TPE conjugated islet amyloid polypeptide probe for detection of peptide oligomers.

Islet amyloid polypeptide (IAPP), also known as amylin, is a polypeptide hormone co-secreted with insulin by pancreatic ß-cells. In general, IAPP is soluble and lacks a defined structure. However, under certain conditions, these peptides tend to aggregate into soluble oligomers, eventually forming insoluble amyloid fibrils with typical cross-ß-sheet structures. Amylin aggregates, therefore, have been regarded as one of the hallmarks of type II diabetes (T2D). Among these aggregated species, oligomers were shown to exhibit significant cytotoxicity, leading to impaired ß-cell function and reduced ß-cell mass. Monitoring of oligomer appearance during IAPP fibrillation is of particular interest. In this study, we successfully grafted an aggregation-induced emission molecule, tetraphenylethylene (TPE), at the N-terminus of IAPP. By mixing a small amount of TPE-labeled IAPP with unlabeled IAPP, we were able to detect an increase in TPE fluorescence during the nucleation phase of IAPP aggregation in vitro. It may enable real-time monitoring of IAPP oligomer formation and is further applied in the diagnosis of T2D.

Piperic acid derivative as a molecular modulator to accelerate the IAPP aggregation process and alter its antimicrobial activity.

Piperic acid derivatives were found to affect the islet amyloid polypeptide (IAPP) aggregation process. Structure-activity relationship studies revealed that PAD-13 was an efficient molecular modulator to accelerate IAPP fibril formation by promoting primary and secondary nucleation and reducing its antimicrobial activity.

Investigating the inhibitory property of DM hCT on hCT fibrillization via its relevant peptide fragments.

The irreversible aggregation of proteins or peptides greatly limits their bioavailability; therefore, effective inhibition using small molecules or biocompatible materials is very difficult. Human calcitonin (hCT), a hormone polypeptide with 32 residues, is secreted by the C-cells of the thyroid gland. The biological function of this hormone is to regulate calcium and phosphate concentrations in the blood via several different pathways. One of these is to inhibit the activity of osteoclasts; thus, calcitonin could be used to treat osteoporosis and Paget's disease of the bone. However, hCT is prone to aggregation in aqueous solution and forms amyloid fibrils. Salmon and eel calcitonin are currently used as clinical substitutes for hCT. In a previous study, we found that the replacement of two residues at positions 12 and 17 of hCT with amino acids that appear in the salmon sequence can greatly suppress peptide aggregation. The double mutations of hCT (DM hCT) also act as good inhibitors by disrupting wild-type hCT fibrillization, although the inhibition mechanism is not clear. More importantly, we demonstrated that DM hCT is biologically active in interacting with the calcitonin receptor. To further understand the inhibitory effect of DM hCT on hCT fibrillization, we created four relevant peptide fragments based on the DM hCT sequence. Our examination revealed that the formation of a helix of DM hCT was possibly a key component contributing to its inhibitory effect. This finding could help in the development of peptide-based inhibitors and in understanding the aggregation mechanism of hCT.

An environmentally sensitive molecular rotor as a NIR fluorescent probe for the detection of islet amyloid polypeptide.

The deposits of human islet amyloid polypeptide (IAPP), also called amylin, in the pancreas have been postulated to be a factor of pancreatic ß-cell dysfunction and is one of the common pathological hallmarks of type II diabetes mellitus (T2DM). Therefore, it is imperative to gain an in-depth understanding of the formation of these aggregates. In this study, we demonstrate a rationally-designed strategy of an environmentally sensitive near-infrared (NIR) molecular rotor utilizing thioflavin T (ThT) as a scaffold for IAPP deposits. We extended the π delocalized system not only to improve the viscosity sensitivity but also to prolong the emission wavelength to the NIR region. A naphthalene moiety was also introduced to adjust the sensitivity of our designed probes to differentiate the binding microenvironment polarity of different targeted proteins. As a result, a novel NIR fluorogenic probe toward IAPP aggregates, namely AmySP-4-Nap-Ene, was first developed. When attached to different protein aggregates, this probe exhibited distinct fluorescence emission profiles. In a comparison with ThT, the fluorescence emission of non-ionic AmySP-4-Nap-Ene exhibits a significant difference between the presence of non-fibrillar and fibrillar IAPP and displays a higher binding affinity toward IAPP fibrils. Further, the AmySP-4-Nap-Ene can be utilized to monitor IAPP accumulating process and image fibrils both in vitro and in living cells.

