Insulin Amyloid Fibrils Research Articles

Amyloid jams: Mechanical and dynamical properties of an amyloid fibrillar network

Amyloid fibrils have well known pathological implications as well as a clear functional role in different biological systems due to their peculiar structural and mechanical properties. We had previously shown the appearance of elastic properties during the formation of a gel of insulin amyloid fibrils. Here, we study the morphological, rheological and dynamical behaviour of this jammed system. We observe different non-diffusive relaxation processes over a wide length and time interval, suggesting the formation of an elastic transient network of fibrils, and evidencing the structural heterogeneity of the gel matrix and the peculiarity of this potentially new material.

Toxicity of insulin-derived amyloidosis: a case report

BackgroundInsulin-derived amyloidosis is a skin-related complication of insulin therapy that interferes with insulin therapy. Although toxicities of in vitro-formed insulin amyloid fibrils have been well studied, the toxicity of insulin-derived amyloidosis remains to be clarified.Case presentationA 58-year-old man with type 2 diabetes mellitus underwent a lower limb amputation due to diabetic gangrene. Several antibiotics including minocycline were administered for infection and sepsis. A hard mass at the insulin injection sites in the lower abdomen was discovered by chance four months later. Although no abnormal findings in the surface skin of the mass were observed, necrotic tissue was seen around the mass when a biopsy was performed. Histological and toxicity studies were performed for this patient and four other patients with abdominal masses at insulin injection sites. Histological and immunohistochemical studies showed that the masses had typical characteristics of amyloid deposits in all cases, whereas necrotic findings were seen adjacent to the amyloid deposit only in the case presented. Toxicity studies indicated that the amyloid tissue from the present case had significant cell toxicity compared to the control skin tissue or the amyloid tissues from the other four cases.ConclusionsThis report showed that toxic insulin-derived amyloidosis can occur. In addition, this report suggested that toxic insulin-derived amyloidosis may cause necrosis in the surrounding tissue. Although the toxic amyloid deposit of insulin-derived amyloidosis was found in only one patient, no structural differences between toxic and non-toxic deposits were seen on histological and immunohistochemical studies.

Insulin amyloid fibrils-templated rational self-assembly of vine-tree-like PtRh nanocatalysts for efficient methanol electrooxidation

Shape-controlled synthesis of platinum-based alloy nanocatalysts with well-designed size and structure is essential for developing highly efficient catalysts. Here, a biomolecule-based strategy for the synthesis of vine-tree-like PtRh nanowires (VT-PtRhNWs) using insulin amyloid fibrils (INSAFs) as sacrificial templates is presented. In this structure, the inner PtRh nanowires behave as the rigid ‘tree’ and the outer PtRh nanoparticles interconnects to form a spiral chain-like structure as the flexible ‘vine’. Owning to the limitation effect of biological template, both of nanoparticles and nanowires present a very small size of less than 2 nm, and thus leading to a large electrochemical active surface area of 79.20 m2/g. As a result, the as-prepared VT-PtRhNWs exhibit a much higher catalytic activity toward methanol oxidation compared with the commercial Pt/C catalyst, and also an improved CO tolerance, indicating a great potential as excellent electrocatalysts for fuel cells.

Effects of Eugenol on Alzheimer’s Disease-like Manifestations in Insulin- and Aβ-Induced Rat Models

Eugenol is a phenylpropene having antidepressant properties. We investigated the effect of eugenol on amyloid plaques as a hallmark of Alzheimer’s disease (AD) in two AD rat models. In the latter, betaamyloid (Aβ) and insulin amyloid fibrils were injected into the rat hippocampus. After a one week lapse, the rats were treated with 0.01 and 0.02 mg/kg eugenol for two weeks. Passive avoidance learning and memory performance was assessed by measuring the step-through latency (STL). Amyloid plaques in the hippocampus were quantitatively evaluated and statistically analyzed using image processing software and ANOVA. Aβ amyloid was a more potent inducer of the signs related to Alzheimer’s disease than insulin. Eugenol at a 0.01 mg/kg dose improved memory and reduced the number of amyloid plaques significantly (P < 0.001). Possible mechanisms are proposed for the inhibitory effects of eugenol on hippocampus plaques.

Fluorescent silicon nanoparticles inhibit the amyloid fibrillation of insulin.

