CA2242851A1 - Protein fibril assembly assay - Google Patents

Abstract

The invention is a method for in vitro monitoring of peptide or protein fibril assembly. In order to provide sensitivity at nanomolar concentrations on the order of that observed in vivo, the method makes use of fluorescent energy transfer between closely juxtaposed donor and acceptor fluorophores. Accordingly, the invention requires attaching a donor fluorophore to a fibrillogenic peptide or protein, and attaching an acceptor fluorophore to a second fibrillogenic peptide or protein. The donor and acceptor fluorophores are located on the first and second peptides or proteins so that they are juxtaposed to permit energy transfer between them upon fibril formation. The fluorophore-containing peptides or proteins are mixed in solution at a normal physiological concentration, and fibril formation is monitored by observing the fluorescence energy transfer between the donor and acceptor fluorophores. Mixing is preferably done by providing equimolar amounts of peptides or proteins in a denaturant solution, and monitoring of fibril formation is initiated by diluting out the denaturant.

Description

CA 022428~1 1998-07-13 w ~ 97/a7~a~ PCT/CA9~'0~'5 PROTEIN FIBRIL ASSFMRLY ASSAY

The invention is a method for in vi tro monitoring of ~ protein fibril assembly at physiologically relevant concentrations. The invention i5 readily adaptable for in vitro monitoring of fibril assembly processes associated with various amyloidosis disorders, such as Al~hetm~'s disease, multiple myeloma, rheumatoid arthritis, diabetes, and prion disorders. The invention has particular utility as a screening assay for potential inhibitors of fibril formation, which in turn may be candidates for treatment o~ Alzheimer's disease or other amyloidosis disorders.

Alzheimer's disease is a neurodegenerative disorder of the elderly. While neurodegeneration in Al~h~;m~r's disease begins slowly, its progression follows an exponential course, and it finally culminates in dementia and death (Muller-Hill and Beyreuther, 1989). Key pathological features of Al~h~;m~-'s disease found in the brains of afflicted patients include the following: a) senile plaques, which are extracellular amyloid deposits in close contact with neurons, b) neurofibrillary tangles, which are intraneuronal deposits of paired helical filaments, and c) cerebrovascular amyloid, which are amyloid deposits in the walls of cortical and meningeal vessels (Selkoe, 1991; Katzman and Saitoh, 1991). The link between the symptoms of Alzheimer's disease and its pathology arises from the plaques and neurofibrillary tangles being concentrated around the hippocampus, a region of the brain responsible for memory and learning.
.

A major component of senile plaques and cerebrovascular amyloid is Al~hPtm~r/s ~-amyloid peptide (A~), a 39-43 residue peptide that folds into a ~-sheet structure and assembles into fibrils 60-goA in diameter CA 022428~1 1998-07-13 2 PCT/CA9~ S'5 (Fraser et al., 1993). A~ is a fragment of a much larger integral membrane protein called amyloid precursor protein (APP). APP possesses one transmembrane segment, a large extracellular ~om~; n, a small intracellular ~om~; n, several glycosylation and sulfation sites, and a Kunitz protease inhibitor ~om~; n . A~' is derived from residues 672 to 715 of APP, and it encompasses the C-terminal region of the extracellular ~om~; n of APP and the N-terminal half of the transmembrane ~o~;n of APP.
Other proteins found in senile plaques include: serum complement proteins (Clq, C3d, and C4d), serum amyloid P, ~1-antichymotrypsin, Apo-E, and heparan sulfate proteoglycans. The relative amounts of these proteins in senile plaques are, however, much lower than that of A~.

The other characteristic feature of Alzheimer's disease are neurofibrillary tangles, which are intraneuronal deposits of paired helical filaments.
Paired helical filaments are not as well characterized as senile plaques (Crowther, 1991). The protein components of paired helical filaments discovered so far include:
neuro~ilament protein, a microtubule associated protein (MAP) called tau, MAP2, and MAP5. Recent evidence suggests that paired helical filament formation results, at least in part, from abnormal phosphorylation of tau.

The molecular pathogenesis of Alzheimer's disease is not well established. However, there is accumulating evidence that deposition of A~'-fibrils in senile plaques may account for much of the neurotoxicity of Alzheimer's disease (Selkoe, 1993). Neurons which contact A~-fibrils are dystrophic. Patients that have three copies of the APP gene (i.e. Down's syndrome patients) produce a greater number of senile plaques and develop Alzheimer's disease when they reach their late thirties. Mutations in the APP gene are often seen in cases of familial Alzheimer's disease, and related genetic diseases (Goate CA 022428~1 1998-07-13 W~ 97~7402 P ~CA9~ 5 et al., 1991). A~-fibrils are toxic to neurons in culture (Yankner et al., 1989; Pike et al., 1993).
Alzheimer-type neuropathology is induced in transgenic mice overexpressing a mutant form of APP (Games et al., 1995). These findings all point to A~ as the primary ~ neurotoxic agent o~ Alzheimer's disease. The mechanism of A~ neurotoxicity is also not well understood, but it appears to be related to calcium ion homeostasis in neurons (Mattson et al., 1993).

Two experimental findings that must be reconciled by any proposed model o~ Alzheimer's disease pathogenesis is that A~ is a product of normal cells (Haass et al., 1992), and that it is present in cerebrospinal fluid and blood plasma o~ healthy individuals (Seubert et al., 1992). Experimental evidence suggests that there may be multiple primary causes that ultimately lead to A~
deposition in senile plaques and Alzheimer's disease (Selkoe, 1993). While certain individuals may develop Alzheimer's disease by producing greater amounts o~ A~, others might develop the disease by producing a ~orm o~
A~ that is especially prone to fibril formation. Thus, the concentration of A~ in the brain as well as its intrinsic tendency to aggregate are both integral to the process of A~-fibril deposition in Alzheimer's disease.