Dihydrocaffeic Acid-Decorated Iron Oxide Nanomaterials Effectively Inhibit Human Calcitonin Aggregation.

To date, more than 30 human peptides or proteins have been found to form amyloid fibrils, most of which are associated with human diseases. However, currently, no cure for amyloidosis exists. Therefore, development of therapeutic strategies to inhibit amyloid formation is urgently required. Although the role of some amyloidogenic proteins has not been identified in certain diseases, their self-assembling behavior largely affects their bioactivity. Human calcitonin (hCT) is a hormone peptide containing 32 amino acids and is secreted by the parafollicular cells of the thyroid gland in the human body. It can regulate the concentration of calcium ions in the blood and block the activity of osteoclasts. Therefore, calcitonin has also been considered a therapeutic peptide. However, the aggregation of hCT hinders this process, and hCT has been replaced by salmon calcitonin in drug formulations. Recently, iron oxide nanomaterials have been developed as potential materials for various applications owing to their high biocompatibility, low toxicity, and ease of functionalization. In this study, nanoparticles (NPs) were prepared using a simple chemical coprecipitation method. We first demonstrated that dopamine-conjugated Fe3O4 inhibited hCT aggregation, similar to what we found when carbon dots were used as core materials in the previous study. Later, we continued to simplify the preparation process, that is, the mixing of dihydrocaffeic acid (DCA) and iron oxide NPs, to maintain their stability and inhibitory effect against hCT aggregation. Furthermore, DCA-decorated Fe3O4 can dissociate preformed hCT amyloid fibrils. This appears to be one of the most promising ways to stabilize hCT in solution and may be helpful for amyloidosis treatment.

Dopamine-Conjugated Carbon Dots Inhibit Human Calcitonin Fibrillation.

The development of biocompatible nanomaterials has become a new trend in the treatment and prevention of human amyloidosis. Human calcitonin (hCT), a hormone peptide secreted from parafollicular cells, plays a major role in calcium-phosphorus metabolism. Moreover, it can be used in the treatment of osteoporosis and Paget's disease. Unfortunately, it tends to form amyloid fibrils irreversibly in an aqueous solution, resulting in a reduction of its bioavailability and therapeutic activity. Salmon calcitonin is the replacement of hCT as a widely therapeutic agent due to its lower propensity in aggregation and better bioactivity. Herein, we used citric acid to synthesize carbon dots (CDs) and modified their surface properties by a variety of chemical conjugations to provide different functionalized CDs. It was found that dopamine-conjugated CDs can effectively inhibit hCT aggregation especially in the fibril growth phase and dissociate preformed hCT amyloids. Although the decomposition mechanism of dopamine-conjugated CDs is not clear, it seems to be specific to hCT amyloids. In addition, we also tested dopamine-conjugated mesoporous silica nanoparticles in preventing hCT fibrillization. They also can work as inhibitors but are much less effective than CDs. Our studies emphasized the importance of the size and surface functionalization of core materials in the development of nanomaterials as emerging treatments for amyloidosis. On the other hand, proper functionalized CDs would be useful in hCT formulation.

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.

A Fluorogenic Molecule for Probing Islet Amyloid Using Flavonoid as a Scaffold Design.