Amyloid fibrillation of proteins is likely a key factor leading to the development of amyloidosis-associated diseases. Inhibiting amyloid fibrillation has become a crucial therapeutic strategy. Water-soluble, fluorescent silicon nanoparticles (SiNPs) have great potential in biomedicine for various therapeutic and diagnostic purposes; however, it is unclear whether SiNPs have the ability to inhibit amyloid fibrillation. Herein, insulin was chosen as a protein model, and SiNPs of varying sizes were synthesized upon UV irradiation. The influence of size and concentration of the SiNPs on insulin fibrillation was investigated, and it has been observed that these variables were crucial in regulating insulin fibrillation. Using an average particle size of 6.6 nm and increasing the concentration of the SiNPs to 5.0 μg mL-1, the Thioflavin T (ThT) fluorescence intensity decreased significantly by 90%, with an increased lag time of 76.8 h, compared to that of the control. Insulin aggregates were short, thin fibrils or clusters when incubated with SiNPs, compared to the long, thick fibrils formed for insulin alone. Additionally, we found that the SiNPs prevent the conformational transition of insulin from its initial structure to β-sheets, and thus inhibit nucleation, which is necessary for the formation of large fibrils. The inhibitory activity is attributed to the interactions between the SiNPs and insulin during the nucleation period. Our results demonstrate that the SiNPs disrupt insulin amyloid fibrillation, and thus, may play a useful role in new therapeutic and diagnostic strategies for amyloid-related disorders.

A molecular rotor-based turn-on sensor probe for amyloid fibrils in the extreme near-infrared region.

A fluorescence turn-on probe for amyloid detection in the extreme near-infrared region (>750 nm) is a highly desirable technological evolution from the view point of potential in vivo applications. Herein, we report a molecular rotor-based amyloid sensor probe which, on binding to the insulin amyloid fibril, registers a large turn-on emission in the near-infrared region, and records an exceptionally large red-shifted emission wavelength of ∼770 nm along with a Stokes' shift of ∼150 nm, the highest reported to date for any amyloid sensor probe, in the insulin fibril bound form, in the near-infrared region. Importantly, when bound to insulin fibrils, this probe also exhibits an exceptionally large red-shift of ∼120 nm in the absorption spectra, which enables the naked eye in vitro detection of amyloid fibrils.

Three-Step Resonance Energy Transfer in Insulin Amyloid Fibrils

The applicability of the three-step Förster resonance energy transfer (FRET) to detection of insulin amyloid fibrils was evaluated, using the chromophore system, containing Thioflavin T (ThT), 4-dimethylaminochalcone (DMC), and two squaraine dyes, referred to here as SQ1 and SQ4. The mediator chromophore DMC was found to enhance the fluorescence intensity of the terminal acceptor, SQ1, excited at 440 nm (at the absorption maximum of the principal donor, ThT), in fibrillar insulin compared to the system without DMC, providing the evidence for the cascade energy transfer in the chain ThT→DMC→SQ4→SQ1. Furthermore, the resulting Stokes shift in the four-chromophore system was 240 nm, as compared to 45 nm for the fibril-bound ThT, suggesting that higher signal-to-noise ratio is the advantage of amyloid fibril detection by multistep FRET. The maximum efficiencies of energy transfer in the insulin fibrils estimated from the quenching of the donor fluorescence in the presence of acceptor for the donor-acceptor pairs ThT-DMC, DMC-SQ4 and SQ4-SQ1 were 40%, 60% and 30% respectively, while negligible FRET occurred in the non-fibrillized protein. The most pronounced differences between fibrillar and non-fibrillized insulin were observed in the 3D fluorescence spectra. Specifically, two intensive spots centered at the emission wavelengths ~ 650 nm (SQ4) and ~ 685 nm (SQ1) were revealed at the excitation wavelength ~ 440 nm in the 3D patterns of insulin amyloid aggregates. In contrast, in the case of the non-fibrillized protein, the barely noticeable spots centered at the same wavelengths, as well as higher fluorescence intensities at the excitation above 550 nm were observed, suggesting the predominant impact of the direct excitation of SQ1 and SQ4 on their fluorescence responses. The inter-chromophore distances calculated from the experimental values of the energy transfer efficiency assuming the isotropic rotation of the dyes, were found to be 2.4, 4.5 and 4.3 nm for the ThT-DMC, DMC-SQ4 and SQ4-SQ1 pairs, respectively, revealing the different fibril binding sites for the examined dyes. The quantum-chemical calculations and simple docking studies provided evidence for the SQ1, SQ4 and ThT, DMC binding to the wet and dry interface of the insulin amyloid protofilament, respectively. The dye-protein complexes are likely to be stabilized by the hydrophobic, van der Waals, aromatic and electrostatic interactions. In summary, the above technique based on the multistep FRET can be employed for the identification and characterization of amyloid fibrils in vitro along with the classical ThT assay, allowing the increase of the amyloid detection sensitivity and lowering the probability of the pseudo-positive result. The applicability of the multistep FRET for amyloid visualization in vivo can be also tested by the involvement of the near-infrared fluorescent dyes to the cascade.