Given the possibility that fibril formation by A~
may account for the neurotoxicity in Alzheimer's disease, it follows that molecules that inhibit fibril formation should be good candidates for therapeutic agents for the treatment of Alzheimer's disease. One critical research tool for evaluating therapeutic inhibitors is an assay ~ ~or A~-fibril formation. Current methods that monitor A~
fibril formation employ various biophysical techniques, ~ such as electron microscopy, X-ray diffraction, ~ourier transform infrared (FTIR) spectroscopy, circular dichroism (CD) spectroscopy, and nuclear magnetic CA 022428~1 1998-07-13 WO 97/07402 PCT/CA95.~ C'5 resonance (NMR) spectroscopy. Studies utilizing these biophysical techniques have shown that A~ fibrils possess remarkable intrinsic stability. Even after altering much of the sequence of A~, fibril formation still occurs (Fraser et al., 1992; Hilbich et al., 1992; Fraser et al., 1994). Asp23 of A~, however, is a unique residue;
since when it is replaced with Lys a significant reduction in the stability of the fibril is observed (Fraser et al., 1994). Two regions of the A~ sequence show a high propensity to form ~-structure: residues 9 to 28 (Fraser et al., 1991) and residues 34 to 42 (Halverson et al., 1990). One factor which has a major effect on fibrillogenesis is pH; at low (~ 3) and high (~10) pH, fibrillogenesis by A~ analogues is both slower and less stable (Fraser et al., 1991; 1992). It appears that hydrogen bond, hydrophobic, and electrostatic interactions all act in concert to stabilize the A~
fibril; however, a high resolution structural model of the A~ fibril has yet to be developed.

The studies on the mechanism of fibrillogenesis mentioned have employed methods of measuring fibril formation which require sample concentrations that are 200,000 to 5,000,000-fold higher than the physiological concentration. Fibril assembly is a bimolecular reaction: A~ + (A~)n z (A~)n~l. One distinguishing feature of bimolecular reactions is that both their kinetics and thermodynamics are concentration-dependent.
It follows, therefore, that the concentration of A~ in the brain and cerebrovasculature controls the rate of amyloid fibril formation as well as the intrinsic stability of amyloid plaques. Consequently, the mechanism of fibril assembly at low physiological concentrations may differ from the mechanism that operates at the high concentrations required by the above mentioned biophysical techniques. Any attempt to model Alzheimer's disease in vitro should take into .

CA 022428~1 1998-07-13 W O 97/07402 PCT/CA9~C'S

consideration the relatively low concentrations of A~ in the brain and cerebrovasculature.

- Another limitation of using biophysical techniques, such as electron microscopy, FTIR, CD, and X-ray di~fraction, to follow fibril formation is the technical complexity of such techniques. Specialized training and involved procedures are required; thus, limiting the usefulness of these techniques as screening tests for fibrillogenesis inhibitors.

In contrast to prior techniques, the invention provides a very sensitive fluorescence technique ~or monitoring fibrillogenesis by A~ at concentrations close to the physiological concentration of A~ in the brain.
This fluorescence techni~ue can be adapted into a rapid, technically simple, standardized assay to simultaneously screen many compounds that could potentially inhibit fibril formation.

The skilled person will appreciate that the method as applied to A~ fibrillogenesis has general application for the assaying of fibrillogenesis associated with other amyloidosis disorders as mentioned above.

Accordingly, the invention provides a method for in vitro monitoring of peptide or protein fibril assembly, comprising the steps of:

1) attaching a donor ~luorophore to a first fibrillogenic peptide or protein;

2) attaching an acceptor ~luorophore to a second fibrillogenic peptide or protein, the donor and acceptor fluorophores being located on said first and second peptides or proteins so that they are juxtaposed to permit energy transfer between them upon fibril CA 022428~1 1998-07-13 W O 97/07402 PCT/CA9~ 55 formation;

3) mixing the first and second fluorophore-containing peptides or proteins in solution at an approximately normal physiological concentration; and 4) monitoring formation of fibrils by observing the occurrence of fluorescence energy transfer between the donor and acceptor fluorophores upon excitation of the donor fluorophores.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows fluorescence emission spectra of fibrils composed of Trp-A~(9-25) and AEDANS-A~(9-25).

Figure 2 is a graph showing pH dependence of fibril formation monitored by fluorescent fibrillogenesis assay, and attenuation of the titration curve in the presence of 0.5 M NaCl.

Figure 3 is a graph of the kinetics of mnnom~r fibril exchange.

Figure 4 is a graph showing the promotion of fibril formation by heparin as monitored by the fluorescence assay of the invention.

The invention will be described in relation to a specific assay for A~ fibril assembly. The following description while specific for A~ fibrillogenesis illustrates the general principles of the invention which may be used to assay fibril assem-bly processes in other amyloidosis disorders.

Develop~nent of a fluorescent fibrillogenesis assay.
Fluorescence spectroscopy is a very sensitive CA 022428~1 1998-07-13 W O 9~J074~2 PCT/CA9~ '5 technique, and depending on the quantum yield of the fluorophore, the technique may be sensitive down to the nAnom~lar concentration range. The physiological - concentration of A~ in the brain is estimated to be in the nanomolar range; therefore, the sensitivity of ~1uorescence spectroscopy is in principle sufficient to detect physiological concentrationS of A~. Unfortunately, fluorescence cannot be used to ~Am;ne fibril formation by native A~ because the only intrinsic fluorophore present in A~ is the phenolic side chain of TyrlO, and its very low quantum yield is ;nA~e~uate ~or sensitive measurements. Extrinsic fluorescent groups, however, can be chemically attached to A~, and the techni~ue of nonradiative fluoresence energy transfer can be used to measure fibril assembly by A~ in the nanomolar concentration range.