Aggregation of polypeptides and proteins is commonly associated with human and other vertebrate diseases. For example, amyloid plaques consisting of amyloid-ß proteins are frequently identified in Alzheimer's disease and islet amyloid formed by islet amyloid polypeptide (IAPP, amylin) can be found in most patients with type 2 diabetes (T2D). Although many fluorescent dyes have been developed to stain amyloid fibrils, very few examples have been designed for IAPP. In this study, a series of environmentally sensitive fluorescent probes using flavonoid as a scaffold design are rationally designed and synthesized. One of these probes, namely 3-HF-ene-4'-OMe, can bind to IAPP fibrils but not nonfibrillar IAPP by exhibiting a much stronger fluorescent enhancement at 535 nm. In addition, this probe shows better detection sensitivity to IAPP fibrils compared with that of conventionally used thioflavin-T. We demonstrate that 3-HF-ene-4'-OMe can be used to monitor the kinetics of IAPP fibril formation in vitro even in the presence an amyloid inhibitor. To test the specificity of the probe, we attempt to incubate this probe with amyloid fibrils formed from other amyloidogenic proteins. Interestingly, this probe shows different responses when mixed with these fibrils, suggesting the mode of binding of this probe on these fibrils could be different. Moreover, we show that this probe is not toxic to pancreatic mouse ß-cells. Further structural optimization based on the structure of 3-HF-ene-4'-OMe may yield a specific probe for imaging islet amyloid in the pancreas. That would improve our understanding of the relationship between islet amyloid and T2D.

Inhibiting Human Calcitonin Fibril Formation with Its Most Relevant Aggregation-Resistant Analog.

The most common obstacles to the development of therapeutic polypeptides are peptide stability and aggregation. Human calcitonin (hCT) is a 32-residue hormone polypeptide secreted from the C-cells of the thyroid gland and is responsible for calcium and phosphate regulation in the blood. hCT reduces calcium levels by inhibiting the activity of osteoclasts, which are bone cells that are mainly responsible for breaking down the bone tissue or decreasing the resorption of calcium from the kidneys. Thus, calcitonin injection has been used to treat osteoporosis and Paget's disease of bone. hCT is an aggregation-prone peptide with a high tendency to form amyloid fibrils. As a result, salmon calcitonin (sCT), which is different from hCT at 16-residue positions and has a lower propensity to aggregate, has been chosen as a clinical substitute for hCT. However, significant side effects, including immune reactions, have been shown with the use of sCT injection. In this study, we found that two residues, Tyr-12 and Asn-17, play key roles in inducing the fibrillization of hCT. Double mutation of hCT at these two crucial sites could greatly enhance its resistance to aggregation and provide a peptide-based inhibitor to prevent amyloid formation by hCT. Double-mutated hCT retains its ability to interact with its receptor in vivo. These findings suggest that this variant of hCT would serve as a valuable therapeutic alternative to sCT.

Protein Glycation by Glyoxal Promotes Amyloid Formation by Islet Amyloid Polypeptide.

Protein glycation, also known as nonenzymatic glycosylation, is a spontaneous post-translational modification that would change the structure and stability of proteins or hormone peptides. Recent studies have indicated that glycation plays a role in type 2 diabetes (T2D) and neurodegenerative diseases. Over the last two decades, many types of advanced glycation end products (AGEs), formed through the reactions of an amino group of proteins with reducing sugars, have been identified and detected in vivo. However, the effect of glycation on protein aggregation has not been fully investigated. In this study, we aim to elucidate the impact of protein glycation on islet amyloid polypeptide (IAPP, also known as amylin) aggregation, which was strongly associated with T2D. We chemically synthesized glycated IAPP (AGE-IAPP) to mimic the consequence of this hormone peptide in a hyperglycemia (high blood sugar) environment. Our data revealed that AGE-IAPP formed amyloid faster than normal IAPP, and higher-molecular-weight AGE-IAPP oligomers were also observed in the early stage of aggregation. Circular dichroism spectra also indicated that AGE-IAPP exhibited faster conformational changes from random coil to its ß-sheet fibrillar states. Moreover, AGE-IAPP can induce normal IAPP to expedite its aggregation process, and its fibrils can also act as templates to promote IAPP aggregation. AGE-IAPP, like normal IAPP, is capable of interacting with synthetic membranes and also exhibits cytotoxicity. Our studies demonstrated that glycation modification of IAPP promotes the amyloidogenic properties of IAPP, and it may play a role in accumulating additional amyloid during T2D progression.

Rationally designed divalent caffeic amides inhibit amyloid-ß fibrillization, induce fibril dissociation, and ameliorate cytotoxicity.

