Monomeric Insulin Research Articles

Monitoring insulin fibrillation kinetics using chromatographic analysis

Insulin is a small protein widely used to treat patients with diabetes and is a commonly used model for protein fibrillation studies. Under specific conditions, such as low pH and high temperature, insulin monomers aggregate to form fibrils. This aggregation is problematic for manufacturing and storage of insulin. The thioflavin T (ThT) assay is commonly used to study amyloid fibrillation but suffers from several limitations, such as the effect of protein concentration, the size of the amyloid fibrillar bundles, competitive binding, and fibril aggregation, all of which hinder precise quantitative analysis. Here, we present a method for studying the kinetics of insulin fibrillation utilizing ultra-performance liquid chromatography (UPLC). This method enables the quantitative detection of soluble insulin components, including chemically modified components. The formation of a deamidated species could be monitored at the early stage of fibrillation, and this species was likely included in the fibrils. In addition, in the presence of inhibitors known to compete with ThT for binding to fibrils, UPLC analysis showed the disappearance of soluble components even though the ThT assay did not indicate the presence of fibrils. These results suggest that the UPLC-based analysis presented here can complement the ThT assay for investigating the kinetics of protein fibrillation.

Inhibiting insulin aggregation by chaperone-like green cholinium-based DESs as additives

Protein microenvironment is critical to study protein function and stability. In the current study, the ability of synthesized choline-based deep eutectic solvents (DESs) as additives to inhibit and disaggregate amyloid fibrils of human insulin was investigated. The DESs synthesized in this study were nontoxic and can be classified as green chemistry. Utilizing various biophysical methods indicated that choline-chloride glycine (Ch/Gly) and choline-chloride ascorbic acid (Ch/Asb) exhibited chaperone-like properties. This was attributed to their inhibitory effects on the lag phase by stabilizing insulin monomers and on the saturation phase by disaggregating mature insulin fibrils. In contrast, choline-chloride histidine (Ch/His) accelerated the aggregation of insulin. Our results suggest that antioxidant and hydrophobic properties, combined with lower surface activity and higher hydrogen bond formation ability, are the main features describing the inhibitory effect of DESs on protein aggregation.

Atomic resolution structure of full-length human insulin fibrils

Patients with type 1 diabetes mellitus who are dependent on an external supply of insulin develop insulin-derived amyloidosis at the sites of insulin injection. A major component of these plaques is identified as full-length insulin consisting of the two chains A and B. While there have been several reports that characterize insulin misfolding and the biophysical properties of the fibrils, atomic-level information on the insulin fibril architecture remains elusive. We present here an atomic resolution structure of a monomorphic insulin amyloid fibril that has been determined using magic angle spinning solid-state NMR spectroscopy. The structure of the insulin monomer yields a U-shaped fold in which the two chains A and B are arranged in parallel to each other and are oriented perpendicular to the fibril axis. Each chain contains two β-strands. We identify two hydrophobic clusters that together with the three preserved disulfide bridges define the amyloid core structure. The surface of the monomeric amyloid unit cell is hydrophobic implicating a potential dimerization and oligomerization interface for the assembly of several protofilaments in the mature fibril. The structure provides a starting point for the development of drugs that bind to the fibril surface and disrupt secondary nucleation as well as for other therapeutic approaches to attenuate insulin aggregation.

4S-fluorination of ProB29 in insulin lispro slows fibril formation

Recombinant insulin is a life-saving therapeutic for millions of patients affected by diabetes mellitus. Standard mutagenesis has led to insulin variants with improved control of blood glucose; for instance, the fast-acting insulin lispro contains two point mutations that suppress dimer formation and expedite absorption. However, insulins undergo irreversible denaturation, a process accelerated for the insulin monomer. Here we replace ProB29 of insulin lispro with 4R-fluoroproline, 4S-fluoroproline, and 4,4-difluoroproline. All three fluorinated lispro variants reduce blood glucose in diabetic mice, exhibit similar secondary structure as measured by circular dichroism, and rapidly dissociate from the zinc- and resorcinol-bound hexamer upon dilution. Notably, however, we find that 4S-fluorination of ProB29 delays the formation of undesired insulin fibrils that can accumulate at the injection site in vivo, and can complicate insulin production and storage. These results demonstrate how subtle molecular changes achieved through non-canonical amino acid mutagenesis can improve the stability of protein therapeutics.

Understanding the Reversible Binding of a Multichain Protein-Protein Complex through Free-Energy Calculations.

