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J Biol Chem, Vol. 275, Issue 3, 1673-1678, January 21, 2000


Controlling Polymerization of beta -Amyloid and Prion-derived Peptides with Synthetic Small Molecule Ligands*

Pascal KunerDagger , Bernd BohrmannDagger , Lars O. Tjernberg§, Jan Näslund, Gerda HuberDagger , Suna CelenkDagger , Fiona Grüninger-LeitchDagger , J. Grayson RichardsDagger , Roland Jakob-RœtneDagger , John A. KempDagger , and Christer NordstedtDagger ∥

From Dagger  F. Hoffmann-La Roche AG, Pharma Division, Preclinical Research, CH-4070 Basel, Switzerland, the § Laboratory of Biochemistry and Molecular Pharmacology, Section of Drug Dependence Research, Department of Clinical Neuroscience, Karolinska Hospital, S-171 77 Stockholm, Sweden, and the  Department of Clinical Neuroscience, Occupational Therapy, and Elderly Care Research, Karolinska Institute, KFC Novum plan 4, S-141 86 Huddinge, Sweden

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Alzheimer beta -amyloid peptide (Abeta ) and a fragment of the prion protein have the capacity of forming amyloid-like fibrils when incubated under physiological conditions in vitro. Here we show that a small amyloid ligand, RO-47-1816/001, enhances this process severalfold by binding to amyloid molecules and apparently promote formation of the peptide-to-peptide bonds that join the monomers of the amyloid fibrils. This effect could be antagonized by other ligands, including analogues of RO-47-1816/001, as well as the structurally unrelated ligand Congo red. Analogues of RO-47-1816/001 with low affinity for amyloid did not display any antagonistic effect. In conclusion, these data suggest that synthetic molecules, and possibly also small natural substances present in the brain, may act in a chaperone-like fashion, promoting Abeta polymerization and growth of amyloid fibrils in vitro and possibly also in vivo. Furthermore, we demonstrate that small organic molecules can be used to inhibit the action of amyloid-enhancing compounds.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The amyloid deposits in brain vasculature and parenchyma that are the main histopathological hallmarks of Alzheimer's disease (1) are composed of large polymers of Alzheimer beta -amyloid peptide (Abeta ).1 This peptide is present in two major forms, one being 40 amino acids long, and the other, more aggregable form being 42 amino acids long (2-4). The Abeta peptide is secreted by numerous cell types in the body but the amyloid deposits are only present in the central nervous system.

The amyloid deposits are formed by the aggregation of individual monomers of Abeta peptide into very large polymers which have a fibril-like appearance when observed in an electron microscope (5). These polymers can also be observed by light microscopy following staining with certain histological dyes such as Congo red and thioflavine T (6).

Soluble Abeta peptide can be detected in blood and cerebrospinal fluid. The levels are, however, very low, usually in the low nano- to picomolar range and at these concentrations the peptide polymerizes at a very slow rate (7). A number of recent studies on the mechanisms of amyloid formation have conclusively shown that the presence of preformed oligo- or polymers of the amyloid peptide in the reaction mixture increases the polymerization rate dramatically (7). These multimers serve as templates for the reaction and, as a result, the initial, slow phase of primary nucleation is eliminated.

It has also been proposed that charged molecules, such as gangliosides (8) and metal ions, including Zn2+and Cu2+ (9), may enhance formation of amyloid in vitro and in vivo. Apolipoprotein E4, a well established risk factor for Alzheimer's disease (10) has also been suggested to enhance amyloid formation by serving as a "pathological chaperone" (11-13). In the case of apolipoprotein E, there are conflicting data suggesting that the protein can enhance as well as inhibit amyloidogenesis (14, 15).

The existence of putative co-factors capable of enhancing amyloid formation potentially offers new targets for pharmaceuticals for use in the treatment of amyloid disorders. However, so far very little is known about how such compounds might work, and it is therefore difficult to develop assays for finding candidate pharmaceuticals and to predict their molecular mode of action.