One of the pathologic hallmarks in Alzheimer's disease (AD) is extracellular senile plaques composed of amyloid-ß (Aß) fibrils. Blocking Aß self-assembly or disassembling Aß aggregates by small molecules would be potential therapeutic strategies to treat AD. In this study, we synthesized a series of rationally designed divalent compounds and examined their effects on Aß fibrillization. A divalent amide (2) derived from two molecules of caffeic acid with a propylenediamine linker of ∼5.0 Šin length, which is close to the distance of adjacent ß sheets in Aß fibrils, showed good potency to inhibit Aß(1-42) fibrillization. Furthermore, compound 2 effectively dissociated the Aß(1-42) preformed fibrils. The cytotoxicity induced by Aß(1-42) aggregates in human neuroblastoma was reduced in the presence of 2, and feeding 2 to Aß transgenic C. elegans rescued the paralysis phenotype. In addition, the binding and stoichiometry of 2 to Aß(1-40) were demonstrated by using electrospray ionization-traveling wave ion mobility-mass spectrometry, while molecular dynamic simulation was conducted to gain structural insights into the Aß(1-40)-2 complex.

Understanding co-polymerization in amyloid formation by direct observation of mixed oligomers.

Although amyloid assembly in vitro is commonly investigated using single protein sequences, fibril formation in vivo can be more heterogeneous, involving co-assembly of proteins of different length, sequence and/or post-translational modifications. Emerging evidence suggests that co-polymerization can alter the rate and/or mechanism of aggregation and can contribute to pathogenicity. Electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) is uniquely suited to the study of these heterogeneous ensembles. Here, ESI-IMS-MS combined with analysis of fibrillation rates using thioflavin T (ThT) fluorescence, is used to track the course of aggregation of variants of islet-amyloid polypeptide (IAPP) in isolation and in pairwise mixtures. We identify a sub-population of extended monomers as the key precursors of amyloid assembly, and reveal that the fastest aggregating sequence in peptide mixtures determines the lag time of fibrillation, despite being unable to cross-seed polymerization. The results demonstrate that co-polymerization of IAPP sequences radically alters the rate of amyloid assembly by altering the conformational properties of the mixed oligomers that form.

A Free Energy Barrier Caused by the Refolding of an Oligomeric Intermediate Controls the Lag Time of Amyloid Formation by hIAPP.

Transiently populated oligomers formed en route to amyloid fibrils may constitute the most toxic aggregates associated with many amyloid-associated diseases. Most nucleation theories used to describe amyloid aggregation predict low oligomer concentrations and do not take into account free energy costs that may be associated with structural rearrangements between the oligomer and fiber states. We have used isotope labeling and two-dimensional infrared spectroscopy to spectrally resolve an oligomeric intermediate during the aggregation of the human islet amyloid protein (hIAPP or amylin), the protein associated with type II diabetes. A structural rearrangement includes the F23G24A25I26L27 region of hIAPP, which starts from a random coil structure, evolves into ordered ß-sheet oligomers containing at least 5 strands, and then partially disorders in the fibril structure. The supercritical concentration is measured to be between 150 and 250 µM, which is the thermodynamic parameter that sets the free energy of the oligomers. A 3-state kinetic model fits the experimental data, but only if it includes a concentration independent free energy barrier >3 kcal/mol that represents the free energy cost of refolding the oligomeric intermediate into the structure of the amyloid fibril; i.e., "oligomer activation" is required. The barrier creates a transition state in the free energy landscape that slows fibril formation and creates a stable population of oligomers during the lag phase, even at concentrations below the supercritical concentration. Largely missing in current kinetic models is a link between structure and kinetics. Our experiments and modeling provide evidence that protein structural rearrangements during aggregation impact the populations and kinetics of toxic oligomeric species.

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

Islet amyloidosis by IAPP contributes to pancreatic ß-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 ß-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 ß-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 ß-cell death.

Islet Amyloid Polypeptide: Structure, Function, and Pathophysiology.

