Insulin Fibril Formation Research Articles

Insulin amyloid fibril formation reduction by tripeptide stereoisomers.

Insulin fibrillation is a problem for diabetic patients that can occur during storage and transport, as well as at the subcutaneous injection site, with loss of bioactivity, inflammation, and various adverse effects. Tripeptides are ideal additives to stabilise insulin formulations, thanks to their low cost of production and inherent cytocompatibility. In this work, we analysed the ability of eight tripeptide stereoisomers to inhibit the fibrillation of human insulin in vitro. The sequences contain proline as β-breaker and Phe-Phe as binding motif for the amyloid-prone aromatic triplet found in insulin. Experimental data based on spectroscopy, fluorescence, microscopy, and calorimetric techniques reveal that one stereoisomer is a more effective inhibitor than the others, and cell live/dead assays confirmed its high cytocompatibility. Importantly, in silico data revealed the key regions of insulin engaged in the interaction with this tripeptide, rationalising the molecular mechanism behind insulin fibril formation reduction.

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.

Hesperidin attenuate fibrillation and cytotoxicity of human insulin: Role of secondary structure and its hydrophobicity in protein aggregation

Protein aggregation occurs when proteins are subjected to abnormal physical and chemical conditions; the subsequent aggerate can lead to multiple debilitating and deadly neurological conditions, such as Alzheimer's, Parkinson's, type II diabetes, and Huntington's disease. As such, we must further examine possible therapeutic agents such as polyphenols for possible utilization in the treatment of aggerate – born – illness. In this study, hesperidin (bioflavonoid) was examined for its anti-aggregating potential using human insulin as model protein. Our results show protein denaturing conditions (pH =2.0, Temperature 40 °C, NaCl 50 mM, agitation 1200 rpm) resulted in the formation of insulin fibrils as displayed by various spectroscopic and microscopic techniques. At 20 & 50 µM concentrations of hesperidin, aggregation of insulin decreased in a nucleation-independent manner. Far UV-CD measurement illustrated transition of α-helix to cross β-sheet during formation of amyloid. However, in the presence of hesperidin stabilization of α-helix along with concomitant decrease of β-sheet was observed. The hydrophobicity measurement suggested exposure of hydrophobic residues during fibrillation. Moreover, in the presence of hesperidin hydrophobicity was significantly dropped. Cytotoxicity of fibrillated insulin shows dose dependent inhibition of growth of SH-SY5Y cells and their protection by hesperidin. Molecular docking analysis revealed strong binding energy of 6.61 kcal mol−1 between insulin-hesperidin complex. Also, hesperidin binds to both A (Leu13, Tyr14, Glu17) and B (Phe1, Leu6, HIS10, Ala14, Glu13, Ala14, Leu17) chains of human insulin involving hydrogen and hydrophobic interactions. Conclusively, we demonstrated that hesperidin inhibited the formation of insulin amyloid and should be further exploited for insulin amyloidosis and other protein-misfolding diseases.

Chickpea Peptide: A Nutraceutical Molecule Corroborating Neurodegenerative and ACE-I Inhibition.

Chickpea seeds are the source of proteins in human nutrition and attribute some nutraceutical properties. Herein, we report the effects of chickpea seed bioactive peptide on albumin, insulin, lactoglobulin and lysozyme amyloid fibril formation. Employing thioflavin T (ThT) assays and circular dichroism (CD), amyloid structural binding transition was experimented to analyze the inhibition of amyloid fibril formation. The purified active peptide with a molecular mass of 934.53 Da was evaluated in vitro for its ACE-I inhibitory, antibacterial, antifungal and antidiabetic activities. Further, in vivo animal studies were carried out in wistar rats for blood pressure lowering action. In hypertensive rats, chickpea peptide decreased 131 ± 3.57 mm of Hg for systolic blood pressure and 86 ± 1.5 mm of Hg for diastolic blood pressure after 8 h intraperitoneal administration. Additionally, the peptide suppressed the fibrillation of amyloid and destabilized the preformed mature fibrils. Data emphasize efficacy of chickpea peptide vis-a-vis ACE-Inhibitory, antibacterial, antifungal, antidiabetic and anti-amyloidogenic activities, allowing us to propose this novel peptide as a suitable candidate for nutraceutical-based drugs and seems the first kind of its nature.

