Fanling Meng

The flavanol (-)-epigallocatechin 3-gallate inhibits amyloid formation by islet amyloid polypeptide, disaggregates amyloid fibrils, and protects cultured cells against IAPP-induced toxicity.

Islet amyloid polypeptide (IAPP, amylin) is the major protein component of the islet amyloid deposits associated with type 2 diabetes. The polypeptide lacks a well-defined structure in its monomeric state but readily assembles to form amyloid. Amyloid fibrils formed from IAPP, intermediates generated in the assembly of IAPP amyloid, or both are toxic to β-cells, suggesting that islet amyloid formation may contribute to the pathology of type 2 diabetes. There are relatively few reported inhibitors of amyloid formation by IAPP. Here we show that the tea-derived flavanol, (-)-epigallocatechin 3-gallate [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate] (EGCG), is an effective inhibitor of in vitro IAPP amyloid formation and disaggregates preformed amyloid fibrils derived from IAPP. The compound is thus one of a very small set of molecules which have been shown to disaggregate IAPP amyloid fibrils. Fluorescence-detected thioflavin-T binding assays and transmission electron microscopy confirm that the compound inhibits unseeded amyloid fibril formation as well as disaggregates IAPP amyloid. Seeding studies show that the complex formed by IAPP and EGCG does not seed amyloid formation by IAPP. In this regard, the behavior of IAPP is similar to the reported interactions of Aβ and α-synuclein with EGCG. Alamar blue assays and light microscopy indicate that the compound protects cultured rat INS-1 cells against IAPP-induced toxicity. Thus, EGCG offers an interesting lead structure for further development of inhibitors of IAPP amyloid formation and compounds that disaggregate IAPP amyloid.

Rifampicin Does Not Prevent Amyloid Fibril Formation by Human Islet Amyloid Polypeptide but Does Inhibit Fibril Thioflavin-T Interactions: Implications for Mechanistic Studies of β-Cell Death†

Amyloid formation has been implicated in more than 20 different human diseases, including Alzheimers disease, Parkinsons disease, and type 2 diabetes. The development of inhibitors of amyloid is a topic of considerable interest, both because of their potential therapeutic applications and because they are useful mechanistic probes. Recent studies have highlighted the potential use of rifampicin as an inhibitor of amyloid formation by a variety of polypeptides; however, there are conflicting reports on its ability to inhibit amyloid formation by islet amyloid polypeptide (IAPP). IAPP is the cause of islet amyloid in type 2 diabetes. We show that rifampicin does not prevent amyloid formation by IAPP and does not disaggregate preformed IAPP amyloid fibrils;, instead, it interferes with standard fluorescence-based assays of amyloid formation. Rifampicin is unstable in aqueous solution and is readily oxidized. However, the effects of oxidized and reduced rifampicin are similar, in that neither prevents amyloid formation by IAPP. Furthermore, use of a novel p-cyanoPhe analogue of IAPP shows that rifampicin does not significantly affect the kinetics of IAPP amyloid formation. The implications for the development of amyloid inhibitors are discussed as are the implications for studies of the toxicity of islet amyloid. The work also demonstrates the utility of p-cyanoPhe IAPP for the screening of inhibitors. The data indicate that rifampicin cannot be used to test the relative toxicity of IAPP fibrils and prefibril aggregates of IAPP.

The ability of rodent islet amyloid polypeptide to inhibit amyloid formation by human islet amyloid polypeptide has important implications for the mechanism of amyloid formation and the design of inhibitors.

Islet amyloid polypeptide (IAPP) is a 37-residue polypeptide hormone that is responsible for islet amyloid formation in type II diabetes. Human IAPP is extremely amyloidogenic, while rat IAPP and mouse IAPP do not form amyloid in vitro or in vivo. Rat IAPP and mouse IAPP have identical primary sequences, but differ from the human polypeptide at six positions, five of which are localized between residues 20 and 29. The ability of rat IAPP to inhibit amyloid formation by human IAPP was tested, and the rat peptide was found to be an effective inhibitor. Thioflavin-T fluorescence-monitored kinetic experiments, transmission electron microscopy, and circular dichroism showed that rat IAPP lengthened the lag phase for amyloid formation by human IAPP, slowed the growth rate, reduced the amount of amyloid fibrils produced in a dose-dependent manner, and altered the morphology of the fibrils. The inhibition of human IAPP amyloid formation by rat IAPP can be rationalized by a model that postulates formation of an early helical intermediate during amyloid formation where the helical region is localized to the N-terminal region of IAPP. The model predicts that proline mutations in the putative helical region should lead to ineffective inhibitors as should mutations that alter the peptide-peptide interaction interface. We confirmed this by testing the ability of A13P and F15D point mutants of rat IAPP to inhibit amyloid formation by human IAPP. Both these mutants were noticeably less effective inhibitors than wild-type rat IAPP. The implications for inhibitor design are discussed.

