Exploration of Isoquinoline Alkaloids as Potential Inhibitors against Human Islet Amyloid Polypeptide.

Type-2 diabetes mellitus (T2DM) is one of the most concerning public health problems because of its high incidence, multiple complications, and difficult treatment. Human islet amyloid polypeptide (hIAPP) is closely linked to T2DM because its abnormal self-assembly causes membrane damage and cell dysfunction. The development of potential inhibitors to prevent hIAPP fibrillation is a promising strategy for the intervention and treatment of diabetes. Natural isoquinoline alkaloids are used as effective medication that targets different biomolecules. Although studies explored the efficacy of berberine, jatrorrhizine, and chelerythrine in diabetes, the underlying mechanism remains unclear. Herein, three isoquinoline alkaloids are selected to reveal their roles in hIAPP aggregation, disaggregation, and cell protection. All three compounds displayed good inhibitory effects on peptide fibrillation, scattered the preformed fibrils into small oligomers and most monomers, and upregulated cell viability by reducing hIAPP oligomerization. Moreover, combined biophysical analyses indicated that the compounds affected the ß-sheet structure and hydrophobicity of polypeptides significantly, and the benzo[c]phenanthridine structure of chelerythrine was beneficial to the inhibition of hIAPP aggregation and their hydrophobic interaction, compared with that of berberine and jatrorrhizine. Our work elaborated the effects of these alkaloids on hIAPP fibrillation and reveals a possible mechanism for these compounds against T2DM.

Substoichiometric Inhibition of Insulin against IAPP Aggregation Is Attenuated by the Incompletely Processed N-Terminus of proIAPP.

Substoichiometric aggregation inhibition of human islet amyloid polypeptide (IAPP), the hallmark of type 2 diabetes impacting millions of people, is crucial for developing clinic therapies, yet it remains challenging given that many candidate inhibitors require high doses. Intriguingly, insulin, the key regulatory polypeptide on blood glucose levels that are cosynthesized, costored, and cosecreted with IAPP by pancreatic ß cells, has been identified as a potent inhibitor that can suppress IAPP amyloid aggregation at substoichiometric concentrations. Here, we computationally investigated the molecular mechanisms of the substoichiometric inhibition of insulin against the aggregation of IAPP and the incompletely processed IAPP (proIAPP) using discrete molecular dynamics simulations. Our results suggest that the amyloid aggregations of both IAPP and proIAPP might be disrupted by insulin through its binding with the shared amyloidogenic core sequences. However, the N-terminus of proIAPP competed with the amyloidogenic core sequences for the insulin interactions, resulting in attenuated inhibition by insulin. Moreover, insulin preferred to bind the elongation surfaces of IAPP seeds with fibril-like structure, with a stronger affinity than that of IAPP monomers. The capping of elongation surfaces by a small amount of insulin sterically prohibited the seed growth via monomer addition, achieving the substoichiometric inhibition. Together, our computational results provided molecular insights for the substoichiometric inhibition of insulin against IAPP aggregation, also the weakened effect on proIAPP. The uncovered substoichiometric inhibition by capping the elongation of amyloid seeds or fibrils may guide the rational designs of new potent inhibitors effective at low doses.

Anti-diabetic activity of a sulfated galactoarabinan with unique structural characteristics from Cladophora oligoclada (Chlorophyta).

The polysaccharide from green alga Cladophora oligoclada, OHSS2, was a sulfated galactoarabinan which was constituted by a backbone of (1 â†’ 4)-ß-l-arabinopyranose units with partial sulfate at C-3 of (1 â†’ 4)-ß-l-arabinopyranose units. The side chains containing (1 â†’ 4)-ß-l-arabinopyranose, (1 â†’ 4)-ß-d-galactopyranose and/or (1 â†’ 4,6)-ß-d-galactopyranose units were in C-2/C-3 of (1 â†’ 4)-ß-l-arabinopyranose units. OHSS2 had strong anti-diabetic activity in vitro assessed by inhibition of human islet amyloid polypeptide (hIAPP) aggregation. The mechanism analysis of anti-diabetic activity showed that OHSS2 diminished the production of intracellular reactive oxygen species and alleviated hIAPP aggregation-induced oxidative stress in NIT-1 cells. OHSS2 stabilized mitochondrial membrane potential, and enhanced the mitochondrial complex I, II or III activity and ATP level. Thus, OHSS2 effectively protected mitochondria from hIAPP aggregation-induced damage. Furthermore, OHSS2 was co-localized with mitochondria and could have a direct influence on mitochondrial function. These results revealed that OHSS2 had potential as a novel anti-diabetic agent.