A number of molecules which function as amyloid ligands have been described. These include the textile dyes Congo red and thioflavine T that bind to amyloid fibrils but not to nonpolymerized peptide. These compounds, therefore, have found widespread use in histopathological diagnosis of amyloid disorders. The elements in the amyloid fibril that these compounds bind to are probably the novel surfaces or binding pockets generated as a result of amyloid peptide polymerization (16). In addition to these small organic molecules there are also peptide-based ligands. The rationale behind such ligands is that during polymerization short stretches of amino acid residues interact and form beta -strands that join the monomers encompassing the fibril (17-19). It was speculated that short peptides corresponding to one of the strands in the beta -sheet can bind to the amyloid peptide/protein. With this approach, ligands capable of interfering with amyloid formation in vitro and possibly also in vivo have been identified (20-23).

Here, we first investigated if nonpeptidic, synthetic molecules of low molecular weight can have similar effects as the larger natural substances described above. By having access to molecules that easily can be altered structurally, the mechanism through which small molecules regulate the higher order structure of the Abeta peptide can be probed with a more rational, structure based, approach than otherwise. Moreover, small synthetic molecules with pharmacokinetic and toxicological properties allowing them to be easily administered to animals, may also be useful in studies of amyloidogenesis in vivo.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Synthetic Abeta 1-40, Abeta 1-42, and PrP106-126 biotinylated at the N terminus were obtained from ANAWA (Wangen, Switzerland). Nonlabeled Abeta 1-40, Abeta 1-42, and PrP106-126 were obtained from Bachem (Bubendorf, Switzerland). The peptides were stored in dimethyl sulfoxide (Me2SO) at -20 °C. Human serum albumin, fatty acid-free, 99% purity, was from Sigma. Streptavidin-peroxidase was bought from Roche Molecular Biochemicals (Mannheim, Germany). Antibody 6E10 was bought from Senetek PLC. All other reagents were from Sigma.

Analysis of Abeta Peptide Polymerization-- This assay was carried out essentially as described previously (24). Briefly, 96-well plates (Maxisorp, Nunc) were coated with peptide by incubating them with a solution of Abeta 1-42 or Abeta 1-40 (2.5 µM) in Tris-buffered saline (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, and 0.05% NaN3). To each well, 100 µl of the solution was added, and the plates were incubated at 37 °C with shaking for 48 h. The peptide solution was then decanted. Staining with a solution of Congo red showed that the polymeric peptide had bound to the wells. After removal of the nonbound peptide, the plates were allowed to dry. Coated plates were stored at -20 °C in a desiccator until used. On the day of experiment, the plates were blocked by addition of 300 µl of PBS containing 0.05% (v/v) Tween 20 (PBS-T) and 1% bovine serum albumin/well for 2 h at room temperature. The plates were then washed with PBS-T, and the fluid was decanted. Biotin-Abeta 1-40 or biotin-Abeta 1-42 was dissolved in Me2SO and diluted in Tris-buffered saline with NaN3 (0.05%) to a final concentration of 20 nM. The plates were incubated 3 h, unless stated otherwise, at 37 °C with shaking. Nonbound peptide was removed by washing the plates three times with PBS-T (300 µl/well). Streptavidin-peroxidase was diluted with PBS-T containing 1% bovine serum albumin and added to the plates (200 µl/well). After incubation (30 min at room temperature) the solution was flicked off, and the plates were washed four times with PBS-T. Tetramethylbenzidine was used as chromogenic substrate for the peroxidase. After termination of the reaction with sulfuric acid (0.33 M), absorbance was measured at 455 nm using a SpectraMAX 250, 96-well plate reader. Nonspecific binding is defined as binding of biotin-Abeta to wells that had not been coated with Abeta . There was a linear relationship between peroxidase activity and the amount of peptide bound (data not shown). Nonspecific binding was, on average, approximately 15% of total binding (data not shown).

