Toxic Oligomers Research Articles

Treatment of Alzheimer’s disease by promoting the aggregation of small β-amyloid peptides

<p indent="0mm">Alzheimer’s disease (AD) is a type of senile dementia with complex pathological features. The amyloid cascade and oligomer toxicity hypotheses play a dominant role in explaining the pathological mechanism of AD. A large number of patients develop AD, but no effective treatment is available, and most drugs have not had good results in phase III clinical trials. In this study, research progress on the aggregation of β-amyloid (Aβ) is summarized, and the possible mechanism of small molecule compounds accelerating the formation of Aβ fibers is described. This process contrasts with traditional therapy but has achieved quite good efficacy in animal models, suggesting that promoting Aβ aggregation may be a new strategy for treating AD and indicating a new direction for drug development.

Phosphatidylcholine and Phosphatidylserine Uniquely Modify the Secondary Structure of α-Synuclein Oligomers Formed in Their Presence at the Early Stages of Protein Aggregation.

Abrupt aggregation of α-synuclein (α-Syn) leads to a formation of highly toxic protein oligomers. These aggregates are the underlying molecular cause of an onset of the irreversible degeneration of dopaminergic neurons in midbrain, hypothalamus, and thalamus, a pathology known as Parkinson's disease. The transient nature of oligomers, as well as their structural and morphological heterogeneity, limits the use of cryo-electron microscopy and solid-state NMR, classical tools of structural biology, for elucidation of their secondary structure. Despite this limitation, numerous pieces of experimental evidence suggest that phospholipids can uniquely alter the structure and toxicity of oligomers. In this study, we utilize an innovative nano-infrared imaging technique, also known as atomic force microscopy infrared (AFM-IR) spectroscopy, to examine the structure of individual α-Syn oligomers grown in the presence of phosphatidylcholine (α-Syn:PC) and phosphatidylserine (α-Syn:PS). We determined the amount of the parallel and the antiparallel β-sheets, as well as the amount the α-helix and the unordered protein, in the secondary structure of α-Syn:PC and α-Syn:PS formed at day 2 (D2), 8 (D8), and 15 (D15) after initiation of protein aggregation. We found a gradual decrease in the amount of the parallel β-sheet in both α-Syn:PC and α-Syn:PS from D2 to D15 together with an increase in the α-helix and the unordered protein secondary structure. We infer that this is due to the presence of lipids in the structure of oligomers that prevent an expansion of the parallel β-sheet upon interaction of the oligomers with monomeric α-Syn.

Clusterin Binding Modulates the Aggregation and Neurotoxicity of Amyloid-β(1-42).

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) aggregates in the brain. Clusterin (CLU), also known as apolipoprotein J, is a potent risk factor associated with AD pathogenesis, in which Aβ aggregation is essentially involved. We observed close colocalization of CLU and Aβ(1-42) (Aβ42) in parenchymal amyloid plaques or vascular amyloid deposits in the brains of human amyloid precursor protein (hAPP)-transgenic Tg2576 mice. Therefore, to elucidate the binding interaction between CLU and Aβ42 and its impact on amyloid aggregation and toxicity, the two synthetic proteins were incubated together under physiological conditions, and their structural and morphological variations were investigated using biochemical, biophysical, and microscopic analyses. Synthetic CLU spontaneously bound to different possible variants of Aβ42 aggregates with very high affinity (Kd = 2.647nM) in vitro to form solid CLU-Aβ42 complexes. This CLU binding prevented further aggregation of Aβ42 into larger oligomers or fibrils, enriching the population of smaller Aβ42 oligomers and protofibrils and monomers. CLU either alleviated or augmented Aβ42-induced cytotoxicity and apoptosis in the neuroblastoma-derived SH-SY5Y and N2a cells, depending on the incubation period and the molar ratio of CLU:Aβ42 involved in the reaction before addition to the cells. Thus, the effects of CLU on Aβ42-induced cytotoxicity were likely determined by the extent to which it bound and sequestered toxic Aβ42 oligomers or protofibrils. These findings suggest that CLU could influence amyloid neurotoxicity and pathogenesis by modulating Aβ aggregation.