We demonstrate that the binding affinity of a multichain protein-protein complex, insulin dimer, can be accurately predicted using a streamlined route of standard binding free-energy calculations. We find that chains A and C, which do not interact directly during binding, stabilize the insulin monomer structures and reduce the binding affinity of the two monomers, therefore enabling their reversible association. Notably, we confirm that although classical methods can estimate the binding affinity of the insulin dimer, conventional molecular dynamics, enhanced sampling algorithms, and classical geometrical routes of binding free-energy calculations may not fully capture certain aspects of the role played by the noninteracting chains in the binding dynamics. Therefore, this study not only elucidates the role of noninteracting chains in the reversible binding of the insulin dimer but also offers a methodological guide for investigating the reversible binding of multichain protein-protein complexes utilizing streamlined free-energy calculations.

Effects of Hydrogen Peroxide on the Hydrogen Bonding Structure and Dynamics of Water and Its Influence on the Aqueous Solvation of the Insulin Monomer.

The hydrogen bond structure and dynamics of water and hydrogen peroxide (H2O2) in their binary mixtures have been studied at 298 K by classical molecular dynamics simulations. Twelve different concentrations of aqueous-H2O2 solutions are considered for this study. We have analyzed the interactions between water and H2O2 by site-site pair correlation functions and observed that the probability of formation of OW···HP hydrogen bonds are higher compared to OP···HW. The second solvation shell of water is strongly affected by increasing H2O2 concentrations (XP > 0.50), which signifies the destruction of the tetrahedral network structure of water. The translational and rotational dynamics of water and H2O2 do not significantly change up to 25% of H2O2 in aqueous mixtures. The hydrogen bond lifetime of water-water, water-H2O2, and H2O2-H2O2 in the aqueous-H2O2 solutions shows a very minimal change with increasing H2O2 concentrations. In addition to this, we also investigated the effect of H2O2 on the insulin monomer and observed that higher concentrations of H2O2 (XP = 0.10) change the secondary structure. The influence of H2O2 is more on chain-B than that on chain-A in the insulin monomer. The H2O2 occupancy at the protein surface is higher for negatively charged (GLU) and polar (ASN and THR) amino acid residues compared with that for positively charged and neutral residues in the solutions.

Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies

Insulin formulations with diverse oligomerization states are the hallmark of interventions for the treatment of diabetes. Here using single-molecule recordings we firstly reveal that insulin oligomerization can operate via monomeric additions and secondly quantify the existence, abundance and kinetic characterization of diverse insulin assembly and disassembly pathways involving addition of monomeric, dimeric or tetrameric insulin species. We propose and experimentally validate a model where the insulin self-assembly pathway is rerouted, favoring monomeric or oligomeric assembly, by solution concentration, additives and formulations. Combining our practically complete kinetic characterization with rate simulations, we calculate the abundance of each oligomeric species from nM to mM offering mechanistic insights and the relative abundance of all oligomeric forms at concentrations relevant both for secreted and administrated insulin. These reveal a high abundance of all oligomers and a significant fraction of hexamer resulting in practically halved bioavailable monomer concentration. In addition to providing fundamental new insights, the results and toolbox presented here can be universally applied, contributing to the development of optimal insulin formulations and the deciphering of oligomerization mechanisms for additional proteins.

Characterizing protein conformational ensembles and dynamics with 2D IR spectroscopy.

Advances in protein two-dimensional infrared spectroscopy and computational spectroscopy of amide I vibrations have provided a way to characterize the conformational variation and disorder in protein structure. These techniques use isotope-edited amide I spectra in combination with spectra computed from Markov state models built from molecular dynamics simulations to infer variation in local configuration and to refine ensembles for peptides and proteins. This talk will describe these tools drawing on examples from studies of insulin monomer and dimer structure and the dynamic interactions in the monomer-dimer equilibrium.

Dietary anthocyanins inhibit insulin fibril formation and cytotoxicity in 3T3-L1 preadipocytes

Insulin fibril formation decreases the effectiveness of insulin therapy and causes amyloidosis in diabetes. Studies suggest that phytochemicals are capable of inhibiting fibril formation. Herein, we investigated the inhibitory effects of anthocyanins, including cyanidin, cyanidin-3-glucoside (C3G), cyanidin-3-rutinoside (C3R), malvidin, and malvidin-3-glucoside (M3G) on fibril formation. Our results revealed that anthocyanins (50–200 μM) significantly reduced the formation of insulin fibrils by increasing lag times and decreasing ThT fluorescence at the plateau phase. These findings were confirmed by TEM images, which showed reduced fibril length and number. Furthermore, FTIR analysis indicated that anthocyanins reduced the secondary structure transition of insulin from α-helix to β-sheet. Anthocyanins interacted with monomeric insulin (residues B8–B30) via H-bonds, van der Waals, and hydrophobic interactions, covering the fibril-prone segments of insulin (residues B12–B17). Based on the structure-activity analysis, the presence of glycosides and hydroxyl groups on phenyl rings increased intermolecular interaction, mediating the inhibitory effect of anthocyanins on fibril formation in the order of malvidin < cyanidin < M3G < C3G < C3R. Moreover, anthocyanins formed H-bonds with preformed insulin fibrils, except for malvidin. In preadipocytes, C3R, C3G, and cyanidin attenuated insulin fibril-induced cytotoxicity. In conclusion, anthocyanins are effective inhibitors of insulin fibril formation and cytotoxicity.