Fluorescence Equilibrium Binding-- In these experiments, the test compounds (0.5 µM) were incubated in white 96-well plates in the presence of polymerized Abeta 1-42 or Prp106-126 at concentrations of 123, 61.5, 30.8, 15.4, 7.7, 3.8, 1.9, or 0 µM (in quadruplicate) for approximately 10 min at room temperature with agitation. Fluorescence was measured in a Perkin-Elmer LS-50-B equipped with a well-plate reader at the following emission and excitation wavelengths (nm). RO-47-1816/001: 410, 510; RO-65-8564/001: 410, 510; RO-65-7560/000: 415, 500; RO-65-8815/001; 410, 480; RO-65-3537/000: 410, 480; RO-65-5780/000: 380, 440. As presented in Fig. 1C, F0 represents fluorescence of the compound alone, F is the observed fluorescence, Finfinity is the maximal fluorescence (i.e. fluorescence in the presence of the highest concentration of peptide used), and L is the concentration of the peptide.

Western Blot Assay-- For aggregation analysis of Abeta 1-40 monomers, 20 µM Abeta 1-40 were co-incubated with the indicated concentrations of RO-47-1816/001 for 19 h at 37 °C. After incubation, samples were mixed with NuPAGE LDS sample buffer (4 ×) from Novex (San Diego, CA) and boiled for 5 min. Samples, 30 µl/lane, were loaded on Novex 4-12% BisTris PAGE gel, separated, and blotted on polyvinylidene difluoride membranes (Novex, San Diego, CA). Membranes were washed once with PBS-T blocked with 5% (w/v) nonfat milk in PBS-T for 1 h, washed again three times with PBS-T, and probed with 6E10 IgG from Senetek PLC (1 µg/ml in PBS-T with nonfat milk) for 1 h at room temperature. Membranes were washed again three times with PBS-T followed by a 30-min incubation of a second antibody linked to horseradish peroxidase (Amersham Pharmacia Biotech, Little Chalfont, UK, 1:25.000 dilution). Bands were detected using Lumi-LightTM (Roche Molecular Biochemicals, Mannheim, Germany) and Hyperfilms (Pharmacia Amersham Biochem).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RO-47-1816/001 Binds to Abeta -amyloid-- RO-47-1816/001, a compound of the pyridone class (Fig. 1A), was identified through random screening of a large library of small organic molecules. Equilibrium fluorescence binding (25) demonstrated that this compound bound to Abeta 1-42 fibrils (Fig. 1B) with a Kd of approximately 6.2 µM (Fig. 1C). In the same type of experiments it could also be shown that the compound binds to Abeta 1-40 with a similar affinity (data not shown).


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  		Fig. 1.   The structures of compounds used in the study and binding of RO-47-1816/001 to Abeta 1-42 polymers. A, the structures of compounds used in the study. B, emission spectra of RO-47-1816/001 in the presence of increasing concentrations of Abeta 1-42-amyloid. The compound was incubated at a concentration of 0.5 µM in the presence of (from top to bottom) 123, 61.5, 30.8, 15.4, 7.7, 3.8, 1.9, or 0 µM polymerized Abeta 1-42 in Tris-buffered saline. Excitation wavelength was 410 nm, and slit widths were 5 nm for both excitation end emission. C, saturation binding (left panel) and the corresponding Scatchard plot (right panel) of Abeta 1-42 to RO-47-1816/001. Measurements were performed in quadruplicate as described under "Experimental Procedures."

RO-47-1816/001 Enhances Binding of Soluble Abeta to Immobilized Polymers-- Here, an assay was employed where binding of biotinylated Abeta 1-40 or Abeta 1-42 to immobilized nonlabeled Abeta 1-42 was used as a measure for Abeta polymerization (24). When RO-47-1816/001 was added to the assay mixture, polymerization increased severalfold (Fig. 2A). As seen in the figure, the compound also increased binding to control wells to which no nonlabeled peptide had been attached, but only to a small extent. A reasonable explanation is that the test compound increased binding of soluble biotinylated Abeta 1-40 to the small amounts of biotinylated Abeta 1-40 that had been nonspecifically absorbed to the plastic material of the wells.


