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Ah fibril formation; an identifiable nucleating species has yet be isolated.

Direct observation has been made difficult by the small size of the h

peptide, which has an effective hydrodynamic radius of 4 nm [98–100], and

by the apparent low abundance of nucleating species due to the low

probability of their formation. Such species would be formally akin to

an enzyme transition state that is usually kinetically inferred or sometimes

trapped with certain kinds of inhibitor. In disaggregated, ultrafiltered

(20 nm pore size) preparations, less than 1% of the molar peptide concentration is inferred to be present as ‘‘seeds’’ or nuclei determined by the

kinetics of fibril formation [101].

There is, however, ‘‘hard’’ evidence for the involvement of transient

species in h-peptide fibril formation. Recent atomic force microscopy

(AFM) [93,94,102–104] and electron microscopic observations [105,106]

have characterized rope like species intermediate between nucleation and

fibril extension. Designated as protofibrils, these species appear to anneal

and to wind around each other. Such a model is consistent with oriented

x-ray fiber diffraction patterns of a triple or higher helix of h sheets

producing a h-helix quaternary fibril structure [107–113]. These protofibrils are on-pathway intermediates in amyloid fibril formation [93,94]

containing h-sheet structure. They are negative with respect to thioflavin

T and apparently toxic to cultured cells [105]. A stiffer, more compact

fibril species (type I) is eventually formed from the initial type II fibril,

10–20 nm high. The dominant fibril form observed is dependent on the

environmental conditions and the initial conformational state of the

peptide [103]. Recent AFM studies indicate that amylin fibrils grow

bidirectionally, from both ends at roughly equal rates [114]. Branched

fibrils and heterogeneous catalysis along the edge of the fibrils [70,71]

have also been observed [94].

The variety of structures of h-peptide species observed by electron

microscopy and by AFM suggest that different surfaces would be available

to bind inhibitors on each species; moreover, the ability of a given inhibitor

to block the fibrillization reaction should depend on the peptide species

present in a particular situation. The rate-limiting species in vivo is

unknown at present. It is also possible for more than one fibrillization

pathway to operate concurrently, depending on the in vitro and in vivo

reaction conditions. A host of molecules have been claimed to inhibit hpeptide amyloid fibril formation on the basis of a variety of assays for

activity. The diversity of structures is represented in Figure 2. Their efficacy

is in general low (IC50 tens of micromolar or higher), corresponding

roughly to the order of magnitude of the amount of peptide present in

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Figure 2 Reported inhibitors of Ah aggregation: 1, nicotinamide [156]; 2, Anthranilates [59]; 3, N-alkyl-N-methylpiperidinium bromides

[157]; 4, benzothiazoles [U.S. patent 6,001,331]; 5, Congo Red [142]; 6, melatonin [158]; 7, PPI-558 (Praecis Pharmaceuticals, Inc. patent

WO 9628471); 8, anthracyclines (IDOX) [159]; 9, aza-anthracyclones (WO 9832754-A); 10, iminoaza-anthracyclinones (WO 9832754-A);

11, acridinones (U.S. patent 5,972,956); 12, naphthyl monoazo compounds [U.S. patent 5,955,472]; 13, porphyrins [160]; 14,

naphthalenes (Japanese patent 090954222, Teijin KK); 15, rifamycins [161]; 16, rifampicins [161]; 17, alkylsulfonates/sulfates [162].

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

most assays, implying a 1:1 compound-to-peptide stoichiometry. In most

cases, however, the stoichiometry for inhibition has not been determined.