Inhibition of amyloid formation of amyloid ß (1-42), amylin and insulin by 1,5-diazacyclooctanes, a spermine-acrolein conjugate.

Amyloid aggregates of proteins are known to be involved in various diseases such as Alzheimer's disease (AD). It is therefore speculated that the inhibition of amyloid formation can play an important role in the prevention of various diseases involving amyloids. Recently, we have found that acrolein reacts with polyamines, such as spermine, and produces 1,5-diazacyclooctane, such as cyclic spermine (cSPM). cSPM could suppress the aggregation of amyloid ß 1-40 (Aß40), one of the causative proteins of AD. This result suggests the potential inhibitory effect of cSPM against Aß 1-42 (Aß42) and other amyloid protein aggregation which are the main pathological features of AD and other diseases. However, the effect on the aggregation of such proteins remains unclear. In this study, the effect of cSPM on the amyloid formation of Aß42, amylin, and insulin was investigated. These three amyloidogenic proteins forming amyloids under physiological conditions (pH 7.4 and 37℃) served as model and are thought to be the causative proteins of AD, type 2 diabetes, and insulin-derived amyloidosis, respectively. Our results indicate that cSPM can suppress the amyloid aggregation of these proteins and reduce cytotoxicity. This study contributes to a better understanding of means to potentially counteract diseases by the means of polyamine and acrolein.

Development of Cagrilintide, a Long-Acting Amylin Analogue.

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

Mechanisms of Transthyretin Inhibition of IAPP Amyloid Formation.

Amyloid-formation by the islet amyloid polypeptide (IAPP), produced by the ß-cells in the human pancreas, has been associated with the development of type II diabetes mellitus (T2DM). The human plasma-protein transthyretin (TTR), a well-known amyloid-inhibiting protein, is interestingly also expressed within the IAPP producing ß-cells. In the present study, we have characterized the ability of TTR to interfere with IAPP amyloid-formation, both in terms of its intrinsic stability as well as with regard to the effect of TTR-stabilizing drugs. The results show that TTR can prolong the lag-phase as well as impair elongation in the course of IAPP-amyloid formation. We also show that the interfering ability correlates inversely with the thermodynamic stability of TTR, while no such correlation was observed as a function of kinetic stability. Furthermore, we demonstrate that the ability of TTR to interfere is maintained also at the low pH environment within the IAPP-containing granules of the pancreatic ß-cells. However, at both neutral and low pH, the addition of TTR-stabilizing drugs partly impaired its efficacy. Taken together, these results expose mechanisms of TTR-mediated inhibition of IAPP amyloid-formation and highlights a potential therapeutic target to prevent the onset of T2DM.

Identification of Crocin as a New hIAPP Amyloid Inhibitor via a Simple Yet Highly Biospecific Screening System.

Amylin (hIAPP) amyloid formation plays an important role in the pathogenesis of type 2 diabetes (T2D), which makes it a promising therapeutic target for T2D. In this study, we established a screening tool for identifying chemicals affecting hIAPP amyloid formation based on a reported genetic tool, which constantly tracks protein aggregates in Saccharomyces cerevisiae. In order to obtain the hIAPP with better aggregation ability, the gene of hIAPP was tandemly ligated to create 1×, 2×, 4× or 6×-hIAPP expressing strains. By measuring the cell density and fluorescence intensity of green fluorescent protein (GFP) regulated by the aggregation status of hIAPP, it was found that four intramolecular ligated hIAPP (4×hIAPP) could form obvious amyloids with mild toxicity. The validity and reliability of the screening tool were verified by testing six reported hIAPP inhibitors, including curcumin, epigallocatechin gallate and so on. Combined with surface plasmon resonance (SPR) and the screening tool, which could be a screening system for hIAPP inhibitors, we found that crocin specifically binds to hIAPP and acts inhibit amyloid formation of hIAPP. The effect of crocin was further confirmed by Thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM) analysis. Thus, a screening system for hIAPP amyloid inhibitors and a new mechanism of crocin on anti-T2D were obtained as a result of this study.

