The present invention relates to novel compositions useful for elucidating the onset or progress of diseases such as Huntington's disease, that are associated with the formation of fibrils or protein aggregates. Further, the present invention relates to methods for monitoring formation of fibrils or protein aggregates as well as to methods for identifying inhibitors of fibril or protein aggregate formation. Additionally, the invention relates to inhibitors of the formation of fibrils or protein aggregates identified by the method of the invention as well as to pharmaceutical compositions that include the inhibitors.

SUMMARY OF THE INVENTION

The above technical problem is solved by the embodiment characterized in the claims. Accordingly, the present invention relates to a composition comprising:

(a) a nucleic acid molecule encoding a fusion protein comprising: (aa) a (poly)peptide that enhances solubility and/or prevents aggregation of said fusion protein; and (ab) an amyloidogenic (poly)peptide that has the ability to self-assemble into amyloid-like fibrils or protein aggregates; (b) a vector containing the nucleic acid molecule of (a); (c) a host transformed with the vector of (b); (d) a fusion protein encoded by the nucleic acid of (a) or a functional derivative thereof; and/or (e) an antibody specific for the fusion protein of (d).

As used herein, the term "(poly)peptide" relates to a polypeptide or a peptide depending on the length of the amino acid string. Said (poly)peptide has the ability to enhance solubility of a fusion partner in said fusion protein and thus of the fusion protein itself. Additionally, or alternatively, said (poly)peptide prevents the aggregation of the fusion partner and thus of the fusion protein. Said (poly)peptide is combined within said fusion protein with an amyloidogenic (poly)peptide having the above recited features. The connection of both (poly)peptides may be via a linker or by a direct attachment. It is preferred that either the linker or either (poly)peptide comprises a cleavable site. Said cleavable site should render both (poly)peptides essentially intact. Alternatively, said fusion protein may comprise a number of cleavage sites. Upon cleavage, which may be exhaustive or under limiting conditions, the amyloidogenic (poly)peptide should, when used for the purposes of the present invention, retain the ability to self-assemble. The person skilled in the art is in the position to determine appropriate conditions for a corresponding limited cleavage. The composition of the invention may comprise one, several, or all of the compounds recited in features (a) to (e), above.

The term "functional derivative" refers to a fusion protein which comprises, for example, modified amino acids or amino acid substitutions and retains the functions of the fusion protein detailed herein above.

The term "antibody specific for the fusion protein" comprised in the composition of the invention is intended to mean that said antibody is only specific for the fusion protein but not for either of the above cited components of said fusion protein.

In accordance with the present invention, it could surprisingly be shown that the composition comprising the above recited components can be used for the elucidation of amyloid-like fibril or protein aggregate formation. The components of the composition can be used in varying combinations to test, for example, for specific conditions under which amyloids are formed in vitro. The in vitro data obtained with the composition of the invention may then be compared to or brought into relation with the in vivo situation and appropriate conclusions may be drawn therefrom.

The in vitro systems that can be established with the composition of the invention allow formation of highly stable amyloid-like protein aggregates. Such aggregates may be obtained, for example, by proteolytic cleavage of GST fusion proteins comprising exon 1 of the HD gene and containing expanded polygln sequences. Alternatively, such aggregates may be obtained by lowering the pH value from 8 to 5 or by increasing the protein concentration. The arrays of fibrillar structures of varying sizes and shapes observed by electron microscopy surprisingly clearly resemble those of purified amyloids. Furthermore, the polarization microscopic properties of the fibrils stained with Congo red are strikingly similar to those described for amyloids. The green-gold birefringence of the amyloid-like fibrils indicates that the polymers have common structural features. Although the Congo red staining does not determine conclusively whether the fibrils consist of .beta.-pleated sheets, the method suggests that this is likely in view of experience gained with other protein polymers (Caputo et al., 1992). However, it has been generally accepted that naturally occurring mammalian protein polymers that exhibit fibrillar structures and green birefringence after Congo red staining should be classified as amyloids (Glenner, 1980). Instead of the HD gene, other nucleic acid sequences encoding amyloidogenic (poly)peptides may be used to generate said fusion proteins. Preferably, the composition of the invention is a diagnostic composition. The composition of the invention may also be a kit. The diagnostic composition can advantageously be employed in the assessment of a disease state whereas the kit may rather be employed in the development of, for example, inhibitors or in the elucidation of amyloid formation.

