Peptides for detecting amyloid fibril aggregates

Abstract

The present invention relates to molecules capable of binding amyloid fibril aggregates. The present invention further describes methods for detecting the presence of amyloid fibril aggregates in a sample or subject. The present invention even further describes methods for treating pathologies caused by amyloid fibril aggregates.

Description

Technical Field

[0001] This invention relates to the fields of medicine and molecular diagnostics. In particular, this invention relates to molecules that specifically bind to amyloid fibrillary aggregates. The invention further relates to methods for detecting the presence of amyloid fibrillary aggregates in samples or subjects, and methods for treating diseases caused by amyloid fibrillary aggregates. Background Technology

[0002] Protein aggregation into fibrils is a hallmark of the development of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), which remain incurable due to a lack of understanding of their mechanisms. In these diseases, aggregated proteins are thought to be causally linked to disease onset. Therefore, understanding the aggregation process is crucial. Current diagnosis relies on phenotypic behavior, family history, and genetic testing, and sometimes on techniques such as PET tracking and brain imaging, which are based on the large accumulation of aggregated proteins that form amyloid fibrils or brain degeneration that occurs later in the disease progression (“2024 Alzheimer's disease facts and figures,” 2016; Thal et al., 2013; Jack et al., 2024).

[0003] A range of techniques exist for detecting amyloid fibrils postmortemly or in vitro. Electron microscopy and negative staining can be used to assess the morphology of formed fibrils, as well as their branching, width, and length (Shi et al., 2021; Sunde and Blake, 1997). Dyes such as thiosulfate T (ThT) and Congo red are qualitative methods for identifying the structure and histological staining of amyloid material in vitro (Elghetany and Saleem, 1988; Howie et al., 2008; Vassar and Culling, 1959). Antibodies (large biological agents of 150 kDa) can recognize specific proteins that form aggregates but generally cannot cross the blood-brain barrier (Ko et al.; Ksiezak-Reding et al., 1987). There is a strong medical need for diagnostic tracers (such as Tau tracers) for the early identification of pathological protein fibrils in patients. We anticipate eventually being able to detect protein fibrils in bodily fluids, including plasma, CSF, or tears.

[0004] Seed amplification assays (SAAs) have been developed to detect and amplify the signal of protofibrils in fluids (Vaneyck et al. 2023; Concha-Marambio et al. 2023). These assays rely on the prion-like properties of amyloid protofibrils, which can multiply in the presence of available monomers. One such assay is RT-Quic (Okuzumi et al. 2023; Atarashi et al. 2023). After a detectable increase in signal is observed, if the protofibrils are available, these protofibrils become seeds for the formation of more protofibrils. These assays are limited by determining the presence of protofibril seeds, and they cannot determine which species are responsible for reproductive aggregation or provide information about protofibril length. Moreover, monomer selection can bias the results towards specific diseases.

[0005] Fibrous tracers require the development of molecules that specifically bind to fibrils. Tau accumulation is associated with a variety of Tau protein diseases, including Alzheimer's disease (AD). Tracers are designed to identify these aberrant accumulations and aid in the diagnosis of these Tau protein diseases. In 2020, the imaging agent Tauvid ([18F]-flutoxetine) received FDA approval (Commissioner, 2020; Jie et al., 2021; Xia et al., 2013). Tauvid is currently the only approved Tau tracer for diagnosing Tau in living patients, with results that can differentiate between disease and disease stage (Bejanin et al., 2017; Iaccarino et al., 2021; Jack et al., 2019; Merz et al., 2023; Ossenkoppele et al., 2018; Ossenkoppele et al., 2016; Leuzy et al., 2020; Jie et al., 2021; Mohammadi et al., 2023). Tracers for other aggregated proteins are also under development. For huntingtin (a protein that aggregates in HD), tracers have passed in vitro sample testing (Delva et al., 2022; Herrmann et al., 2021; Liu et al., 2021; Liu et al., 2020; Matlahov et al., 2022). To date, tracer development has focused on identifying aggregates of a single protein. Currently available fibril tracers are typically limited to binding to a specific fibril type. It is extremely important to develop techniques that specifically identify fibril structures and the type of early aggregation (rather than monomers) to allow for (early) diagnosis and monitoring of disease progression in patients.

[0006] Recently, diagnostic strategies for neurodegenerative diseases have shifted towards fluid biomarkers in the CSF and blood (Ferreira, Therriault et al. 2023, Gonzalez-Ortiz, Kac et al. 2023, Johnson, Suárez-Calvet et al. 2023, Dubbelman, Hendriksen et al. 2024, Tijms, Vromen et al. 2024). These offer several advantages over PET tracers because they can predict disease in the pre-symptomatic stage. Furthermore, they are cheaper and less invasive, making them more readily available. In the CSF, fibrillary length has been shown to indicate disease progression (Nirmalra et al. 2023). Therefore, studying the presence of amyloid fibrils in a patient's body fluids can provide valuable information for understanding patient progression and can also be used to monitor the effects of new therapies. Summary of the Invention

[0007] In a first aspect, the present invention relates to a polypeptide comprising: i) Amino acid sequence: PWWX 1 -X 2 -PWWPWHHPX 3 in X 1 It is R or K, preferably R; X 2 It is R or K, preferably R; X 3 It is H or W, preferably H; or ii) Amino acid sequence: PWWRRPWWPWHHPH; or WWHPEPPHVRSWSWWRHGRGE-D The amino acid sequence contains no more than 4, 3, 2, or 1 amino acid substitutions, deletions, or insertions. Furthermore, the amino acid sequences of i) and ii) are bound to aggregates or oligomeric precursors of amyloid fibrils.

[0008] In a second aspect, the present invention relates to a nucleic acid sequence that encodes a polypeptide as described herein.

[0009] In a third aspect, the present invention relates to a pharmaceutical composition comprising a polypeptide according to any one of the preceding claims, optionally further comprising a pharmaceutical excipient.

[0010] In a fourth aspect, the present invention relates to a method for diagnosing and / or monitoring the progression of a pathological condition caused by amyloid fibrillation in a subject, or for identifying a disease state of a pathological condition caused by amyloid fibrillation or a predisposition to a condition caused by amyloid fibrillation.

[0011] In one embodiment, the present invention relates to a method for diagnosing, in a subject, a pathological condition caused by amyloid fibrillation or a predisposition to such a condition, the method comprising: a) Provide a sample from the subject; b) Contact the sample with a polypeptide as described herein, which specifically binds to aggregated amyloid fibrils; and c) Detect the presence of aggregated amyloid fibrils bound to the polypeptide. The detection of aggregated amyloid fibrils bound to the polypeptide indicates the presence of aggregated amyloid fibrils.

[0012] In another embodiment, the present invention relates to a method for monitoring the progression of a pathological condition caused by amyloid fibrillation in a subject or for identifying a disease state caused by amyloid fibrillation, the method comprising: a) Provide a sample from the subject; b) Contact the sample with a polypeptide as described herein, which specifically binds to aggregated amyloid fibrils; and c) Determine at least one of the following: the length of the amyloid fibrils bound to the polypeptide, the hydrodynamic radius, and the location of the fibrils. The length of the fibril, its hydrodynamic radius, and / or its location provide an indication of progression or identification of a disease state.

[0013] In a fifth aspect, the present invention relates to polypeptides, nucleic acids, or pharmaceutical compositions as described herein, for use as medicines, preferably for use as medicines for treating pathological conditions caused by amyloid fibrillary aggregation.

[0014] In a sixth aspect, the present invention provides the use of polypeptides, nucleic acids, or pharmaceutical compositions as described herein for the detection of aggregated amyloid fibrils. Detailed Implementation

[0015] definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Those skilled in the art will recognize that many methods and materials similar to or equivalent to those described and used herein can be employed in the practice of this invention. In fact, this invention is by no means limited to these methods.

[0016] In this document and its claims, the verb "comprising" and its inflections are used in their non-limiting sense to mean including the items that follow the word, but not excluding items not specifically mentioned. Furthermore, unless the context explicitly requires the presence of one / a and only one / a type of element, mentioning an element by the indefinite article "a / a (a or an)" does not preclude the possibility of more than one / a type of element. Therefore, the indefinite article "a / a (a or an)" generally means "at least one / a type".

[0017] As used herein, the term “and / or” indicates that one or more of the stated circumstances may occur individually or in combination with at least one of the stated circumstances, up to and including all of the stated circumstances.

[0018] As used in this article, “at least” a specific value means that specific value or more. For example, “at least 2” is understood as the same as “2 or more” (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.).

[0019] The word “about” or “approximately” when used in conjunction with a numerical value (e.g., about 10) preferably means that the value can be a given value (10) plus or minus 0.1% of that value.

[0020] The terms “subject” and “patient” are used interchangeably throughout the specification and describe animals, humans or non-humans to whom the methods according to the invention are provided for treatment, diagnosis or monitoring of disease progression.

[0021] The terms “protein” and “polypeptide” are used interchangeably and refer to molecules composed of chains of amino acids without regard to a particular mode of action, size, three-dimensional structure or origin. Negatively charged polypeptides are also included in this definition.

[0022] "Amino acid" refers to one of the twenty biologically existing amino acids encoded by the universal genetic code, as well as synthetic amino acids (including D / L optical isomers).

[0023] "Disease" or "pathology" is a health condition in which the subject is unable to maintain homeostasis, and where the subject's health condition will continue to deteriorate if the disease is not improved. In contrast, a "disorder" in the subject is a health condition in which the subject is able to maintain homeostasis, but where the subject's health condition is not as good as it would be without the disorder. Without treatment, a disorder does not necessarily lead to a further decline in the subject's health condition.