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  		Fig. 2.   RO-47-1816/001 enhances amyloid peptide polymerization. A, biotin-Abeta 1-40 (circles) or biotin-Abeta 1-42 (triangles), at a concentration of 20 nM, was incubated in wells coated with Abeta 1-42 (filled symbols) or blocking buffer only (open symbols) in the presence of the indicated concentrations of RO-47-1816/001. Following incubation at 37 °C for 3 h, the reaction was terminated by washing, and the plate processed as described under "Experimental Procedures." B, RO-47-1816/001 increases the formation of multimeric Abeta . Abeta 1-40 was incubated at a concentration of 20 µM at 37 °C for 19 h with agitation in the presence of the indicated concentrations of RO-47-1816/001. Following incubation, the peptide mixture was separated by SDS-PAGE, and Abeta immunoreactivity was visualized by Western blotting using antibody 6E10.

By coating the plates with Abeta 1-40, instead of Abeta 1-42 polymers, and studying binding of biotin-Abeta 1-40, virtually identical results were obtained (data not shown). This demonstrates that the two C-terminal residues of Abeta 1-42 are not required for the effect of RO-47-1816/001. The results also show that the compound requires immobilized polymers for its effect in the assay and that the increase in biotin signal reflects enhanced incorporation of soluble peptide into the immobilized polymers. We also studied how addition of RO-47-1816/001 affected the resistance of the polymers to trypsin (26) and to solubilization with high concentrations of urea. Addition of 30 µM RO-47-1816/001 did not have any significant effect on these parameters, suggesting that polymers formed in the presence of the compound had a similar or identical sensitivity toward the protease and chaotrope solubilization. Because the amyloid formed in the presence of RO-47-1816/001 shared these features of genuine amyloid (i.e. relative resistance to proteolysis and chaotropes), it is likely that it is very similar, or identical, to amyloid formed in the absence of the compound.

It has previously been demonstrated that at least a fraction of polymeric Abeta remains associated when separated under the relative harsh denaturing conditions employed in SDS-PAGE (27). We used this feature of the peptide to study if addition of RO-47-1816/001 to nonlabeled Abeta 1-40 increased formation of high molecular weight forms of the peptide. In the absence of RO-47-1816/001, immunoreactive bands with apparent molecular masses of approximately 4, 8 and 16 kDa, probably corresponding to Abeta mono-, di-, and tetramers, respectively, were obtained (Fig. 2B). Addition of RO-47-1816/001 increased formation of the 16-kDa form dose-dependently and induced formation of even larger polymers. This directly demonstrates that the compound enhances association of Abeta into large multimers.

In a similar experiment, nonlabeled Abeta was incubated with increasing concentrations of RO-47-1816/001. After 19 h of incubation at 37 °C, it was observed that the compound dose-dependently increased the amount of polymerized peptide as measured by Congo red staining (28), suggesting that the compound had stimulated formation of fibrils fulfilling this criteria for amyloid.

Finally, we also studied if addition of RO-47-1816/001 affected the morphology of fibrils formed by nonlabeled Abeta 1-40 and Abeta 1-42. The peptides were incubated in the presence of 30 µM RO-47-1816/001 for 20 h and then subjected to electron microscopy as described previously (24). As seen in Fig. 3, addition of the compound to the polymerization mixture had no apparent effect on the morphology of the fibrils (Fig. 3, compare A with B and C with D). A reasonable interpretation of this experiment is that RO-47-1816/001 enhances polymerization without affecting the ultrastructural appearance of the Abeta fibrils.


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  		Fig. 3.   RO-47-1816/001 has no apparent effect on the ultrastructural appearance of Abeta fibrils. Abeta 1-40 (A and B) and Abeta 1-42 (C and D) at a concentration of 20 µM were incubated in Tris-buffered saline for 24 h in the absence (A and C) or presence (B and D) of 30 µM RO-47-1816/001. After the incubation samples were negatively stained, they were viewed in an electron microscope.

