Insulin is a small globular protein hormone secreted by the pancreas to lower blood glucose levels. Subcutaneous injection of insulin has long been established as a treatment for diabetes mellitus in which either the pancreas ceases to produce insulin (type 1) or in which the tissues become insensitive to the action of insulin (type 2).
Structurally, the Insulin monomer consists of two chains, an A chain, containing 21 residues and a B-chain containing 30 residues (fig. 1a).
Figure 1. (a) The disposition of the two chains in the insulin molecule indicating the three disulphide bridges. (b) The hydrogen-bonds (dashed lines) of the antiparallel b-sheet formed between the two molecules of the insulin dimer and the cluster of non-polar residues at the interface .
Both chains pack to form a compact globular domain stabilized by three disulphide bridges (A6-A11, A7-B7, and A20-B19). PheB24 lies at the classical receptor-binding surface with its aromatic ring packed against the hydrophobic core (fig. 1b) and has been proposed to direct a change in conformation on receptor binding, with residues B24–B30 detaching from the core (a). It is this propensity for conformational change which makes the insulin monomer susceptible to fibrillation whereupon the structure changes from a predominantly α-helical state to a β-sheet rich conformation (fig. 2).
Figure 2. (a) Native structure of the insulin dimer. (b) View of insulin fibril model, looking down fibril axis . (c) The protofilament structure of insulin amyloid fibrils . (d) Insulin fibrils formed after incubation at 65°C for 2 days in the presence of 1 mM NaCl (pH 2.0).
Possibly for this reason, in the pancreatic β-cell insulin has evolved to be stored as Zn2+-stabilized hexamers, arranged in crystalline arrays within mature storage granules (b).
Figure 3. The 2-zinc insulin hexamer of monomers (or a trimer of dimers). Each axial Zn2+ ion is coordinated by three B10 His side chains .
Upon secretion from the pancreas in response to rising blood glucose levels, the insulin Zn2+-hexamers dissociate into Zn2+-free dimers and monomers for immediate passage into blood capillaries. The rate of absorption of injected insulin is also limited by the time required for dissociation of the hexamers into the monomer. Recombinant DNA technology has made it possible to prepare rapid-acting insulin analogs with accelerated heaxmeric disassembly that remain dimeric or even monomeric at high concentration by introducing amino acid substitutions into the molecule.
Insulin KP (lispro – Humalog) is one such rapid-acting analog developed by Eli Lilly that can be injected just before meals. Lispro contains the substitutions ProB28→Lys and LysB29→Pro, which destabilise the classical dimer-forming C-terminal anti-parallel β-sheet, an inversion that mimics the sequence of the homologous insulin-Iike growth factor-1 (IGF-1)(Fig. 4).
Fig. 4. The hydrogen-bonds of the antiparallel b-sheet formed at the dimer interface of native insulin and insulin lispro.
However, despite destabilisation of the dimer interface insulin lispro still forms Zn2+ insulin hexamers in the presence of the phenolic excipients present in commercial pharmaceutical formulations (c). This provides the necessary stability against fibrillation during storage in the vial but absorption is still not as rapid as could be provided by a zinc-free formulation. Additionally lispro and other meal-time insulin analogs have reduced shelf life upon dilution by the patient or health-care provider.
There is a great need for an insulin analog which augments the stability of the insulin monomer while retaining the weakened dimer-related β-sheet of lispro.
Halogen stabilization in medicinal chemistry
Halogen atoms have long been used for compound optimization in medicinal chemistry (d). The utility of halogen substitutions in amino acids is also well established in medicinal chemistry. Of all the halogens, the effect of fluorine (with an atomic radius of 42 pm similar to that of hydrogen at 53 pm) incorporation on the physical and chemical properties of proteins is the most characterized in the scientific literature (e). Such observations have motivated the study of fluorinated amino acids for the structural stabilization of proteins, with the provisio that in the case of a biologically active polypeptide at least a significant proportion of the activity must also be maintained.
Fluorolog (f) is a rapid-acting, ultra-concentrated insulin that is in pre-clinical development by Thermalin Diabetes (Cleveland, Ohio). In this lispro analog B24 Phe has been substituted with ortho-monofluorophenylalanine (2F-Phe)(fig. 5).
Figure 5. (a) A vial of rapid-acting, ultraconcentrated U-500 Fluorolog. (b) Ortho-monofluoro-phenylalanine (2F-Phe). (c) The disposition of 2F-Phe against the body of the Fluorolog molecule .