Extreme Adhesion Activity of Amyloid Fibrils Induces Subcutaneous Insulin Resistance.

Insulin-derived amyloidoma, also called an insulin ball, is a skin-related complication of insulin therapy caused by repeated insulin injections at the same site, where native folded insulin changes into amyloid fibrils and forms a mass with a granulomatous reaction. Insulin-derived amyloidoma is a clinically important condition because of its association with subcutaneous insulin resistance, but the precise effect and mechanism of the insulin absorption impairment have not been clarified. We generated insulin-derived amyloidomas in mouse skin, with the amyloidomas large enough to perform insulin tolerance tests in the mass by repeated injections of highly concentrated insulin amyloid fibrils. We demonstrated that the insulin-derived amyloidomas inhibit insulin absorption. By simultaneous administration of insulin and insulin amyloid fibrils, we showed that this effect is due to the amyloid fibril itself in the absence of a granulomatous reaction. In vitro studies revealed that insulin amyloid fibrils have extremely strong adhesion to native human insulin and various insulin analogs. Furthermore, we showed that native insulin that had adhered to insulin amyloid forms amyloid fibrils at physiological pH. These results suggest that the extreme adhesion of insulin amyloid to native insulin is the main mechanism of impaired insulin absorption and amyloidoma growth.

Dual effect of non-ionic detergent Triton X-100 on insulin amyloid formation.

Atomic force microscopy, Thioflavin T (ThT) fluorescence assay, circular dichroism spectroscopy, differential scanning calorimetry, and molecular modeling techniques have been employed to investigate the amyloid aggregation of insulin in the presence of non-ionic detergent, Triton X-100 (TX-100). In contrast to recently described inhibition of lysozyme amyloid formation by non-ionic detergents (Siposova, 2017), the amyloid aggregation of insulin in the presence of sub-micellar TX-100 concentration exhibits two dissimilar phases. The first, inhibition phase, is observed at the protein to detergent molar ratio of 1:0.1 to 1:1. During this phase, the insulin amyloid fibril formation is inhibited by TX-100 up to ∼60%. The second, "morphological" phase, is observed at increasing detergent concentration, corresponding to protein:detergent molar ratio of ∼1:1 - 1:10. Under these conditions a significant increase of the steady-state ThT fluorescence intensities and a dramatically changed morphology of the insulin fibrils were observed. Increasing of the detergent concentration above the CMC led to complete inhibition of amyloidogenesis. Analysis of the experimental and molecular modeling results suggests an existence of up to six TX-100 binding sites within dimer of insulin with different binding energy. The physiological relevance of the results is discussed.

Protective effects of silibinin on insulin amyloid fibrillation, cytotoxicity and mitochondrial membrane damage

A growing body of evidence suggests that secretion and assembly of insulin to amyloid fibrils reduce its efficacy in treating type II diabetes and may lead to dysfunctioning of several organs. The research presented here explores the effects of silibinin on the in vitro amyloid fibrillation and cytotoxicity of bovine insulin fibrils on SH-SY5Y human neuroblastoma cells. Interaction of the resulting structures with rat brain mitochondria was also investigated. Using a range of methods for amyloid detection we showed that insulin fibrillation was significantly inhibited by silibinin in a dose-dependent fashion. Moreover, we found that silibinin was very effective in attenuating insulin fibril-induced neuronal toxicity characterized by decrease of cell viability, the release of lactate dehydrogenase, intracellular reactive oxygen species enhancement, morphological alterations, and apoptotic cell death induction. While insulin fibrillation products showed the capacity to damage mitochondria, the resultant structures produced in the presence of silibinin were totally ineffective. Together, results demonstrate the capacity of insulin fibrils to cause SH-SY5Y cell death by inducing necrosis/apoptosis changes and suggest how silibinin may afford protection. It is concluded that elucidation of such protection may provide important insights into the development of preventive and therapeutic agents for amyloid-related diseases.