The principle of fluore~cence energy transfer is that when appropriate donor and acceptor fluorophores are close in space, light energy absorbed by the donor can be transferred to the acceptor, and the efficiency of energy transfer depends on the distance separating the donor and acceptor (see Fairclough and Cantor, 1978). The following example outlines the general method of energy transfer measurements. The fluorescent amino acid tryptophan, absorbs light of 281 nm, and after absorption it emits light of 337 nm. The fluorescent compound 5-acetylethyl-di~minonAphthalene-l-sulfonic acid (AEDANS), on the other hand, absorbs light of 281 nm very poorly but it absorbs light of 337 nm very strongly; after absorption AEDANS
emits light of 490 nm. Since the absorption band of AEDANS coincides with the emission band of Trp, Trp and AEDANS can be used as donor and acceptor, respectively, in a fluorescence energy transfer experiment. To ~ illustrate the method consider a solution contA;n;ng a mixture of Trp and AEDANS, if the mixture is irradiated with light of 281 nm then the wavelength of the emitted CA 022428~1 1998-07-13 W O 97/07402 PCT/CA~6/Q0~C5 light depends on the distance separating the Trp and AEDANS fluorophores. If the two fluorophores are close in space (~ 30 A) then the emitted light will be of 490 nm pre~om;n~ntly; on the other hand, if the chromophores are well separated in space the emitted light will be of 337 nm pre~om;n~ntly. Using Trp and AEDANS as the donor-acceptor pair, a method for monitoring fibril formation by A~ using nonradiative fluorescence energy transfer was devised.

The fluorescent fibrillogenesis assay of the invention uses a fragment of A~ corresponding to residues 9 to 25. This fragment of A~ forms fibrils that display similar morphology, pH-dependence, X-ray diffraction pattern, and secondary structure as the full length A~
molecule (Fraser et al., 1994). Adapting the assay procedure to the full length A~ molecule is very straightforward and simple.

Two forms of the fibrillogenic fragment A~(9-25), SEQ ID NO:1, were synthesized: one form had a Trp residue appended to the N-terminus of A~(9-25) (denoted Trp-A~(9-25)SEQ ID NO:2), and a second form (denoted AEDANS-A~(9-25)SEQ ID NO:3) had a cysteine residue appended to the N-terminus of A~(9-25), and the AEDANS
group chemically linked to the sulfhydryl side chain of cysteine (Table 1). The basis of the method is that when Trp-A~(9-25) is mixed with AEDANS-A~(9-25), fibrils composed of both forms of A~ will assemble, and in the ~ibril state the Trp and AEDANS groups will be closer in space than in the nonfibril state. Since fluorescence energy transfer between Trp and AEDANS increases when the two ~luorophores are close in space, the ef~iciency of energy transfer between Trp and AEDANS will increase as Trp-A~ (9-25) and AEDANS-A~ (9-25) assemble into fibrils.
At a separation distance of 22 A energy transfer between Trp and AEDANS is easily measurable (Fairclough and CA 022428~1 1998-07-13 WO 971074n2 PCT/CA9~ r5 _g _ Cantor, 1978; the upper limit of energy transfer is ~40A).

~ TABLE 1 Peptide Amino acid sequence A~(9-25) ~Y~v~QKLVFFAEDVG (SEQ ID NO:1) Trp-A~(9-25) Ac-~W Y~v~QKLVFFAEDVG-CONH2 (SEQ ID NO:2) AEDANS-A~(9-25) Ac-C(AEDANS)G~Y~v~QKLVFFAEDVG-CONH2 (SEQ ID NO:3) Trp and AEDANS are relatively large chemical structures. There is a possibility, therefore, that introduction of these large groups into A~ may disrupt or alter fibril formation through steric mechanisms. For this reason, it was decided to place the fluorescent groups at the N-terminus of the A~ (9-25) sequence rather than at an internal position, because placement in the interior could potentially disrupt certain interactions which are known to stabilize fibril formation (Halverson et al., 1990; Hilbich et al., 1992; Fraser et al., 1994).
In addition, a glycine residue was inserted between the fluorescent group and the rest of the A~ (9-25) sequence.
Glycine is the smallest amino acid, it lacks a side chain, and it should act like a flexible tether for the fluorescent group; therefore, as the fluorescent-labeled A~ (9-25) molecule assembles into fibrils the flexible glycine tether will act like a hinge and allow the fluorescent group to be pushed out of the way if steric clashes occur with other parts of the fibril.

For the fluorescence method of the invention to operate as intended, the fluorescent-A~ molecules must be prevented from forming fibrils before they are mixed CA 022428~1 1998-07-13 WO 97/07402 PCT/CA9~ 5S~

together. One approach to achieving this objective is to mix the peptides in solid lyophilized form before dissolving them in buffer. The shortcoming of this method, however, is that the relative amounts of each peptide will have to be determined using dry weights, which is a very inaccurate method. An alternative approach is to first dissolve the two peptides in a solution containing a denaturant that prevents fibril formation, and then to initiate the fibril assembly process by diluting out the denaturant. This method will involve: a) preparing concentrated stock solutions of Trp-A~ and AEDANS-A~ in denaturant and accurately determining the peptide concentrations by W absorbance using the absorption coefficients of Trp and AEDANS, b) mixing appropriate amounts of each peptide solution to produce equimolar mixtures of both peptides in denaturant solution, c) initiate fibril assembly by diluting out the denaturant, and d) simultaneously starting to monitor the evolution of fluorescence energy transfer in the fluorimeter. This method should be very rapid and it should allow evaluation of the kinetics and thermo-dynamics of fibril assem.bly. The kinetic measurement will be limited only by the dead-time required for diluting out the denaturant, which for m~nl~l mixing is around 10 seconds. If the kinetics of fibril assem.bly occur too rapidly using the m~nll~l mixing method, then this method can easily be adapted into a stopped-flow fluorimetric method which has a dead-time of 30 milliseconds.

As regards the choice of denaturant, one possibility is to use formic acid which is known to prevent fibril assembly (Halverson et al., 1990). Initiating fibril assem~ly, however, would then require neutralization of the acid, which in turn would cause an increase in ionic strength of the solution. Thus fibril assembly at low ionic strengths cannot be explored using an acid as the denaturant. In addition, by placing the peptides in acid CA 022428~1 1998-07-13 W O 97/07402 P ~/CA~S~

solutions the risks of acid hydrolysis, deamidations, and formylations exist. Therefore, formic acid is not an ideal denaturant for the purposes of the invention. The organic solvent 40~ trifluoroethanol has been shown to maintain A~ in a soluble monomeric helical state at very ~ high peptide concentrations and at pH values between 1.3 and 10 (Barrow and Zagorski, 1991). Accordingly, this solvent was chosen as the preferred denaturant because it does not cause any harmful side reactions, it is nonviscous, so mixing is e~ficient; and it is volatile allowing for easy ~ ov~l.