A high concentration of Ah peptide is generally used to overcome the

unfavorable kinetics of multiple peptides interacting to form a nucleus

capable of supporting the addition of monomeric peptide. Such reactions

exhibit a lag phase until the nucleus is formed (Fig. 3, curve c). Inhibitors

can affect either the lag phase, the maximal extent of the reaction, or both

(Fig. 3, curve d). Unless both the nucleation and extension reactions are

monitored, inhibitors prolonging the lag phase are poorly distinguished

from those blocking extension from the nucleus, thus muddying any

structure–activity relationships. Quantitative treatment of the reaction

has been proposed to mathematically separate the nucleation and extension reactions [62,115]. Distinguishing true nucleation from various exponential growth mechanisms is actually quite difficult, requiring precise rate

Figure 3 Effect of seeding and inhibitors on aggregation reaction. The lag phase

(curve c) is characteristic of reactions in which formation of nuclei for

polymerization is an unfavorable process. Addition of preformed nuclei or

‘‘seeds’’ (curve a) abolishes the lag phase. Inhibitors may affect the formation of

nuclei and influence either the lag phase, the extension of the nuclei changing the

growth phase, or both (curve d). The inhibitor example (curve d) acts more

strongly at nuclei formation than on the slope or plateau level of the growth phase.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

determinations of the first 10% of the reaction over a range of reactant

concentrations [116]. Such measurements have not been achieved with the

h peptide.

An extension reaction from a nucleus (Fig. 3, curve a) is pseudo–first

order in peptide concentration and thus more easily analyzed. Inhibitors of

extension would be expected to decrease the reaction rate and/or extent

(Fig. 3, curve b). The nature of the nucleus, however, is a variable as well.

Preformed fibrils can act as one kind of nucleus, adding monomers at the

ends or laterally, which can give rise to more complex kinetics [70].

Branching vs linear addition reactions can be distinguished in some

situations by dynamic light-scattering methods. Another type of nucleus

seems to be present in solubilized aqueous peptide solutions that can pass

through a filter having a pore size of 200 nm, but not 20 nm [101] and

displays linear kinetics of fibril extension [117]. These species are present in

too low an abundance to be observed directly, though they are detectable


Distinguishing the preformed and endogenous nucleus forms is

problematic. The behavior of the accretion of soluble peptide onto AD

plaques in tissue sections [118] or onto sonicated fibrils [119] is kinetically

similar to that of spontaneous soluble nuclei. However, the endogenous

soluble nuclei are not equivalent on a molecular level to fibril or AD plaque

nuclei, since molecules such as Congo Red inhibit endogenous soluble

nuclei extension at 0.25 AM [97], while over 700 AM is required to block

accretion of monomer h peptide onto fibrils or plaques [119]. An assemblycompetent form of the h peptide, h10–35, has been shown to interact with

fibrils and plaques [120] and to adopt a particular conformation in solution

as determined by NMR spectroscopy [121], although no high affinity

inhibitors of this accretion reaction have been reported. Alternatively,

low abundance conformational forms of monomeric peptide may be the

actively associating form of the peptide to endogenous seeds, accounting

for the high (micromolar) amounts of peptide required in vitro for the

extension reaction. These forms may be more abundant in biological

systems, allowing fibril formation to occur at the low bulk concentrations

(nanomolar) of Ah peptides found in vivo.

As a result of the confusion over the identity of nucleating h-peptide

species, prenucleation events remain poorly defined. A variety of methods

possessing different degrees of resolution have been employed to look at

these early stages in fibril formation. Chemical [122] and enzymatic

(transglutaminase) [123,124] cross-linking, electron microscopy (EM)

[105,106,125], AFM [93,94,102,103], ultracentrifugation, dynamic light

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

scattering, and fluorescence resonance energy transfer (FRET) with modified beta peptides [89,105] have probed the oligomerization process

preceding nucleus formation but have not yielded definitive structural

information on the species present or the extent of their participation in

nucleus formation. Difficulties arise from the small size (4.3 kDa) of the

monomeric peptide unit, the simultaneous presence of multiple species of

peptide, both conformational and association states, and their transient

nature (since they rapidly form amyloid fibrils as their concentrations

increase). As specific inhibitors of early stages in fibril growth are discovered, peptide species will be better defined, particularly if the intermediates

can be trapped and their structures determined.