α-Synuclein Conformational Plasticity: Physiologic States, Pathologic Strains, and Biotechnological Applications.

α-Synuclein (αS) is remarkable for both its extensive conformational plasticity and pathologic prion-like properties. Physiologically, αS may populate disordered monomeric, helically folded tetrameric, or membrane-bound oligomeric states. Pathologically, αS may assemble into toxic oligomers and subsequently fibrils, the prion-like transmission of which is implicated in a class of neurodegenerative disorders collectively termed α-synucleinopathies. Notably, αS does not adopt a single “amyloid fold”, but rather exists as structurally distinct amyloid-like conformations referred to as “strains”. The inoculation of animal models with different strains induces distinct pathologies, and emerging evidence suggests that the propagation of disease-specific strains underlies the differential pathologies observed in patients with different α-synucleinopathies. The characterization of αS strains has provided insight into the structural basis for the overlapping, yet distinct, symptoms of Parkinson’s disease, multiple system atrophy, and dementia with Lewy bodies. In this review, we first explore the physiological and pathological differences between conformational states of αS. We then discuss recent studies on the influence of micro-environmental factors on αS species formation, propagation, and the resultant pathological characteristics. Lastly, we review how an understanding of αS conformational properties has been translated to emerging strain amplification technologies, which have provided further insight into the role of specific strains in distinct α-synucleinopathies, and show promise for the early diagnosis of disease.

Structural basis of the 24B3 antibody against the toxic conformer of amyloid β with a turn at positions 22 and 23

Amyloid β-protein (Aβ) oligomers are involved in the early stages of Alzheimer's disease (AD) and antibodies against these toxic oligomers could be useful for accurate diagnosis of AD. We identified the toxic conformer of Aβ42 with a turn at positions 22/23, which has a propensity to form toxic oligomers. The antibody 24B3, developed by immunization of a toxic conformer surrogate E22P-Aβ9-35 in mice, was found to be useful for AD diagnosis using human cerebrospinal fluid (CSF). However, it is not known how 24B3 recognizes the toxic conformation of wild-type Aβ in CSF. Here, we report the crystal structure of 24B3 Fab complexed with E22P-Aβ11-34, whose residues 16–26 were observed in electron densities, suggesting that the residues comprising the toxic turn at positions 22/23 were recognized by 24B3. Since 24B3 bound only to Aβ42 aggregates, several conformationally restricted analogs of Aβ42 with an intramolecular disulfide bond to mimic the conformation of toxic Aβ42 aggregates were screened by enzyme immunoassay. As a result, only F19C,A30homoC-SS-Aβ42 (1) bound significantly to 24B3. These data provide a structural basis for its low affinity to the Aβ42 monomer and selectivity for its aggregate form.

Structure elucidation and toxicological evaluation of cyclic Polyethersulfone oligomers present in extracts of membrane filters

Polyethersulfone (PES) is a widely used polymer in consumer and technical products. An important application is PES membranes used in the biopharmaceutical industry for sterilizing-grade filtration and for filtration of food and beverages. For both uses, detailed information about migrating compounds that can be extracted from the polymeric material into a liquid must be gathered. In the pharmaceutical industry, comprehensive extractables studies are required for contact materials, and the data is used in the qualification of the process equipment. PES is generated via polycondensation, which forms cyclic oligomers as a by-product of the reaction. However, no structural information is available for these cyclic oligomers so far. In this publication, we present the analytical determination of PES cyclic oligomers. Their presence in extracts of PES membrane filters is confirmed. The structure of the PES cyclic trimer is elucidated by X-ray and NMR investigation, obtained as crystals from the sublimation of the PES raw material. A strategy is shown to assess the toxicity of such cyclic oligomers and to derive a permitted daily exposure (PDE). The data will reduce the levels of unknowns in extractables and leachables screenings and supports the risk assessment of PES sterile filters.