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.

The intriguing dose-dependent effect of selected amphiphilic compounds on insulin amyloid aggregation: Focus on a cholesterol-based detergent, Chobimalt.

The amyloidogenic self-assembly of many peptides and proteins largely depends on external conditions. Among amyloid-prone proteins, insulin attracts attention because of its physiological and therapeutic importance. In the present work, the amyloid aggregation of insulin is studied in the presence of cholesterol-based detergent, Chobimalt. The strategy to elucidate the Chobimalt-induced effect on insulin fibrillogenesis is based on performing the concentration- and time-dependent analysis using a combination of different experimental techniques, such as ThT fluorescence assay, CD, AFM, SANS, and SAXS. While at the lowest Chobimalt concentration (0.1 µM; insulin to Chobimalt molar ratio of 1:0.004) the formation of insulin fibrils was not affected, the gradual increase of Chobimalt concentration (up to 100 µM; molar ratio of 1:4) led to a significant increase in ThT fluorescence, and the maximal ThT fluorescence was 3-4-fold higher than the control insulin fibril’s ThT fluorescence intensity. Kinetic studies confirm the dose-dependent experimental results. Depending on the concentration of Chobimalt, either (i) no effect is observed, or (ii) significantly, ∼10-times prolonged lag-phases accompanied by the substantial, ∼ 3-fold higher relative ThT fluorescence intensities at the steady-state phase are recorded. In addition, at certain concentrations of Chobimalt, changes in the elongation-phase are noticed. An increase in the Chobimalt concentrations also triggers the formation of insulin fibrils with sharply altered morphological appearance. The fibrils appear to be more flexible and wavy-like with a tendency to form circles. SANS and SAXS data also revealed the morphology changes of amyloid fibrils in the presence of Chobimalt. Amyloid aggregation requires the formation of unfolded intermediates, which subsequently generate amyloidogenic nuclei. We hypothesize that the different morphology of the formed insulin fibrils is the result of the gradual binding of Chobimalt to different binding sites on unfolded insulin. A similar explanation and the existence of such binding sites with different binding energies was shown previously for the nonionic detergent. Thus, the data also emphasize the importance of a protein partially-unfolded state which undergoes the process of fibrils formation; i.e., certain experimental conditions or the presence of additives may dramatically change not only kinetics but also the morphology of fibrillar aggregates.

Experimental and theoretical studies on the anti‐amyloidogenic and destabilizing effects of pyrogallol against human insulin protein

One of the major problems caused by repeated subcutaneous insulin injections in patients with diabetes is insulin amyloidosis. Understanding the molecular mechanism of amyloid fibril formation of insulin and finding effective compounds to inhibit or eliminate them is very important, and extensive research has been done on it. In this study, the anti-amyloidogenic and destabilizing effects of the pyrogallol, as a phenolic compound, on human insulin protein were investigated by CR absorbance, ThT and ANS fluorescence, FTIR spectroscopy, and atomic force microscopy. According to the obtained results, the formation of amyloid fibrils at pH2.0 and 50°C was confirmed by CR, ThT, ANS, and FTIR assays. Microscopic images also showed the twisted and long structures of amyloid fibrils. Simultaneous incubation of the protein with pyrogallol at different concentrations reduced the intensities of CR, ThT, and ANS in a dose-dependent manner, and no trace of fibrillar structures was observed in the microscopic images. FTIR spectroscopy also showed that the position of the amide I band in the spectrum of samples containing pyrogallol was shifted. Based on the findings of this study, it can be concluded that pyrogallol can be effective in preventing and suppressing human insulin amyloid fibrils. PRACTICAL APPLICATIONS: In recent years, finding a strategy for the treatment of amyloid diseases has been considered by many researchers. Targeting protein aggregates by small organic molecules such as polyphenols is one of the most desirable and effective strategies to prevent and improve amyloid disease, which has received much attention in recent years. 1,2,3-Trihydroxybenzene, commonly known as pyrogallol (Py), is a phenolic compound like other natural polyphenols that are present in human food sources, including fruits and vegetables, and a variety of edible and medicinal plants. So far, many beneficial activities for pyrogallol such as anti-cancer, antioxidant, antibacterial, antiviral, and antifungal have been reported in various studies. Since various studies have shown that natural polyphenols have special properties to prevent amyloid disease, the present study could be useful in advancing the design purposes of new anti-amyloid drugs in the future.