The large inductive effects of the fluorine atom act to thermodynamically stabilize the entire molecule yielding an analog that no longer needs to form a hexamer to be stable. Fluorolog is not prone to clumping and can be formulated at high concentrations (500 units/mL) without the risk of fibrillation and targets three market niches :
1) Fibrillation of insulin is enhanced by agitation and can result in blockage of insulin pumps used by some type 1 diabetics. In miniaturized insulin pumps, pump size is currently constrained by the size of the insulin reservoir which if shrunk by 80% could yield a pump that lasts a whole week. Insulin fibrillation is of even greater concern in implantable insulin pumps, where the insulin may be contained for up to 3 months at high concentration and at physiological temperature.
2) In developed countries some highly insulin resistant type 2 diabetics – often members of underprivileged minority communities- currently inject several hundred units of insulin each day (g). Injecting up to 0.5 mL of the standard lispro 100 units/mL (U-100) formulation is not only uncomfortable but also delays mealtime insulin absorption. Highly concentrated Fluorolog will allow these diabetics to inject smaller quantities at one site and closer to meals.
3) Because fibrillation is also enhanced at higher temperatures, insulin must optimally be kept refrigerated prior to use. For both type 1 and type 2 diabetics in underdeveloped parts of Africa and the Middle East there is a great need for an insulin that does not degrade without refrigeration. Fluorolog is stable for up to 3 months in warm climates without refrigeration.(h)
Phase 1 & 2 trials
Phase I studies have confirmed that the introduction of a single fluorine atom at the receptor-binding surface of Fluorolog stabilizes the monomer and protects it from degradation. Fluorolog exhibited the expected rapid-acting pharmacokinetic properties (even at U-500 formulation) in contrast to the impaired pharmacokinetic properties of native insulin. Furthermore, fluorination of B24Phe was able to mitigate the untoward effects of AspB10 in the DKP homolog on cellular proliferation in culture and on cross-binding to the IGF-1 receptor.
For its phase 2 trial Thermalin Diabetes is seeking to extend the pilot stability data to include individual aspects of chemical and physical degradation (such as disulphide cleavage, covalent polymer formation, and fibrillation) in preparation for an Investigational New Drug (IND) Application
a. In single-chain insulin (SCI: the recombinant precursor expressed in yeast) B29Lys and A1Gly are linked by a peptide bond. SCI crystallizes with the same fold as native insulin but is inactive.
b. In the early days insulin could only be crystallized if acid-ethanol extracted from pancreatic tissue in a galvanized bucket.
c. DKP- insulin with the additional HisB10→Asp substitution is monomeric under a wide range of conditions. Although this insulin-like growth factor-1 equivalent substitution eliminates zinc binding it does raise the possibility of mitogenicity.
d. The activities of the widely prescribed statin atorvastatin (Liptor) and the SSRI antidepressant fluoxetine hydrochloride (Prozac) are both enhanced by the covalent incorporation of a fluorine atom.
e. Fluorine is distinguished from the normal C,H,O,N and S constituents of proteins by its atomic radius, electronegativity, stereo-electronic distribution of partial charges, and transmitted effects on the stereo-electronic properties of neighboring atoms.
f. US Patent 20140128319 A1 (2008).
g. As pre-filled insulin pens can only inject 60–80 units of insulin at a time, some highly insulin-resistant diabetics must inject up to 9 times a day or more.
h. Ideal for airmail.
Blog written by Raj Gill.
- Gill, R. & Wood, S. (2003) Structure and Phylogeny of Insulin. Chapter 12 in International Textbook of Diabetes Vol. 1, ed. Pickup, J.C. & Williams, G., Blackwell Scientific, Oxford.
- Ivanova, M.I., Sievers, S.A., Sawaya, M.R., Wall, J.S. & Eisenberg, D. (2009) Molecular Basis for Insulin Fibril Assembly. Proc Natl Acad Sci USA 106, 18990–18995.
- Landreh, M., Johansson, J., Rising, A., Presto, J. &Jörnvall, H. (2012) Control of Amyloid Assembly by Autoregulation. Biochemical J. 447, 185-192.
- Dyukov, M.I., Grudinin, M.P., Sirotkin, A.K., & Kiselev, O.I. (2008) Insulin Fibrillogenesis In vitro. Doklady Biochem. Biophys. 419, 79-81.