The basic protocol of the fluorescence method of the invention using the described A~ analogs is: 1) Trp-A~(9-25) and AEDANS-A~(9-25) are dissolved separately in 40~ trifluoroethanol at millimolar concentrations.
Under these conditions the A~ analogues will adopt mono~riC helical conformations. 2) After determining the concentration of the stock peptide solutions by W
absorbance, an equimolar mixture of the peptides at micromolar concentration is prepared. 3) 20 to 30 ~l of the equimolar peptide mixture is delivered into a cuvette in the fluorimeter which contains 3 ml of aqueous buffer, after rapid mixing, fluorescence emission between 300 nm to 550 nm is monitored (excitation ~ = 281 nm).

Induction of energy transfer by fibril formation.
Fibrils composed of Trp-A~(9-25) alone, AEDANS-A~(9-25) alone, and equimolar mixtures of the two peptides were formed by diluting concentrated solutions of the peptides dissolved in 40~ TFE into aqueous buffer.
The fluorescence emission spectrum of these peptide preparations are shown in Figure 1. The emission of the Trp fluorophore is significantly quenched in the mixed ~ fibril preparation compared to the Trp-emission of fibrils composed of Trp-A~(9-25) alone. The AEDANS-emission, on the other hand, is significantly CA 022428~l l998-07-l3 WO 97/07402 PCT/CA~Gi'~SC5 enhanced in the mixed fibril preparation, relative to the emission of fibrils composed of AEDANS-A~(9-25) alone.
Donor quenching of Trp and the sensitized emission of AEDANS are typical features of energy transfer.

pH dependence of fibril fo~ation.
It is known that fibril formation is pH dependent.
To study this e~fect using fluorescence energy transfer, spectra were collected at varying pH. AEDANS intensities ~rom 425-525 nm were integrated and this value was expressed as a ratio versus the integrated intensities o~
Trp from 340-375 nm. The degree of fibril formation would thus be directly proportional to this acceptor/donor ratio. The advantages of this manipulation are two-fold: 1) taking the ratios ~; m; n; shes the within experiment errors such as noise in the intensity measurements, and 2) results can now be compared between experiments because the acceptor/donor ratio is concentration-independent and it depends only on the fraction of A~t9-25) molecules in multimeric states.

Fibril formation reached a m,i~;mllm at pH 5 and decreased both towards more acidic and more basic pH
(Figure 2). Basal acceptor/donor ratios occurred below pH 2 in the acidic limb and above pH 8 in the basic limb of the titrations suggesting very few fibrils were present beyond these values. The pH dependence of fibril formation was modelled as a sequential equilibrium between 4 ionic species, and the data were fit to the following equation (I) which relates the acceptor/donor ratio to the concentrations of each ionic species and 3 pKa constants:
(I) ¦'10 ~ pH-pK i)i~lt~eii~e2 + 10(-pH-p~l-pK2)il~ eli e3 + 1o~ -pl~ l-pK 2-pEC3~2elirle4 + 1o(-3pH)b~eli el ~cccptor ~ ~io 10 + 10( pH p~l-p}C2~ + 1o(-plCl-plC2-p~3~ + 10(-3pH) CA 022428~l l998-07-l3 W O 971~740~ PCT~CAg~ C'~

The fitted equation gave apparent pKa's of 3.98, 5.86 and 7.62.

The pH titration in the presence of 500 mM NaCl showed a dramatic decrease in the level of m~; mllm fibrillogenesis (Figure 2). The salt titration curve at pH 5.25 ~Pm~n~trated the inverse relationship between NaCl concentration and fibril formation (Figure 2 inset).

The effect of pH on fibrillogenesis was also studied using electron microscopy. The analysis indicates that equimolar mixtures of Trp/AEDANS-A~(9-25) give rise to fibrils that are several mm long and approximately 60 A
in diameter. The morphology of these fibrils are very similar to fibrils formed by A~(11-25) (Fraser et al., 1994) and by full-length A~ (Fraser et al., 1991). The amount of fibrils in the micrograph~ at three dif~erent pH values 2, 5 and 8 paralleled the observations made using fluorescence, with a great number of fibrils present at pH 5 and few at pH 2 and pH 8. Furthermore, continued aging o~ the fibrils for 1 week showed a time-dependent change in morphology from loose short aggregates to well-structured, long fibrils with a "braided" appearance. These braided fibrils were most evident in the pH 5 samples.

Kinetics of exchange between mo~nm~s and fibrils.
Fibrils composed of Trp-A~(9-25) alone and AEDANS-A~(9-25) alone were formed separately, aged for 24 hrs, mixed, and then fluorescence emission spectra of the mixed fibril preparation were acquired at timed intervals for the next 8 hrs. The acceptor/donor ratio as a function of time after mixing is shown in Figure 3.

Fluorescent fibrillogenesis assay.
The successful development of a fluorescent fibrillogenesis assay depends on two prerequisites: a) CA 022428~1 1998-07-13 W O 97/07402 PCT/CA96,'~C~'5 the extrinsic fluorescent groups must not interfere with the process of fibril formation, and b) significant energy transfer must occur between donor and acceptor fluorophores in the fibril state. The data presented show that both prerequisites have been met. The fluorescent-labelled A~(9-25) peptides formed fibrils, as observed by electron microscopy, that had very similar morphology to fibrils formed by native A~ peptides and A~
fragments (Fraser et al., 1991; Fraser et al., 1994).
Significant donor qu~nrhing of Trp and sensitized emission of AEDANS were observed with fibrils composed of equimolar mixtures of Trp- and AEDANS-A~(9-25), compared to ~ibrils composed of Trp-A~(9-25) alone and AEDANS-A~(9-25) alone (Figure 1). These features are typical of energy transfer processes; therefore, supporting the conclusion that energy transfer was induced by fibril formation in the mixed fibril preparation. The dependence of the acceptor/donor ratio on pH (Figure 2) correlated with the electron microscopic observations of the number of fibrils present in solutions of Trp- and AEDANS-A~(9-25) at different values of pH. This correlation cross-validates the fluorescent fibrillogenesis assay with electron microscopy, and it indicates that the acceptor/donor ratio is a ~uantitative measure of the fraction of A~(9-25) molecules present in the fibril state.