[0024] The disease or disorder is considered "relieved" or "improved" if the severity of the symptoms, the frequency with which the patient experiences such symptoms, or both decrease. This also includes halting the progression of the disease or disorder. The disease or disorder is considered "cured" if the severity of the symptoms, the frequency with which the patient experiences such symptoms, or both disappear.

[0025] The inventors have unexpectedly developed peptides (described herein as "fibril paint") that specifically recognize protein fibrils. These peptides are characterized by numerous nitrogen-carbon delocalized side chains (such as W, H, and R). These residues are capable of participating in π-stacking and aromatic interactions with fibrils. These peptides can fluorescently stain amyloid fibrils, but cannot fluorescently stain the non-aggregated monomeric forms of Tau and Huntington proteins, which differ in structure and sequence. These peptides enable the measurement of aggregate and fibril sizes in both recombinant and patient-derived materials throughout the aggregation process. These peptides are particularly useful because they provide a method for not only diagnosing but also monitoring the progression of diseases caused by amyloid fibril aggregation. Advantageously, these peptides can also be used to treat pathologies caused by amyloid fibrils.

[0026] Therefore, in a first aspect, the present invention provides a polypeptide comprising: i) Amino acid sequence: PWWX 1 -X 2 -PWWPWHHPX 3 (SEQ ID NO: 1) in X 1 It is R or K, and in some embodiments, X 1 It is R; X 2 It is R or K, preferably R, and in some embodiments, X 2 It is R; X 3 It is H or W, preferably H, and in some embodiments, X 3 It is H; Or ii) a polypeptide containing the following amino acid sequence: PWWRRPWWPWHHPH (SEQ ID NO: 2) or WWHPEPPHVRSWSWWRHGRGE-D (SEQ ID NO: 3) The amino acid sequence contains no more than 4, 3, 2, or 1 amino acid substitutions, deletions, or insertions. Furthermore, the amino acid sequence of i) or ii) is bound to the aggregates or oligomeric precursors of amyloid fibrils.

[0027] In one embodiment, the polypeptide of the present invention comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 2, 3, 8, 9 and 10.

[0028] These peptides have a strong enough affinity that they are not significantly released from the fibrils during detection runs in microfluidic devices such as the FIDA1 instrument (e.g., runs of at least 1 hour or longer).

[0029] According to embodiments of the invention, the term "amyloid" or "amyloid aggregate" may refer to amyloid peptides and amyloid proteins. This term should encompass tau, IAPP, β-amyloid protein, α-synuclein, huntingtin-Tau protein, and / or α-synuclein and / or other protofibrillating proteins. According to embodiments, amyloid or amyloid aggregate may encompass or be defined as an aggregate of proteins and / or peptides composed of the aberrant association of two or more protein molecules, having a globular and / or fibrous morphology or protofibrillary structure, and particularly comprising oligomers with a diameter of 3 nm-15 nm and protofibrils with a diameter of 6 nm-15 nm, as well as β-sheet secondary structures or, in other words, crossed β-structures. Furthermore, the protofibrils may be capable of being stained with specific dyes (such as Congo red).

[0030] The polypeptides of the present invention may be "modified" and contain deletions, insertions, or substitutions of amino acid residues that result in functional equivalence. Intentional amino acid substitutions may be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the residues, as long as biological activity is preserved. For example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values ​​may include leucine, isoleucine, and valine, glycine and alanine, asparagine and glutamine, serine and threonine, and phenylalanine and tyrosine.

[0031] In this context, the term "substitution" or "substituted" means the presence of an alternative amino acid at a specified position compared to the corresponding parent molecule, which is a polypeptide containing the amino acid sequence PWWRRPWWPWHHPH (SEQ ID NO: 2) or WWHPEPPHVRSWSWWRHGRGE-D (SEQ ID NO: 3). Such a parent molecule can exist physically as a polypeptide or as a nucleic acid encoding such a polypeptide, but it can also exist only as an amino acid sequence or the corresponding nucleic acid sequence encoding that amino acid sequence in a computer or on paper. Therefore, in this context, for example, substitution is considered present if a protein is expressed by a synthetic nucleic acid that results in a mutation or substitution in its encoding, even if the nucleic acid encoding the corresponding parent molecule was not initially actually prepared during the process (e.g., when the nucleic acid molecule is prepared entirely by chemical synthesis).

[0032] In some embodiments, the peptides of the present invention may be chemically modified. For example, the peptides may be mutated to alter peptide properties such as detectability, stability, biodistribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation sites, pH, function, specificity, etc. N-methylation is an example of methylation that can occur in the peptides disclosed herein. In some embodiments, the peptides may be modified by methylating a free amine (e.g., by reductive methylation with formaldehyde and sodium cyanoborohydride).

[0033] In one embodiment, the polypeptide according to the invention comprises at least one detectable marker. As used herein, a detectable marker or detectable tag is intended to refer to a single measurable portion, such as a radioisotope, fluorescent dye, dye, or other portion known in the art that can be measured by analytical methods. The detectable marker or detectable tag can be attached to the polypeptide using methods well known in the art.

[0034] In one embodiment, the polypeptide as described herein comprises a second label. In one embodiment, the second label is a negatively charged amino acid sequence. In some embodiments, the second label is a protein recognition system. In one example, the second label is a protein quality control system such as an E3-ligase CHIP, for example, containing a negatively charged amino acid sequence such as SEQ ID NO: 5.

[0035] As defined herein, peptides can be used as warheads in bifunctional (proteolytically targeted chimeras) PROTAC molecules. PROTACs are small molecules that inhibit the function of their target proteins by targeting them for degradation by the ubiquitin-proteasome system. The design of PROTACs and their ability to selectively and instantaneously degrade target proteins are well described in the art, as in (Winter, GE et al. DRUG DEVELOPMENT. Phthalimide conjugation as astrategy for in vivo target protein degradation. Science 348, 1376-1381 (2015), Bondeson, DP et al. Catalytic in vivo protein knockdown by small-molecule PROTACs. Nat. Chem. Biol. 11, 611-617 (2015)). In a preferred embodiment, when used in a PROTAC molecule, the polypeptide, as defined herein, comprises a negatively charged amino acid sequence containing SEQ ID NO: 5 for recognition by CHIP.

[0036] Peptides, as defined herein, can also be modified for use in other forms of targeted protein degradation (via at least one of proteasome degradation, autophagy, or other degradation pathways) (see [link to relevant documentation]). Figure 12 ). Such modifications can be applied to a second marker, namely an autophagy signal marker (called AUTAC, ATTEC, AUTOTAC, LYTAC, etc.). For example, an instance of such a marker is targeted at p62 / SQSTM1.

[0037] In one embodiment, at least one of the detectable marker and the second marker is linked to an amino acid sequence or peptide via a flexible linker.

[0038] As used in this invention, the term "linker" refers to both amino acid sequences (i.e., peptide linkers) and non-peptide linkers. A peptide linker contemplated by this invention is a peptide or polypeptide linker with a length of at least one amino acid. Preferably, the linker length is from 1 to 100 amino acids. More preferably, the linker length is from 5 to 50 amino acids, and even more preferably, the linker length is from 10 to 20 amino acids. It is well known to those skilled in the art that the properties of the linker (i.e., its length and / or amino acid sequence) can alter or enhance the stability and / or solubility of the molecule. Therefore, the length and sequence of the linker depend on the composition of the corresponding portion of the fusion protein. In some embodiments, the linker is a flexible amino acid linker. As used herein, the term "flexible linker" refers to a peptide comprising at least a portion of flexible amino acid residues that allow adjacent modules to move relative to each other. In one embodiment, the flexible linker is GSGS (SEQ ID NO: 4).

[0039] In one embodiment, a detectable marker and a second marker are attached to opposite ends of the polypeptide.

[0040] In one embodiment, the detectable marker is selected from the group consisting of: radionuclides, isotopes, optical tags, magnetic materials, affinity markers, and any combination thereof.

[0041] In some embodiments, the detectable marker is a radionuclide selected from the group consisting of: 110 In、 111 In、 177 Lu、 118 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 86 Y、 90 Y、 89 Zr、 94m Tc, 99 Tc, 99m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C 13 N、 15 O、 186 Re、 188 Re、 51 Mn, 52m Mn, 55 Co、 72 As、75 Br、 76 Br、 82m Rb、 83 Sr, or other gamma emitters, beta emitters, or positron emitters.

[0042] Examples of detectable tags may include, but are not limited to, optical reporter molecules or optical tags. Suitable optical reporter molecules or optical tags include, but are not limited to, fluorescent reporter molecules and chemiluminescent groups.

