Identification of the molecular interaction site of amyloid beta peptide by using a fluorescence assay.

Beta-amyloid peptides (Abeta) are the main protein components of neuritic plaques and are important in the pathogenesis of Alzheimer's disease. It is reported that Abeta itself is not toxic; however, it becomes toxic to neuronal cells once it has aggregated into amyloid fibrils by peptide-peptide interactions. In this study, to specify the molecular mechanism of aggregation, a novel fluorescence assay was designed. For this purpose, possible partial peptides (38 types of 5-mer) were synthesized on solid-phase. The molecular interactions were examined by a fluorescence probe possessing Lys-Leu-Val-Phe-Phe (KLVFF) as a molecular recognition site. KLVFF is known to be a minimum sequence for formation of the Abeta aggregate. A specific interaction was observed between labeled and immobilized KLVFF. It suggests that the aggregation of Abeta was controlled by the recognition of KLVFF itself by hydrophobic and electrostatic interactions.     

Designing peptide inhibitors for oligomerization and toxicity of Alzheimer's beta-amyloid peptide

Convergent biochemical and genetic evidence suggests that the formation of beta-amyloid (Abeta) deposits in the brain is an important and, probably, seminal step in the development of Alzheimer's disease (AD). Recent studies support the hypothesis that Abeta soluble oligomers are the pathogenic species that prompt the disease. Inhibiting Abeta self-oligomerization could, therefore, provide a novel approach to treating the underlying cause of AD. Here, we designed potential peptide-based aggregation inhibitors containing Abeta amino acid sequences (KLVFF) from part of the binding region responsible for Abeta self-association (residues 16-20), with RG-/-GR residues added at their N- and C-terminal ends to aid solubility. Two such peptides (RGKLVFFGR, named OR1, and RGKLVFFGR-NH2, named OR2) were effective inhibitors of Abeta fibril formation, but only one of these peptides (OR2) inhibited Abeta oligomer formation. Interestingly, this same OR2 peptide was the only effective inhibitor of Abeta toxicity toward human neuroblastoma SH-SY5Y cells. Our data support the idea that Abeta oligomers are responsible for the cytotoxic effects of Abeta and identify a potential peptide inhibitor for further development as a novel therapy for AD.     

Structure-function relationships for inhibitors of beta-amyloid toxicity containing the recognition sequence KLVFF.

Beta-amyloid (Abeta), the primary protein component of Alzheimer's plaques, is neurotoxic when aggregated into fibrils. We have devised a modular strategy for generating compounds that inhibit Abeta toxicity. These compounds contain a recognition element, designed to bind to Abeta, linked to a disrupting element, designed to interfere with Abeta aggregation. On the basis of this strategy, a hybrid peptide was synthesized with the sequence KLVFF (residues 16-20 of Abeta) as the recognition element and a lysine hexamer as the disrupting element; this compound protects cells in vitro from Abeta toxicity [Pallitto, M. M., et al. (1999) Biochemistry 38, 3570]. To determine if the length of the disrupting element could be reduced, peptides were synthesized that contained the KLVFF recognition element and a sequence of one to six lysines as disrupting elements. All compounds enhanced the rate of aggregation of Abeta, with the magnitude of the effect increasing as the number of lysines in the disrupting element increased. The greatest level of protection against Abeta toxicity was achieved with compounds containing disrupting elements of three or more lysines in sequence. A peptide with an anionic disrupting element, KLVFFEEEE, had activity similar to that of KLVFFKKKK, in both cellular toxicity and biophysical assays, whereas a peptide with a neutral polar disrupting element, KLVFFSSSS, was ineffective. Protective compounds retained activity even at an inhibitor:Abeta molar ratio of 1:100, making these some of the most effective inhibitors of Abeta toxicity reported to date. These results provide critical insight needed to design more potent inhibitors of Abeta toxicity and to elucidate their mechanism of action.     

Synthesis and evaluation of stilbenylbenzoxazole and stilbenylbenzothiazole derivatives for detecting beta-amyloid fibrils

This paper describes a novel series of stilbenylbenzoxazole (SBO) and stilbenylbenzothiazole (SBT) derivatives for beta-amyloid specific binding probes. These 24 compounds were synthesized and evaluated by competitive binding assay against beta-amyloid 1-42 (Abeta42) aggregates using [(125)I]TZDM. All the derivatives displayed higher binding affinities with K(i) value in the subnanomolar range (0.10-0.74 nM) than Pittsburgh Compound-B (PIB) (0.77 nM). Among these derivatives, SBT-2, 5-fluoroethoxy-2-{4-[2-(4-methylaminophenyl)vinyl]phenyl}benzothiazole, showed lowest K(i) value (0.10 nM). In conclusion, the preliminary results suggest that these compounds are implying a possibility as a probe for detection of Abeta fibrils in Alzheimer's disease (AD) patients.     