Glucose enrichment impair neurotransmission and induce Aβ oligomerization that cannot be reversed by manipulating O-β-GlcNAcylation in the C. elegans model of Alzheimer's disease

Amyloid beta (Aβ) plaques formation and impaired neurotransmission and neuronal behaviors are primary hallmarks of Alzheimer's disease (AD) that are further associated with impaired glucose metabolism in elderly AD's patients. However, the exact role of glucose metabolism on disease progression has not been elucidated yet. In this study, the effect of glucose on Aβ-mediated toxicity, neurotransmission and neuronal behaviors has been investigated using a C. elegans model system expressing human Aβ. In addition to regular diet, worms expressing Aβ were supplemented with different concentrations of glucose and glycerol and 5 mM 2-deoxyglucose to draw any conclusions. Addition of glucose to the growth medium delayed Aβ-associated paralysis, promoted abnormal body shapes and movement, unable to restore impaired acetylcholine neurotransmission, inhibited egg laying and hatching in pre-existing Aβ-mediated pathology. The harmful effects of glucose may associate with an increase in toxic Aβ oligomers and impaired neurotransmission. O-β-GlcNAcylation (O-GlcNAc), a well-known post-translational modification is directly associated with glucose metabolism and has been found to ameliorates the Aβ- toxicity. We reasoned that glucose addition might induce O-GlcNAc, thereby protect against Aβ. Contrary to our expectations, induced glucose levels were not protective. Increasing O-GlcNAc, either with Thiamet-G (TMG) or by suppressing the O-GlcNAcase (oga-1) gene does interfere with and, therefore, reduce Aβ- toxicity but not in the presence of high glucose. The effects of glucose cannot be effectively managed by manipulating O-GlcNAc in AD models of C. elegans. Our observations suggest that glucose enrichment is unlikely to be an appropriate therapy to minimize AD progression.

Wild-Type α-Synuclein and Variants Occur in Different Disordered Dimers and Pre-Fibrillar Conformations in Early Stage of Aggregation.

α-Synuclein is a 140 amino-acid intrinsically disordered protein mainly found in the brain. Toxic α-synuclein aggregates are the molecular hallmarks of Parkinson’s disease. In vitro studies showed that α-synuclein aggregates in oligomeric structures of several 10th of monomers and into cylindrical structures (fibrils), comprising hundred to thousands of proteins, with polymorphic cross-β-sheet conformations. Oligomeric species, formed at the early stage of aggregation remain, however, poorly understood and are hypothezised to be the most toxic aggregates. Here, we studied the formation of wild-type (WT) and mutant (A30P, A53T, and E46K) dimers of α-synuclein using coarse-grained molecular dynamics. We identified two principal segments of the sequence with a higher propensity to aggregate in the early stage of dimerization: residues 36–55 and residues 66–95. The transient α-helices (residues 53–65 and 73–82) of α-synuclein monomers are destabilized by A53T and E46K mutations, which favors the formation of fibril native contacts in the N-terminal region, whereas the helix 53–65 prevents the propagation of fibril native contacts along the sequence for the WT in the early stages of dimerization. The present results indicate that dimers do not adopt the Greek key motif of the monomer fold in fibrils but form a majority of disordered aggregates and a minority (9–15%) of pre-fibrillar dimers both with intra-molecular and intermolecular β-sheets. The percentage of residues in parallel β-sheets is by increasing order monomer disordered dimers pre-fibrillar dimers. Native fibril contacts between the two monomers are present in the NAC domain for WT, A30P, and A53T and in the N-domain for A53T and E46K. Structural properties of pre-fibrillar dimers agree with rupture-force atomic force microscopy and single-molecule Förster resonance energy transfer available data. This suggests that the pre-fibrillar dimers might correspond to the smallest type B toxic oligomers. The probability density of the dimer gyration radius is multi-peaks with an average radius that is 10 Å larger than the one of the monomers for all proteins. The present results indicate that even the elementary α-synuclein aggregation step, the dimerization, is a complicated phenomenon that does not only involve the NAC region.

Monosialotetrahexosylganglioside Promotes Early Aβ42 Oligomer Formation and Maintenance.

