Search

A hallmark of the pancreatic hormone amylin is its high propensity toward the formation of amyloid fibrils, which makes it a challenging drug design effort. The amylin analogue pramlintide is commercially available for diabetes treatment as an adjunct to insulin therapy but requires three daily injections due to its short half-life. We report here the development of the stable, lipidated long-acting amylin analogue cagrilintide (23) and some of the structure-activity efforts that led to the selection of this analogue for clinical development with obesity as an indication. Cagrilintide is currently in clinical trial and has induced significant weight loss when dosed alone or in combination with the GLP-1 analogue semaglutide.

The insulin molecule was discovered in 1921. Shortly thereafter, its propensity towards amyloid fibril formation, fibrillation, was observed and described in the literature as a "precipitate." In the past decades, the increased incidence of type 2 diabetes has reached global epidemic proportions. This has emphasized the demands for both insulin production and the development of modern insulin products for unmet medical needs. Bringing such new insulin drug products to the market for the benefit of patients requires that many CMC-related processes are understood, described, and controlled. One potential undesired process is insulin fibril formation. The compound thioflavin T (ThT) is known as a fluorescent probe for amyloid fibrils. As such, ThT is utilized in a versatile research assay in microtiter plate format, the ThT assay. This review will describe an experimental set-up using not only a ThT microtiter plate assay but also two orthogonal methods. The use of the ThT assay in research and characterization of insulin analogues, as well as formulations of insulin, is described by cases drawn from the scientific literature and patents. The ThT assay is compared to other physical stability tests and in conclusion the advantages and limitations of the assay are compared.

The structure of insulin, a glucose homeostasis-controlling hormone, is highly conserved in all vertebrates and stabilized by three disulfide bonds. Recently, we designed a novel insulin analogue containing a fourth disulfide bond located between positions A10-B4. The N-terminus of insulin's B-chain is flexible and can adapt multiple conformations. We examined how well disulfide bond predictions algorithms could identify disulfide bonds in this region of insulin. In order to identify stable insulin analogues with additional disulfide bonds, which could be expressed, the Cβ cut-off distance had to be increased in many instances and single X-ray structures as well as structures from MD simulations had to be used. The analogues that were identified by the algorithm without extensive adjustments of the prediction parameters were more thermally stable as assessed by DSC and CD and expressed in higher yields in comparison to analogues with additional disulfide bonds that were more difficult to predict. In contrast, addition of the fourth disulfide bond rendered all analogues resistant to fibrillation under stress conditions and all stable analogues bound to the insulin receptor with picomolar affinities. Thus activity and fibrillation propensity did not correlate with the results from the prediction algorithm. Statement: A fourth disulfide bond has recently been introduced into insulin, a small two-chain protein containing three native disulfide bonds. Here we show that a prediction algorithm predicts four additional four disulfide insulin analogues which could be expressed. Although the location of the additional disulfide bonds is only slightly shifted, this shift impacts both stability and activity of the resulting insulin analogues.

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.

Small molecules with insulin mimetic effects and oral availability are of interest for potential substitution of insulin injections in the treatment of diabetes. We have searched databases for compounds capable of mimicking one epitope of the insulin molecule known to be involved in binding to the insulin receptor (IR). This approach identifies thymolphthalein, which is an apparent weak agonist that displaces insulin from its receptor, stimulates auto- and substrate phosphorylation of IR, and potentiates lipogenesis in adipocytes in the presence of submaximal concentrations of insulin. The various effects are observed in the 10(-5)-10(-3) M range of ligand concentration and result in partial insulin activity. Furthermore, analogues of the related phenol red and fluorescein molecules fully displace insulin from the IR ectodomain, however, without insulin agonistic effects. The interactions are further characterized by NMR, UV-vis, and fluorescence spectroscopies. It is shown that both fluorescence and UV-vis changes in the ligand spectra induced by IR fragments occur with Kd values similar to those obtained in the displacement assay. Nevertheless, insulin itself cannot completely abolish binding of the small molecules. Determination of the binding stoichiometry reveals multiple binding sites for ligands of which one overlaps with the insulin binding site on the receptor.