RO-47-1816/001 Increases the Rate of Abeta Polymerization-- The binding of biotin-Abeta to immobilized polymers is time- dependent (24, 29). We therefore decided to study how RO-47-1816/001 affects polymerization at various time points over an interval of 5 h. As seen in Fig. 4, incorporation of biotin-Abeta 1-40 was essentially linear over time in the studied time interval in the absence of RO-47-1816/001 in agreement with previous studies (30). Addition of the compound changed this pattern, most notably in the presence of the highest concentration used (50 µM) where maximal binding was observed already after 1-h incubation. A reasonable interpretation of these data is that the compound increases the rate through which soluble peptide is incorporated into the immobilized polymers.


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  		Fig. 4.   RO-47-1816/001 increases the rate of binding of biotin-Abeta 1-40 to immobilized Abeta 1-42 polymers. Biotin-Abeta 1-40 was incubated with 0 (filled circle), 0.5 (open circle), 5 (filled triangle), or 50 µM (open triangle) RO-47-1816/001 for the indicated time periods.

RO-47-1816/001 Enhances Binding of Soluble Prion Protein Fragment to Immobilized Polymers-- Here, the specificity of the compound for Abeta -amyloid was addressed. A fragment of the prion protein corresponding to amino acids 106-126 (PrP106-126) spontaneously polymerizes into amyloid-like fibrils, morphologically indistinguishable from those formed by Abeta (31-33). First, it was studied if RO-47-1816/001 can bind to the prion fragment. As seen in Table I, RO-47-1816/001 bound to PrP106-126 with an affinity similar to that displayed for Abeta 1-42, demonstrating that RO-47-1816/001 is not a specific Abeta ligand but also binds other types of amyloid. Using the same type of assay as employed for Abeta , the effect of RO-47-1816/001 on PrP106-126 polymerization was investigated. In Fig. 5 is shown that the compound dose-dependently increased biotin-PrP106-126 binding. Its potency was, however, lower here than in the Abeta assay (compare Figs. 2A and 5). The conclusion is therefore that the effect of the compound is not specific for Abeta -amyloid and thus can be used to enhance the polymerization of at least one other, structurally unrelated, amyloid peptide.

                              
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Table I
Dissociation constants (Kd) for ligand-peptide combinations


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  		Fig. 5.   RO-47-1816/001 increases polymerization of the prion protein-derived peptide PrP106-126. Biotin-PrP106-126 was allowed to bind to immobilized PrP106-126 in the presence of the indicated concentrations of RO-47-1816/001 for 3 h. The reactions was stopped by washing and processed as described under "Experimental Procedures."

Abeta Ligands Structurally Related to RO-47-1816/001 Can Antagonize Its Amyloid Formation-enhancing Effect-- A selection of five analogues of RO-47-1816/001 (Fig. 1A) were tested for their affinity toward polymerized Abeta 1-42 (Table I). Three of the compounds bound to Abeta with an affinity similar to that of RO-47-1816/001. The two others had low affinity for the peptide (RO-47-3537/000 and RO-65-5780/000) with approximately 10 times higher Kd. Clearly, the two compounds with the lowest affinity are less lipophilic than the four with the highest affinity. There are also other structural differences. The polymerization accelerating compound RO-47-1817/001 is an ester, all the other molecules are amides. In addition, the phenyl ring of RO-47-1816/001 is unsubstituted, whereas the antagonists carry methoxy or benzyloxy substituents.

When tested in the Abeta polymerization assay, none of the RO-47-1816/001 variants had any clear effect on biotin-Abeta 1-40 binding (Fig. 6, left panel). This was surprising considering that: (i) three out of five compounds bound to the amyloid with an affinity in the same range as RO-47-1816/001 and (ii) they had striking structural similarities to RO-47-1816/001. This prompted us to investigate if the analogues, by competing with RO-47-1816/001 binding, interfered with its capability to enhance polymerization. As seen in Fig. 6 (right panel), three of the five tested compounds dose-dependently reduced binding of biotin-Abeta 1-40 in the presence of 30 µM RO-47-1816/001. These three antagonists were the compounds displaying the highest affinity for Abeta , whereas the low affinity substances failed to antagonize the action of RO-47-1816/001 (Table I).