The aggregation of the amyloid beta (Aβ) peptide is associated with Alzheimer's disease (AD) pathogenesis. Cell membrane composition, especially monosialotetrahexosylganglioside (GM1), is known to promote the formation of Aβ fibrils, yet little is known about the roles of GM1 in the early steps of Aβ oligomer formation. Here, by using GM1-contained liposomes as a mimic of the neuronal cell membrane, we demonstrate that GM1 is a critical trigger of Aβ oligomerization and aggregation. We find that GM1 not only promotes the formation of Aβ fibrils but also facilitates the maintenance of Aβ42 oligomers on liposome membranes. We structurally characterize the Aβ42 oligomers formed on the membrane and find that GM1 captures Aβ by binding to its arginine-5 residue. To interrogate the mechanism of Aβ42 oligomer toxicity, we design a new liposome-based Ca2+-encapsulation assay and provide new evidence for the Aβ42 ion channel hypothesis. Finally, we determine the toxicity of Aβ42 oligomers formed on membranes. Overall, by uncovering the roles of GM1 in mediating early Aβ oligomer formation and maintenance, our work provides a novel direction for pharmaceutical research for AD.

A rare natural lipid induces neuroglobin expression to prevent amyloid oligomers toxicity and retinal neurodegeneration.

Most neurodegenerative diseases such as Alzheimer's disease are proteinopathies linked to the toxicity of amyloid oligomers. Treatments to delay or cure these diseases are lacking. Using budding yeast, we report that the natural lipid tripentadecanoin induces expression of the nitric oxide oxidoreductase Yhb1 to prevent the formation of protein aggregates during aging and extends replicative lifespan. In mammals, tripentadecanoin induces expression of the Yhb1 orthologue, neuroglobin, to protect neurons against amyloid toxicity. Tripentadecanoin also rescues photoreceptors in a mouse model of retinal degeneration and retinal ganglion cells in a Rhesus monkey model of optic atrophy. Together, we propose that tripentadecanoin affects p‐bodies to induce neuroglobin expression and offers a potential treatment for proteinopathies and retinal neurodegeneration.

Quantitative detection of α-Synuclein and Tau oligomers and other aggregates by digital single particle counting

The pathological hallmark of neurodegenerative diseases is the formation of toxic oligomers by proteins such as alpha-synuclein (aSyn) or microtubule-associated protein tau (Tau). Consequently, such oligomers are promising biomarker candidates for diagnostics as well as drug development. However, measuring oligomers and other aggregates in human biofluids is still challenging as extreme sensitivity and specificity are required. We previously developed surface-based fluorescence intensity distribution analysis (sFIDA) featuring single-particle sensitivity and absolute specificity for aggregates. In this work, we measured aSyn and Tau aggregate concentrations of 237 cerebrospinal fluid (CSF) samples from five cohorts: Parkinson’s disease (PD), dementia with Lewy bodies (DLB), Alzheimer’s disease (AD), progressive supranuclear palsy (PSP), and a neurologically-normal control group. aSyn aggregate concentration discriminates PD and DLB patients from normal controls (sensitivity 73%, specificity 65%, area under the receiver operating curve (AUC) 0.68). Tau aggregates were significantly elevated in PSP patients compared to all other groups (sensitivity 87%, specificity 70%, AUC 0.76). Further, we found a tight correlation between aSyn and Tau aggregate titers among all patient cohorts (Pearson coefficient of correlation r = 0.81). Our results demonstrate that aSyn and Tau aggregate concentrations measured by sFIDA differentiate neurodegenerative disease diagnostic groups. Moreover, sFIDA-based Tau aggregate measurements might be particularly useful in distinguishing PSP from other parkinsonisms. Finally, our findings suggest that sFIDA can improve pre-clinical and clinical studies by identifying those individuals that will most likely respond to compounds designed to eliminate specific oligomers or to prevent their formation.

Neglected N-Truncated Amyloid-β Peptide and Its Mixed Cu-Zn Complexes.