[0043] A wide variety of fluorescent dyes are known in the art. Typically, the fluorophore is an aromatic or heteroaromatic compound, and can be pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzoindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylates, anthranilates, coumarin, fluorescein, rhodamine, or other similar compounds. Suitable fluorescent reporter molecules include xanthine dyes, such as fluorescein or rhodamine dyes, including but not limited to Alexa Fluor® dyes (Invitrogen Corp.; Carlsbad, CA), fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™, rhodamine, Texas Red, tetrarhodamine isothiocyanate (TRITC), 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N,N'-tetramethyl-6-carboxyrhodamine (TAMRA), and 6-carboxy-X-rhodamine (ROX). Suitable fluorescent reporter molecules also include naphthylamine dyes with an amino group at the α or β position. For example, naphthylamine compounds include 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthylsulfonate, 2-p-toluidine-6-naphthylsulfonate, and 5-(2'-aminoethyl)aminonaphthyl-1-sulfonic acid (EDANS). Other fluorescent reporter dyes include coumarins, such as 3-phenyl-7-isocyanate coumarin; acridines, such as 9-isothiocyanate acridine and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; cyanines, such as Cy2, indole-dicarbocyanine 3 (Cy3), indole-dicarbocyanine 5 (Cy5), indole-dicarbocyanine 5.5 (Cy5.5), 3-(-carboxy-pentyl)-3'ethyl-5,5'-dimethyloxacarbocyanine (CyA); 1H,5H,11H,15H-oxaanthra[2,3,4-ij:5,6,7-i'j']diquinazine-18-onium, 9-[2(or 4)-[[[6-[2,5-dioxo-1-pyrrolyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or 2)-sulfonylphenyl]-2,3,6,7,12,13,16,17 octahydro-internal salt (TR or Texas Red); BODIPY™ dyes; benzoxadiazole; stilbene; pyrene, etc. Many suitable forms of these fluorescent compounds are available and can be used.

[0044] Examples of fluorescent proteins suitable for use as imaging agents include, but are not limited to, green fluorescent protein, red fluorescent protein (e.g., DsRed), yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein and variants thereof (see, for example, U.S. Patent Nos. 6,403,374, 6,800,733 and 7,157,566). Specific examples of GFP variants include, but are not limited to, enhanced GFP (EGFP), destabilized EGFP, GFP variants described in Doan et al., Mol. Microbiol, 55:1767-1781 (2005), GFP variants described in Crameri et al., Nat. Biotechnol, 14:315319 (1996), sky blue fluorescent protein described in Rizzo et al., Nat. Biotechnol, 22:445 (2004), and Tsien, Annu. Rev. Biochem., 67:509 (1998), and yellow fluorescent protein described in Nagal et al., Nat. Biotechnol, 20:87-90 (2002). DsRed variants are described, for example, in Shaner et al., Nat. Biotechnol., 22:1567-1572 (2004), and include mStrawberry, mCherry, morange, mBanana, mHoneydew, and mTangerine. Other DsRed variants are described, for example, in Wang et al., Proc. Natl. Acad. Sci. USA, 101:16745-16749 (2004), and include mRaspberry and mPlum. Other examples of DsRed variants include mRFPmar described in Fischer et al., FEBS Lett. [Federation of European Biochemical Societies Letters], 577:227-232 (2004) and mRFPruby described in Fischer et al., FEBS Lett. [Federation of European Biochemical Societies Letters], 580:2495-2502 (2006).

[0045] Extremely small particles (called nanoparticles) can also be used as detectable labels for peptides, as described in this article. These particles range in size from 1 to 1000 nm and include different chemical structures, such as gold and silver particles as well as quantum dots.

[0046] Another type of nanoparticle that can be used as a detectable label is quantum dots. Quantum dots are fluorescent crystals with a diameter of 1–5 nm that can be excited by light over a wide range of wavelengths. These crystals emit light (such as monochromatic light) with wavelengths depending on their chemical composition and size. Quantum dots such as CdSe, ZnSe, InP, or InAs possess unique optical properties.

[0047] In some embodiments, the magnetic material is selected from the group consisting of: nanoparticles containing nanocomposite materials, superparamagnetic iron oxide nanoparticles, and any combination thereof.

[0048] In some embodiments, the affinity marker is selected from the group consisting of: epitopes, streptavidin, or avidin-binding peptides, biotin, and oligohistidine sequences.

[0049] In a second aspect, the present invention provides a nucleic acid sequence that encodes a polypeptide as described herein.

[0050] In a third aspect, the present invention provides a pharmaceutical composition comprising a polypeptide as described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutical excipient.

[0051] Pharmaceutically acceptable excipients (such as adjuvants or mediators) are used to administer the peptide to a subject. The pharmaceutical composition can be used in the treatment methods described below by administering an effective amount of the composition to a subject in need.

[0052] As used herein, the term "pharmaceuticalally acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption-delaying agents compatible with drug administration (see, for example, "Handbook of Pharmaceutical Excipients," Rowe et al., 7th ed., 2012, www.pharmpress.com). The use of such media and agents for the active pharmaceutical ingredient is well known in the art. Their use in a composition should be considered unless any conventional media or agent is incompatible with the active compound. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexachlorocyclohexane quaternary ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) peptides; proteins, Examples of active ingredients include serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming anti-charge ions such as sodium; metal complexes (e.g., Zn2+-protein complexes); and / or nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0053] In a fourth aspect, the present invention relates to a method for diagnosing and / or monitoring the progression of a pathological condition caused by amyloid fibrillation in a subject, or for identifying a disease state of a pathological condition caused by amyloid fibrillation or a predisposition to a condition caused by amyloid fibrillation.

[0054] In one embodiment, the present invention relates to a method for diagnosing, in a subject, a pathological condition caused by amyloid fibrillation or a predisposition to such a condition, the method comprising: a) Provide a sample from the subject; b) Contact the sample with a polypeptide as described herein, which specifically binds to aggregated amyloid fibrils; and c) Detect the presence of aggregated amyloid fibrils bound to the polypeptide. The detection of aggregated amyloid fibrils bound to the polypeptide indicates the presence of aggregated amyloid fibrils.

[0055] In another embodiment, the present invention relates to a method for monitoring the progression of a pathological condition caused by amyloid fibrillation in a subject or for identifying a disease state caused by amyloid fibrillation, the method comprising: a) Provide a sample from the subject; b) Contact the sample with a polypeptide as described herein, which specifically binds to aggregated amyloid fibrils; and c) Determine at least one of the following: the length of the amyloid fibrils bound to the polypeptide, the hydrodynamic radius, and the location of the fibrils. The length of the fibril, its hydrodynamic radius, and / or its location provide an indication of progression or identification of a disease state.

[0056] As used in this article, “diagnosis” typically involves determining whether a subject is likely to be affected by a given disease, disorder, or functional impairment. Technicians typically make diagnoses based on one or more diagnostic indicators (i.e., biomarkers), the presence, absence, frequency, or amount of which indicates the presence or absence of a disease, disorder, or functional impairment.

[0057] Subjects may be symptomatic (e.g., subjects presenting with symptoms associated with Tau protein disorders (e.g., AD, AGD, CBD, PiD, PSP),) such as changes in personality, behavior, sleep patterns, and executive function; memory loss; confusion; inability to learn new things; difficulty performing multi-step tasks; problems coping with new situations; hallucinations; delusions and paranoia; impulsive behavior; inability to communicate; weight loss; seizures; skin infections; dysphagia; groaning; humming; snoring; increased sleep; loss of bowel and bladder control; word-finding disorder; dyslexia; disorientation; supranuclear palsy; wide-eyed appearance; dysphagia; unfounded anxiety; irrational fears; pursuant to purchase; impaired social norms (e.g., breaches of etiquette, vulgar language, inappropriate behavior, disinhibition, erroneous perceptions); passivity; low motivation (lack of will); lethargy; hyperactivity; pacing and wandering; etc. Subjects may be asymptomatic (e.g., subjects not presenting with symptoms associated with Tau protein disorders, or whose symptoms have not yet been identified).

[0058] In some embodiments, the subjects are patients with prodromal symptoms. "Dementia patients with prodromal symptoms" are people who do not have Alzheimer's disease as defined above but have an increased likelihood of developing Alzheimer's disease. Similarly, "Alzheimer's disease patients with prodromal symptoms" are people who do not have AD but have an increased likelihood of developing AD.

[0059] In some embodiments, the subjects had preclinical dementia.

[0060] In one embodiment, the detected aggregates are pathological aggregates. The term "pathological aggregate" refers to aggregates of amyloid and non-amyloid substances. Amyloid aggregates can be further distinguished into amyloid fibrils and amyloid oligomers. In a preferred embodiment, the pathological aggregates are intracellular aggregates. Many diseases known to be associated with pathological aggregates classified as forming extracellular pathological aggregates also form intracellular aggregates. Indeed, IAPP and β-amyloid have both intracellular and extracellular aggregates.

[0061] As used herein, “detecting” or “detection”, “assay”, etc., should be understood as an assay performed to identify pathological aggregates. Assays for detecting such protein complexes are well known in the art and are typically based on the detection of labels or markers, such as any radioactive, fluorescent, biological, or enzyme-based labels or tags used standardly in the art. Examples of methods for measuring the fluorescence intensity of individual molecules in a sample solution include fluorescence correlation spectroscopy (FCS) and fluorescence intensity distribution analysis (FIDA). “Detection limit” should be understood as the minimum concentration at which the desired pathological aggregate is detected and is determined by the binding constant of the indicator-ligand interaction, the detection limit of the indicator (typically in the picomolar range for many fluorophores using current instruments), the difference in diffusion rate between the unbound indicator and the indicator-analyte complex, and the standard deviation used to determine peak distortion. As an example, in cases of strong selective interactions, achieving sub-nanomolar to picomolar sensitivity in FIDA methods should be feasible.

[0062] In a preferred embodiment, the detection method used in the method of the present invention is performed by FIDA, which detects fluctuations in molecular fluorescence intensity present in the focused region of an optical detector. Fitting data reveals the average hydrodynamic radius of the particles as a measure of size. Furthermore, the detection of individual peaks by the detector allows for the inference of the number of fibrils in the sample.