Myricetin protects pancreatic ß-cells from human islet amyloid polypeptide (hIAPP) induced cytotoxicity and restores islet function.

The aberrant misfolding and self-assembly of human islet amyloid polypeptide (hIAPP)-a hormone that is co-secreted with insulin from pancreatic ß-cells-into toxic oligomers, protofibrils and fibrils has been observed in type 2 diabetes mellitus (T2DM). The formation of these insoluble aggregates has been linked with the death and dysfunction of ß-cells. Therefore, hIAPP aggregation has been identified as a therapeutic target for T2DM management. Several natural products are now being investigated for their potential to inhibit hIAPP aggregation and/or disaggregate preformed aggregates. In this study, we attempt to identify the anti-amyloidogenic potential of Myricetin (MYR)- a polyphenolic flavanoid, commonly found in fruits (like Syzygium cumini). Our results from biophysical studies indicated that MYR supplementation inhibits hIAPP aggregation and disaggregates preformed fibrils into non-toxic species. This protection was accompanied by inhibition of oxidative stress, reduction in lipid peroxidation and the associated membrane damage and restoration of mitochondrial membrane potential in INS-1E cells. MYR supplementation also reversed the loss of functionality in hIAPP exposed pancreatic islets via restoration of glucose-stimulated insulin secretion. Molecular dynamics simulation studies suggested that MYR molecules interact with the hIAPP pentameric fibril model at the amyloidogenic core region and thus prevents aggregation and distort the fibrils.

Synthesis of Yakuchinone B-Inspired Inhibitors against Islet Amyloid Polypeptide Aggregation.

Type 2 diabetes mellitus (T2DM) is associated with pancreatic ß-cell dysfunction and insulin resistance. Islet amyloid polypeptide (IAPP) aggregation is found to induce islet ß-cell death in T2DM patients. Recently, we demonstrated that yakuchinone B derivative 1 exhibited inhibitory activity against IAPP aggregation. Thus, in this study, a series of synthesized yakuchinone B-inspired compounds were tested for their anti-IAPP aggregation activity. Four of these compounds, 4e-h, showed greater activity than the lead compound 1, in the sub-µM range (IC50 = 0.7-0.8 µM). The molecular docking analysis revealed crucial hydrogen bonds between the compounds and residues S19 and N22 and important hydrophobic interactions with residue I26. Notably, compounds 4g and 4h significantly protected ß-cells against IAPP-induced toxicity with EC50 values of 0.1 and 0.2 µM, respectively. In contrast, the protective activities of compounds 4e and 4f were weak. Overall, these results suggest that the compounds exhibiting IAPP aggregation-inhibiting activity have the potential to treat T2DM.

Washingtonia filifera seed extracts inhibit the islet amyloid polypeptide fibrils formations and α-amylase and α-glucosidase activity.

Washingtonia filifera seeds have revealed to possess antioxidant properties, butyrylcholinesterase and xanthine oxidase inhibition activities. The literature has indicated a relationship between Alzheimer's disease (AD) and type-2 diabetes (T2D). Keeping this in mind, we have now evaluated the inhibitory properties of W. filifera seed extracts on α-amylase, α-glucosidase enzyme activity and the Islet Amyloid Polypeptide (IAPP) fibrils formation. Three extracts from seeds of W. filifera were evaluated for their enzyme inhibitory effect and IC50 values were calculated for all the extracts. The inhibition mode was investigated by Lineweaver-Burk plot analysis and the inhibition of IAPP aggregate formation was monitored. W. filifera methanol seed extract appears as the most potent inhibitor of α-amylase, α-glucosidase, and for the IAPP fibril formation. Current findings indicate new potential of this extract that could be used for the identification or development of novel potential agents for T2D and AD.