It is a preferred embodiment of the composition of the invention, that said amyloidogenic (poly)peptide comprises a polyglutamine expansion. In the prior art it has been shown by X-ray diffraction studies that synthetic peptides containing polyglns form .beta.-sheets strongly held together by hydrogen bonds (Perutz et al., 1994). Because synthetic poly(L-glutamine) is insoluble in water, a synthetic peptide with the sequence Asp.sub.2-Gln.sub.15-Lys.sub.2 was used in that study. A stretch of 10 glutamines was also inserted into the loop of chymotrypsin inhibitor-2 (Cl2), and it was demonstrated by analytical ultracentrifugation that the recombinant protein, in addition to monomers formed dimers and trimers, whereas wild-type Cl2 was present only in the monomeric form (Stott et al., 1995). It has been proposed that the polygln stretch functions as a polar zipper, joining proteins together. However, the hypothesis that glutamine repeats in proteins form .beta.-pleated sheets and induce protein aggregation by a mechanism similar to that observed in spongiform encephalopathy (TSE) diseases (Caughey and Chesebro, 1997) could not be proven with this recombinant protein. Most likely, the length of the polygln sequence inserted into Cl2 was too short. Accordingly, the experimental data actually obtained teach away from the above recited hypothesis.

In a particularly preferred embodiment said polyglutamine expansion comprises at least 35 glutamines. In a further particularly preferred embodiment said polyglutamine expansion comprises at least 51 glutamines.

Our studies with the GST-HD fusion proteins containing polygln sequences of varying lengths demonstrate that a certain length of the polygln stretch is necessary for the formation of amyloid-like fibrils in vitro. When the purified fusion proteins were analyzed by SDS-PAGE, insoluble high molecular weight protein aggregates were only detected with the proteins containing 81 and 122 glutamines (FIGS. 1a and b), whereas the protein with 51 glutamines was soluble and no fibrillar structures were detected by electron microscopy (FIG. 4a). This indicates that the critical length of the polygln stretch in the fusion proteins leading to the formation of aggregates is greater than 51 glutamines. Accordingly, fusion proteins of the invention with a polyglutamine expansion of more than 51 glutamines, such as 81 or 122 glutamines, may be employed in studies for the formation of aggregates that render a cleavage reaction unnecessary. However, when the GST-tag, which is known to enhance the solubility of many proteins (Smith and Johnson, 1988), was cleaved by limited digestion with trypsin, the liberated HD exon 1 protein with 51 glutamines also started to form aggregates (FIG. 3a) and the amount of these aggregates increased when the GST-tag was totally degraded with trypsin (FIG. 3b). This indicates that in the HD exon 1 protein 51 glutamines are sufficient to form aggregates, whereas 20 and 30 glutamines under the same conditions are not. The minimum critical length essential for the development of amyloid-like structures after removal of the GST-tag is not known and has to be determined. However, preliminary experiments in our laboratory suggest that the threshold for the formation of HD exon 1 protein aggregates is between 35 48 glutamines. This result is strikingly similar to the pathological threshold in HD, SBMA, DRPLA, SCA1, SCA2, SCA3 and SCA7. In all of these neurodegenerative polygln diseases a pathological phenotype was found when more than 41 repeats were present, suggesting that the elongation of the polygln repeat beyond a certain length may lead to a phase change in the affected proteins. This could, for example, be a change from random coils to hydrogen-bonded hairpins in the polygln stretch, see Perutz (1996).