[0063] In another aspect, the present invention provides a method for imaging the presence of pathological aggregates. Using this method, a reliable test can be established to confirm the presence or absence of nanoscale amyloid aggregates. The imaging method includes: a) Provide a sample from the subject; b) Contact the sample with a polypeptide as described herein, which specifically binds to aggregated amyloid fibrils; and c) Detect the presence of aggregated amyloid fibrils bound to the polypeptide; and d) Optionally determine at least one of the following: the state, conformation, length, and concentration of amyloid fibrils and proteins within them. The detection of aggregated amyloid fibrils bound to the polypeptide indicates the presence of aggregated amyloid fibrils, and the state, conformation, length, and concentration of proteins within the amyloid fibrils provide an indication of progression or identification of a disease state.

[0064] In a preferred embodiment, the polypeptide that specifically binds to aggregated amyloid fibrils as described herein is detected in aggregated amyloid fibrils at fibril concentrations of less than 10 nM, 5 nM, 2 nM, 1 nM, 500 pM, 400 pM, or 200 pM, wherein the aggregated amyloid fibrils are preferably detected in (human) serum using the FIDA method.

[0065] In some embodiments, the sample is a “biological sample.” As used herein, the term “biological sample” or “sample” refers to a sample obtained from or derived from a subject. By way of example, a sample may be selected from the group consisting of: bodily fluids, blood, whole blood, plasma, serum, mucus secretions, tears, urine, or saliva. In some embodiments, the sample is or contains a blood sample. In some embodiments, the sample is collected from the subject’s brain (e.g., brain tissue) or pancreatic tissue. In some embodiments, the biological source used to detect biomarkers is a blood sample, serum sample, or plasma sample. In some embodiments, the sample is cerebrospinal fluid (CSF) or urine. In some embodiments, the sample is pancreatic tissue or brain tissue.

[0066] In some embodiments, samples are collected from a biopsy. A biopsy is a sample of tissue taken from a living subject. A biopsy sometimes also refers to a medical procedure that removes tissue from a living subject. In some embodiments, samples can be collected via a needle biopsy. A needle biopsy is performed using a round blade ranging in size from 1 mm to 8 mm. In some embodiments, samples can be collected from a fine-needle aspiration biopsy (FNAB or FNA). A fine-needle aspiration biopsy is a procedure used to examine superficial (just under the skin) masses or lumps. In some embodiments, a thin, hollow needle is inserted into the body to collect the sample.

[0067] In some embodiments, the sample is obtained from a living subject. For example, the sample may be collected from the subject during a medical procedure, such as surgery.

[0068] In some embodiments, samples are collected from post-mortem specimens (e.g., post-mortem human brain specimens).

[0069] In one embodiment, the detected amyloid aggregates originate from amyloid-forming proteins such as Tau, IAPP, β-amyloid, α-synuclein, huntingtin-Tau protein, and / or α-synuclein. Further examples of proteins that form pathological aggregates (particularly amyloid oligomers and amyloid fibrils) and related pathologies are summarized in Table 1 on pages 32, 33, and 34 of Chiti F and Dobson CM (2017) Annu. Rev. Biochem. [Annals of Biochemistry] 86: 27-68.

[0070] In another aspect, the present invention provides polypeptide, nucleic acid, or pharmaceutical compositions as described herein for use as medicines.

[0071] In another aspect, the present invention provides a method for treating a pathological condition caused by amyloid fibrillary aggregation in a subject in need, the method comprising the step of administering a polypeptide, nucleic acid, or pharmaceutical composition as described herein to the subject.

[0072] As used herein, “treatment of a pathological condition caused by amyloid fibrillation” means reducing the frequency or severity of at least one sign or symptom of a disease or disorder caused by amyloid fibrillation. The terms “treat,” “treating,” or “treatment” are used interchangeably herein to describe reversing, alleviating, or inhibiting the progression of a disease and / or lesion, or one or more symptoms of such a disease to which such terms apply. Depending on the subject’s condition, the term also refers to disease prevention and includes prevention of disease onset or prevention of symptoms associated with the disease. Treatment can be carried out in an acute or chronic manner. The term also refers to reducing the severity of a disease or symptoms associated with such a disease prior to its onset. Such prevention of disease or reduction of disease severity prior to onset refers to administration of the pharmaceutical composition to a subject who is not yet ill at the time of administration. “Prevention” also refers to prevention of recurrence of a disease or one or more symptoms associated with such a disease. “Treatment” and “therapeutic” refer to the act of treatment, as defined above as “treating.”

[0073] In one embodiment, the polypeptide, nucleic acid, or pharmaceutical composition as described herein is used as a medicament for treating a pathological condition caused by amyloid fibrillary aggregation. In one embodiment, the polypeptide for use as a medicament or for use in a treatment method as described herein comprises a negatively charged second label as described herein, for example, a negatively charged amino acid sequence comprising SEQ ID NO: 5.

[0074] In one embodiment, the peptide intended for use as a medicine or for use in a treatment method as described herein is used as a warhead in a bifunctional (proteolytic-targeting chimera) PROTAC molecule. In a preferred embodiment, when used in a PROTAC molecule, the peptide, as defined herein, comprises a negatively charged amino acid sequence containing SEQ ID NO: 5 for recognition by CHIP.

[0075] In one embodiment, the pathological condition is amyloidosis such as systemic lysozyme, insulin, hemodialysis amyloidosis, or neurodegenerative diseases including tau protein diseases such as Alzheimer's disease (AD), corticobasal degeneration (CBD), and Pick's disease (PiD), as well as conucleoprotein diseases such as Parkinson's disease (PD) and Lewy body disease, or other neurodegenerative diseases such as prions, auricidal granuloma (AGD), progressive supranuclear palsy (PSP), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS).

[0076] In another aspect, the present invention provides the use of polypeptide, nucleic acid, or pharmaceutical compositions as described herein for the detection of aggregated amyloid fibrils. In some embodiments, the use as described herein is for staining tissue samples (e.g., postmortem tissue).

[0077] In another aspect, the present invention provides the use of peptides or nucleic acids as described herein for inhibiting amyloid fibrillary aggregation. Inhibition of aggregation can occur in vitro (e.g., in an in vitro assay) or in vivo as part of a treatment as described herein.

[0078] All patents and references cited in this specification are incorporated herein by reference in their entirety.

[0079] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. Attached Figure Description

[0080] Figure 1 : FibrilPaint1 is a specific tag for protein fibrils. Inhibition of TauRD (AD) and HttEx1Q44 (EH) aggregation by FibrilPaint1 (A, E), FibrilPaint2 (B, F), FibrilPaint3 (C, G), and FibrilPaint4 (D, H). B) For both TauRD and HttEx1Q44 aggregation, all peptides reduced the terminal plateau in a dose-dependent manner. Fibril paint binds to monomers or fibrils of TauRD (I) or HttEx1Q44 (J). Samples containing fibrils showed a significant increase in Rh (corresponding to fibril size).

[0081] Figure 2 : FibrilPaint1 is specific for amyloid aggregates. A) Binding of 0.2 μM FibrilPaint1 to TauRD fibrils in the presence of 50% cell lysate. FibrilPaint1 has a hydrodynamic radius Rh of 1.7 nm in the presence of cell lysate, and an average Rh of 14 nm when incubated with 4h-TauRD fibrils. B) No binding of 0.2 μM FibrilPaint1 to heat-shocked amorphous aggregates of luciferase. FibrilPaint1 has a size of 1.7 nm, and its size remains unchanged when incubated with heat-shocked luciferase.

[0082] Figure 3 : Aggregation of TauRD and HttEx1Q44 over time The kinetic radius Rh of the TauRD (A) and HttEx1Q44 (D) aggregates was determined using FIDA. In parallel experiments, the aggregation of TauRD and HttEx1Q44 was monitored using ThT measurements (B, E). The final products of TauRD after 24 h (C) and HttEx1Q44 after 6 h (F) were imaged using negative staining electron microscopy.

[0083] Figure 4 : FibrilPaint's inhibition of fibrils over time Aggregation of TauRD (A) and HttEx1Q44 (B) with and without 2 μM FibrilPaint1. With FibrilPaint1, the Rh of TauRD (A) remained at 3.5 nm and that of HttEx1Q44 (B) remained at 28 nm. Both datasets are representative of three independently performed experiments. The final products of TauRD after 24 h (C) with and without FibrilPaint1, and HttEx1Q44 after 6 h (D), were imaged by negative staining electron microscopy.

[0084] Figure 5 : Measurement of patient-derived materials from various Tau protein diseasesA) Dynamic radii of fibrils extracted from patients diagnosed with Alzheimer's disease (AD), frontotemporal dementia (FTD), and corticobasal degeneration (CBD), measured using FibrilPaint1. B) Structural representations of Tau fibrils in different Tau protein disorders, and recombinant Tau fibrils used for comparison. These images were generated in PyMol software based on coordinates of cryo-electron microscopy structures. Functional groups are stained according to the YRB script. PDB codes: AD double-helical filaments, 5O3L; AD straight filaments, 5O3T; FTD thinner filaments, 6GX5; CBD type I, 6TJO; CBD type II, 6VH7; serrated recombinant Tau, 6QJH; twisted recombinant Tau, 6QJM; serrated recombinant Tau, 6QJP. C) Negative-stain electron microscopy (FLTR) of fibrils derived from patients with AD, CBD, and FTD. D) Examples of AD fibrils are divided into two groups: PHF and fibrils that cannot be clearly identified as is. E) Structural representation of an AD double-helical filament consisting of 342 layers (left). The image was generated in PyMol software based on coordinates of the cryo-electron microscopy structure. Functional groups are colored according to the YRB script. F) Prediction of the hydrodynamic radius of different layers of the AD double-helical filament using the FIDAbio hydrodynamic radius prediction tool (right) (left). The lines in the prediction indicate the average HR of the found AD fibrils, similar to 1100 layers.