Recognition sequence design for peptidyl modulators of beta-amyloid aggregation and toxicity

beta-Amyloid (Abeta), the primary protein component of Alzheimer's plaques, is neurotoxic when aggregated into fibrils. We have devised a modular strategy for generating compounds that inhibit Abeta toxicity, based on linking a recognition element for Abeta to a disrupting element designed to interfere with Abeta aggregation. One such compound, with the 15-25 sequence of Abeta as the recognition element and a lysine hexamer as the disrupting element, altered Abeta aggregation kinetics and protected cells from Abeta toxicity [Ghanta et al. (1996) J. Biol. Chem. 271, 29525]. To optimize the recognition element, peptides of 4-8 residues composed of overlapping sequences within the 15-25 domain were synthesized, along with hybrid compounds containing those recognition sequences coupled to a lysine hexamer. None of the recognition peptides altered Abeta aggregation kinetics and only two, KLVFF and KLVF, had any protective effect against Abeta toxicity. The hybrid peptide KLVFF-KKKKKK dramatically altered Abeta aggregation kinetics and aggregate morphology and provided significantly improved protection against Abeta toxicity compared to the recognition peptide alone. In contrast, FAEDVG-KKKKKK possessed only modest inhibitory activity and had no marked effect on Abeta aggregation. The scrambled sequence VLFKF was nearly as effective a recognition domain as KLVFF, suggesting the hydrophobic characteristics of the recognition sequence are critical. None of the cytoprotective peptides prevented Abeta aggregation; rather, they increased aggregate size and altered aggregate morphology. These results suggest that coupling recognition with disrupting elements is an effective generalizable strategy for the creation of Abeta inhibitors. Significantly, prevention of Abeta aggregation may not be required for prevention of toxicity.     

Inhibitors of fibril formation and cytotoxicity of beta-amyloid peptide composed of KLVFF recognition element and flexible hydrophilic disrupting element.

Beta-Amyloid peptide (Abeta) is the main protein components of neuritic plaques and its neurotoxicity would be exposed by formation of aggregate. The aggregation inhibitors composed of an Abeta recognition element (KLVFF) and a flexible hydrophilic disrupting element (aminoethoxy ethoxy acetate and aspartate) are designed and chemically synthesized. The inhibitory effects are examined by a pigment binding assay using Congo red or thioflavin T. The present compounds suppress the formation of aggregate, and the compound DDX3 is an especially effective inhibitor. In addition, the synthesized compounds efficiently suppress the cytotoxicity of Abeta against IMR-32 neuroblastoma cells in vitro.     

Amyloid-beta(1-42) rapidly forms protofibrils and oligomers by distinct pathways in low concentrations of sodium dodecylsulfate

Alzheimer's disease (AD) is characterized by large numbers of senile plaques in the brain that consist of fibrillar aggregates of 40- and 42-residue amyloid-beta (Abeta) peptides. However, the degree of dementia in AD correlates better with the concentration of soluble Abeta species assayed biochemically than with histologically determined plaque counts, and several investigators now propose that soluble aggregates of Abeta are the neurotoxic agents that cause memory deficits and neuronal loss. These endogenous aggregates are minor components in brain extracts from AD patients and transgenic mice that express human Abeta, but several species have been detected by gel electrophoresis in sodium dodecylsulfate (SDS) and isolated by size exclusion chromatography (SEC). Endogenous Abeta aggregation is stimulated at cellular interfaces rich in lipid rafts, and anionic micelles that promote Abeta aggregation in vitro may be good models of these interfaces. We previously found that micelles formed in dilute SDS (2 mM) promote Abeta(1-40) fiber formation by supporting peptide interaction on the surface of a single micelle complex. In contrast, here we report that monomeric Abeta(1-42) undergoes an immediate conversion to a predominant beta-structured conformation in 2 mM SDS which does not proceed to amyloid fibrils. The conformational change is instead rapidly followed by the near quantitative conversion of the 4 kDa monomer SDS gel band to 8-14 kDa bands consistent with dimers through tetramers. Removal of SDS by dialysis gave a shift in the predominant SDS gel bands to 30-60 kDa. While these oligomers resemble the endogenous aggregates, they are less stable. In particular, they do not elute as discrete species on SEC, and they are completed disaggregated by boiling in 1% SDS. It appears that endogenous oligomeric Abeta aggregates are stabilized by undefined processes that have not yet been incorporated into in vitro Abeta aggregation procedures.     