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  		Fig. 6.   Structural variants of RO-47-1816/001 antagonize its effect on Abeta polymerization. The effect of structural variants of RO-47-1816/001 on the binding of 20 nM biotin-Abeta 1-40 to immobilized Abeta 1-42 was studied in the absence (left panel) or presence (right panel) of RO-47-1816/001 at a concentration of 30 µM. The incubation was performed for 3 h. RO-65-5780/000 (open circle), RO-65-3537/000 (filled circle), RO-65-8815/001 (open triangle), RO-65-7560/000 (filled triangle), RO-65-8564/001 (filled square).

Congo Red Inhibits the Effect of RO-47-1816/001-- Congo red is an amyloid ligand that changes its spectral properties upon binding to amyloid fibrils and therefore is a useful tool in histochemical diagnosis of Alzheimer's disease and other amyloid disorders (6). It has previously been shown to interact with the central core region of Abeta as well as to regions with similar structural properties in other amyloid-forming proteins and peptides (16). Structurally, Congo red is very different from the hydrophobic pyridone derivatives described here (Fig. 1A).

Fig. 7 shows the effect of Congo red on the binding of biotin-Abeta 1-40 both in the presence and in the absence of RO-47-1816/001. In the absence, Congo red slightly enhanced binding at the highest concentrations employed, which is in agreement with previous published data (29). In the presence of 10 µM RO-47-1816/001, Congo red reduced binding similar to that of the previously analyzed RO-47-1816/001 analogues. As seen in the figure, Congo red only reduced the effect of RO-47-1816/001 and was not capable of reducing polymerization below that obtained in the absence of RO-47-1816/001. At the highest concentrations used, the level of binding was essentially identical to that in the absence of RO-47-1816/001. This suggests that RO-47-1816/001 and Congo red interact with Abeta at the same binding site.


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  		Fig. 7.   Congo red inhibits the action of RO-47-1816/001 but does not affect basal binding of biotin-Abeta 1-40 to immobilized Abeta 1-42 polymers. Biotin-Abeta 1-40 was incubated in the presence (open circles) or absence (filled circles) of RO-47-1816/001 (10 µM) together with the indicated concentrations of Congo red.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here it has been shown that RO-47-1816/001, a synthetic compound with a molecular mass of 592 Da accelerates polymerization of amyloid peptides several times its own size. There are only a few known examples of small molecule compounds regulating the state of polymerization of proteins. The most well known compounds of this class are probably colchicine and paclitaxel (previously taxol), that induce disassembly and assembly, respectively, of microtubuli and are used clinically to treat gout and neoplasms (34).

Binding studies showed that RO-47-1816/001 displayed affinity both to Abeta -amyloid as well as to amyloid formed by a peptide derived from the prion protein (Prp106-126). This is a feature shared by several other organic compounds, including Congo red and thioflavine T. It is believed that the novel surfaces generated as a result of the adoption of beta -pleated sheet structures during polymerization serve as binding sites for these amyloid ligands (35). This property of the compounds is widely used to histochemically distinguish amyloid from normal tissue. Because Congo red could interfere with the action of RO-47-1816/001, it is reasonable to assume that the latter interacted with the same binding site(s) as the former and that this site(s) therefore can be of importance for putative amyloid-enhancing compounds and their antagonists.