With the understanding that the applicant is not bound by any scientific theory, a mechanism is proposed for the fibril formation induced by proteolytic: cleavage of GST-HD51 as shown in FIG. 7. Based on the known crystal structure of GST with a C-terminal fusion peptide (Lim et al., 1994) and the fact that the purified GST protein is a dimer we suppose that native GST-HD51 exists as a dimer with two expanded polygln sequences which form stable hairpins consisting of antiparallel .beta.-strands strongly held together by hydrogen bonds between the main chain and the side chain amides. In the native protein both hairpins are tightly bound to the surface of GST and not accessible for protein--protein interactions with other polygln sequences. As a result of the cleavage with a site-specific protease, both hairpins become accessible and .beta.-sheets with hairpins from other cleaved protein molecules are formed. This transient population of intermediates consisting of GST molecules and hairpins leads to the formation of polygln-containing .beta.-sheet fibrils and free GST molecules. This model is supported by the finding of potential intermediate structures present on one or both ends of the growing fibrils (FIGS. 4c and d). These clots of varying sizes were not detected when GST-HD51 was digested to completion with trypsin, which totally degrades the GST-tag, whilst they were detectable upon limited digestion, leaving the GST moiety largely intact (FIG. 4d). This indicates that these structures are transient intermediates.

A model of the formation of amyloid-like fibrils via transient intermediates is not without precedent. Booth et al. (1997) have shown that amyloidogenic lysosome variants aggregate on heating, unlike the wild-type protein, and that the lysozyme fibrils are formed from potential precursor proteins. It is possible that the transient GST-HD intermediates function as nuclei for ordered protein aggregation, very similarly to protein crystallization and microtubule formation, which are nucleation-dependent polymerisations (Jarrett and Lansburry, 1993). Once a nucleus is formed, the further addition of monomers becomes thermodynamically favorable and results in rapid polymerization. An important feature of a nucleation-dependent process is a lag time before the aggregates are detectable. During this period, dimers and trimers are formed. FIG. 3a shows that during proteolytic cleavage of GST-HD51 dimers of the released HD portion are formed, the concentration of which then decreases upon prolonged incubation concomitant with an increase in the formation of large protein aggregates (FIG. 3b). Although additional kinetic studies will be necessary to prove this assumption, preliminary results in our laboratory suggest that a "one-dimensional" crystallization leads to the formation of in vitro amyloid-like huntingtin aggregates.

Accordingly, the present invention provides both the possibilities to analyze aggregation of amyloid-like aggregates using defined cleavage conditions or using fusion proteins comprising long polyglutamine stretches that render the cleavage unnecessary. Whereas the cleavage of the fusion protein enables to set a distinct starting point of the reaction and therefore of the aggregate formation, the second alternative has the advantage that the use of a cleaving agent is rendered obsolete.

In a further particularly preferred embodiment of the invention said (poly)peptide defined in (ab) is huntingtin, androgen receptor, atropin, TATA binding protein, or ataxin-1, -2, -3, -6 or -7 or a fragment or derivative thereof.