The oxidative stress metabolite 4-hydroxynonenal promotes Alzheimer protofibril formation

4-Hydroxynonenal (4-HNE), formed as a consequence of oxidative stress, exists at increased concentrations in Alzheimer's disease (AD) patients and is found in amyloid beta peptide (Abeta) plaques associated with AD. Although it remains an open question as to whether oxidative stress is a causative factor or a consequence of AD, we show here that 4-HNE, putatively resulting from the peroxidation of lipids, covalently modifies Abeta, triggering its aggregation. These Abeta modifications result from 1,4 conjugate addition and/or Schiff base formation, they occur at multiple locations on a single Abeta peptide, and they result in covalent cross-linking of Abeta peptides. The consequence of these reactions is that 4-HNE accelerates the formation of Abeta protofibrils while inhibiting the production of straight, mature fibrils. Recent studies implicating Abeta oligomers and protofibrils in the neurotoxic process that ultimately leads to AD suggest that the Abeta aggregates induced by 4-HNE may be important in the pathogenesis of AD. These results provide further incentive to understand the role of oxidative stress and small-molecule Abeta modifications in sporadic AD.     

Immunotherapeutic strategies for prevention and treatment of Alzheimer's disease.

Pathologic examination in Alzheimer's disease (AD) shows a significant correlation between beta-amyloid peptide (AbetaP) deposition and the clinical severity of dementia. Formation of beta-amyloid (Abeta) is a complex kinetic and thermodynamic process, dependent on peptide-peptide interactions that may be modulated by other proteins. We found that site-directed antibodies toward peptide EFRH sequences 3-6 of the N-terminal region of AbetaP suppress in vitro formation of Abeta and dissolve already-formed fibrillar amyloid. These so-called chaperone-like properties of monoclonal antibodies led to the development of a new immunologic approach to AD treatment. The immunization procedure, based on phages displaying the EFRH epitope as antigen, induced anti-AbetaP antibodies that recognized the whole AbetaP and exhibited antiaggregating properties similar to those of antibodies obtained by injection of Abeta fibrils. Production and performance of anti-beta-amyloid antibodies in the transgenic mouse model of AD showed that these antibodies may be delivered from the periphery to the central nervous system, preventing the formation of Abeta and dissolving already-present aggregates. Moreover, immunization with Abeta protected transgenic mice from the learning and age-related memory deficits that occur in AD. These data support the hypotheses that Abeta plays a central role in AD and that site-directed antibodies that modulate Abeta conformation may provide immunotherapy of the disease.     

(18)F]-labeled isoindol-1-one and isoindol-1,3-dione derivatives as potential PET imaging agents for detection of beta-amyloid fibrils

In this study a novel series of isoindol-1-one and isoindol-1,3-dione derivatives for beta-amyloid-specific binding agents is described. Twelve compounds were synthesized and evaluated via a competitive binding assay with [(125)I]TZDM against beta-amyloid 1-42 (Abeta42) aggregates. Two new [(18)F]-labeled isoindole derivatives were synthesized and evaluated as potential beta-amyloid imaging probes based on the in vivo pharmacokinetic profiles. The preliminary results suggest that these [(18)F]18b and [(18)F]18c are promising positron emission tomography (PET) imaging probes for studying accumulation of Abeta fibrils in the brains of Alzheimer's disease (AD) patients.     