[0085] Figure 6 : FibrilPaint1 is a specific tag for protein fibrils. FibrilPaint binds to A) α-syn and Aβ (FibrilPaint peptides are blue; α-syn fibrils are light green; Aβ fibrils are light red) and B) IAPP fibrils.

[0086] Figure 7 : FibrilPaint1 is specific for amyloid aggregates.(A) Binding of 0.2 μM FibrilPaint1 to TauRD fibrils in buffer or 50% cell lysate. In buffer, FibrilPaint1 has a size of 1.7 nm, and an average size of 54 nm when incubated with TauRD fibrils. In 50% cell lysate, FibrilPaint1 has a size of 1.7 nm, and an average size of 39 nm when incubated with TauRD fibrils. (B) Binding of 0.2 μM FibrilPaint1 to TauRD fibrils in buffer or 50% human serum. In the presence of buffer, FibrilPaint1 has a size of 1.7 nm, and an average size of 52 nm when incubated with TauRD fibrils. In 50% human serum, FibrilPaint1 has a size of 1.7 nm, and an average size of 54 nm when incubated with TauRD fibrils. (FibrilPaint1 in buffer is dark blue; folded luciferase is dark green; heat shock luciferase is light green; TauRD fibrils in buffer are light blue; FibrilPaint1 in cell lysates is dark purple; TauRD fibrils in cell lysates are light purple; FibrilPaint1 in human serum is light yellow; TauRD fibrils in human plasma are dark yellow.) C) FibrilPaint1 binds to fibrils in Htt brain supernatant.

[0087] Figure 8 ： FibrilPaint1 binds to amyloid fibrils and the E3 ligase CHIP. FibrilPaint1 is designed as a PROTAC as follows: the C-terminal side of the peptide binds to amyloid fibrils (A), while the EEVD motif binds to the ubiquitin E3-ligase CHIP. B) Schematic diagram of FibrilPaint1, where its C-terminal amino acid targets amyloid fibrils, and its EEVD motif recruits the E3 ligase CHIP, subsequently forming a complex, and ubiquitin is transferred to the amyloid fibrils (C). FibrilPaint1 binds to TauRD fibrils but not to monomers. C) HttEx1Q44. FibrilPaint1 binds to CHIP with an affinity of 3 µM. Without the EEVD motif, FP1 cannot bind to CHIP.

[0088] Figure 9 ： FibrilPaint1 acts as a PROTAC for protofibril ubiquitination. Ubiquitination of TauRD (A), HttEX1Q44 (B), α-synuclein (C), and β-amyloid (D) in buffer (pale purple) and after the addition of urea (purple). In the absence of ATP (yellow), the size was subsequently maintained only at the ubiquitinated size.

[0089] Figure 10 FibrilPaint1 ubiquitinated fibrils are derived from patients.

[0090] Figure 11 FibrilPaint peptides bound to fibrils. FibrilPaint binds to either TauRD (A) or HttEx1Q44 (B) fibrils. FibrilPaint peptides alone appear as dark gray, light gray in the presence of TauRD, and light gray in the presence of HttEx1Q44 (FP8: not determined). The type of binding shows an increased hydrodynamic radius corresponding to the radius of the bound fibril.

[0091] Figure 12 : A schematic diagram of fibril-paint marker alterations that can lead to targeted protein degradation in fibrils. The markers can be altered to mediate ubiquitination or other modifications to fibril signaling for protein degradation, whether via proteasome degradation, autophagy, or other cellular systems. The markers can also be altered to directly signal the recruitment of degradation systems.

[0092] Table 1: Sequences Example Methods and Materials Expression and purification of Tau and Huntington proteins We generated human Tau-RD (Q244-E372, with the pro-aggregation mutation ΔK280) in *E. coli* BL21 Rosetta 2 (Shanghai Novagen Biotechnology Co., Ltd.) carrying an additional N-terminal FLAG marker with a removable N-terminal His6-Smt-tagged label (MGHHHHHHGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG - SEQ ID NO: 6) with an N-terminal FLAG marker carrying an additional removable N-terminal His6-Smt-tagged label (MGHHHHHHGSDSEVNQEAKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG - SEQ ID NO: 6) by adding 0.15 mM IPTG and incubating overnight at 18°C. 600Expression was induced at 0.8°C. Cells were harvested by centrifugation, resuspended in 25 mM HEPES-KOH (pH 8.5) and 50 mM KCl, rapidly frozen in liquid nitrogen, and maintained at -80°C until further use. The precipitate was thawed at 37°C, and then ½ tablet / 50 ml of EDTA-free protease inhibitor and 5 mM β-mercaptoethanol were added. Cells were lysed using an EmulsiFlex-C5 cell disruptor, and the lysates were clarified by centrifugation. The supernatant was filtered through a 0.22 μm polypropylene filter and purified using an ÄKTA purification chromatography system. The sample was loaded onto a POROS 20MC affinity purification column containing 50 mM HEPES-KOH (pH 8.5) and 50 mM KCl, and eluted with 0.5 M imidazole at a 0-100% linear gradient of 5 CV. The target fraction was collected and concentrated to 2.5 ml using a buffer concentration column (vivaspin, MWCO 10 kDa), and then desalted using a PD-10 desalting column to HEPES pH 8.5, ½ tablet / 50 ml Complete protease inhibitor, and 5 mM β-mercaptoethanol. His6-Smt-labeling was removed by overnight shaking with Ulp1 at 4°C. The next day, the sample was loaded onto a POROS 20HS column containing HEPES pH 8.5 and eluted with a 0-100% linear gradient of 1 M KCl at 12 CV. The target fraction was collected and loaded onto a Superdex 26 / 60, 200 pg size size size size size column containing 25 mM HEPES-KOH pH 7.5, 75 mM NaCl, and 75 mM KCl. The target fraction was concentrated to the desired concentration using a concentrator (vivaspin, MWCO 5 kDa). Protein concentration was measured using a Nanodrop UV / Vis spectrophotometer, and purity was assessed by SDS-PAGE. Proteins were aliquoted and stored at -80°C.

[0093] Expression and purification of HttEx1Q44.

[0094] We generated HttEx1Q44 (MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGP) with additional N-terminal MBP- and C-terminal His6- tags in E. coli BL21 Rosetta 2 (Shanghai Nuojin Biotechnology Co., Ltd.). WAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDAALAAAQTNAAA ASEFSSNNNNNNNNNNLGIEGRMATLEKLMKAFESLKSFQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQPPPPPPPPPPPQLPQPPPQAQPLLPQPQPPPPPPPPPGPAVAEEPLHRPSGSHHHHHH - SEQ ID NO: 7). By adding 0.15 mM IPTG and incubating overnight at 18°C, the OD 600Expression was induced at 0.8°C. Cells were harvested by centrifugation, resuspended in 50 mM HEPES-KOH (pH 8.5) and 50 mM KCl, rapidly frozen in liquid nitrogen, and maintained at -80°C until further use. The precipitate was thawed at 37°C, and then ½ tablet / 50 ml of EDTA-free protease inhibitor and 5 mM β-mercaptoethanol were added. Cells were lysed using an EmulsiFlex-C5 cell disruptor, and the lysates were clarified by centrifugation. The supernatant was filtered through a 0.22 μm polypropylene filter and purified using an ÄKTA purification chromatography system. The sample was loaded onto a POROS 20MC affinity purification column containing 50 mM HEPES-KOH (pH 8.5) and 100 mM KCl, and eluted with a 0-100% linear gradient of 10 CV of 0.5 M imidazole and 30 mM NaCl. The target fraction was collected and concentrated to the desired concentration using a concentrator column (vivaspin, MWCO 30 kDa). Protein concentration was measured using a Nanodrop UV / Vis spectrophotometer, and purity was assessed by SDS-PAGE. Proteins were aliquoted and stored at -80°C.

[0095] Expression and purification of α-synuclein Wild-type human α-synuclein (α-Syn) was recombinantly generated as previously described (Stöckl et al., 2011). Briefly, the protein was generated in *E. coli* BL21(DE3) cells using the pT77 expression system. Following IPTG induction, cells were harvested by centrifugation and resuspended in a mixture of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 1 mM Pefabloc protease inhibitor. Cells were lysed by sonication, and the lysate was clarified by centrifugation. Nucleotides were removed by adding 1% w / v streptomycin sulfate followed by centrifugation. α-Syn was precipitated with 0.3 g / ml ammonium sulfate and subsequently centrifuged. The precipitate was dissolved in 10 mM Tris-HCl (pH 7.4), 1 mM Pefabloc, and filtered through a 0.2 μm membrane. The sample was loaded onto a Resource Q anion exchange column (GE) coupled with an ÄKTA purification chromatography system. The sample was eluted using a 0–1 M salt gradient. The target fraction was collected and dialyzed against 10 mM HEPES and 50 mM NaCl (pH 7.4). Concentration was measured using a NanoDrop UV / Vis spectrophotometer, and purity was assessed by SDS-PAGE. The purified protein was aliquoted, rapidly frozen in liquid nitrogen, and stored at -80°C in 10 mM TRIS pH 7.4 until further use.

[0096] Preparation of β-amyloid (1-42) monomers and fibrils β-amyloid peptide 1-42 (Aβ42) was purchased from Sigma-Aldrich (A9810). The monomer was dissolved in PBS (pH 7.4) according to a well-established protocol (Vandersteen et al., 2012). 10 μM aliquots of the peptide were stored at -20°C. The monomer was freshly thawed before the experiment.