The hormone islet amyloid polypeptide (IAPP, or amylin) plays a role in glucose homeostasis but aggregates to form islet amyloid in type-2 diabetes. Islet amyloid formation contributes to ß-cell dysfunction and death in the disease and to the failure of islet transplants. Recent work suggests a role for IAPP aggregation in cardiovascular complications of type-2 diabetes and hints at a possible role in type-1 diabetes. The mechanisms of IAPP amyloid formation in vivo or in vitro are not understood and the mechanisms of IAPP induced ß-cell death are not fully defined. Activation of the inflammasome, defects in autophagy, ER stress, generation of reactive oxygen species, membrane disruption, and receptor mediated mechanisms have all been proposed to play a role. Open questions in the field include the relative importance of the various mechanisms of ß-cell death, the relevance of reductionist biophysical studies to the situation in vivo, the molecular mechanism of amyloid formation in vitro and in vivo, the factors which trigger amyloid formation in type-2 diabetes, the potential role of IAPP in type-1 diabetes, the development of clinically relevant inhibitors of islet amyloidosis toxicity, and the design of soluble, bioactive variants of IAPP for use as adjuncts to insulin therapy.

Matrix Metalloproteinase-9 Protects Islets from Amyloid-induced Toxicity.

Deposition of human islet amyloid polypeptide (hIAPP, also known as amylin) as islet amyloid is a characteristic feature of the pancreas in type 2 diabetes, contributing to increased ß-cell apoptosis and reduced ß-cell mass. Matrix metalloproteinase-9 (MMP-9) is active in islets and cleaves hIAPP. We investigated whether hIAPP fragments arising from MMP-9 cleavage retain the potential to aggregate and cause toxicity, and whether overexpressing MMP-9 in amyloid-prone islets reduces amyloid burden and the resulting ß-cell toxicity. Synthetic hIAPP was incubated with MMP-9 and the major hIAPP fragments observed by MS comprised residues 1-15, 1-25, 16-37, 16-25, and 26-37. The fragments 1-15, 1-25, and 26-37 did not form amyloid fibrils in vitro and they were not cytotoxic when incubated with ß cells. Mixtures of these fragments with full-length hIAPP did not modulate the kinetics of fibril formation by full-length hIAPP. In contrast, the 16-37 fragment formed fibrils more rapidly than full-length hIAPP but was less cytotoxic. Co-incubation of MMP-9 and fragment 16-37 ablated amyloidogenicity, suggesting that MMP-9 cleaves hIAPP 16-37 into non-amyloidogenic fragments. Consistent with MMP-9 cleavage resulting in largely non-amyloidogenic degradation products, adenoviral overexpression of MMP-9 in amyloid-prone islets reduced amyloid deposition and ß-cell apoptosis. These findings suggest that increasing islet MMP-9 activity might be a strategy to limit ß-cell loss in type 2 diabetes.

Insights into the consequences of co-polymerisation in the early stages of IAPP and Aß peptide assembly from mass spectrometry.

The precise molecular mechanisms by which different peptides and proteins assemble into highly ordered amyloid deposits remain elusive. The fibrillation of human amylin (also known as islet amyloid polypeptide, hIAPP) and the amyloid-beta peptide (Aß-40) are thought to be pathogenic factors in Type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD), respectively. Amyloid diseases may involve co-aggregation of different protein species, in addition to the self-assembly of single precursor sequences. Here we investigate the formation of heterogeneous pre-fibrillar, oligomeric species produced by the co-incubation of hIAPP and Aß-40 using electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS)-based methods. Conformational properties and gas-phase stabilities of amyloid oligomers formed from hIAPP or Aß40 alone, and from a 1 : 1 mixture of hIAPP and Aß40 monomers, were determined and compared. We show that co-assembly of the two sequences results in hetero-oligomers with distinct properties and aggregation kinetics properties compared with the homo-oligomers present in solution. The observations may be of key significance to unravelling the mechanisms of amyloid formation in vivo and elucidating how different sequences and/or assembly conditions can result in different fibril structures and/or pathogenic outcomes.

Mutational analysis of the ability of resveratrol to inhibit amyloid formation by islet amyloid polypeptide: critical evaluation of the importance of aromatic-inhibitor and histidine-inhibitor interactions.