Destroying activity of glycine coated magnetic nanoparticles on lysozyme, α-lactalbumin, insulin and α-crystallin amyloid fibrils

Abstract A great variety of human protein deposition and protein aggregation diseases (Alzheimer’s disease, diabetes mellitus, cataract, systemic amyloidosis and other) have been associated with the accumulation of amyloid fibrils in different tissues. Therefore, development of the agents able to reduce amyloid deposits represents an attractive strategy for their treatment. We have investigated ability of glycine coated magnetic nanoparticles (Gly-MNPs) to destroy protein amyloid fibrils. The properties of Gly-MNPs were characterized with the aim to identify the optimized conditions for the glycine adsorption on surface of MNPs. It was found that Gly-MNPs have superparamagnetic behavior and their size, isoelectric point and stability depend on the amount of the glycine in the samples. The obtained results suggest that optimal weight ratio (w/w) for the modification of MNPs by glycine (Gly/Fe3O4) is equal to 5/1. The selected Gly5-MNPs1 were used for the study of their effect on amyloid fibrils of four globular proteins, namely lysozyme, bovine α-lactalbumin, insulin and α-crystallin. It was found that Gly5-MNPs1 destroy lysozyme, α-lactalbumin and insulin amyloid fibrils in concentration dependence manner. However, Gly5-MNPs1 were not able significantly destroy bovine α-crystallin amyloid fibrils. We assume that obtained results represent important contribution for rational design of potential therapeutics of amyloid diseases based on nanoparticles.

Immobilization of tobacco etch virus (TEV) protease on a high surface area protein nanofibril scaffold.

Tobacco etch virus (TEV) protease is widely used for the removal of poly-histidine affinity tags from proteins. In solution, it is a one-time use enzyme for tag cleavage that has low stability, and is therefore a good candidate for immobilization. Amyloid fibrils can act as a versatile nanoscaffold by providing a large surface area for biomolecule immobilization. Immobilization of TEV protease to amyloid fibrils grown from the surface of a small glass bead, using physisorption, successfully immobilized active TEV protease. The bead retained activity over several uses and successfully cleaved a poly-histidine tag from several his-tagged proteins. This is first time that TEV protease has been immobilized to insulin amyloid fibrils, or any protein based support. Such functionalized surface assembled amyloid fibrils show promise as a novel nanosupport for the creation of functional bionanomaterials, for example, active surface coatings for the production of fine chemicals, chemical detoxification, or biosensing. Insulin amyloid fibrils provide a new nanosupport for the immobilization of TEV protease, which could allow for the reuse of the enzyme, saving on production costs for recombinantly expressed poly-histidine tagged proteins. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1506-1512, 2018.

Metal-enhanced fluorescence of the trimethine cyanine dyes complexed with amyloid fibrils

In this work, we present the investigation of metal enhancement effect of silver island films (SIF) on the fluorescence intensity and correspondingly reporting properties of amyloid- sensitive trimethine cyanine dyes. The benzothiazole dyes zwitterionic D-51 and multiply positively charged D-151 as well as their complexes with amyloid fibrils of proteins insulin and lysozyme were examined. The obtained data revealed that the presence of SIF causes aggregation of the positively charged dye D-151 and the destruction of the dye-fibril complex. That could be explained by electrostatic interactions between multiply charged molecules and electrically polarized silver islands. For the zwitterionic dye D-51, the presence of SIF caused metal enhancement of fluorescence emission intensity in 5.2, 6 and 3.4 times for the free dye and its complexes with fibrils of insulin and lysozyme respectively. Thus metal enhancement as emission intensity enhancement method offers a potential platform to increase the sensitivity of probes detecting protein amyloid fibrils.