Study of the kinetics and mechanism of ferrocene-tripeptide inhibiting insulin aggregation.

Peptides are gaining popularity as neurodegenerative disease-targeting drugs due to their medicinal value and simplicity in the biomedical and pharmaceutical industry fields. Herein, based on previously studied ferrocene-modified dipeptide (Fc-dipeptide) ferrocene-L-Phe-L-Phe (Fc-FF), we developed four Fc-tripeptides, i.e., Fc-FFY, Fc-FFF, Fc-FFD, and Fc-FFK, to further study the anti-amyloid effects of peptide-based inhibitors. The results showed that all Fc-tripeptides inhibited the formation of insulin fibrils in a dose-dependent manner more responsive than Fc-FF. Meanwhile, Fc-FFY and Fc-FFF had a more significant inhibitory effect on insulin amyloid fibrillation than Fc-FFD and Fc-FFK. In addition, molecular dynamics simulations indicated that Fc-FFY and Fc-FFF were contacted mainly with the hydrophobic core residues of insulin chain A and chain B, respectively. The research indicated that Fc-tripeptides have potential effects in preventing protein misfolding diseases and could be further used to design effective anti-amyloidosis compounds.

Hydroxy-Porphyrin as an Effective, Endogenous Molecular Clamp during Early Stages of Amyloid Fibrillization.

Amyloid fibril formation of proteins is of great concern in neurodegenerative disease and can be detrimental to the storage and stability of biologics. Recent evidence suggests that insulin fibril formation reduces the efficacy of type II diabetes management and may lead to several complications. To develop anti-amyloidogenic compounds of endogenous origin, we have utilized the hydrogen bond anchoring, π stacking ability of porphyrin, and investigated its role on the inhibition of insulin amyloid formation. We report that hydroxylation and metal removal from the heme moiety yields an excellent inhibitor of insulin fibril formation. Thioflavin T, tyrosine fluorescence, Circular Dichorism (CD) spectroscopy, Field emission scanning electron microscopy (FESEM) and molecular dynamics (MD) simulation studies suggest that hematoporphyrin (HP) having hydrogen bonding ability on both sides is a superior inhibitor compared to hemin and protoporphyrin (PP). Experiments with hen egg white lysozyme (HEWL) amyloid fibril formation also validated the efficacy of endogenous porphyrin based small molecules. Our results will help to decipher a general therapeutic strategy to counter amyloidogenesis.

Insulin Analogues with Altered Insulin Receptor Isoform Binding Specificities and Enhanced Aggregation Stabilities.

Insulin is a lifesaver for millions of diabetic patients. There is a need for new insulin analogues with more physiological profiles and analogues that will be thermally more stable than human insulin. Here, we describe the chemical engineering of 48 insulin analogues that were designed to have changed binding specificities toward isoforms A and B of the insulin receptor (IR-A and IR-B). We systematically modified insulin at the C-terminus of the B-chain, at the N-terminus of the A-chain, and at A14 and A18 positions. We discovered an insulin analogue that has Cα-carboxyamidated Glu at B31 and Ala at B29 and that has a more than 3-fold-enhanced binding specificity in favor of the "metabolic" IR-B isoform. The analogue is more resistant to the formation of insulin fibrils at 37 °C and is also more efficient in mice than human insulin. Therefore, [AlaB29,GluB31,amideB31]-insulin may be interesting for further clinical evaluation.

Sulfated polysaccharide ascophyllan prevents amyloid fibril formation of human insulin and inhibits amyloid-induced hemolysis and cytotoxicity in PC12 cells.