Amyloid-β (Aβ) peptides are involved in Alzheimer's disease (AD) development. The interactions of these peptides with copper and zinc ions also seem to be crucial for this pathology. Although Cu(II) and Zn(II) ions binding by Aβ peptides has been scrupulously investigated, surprisingly, this phenomenon has not been so thoroughly elucidated for N-truncated Aβ4-x-probably the most common version of this biomolecule. This negligence also applies to mixed Cu-Zn complexes. From the structural in silico analysis presented in this work, it appears that there are two possible mixed Cu-Zn(Aβ4-x) complexes with different stoichiometries and, consequently, distinct properties. The Cu-Zn(Aβ4-x) complex with 1:1:1 stoichiometry may have a neuroprotective superoxide dismutase-like activity. On the other hand, another mixed 2:1:2 Cu-Zn(Aβ4-x) complex is perhaps a seed for toxic oligomers. Hence, this work proposes a novel research direction for our better understanding of AD development.

Cucurbit[7]uril Inhibits Islet Amyloid Polypeptide Aggregation by Targeting NTerminus Hot Segments and Attenuates Cytotoxicity.

Two "hot segments" within an islet amyloid polypeptide are responsible for its self-assembly, which in turn is linked to the decline of β-cells in type 2 diabetes (T2D). A readily available water-soluble, macrocyclic host, cucurbit[7]uril (CB[7]), effectively inhibits islet amyloid polypeptide (IAPP) aggregation through ion-dipole and hydrophobic interactions with different residues of the monomeric peptide in its random-coil conformation. A HSQC NMR study shows that CB[7] likely modulates IAPP self-assembly by interacting with and masking major residues present in the "hot segments" at the N terminus. CB[7] also prevents the formation of toxic oligomers and inhibits seed-catalyzed fibril proliferation. Importantly, CB[7] recovers rat insulinoma cells (RIN-m) from IAPP-assembly associated cytotoxicity.

Novel Stilbene-Nitroxyl Hybrid Compounds Display Discrete Modulation of Amyloid Beta Toxicity and Structure.

Several neurodegenerative diseases are driven by misfolded proteins that assemble into soluble aggregates. These “toxic oligomers” have been associated with a plethora of cellular dysfunction and dysregulation, however the structural features underlying their toxicity are poorly understood. A major impediment to answering this question relates to the heterogeneous nature of the oligomers, both in terms of structural disorder and oligomer size. This not only complicates elucidating the molecular etiology of these disorders, but also the druggability of these targets as well. We have synthesized a class of bifunctional stilbenes to modulate both the conformational toxicity within amyloid beta oligomers (AβO) and the oxidative stress elicited by AβO. Using a neuronal culture model, we demonstrate this bifunctional approach has the potential to counter the molecular pathogenesis of Alzheimer’s disease in a powerful, synergistic manner. Examination of AβO structure by various biophysical tools shows that each stilbene candidate uniquely alters AβO conformation and toxicity, providing insight towards the future development of structural correctors for AβO. Correlations of AβO structural modulation and bioactivity displayed by each provides insights for future testing in vivo. The multi-target activity of these hybrid molecules represents a highly advantageous feature for disease modification in Alzheimer’s, which displays a complex, multifactorial etiology. Importantly, these novel small molecules intervene with intraneuronal AβO, a necessary feature to counter the cycle of dysregulation, oxidative stress and inflammation triggered during the earliest stages of disease progression.

The Small Molecule GAL-201 Efficiently Detoxifies Soluble Amyloid β Oligomers: New Approach towards Oral Disease-Modifying Treatment of Alzheimer's Disease.