The process of amyloid formation by the normally soluble hormone islet amyloid polypeptide (IAPP) contributes to ß-cell death in type 2 diabetes and in islet transplants. There are no clinically approved inhibitors of islet amyloidosis, and the mode of action of existing inhibitors is not well-understood. Resveratrol, a natural polyphenol, has been reported to inhibit amyloid formation by IAPP and by the Alzheimer's disease Aß peptide. The mechanism of action of this compound is not known, nor is its mode of interaction with IAPP. In this study, we use a series of IAPP variants to examine possible interactions between resveratrol and IAPP. Fluorescence assays, transmission electron microscopy, and mass spectrometry demonstrate that resveratrol is much less effective as an inhibitor of IAPP amyloid formation than the polyphenol (-)-epigallocatechin 3-gallate (EGCG) and, unlike EGCG, does not significantly disaggregate preformed IAPP amyloid fibrils. Resveratrol is also shown to interfere with thioflavin-T assays. His-18 mutants, a truncation mutant, mutants of each of the aromatic residues, and mutants of Arg-11 of IAPP were examined. Mutation of His to Gln or Leu weakens the ability of resveratrol to inhibit amyloid formation by IAPP, as do mutations of Arg-11, Phe-15, or Tyr-37 to Leu, and truncation to form the variant Ac 8-37-IAPP, which removes the first seven residues to eliminate Lys-1 and the N-terminal amino group. In contrast, replacement of Phe-23 with Leu has a smaller effect. The data highlight Phe-15, His-18, and Tyr-37 as being important for IAPP-resveratrol interactions and are consistent with a potential role of the N-terminus and Arg-11 in polypeptide-resveratrol interactions.

Screening and classifying small-molecule inhibitors of amyloid formation using ion mobility spectrometry-mass spectrometry.

The search for therapeutic agents that bind specifically to precursor protein conformations and inhibit amyloid assembly is an important challenge. Identifying such inhibitors is difficult because many protein precursors of aggregation are partially folded or intrinsically disordered, which rules out structure-based design. Furthermore, inhibitors can act by a variety of mechanisms, including specific or nonspecific binding, as well as colloidal inhibition. Here we report a high-throughput method based on ion mobility spectrometry-mass spectrometry (IMS-MS) that is capable of rapidly detecting small molecules that bind to amyloid precursors, identifying the interacting protein species and defining the mode of inhibition. Using this method we have classified a variety of small molecules that are potential inhibitors of human islet amyloid polypeptide (hIAPP) aggregation or amyloid-beta 1-40 aggregation as specific, nonspecific, colloidal or non-interacting. We also demonstrate the ability of IMS-MS to screen for inhibitory small molecules in a 96-well plate format and use this to discover a new inhibitor of hIAPP amyloid assembly.

Aspirin, diabetes, and amyloid: re-examination of the inhibition of amyloid formation by aspirin and ketoprofen.

The loss of ß-cell function and ß-cell death are key features of diabetes. A range of mechanisms are thought to contribute to ß-cell loss, including islet amyloid formation by the neuropancreatic hormone amylin (islet amyloid polypeptide, IAPP). Islet amyloid deposition also contributes to the failure of islet transplants. There are no therapeutic strategies for the treatment or prevention of islet amyloidosis. Aspirin and the nonsteroid anti-inflammatory drug (NSAID) ketoprofen, at clinically relevant doses, have been proposed to inhibit amyloid formation by amylin and thus may hold promise for treatment of islet amyloidosis. These compounds are potentially attractive given the importance of inflammation in islet amyloidosis and given the fact that there are no anti-islet amyloid agents in the clinic. We show that aspirin, even in 20-fold excess, has no effect on the kinetics of amyloid formation by amylin as judged by thioflavin-T binding, right angle light scattering, and transmission electron microscopy, nor does it alter the morphology of resulting amyloid fibrils. Aspirin showed no ability to disaggregate preformed amylin amyloid fibrils under the conditions of these studies, 25 °C and pH 7.4. Ketoprofen is similarly ineffective at inhibiting amylin amyloid formation. The compounds do, however, interfere with circular dichroism- and Congo Red-based assays of amylin amyloid formation. This study highlights the importance of using multiple methods to follow amyloid formation when screening inhibitors.