Small molecule induced toxic human-IAPP species characterized by NMR.

In this study, the effect of CurDAc, a water-soluble curcumin derivative, on the formation and stability of amyloid fibers is revealed. CurDAc interaction with amyloid is structurally selective, which is reflected in a strong interference with hIAPP aggregation while showing weaker interactions with human-calcitonin and amyloid-ß1-40 in comparison. Remarkably, CurDAc also exhibited potent fiber disaggregation for hIAPP generating a toxic oligomeric species.

Role of lysine residue of islet amyloid polypeptide in fibril formation, membrane binding, and inhibitor binding.

The aggregation of islet amyloid polypeptide (IAPP) is implicated in the pathogenesis of type 2 diabetes (T2D). In T2D, this peptide aggregates to form amyloid fibrils; the mechanism responsible for islet amyloid formation is unclear. However, it is known that the aggregation propensity of IAPP is highly related to its primary sequence. Several residues have been suggested to be critical in modulating IAPP amyloid formation, but role of the sole lysine residue at position 1 (Lys-1) in IAPP has not been discussed. In our previous study, we found that glycated IAPP can form amyloid faster than normal IAPP and induce normal IAPP to expedite the aggregation process. To gain more insight into the contribution of Lys-1 in the kinetics of fibril formation, we synthesized another two IAPP variants, K1E-IAPP and K1Nle-IAPP, in which the Lys residue was mutated to glutamate and norleucine, respectively. Interestingly, we observed that the negative or neutral charged side chain at this position was preferred for amyloid formation. The findings suggested this residue may take part in the inter- or intra-molecular interaction during IAPP aggregation, even though it was proposed not to be in part of fibril core structure. Our data also revealed that the inhibitory mechanism of some inhibitors for IAPP aggregation require reaction with Lys-1. Modifications of Lys-1, such as protein glycation, may affect the effectiveness of the inhibitory action of some potential drugs in the treatment of amyloidosis.

Cryo-EM structure and inhibitor design of human IAPP (amylin) fibrils.

Human islet amyloid polypeptide (hIAPP) functions as a glucose-regulating hormone but deposits as amyloid fibrils in more than 90% of patients with type II diabetes (T2D). Here we report the cryo-EM structure of recombinant full-length hIAPP fibrils. The fibril is composed of two symmetrically related protofilaments with ordered residues 14-37. Our hIAPP fibril structure (i) supports the previous hypothesis that residues 20-29 constitute the core of the hIAPP amyloid; (ii) suggests a molecular mechanism for the action of the hIAPP hereditary mutation S20G; (iii) explains why the six residue substitutions in rodent IAPP prevent aggregation; and (iv) suggests regions responsible for the observed hIAPP cross-seeding with ß-amyloid. Furthermore, we performed structure-based inhibitor design to generate potential hIAPP aggregation inhibitors. Four of the designed peptides delay hIAPP aggregation in vitro, providing a starting point for the development of T2D therapeutics and proof of concept that the capping strategy can be used on full-length cryo-EM fibril structures.

Inhibitory activities of phenylpropanoids from Lycopus lucidus on amyloid aggregation related to Alzheimer's disease and type 2 diabetes.

The number of patients with Alzheimer's disease (AD) and type 2 diabetes (T2D) is increasing rapidly, and thus more research has been focused on the relationship between these two age-related chronic diseases. According to the amyloid hypothesis, prevention of the aggregation of amyloid ß (Aß) and human islet amyloid polypeptide (hIAPP) is a promising strategy for AD and T2D. In this study, thioflavin-T assay and transmission electron microscopy were performed to evaluate the inhibitory effect of three phenylpropanoids isolated from Lycopus lucidus-schizotenuin A and lycopic acids A and B-on both Aß and hIAPP fibrillization. All tested compounds exhibited similarly strong inhibitory activity toward amyloid aggregation. These results suggested that catechol moieties play important roles in the inhibition of amyloid plaque formation.