pH dependence of f~ibrillogenesis FTIR and electron microscopy studies have established that A~ fibrillogenesis is very pX dependent;
thus, it was concluded that fibrillogenesis is stabilized, at least in part, by electrostatic interactions (Fraser et al., 1991; Fraser et al., 1994).
However, the number and type of ionizable groups involved in the electrostatic interaction has not been determined.
The sensitive and quantitative nature of the fluorescent fibrillogenesis assay allows such quantitation of the pH

CA 022428~l l998-07-l3 W O 97/07402 P ~CA~ ~S

dependence. The simplest model (equation I) that accurately describes the pH effects shown in Figure 2 is a sequential equilibrium between 4 ionic species, where ~ each species differ in the ionization state of three chemical groups. The ~itted equation gave apparent pKa's - of 3.98, 5.86 and 7.62. The apparent pKa of 3.98 is suggestive of carboxyl groups on Asp and Glu. The apparent pKa's of 5. 86 and 7.62 is suggestive of imidazole groups on His. In light of this information one can conclude that fibrillogenesis in A~ (9-25) is m~;m~l when His13, His 14, Glull, Glu22 and Asp23 are in their ionized states. The involvement of Lysl6 cannot be assessed because fibril formation does not occur around pH 10 where titration of Lys sidechain occurs.

As regards the mechanism of electrostatic stabilization of fibrillogenesis, two different scenarios could apply: the pH dependence could be caused by either isoelectric precipitation or formation of salt bridges.
In the case of isoelectric precipitation, the ~im~l fibrillogenesis at pH 5.00 (Figure 2) is a result of the peptide showing a greater tendency to associate at its isoelectric point. Alternatively, the pH dependence could be caused by formation of imidazole-carboxylate salt bridges between His side~h~; n~ and sidech~i ns of Asp and/or Glu. To differentiate between these two possibilities the effect of counter ion screening by NaCl was ~m; ned~ If the pH dependence of fibrillogenesis is an isoelectric effect, then addition of NaCl to the solution should screen the repulsive interactions between peptides at pH values that are away from the isoelectric point causing a bro~n;ng of the pH titration curve.
If, on the other hand, the pH dependence is caused by formation of imidazole-carboxylate salt bridges, then ~ addition of NaCl will screen these attractive interactions and the pH titration curve will be attenuated. Addition of 0.5 M NaCl resulted in CA 022428~l l998-07-l3 attenuation of the pH titration curve, supporting the conclusion that imidazole-carboxylate salt bridges contribute to the stability of the fibril formed by A~(9-25). This conclusion was further substantiated by ~m~n~trating that NaCl titration causes a reduction in fibrillogenesis (Figure 2 inset). In addition, since two imidazole titrations were detected, it can also be concluded that there are two stabilizing imidazole-carboxylate salt bridges per monQm~r Kinetics of exchange between monom~rs and fibrils.
In the ~ch~nge experiments, fibrils of Trp-A~(9-25) and AEDANS-A~(9-25) were formed separately and then mixed. Upon mixing these fibrils, little if any energy transfer was expected because ~ibrils would be composed of either Trp-A~(9-25) or AEDANS-A~(9-25), but not both.
Interaction between fluorophores would be limited by fibril structure. This indeed was the case; ;mm~ately after mixing, the acceptor/donor ratio was only slightly greater than the basal ratio (Figure 3). However, the acceptor/donor ratio increased signi~icantly over the next 8 hrs, which suggested that fibrils, though insoluble, existed in a dynamic state in which monomers were in constant exchange between fibrils. The kinetics of exchange was ade~uately fit to two exponentials, which had relaxation times of 21 seconds and 77 minutes (Figure 3).

The use of amyloid-enhancing factors to promote fibril formation under physiological conditions.
Studies of the pH dependence of A~ fibrillogenesis have revealed that fibrils do not readily form at the physiological pH of 7.00. Amyloid-enhancing factors such as heparin are known, however, to promote fibril formation under physiological conditions (Fraser et al., 1993). Thus, by using the fluorescent fibrillogenesis assay in the presence of an amyloid-enhancing factor, A~

CA 022428~l l998-07-l3 W O 97/07402 PCT/CA9~ 5 ~ibrillogenesis can be investigated at the physiological pH of 7.00. As ~hown in Figure 4, increasing the amount of heparin present results in an increased level o~
fluorescence energy transfer as evidenced by a decrease in donor fluorescence. The data shown in Figure 4 were - obtained using a concentration of Trp/AEDANS-A~(9-25) of 100 nM, and the buf~er was phosphate-buffered saline, pH
7.00.

The invention is a new technique for monitoring fibrillogenesis by A~. Using this technique, it has been ~Pmon~trated that fibril formation by Trp-A~(9-25) and AEDANS-A~(9-25) is stabilized by 2 imidazole-carboxylate salt bridges. It has also been ~Pmon~trated that monom~rs that compose the fibril are not kinetically trapped in the fibril state, and that they exchange with the soluble fraction. The exchange kinetics are multiphasic. This information will be extremely valuable when designing pharmacological agents that inhibit fibril formation. By obt~in;ng a greater underst~n~;ng of the fibril assembly process under physiological conditions, a better idea of which features of the process are the most susceptible to attack can be obtained and exploited.

The st~n~rdized fluorescence assay for fibrillogenesis of the invention should also be of immediate value for screening compounds that have potential for being inhibitors of fibril formation.