The fibrillar structures formed by proteolytic cleavage of purified GST-HD51 in vitro and also in the brains of mice transgenic for the HD mutation are very similar to structures detected in brain sections or purified protein fractions of Alzheimer's disease (AD), Creutzfeldt-Jakob disease (CJD), Parkinson's Disease, Gerstmann-Stratssler-Scheinker syndrome (GSS), fatal familial isomnia (FFI), kuru, bovine spongiform encephalopathy (BSE) and scrapie (Caughey and Chesebro, 1997). In all these disorders, the accumulation of amyloid-like fibrils in the central nervous system is accompanied by loss of nerve cells and a neuropathological phenotype. However, the molecular basis of these diseases is not known. For the first time, our results raise the possibility that HD, DRPLA, SBMA, SCA1, SCA2, SCA3 and SCA7 are also the result of a toxic amyloid fibrillogenesis. Although the detection of amyloid-like fibrils has not previously been reported in these inherited diseases, our results strongly suggest that polygln-containing polymers are also formed in vivo by their detection in a transgenic model of polyglutamine disease. The high molecular weight aggregates were exclusively detected in the nuclear fraction prepared from transgene brain material, which is in good agreement with the results of Davies et al. (1997), who demonstrated the presence of the transgene protein and ubiquitin in neuronal intranuclear inclusions, from a time prior to the development of a neurological phenotype. Strikingly, ultrastuctural analysis has shown similar intranuclear inclusions to be present in the cortical and striatal biopsy material from HD patients (Roizin et al., 1979) some of which showed clear evidence of intranuclear fibrils of up to 1 .mu.m in length. Preliminary experiments with nuclear protein fractions prepared from HD brain material indicated that insoluble huntingtin aggregates are indeed present in these fractions. However, additional control experiments have to be performed to substantiate these results.

One possible explanation for the absence of detection of high molecular weight huntingtin protein aggregates in HD brains could be that the aggregates consist mainly of polygln-containing peptides which have been cleaved from the full length protein. In such a case, only an antibody raised against an N-terminal huntingtin fragment, containing the polygln sequence, would be able to detect the aggregates in the nucleus. In most of the previous immunohistochemical studies, antibodies raised against the central or C-terminal portion of huntingtin have been used, which detect the full length protein (350 kDa) in the cytosol and in the membrane containing fractions (DiFiglia et al., 1995; Sharp et al., 1995; Trottier et al., 1995a). However, antibodies raised against peptides and fusion proteins from the N- and C-terminus of huntingtin also detected the protein in the nucleus (de Rooij et al., 1996; Hoogeveen et al., 1993), indicating that huntingtin is also present in this subcellular compartment. There are several lines of evidence to implicate a shorter polygln-containing peptide/protein fragment of huntingtin in the pathology of HD. Ikeda et al. (1996) showed that a short fragment of the MJD1 protein containing 79 polyglns (Q79C) but not the full length protein with the elongated repeat induced apoptotic cell death in COS cells. The polygln-containing protein fragment migrated in SDS-gels at a position much higher than expected from its molecular weight, even after boiling in the presence of 2% SDS. These results are in good agreement with our data obtained using the GST-HD fusion proteins containing elongated polygln sequences. FIGS. 1a and b show that the expression of GST-HD83 and GST-HD122 in E. coli was dramatically reduced compared to the fusion proteins containing 20 51 repeats, and additional studies have indicated that the elongated glutamines are toxic for E. coli cells.

The possibility that polygln-containing cleavage products of huntingtin cause neurodegeneration in HD is substantiated by the finding of Goldberg et al. (1996) who showed that an N-terminal 80 kDa huntingtin fragment is cleaved from the full length protein by apopain, a proapoptotic cysteine protease. This indicates that the N-terminus of huntingtin is primarily accessible for proteases and distinct proteolytic cleavage products can be formed in vitro and in vivo. In addition, there is strong evidence that the mutated huntingtin somehow induces apoptotic cell death in HD, but the underlying molecular mechanism is not known (Duyao et al., 1995; Portera-Cailliau et al., 1995). Our data suggest that a proteolytic cleavage product of huntingtin, which is transported into the nucleus by an unknown mechanism, causes selective neuronal cell death by the formation of insoluble amyloid-like fibrils. It is possible that the transport to the nucleus is facilitated by a specific nuclear transport mechanism which is unique to certain neuronal cells and involves abnormal protein--protein interactions related to the elongated polygln. Alternatively, there may be specific nuclear proteins in the affected neurons which enhance the huntingtin protein aggregation.