Formulation Excipients and Their Role in Insulin Stability and Association State in Formulation.

While excipients are often overlooked as the "inactive" ingredients in pharmaceutical formulations, they often play a critical role in protein stability and absorption kinetics. Recent work has identified an ultrafast absorbing insulin formulation that is the result of excipient modifications. Specifically, the insulin monomer can be isolated by replacing zinc and the phenolic preservative metacresol with phenoxyethanol as an antimicrobial agent and an amphiphilic acrylamide copolymer excipient for stability. A greater understanding is needed of the interplay between excipients, insulin association state, and stability in order to optimize this formulation. Here, we formulated insulin with different preservatives and stabilizing excipient concentrations using both insulin lispro and regular human insulin and assessed the insulin association states using analytical ultracentrifugation as well as formulation stability. We determined that phenoxyethanol is required to eliminate hexamers and promote a high monomer content even in a zinc-free lispro formulation. There is also a concentration dependent relationship between the concentration of polyacrylamide-based copolymer excipient and insulin stability, where a concentration greater than 0.1g/mL copolymer is required for a mostly monomeric zinc-free lispro formulation to achieve stability exceeding that of Humalog in a stressed aging assay. Further, we determined that under the formulation conditions tested zinc-free regular human insulin remains primarily hexameric and is not at this time a promising candidate for rapid-acting formulations.

Exchange Broadening Underlies the Enhancement of IDE-Dependent Degradation of Insulin by Anionic Membranes.

Insulin-degrading enzyme (IDE) is an evolutionarily conserved ubiquitous zinc metalloprotease implicated in the efficient degradation of insulin monomer. However, IDE also degrades monomers of amyloidogenic peptides associated with disease, complicating the development of IDE inhibitors. In this work, we investigated the effects of the lipid composition of membranes on the IDE-dependent degradation of insulin. Kinetic analysis based on chromatography and insulin’s helical circular dichroic signal showed that the presence of anionic lipids in membranes enhances IDE’s activity toward insulin. Using NMR spectroscopy, we discovered that exchange broadening underlies the enhancement of IDE’s activity. These findings, together with the adverse effects of anionic membranes in the self-assembly of IDE’s amyloidogenic substrates, suggest that the lipid composition of membranes is a key determinant of IDE’s ability to balance the levels of its physiologically and pathologically relevant substrates and achieve proteostasis.

Insulin biomolecular condensate formed in ionic microenvironment modulates the structural properties of pristine and magnetic cellulosic nanomaterials

The study reports the formation of a unique molecular condensate of insulin molecules with Zn2+ at a molar ratio of 1:1 under thermal stress. The condensate was cross-linked, viscoelastic, and structurally reversible by pH modulation. This unique state was formed by isoelectric condensation of insulin conformational variants (CVs) through helix unwinding, random coil relaxation, and subsequently transition to a β-sheet rich aggregate-like structure. The ions (Na+, Cl- and Zn2+) were found to promote the macromolecular organization through localized ionic interactions. Interestingly, this viscous aggregate instantaneously dissolved upon pH reduction, releasing monomeric insulin. Next, large-length cellulose nanofibers (CNF) and rod-shaped Fe-functionalized cellulose nanocrystals (Fe-CNC) were independently co-incubated with [Zn2+]:[Insulin]. The micrometer-length CNFs were reduced to short-length cellulose nanorods (CNRs), whereas, Fe-CNCs were reduced to ultrathin nanothreads which interacted together and formed spherical Fe-CNCs within insulin microspheres. The CNR-Ins condensate and Fe-CNC-Ins microsphere promoted the adhesion and growth of BHK-21 cells by 74.5 % and 72.6 %, respectively. This unique insulin condensate and its specialized hybrid structures might have an enormous impact on material science.