Development of novel beta-amyloid probes based on 3,5-diphenyl-1,2,4-oxadiazole

In the search for novel probes for the imaging in vivo of beta-amyloid plaques in Alzheimer's disease (AD) brain, we have synthesized and evaluated a series of 3,5-diphenyl-1,2,4-oxadiazole (DPOD) derivatives. The affinity for beta-amyloid plaques was assessed by an in vitro-binding assay using pre-formed synthetic Abeta42 aggregates. The new series of DPOD derivatives showed excellent affinity for Abeta aggregates with K(i) values ranging from 4 to 47nM. In biodistribution experiments using normal mice, [(125)I]12, [(125)I]13, [(125)I]14, and [(125)I]15 examined displayed sufficient uptake for imaging, ranging from 2.2 to 3.3% ID/g. But the washout of the four ligands from the brain was relatively slow. Although additional modifications are necessary to improve the uptake and rapid clearance of non-specifically bound radiotracers, the DPOD pharmacophore with high-binding affinity for Abeta aggregates may be useful as a backbone structure to develop novel beta-amyloid imaging agents.     

Modulating amyloid-β aggregation: The effects of peptoid side chain placement and chirality

Alzheimer’s disease (AD) is characterized by the buildup of insoluble aggregated amyloid-β protein (Aβ) into plaques that accumulate between the neural cells in the brain. AD is the sixth leading cause of death in the United States and is the only cause of death among the top ten that cannot currently be treated or cured (Alzheimer’s Association, 2011; Selkoe, 1996). Researchers have focused on developing small molecules and peptides to prevent Aβ aggregation; however, while some compounds appear promising in vitro, the research has not resulted in a viable therapeutic treatment. We previously reported a peptoid-based mimic (JPT1) of the peptide KLVFF (residues 16–20 of Aβ) that modulates Aβ40 aggregation, specifically reducing the total number of fibrillar, β-sheet structured aggregates formed. In this study, we investigate two new variants of JPT1 that probe the importance of aromatic side chain placement (JPT1s) and side chain chirality (JPT1a). Both JPT1s and JPT1a modulate Aβ40 aggregation by reducing total β-sheet aggregates. However, JPT1a also has a pronounced effect on the morphology of fibrillar Aβ40 aggregates. These results suggest that Aβ40 aggregation may follow a different pathway in the presence of peptoids with different secondary structures. A better understanding of the interactions between peptoids and Aβ will allow for improved design of AD treatments.     

Specific compositions of amyloid-beta peptides as the determinant of toxic beta-aggregation

Alzheimer's disease (AD) may be caused by toxic aggregates formed from amyloid-beta (Abeta) peptides. By using Thioflavin T, a dye that specifically binds to beta-sheet structures, we found that highly toxic forms of Abeta-aggregates were formed at the initial stage of fibrillogenesis, which is consistent with recent reports on Abeta oligomers. Formation of such aggregates depends on factors that affect both nucleation and elongation. As reported previously, addition of Abeta42 systematically accelerated the nucleation of Abeta40, most likely because of the extra hydrophobic residues at the C terminus of Abeta42. At Abeta42-increased specific ratio (Abeta40: Abeta42 = 10: 1), on the other hand, not only accelerated nucleation but also induced elongation were observed, suggesting pathogenesis of early-onset AD. Because a larger proportion of Abeta40 than Abeta42 was still required for this phenomenon, we assumed that elongation does not depend only on hydrophobic interactions. Without any change in the C-terminal hydrophobic nature, elongation was effectively induced by mixing wild type Abeta40 with Italian variant Abeta40 (E22K) or Dutch variant (E22Q). We suggest that Abeta peptides in specific compositions that balance hydrophilic and hydrophobic interactions promote the formation of toxic beta-aggregates. These results may introduce a new therapeutic approach through the disruption of this balance.     

Beta-amyloid treatment of two complementary P301L tau-expressing Alzheimer's disease models reveals similar deregulated cellular processes

Alzheimer's disease (AD) is characterized by Abeta peptide-containing plaques and tau-containing neurofibrillary tangles (NFTs). Both pathologies have been combined by crossing Abeta plaque-forming APP mutant mice with NFT-forming P301L tau mutant mice or by stereotaxically injecting beta-amyloid peptide 1-42 (Abeta42) into brains of P301L tau mutant mice. In cell culture, Abeta42 induces filamentous tau aggregates. To understand which processes are disrupted by Abeta42 in the presence of tau aggregates, we applied comparative proteomics to Abeta42-treated P301L tau-expressing neuroblastoma cells and the amygdala of P301L tau transgenic mice stereotaxically injected with Abeta42. Remarkably, a significant fraction of proteins altered in both systems belonged to the same functional categories, i.e. stress response and metabolism. We also identified model-specific effects of Abeta42 treatment such as differences in cell signaling proteins in the cellular model and of cytoskeletal and synapse associated proteins in the amygdala. By Western blotting (WB) and immunohistochemistry (IHC), we were able to show that 72% of the tested candidates were altered in human AD brain with a major emphasis on stress-related unfolded protein responsive candidates. These data highlight these processes as potentially important initiators in the Abeta42-mediated pathogenic cascade in AD and further support the role of unfolded proteins in the course of AD.     