Cichoric acid from witloof inhibit misfolding aggregation and fibrillation of hIAPP.

The misfolding, aggregation and fibrillation of human islet amyloid polypeptide (hIAPP) has been acknowledged as a hallmark event in type-II diabetes. Hence, inhibiting the misfolding, aggregation and fibrillation of hIAPP have been accepted as a vital factor to treat the disease. Here cichoric acid was extracted from witloof to explore its inhibition effects on misfolding, aggregation and fibrillation of hIAPP. Thioflavin-T (ThT) fluorescence assay, dynamic light scattering (DLS) and atomic force microscopy (AFM) images showed that cichoric acid inhibited the aggregation and fibrillation of hIAPP in a dosage-dependent manner. Circular dichroism (CD) spectra showed that cichoric acid inhibited the misfolding of hIAPP from unfolded to ß-sheet. Molecular docking and further experiments revealed interactions between hIAPP and cichoric acid. Cichoric acid could bind to K1 and R11 of hIAPP via electrostatic interaction. In addition, cichoric acid could form π-π stacking with hIAPP residues F15 and F23. These interactions inhibited the misfolding, aggregation and fibrillation of hIAPP. These results, together with cichoric acid's good cytocompatibility and significant protective effects in hIAPP lesioned cell models, not only showed that cichoric acid could be used to fight against amyloidosis, but also brought a new perspective for Chinese herbal medicine as natural compound's medical potential.

Deformation of stable and toxic hIAPP oligomers by liposomes with distinct nanomechanical features and reduced cytotoxicity.

Human islet amyloid polypeptide (hIAPP) oligomers are transient due to rapid aggregation rate in vitro, but play an important role in the pathogenesis of type 2 diabetes mellitus (T2DM). Here we report an easy and robust method to generate toxic hIAPP oligomers, which are stable for at least 8 hours. The toxic hIAPP oligomers are quickly transformed from α-helix to ß-sheet by membrane phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes, exhibiting distinct nanomechanical features from the hIAPP oligomers or pristine fibrils. DOPC liposomes significantly block the cytotoxicity induced by the hIAPP oligomers, which has the potential for new treatment.

Quantification of protein aggregation rates and quenching effects of amylin-inhibitor complexes.

The formation of amyloid aggregates is the hallmark of many protein misfolding diseases, including Type-II diabetes mellitus, which is caused by the fibrillation of amylin protein. It is established that nano-sized ligands such as curcumin, resveratrol and graphene quantum dots can modify protein aggregation rates. In this article, we report a comparative study of these ligands to estimate their protein aggregation rates and fluorescence quenching using various experimental techniques. Through light scattering experiments, the RH of bare amylin was found to increase at a rate of 43% per day, whereas in the presence of the ligands in different molar ratios (A1C10, A1R10 and A1GQDs20), the sizes of the complexes were found to grow at rates of 7%, 8% and 13% per day, respectively. We observed fluorescence quenching using photoluminescence experiments for all three protein-ligand complexes. The protein aggregation rate and fluorescence quenching exhibited a concentration-dependent competitive role in the inhibition process. Interestingly, for graphene quantum dots, the protein aggregation rate is more affected at lower concentrations, while fluorescence quenching dominates at higher concentrations; this is in contrast to curcumin and resveratrol, where fluorescence quenching dominates at all concentrations of the ligands in the complex. The FTIR data showed appreciable conversion of ß-sheets into less aggregation-prone secondary structures for all three amylin-ligand ratios; however, the inhibition performance of curcumin overshadowed those of the other two inhibitors. The inhibition behavior of these three ligands was corroborated by analysis of analytical and high-resolution TEM images of the fibrils.

Molecular dynamics simulation study on the inhibitory effects of choline-O-sulfate on hIAPP protofibrilation.