[0097] Aβ42 fibrils were prepared by static incubation at 37°C for 20 hours in the presence of 0.02 w / v% NaN3. After holding at room temperature (21°C) for 4 hours, the presence of Aβ42 aggregates was verified using AFM. AFM samples were prepared as follows: 10 μl of a 5-fold diluted sample was adsorbed onto mica (silica, V1 quality, EMS) for 45 minutes, followed by careful washing of the mica sheet four times with milliQ, covering, and drying overnight. Aβ42 fibril samples were stored at room temperature (21°C).

[0098] IAPP preparation and aggregation Human islet amyloid peptide (hIAPP) (amyloid trifluoroacetate, product number 4030200) was purchased from Bachem, Switzerland. The peptide was monomerized by dissolving it in hexafluoro-2-propanol to a final concentration of 1 mg / ml. The solution was incubated at room temperature for 15 minutes with occasional stirring. The monomerized hIAPP was aliquoted into glass vials, each containing 50 μg of peptide. A peptide film was formed around the vial by gently blowing a stream of nitrogen while simultaneously rotating the vial. The aliquots with the dried peptide film were stored at -20°C. Before use, the vials were allowed to reach room temperature, and then an aliquot of ice-cold aggregation buffer (20 mM ammonium acetate buffer, pH 6.8, filtered through a 0.2 μm filter immediately before use) was added to achieve a stock concentration of approximately 100 μM. The stock concentration was estimated by absorbance at 280 nm using the calculated molar extinction coefficient of 1,615 M⁻¹ cm⁻¹. To aggregate hIAPP, the concentration was adjusted to 10 μM, 30 μM, or 50 μM by diluting it in aggregation buffer. The sample was then incubated at 25°C for 24 hours.

[0099] Peptide synthesis and purification Peptides were synthesized using a Liberty Blue microwave-assisted peptide synthesizer (CEM) with standard Fmoc chemicals and Oxyma / DIC as coupling reagents. Peptide concentrations were measured by UV spectroscopy. The peptides were labeled with 5(6)-carboxyfluorescein at their N' ends. The peptides were cleaved from the resin using a mixture of 95% (v / v) trifluoroacetic acid (TFA), 2.5% (v / v) triisopropylsilane (TIS), and 2.5% (v / v) triple-distilled water (TDW) with vigorous stirring at room temperature for 3 hours. The volume was reduced by N2 flux, and the peptides were precipitated by adding 4 volumes of diethyl ether at -20°C. The peptides were precipitated at -20°C for 30 minutes, then centrifuged and the diethyl ether was discarded. The peptides were washed three times with diethyl ether and dried by a gentle N2 flux. The solids were dissolved in acetonitrile (ACN):TDW at a volume ratio of 1:2, frozen in liquid nitrogen, and lyophilized. The peptide was purified using a reverse-phase C18 preparative column on a Waters HPLC system with a gradient of ACN / TDW. The identity and purity of the peptide were verified by ESI mass spectrometry and by a Merck Hitachi analytical HPLC system using a reverse-phase C8 column.

[0100] fibrin extraction The brain material for FTD and CBD was obtained from the Dutch Brain Bank (project number 1369). The brain material for AD was donated by Professor JJM Hoozemans of VU University Medical Center Amsterdam.

[0101] PHF and SF were extracted from the gray matter of the prefrontal cortex of patients diagnosed with Alzheimer's disease (AD). Tissues were homogenized at maximum speed using Polytron (PT2500E, Kinematica AG) in 20% (w / v) A68 buffer, which consisted of 20 mM TRIS-HCl pH 7.4, 10 mM EDTA, 1.6 M NaCl, 10% sucrose, 1 tablet / 10 ml Pierce protease inhibitor, and 1 tablet / 10 ml phosphatase inhibitor. The homogenized sample was centrifuged at 14,000 rpm for 20 min at 4 °C. The supernatant was collected, and the precipitate was homogenized in 10% (w / v) A68 buffer. The homogenate was centrifuged again. The supernatants from the two centrifugations were combined and supplied with 10% w / v creatine ester, incubated on a shaker at room temperature for 1 h. The sample was then ultracentrifuged at 100,000 x g at 4 °C for 1 h. Discard the supernatant and incubate the precipitate overnight at 4°C in 20 μl / 0.2 g of starting material in 50 mM MTH-HCl pH 7.4. The next day, dilute the precipitate to a maximum of 1 ml with A68 buffer and resuspend. To remove contamination, centrifuge the sample at 14,000 rpm for 30 min at 4°C. Collect the supernatant and centrifuge again at 100,000 x g for 1 h at 4°C. Resuspend the precipitate in 30 μl of 25 mM HEPES-KOH pH 7.4, 75 mM KCl, and 75 mM NaCl and store at 4°C for up to one month.

[0102] Fine filaments were extracted from the gray matter of the middle gyrus of patients diagnosed with FTD. Fibrous fibers were extracted following the protocol for AD fibrils. After the first ultracentrifugation step, the precipitate was resuspended in 250 μl / 1 g of starting material in 50 mM Tris pH 7.5, 150 mM NaCl, and 0.02% Amphipol A8-35. The sample was centrifuged at 3000 x g and 4 °C for 30 min. The precipitate was discarded, and the supernatant was ultracentrifuged at 100000 x g and 4 °C for 1 h. The precipitate was resuspended in 30 μl of 50 mM Tris-HCl pH 7.4 and 150 mM NaCl and stored at 4 °C for up to one month.

[0103] CBD fibrils were extracted from the upper apical gyrus gray of patients diagnosed with CBD. Tissue was homogenized at maximum speed using Polytron (PT2500E, Kanemerg AG) in 20% w / v 10 mM TRIS-HCl pH 7.5, 1 mM EGTA, 0.8 M NaCl, and 10% sucrose. The homogenate was supplied with 2% w / v creatine ester and incubated at 37°C for 20 min. The sample was centrifuged at 20000 x g and 25°C for 10 min. The supernatant was ultracentrifuged at 100000 x g and 25°C for 20 min. The precipitate was resuspended in 750 μl / 1 g of starting material in 10 mM TRIS-HCl pH 7.5, 1 mM EGTA, 0.8 M NaCl, and 10% sucrose and centrifuged at 9800 x g for 20 min. The supernatant was ultracentrifuged at 100000 x g for 1 h. The precipitate was resuspended in 20 mM TRIS-HCl pH 7.4 and 100 mM NaCl at 25 μl / g of starting material and stored at 4°C for up to one month.

[0104] For Htt measurements, brain material was obtained from the Dutch Brain Bank under project number 1504. Tissue was homogenized with A68 buffer, which consisted of 20 mM TRIS-HCl pH 7.4, 10 mM EDTA, 1.6 M NaCl, 10% sucrose, one tablet / 10 ml Pierce protease inhibitor, and one tablet / 10 ml phosphatase inhibitor. The homogenized sample was centrifuged at 14,000 rpm for 20 minutes at 4°C. The supernatant was collected, and the precipitate was homogenized in 10% (w / v) A68 buffer. The homogenate was centrifuged again. The supernatants from the two centrifugations were combined and diluted to a final concentration of 25% supernatant, 200 nM FibrilPaint1, and 0.5% Prönkel to prepare samples for FIDA studies.

[0105] Thioflavin T Aggregation Assay In the presence of 45 µM thioflavone T, aggregation of 20 µM Tau-RD was induced in 25 mM HEPES-KOH at pH 7.4, 75 mM KCl, 75 mM NaCl, and ½ tablet / 50 ml of protease inhibitor by adding 5 µM low molecular weight heparin. The effect of the peptide was assessed by adding 0.02, 0.2, or 2 µM of the peptide. Fluorescence spectra were recorded every 5 minutes in CLARIOstar® Plus at 37 °C and 600 rpm over 24 hours.

[0106] In the presence of 45 µM thioflavin T, aggregation of 20 µM ttEx1Q44 was induced in 25 mM HEPES-KOH at pH 7.4, 75 mM KCl, 75 mM NaCl, and ½ tablet / 50 ml of protease inhibitor by cleavage of the C-terminal MBP label with factor Xa. The effect of the peptide was assessed by adding 0.02, 0.2, or 2 µM of the peptide. Fluorescence spectra were recorded every 5 minutes in CLARIOstar® Plus at 37 °C and 600 rpm over 24 hours.

[0107] Negative staining electron microscopy (nsEM) Glow discharge was performed on a carbon-coated copper mesh (200 or 300 mesh) for 20 seconds. The fibrils were diluted as needed in buffer solutions (25 mM MEPES-KOH pH 7.4, 75 mM KCl, 75 mM NaCl), and 1 µl of sample was loaded onto the mesh surface and incubated for 60 seconds. Excess sample was blotted dry with filter paper while keeping the mesh vertical. The mesh was then incubated with 100 µL of 2% uranyl acetate droplets for 30 seconds to stain it. The treated mesh was analyzed at 120 kV using a transmission electron microscope (Talos L120C, Thermoscientific, Utrecht, Netherlands). Images were analyzed using ImageJ.

[0108] Dimensional measurement using fluid-induced dispersion analysis (FIDA) The fibril size was determined using fluid-induced dispersion analysis (FIDA) with the hydrodynamic radius as a parameter. FIDA experiments were performed using FIDA 1 with a 480 nm excitation source.