Abeta immunotherapy leads to clearance of early, but not late, hyperphosphorylated tau aggregates via the proteasome

Amyloid-beta (Abeta) plaques and neurofibrillary tangles are the hallmark neuropathological lesions of Alzheimer's disease (AD). Using a triple transgenic model (3xTg-AD) that develops both lesions in AD-relevant brain regions, we determined the consequence of Abeta clearance on the development of tau pathology. Here we show that Abeta immunotherapy reduces not only extracellular Abeta plaques but also intracellular Abeta accumulation and most notably leads to the clearance of early tau pathology. We find that Abeta deposits are cleared first and subsequently reemerge prior to the tau pathology, indicative of a hierarchical and direct relationship between Abeta and tau. The clearance of the tau pathology is mediated by the proteasome and is dependent on the phosphorylation state of tau, as hyperphosphorylated tau aggregates are unaffected by the Abeta antibody treatment. These findings indicate that Abeta immunization may be useful for clearing both hallmark lesions of AD, provided that intervention occurs early in the disease course.     

Amyloid-beta protofibrils differ from amyloid-beta aggregates induced in dilute hexafluoroisopropanol in stability and morphology

The brains of Alzheimer's disease (AD) patients contain large numbers of amyloid plaques that are rich in fibrils composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that fibrillar Abeta and especially soluble Abeta aggregates are important in the etiology of AD. Recent reports also stress that amyloid aggregates are polymorphic and that a single polypeptide can fold into multiple amyloid conformations. Here we demonstrate that Abeta-(1-40) can form soluble aggregates with predominant beta-structures that differ in stability and morphology. One class of aggregates involved soluble Abeta protofibrils, prepared by vigorous overnight agitation of monomeric Abeta-(1-40) at low ionic strength. Dilution of these aggregation reactions induced disaggregation to monomers as measured by size exclusion chromatography. Protofibril concentrations monitored by thioflavin T fluorescence decreased in at least two kinetic phases, with initial disaggregation (rate constant approximately 1 h(-1)) followed by a much slower secondary phase. Incubation of the reactions without agitation resulted in less disaggregation at slower rates, indicating that the protofibrils became progressively more stable over time. In fact, protofibrils isolated by size exclusion chromatography were completely stable and gave no disaggregation. A second class of soluble Abeta aggregates was generated rapidly (<10 min) in buffered 2% hexafluoroisopropanol (HFIP). These aggregates showed increased thioflavin T fluorescence and were rich in beta-structure by circular dichroism. Electron microscopy and atomic force microscopy revealed initial globular clusters that progressed over several days to soluble fibrous aggregates. When diluted out of HFIP, these aggregates initially were very unstable and disaggregated completely within 2 min. However, their stability increased as they progressed to fibers. Relative to Abeta protofibrils, the HFIP-induced aggregates seeded elongation by Abeta monomer deposition very poorly. The techniques used to distinguish these two classes of soluble Abeta aggregates may be useful in characterizing Abeta aggregates formed in vivo.     

Abeta40 protects non-toxic Abeta42 monomer from aggregation

Abeta40 and Abeta42 are the predominant Abeta species in the human body. Toxic Abeta42 oligomers and fibrils are believed to play a key role in causing Alzheimer's disease (AD). However, the role of Abeta40 in AD pathogenesis is not well established. Emerging evidence indicates a protective role for Abeta40 in AD pathogenesis. Although Abeta40 is known to inhibit Abeta42 fibril formation, it is not clear whether the inhibition acts on the non-toxic monomer or acts on the toxic Abeta42 oligomers. In contrast to conventional methods that detect the appearance of fibrils, in our study Abeta42 aggregation was monitored by the decreasing NMR signals from Abeta42 monomers. In addition, differential NMR isotope labelling enabled the selective observation of Abeta42 aggregation in a mixture of Abeta42 and Abeta40. We found Abeta40 monomers inhibit the aggregation of non-toxic Abeta42 monomers, in an Abeta42/Abeta40 ratio-dependent manner. NMR titration revealed that Abeta40 monomers bind to Abeta42 aggregates with higher affinity than Abeta42 monomers. Abeta40 can also release Abeta42 monomers from Abeta42 aggregates. Thus, Abeta40 likely protects Abeta42 monomers by competing for the binding sites on pre-existing Abeta42 aggregates. Combining our data with growing evidence from transgenic mice and human genetics, we propose that Abeta40 plays a critical, protective role in Alzheimer's by inhibiting the aggregation of Abeta42 monomer. Abeta40 itself, a peptide already present in the human body, may therefore be useful for AD prevention and therapy.     