A novel IONP-decorated two-dimensional [Zn2+]:[Insulin] nanosheet with ordered array of surface channels and cellular uptake potential

The study presents the novel design and potential function of a two-dimensional nanosheet formed at the liquid-liquid interface between FeII,IIIoxide nanoparticles (IONPs) and [Zn2+]:[Insulin]. Under thermal stress, the supramolecular organization of [Zn2+]:[Insulin] shifted from hydrophilic phase to hydrophobic, which gradually dissolved with the IONP hydrophobic layer. As a consequence, insulin monomers assembled into two-dimensional nanosheets by intercalating Zn2+-bridges, whereas, the IONPs (18–20 nm) were oxidized from Fe3O4 to Fe2O3 crystals, and decomposed into ultra-small crystallites of 2–5 nm. These tiny crystallites were found adsorbed on the nanosheet surface, creating IONP-decorated insulin nanosheet. To our knowledge, the ability of insulin to form nanosheet-structure has never been reported earlier. Adding to its uniqueness, the IONP-insulin nanosheets formed ordered arrays of superimposed surface wrinkles, giving rise to geometrically arranged channels. These surface properties have been previously demonstrated with graphene, but the ability of proteins to exhibit such material properties has not been reported earlier. Thus, the study demonstrates an entirely new potential of proteins, in this case insulin, and is believed to be a new advancement in protein-based bio-nanomaterials. Further, cellular uptake of these nanosheets was observed when BHK-21 cells were cultured on its layer, thereby displaying cell-targeting potential.

Functionally selective signaling and broad metabolic benefits by novel insulin receptor partial agonists

Insulin analogs have been developed to treat diabetes with focus primarily on improving the time action profile without affecting ligand-receptor interaction or functional selectivity. As a result, inherent liabilities (e.g. hypoglycemia) of injectable insulin continue to limit the true therapeutic potential of related agents. Insulin dimers were synthesized to investigate whether partial agonism of the insulin receptor (IR) tyrosine kinase is achievable, and to explore the potential for tissue-selective systemic insulin pharmacology. The insulin dimers induced distinct IR conformational changes compared to native monomeric insulin and substrate phosphorylation assays demonstrated partial agonism. Structurally distinct dimers with differences in conjugation sites and linkers were prepared to deliver desirable IR partial agonist (IRPA). Systemic infusions of a B29-B29 dimer in vivo revealed sharp differences compared to native insulin. Suppression of hepatic glucose production and lipolysis were like that attained with regular insulin, albeit with a distinctly shallower dose-response. In contrast, there was highly attenuated stimulation of glucose uptake into muscle. Mechanistic studies indicated that IRPAs exploit tissue differences in receptor density and have additional distinctions pertaining to drug clearance and distribution. The hepato-adipose selective action of IRPAs is a potentially safer approach for treatment of diabetes.

Predicting the evolution of number of native contacts of a small protein by using deep learning approach

Native contacts (NCs) are one of the most vital parameters in order to define the resemblance of a protein conformation with its native state. Prediction of number of native contacts in a protein is useful in protein folding mechanism. In this work, we focused to predict the time series of the number of NCs of a small protein, insulin monomer by using three neural network based models, namely; Multi-Layer Perceptron (MLP), Long Short Term Memory (LSTM) and Gated Recurrent Unit (GRU). The input data used in the study was the time evolution of NC values of the folded and unfolded protein conformations computed from the equilibrated trajectories of atomistic molecular dynamics (MD) simulations performed with the aqueous solution of the protein at ambient as well as at an elevated temperature. The evolutionary prediction accuracy of the three models was tested by calculating two error parameters; Root Mean Square Error (RMSE) and Mean Absolute Error (MAE). Our study revealed that, although these three models are successful in forecasting the time evolutions of the NCs in terms of lower RMSE and MAE, the prediction through static memoryless artificial neural network, MLP was relatively less precise as compared to other two recurrent units, LSTM and GRU. The study infers that by using the available input data generated from the MD trajectories; these neural network based models could be used to predict the complex evolution pattern of distanced based structural parameters of a protein with a satisfactory level.

Insulin Complexation with Cyclodextrins-A Molecular Modeling Approach.

Around 5% of the population of the world is affected with the disease called diabetes mellitus. The main medication of the diabetes is the insulin; the active form is the insulin monomer, which is an instable molecule, because the long storage time, or the high temperature, can cause the monomer insulin to adapt an alternative fold, rich in β-sheets, which is pharmaceutically inactive. The aim of this study is to form different insulin complexes with all the cyclodextrin used for pharmaceutical excipients (native cyclodextrin, methyl, hydroxyethyl, hydroxypropyl and sulfobutylether substituted β-cyclodextrin), in silico condition, with the AutoDock molecular modeling program, to determine the best type of cyclodextrin or cyclodextrin derivate to form a complex with an insulin monomer, to predict the molar ratio, the conformation of the complex, and the intermolecular hydrogen bonds formed between the cyclodextrin and the insulin. From the results calculated by the AutoDock program it can be predicted that insulin can make a stable complex with 5–7 molecules of hydroxypropyl-β-cyclodextrin or sulfobutylether-β-cyclodextrin, and by forming a complex potentially can prevent or delay the amyloid fibrillation of the insulin and increase the stability of the molecule.