Recently, the formation of neuronal intranuclear inclusions in mice transgenic for the SCA1 mutation have been detected, indicating that polygln-containing polymers are also formed in spinocerebellar ataxia type 1. Furthermore, the accumulation of polyglutamine-containing protein aggregates in neuronal intranuclear inclusions (NIIs) has been demonstrated for several progressive neurodegenerative diseases such as Huntington's disease (HD) (M. DiFiglia et al., 1997; M. W. Becher et al., 1997), dentatorubral pallidoluysian atrophy (DRPLA) (M. W. Becher et al., 1997; S. Igarashi et al., 1998) and the spinocerebellar ataxia (SCA) types 1 (P. J. Skinner et al., 1997; A. Matilla et al., 1997), 3 (H. L. Paulson et al., 1997) and 7 (M. Holmberg et al., 1998).

The components of the composition of the invention may be packaged in containers such as vials, optionally in buffers and/or solutions. If appropriate, one or more of said components may be packaged in one and the same container.

In an additional preferred embodiment of the composition of the present invention, said amyloidogenic (poly)peptide self-assembles subsequent to release from said fusion protein.

As has been pointed out herein before, the self-assembly of said (poly)peptides only subsequent to the release from the fusion protein provides the advantage that an exact time point of the start of the formation can be set. This has a number of advantages. For example, inhibitors of aggregate formation can be tested as regards their efficacy as a function of time. In addition, obtainment of data is facilitated in view of the fact that for amyloid formation a premix may be set up to which only the cleaving agent must be added.

In a further preferred embodiment of the composition of the invention, said amyloidogenic (poly)peptide is the amyloid precursor protein (APP), .beta.-protein, an immunoglobulin light chain, serum amyloid A, transthyretin, cystatin C, .beta.2-microglobulin, apolipoprotein A-1, gelsoline, islet amyloid polypeptide (IAPP), calcitonin, a prion, atrial natriuretic factor (ANF), lysozyme, insulin, fibrinogen, tau proteins or .alpha.-synuclein or a fragment or derivative thereof.

Deposits of .beta.-amyloid in neuritic plaques and blood vessel walls are the principal pathological feature in the brains of patients with Alzheimer's disease. These amyloid deposits contain the 39 43 amino acid .beta.-amyloid peptide which is derived by proteolytic cleavage from the larger precursor, the amyloid precursor protein (APP). There is strong evidence that the formation and aggregation of .beta.-amyloids into fibrils is the primary pathogenic event leading to amyloid deposition in Alzheimer's disease.

An additional preferred embodiment relates to a composition wherein said (poly)peptide defined in (aa) is glutathione S-transferase (GST), intein, thioredoxin, dihydrofolate reductase (DHFR) or chymotrypsin inhibitor 2 (Cl2) or a functional fragment or derivative thereof.

All of these (poly)peptides may be advantageously used to enhance the solubility and/or prevent aggregation of the fusion proteins of the invention. Particularly preferred is to employ intein in said composition because the protein has been modified such that it undergoes a self-cleavage reaction at its N-terminus at low temperatures in the presence of thiols such as DTT (MPACT.TM. I System/New England Biolabs). Also comprised by this embodiment are functional fragments of any of these above recited proteins. The term "functional fragment" as used herein is intended to denote the capability of said fragment to confer solubility or prevent aggregation.

Further preferred is that the nucleic acid contained in the composition of the invention is DNA. Particularly preferred is that said DNA is cDNA, synthetic DNA or (semi)synthetic DNA.

Also preferred is that the vector that may be contained in the composition of the invention is an expression vector or a gene targeting vector. These vectors may advantageously be used for transfecting hosts that may or may not be contained in the composition of the invention.

Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions. Preferably, the nucleic acid molecule of the invention is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells. Expression of said nucleic acid molecule comprises transcription of the nucleic acid molecule into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the PL, lac, trp or tac promoter in E. coli, and examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the nucleic acid molecule. Furthermore, depending on the expression system used leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the nucleic acid molecule of the invention and are well known in the art. The leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), or pSPORT1 (GIBCO BRL). Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used.