Type 2 diabetes mellitus (T2Dm) is a neurodegenerative disease, which occurs due to the self-association of human islet amyloid polypeptide (hIAPP), also known as human amylin. It was reported experimentally that choline-O-sulfate (COS), a small organic molecule having a tertiary amino group and sulfate group, can prevent the aggregation of human amylin without providing the mechanism of the action of COS in the inhibition process. In this work, we investigate the influence of COS on the full-length hIAPP peptide by performing 500 ns classical molecular dynamics simulations. From pure water simulation (without COS), we have identified the residues 11-20 and 23-36 that mainly participate in the fibril formation, but in the presence of 1.07 M COS these residues become totally free of ß-sheet conformation. Our results also show that the sulfate oxygen of COS directly interacts with the peptide backbone, which leads to the local disruption of peptide-peptide interaction. Moreover, the presence of favorable peptide-COS vdW interaction energy and high coordination number of COS molecules in the first solvation shell of the peptide indicates the hydrophobic solvation of the peptide residues by COS molecules, which also play a crucial role in the prevention of ß-sheet formation. Finally, from the potential of mean force (PMFs) calculations, we observe that the free energy between two peptides is more negative in the absence of COS and with increasing concentration of COS, it becomes unfavorable significantly indicating that the peptide dimer formation is most stable in pure water, which becomes less favorable in the presence of COS. © 2019 Wiley Periodicals, Inc.

Influence of methionine-ruthenium complex on the fibril formation of human islet amyloid polypeptide.

The abnormal aggregation and deposition of human islet amyloid polypeptide (hIAPP) are implicated in the pathogeny of type 2 diabetes mellitus (T2DM). Many aromatic ring-containing Ru complexes inhibit the aggregation of hIAPP. A new Ru complex Ru(bipy)(met)2·3H2O (1), where bipy is 2,2'-bipyridine and met is methionine, was synthesized and employed to resist the fibril formation of hIAPP and to promote the biocompatibility of metal complexes. Two polypyridyl Ru complexes, namely [Ru(bipy)3]Cl2(2) and Ru(bipy)2Cl2(3), were used for comparison. Results reveal that the three Ru complexes can inhibit hIAPP aggregation and depolymerize mature hIAPP fibrils. Interaction studies show that Ru complexes bind to hIAPP through metal coordination, hydrophobic interaction, and other intermolecular forces. The binding of the three compounds is spontaneous and exothermic. The compounds also rescue peptide-induced cytotoxicity to some extent. Similar to 3, the novel methionine-Ru complex 1 exhibits an enhanced inhibitory effect and binding affinity to hIAPP possibly because of the smaller steric hindrance and more profitable molecular configuration of 1 than those of 2. The newly designed amino acid-Ru complex may provide new insights into the treatment of T2DM and related amyloidosis diseases. Methionine-Ru complex effectively impedes the fibril formation of human islet amyloid polypeptide.

Synthesis and identification of novel pyridazinylpyrazolone based diazo compounds as inhibitors of human islet amyloid polypeptide aggregation.

We have carried out a docking inspired synthesis and screening of a library of diazenyl-derivatives of pyridazinylpyrazolone molecules for their ability to modulate the amyloidogenic self-assembly of human islet amyloid polypeptide (hIAPP). hIAPP is a 37-residue peptide which is involved in glycemic control along with insulin. Its extracellular fibrillar assemblies in pancreatic ß-cells are responsible for type 2 diabetes. A three-step synthetic scheme was used to prepare these novel compounds using 2-(6-chloropyridazin-3-yl)-5-methyl-2,4-dihydro-3H-pyrazol-3-one as a key intermediate that was reacted with various diazo electrophiles to generate a library of compounds with yields ranging from 64 to 85%. The effect of the compounds on hIAPP amyloid fibril formation was evaluated with a thioflavin T (ThT) fluorescence-based kinetic assay. Furthermore, TEM imaging was carried out to corroborate the interactions of the compounds with hIAPP and subsequent hIAPP inhibition at the different level of fibrillization. The CD spectroscopy showed that upon incubation with SSE15314 for 12 h, the percentage of α-helices was maintained to a level of hIAPP at 0 h. The current study presents identification and characterization of SSE15314 as the hit, which completely inhibited the fibril formation and can be further optimized into a lead compound.