We found that ascophyllan significantly inhibited the fibrillation of human insulin and was the most effective among the sulfated polysaccharides tested. Gel-filtration analysis suggested that ascophyllan was capable of forming a complex with insulin through a weak interaction. Secondary structure transition from native α-helix to β-sheet predominant structure of insulin under the fibrillation conditions was suppressed in the presence of ascophyllan. Interestingly, ascophyllan attenuated insulin fibril-induced hemolysis of human erythrocytes. Moreover, ascophyllan attenuated insulin amyloid-induced cytotoxicity on rat pheochromocytoma PC12 cells and reduced the level of intracellular reactive oxygen species. This is the first report indicating that a sulfated polysaccharide, ascophyllan, can suppress the insulin amyloid fibril formation and inhibit the fibril-induced detrimental bioactivities.

Insulin Derived Fibrils Induce Cytotoxicity in vitro and Trigger Inflammation in Murine Models.

Effective exogenous insulin delivery is the cornerstone of insulin dependent diabetes mellitus management. Recent literature indicates that commercial insulin-induced tissue reaction and cellular cytotoxicity may contribute to variability in blood glucose as well as permanent loss of injection or infusion site architecture and function. It is well accepted that insulin formulations are susceptible to mechanical and chemical stresses that lead to insulin fibril formation. This study aims to characterize in vitro and in vivo toxicity, as well as pro-inflammatory activity of insulin fibrils. In vitro cell culture evaluated cytotoxicity and fibril uptake by macrophages and our modified murine air-pouch model quantified inflammatory activity. The latter employed FLOW cytometry and histopathology to characterize fibril-induced inflammation in vivo, which included fibril uptake by inflammatory phagocytes. These studies demonstrated that insulin derived fibrils are cytotoxic to cells in vitro. Furthermore, inflammation is induced in the murine air-pouch model in vivo and in response, macrophages uptake fibrils both in vitro and in vivo. Administration of insulin fibrils can lead to cytotoxicity in macrophages. In vivo data demonstrate insulin fibrils to be pro-inflammatory which over time can lead to cumulative cell/tissue toxicity, inflammation, and destructive wound healing. Long term, these tissue reactions could contribute to loss of insulin injection site architecture and function.

Insulin Fibril Formation Caused by Mechanical Shock and Cavitation.

Cavitation can occur when liquids are exposed to pressure waves of sufficient amplitude, producing rapidly expanding and collapsing gas bubbles that generate localized regions of high energy dissipation. When vials containing insulin were subjected to mechanical shock or when ultrasound was applied to the vials, the resulting cavitation events induced formation of insulin amyloid fibril nuclei that were detected by transmission electron microscopy and quantified by fluorescence spectroscopy following staining with the amyloid-sensitive dye thioflavin-T. Dropping insulin solutions in glass vials produced only minute amounts of insulin fibril nuclei, which could be detected by allowing the nuclei to grow. Cavitation-induced formation of amyloid aggregates may be relevant for iatrogenic insulin deposition disease, where insulin fibrils formed in vitro prior to administration to patients could serve as nuclei for growing fibril deposits in vivo.

Alizarin and Purpurin from Rubia tinctorum L. Suppress Insulin Fibrillation and Reduce the Amyloid-Induced Cytotoxicity.

Alizarin (1,2-dihydroxyanthraquinone) and purpurin (1,2,4-trihydroxyanthraquinone), natural anthraquinone compounds from Rubia tinctorum L., are reported to have diverse biological effects including antibacterial, antitumor, antioxidation, and so on, but the inhibition activity against amyloid aggregation has been rarely reported. In this study, we used insulin as a model protein to explore the anti-amyloid effects of the two compounds. The results showed that alizarin and purpurin inhibited the formation of insulin fibrils in a dose-dependent manner and reduced insulin-induced cytotoxicity. Meanwhile, purpurin had a more significant inhibitory effect on insulin amyloid fibrils compared with alizarin. In addition, computer simulations indicated that the two compounds interacted mainly with the hydrophobic residues of insulin chain B and interfered with the binding of phenylalanine residues. The research indicated that natural anthraquinone compounds had potential effects in preventing protein misfolding diseases and could be further used to design effective antiamyloidosis compounds.

Modification of insulin amyloid aggregation by Zr phthalocyanines functionalized with dehydroacetic acid derivatives.