Gene therapy, which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534 539; Schaper, Circ. Res. 79 (1996), 911 919; Anderson, Science 256 (1992), 808 813; Isner, Lancet 348 (1996), 370 374; Muhlhauser, Circ. Res. 77 (1995), 1077 1086; Wang, Nature Medicine 2 (1996), 714 716; WO94/29469; WO 97/00957 or Schaper, Current Opinion in Biotechnology 7 (1996), 635 640, and references cited therein. The nucleic acid molecules and vectors of the invention may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell. Preferably, said cell is a germ line cell, embryonic cell, or egg cell or derived therefrom, most preferably said cell is a stem cell.

Said hosts may then be used for the production of the fusion protein comprised in the composition of the invention.

Said host cell may be a prokaryotic or eukaryotic cell. The nucleic acid molecule or vector of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally.

Preferably, said host is a bacterial, preferably an E. coli, an animal-, preferably a mammalian, an insect-, a plant-, a fungal, preferably a yeast- and most preferably a Saccharomyces or Aspergillius cell, a Pichia pastoris cell, a transgenic animal or a transgenic plant.

The present invention also relates to a method of producing a fusion protein as defined in the diagnostic composition of any of the preceding claims comprising culturing or raising the host as defined in claim 11 and isolating said fusion protein. Preferably the bacterial host E. coli is used for the expression of the GST-HD fusion proteins. The cDNA fragments containing CAG repeats in the normal or pathological range may, for example, be cloned into pGEX-5X-1 (Pharmacia) and the resulting plasmids expressing fusion proteins with polygln-sequences used for protein purification. The resulting proteins may be purified under native conditions by affinity chromatography on glutathione agarose (Smith and Johnson, 1988).

Additionally, the present invention relates in a preferred embodiment to a composition wherein said antibody is a monoclonal antibody, polyclonal antibody, phage display antibody or a fragment or derivative thereof.

The above recited fragments or derivatives comprise Fv-, Fab-, F(ab)'.sub.2-fragments, single-chain antibodies or single-chain antibody domains. These antibodies may advantageously be used in experiments such as Western blotting experiments to determine the presence of the protein on, for example, nitrocellulose membrane.

Alternatively, the antibody, derivative or fragment thereof may be used in immunometric assays such as ELISAs or RIAs or may be coupled to columns in order to retain the fusion protein from, for example, mammalian sources on said column for further detection or purification.

Alternative uses and advantages of the antibody or fragment or derivative contained in the composition of the invention are, on the basis of the teachings of this invention, clear to the person skilled in the art.

The invention further relates to an in vitro method of producing amyloid aggregates comprising (a) at least partially cleaving the fusion protein comprised in the diagnostic composition of the invention wherein the (poly)peptide that is released has the ability to self-assemble into amyloid-like fibrils or protein aggregates or (b) inducing self-assembly into amyloid-like fibrils or protein aggregates by heating the fusion protein comprised in the composition of the present invention or an amyloidogenic (poly)peptide that has a ability to self-assemble into amyloid-like fibrils or protein aggregates by inducing a pH change in a solution comprising said fusion protein/(poly)peptide or by treating said fusion protein/(poly)peptide with a denaturing agent.

The method of the invention may advantageously be used to study in more detail the process that leads to the formation of amyloid-like fibrils or protein aggregates from amyloidogenic (poly)peptides. Using the method of the invention, the onset of, for example, HD or AD can be examined in an in vitro situation. It is important that the polypeptide that is released by the cleaving agent still retains the possibility to form amyloid-like fibrils or protein aggregates. The formation of such fibrils or aggregates may be monitored, for example, by electron or light microscopy. Varying the cleaving conditions in cases where more than one cleavage site is present on the fusion protein may be used to further elaborate the minimal requirements for said amyloid-like fibril or protein aggregate formation.