[0109] Pre-formed Tau-RD or HttEx1Q44 fibrils at different time points were diluted to a final concentration of 2 µM in 25 mM HEPES-KOH pH 7.5, 75 mM KCl, 75 mM NaCl, and 0.5% Prömnick (for Tau-RD) or 50 mM HEPES-KOH pH 7.5, 150 mM KCl, and 0.5% Prömnick (for HttExQ4) along with 200 nM FibrilPaint1. For patient-derived Tau filaments, AD, CBD, or FTD fibrils were diluted to a final concentration of 2 µM in 20 mM TRIS-HCl pH 7.4, 100 mM NaCl, and 0.5% Prömnick (for AD and CBD) or 50 mM TRIS-HCl pH 7.4, 150 mM NaCl, and 0.5% Prömnick (for FTD). The operating mode is capillary dissociation (Capdis). In this mode, the capillary is equilibrated with buffer solution and then the sample is injected. Only the indicator sample contains fibrils to minimize viscosity to the capillary.

[0110] ubiquitination of amyloid fibrils For the ubiquitination reaction, with or without 5 mM ATP, the enzyme was diluted in ubiquitination buffer (40 mM MTTRIS pH = 8, 5 mM MgCl2, 0.05% TWEEN-20, and 1 mM DTT) to final concentrations of 500 nM E1, 500 nM E2, 500 nM E3, 10 nM fluorescein-ubiquitin, 500 nM FibrilPaint, and 5 μM fibrils. The reaction was then incubated at 30 °C for 1 h. To confirm ubiquitination, the sample was loaded onto a FIDA1 with a 480 nm excitation source. The operating mode used was capillary dissociation (Capdis). In this case, the capillary was equilibrated with buffer and then the sample was injected (Table 2).

[0111] Table 2. R up to 75 nm h Experimental parameters for Capdis analysis of protein fibril size.

[0112] Micro-thermophoretic binding assay The combination of FibrilPaint1 or FibrilPaint6 with CHIP was analyzed using a Monolith (NanoTemper Technologies) Micro Thermophoresis System (MST). Thermophoresis was monitored at concentrations of 50 nM FibrilPaint and CHIP in 25 mM HEPES-KOH pH 7.5, 75 mM KCl, and 75 mM NaCl. Samples were transferred to high-quality capillaries (NanoTemper Technologies) and measurements were performed at 37°C using a medium blue LED power and 50% of the MST infrared laser power to induce thermophoretic motion. The infrared laser was turned on 1 second after the start of the measurement and continued for 20 seconds. The data point used is the MST response at 5 seconds.

[0113] result Development of a multi-targeting method for determining amyloid size .

[0114] We set out to develop a method for determining the size of various amyloid fibrils. Our strategy was built using two sequence- and structurally unrelated aggregating proteins, Tau and Huntington's protein (Htt). Htt fibrillation is directly associated with the onset and progression of Huntington's disease (HD), an autosomal inherited disorder caused by glutamine sequence expansion in exon 1 of the Htt protein. Tau is an intrinsically disordered protein that undergoes amyloid aggregation under various pathologies. Tau fibrillation is highly associated with the progression of tau protein disorders, including Alzheimer's disease (AD), frontotemporal dementia (FTD), or corticobasal degeneration (CBD).

[0115] Our previous work (Garfagnini et al., 2023) described the development of a family of peptides that inhibit the aggregation of several unrelated proteins in the early stages. We used these peptides as a starting point for developing fibril paint. To enhance peptide binding to aggregates, we increased the density of π-stacking and H-bonded residues (Ferrari et al., 2020) and varied the net charge and the number of aromatic compounds in the peptides. A fluorescein group (Fl-) was added to the N-terminus to facilitate detection. In some peptides, a negative EEVD sequence with a GSGS spacer was added to the C-terminus, resulting in oppositely charged portions (with a positively charged N-terminal region and a negatively charged C-terminus). The fluorescein group (Fl-) was added to the N-terminus to facilitate detection. The net charge and the number of aromatic compounds in the peptides varied.

[0116] This resulted in a group of peptides with different charges, numbers of aromatic compounds, and uneven distribution of residues of different types (Table 3). Intended to make the fibrils visible, we call these peptides fibril paint.

[0117] Table 3: List of FibrilPaint “(FI)” indicates fluorescein. Four peptides inhibit the aggregation of Tau and Htt amyloid proteins. We evaluated whether the designed fibril paint peptides could interact with multiple types of aggregated proteins. To this end, we used a ThT assay to test the inhibitory effect of the peptides on amyloid aggregation of two unrelated proteins: a Tau repeat domain (Q244-E372, TauRD) with a pro-aggregation mutation ΔK280 and a huntingtin exon 1 (HttEx1Q44) containing a 44-residue polyglutamine sequence. This is a method for monitoring protein fibril maturation using the fluorescent dye thioflavin-T (ThT), which fluoresces upon binding to fibrils. Data for four fibril paints are presented in this paper. The four fibril paint peptides strongly inhibited Tau-RD aggregation in a dose-dependent manner at substoichiometric concentrations (…). Figure 1 (AD). FibrilPaint1 was the most effective, reducing fluorescence intensity in the platform by 92%, followed by FibrilPaint4 (89%), FibrilPaint2 (82%), and FibrilPaint3 (73%).

[0118] All fibril paints also inhibited HttEx1Q44 aggregation in a dose-dependent manner, but not as effectively as for Tau. Figure 1 (EH). Here, FibrilPaint4 exhibited the strongest effect, reducing the fluorescence intensity of the platform by 74%. FibrilPaint2 (55%), FibrilPaint3 (54%), and FibrilPaint1 (30%) also inhibited the aggregation of polyglutamine proteins to some extent. Overall, the peptides tested inhibited the amyloid aggregation of both Tau-RD and HttEx1Q44 at substoichiometric concentrations. This is remarkable because these two proteins are completely unrelated in sequence, with their only common characteristic being the formation of fibrils.

[0119] FibrilPaint1 binds to Tau and Htt fibrils Next, we screened for peptides that bind to TauRD and HttEx1Q44 fibrils. Figure 1 (IJ). To this end, we developed an application of FIDA to visualize the size of amyloid aggregates. With FIDA, the target fluorescently labeled sample is passed under pressure through a long capillary and finally through a detector, generating a fluorescent signal. Smaller species diffuse faster, while larger species diffuse more slowly, resulting in narrower or wider dispersion of the fluorescent signal, respectively. Because FIDA relies on the physical diffusion properties of the sample, we can calculate the hydrodynamic radius (Rh) of the labeled species based on the fluorescence signal. The average Rh value is an ensemble value representing the average value of all labeled particles.

[0120] Rh is a parameter corresponding to the size of molecules or complexes in solution. It is the radius of the sphere produced by tumbling particles. Rh values ​​can be estimated from structural coordinates by experimental methods or predictions (such as AlphaFold) (Table 4).

[0121] Table 4 Protein structure and its predicted hydrodynamic radius. The number of residues and molecular weight of the total structure, as available in the PDB, are given. If the structure is available, the included 1-layer Rh is the Rh of the structure that can fold into its fibrillary conformation using only one layer.

[0122] Determining the Rh value for aggregate species requires a compound that can label the aggregates without altering their properties. Aggregated proteins were flowed through a capillary at 37°C to increase the flow rate without exceeding physiological temperature. We used low pressure (75 bar) and long flow time (35 minutes) to maintain Taylor conditions for larger species. To minimize the volume of incoming fibrils to sub-microliter and reduce potential interactions within the capillary, we used a capillary dissociation setting. Here, we premixed the fibril paint peptide with the fibrils and allowed the mixture to diffuse only in the buffer. The binding of the fluorescently labeled fibril paint peptide to the protein fibrils increases the average Rh value because the peptide now tumbles with the larger fibrils.

[0123] Testing of the binding of fibril paint peptide to TauRD fibrils aggregated for 4 h showed that fibril paint 1 binds effectively, with the average Rh value increasing from 1.2 nm to 45 nm. Figure 1I). Importantly, incubation of fibril paint 1 with TauRD monomers did not result in an increase in Rh values, indicating that it specifically binds to fibrils but not to monomers. The binding of fibril paint 2–4 and 9–13 was not stable enough to allow for efficient fluorescent labeling of TauRD fibrils. Interestingly, while all fibril paint peptides are assumed to inhibit TauRD aggregation, not all peptides act as non-covalent tags. This suggests that labeling and aggregation prevention are two distinct properties.

[0124] Testing with fibril paint peptides used to label HttEx1Q44 fibrils yielded very similar results. In the presence of HttEx1Q44 fibrils, the average Rh of fibril paint1 increased, while it did not bind to monomers ( Figure 1 J). Fibril paint peptides 2 to 4 and 9-13 2-4 also failed to bind effectively to HttEx1Q44 ( Figure 1 J shows the data for FibrilPaint2-4.

[0125] Next, to demonstrate FibrilPaint1's ability to recognize various amyloid substances, we tested its binding to α-Syn, Aβ42 fibrils, and peptides (IAPP, amyloidin). However, α-Syn fibrils are associated with conucleoprotein diseases such as Parkinson's disease (Goedert et al., 2017; Spillantini et al., 1997), plaque-forming Aβ peptide deposition is associated with the development of Alzheimer's disease (Hardy and Higgins, 1992; Long and Holtzman, 2019), and IAPP forms amyloid fibrils in type II diabetes mellitus (TIIDM). Notably, FibrilPaint1 bound to α-Syn, Aβ42, and IAPP fibrils at 33 nm, 50 nm, and 30 nm, respectively. Figure 6 A) and 10nm ( Figure 6 The average size of B) is shown. This demonstrates the detection of various protein fibrils by FibrilPaint1.