Fibril extension from nuclei preformed under defined conditions

has been characterized through a series of nucleus-dependent kinetic

assays. The process of fibril formation from a nucleus in equilibrium with

soluble, mostly monomeric peptide has proved much more amenable to

study than the formation of the nucleus itself. Fibrillar species are readily

detected by growth in size (filtration, sedimentation, static light scattering–turbidity), amyloid-specific reactivity with the optical probes Congo

Red and thioflavin S and T, and by EM and AFM. Endogenous soluble

nuclei or seeds form in aqueous solution, accumulating slowly at low

temperature. Brief treatment with denaturants, organic solvents, and

treatment with neat trifluoroacetic acid (TFA) or concentrated formic

acid breaks down these seed structures, restoring the lag period of

unseeded fibril formation.

The processes of both seed formation and fibril extension are

dependent on temperature and on peptide concentration, with 37jC

being required for establishing equilibrium within 24 h with 30 AM

h1–40. A full description of the assay system may be found elsewhere

[97,117]. A 4 h reaction time is typically within the linear portion of the

time course. This nucleus-dependent assay detects mainly inhibitors that

are substoichiometric with the monomeric peptide, which is present at

high concentration. It is relatively insensitive to inhibitors that target

the monomeric peptide. Whether the inhibitors interact with the growing end of a seed or with a low abundance conformational form of the

h peptide that is competent to add to the seed is difficult to determine at

this time. Similar dose–response curves are obtained for Congo Red as

an inhibitor with either thioflavin T (ThT) fluorescence or filtration of

radioiodinated peptide readouts (Fig. 4) Caveats in the interpretation of

both the ThT and radiometric filtration assays for the evaluation of putative inhibitors are discussed elsewhere [97].

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Figure 4 Inhibition of nucleated fibril extension by Congo Red: h1–40 fibril

formation detected by filtration of either radiolabeled peptide (circles) or

thioflavin T (ThT) reactivity, (triangles) is inhibited by Congo Red with similar

potency. For assay details, see Ref. 97.

The prediction that fibrillization reactions proceeding via different folding pathways governed by different rate-limiting steps could be

subject to different modes of inhibition appears to be substantiated.

The endogenously seeded type of assay identifies types of inhibitor

different from unseeded assays by using light scattering or turbidity

detection. With the exception of the naphthyl monoazo benzo compounds (12) and the acridinone series (11), the molecules reported in

Figure 2 are ineffective (IC50 > 100 AM) in the presence of 30 AM

h1–40 in seeded assays. In particular, short peptide sequences derived from

the h16–25 amyloidogenic core of the h peptide KLVFFA are ineffective under the seeded assay conditions, although many modifications

of this sequence have been studied [126–129], some of which (e.g., 7)

are being developed as therapeutics. Inhibitors effective in the seeded

assay format such as Congo Red are inactive in an accretion assay

onto immobilized fibrils [119]. Rifampicin and daunomycin are very

weakly active against accretion [130].

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.


One of the reported biological effects of h-amyloid and amylin fibrils

is cellular toxicity, inferred in vivo and modeled in various tissue

culture systems. While amyloid fibrils were initially thought to be the

toxic species, it has become increasingly clear that some other entity,

probably soluble oligomers of the h peptide [131,132] that are in

equilibrium with fibrils, are the culprit. Thus, in developing aggregation inhibitors that would be therapeutically useful, it is important to