Amyloid fibrils are widely studied both as target in conformational disorders and as basis for the development of protein-based functional materials. The three Zr phthalocyanines bearing dehydroacetic acid residue (PcZr(L1)2) and its condensed derivatives (PcZr(L2)2 and PcZr(L3)2) as out-of-plane ligands were synthesized and their influence on insulin fibril formation was studied by amyloid-sensitive fluorescent dye based assay, scanning electron microscopy, fluorescent and absorption spectroscopies. The presence of Zr phthalocyanines was shown to modify the fibril formation. The morphology of fibrils formed in the presence of the Zr phthalocyanines differs from that of free insulin and depends on the structure of out-of-plane ligands. It is shown that free insulin mostly forms fibril clusters with the length of about 0.3–2.1 μm. The presence of Zr phthalocyanines leads to the formation of individual 0.4–2.8 μm-long fibrils with a reduced tendency to lateral aggregation and cluster formation (PcZr(L1)2), shorter 0.2–1.5 μm-long fibrils with the tendency to lateral aggregation without clusters (PcZr(L2)2), and fibril-like 0.2–1.0 μm-long structures (PcZr(L3)2). The strongest influence on fibrils morphology made by PcZr(L3)2 could be explained by the additional stacking of phenyl moiety of the ligand with aromatic amino acids in protein. The evidences of binding of studied Zr phthalocyanines to mature fibrils were shown by absorption spectroscopy (for PcZr(L1)2 and PcZr(L2)2) and fluorescent spectroscopy (for PcZr(L3)2). These complexes could be potentially used as external tools allowing the development of functional materials on protein fibrils basis.

Side-Chain Proline-Based Polymers as Effective Inhibitors for In Vitro Aggregation of Insulin.

Insulin fibril formation is considered as the hallmark of several debilitating pathological conditions. To develop effective therapeutics that are able to control the amyloidogenesis process and inhibit fibril formation, herein we have designed a side-chain proline (Pro)-based homopolymer and block copolymers through the reversible addition-fragmentation chain transfer (RAFT) polymerization technique and further explored their obligatory role in the in vitro insulin fibrillation process. Using a variety of biophysical tools, including turbidity measurements, thioflavin T (ThT) fluorescence kinetics, tyrosine (Tyr) fluorescence study, Nile red (NR) fluorescence assay, dynamic light scattering (DLS) study, circular dichroism (CD) measurements, and isothermal titration calorimetry (ITC) techniques, we demonstrated that Pro-based polymers can significantly inhibit the insulin fibrillation process. Among them, the Pro-based homopolymer acts as the most potent inhibitor of insulin fibrillation as confirmed by ThT assay, CD study, and transmission electron microscopic (TEM) analysis. Tyrosine fluorescence measurements and NR fluorescence assay revealed that hydrophobic interactions are the crucial factor that mainly controls the inhibition process. Apart from hydrophobic interactions, polar interactions may also be responsible for the inhibition process as evaluated by ITC study.

985-P: Insulin Excipient Removal Is Counterbalanced by Insulin Fibril Formation

Objective: Insulin infusion pump sets are only FDA approved for 3 days as device failure often ensues from an inflammatory response driven by insulin excipients. However, removal of excipients can result in insulin fibril formation, which is more toxic than excipients alone. Our objective was to determine excipient and fibril toxicity and optimize the parameters of insulin excipient removal. Methods: A modified murine air pouch model with an inline filter removed excipients prior to insulin infusion/injection. Protein analysis by circular dichroism (CD) measured protein stability upon excipient removal. A separate analysis determined cytotoxicity for insulin fibrils in cell and tissue. These studies were also extended into a porcine model. Results: Excipient removal significantly reduced the inflammatory response in vivo. However, this resulted in a loss of insulin oligomeric state. In vitro and in vivo studies demonstrated toxicity of excipients (in vitro and in vivo) and fibrils (in vivo) (Figures 1A-D). Conclusions: These studies demonstrate that insulin excipients result in cell and tissue toxicity. Complete removal of these excipients alters protein stability. Since inflammation is minimized with removal of excipients, current studies are directed at optimizing the infusion parameters and excipient concentrations. Disclosure B. Lewis: None. A. Mulka: None. C. Kesserwan: None. D. Kreutzer: Other Relationship; Self; Abbott Laboratories, cell and molecualar tissue enginnering (cmte). U. Klueh: Research Support; Self; Abbott. Other Relationship; Self; Cell and Molecular Tissue Engineering, LLC. Funding The Leona M. and Harry B. Helmsley Charitable Trust