It was interesting to observe that analogues of the amyloid ligand RO-47-1816/001 failed to produce an accelerating effect on amyloid formation despite having similar affinities to Abeta as the original compound. This initially surprising finding was later explained when it was revealed that the inactive ligands instead antagonized the effect of RO-47-1816/001. The present finding therefore suggests that RO-47-1816/001 exerts its action not only by binding to the amyloid polymer but that it also has an additional effect that the antagonists do not possess. It is tempting to speculate that RO-47-1816/001, after it has bound to the amyloid fibril, binds to the soluble peptide and brings it into close proximity to the fibril (Fig. 8), thereby facilitating docking of the peptides via their specific binding sequences (20, 21, 36). Whether cross-linking is mediated directly by RO-47-1816/001 or by a composite surface of RO-47-1816/001 and the Abeta polymer is not clear. The latter mechanism is, however, more favored in nature. The most well known example is probably the interaction of the T-cell receptor with its antigen. The affinity of the receptor for a peptide antigen per se is low but when bound to and presented by the major histocompatibility complex, the affinity increases dramatically. Under these circumstances, the T-cell receptor can bind both to the antigen and the major histocompatibility complex (37, 38), and the increased number of contacts leads to increased affinity.


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  		Fig. 8.   Proposed mechanism to explain how the amyloid ligand RO-47-1816/001 accelerates incorporation of soluble Abeta peptide into preexisting polymers. A, soluble Abeta directly interacts and binds to the immobilized polymer (indicated as a trimer in the figure). In addition to the sites were the peptide-to-peptide interactions take place, there is a binding site for the ligand RO-47-1816/001. After the ligand has bound to the Abeta polymer, either a novel surface is formed (a composite of the ligand and the polymer), to which the soluble peptide can bind or alternatively, the protruding part of RO-47-1816/001 per se binds the soluble peptide and cross-links it to the polymer. It is not clear if the ligand remains associated with the polymer (lower part of the figure) after the soluble peptide has bound to the polymer or if it dissociates from the peptide complex (upper part of the figure). B, the antagonists also bind to the polymers but fail to cross-link the polymer to the soluble peptide. Instead it will occupy the binding site and inhibit the binding and the effect of RO-47-1816/001 but without affecting the direct peptide-to-peptide interaction.

There are also examples of natural compounds being used as pharmaceuticals that employ this strategy in mediating their specific action. Cyclosporin A and FK506 both inhibit calcineurin by forming a complex with cyclophilin and FK506-binding protein, respectively, that display high affinity toward the phosphatase (39, 40). Recently, this mechanism was elegantly used to increase the affinity of a bifunctional ligand designed to induce heterodimerization of the FK506-binding protein and an SH2 domain (41).

It is not clear if the ability to enhance polymerization of amyloid peptides is a common feature in small organic molecules or if it is restricted to a very small number of substances. We do, however, want to point to the possibility that clinically used pharmaceuticals may have this property and, at least theoretically, may promote amyloidogenesis in vivo.

It can be speculated that the present results suggest that the brain might contain small endogenous compounds having similar effects as RO-47-1816/001. Apolipoprotein E (apoE) and gangliosides have been mentioned previously, but it is still possible that there are other natural compounds with similar effects. An interesting observation by Selkoe and co-workers (42) is that Congo red stabilizes monomeric Abeta in cell culture supernatant, which may suggest that compounds with similar effects as those of RO-47-1816/001 may be secreted by cells. Finally, the finding presented here, that some compounds, including Congo red, displaying affinity for amyloid can antagonize the action of amyloid enhancers, may therefore be of therapeutic significance.

    ACKNOWLEDGEMENT

We thank Dr. Patrick Nef for valuable suggestions during preparation of the manuscript.

    FOOTNOTES

* This work was supported by F. Hoffmann-La Roche AG, The Swedish Medical Research Council, and the Swedish Foundation for Strategic Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

par To whom correspondence should be addressed. Tel.: 41-61-688-39-31; Fax: 41-61-688-17-20; E-mail: christer.nordstedt@roche.com.

    ABBREVIATIONS

The abbreviations used are: Abeta , Alzheimer amyloid beta  peptide; Prp, prion protein; BisTris, 2[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Selkoe, D. J. (1994) Annu. Rev. Cell Biol. 10, 373-403[CrossRef]
2. Glenner, G. G., and Wong, C. W. (1984) Biochem. Biophys. Res. Commun. 122, 1131-1135[CrossRef][Medline] [Order article via Infotrieve]
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