MAT~212 T~T-.S AND I~sl~~Ol~S
Peptide synthesis and fluorescent labeling Peptides were synthesized, by the solid phase method on the Milligen 9050~ peptide synthesizer, as peptide-amides using Rink-resin (Advanced Chemtech). An active ester coupling procedure, employing pentafluorophenyl esters of 9-fluorenylmethoxycarbonyl amino acids, was used. The N-termini was acetylated with acetic anhydride.
-CA 022428~l l998-07-l3 W O 97/07402 PCT/CA~ '5 The peptides were cleaved from the resin with 95:2.5: 2. 5 trifluoroacetic acid: thioanisole: eth~n~;thiol mixture.
The peptides were purified by C18 reverse phase chromatography, and peptide identity was confirmed by FAB
mass spectrometry and amino acid analysis. Peptide purity was assessed by analytical C18 reverse phase chromatography using the Pharmacia FPLC system.

5-Acetylethyl~; ~m; nonaphthalene-1-sulfonic acid (AEDANS) labelling was perfonmed by incubation of 1 mM
Cys-contA;n;ng peptide with 5 mM (1,5)-IAEDANS (Molecular Probes) in 50 mM Tris-HCl, 6 M guanidine hydrochloride, pH 8.0 for 18 hrs. AEDANS-labelled peptide was separated from unreacted peptide and 1,5-IAEDANS by Cl8 reverse phase chromatography.

Preparation of peptide stock solutions The lyophilised Trp-A,B(9-25) was dissolved in a 2 ml solution of 40~ (v/v) trifluoroethanol (TFE) cont~;n;ng 10 mM acetic acid. This stock solution was diluted 1:10 with 7.5 M guanidine hydrochloride (GdnHCl) and the peptide concentration was determined via tryptophan absorbance at 280 nm with an absorbance coefficient (~) of 5690 M~1cm~1 (~lhochl 1967).

Lyophilised AEDANS-A,~ (9-25) was dissolved in a 2ml solution of 40~ (v/v) TFE adjusted to pH 9 with ammonium hydroxide. Hudson and Weber have shown that ~ for AEDANS
in 40~ ethanol is 6500 M~lcm~l and the absorbance A~ is 338.0 nm. It was found that the difference in absorbance between AEDANS in ethanol and TFE is less than 1~, well within experimental error; therefore, an ~ of 6500 M~1cm~
were used in all concentration determinations of AEDANS-A~(9-25) in 40~ TFE.

Absorbance measurements were made on a Perkin Elmer Lambda 3B~ spectrophotometer. The stock solutions were CA 022428~1 1998-07-13 WO 97/07402 PCT/CA9C,'~ ~'=5 ~tored at -20 C.

Electron Microscopy ~ Negatively stained ~ibrils were prepared by floating charged pioloform, carbon-coated grids on peptide solutions (0.025 mg/mL Trp:A~(9-25) and 0.025 mg/mL
AEDANS:A~(9-25), pH2-pH8). These solutions were pre-aged 24 h and 1 week. To control pH, the peptide solutions were made using a bu~er of 1 mM borate, 1 mM citrate and 1 mM phosphate. After the grids were blotted and air-dried, the samples were stained with 1~ (w/v) phosphotungstic acid which was prepared using the same borate, citrate, phosphate buf~er and pH adjusted to correspond to the respective samples. The Hitachi H-7000~ instrument used ~or visualization was operated at 75 kV. The samples were inspected and images of representative fibrils were recorded.

Fluorescence Spectroscopy Steady-state ~luorescence was measured at room temperature using a Photon Technologies International QM-1~ fluorescence spectrophotometer equipped with excitation intensity correction, a temperature controlled cell holder and a magnetic stirrer. Emission spectra from 300nm to 550 nm were collected with excitation set at 281 nm. Both the excitation and emission monochromator slit widths were 4 nm.

In the energy transfer experiments, four spectra were taken: a blank of the bu~fer containiny 1 mM
borate, 1 mM citrate and 1 mM phosphate; 3 ~M
Trp:A~(9-25) alone; 3 ~M AEDANS:A~(9-25) alone; and a mixture of 3~M of each labeled peptide. The samples were pre-aged at pH 5 ~or 18 to ~4 hours prior to spectral acquisition. The pH titrations were performed using hydrochloric acid and sodium hydroxide. For the salt studies, samples were pre-aged with the appropriate -WO 97/07402 PCT/CA96~'~~5~' concentration o~ sodium chloride.

In the exchange experiments 6 ~M Trp:A~(9-25) and 6 ~M AEDANS:A,B (9-25) were incubated separately overnight at pH 5. These solutions were mixed giving a final concentration of 3 ~M each labeled peptide at which time ~pectra were collected.

CA 022428~l l998-07-l3 WO 97/07402 PCTtCA96i'~ 5 K~ ~S

Barrow, C.J., and Zagor~ki, M.G. (1991) Solution structures of ~-peptide and its constituent fragments:
relation to amyloid deposition. Science 253, 179-182.

Crowther, R.A. (1991) Structural aspects of pathology in Alzheimer's disease. Biochim. Biophys. Acta 1096, 1-9.

~APlhoch~ H. (1967) Biochemistry 6, 1948 - 1954.

Fairclough, R.H., and Cantor, C.R. (1978) The use of singlet-singlet energy transfer to study macromolecular assemblies. Methods Enzymol. 48, 347-379.

Fraser, P.E., Nguyen, J.T., Surewicz, W.K., and Kirschner, D.A. (1991) pH-dependent structural transitions of Al~he~mPr amyloid ~/A4 peptides.
Biophysical J. 60, 1190-1201.

Fraser, P.E., Nguyen, J.T., Inouye, H., Surewicz, W.K., Selkoe, D.J., Podlisny, M.B., and Kirschner, D.A. (1992) Fibril formation by primate, rodentl and Dutch-hemorrahagic analogues of Alzheimer amyloid ~-protein. Biochemistry 31, 10716-10723.

Fraser, P.E., Leves~ue, L., and McLachlan, D.R. (1993) Biochemistry of Alzheimer's Disease Amyloid Plaques.
Clin. Biochem. 26, 339-349.