Effect of fibrillation conditions on the anti-amyloidogenic properties of polyphenols and their involved mechanisms

In the present study, we have investigated the effects of protein concentration and stirring on the in vitro assembly of Hen Egg White Lysozyme (HEWL), particularly with regard to the aggregate morphology and anti-amyloidogenic properties of two naturally occurring polyphenols, taxifolin and silibinin. The results obtained clearly demonstrated that applying stirring and concentration enhancement alter the amount as well as morphology of amyloid fibrils formed. Additionally, latter aggregates exhibited higher affinity for amyloid-specific dyes. The second part of the present investigation was devoted to studies involving anti-amyloidogenic properties of selected polyphenols. Importantly, we found that the potency of polyphenols to inhibit HEWL amyloid fibrillation and related toxicity is strongly dependent on the amyloidogenic conditions in which amyloid fibrils are produced. Based on obtained data, under condition where the rate of protein assembly is high (higher protein concentration and stirring), the capacity of polyphenols to inhibit HEWL fibrillogenesis and related cytotoxicity may dramatically decrease. Similar results were obtained when we used taxifolin to inhibit bovine insulin amyloid fibrillation. Additionally, amyloidogenic conditions may also affect the mechanism by which these molecules inhibit HEWL fibrillation. The possible mechanism by which selected polyphenols exert their inhibitory effects, under various experimental conditions, is also discussed.

Trans-2-[4-(dimethylamino)styryl]-3-ethyl-1,3-benzothiazolium perchlorate - New fluorescent dye for testing of amyloid fibrils and study of their structure

We synthesized the benzothiazole dye trans-2-[4-(dimethylamino)styryl]-3-ethyl-1,3-benzothiazolium perchlorate (DMASEBT) that is a new analogue of the widely used for amyloid fibrils detection fluorescent probe thioflavin T (ThT). The spectra of DMASEBT are shifted about 100 nm closer to the "transparent window" of biological tissues than that of ThT. Quantum-chemical calculations proved the molecular rotor nature of DMASEBT that underlies the similarity of its photophysical properties with ThT: high level of fluorescence anisotropy in aqueous solutions; fluorescence intensity and lifetime increase during their binding to amyloid fibrils. Using equilibrium microdialysis for sample preparation, two modes of DMASEBT binding to insulin amyloid fibrils, characterized by the different affinity and stoichiometry of interaction, as well as photophysiсal characteristics of the bound to fibrils dye were detected. The obtained results proved the change in DMASEBT photophysical properties when the dye binds to amyloid fibrils (what is critical point for a fluorescent probe), similar to ThT mechanism of the dye binding to fibrils and its improved spectral properties in comparison to ThT. These points make DMASEBT a perspective instrument for amyloid fibrils testing and examining their structure.

Capreomycin inhibits the initiation of amyloid fibrillation and suppresses amyloid induced cell toxicity

Protein aggregation and amyloid fibrillation are responsible for several serious pathological conditions (like type II diabetes, Alzheimer's and Parkinson's diseases etc.) and protein drugs ineffectiveness. Therefore, a molecule that can inhibit the amyloid fibrillation and potentially clear amyloid fibrils is of great therapeutic value. In this manuscript, we investigated the antiamyloidogenic, fibril disaggregating, as well as cell protective effect of an anti-tuberculosis drug, Capreomycin (CN). Aggregation kinetics data, as monitored by ThT fluorescence, inferred that CN retards the insulin amyloid fibrillation by primarily targeting the fibril elongation step with little effect on lag time. Increasing the dose of CN boosted its inhibitory potency. Strikingly, CN arrested the growth of fibrils when added during the elongation phase, and disaggregated mature insulin fibrils. Our Circular Dichroism (CD) results showed that, although CN is not able to maintain the alpha helical structure of protein during fibrillation, reduces the formation of beta sheet rich structure. Furthermore, Dynamic Light Scattering (DLS) and Transmission Electronic Microscopy (TEM) analysis confirmed that CN treated samples exhibited different size distribution and morphology, respectively. In addition, molecular docking results revealed that CN interacts with insulin through hydrophobic interactions as well as hydrogen bonding, and the Hemolytic assay confirmed the non-hemolytic activity of CN on human RBCs. For future research, this study may assist in the rational designing of molecules against amyloid formation.