Engineering of a Biologically Active Insulin Dimer.

The growing epidemic of diabetes means that there is a need for therapies that are more efficacious, safe, and convenient. Here, we report the efficient synthesis of a novel disulfide dimer of human insulin tethered at the N-terminus of its B-chain through placement of a cysteine residue. The resulting peptide was shown to bind to both the insulin receptor isoform B and insulin-like growth factor-1 receptor with comparable affinity to native insulin. In in vivo insulin tolerance tests, the dimer was equipotent to Actrapid insulin and possessed a sustained duration of action greater than that of Actrapid and Glargine. While the secondary structure of our dimeric insulin was similar to that of insulin, it was more resistant to proteolysis. More importantly, our analogue was produced in quantitative yield from a monomeric thiol insulin scaffold. Our results suggest that this dimer has significant potential to address the clinical needs in the treatment of diabetes.

Conformational Flexibilities and Solvation Properties of Insulin in Aromatic Amino Acid Solutions: A Molecular Dynamics Study Using CHARMM Drude Polarizable Model

Aromatic amino acids (AAA) play a crucial role in the structure and function of proteins. A higher level of AAA causes several diseases, controls insulin levels. In this work, we carried out atomistic molecular dynamics simulations by using CHARMM Drude polarizable force field to investigate the conformational properties of insulin monomer in 2M phe, tyr, trp solutions as well as in pure aqueous solution to compare the relative changes of protein conformations, its solvation properties and the interactions of the free AAA with insulin. Although insulin’s native folded form was intact in all the solutions within the simulation length scale, we observed that the protein is a little more flexible and less compact in phe solution than in tyr/trp solutions. The free AAAs identified to self-aggregate around the protein surface and form clusters of different sizes. They interacted with insulin, significantly through cation/anion–π and π–π stacking, and partly through hydrogen bonded interactions. Among the three, trp was prone to interact through cation–π interactions while phe and tyr interacted through π–π stacking with insulin. Despite a significant number of free AAA molecules in the solvation shell, insulin was observed to be sufficiently hydrated and formed hydrogen bonds with water. Some of our findings agreed with the available experimental results that establish the reliability of the chosen force field. Our findings would interpret the interactions between the free AAA and insulin in solution, helpful to recognize the microscopic details of AAA governed biological processes in living organisms.

Structural Stability of Insulin Oligomers and Protein Association-Dissociation Processes: Free Energy Landscape and Universal Role of Water.

Association and dissociation of proteins are important biochemical events. In this Feature Article, we analyze the available studies of these processes for insulin oligomers in aqueous solution. We focus on the solvation of the insulin monomer in water, stability and dissociation of its dimer, and structural integrity of the hexamer. The intricate role of water in solvation of the dimer- and hexamer-forming surfaces, in long-range interactions between the monomers and the stability of the oligomers, is discussed. Ten water molecules inside the central cavity stabilize the structure of the insulin hexamer. We discuss how different order parameters can be used to understand the dissociation of the insulin dimer. The calculation of the rate using a recently computed multidimensional free energy provides considerable insight into the interplay between protein and water dynamics.

Structural Ensemble of the Insulin Monomer.

Experimental evidence suggests that monomeric insulin exhibits significant conformational heterogeneity, and modifications of apparently disordered regions affect both biological activity and the longevity of pharmaceutical formulations, presumably through receptor binding and fibrillation/degradation, respectively. However, a microscopic understanding of conformational heterogeneity has been lacking. Here, we integrate all-atom molecular dynamics simulations with an analysis pipeline to investigate the structural ensemble of human insulin monomers. We find that 60% of the structures present at least one of the following elements of disorder: melting of the A-chain N-terminal helix, detachment of the B-chain N-terminus, and detachment of the B-chain C-terminus. We also observe partial melting and extension of the B-chain helix and significant conformational heterogeneity in the region containing the B-chain β-turn. We then estimate hydrogen-exchange protection factors for the sampled ensemble and find them in line with experimental results for KP-insulin, although the simulations underestimate the importance of unfolded states. Our results help explain the ready exchange of specific amide sites that appear to be protected in crystal structures. Finally, we discuss the implications for insulin function and stability.