Fraser, P.E., McLachlan, D.R., Surewicz, W.K., Mizzen, C.A., Snow, A.D., Nguyen, J.T., and Kirschner, D.A.
(1994) Conformation and fibrillogenesis of Al~h~;mer A~
peptides with selected substitutions of charged residues.
J. Mol. Biol. (in the press).

CA 022428~1 1998-07-13 Games, D., Adams, D., Alessandrini, R., Barbour, R., Berthelette, P., Blackwell, C., Carr, T., Clemens, J., Donaldson, T., Gillespie, F., et al. (1995) Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein.
Nature 373, 523-527.

Goate A. Chartier-Harlin MC. Mullan M. Brown J. Crawford F. Fidani L. Giu~fra L. Haynes A. Irving N. James L. et al. (1991) Segregation of a missense mutation in the amyloid precursor protein gene with familial Al~he;m~r's disea~e. Nature 349, 704-706.

Haass, C., Schlossmacher, M.G., Hung, A.Y., Vigo-Pelfrey, C., Mellon, A., Ostaszewski, B.L., Lieberburg, I., Koo, E.H., Schenk, D., Teplow, D.B., and Selkoe, D.J. (1992) Amyloid ~-protein is produced by cultured cells during normal metabolism. Nature 359, 322-325.

Halverson, K., Fraser, P.E., Kirschner, D.A., and Lansbury, P.T. (1990) Molecular determinants of amyloid deposition in Alzheimer's disease: conformational studies of synthetic beta-protein fragments.
Biochemistry 29, 2639-2644.

Hilbich, C., Kister-Wolke, B., Reed, J., Masters, C.L., and Beyreuther, K. (1992) Substitutions of hydrophobic amino acids reduce the amyloidogenicity of Alzheimer's disease ~A4 peptides. J. Mol. Biol. 228, 460-473.

Mattson, M.P., Barger, S.W., Cheng, B., Lieberburg, I., Smith-Swintosky, V.L., and Rydel, R.E. (1993) ~-Amyloid precursor protein metabolites and loss of neuronal Ca homeostasis in Alzheimer's disease. Trends Neurochem.
Sci. 16, 409-414.

CA 0224285l l998-07-l3 W 097~07402 PCT~CAgC~a'~

Pike C.J., Burdick, D., Walencewicz, A.J., Glabe, C.G., Cotman, C.W., (1993) Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state. J. Neurosci. 13, 1676-1687.

Selkoe, D.J. (1993) Physiological production o~ the ~-amyloid protein and the mechanism of Alzheimer's disease. Trends Neurochem. Sci. 16, 403-409.

Seubert, P., Vigo-Pel~rey, C., Esch, F., Lee, M., Dovey, H., Sinha, S., Schlossm--acher~ M.G., Whaley, J., Swindlehurst, C., McCormack, R., Wol~ert, R., Selkoe, D.J., Lieberburg, I., and Schenk, D. (1992) Isolation and quantification of soluble Al~heimer's ~B-peptide ~rom biological fluids. Nature 359, 325-327.

Yankner, B.~., Dawes, L.R., Fisher, S., et al. (1989) Neurotoxicity of a ~ragment o~ the amyloid precursor associated with Alzheimer's disea~e. Science 245, 417-420.

CA 022428~1 1998-07-13 WO 97/07402 PCT/CA9f/~CS'' SEQUENCE LISTING

(1) GENERAL INFORMATION:
.

(1) APPLICANT: THE ONTARIO CANCER lNS'l'l'l'U'l'~;
(ii) TITLE OF INVENTION: PROTEIN FIBRIL ASSEMBLY
ASSAY
(iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Ridout & Maybee (B) STREET: Suite 2400, One Queen Street East (C) CITY: Toronto (D) STATE: Ontario (E) COUNTRY: C~ n A ~ ~
(F) ZIP CODE: M5C 3B1 (v) COM~uL~ READABLE FORM:
(A) MEDIUM TYPE: Diskette - 3.5 inch, 1.4 Mb storage (B) COM~u~L~: IBM PC Compatible (C) OPERATING ~:iY~L~:M: MS-DOS 6.00 (D) SOFTWARE: Wordperfect 5.1 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Lake, James R.
(B) REFERENCE/DOCKET NUMBER: ONTCA/46B
(ix) TELECOMMUNICATION INFORMATION:
(A) TEL~O~: (416) 868-1482 (B) TELEFAX: (416) 362-0823 (2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

Gly Tyr Glu Val His His Glu Lys Leu Val Phe Phe Ala Glu Asp Val Gly W O 97/07402 PCT/CA9~ '5 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQ~N~ CHARACTERISTICS:
(A) LENGTH: 19 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ix) FEATURE:
(D) OTHER INFORMATION: The N-terminal amino acid residue Xaa is AcTRP, and the C-terminal amino acid residue Xaa is Gly-CONH2.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Xaa Gly Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Xaa (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ix) FEATURE:
(D) OTHER INFORMATION: The N-terminal amino acid residue Xaa is AcCYS (AEDANS), and the C-terminal amino acid residue Xaa is Gly-CONH2.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Xaa Gly Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Xaa

Claims (37)

CLAIMS:

1. A method for in vitro monitoring of peptide or protein fibril assembly, comprising the steps of:

1) attaching a donor fluorophore to a first fibrillogenic protein or peptide;

2) attaching an acceptor fluorophore to a second fibrillogenic protein or peptide, the donor and acceptor fluorophores being located on said first and second peptides or proteins so that they are juxtaposed to permit energy transfer between them upon fibril formation;

3) mixing the first and second fluorophore-containing peptides or proteins in solution at an approximately normal physiological concentration; and 4) monitoring formation of fibrils by observing the occurrence of fluorescence energy transfer between the donor and acceptor fluorophores upon excitation of the donor fluorophores.

2. A method as claimed in claim 1, wherein the donor fluorophore is a tryptophan residue.

3. A method as claimed in claim 2, wherein the donor fluorophore is attached to the N-terminus of a fibrillogenic peptide or protein.

4. A method as claimed in claim 3, wherein the peptide or protein is modified by adding a glycine residue to the N-terminus prior to attaching the fluorophore.