Preferably, the cleavage is effected chemically or enzymatically, or by the intein self-cleavage reaction in the presence of thiols.

In the following, enzymatic cleavage will also be referred to as proteolytic cleavage. The enzymatic cleavage has the advantage that the cleavage reaction can be performed under almost physiological conditions and normally only low amounts of the protease are necessary for the cleavage reaction. Furthermore, said cleavage is highly specific and the enzyme can be regarded as nontoxic. Therefore, one can envisage a wide variety if applications within this invention. The disadvantage of the enzymatic cleavage reaction is that prospective inhibitors might inhibit in some cases the protease and in turn prevent the formation of protein aggregates. In comparison, this is not the case when the cleavage reaction is performed chemically.

Further, the present invention relates to a method of testing a prospective inhibitor of aggregate formation of a fusion protein as defined in the composition of the invention when enzymatically or chemically cleaved or a non-cleaved fusion amyloidogenic (poly)peptide as defined hereinbefore or an amyloidogenic non-fusion (poly)peptide comprising

(a) incubating in the presence of a prospective inhibitor

(aa) said fusion protein in the presence or absence of a cleaving agent; or

(ab) said non-fusion poly(peptide); and

(b) assessing the formation of amyloid-like fibrils or protein aggregates.

This method of the present invention provides a particularly strong impact on the pharmaceutical research related to amyloid-associated diseases. For the first time, an inhibitor of fibril or aggregate formation can conveniently, directly, easily and within a short time be tested in vitro. As has been detailed herein above, aggregate formation may be tested on cleavage products, on non-cleaved fusion proteins or on the above recited non-fusion proteins which have the capacity to aggregate when the temperature is raised, the pH is lowered or the protein is dissolved in urea and the urea is slowly diluted out with a solvent. Additionally, the present invention does not exclude self-assembly under different conditions.

It was shown recently that acid-mediated denaturation of, e.g., transthyretin yields a conformational intermediate that can self-assemble into amyloid (Lai et al., 1996). Booth et al. demonstrated that heat denaturation of human lysozyme variants resulted in instability, unfolding, and amyloid fibrillogenesis.

Preferably, the incubation is effected in the presence of factor Xa, trypsin, endoproteinase Arg-C, endoproteinase Lys-C, proteinase K, thrombin or elastase at a temperature of preferably 25 to 37.degree. C. for 0,5 to 16 hours and the assessment of the formation of fibrils or aggregates in step (b) is preferably effected by a filter assay or by a thioflavine T (ThT) fluorescence assay, in which the fluorescence intensity reflects the degree of aggregation.

As regards the filter assay, a more detailed protocol thereof is explained in the European patent application entitled "Novel method of detecting amyloid-like fibrils or protein aggregates" filed on the same day with the European Patent Office and assigned to the same applicant which is explicitly incorporated herein by reference.

Additionally, the present invention relates to a method for identifying an inhibitor of aggregate formation of a fusion protein as defined in the invention prior to or after proteolytic or chemical cleavage or of a non-fusion amyloidogenic (poly)peptide as described herein above comprising (a) loading a surface or gel with said protein or an aggregate thereof; (b) incubating said surface or gel with a prospective inhibitor; and (c) assessing whether the presence of said prospective inhibitor avoids or reduces aggregate formation or further aggregate formation.

In accordance with this embodiment of the invention, proteolytic or chemical cleavage can be advantageously effected either prior or after the loading of the surface or gel leaving the investigator additional degrees of freedom in devising his experiments. The method of the invention is both useful for investigating the onset of aggregate or fibril formation or assessing the progression of such a process starting from the already existing aggregate or fibril. The latter embodiment is particularly useful in investigating treatment conditions for patients that are already affected by the disease at an early or medium stage thereof.