demonstrate that the nonfibrillar peptide species stabilized by inhibitor

treatment are not toxic to cells. The selection of an appropriate

cellular system is important because the resistance of cell types to

the toxic effects of the Ah peptide varies significantly, often requiring

industrial (50–100 AM) concentrations of peptide or the use of the

nonbiological h25–35 fragment. Mixed neuronal/glial or pure neuronal

embryonic hippocampal or cortical cultures would seem to be the most

relevant cell type, since neuronal cell death and dysfunction are

hallmarks of neurodegenerative disease like AD. Unfortunately, the

embryonic primary cultures are irregularly resistant to the effects of

h1–42 when cell death is monitored. These cultures are heterogeneous

mixtures of neuronal cell types, only some of which seem to be

affected by the Ah peptide. In addition, embryonic mouse neurons

are not the same as the deeply differentiated cells in the brain of an

80-year-old human. Cultured PC12 cell lines have become a favorite

system, with changes in MTT formazan production serving as a

readout. However, the formazan deposition is not related to cell

survival [133–136] and so is not reliable as an indicator of the effects

of amyloid on cell death.

Another prominent site of deposition of h-amyloid fibrils with age

and in AD is within the cerebrovasculature in areas of the brain prone

to parenchymal amyloid deposition [137–139]. The peptide deposits

along the surfaces of the smooth muscle cells of the vascular wall,

resulting in the death of those cells and their replacement by amyloid

fibrils, weakening the vascular wall. Endothelial cells are also affected

[140]. The ‘‘Dutch’’ mutation in the APP precursor protein Q22E,

within the h-peptide sequence, produces a particularly fibrillogenic

and toxic (to smooth muscle cells) peptide associated with primarily

vascular deposition of mutant peptide and hemorrhagic vessel disease

[137]. Thus, in addition to neuronal cells, the brain vascular smooth

muscle cells are a pathologically relevant cell type. While the source of

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

the h peptide in these deposits (brain or smooth muscle cells) is under

debate, the smooth muscle cells in culture generate prodigious amounts

of h peptide and accumulate C-terminal fragments of hAPP [139].

Organotypic cultures of the leptomeningeal blood vessels will accumulate exogenously applied, fluorescently labeled Ah peptide [141]. The

leptomeningeal vascular smooth muscle cells (VSMC) isolated from

either human or canine sources have proved to be reliable indicators for

h-amyloid toxicity. Overnight treatment with 10 AM h1–42 leads to

deposition of fibrillar peptide in ThS-positive strands onto the cell

surface and apoptosis of 70 to 80% of the VSMC assessed by

bisbenzamide staining of condensed nuclei. For this cell type, preformed

fibrils have little effect on cell survival, and the added fibrils remain

scattered over the surface of the culture dish.

Interpreting the effects of amyloid-modulating compounds on

h1–42-induced cellular toxicity and relating the results to in vitro aggregation inhibition is far from straightforward. A number of compounds

are toxic to cells by a variety of routes. Besides interfering with

aggregation, test compounds can block binding to the cell surface,

internalization of h peptide, or any of a myriad of cellular events that

could affect expression of h-peptide toxicity. Lack of effect of a

compound could indicate that it is not blocking the toxic ‘‘site’’ on

the peptide species, that it is not penetrating the cell, or simply that

the compound is adsorbed, sequestered, or metabolized to an inactive

form. In the VSMC system as in other cellular systems [142], Congo

Red blocks both aggregation on the cell surface and h1–42 toxicity at

10 AM, roughly equivalent to the total added peptide concentration.

For optimal effect it must be added either before or along with the h

peptide. Since the IC50 for an antiaggregation effect on 30 AM peptide

in vitro is 0.25 AM, nonspecific adsorption of the compound to

cellular components and to h-peptide fibrils may be mitigating the

effects. Congo Red and other polysulfonate/sulfate polyanions are

known to displace proteins from binding sites on the cell surface

[143]. Congo Red’s practical therapeutic potential is limited because it

does not penetrate the cell membrane or the blood–brain barrier, and

the azo linkages are susceptible to metabolism and carcinogenic

liability. Such difficulties can be addressed by structural modifications

in inhibitors that will likely also improve some of the pharmacokinetic

properties in vivo. However, the connection between cellular effects

and desired in vivo properties of bioavailability and brain penetration

is also not straightforward.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

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