5. A method as claimed in claim 1, wherein the acceptor fluorophore is an AEDANS residue.

6. A method as claimed in claim 5, wherein the acceptor fluorophore is attached to a cysteine sulfhydryl at the N-terminus of a fibrillogenic peptide or protein.

7. A method as claimed in claim 6, wherein the peptide or protein is further modified by providing a glycine residue following the N-terminal cysteine.

8. A method as claimed in claim 1, wherein the mixing step comprises:

a) preparing concentrated stock solutions of said first and second fluorophore-containing peptides or proteins in denaturant and accurately determining the concentrations of the peptides or proteins by ultraviolet (UV) absorbance measurements of the solutions using the known absorption coefficients of the fluorophores; and b) mixing appropriate volumes of each solution to produce equimolar concentrations of each peptide or protein in denaturant solution.

9. A method as claimed in claim 8, wherein the monitoring step includes diluting out the denaturant, thereby initiating fibril assembly.

10. A method as claimed in claim 1, wherein the mixing step includes the addition of a promoter at a concentration sufficient to promote fibril formation.

11. A method as claimed in claim 10, wherein the promoter is heparin.

12. A method as claimed in claim 11, wherein the heparin concentration is 0.1-1 mg/ml.

13. A method as claimed in claim 12, wherein the method is carried out at physiological pH.

14. A method as claimed in claim 1, wherein the monitoring step is carried out in a fluorimeter.

15. A method as claimed in claim 8, wherein the denaturant is approximately 40% trifluoroethanol, and each peptide or fragment is dissolved in the denaturant at a millimolar concentration.

16. A method as claimed in claim 15, wherein the monitoring step includes diluting out the denaturant by quickly adding 20-30 µ1 of the equimolar mixture to about 3 ml of aqueous buffer.

17. A method as claimed in claim 1, comprising the further step of mixing the first and second fluorophore-containing peptides or proteins in the presence of a candidate inhibitor of fibril formation.

18. A method for in vitro monitoring of Alzheimer .beta.-amyloid protein fibril assembly, comprising the steps of:

1) attaching a donor fluorophore to a first Alzheimer .beta.-amyloid peptide or to a fibrillogenic fragment of Alzheimer .beta.-amyloid peptide;

2) attaching an acceptor fluorophore to a second Alzheimer .beta.-amyloid peptide or to a fibrillogenic fragment of Alzheimer .beta.-amyloid peptide, the donor and acceptor fluorophores being located on said first and second peptides or fragments so that they are juxtaposed to permit energy transfer between them upon fibril formation;

3) mixing the first and second fluorophore-containing peptides or fragments in solution at an approximately normal physiological concentration; and 4) monitoring formation of fibrils by observing the occurrence of fluorescence energy transfer between the donor and acceptor fluorophores upon excitation of the donor fluorophores.

19. A method as claimed in claim 18, wherein the donor fluorophore is a tryptophan residue.

20. A method as claimed in claim 19, wherein the donor fluorophore is attached to the N-terminus of Alzheimer .beta.-amyloid peptide or a fibrillogenic fragment of Alzheimer .beta.-amyloid peptide.

21. A method as claimed in claim 20, wherein Trp-A.beta.(9-25), SEQ ID NO:2, is said first fluorophore-containing fragment of Alzheimer .beta.-amyloid peptide.

22. A method as claimed in claim 21, wherein the peptide or peptide fragment is modified by adding a glycine residue to the N-terminus prior to attaching the fluorophore.

23. A method as claimed in claim 18, wherein the acceptor fluorophore is an AEDANS residue.

24. A method as claimed in claim 23, wherein the acceptor fluorophore is attached to a cysteine sulfhydryl at the N-terminus of Alzheimer .beta.-amyloid peptide or a fibrillogenic fragment of Alzheimer .beta.-amyloid peptide.

25. A method as claimed in claim 24, wherein the peptide or peptide fragment is further modified by providing a glycine residue between the N-terminal cysteine and Gly 9 residues.

26. A method as claimed in claim 25, wherein AEDANS-A.beta.(9-25), SEQ ID NO:3, is said second fluorophore-containing fragment of Alzheimer .beta.-amyloid peptide.

27. A method as claimed in claim 18, wherein the mixing step comprises:

a) preparing concentrated stock solutions of said first and second fluorophore-containing peptides or fragments in denaturant and accurately determining the concentrations of the peptides or fragments by ultraviolet (UV) absorbance measurements of the solutions using the known absorption coefficients of the fluorophores; and b) mixing appropriate volumes of each solution to produce equimolar concentrations of each peptide or fragment in denaturant solution.

28. A method as claimed in claim 27, wherein the monitoring step includes diluting out the denaturant, thereby initiating fibril assembly.

29. A method as claimed in claim 18, wherein the mixing step includes the addition of a promoter at a concentration sufficient to promote fibril formation.

30. A method as claimed in claim 29, wherein the promoter is heparin.

31. A method as claimed in claim 30, wherein the heparin concentration is 0.1-1 mg/ml.

32. A method as claimed in claim 31, wherein the method is carried out at physiological pH.

33. A method as claimed in claim 18, wherein the monitoring step is carried out in a fluorimeter.

34. A method as claimed in claim 27, wherein the denaturant is approximately 40% trifluoroethanol, and each peptide or fragment is dissolved in the denaturant at a millimolar concentration.

35. A method as claimed in claim 34, wherein the monitoring step includes diluting out the denaturant by quickly adding 20-30 µl of the equimolar mixture to about 3 ml of aqueous buffer.

36. A method as claimed in claim 35, wherein the fluorophore-containing peptides are Trp-A.beta.(9-25), SEQ ID
NO:2, and AEDANS-A.beta.(9-25), SEQ ID NO:3, and fibril formation is monitored by measuring emission in the range 300-550 nm using a fluorimeter with excitation at 281 nm.

37. A method as claimed in claim 18, comprising the further step of mixing the first and second fluorophore-containing peptides or fragments in the presence of a candidate inhibitor of amyloid fibril formation.