A barley gene encoding a novel DNA-binding protein (HRT) was identified by southwestern screening with baits containing a gibberellin phytohormone response element from an alpha-amylase promoter. The HRT gene contains two introns, the larger of which (5722 base pairs (bp)) contains a 3094-bp LINE-like element with homology to maize Colonist1. In vitro mutagenesis and zinc- and DNA-binding assays demonstrate that HRT contains three unusual zinc fingers with a CX8-9CX10CX2H consensus sequence. HRT is targeted to nuclei, and homologues are expressed in other plants. In vivo, functional tests in plant cells indicate that full-length HRT can repress expression from certain promoters including the Amy1/6-4 and Amy2/32 alpha-amylase promoters. In contrast, truncated forms of HRT containing DNA-binding domains can activate, or derepress, transcription from these promoters. Northern hybridizations indicate that HRT mRNA accumulates to low levels in various tissues. Roles for HRT in mediating developmental and phytohormone-responsive gene expression are discussed.

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.

Through binding to and signaling via the insulin receptor (IR), insulin is involved in multiple effects on growth and metabolism. The current model for the insulin-IR binding process is one of a biphasic reaction. It is thought that the insulin peptide possesses two binding interfaces (sites 1 and 2), which allow it to bridge the two alpha-subunits of the insulin receptor during the biphasic binding reaction. The sequential order of the binding events involving sites 1 and 2, as well as the molecular interactions corresponding to the fast and slow binding events, is still unknown. In this study we examined the series of events that occur during the binding process with the help of three insulin analogues: insulin, an analogue mutated in site 2 (B17A insulin), and an analogue in which part of site 1 was deleted (Des A1-4 insulin), both with and without a fluorescent probe attached. The binding properties of these analogues were tested using two soluble Midi IR constructs representing the two naturally occurring isoforms of the IR, Midi IR-A and Midi IR-B. Our results showed that in the initial events leading to Midi IR-insulin complex formation, insulin site 2 binds to the IR in a very fast binding event. Subsequent to this initial fast phase, a slower rate-limiting phase occurs, consistent with a conformational change in the insulin-IR complex, which forms the final high-affinity complex. The terminal residues A1-A4 of the insulin A-chain are shown to be important for the slow binding phase, as insulin lacking these amino acids is unable to induce a conformational change of IR and has a severely impaired binding affinity. Moreover, differences in the second phase of the binding process involving insulin site 1 between the IR-A and IR-B isoforms suggest that the additional amino acids encoded by exon 11 in the IR-B isoform influence the binding process.

We have applied spectroscopic techniques to study two kinds of ligand binding to the insulin receptor. First, a fluorescently labelled insulin analogue is used to characterize the mechanism of reversible 1 :1 complex formation with a fragment of the insulin receptor ectodomain. The receptor induced fluorescence enhancement of the labelled insulin analogue provides the basis for stopped flow kinetic experiments. The kinetic data are consistent with a bimolecular binding event followed by a conformational change. This emphasizes the importance of insulin induced conformational changes in the activation of the insulin receptor. Second, the binding of fluorescein derivatives to the insulin receptor is studied. These small molecule ligands displace insulin from its receptor with micromolar affinity. The binding is verified by transferred NOESY NMR experiments. Their chromophoric properties are used to measure the affinity by UV-vis and fluorescence difference spectroscopies and the resulting Kdvalues are similar to those observed in the displacement receptor binding assay. However, these experiments and a stoichiometry determination indicate multiple binding sites, of which one overlaps with the insulin binding site. These two examples illustrate how spectroscopy complements biochemical receptor binding assays and provides information on ligand–insulin receptor interactions in the absence of three dimensional structures.

Within the class of insulin and insulin-like growth factor receptors, detailed information about the molecular recognition event at the hormone-receptor interface is limited by the absence of suitable co-crystals. We describe the use of a biologically active insulin derivative labeled with the NBD fluorophore (B29NBD-insulin) to characterize the mechanism of reversible 1:1 complex formation with a fragment of the insulin receptor ectodomain. The accompanying 40 % increase in the fluorescence quantum yield of the label provides the basis for a dynamic study of the hormone-receptor binding event. Stopped-flow fluorescence experiments show that the kinetics of complex formation are biphasic comprising a bimolecular binding event followed by a conformational change. Displacement with excess unlabeled insulin gave monophasic kinetics of dissociation. The rate data are rationalized in terms of available experiments on mutant receptors and the X-ray structure of a non-binding fragment of the receptor of the homologous insulin-like growth factor (IGF-1).