Soluble amyloid β (Aβ) oligomers have been shown to be highly toxic to neurons and are considered to be a major cause of the neurodegeneration underlying Alzheimer’s disease (AD). That makes soluble Aβ oligomers a promising drug target. In addition to eliminating these toxic species from the patients’ brain with antibody-based drugs, a new class of drugs is emerging, namely Aβ aggregation inhibitors or modulators, which aim to stop the formation of toxic Aβ oligomers at the source. Here, pharmacological data of the novel Aβ aggregation modulator GAL-201 are presented. This small molecule (288.34 g/mol) exhibits high binding affinity to misfolded Aβ1-42 monomers (KD = 2.5 ± 0.6 nM). Pharmacokinetic studies in rats using brain microdialysis are supportive of its oral bioavailability. The Aβ oligomer detoxifying potential of GAL-201 has been demonstrated by means of single cell recordings in isolated hippocampal neurons (perforated patch experiments) as well as in vitro and in vivo extracellular monitoring of long-term potentiation (LTP, in rat transverse hippocampal slices), a cellular correlate for synaptic plasticity. Upon preincubation, GAL-201 efficiently prevented the detrimental effect on LTP mediated by Aβ1-42 oligomers. Furthermore, the potential to completely reverse an already established neurotoxic process could also be demonstrated. Of particular note in this context is the self-propagating detoxification potential of GAL-201, leading to a neutralization of Aβ oligomer toxicity even if GAL-201 has been stepwise removed from the medium (serial dilution), likely due to prion-like conformational changes in Aβ1-42 monomer aggregates (trigger effect). The authors conclude that the data presented strongly support the further development of GAL-201 as a novel, orally available AD treatment with potentially superior clinical profile.

Molecular Dynamics Simulations Indicate Aromaticity as a Key Factor in the Inhibition of IAPP(20-29) Aggregation.

Islet amyloid polypeptide (IAPP) is a 37-residue amyloidogenic hormone implicated in the progression of Type II Diabetes (T2D). T2D affects an estimated 422 million people yearly and is a comorbidity with numerous diseases. IAPP forms toxic oligomers and amyloid fibrils that reduce pancreatic β-cell mass and exacerbate the T2D disease state. Toxic oligomer formation is attributed, in part, to the formation of interpeptide β-strands comprised of residues 20-29 (IAPP(20-29)). Flavonoids, a class of polyphenolic natural products, have been found experimentally to inhibit IAPP aggregate formation. Many of these small flavonoids differ structurally only slightly; the influence of functional group placement on inhibiting the aggregation of the IAPP(20-29) has yet to be explored. To probe the role of small-molecule structural features that impede IAPP aggregation, molecular dynamics simulations were performed to observe trimer formation on a model fragment of IAPP(20-29) in the presence of morin, quercetin, dihydroquercetin, epicatechin, and myricetin. Contacts between Phe23 residues were critical to oligomer formation, and small-molecule contacts with Phe23 were a key predictor of β-strand reduction. Structural properties influencing the ability of compounds to disrupt Phe23-Phe23 contacts included aromaticity and carbonyl and hydroxyl group placement. This work provides key information on design considerations for T2D therapeutics that target IAPP aggregation.

Interaction of Therapeutic d-Peptides with Aβ42 Monomers, Thermodynamics, and Binding Analysis.

The aggregation of the amyloid-β (Aβ) peptide is a major hallmark of Alzheimer's disease. This peptide can aggregate into oligomers, proto-fibrils, and mature fibrils, which eventually assemble into amyloid plaques. The peptide monomers are the smallest assembly units and play an important role in most of the individual processes involved in amyloid fibril formation, such as primary and secondary nucleation and elongation. Several d-peptides have been confirmed as promising candidates to inhibit the aggregation of Aβ into toxic oligomers and fibrils by specifically interacting with monomeric species. In this work, we elucidate the structural interaction and thermodynamics of binding between three d-peptides (D3, ANK6, and RD2) and Aβ42 monomers by means of enhanced molecular dynamics simulations. Our study derives thermodynamic energies in good agreement with experimental values and suggests that there is an enhanced binding for D3 and ANK6, which leads to more stable complexes than for RD2. The binding of D3 to Aβ42 is shown to be weakly exothermic and mainly entropically driven, whereas the complex formation between the ANK6 and RD2 with the Aβ42 free monomer is weakly endothermic. In addition, the changes in the solvent-accessible surface area and the radius of gyration support that the binding between Aβ42 and d-peptides is mainly driven by electrostatic and hydrophobic interactions and leads to more compact conformations.