Reduction of Abeta accumulation in the Tg2576 animal model of Alzheimer's disease after oral administration of the phosphatidyl-inositol kinase inhibitor wortmannin.

The abnormal accumulation of the amyloid beta protein (Abeta) has been implicated as an early and critical event in the etiology and pathogenesis of Alzheimer's disease (AD). Compounds that reduce Abeta accumulation may therefore be useful therapeutically. In cell-based screens we detected a significant reduction in Abeta concentration after treatment with the phosphatidylinositol kinase inhibitors wortmannin and LY294002. To determine the effect of this class of compounds on in vivo Abeta accumulation, we administered wortmannin to the Tg2576 mouse model of AD. Oral administration of wortmannin over four months resulted in a significant, non-overlapping 40%-50% reduction in the number of senile plaques, one of the pathological hallmarks of AD. Sandwich ELISA analysis of formic acid extractable Abeta in the brain of treated animals indicates that both Abeta40 and the longer, more amyloidogenic form of the peptide, Abeta42, were significantly reduced. These data provide the first direct evidence that compounds identified by their ability to reduce Abeta concentration in vitro can reduce Abeta accumulation and deposition in the brain, thus establishing a basic paradigm for the identification and evaluation of additional compounds that lower Abeta accumulation.     

Alternate aggregation pathways of the Alzheimer beta-amyloid peptide. An in vitro model of preamyloid

Deposition of amyloid-beta (Abeta) aggregates in the brain is a defining characteristic of Alzheimer's disease (AD). Fibrillar amyloid, found in the cores of senile plaques, is surrounded by dystrophic neurites. In contrast, the amorphous Abeta (also called preamyloid) in diffuse plaques is not associated with neurodegeneration. Depending on the conditions, Abeta will also form fibrillar or amorphous aggregates in vitro. In this present study, we sought to characterize the properties of the amorphous aggregate and determine whether we could establish an in vitro model for amorphous Abeta. CD data indicated that Abeta40 assembled to form either a beta-structured aggregate or an unfolded aggregate with the structured aggregate forming at high peptide concentrations and the unstructured aggregate forming at low Abeta40 levels. The critical concentration separating these two pathways was 10 microm. Fluorescence emission and polarization showed the structured aggregate was tightly packed containing peptides that were not accessible to water. Peptides in the unstructured aggregate were loosely packed, mobile, and accessible to water. When examined by electron microscopy, the structured aggregate appeared as protofibrillar structures and formed classic amyloid fibrils over a period of several weeks. The unstructured aggregate was not visible by electron microscopy and did not generate fibrils. These findings suggest that the unstructured aggregate shares many properties with the amorphous Abeta of AD and that conditions can be established to form amorphous Abeta in vitro. This would allow for investigations to better understand the relationship between fibrillar and amorphous Abeta and could have significant impact upon efforts to find therapies for AD.     

Aurones serve as probes of beta-amyloid plaques in Alzheimer's disease

A novel series of aurone derivatives for in vivo imaging of beta-amyloid plaques in the brain of Alzheimer's disease (AD) were synthesized and characterized. When in vitro binding studies using Abeta(1-42) aggregates were carried out with aurone derivatives, they showed high binding affinities for Abeta(1-42) aggregates at the K(i) values ranging from 1.2 to 6.8 nM. When in vitro plaque labeling was carried out using double transgenic mice brain sections, the aurone derivatives intensely stained beta-amyiloid plaques. Biodistribution studies in normal mice after i.v. injection of the radioiodinated aurones displayed high brain uptake (1.9-4.6% ID/g at 2 min) and rapid clearance from the brain (0.11-0.26% ID/g at 60 min), which is highly desirable for amyloid imaging agents. The results in this study suggest that novel radiolabeled aurones may be useful amyloid imaging agents for detecting beta-amyloid plaques in the brain of AD.