Combination of Kinetically Selected Inhibitors in Trans Leads to Highly Effective Inhibition of Amyloid Formation

Amyloid formation plays a role in over 25 human disorders. A range of strategies have been applied to the problem of developing inhibitors of amyloid formation, but unfortunately, many inhibitors are effective only in molar excess and typically either lengthen the time to the onset of amyloid formation, (the lag time), while having modest effects on the total amount of amyloid fibrils produced, or decrease the amount of amyloid without significantly reducing the lag time. We demonstrate a general strategy whereby two moderate inhibitors of amyloid formation can be rationally selected via kinetic assays and combined in trans to yield a highly effective inhibitor which dramatically delays the time to the appearance of amyloid and drastically reduces the total amount of amyloid formed. A key feature is that the selection of the components of the mixture is based on their effect on the time course of amyloid formation rather than on just the amount of amyloid produced. The approach is validated using inhibitors of amyloid formation by islet amyloid polypeptide, the causative agent of amyloid formation in type 2 diabetes and the Alzheimers disease Aβ peptide.

The Sulfated Triphenyl Methane Derivative Acid Fuchsin is a Potent Inhibitor of Amyloid Formation by Human Islet Amyloid Polypeptide and Protects Against the Toxic Effects of Amyloid Formation

Islet amyloid polypeptide (IAPP), also known as amylin, is responsible for amyloid formation in type 2 diabetes. The formation of islet amyloid is believed to contribute to the pathology of the disease by killing beta-cells, and it may also contribute to islet transplant failure. The design of inhibitors of amyloid formation is an active area of research, but comparatively little attention has been paid to inhibitors of IAPP in contrast to the large body of work on beta-amyloid, and most small-molecule inhibitors of IAPP amyloid are generally effective only when used at a significant molar excess. Here we show that the simple sulfonated triphenyl methane derivative acid fuchsin, 3-(1-(4-amino-3-methyl-5-sulfonatophenyl)-1-(4-amino-3-sulfonatophenyl) methylene) cyclohexa-1,4-dienesulfonic acid, is a potent inhibitor of in vitro amyloid formation by IAPP at substoichiometric levels and protects cultured rat INS-1 cells against the toxic effects of human IAPP. Fluorescence-detected thioflavin-T binding assays, light-scattering, circular dichroism, two-dimensional IR, and transmission electron microscopy measurements confirm that the compound prevents amyloid fibril formation. Ionic-strength-dependent studies show that the effects are mediated in part by electrostatic interactions. Experiments in which the compound is added at different time points during the lag phase after amyloid formation has commenced reveal that it arrests amyloid formation by trapping intermediate species. The compound is less effective against the beta-amyloid peptide, indicating specificity in its ability to inhibit amyloid formation by IAPP. The work reported here provides a new structural class of IAPP amyloid inhibitors and demonstrates the power of two-dimensional infrared spectroscopy for characterizing amyloid inhibitor interactions.

Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics

Islet amyloidosis by IAPP contributes to pancreatic β-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 β-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced β-cell death. DOI: http://dx.doi.org/10.7554/eLife.12977.001

Inhibition of Glycosaminoglycan-Mediated Amyloid Formation by Islet Amyloid Polypeptide and proIAPP Processing Intermediates

Islet amyloid polypeptide (IAPP; also known as amylin) is responsible for islet amyloid formation in type 2 diabetes, and IAPP-induced toxicity is believed to contribute to the loss of β-cell mass associated with the late stages of type 2 diabetes. Islet amyloid formation may also play a role in graft failure after transplantation. IAPP is produced as a prohormone, pro-islet amyloid polypeptide (proIAPP), and processed in the secretory granules of the pancreatic β-cells. Partially processed forms of proIAPP are found in amyloid deposits; most notable is a 48-residue intermediate, proIAPP(1-48), which includes the N-terminal pro-extension, but which has been properly processed at the C-terminus. Incomplete processing may play a role in islet amyloid formation by promoting interactions with sulfated proteoglycans of the extracellular matrix, which, in turn, promote amyloid formation. We show that acid fuchsin (3-(1-(4-amino-3-methyl-5-sulphonatophenyl)-1-(4-amino-3-sulphonatophenyl)methylene)cyclohexa-1,4-dienesulphonic acid), a simple sulfonated triphenyl methyl derivative, is a potent inhibitor of amyloid formation by the proIAPP(1-48) intermediate. The more complicated triphenyl methane derivative fast green FCF {ethyl-[4-[[4-[ethyl-[(3-sulfophenyl)methyl]amino]phenyl]-(4-hydroxy-2-sulfophenyl)methylidene]-1-cyclohexa-2,5-dienylidene]-[(3-sulfophenyl)methyl]azanium} also inhibits amyloid formation by IAPP and the proIAPP processing intermediate. Both compounds inhibit amyloid formation by mixtures of the proIAPP intermediate and the model glycosaminoglycan heparan sulfate. Acid fuchsin also inhibits glycosaminoglycan-mediated amyloid formation by mature IAPP. The ability to inhibit amyloid formation is not simply due to the compounds being sulfonated, since the sulfonated inhibitor of amyloid-β, tramiprosate, is not an inhibitor of amyloid formation by proIAPP(1-48).