Constructing conformational library for amyloid-β42 dimers as the smallest toxic oligomers using two CHARMM force fields

The study of amyloid-β (Aβ) dimers as the smallest toxic aggregates in the human brain suffering from Alzheimer's disease is of great interest. The structural characterization of the dimers, which is important to rationally design inhibitors for Aβ dimerization, is limited by the low stability of these species and their high tendency to aggregate into protofibrils and amyloid fibrils. Therefore, an efficient sampling method is needed for the computational study of the Aβ dimers. In this regard, we build a conformational library of the Aβ42 dimers by a new computational protocol; the blockwise excursion sampling (BES); with the CHARMM27 and CHARMM36m force fields. The CHARMM27 overestimates helix content and underestimates β-sheet content, while secondary structure content for the dimers sampled by the CHARMM36m force field is in reasonably consistent with the circular dichroism data. The CHARMM36m force field also generates more Aβ42 dimers in line with experimentally measured collision cross sections values relative to the CHARMM27 force field. Our results demonstrate that the BES is an efficient protocol for fast generating a heterogeneous conformational library of the Aβ42 dimers in agreement with experimental data. Having a reliable structural library for the Aβ42 dimers is very important to identify binding “hot spots” of the dimers versus potential drug candidates using ensemble docking approach.

Revisiting Small Molecule Inhibitors of Amyloid‐β Aggregation

Alzheimer’s disease (AD) is associated with amyloid formation and accumulation in the brain contributing to neurodegeneration. To date, there are no suitable mitigation strategies that stop or slow AD progression. Novel therapeutic approaches applicable at different stages of AD amyloid formation are direly needed. The long‐term goal of this research is to establish effective treatment strategy for AD. Our overall objective is to develop and validate a new proprietary class of anti‐amyloidogenic molecules to impede the formation of Aβ toxic oligomers. The specific aims of our research are 1) to provide a relevant library of small molecules, 2) to identify the key chemical moieties driving their effect in interfering with oligomer formation (structure‐activity relationship). The potency of at least three different series of small molecules to reduce the formation of amyloid fibrils and toxic oligomers was assessed in vitrousing biophysical methods such as Thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM). Based on the screening of 30 newly synthesized molecules, we discovered KN‐21‐48 and MO‐19‐49 as amyloid inhibitors of Aβ1‐40. A preliminary structure‐activity relationship study indicates that compounds KN‐21‐48 and MO‐19‐49 can serve as a scaffold for further modifications in the design of more potent novel small molecules targeting amyloidogenesis in AD. This project may lead to a better understanding of AD by providing a model compound that may confirm or deny the role of amyloidosis in disease progression.

Diabetes Mellitus Drug Discovery: Insights into Targeting Feline and Human Amylin with Small Molecules

In a majority of diabetic feline and human patients, amyloid deposits have been detected in the islets of Langerhans. These deposits originate from islet amyloid polypeptide (IAPP or amylin), a satiety hormone produced and co‐secreted with insulin by beta‐cells. These islet amyloid deposits have been associated with beta‐cell death during the progression of diabetes. Several molecular entities have been shown to inhibit human IAPP aggregation, including silibinin and resveratrol. However, these agents have poor bioavailability and cause multiple pharmacological effects. An effective means to stop or prevent pancreatic amyloidosis in diabetes mellitus with small drug‐like molecules is still not available. The overall objective of this project was to identify selective and general inhibitors of feline and human IAPP fibril formation. The potency of molecules to reduce the formation of feline and human IAPP fibrils and toxic oligomers was monitored in vitrousing biophysical methods such as Thioflavin T (ThT) fluorescence assays, dynamic light scattering (DLS), and transmission electron microscopy (TEM). We then assessed the potency of the JF analogs on both feline and human IAPP, which differ by only 4 amino acids. JF‐19‐033 selectively inhibited feline IAPP fibril formation. JF‐19‐034 selectively inhibited human IAPP fibril formation. In contrast, JF‐19‐029 non‐selectively inhibited all of the human and feline IAPP peptides we tested. By doing this, we identified selective and non‐selective inhibitors of IAPP fibril formation. This project represents a phenotypic‐based drug design strategy for modulating feline and human IAPP fibril formation based on amino acid variations by small molecules. As a result, it has the potential to point towards new therapeutic strategies for type 2 diabetes.