There is strong evidence that the formation of amyloids is a nucleation-dependent polymerization similar to protein crystallization or microtubule assembly. However, the deposition of a monomer onto a preexisting amyloid template is independent of the nucleation process. Thus, it will be very important to study the deposition of monomers onto a defined template under physiological conditions. With our in vitro system we should be able to monitor the deposition of radiolabelled polygln-containing monomers.

Preferably, said surface employed in the method of the invention is a membrane. Preferably, the membrane should be cellulose acetate and should have a low binding capacity for soluble proteins.

The invention also relates to an inhibitor identifiable or identified by the method of the invention.

The various methods described herein above will give rise to the isolation of a number of inhibitors which are also comprised by the present invention. Once such an inhibitor is known, it is of course not necessary to identify it again by the method of the invention. Rather, said inhibitor can be produced by chemical or recombinant means. In the case that the inhibitor is of proteinaceous material, it is preferred to resynthesize a compound having the or most of the characteristics of said inhibitor by peptidomimetics.

Preferably, a number of compounds or compound classes are tested for their efficacy to inhibit amyloid-like fibril or protein aggregate formation. Said compounds comprise an antibody, 4'-Iodo-4'-deoxydoxorubicin (IDOX), pyronine Y, guanidine hydrochloride, urea, rifampicin and derivatives thereof, myristyltrimethylammonium bromide, hydroquinone, p-benzoquinone, 1,4-dihydroxynaphthalene, p-methoxyphenol, .alpha.-tocopherol, ascorbic acid, .beta.-carotene, anthracycline, doxorubicin, hexadecyl-N-methylpiperidinium, dodecyltrimethyl-ammonium, N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, a (poly)peptide, glutamine or an oligoglutamine peptide. These compounds may be used in the inhibition or reversion of aggregate formation, for example, by formulation into a pharmaceutical composition for the treatment of any of the diseases cited herein.

The present invention also relates to a pharmaceutical composition comprising the inhibitor of the invention and a pharmaceutically acceptable carrier and/or diluent.

The pharmaceutical composition of the invention will find wide applicability in the medical field. Essentially all diseases associated with protein aggregate formation or amyloid-like fibril formation, in particular if they are associated with neuronal tissue or cells, may be effectively treated with the pharmaceutical composition of the invention.

Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 .mu.g (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 .mu.g to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 .mu.g to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 10.sup.6 to 10.sup.12 copies of the DNA molecule. The compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery.

The therapeutically useful compounds identified according to the method of the invention may be administered to a patient by any appropriate method for the particular compound, e.g., orally, intravenously, parenterally, transdermally, transmucosally, or by surgery or implantation (e.g., with the compound being in the form of a solid or semi-solid biologically compatible and resorbable matrix) at or near the site where the effect of the compound is desired.

Finally, the present invention relates to a transgenic mammal or plant comprising a nucleic acid molecule or vector as described in the invention. The transgenic mammal or plant would advantageously be used for the in vivo testing of the efficacy of the inhibitors referred to above. With the pharmaceutical composition of the invention the formation of protein aggregates in the brain or other tissues of a transgenic animal can be monitored quantitatively. Furthermore, in vivo studies relating to the onset or progress of the above recited diseases may be carried out.
 

Claim 1 of 13 Claims

1. A composition comprising (a) a nucleic acid molecule encoding a fusion protein comprising (aa) a glutathione S-transferase (GST) (poly)peptide that enhances solubility and/or prevents aggregation of said fusion protein; and (ab) a huntingtin (poly)peptide that has the ability to self-assemble into fibrils or protein aggregates, wherein connection of polypeptides (aa) and (ab) is via a linker or by a direct attachment, and wherein at least one of the linker, (poly)peptide (aa) and (poly)peptide (ab) includes a cleavable site, and wherein said huntingtin (poly)peptide self-assembles subsequent to release from said fusion protein, (b) a vector containing the nucleic acid molecule of (a); (c) a host transformed with the vector of (b); and/or (d) a fusion protein encoded by the nucleic acid of (a).
 

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