Cyanidin-3-rutinoside reduces insulin fibrillation and attenuates insulin fibrils-induced oxidative hemolysis of human erythrocytes

Insulin is able to form amyloid-like fibrils, a misfolding process by which insulin molecules interact with each other to form aggregates and pathological amyloid deposition. Inhibition of amyloid aggregation using natural products is proposed as a new strategy to prohibit the development of amyloid diseases. Herein, we demonstrated the inhibitory effect of cyanidin-3-rutinoside (C3R), a natural anthocyanin with multiple biological activities, against insulin amyloid fibrillation. The results showed that increased insulin concentration resulted in faster growth and higher amounts of insulin fibrils. C3R (10.6–170μM) concentration dependently decreased insulin fibril growth and increased the duration of lag time of insulin fibril formation. Moreover, C3R directly decreased the secondary structure transition from α-helix to β-sheet structure. C3R (0.31–5μM) attenuated insulin fibrils-induced oxidative hemolysis of human erythrocytes in a concentration-dependent manner. Moreover, C3R reduced insulin fibrils-induced erythrocyte membrane disruption through the inhibition of reactive oxygen species (ROS) generation. The findings also suggest that C3R reduced fibrils-induced membrane lipid peroxidation by maintaining the catalase activity and oxidized/reduced glutathione content (GSH/GSSH) in erythrocytes. These findings suggest that C3R may serve as a potential inhibitory agent against amyloid fibril formation and insulin fibrils-induced oxidative hemolysis.

Amino group of salicylic acid exhibits enhanced inhibitory potential against insulin amyloid fibrillation with protective aptitude toward amyloid induced cytotoxicity.

Protein misfolding and aggregation lead to amyloid generation that in turn may induce cell membrane disruption and leads to cell apoptosis. In an effort to prevent or treat amyloidogenesis, large number of studies has been paying attention on breakthrough of amyloid inhibitors. In the present work, we aim to access the effect of two drugs, that is, acetylsalicylic acid and 5-amino salicylic acid on insulin amyloids by using various biophysical, imaging, cell viability assay, and computational approaches. We established that both drugs reduce the turbidity, light scattering and fluorescence intensity of amyloid indicator dye thioflavin T. Premixing of drugs with insulin inhibited the nucleation phase and inhibitory potential was boosted by increasing the concentration of the drug. Moreover, addition of drugs at the studied concentrations attenuated the insulin fibril induced cytotoxicity in breast cancer cell line MDA-MB-231. Our results highlight the amino group of salicylic acid exhibited enhanced inhibitory effects on insulin fibrillation in comparison to acetyl group. It may be due to presence of amino group that helps it to prolong the nucleation phase with strong binding as well as disruption of aromatic and hydrophobic stacking that plays a key role in amyloid progression.

A sensitive probe for amyloid fibril detection with strong fluorescence and early response.

We synthesized a new probe, 4-[2-(2-naphthyl)-(E)-ethenyl]-benzyl(triphenyl)phosphonium bromide (NEB), to detect the formation of amyloid fibrils of bovine insulin. The fluorescence intensity of NEB in the presence of insulin fibrils was 30 times higher than that before fibrillation, with the fluorescence quantum yield increased from 2.5% to 78%. In comparison with the commercially available probe, thioflavin T (ThT), NEB exhibits a 10 times stronger fluorescence and a shorter identification lag phase for detecting insulin fibrillation, indicating a higher sensitivity in detection of insulin oligomers and fibrils.

Inhibition of insulin amyloid fibrillation by Morin hydrate

We report here the inhibition of amyloid fibrillation of human insulin in vitro by Morin hydrate, a naturally occurring small molecule. Using spectroscopic assays and transmission electron microscopy, we found that Morin hydrate effectively inhibits insulin amyloid fibrillation in a dose dependent manner with more than 80% inhibition occurring even at only a 1:1 concentration. As suggested by fluorescence spectroscopic titration studies, Morin hydrate binds to insulin with a fairly strong affinity of -26.436kJmol−1. Circular dichroism (CD) spectroscopy was used to analyse structural changes of insulin in the presence of Morin hydrate demonstrating the ability of Morin hydrate to bind with the native monomeric protein and/or its near native state, intermediate oligomeric species and amyloid fibrils. Based on computational docking and molecular dynamics study, we propose that Morin hydrate binds to residues having greater aggregation propensity and prevent structural and/or conformational changes leading to amyloid fibrillation. Morin hydrate should also bind to fibrils by hydrogen bonding and/or hydrophobic forces throughout the surface, stabilize them and inhibit the release of oligomeric species which could be nuclei or template for further fibrillation. Overall results provide an insight into the mechanism of inhibition of insulin amyloid fibrillation by Morin hydrate.