics in response to equipotent concentrations of GLP-1 and the stable GLP-1 analogues exendin-4 and liraglutide. Receptor internalization was confirmed in mouse pancreatic islets. GLP-1R is shown to be a recycling receptor with faster recycling rates mediated by GLP-1 as compared to exendin-4 and liraglutide. Furthermore, a prolonged cycling of ligand-activated GLP-1Rs was observed and is suggested to be correlated with a prolonged cAMP signal.