Biopolymer-coated gold nanoparticles inhibit human insulin amyloid fibrillation

Deposits of protein misfolding and/or aggregates are a pathological hallmark of amyloid-related diseases. For instance, insulin amyloid fibril deposits have been observed in patients with insulin-dependent diabetes mellitus after insulin administration. Here, we report on the use of AuNPs functionalized with linear- (i.e. dextrin and chitosan) and branched- (i.e. dextran-40 and dextran-10) biopolymers as potential agents to inhibit insulin fibril formation. Our dynamic light scattering analyses showed a size decrease of the amyloid fibrils in the presence of functionalized AuNPs. Circular dichroism spectroscopy as well as enzyme-linked immunosorbent assay data demonstrated that the secondary structural transition from α-helix to β-sheet (which is characteristic for insulin amyloid fibril formation) was significantly suppressed by all biopolymer-coated AuNPs, and in particular, by those functionalized with linear biopolymers. Both transmission electron microscopy and atomic force microscopy analyses showed that the long thick amyloid fibrils formed by insulin alone become shorter, thinner or cluster when incubated with biopolymer-coated AuNPs. Dextrin- and chitosan-coated AuNPs were found to be the best inhibitors of the fibril formation. Based on these results, we propose a mechanism for the inhibition of insulin amyloid fibrils: biopolymer-coated AuNPsstrongly interact with the insulin monomers and inhibit the oligomer formation as well as elongation of the protofibrils.Moreover, cytotoxicity experiments showed that AuNP-insulin amyloid fibrils are less toxic compared to insulin amyloid fibrils alone. Our results suggest that both dextrin- and chitosan-AuNPs could be used as therapeutic agents for the treatment of amyloid-related disorders.

Small-Molecule Inhibitor Prevents Insulin Fibrillogenesis and Preserves Activity.

Amyloidosis is a well-known but poorly understood phenomenon caused by the aggregation of proteins, often leading to pathological conditions. For example, the aggregation of insulin poses significant challenges during the preparation of pharmaceutical insulin formulations commonly used to treat diabetic patients. Therefore, it is essential to develop inhibitors of insulin aggregation for potential biomedical applications and for important mechanistic insights into amyloidogenic pathways. Here, we have identified a small molecule M1, which causes a dose-dependent reduction in insulin fibril formation. Biophysical analyses and docking results suggest that M1 likely binds to partially unfolded insulin intermediates. Further, M1-treated insulin had lower cytotoxicity and remained functionally active in regulating cell proliferation in cultured Drosophila wing epithelium. Thus, M1 is of great interest as a novel agent for inhibiting insulin aggregation during biopharmaceutical manufacturing.

Mechanism of rutin mediated inhibition of insulin amyloid formation and protection of Neuro-2a cells from fibril-induced apoptosis.

Many metabolic and neurodegenerative diseases are associated with protein misfolding and aggregation. Insulin a key hormone, under certain conditions aggregates and forms pathological amyloid fibrils. Several polyphenols have been studied extensively to elucidate their inhibitory effect on amyloid formation. In the present study, we used insulin as an amyloid model to test the mechanism and efficacy of rutin as an anti-amyloidogenic molecule. By using electron microscopy, dynamic light scattering and circular dichroism spectroscopy, we show that rutin inhibits the insulin aggregate and fibril formation. Further, rutin interacts with insulin directly and inhibits fibril formation in a dose-dependent manner as demonstrated by micro scale thermophoresis experiments. The molecular docking study predicted the potential binding pocket of rutin at the interface of chain A and chain B of insulin thereby preventing it from forming the aggregates. Since, rutin is a natural anti-oxidant, we studied its role in diminishing amyloid fibril induced cytotoxicity and apoptosis. Rutin, decreases the insulin amyloid fibrils-induced Neuro-2a cytotoxicity by reducing reactive oxygen species (ROS) levels which in turn downregulates Bax and upregulates Bcl-2 and pBad proteins. These findings suggest the potential action of rutin in preventing protein misfolding, cell death, and serves as a lead structure to design novel anti-amyloidosis compounds.