[0126] FibrilPaint1 is specific to amyloid. We investigated whether FibrilPaint1 is specific for fibrillar aggregates. First, we tested its ability to specifically recognize TauRD fibrils in the presence of numerous other cellular proteins by adding *E. coli* cell lysates. We incubated pre-formed TauRD fibrils with FibrilPaint1 at a 1:100 ratio in 50% cell lysates. Under these conditions, FibrilPaint1 showed a 14 nm increase in size, unlike when FibrilPaint1 was incubated alone in cell lysates. Figure 2 A). Therefore, FibrilPaint1 specifically recognizes fibrils in complex cellular mixtures.

[0127] Having confirmed FibrilPaint1's ability to bind to protein fibrils rather than monomers, we determined its specificity for amyloid structures. We used luciferase as a mature example of non-amyloid aggregates (Parsell et al., 1994). Luciferase is a 61 kDa globular protein with an Rh of 3.4 nm that forms amorphous aggregates upon heat shock denaturation. We co-incubated FibrilPaint1 with heat-shocked luciferase at a 1:100 ratio. The Rh of FibrilPaint1 in FIDA measurements was unaffected by the presence of luciferase aggregates. Figure 2 B). This indicates that FibrilPaint1 does not bind to amorphous luciferase aggregates. In summary, these data demonstrate that FibrilPaint1 specifically binds to amyloid fibrils, unaffected by the presence of other biomolecules.

[0128] To test FibrilPaint1 in a more physiological and disease-relevant environment, we incubated it in 50% human serum in the absence or presence of pre-formed TauRD fibrils. FibrilPaint1 alone appeared as 1.5 nm in 50% human serum, which is consistent with the 1.4 nm observed in buffer. Figure 7 B). In the presence of pre-formed TauRD fibrils, the size increases to up to 34 nm in 50% human serum and up to 42 nm in buffer. Figure 7 B). These findings enhance the importance of FibrilPaint1 in clinically relevant settings.

[0129] Finally, we tested whether FibrilPaint1 could also target patient fibrils in the heterogeneous environment of the brain. We liquefied brain material derived from Htt patients. We diluted Htt brain supernatant to 25% and added FibrilPaint1. The size increased from 1.5 nm with FibrilPaint1 alone to 4.5 nm, indicating that FibrilPaint1 binds to amyloid fibrils ( Figure 7 C).

[0130] Having confirmed the ability of FibrilPaint1 to determine fibril size in human serum, the inventors aimed to estimate the concentration threshold for such a measurement. Recombinant TauRD fibrils were titrated into serum, and their size was measured using FibrilPaint1 / FIDA at decreasing concentrations. The TauRD fibrils in this experiment had an Rh value of 21 nM. The inventors observed consistent Rh values ​​for fibril samples formed from monomers down to 200 nM. For PHF-shaped fibrils, an Rh value of 21 nm corresponds to 260 layers (…). Figure 7 D). Considering the relationship between Rh and average fibril length of PHF-shaped fibrils ( Figure 7 D), the inventors estimated that the lowest observed fibril concentration corresponds to 400 pM ( Figure 7 D). These findings indicate that FibrilPaintl determined fibril length at sub-nanomolar amyloid concentrations in clinically relevant environments.

[0131] Monitoring fibril dynamics For both TauRD and HttEx1Q44, we now set out to use FibrilPaint1 and FIDA to monitor aggregation kinetics according to the increase of Rh. Figure 3 (A, D). After 0.5 h, the aggregation reaction of TauRD resulted in an increase in average Rh from 1.7 nm to 2 nm. All species measured at t=0.5 h had the same size, indicating that FibrilPaint1 was fully bound without any dissociation, and larger species were not obtainable at this time point. For the ternary aggregates, the full-length recombinant Tau had an Rh between 2.11 nm and 2.78 nm (Zhang et al., 2019). Our TauRD as fibrils has an unknown Rh, but we expect it to have an Rh between 2–2.5 nm. The Rh of FibrilPaint may affect the size of such different extensions depending on the binding sites of FibrilPaint1. However, this species with a binding size of 2 nm is likely the earliest measurable aggregate of FibrilPaint1.

[0132] TauRD aggregation continued and rapidly increased to a size of 10 nm after 2 hours. Figure 3 A). The measured fibril sizes were also more heterogeneous, indicating the presence of aggregates of various lengths. Only the largest species were plotted ( Figure 3 A). From this perspective, the formation of elongated fibrils means that Rh can no longer be directly converted to radius, but must be converted to fibril length through molecular weight and radius of gyration (He and Niemeyer, 2003; Yoshizaki and Yamakaawa, 1980). This means that for radii between 2 and 2.5 nm, fibril sizes reach lengths of 58–64 nm. TauRD aggregation reaches a plateau of 30 nm Rh value after 8 h, corresponding to lengths of 260–280 nm (…). Figure 3 A).

[0133] For HttEx1Q44, Rh increased to 5 nm after 1 h. This exceeds the predicted Rh (estimated at 3.4 nm) for the unfolded monomer structure of HttEx1Q44. Therefore, this structure consists of multiple monomers. Aggregation then increased exponentially, reaching an average Rh of 500 nm after 7 h. Figure 3 E). When the particle size reaches the upper limit of FIDA measurement, we cannot determine whether or when it reaches the plateau value.

[0134] We used the established ThT measurement to monitor the aggregation process of both TauRD and HttExon1Q44 in parallel. Figure 3 B, E). Upon addition of heparin to TauRD, the ThT fluorescence signal immediately increased and rapidly, reaching a plateau after 4 h (B, E). Figure 3 B). This is faster than we observed in FIDA measurements. It suggests that the formed fibrils may have been saturated with ThT, but the length is still increasing. For HttEx1Q44, we obtained similar results. ThT revealed a hysteresis period of approximately 2 hours and a plateau after 5 hours of aggregation (B). Figure 3 E), while the size of the fibrils is still increasing ( Figure 3 D). The aggregation of HttEx1Q44 appears to be more vigorous than that of TauRD, resulting in much larger sizes. This suggests that another process is underway that binds the fibrils together. We conclude that the two methods are complementary, with ThT measurements providing a signal representing the presence of fibrils, while FIDA measurements provide a measurement of fibril length beyond the early aggregation stages.

[0135] Characterizing fibril shape We used negative staining electron microscopy imaging to characterize the shape of TauRD fibrils after 24 h of aggregation and HttEx1Q44 fibrils after 6 h. Figure 3 C, F). TauRD fibrils exhibit a relatively long, single fibril structure (C, F). Figure 3 C). The length of Tau fibrils varies considerably, averaging 500 to 600 nm (data not shown), which is larger than our FIDA observations. This is expected, as smaller fibrils are excluded due to the low detection limit of this technique.

[0136] Conversely, HttEx1Q44 fibrils stick together to form larger fibril clusters ( Figure 3 (F) (Boatz et al., 2020; Isas et al., 2021; Matlahov et al., 2022; Nazarov et al., 2022). We note that these clusters vary greatly in size: from just a few 50-100 nm fibrils to giant clusters several µm wide. A representative shape formed by these clusters is approximately 800 nm long and 200 nm wide. Figure 3 F). Because this shape is closer to a sphere than a single fibril, it will result in an Rh of 400 nm in FIDA. We measured sizes with Rh of 300–500 nm in our FIDA experiments. These clusters illustrate the rapid increase in size detected by FIDA, reaching the upper limit of FIDA measurements.

[0137] FibrilPaint1 determines the size of the suppressed species. In the application of FIDA to size determination, we set out to determine the size at which FibrilPaint inhibits amyloid aggregation, as observed in ThT measurements. Figure 1 A, E). ThT assays showed almost complete inhibition of TauRD aggregation in the presence of FibrilPaint1 (A, B). Figure 1 A), and at a peptide: monomer ratio of 1:10, HttEx1Q44 aggregation was inhibited by 30% ( Figure 1 E). We replicated the same experimental setup and placed TauRD and HttEx1Q44 under aggregation conditions in the presence of FibrilPaint1 to investigate the inhibitory capacity of FibrilPaint1. Next, we sampled at different time points and measured them in FIDA (…). Figure 4 In the presence of FibrilPaint1, TauRD aggregated to a 3.5 nm Rh concentration after 2 h of incubation. This 3.5 nm concentration remained stable over 12 h. Figure 4A). Negative-stained electron microscopy images confirmed that TauRD suppressed by FibrilPaint1 resulted in fibrils that were so small as to be undetectable. Figure 4 C). Under the same suppression conditions, HttEx1Q44 aggregated to an average size of 28 nm in the presence of FibrilPaint1 and remained stable for 24 h. Figure 4 B). Here, Htt fibrils have more space between them, exhibiting more unique fibril shapes ( Figure 4 D).

[0138] By using the size obtained from FibrilPaint1 inhibition, we can determine the stage at which inhibition occurs. This size is expected for this type of dimer PHF, where Rh corresponds to two layers of fibrils, thus consisting of four Tau molecules. Figure 5 F). Alternatively, 3.5 nm Rh also corresponds to a five-layer stack of serpentine reconstituted fibrils (Table 4). Interestingly, the fibrils are too small to be visible in negatively stained electron microscopy images. Figure 4 C). The molecular weight of HttEx1Q44 is 13.8 kDa, resulting in an estimated Rh of 2.1 nm (if the protein folds into a perfect sphere) to 3.5 nm (if the protein is completely unfolded). When bound to FibrilPaint1, the measured size of the inhibited HttEx1Q44 aggregates is approximately 28 nm. Assuming all fimbriae are now completely unique, we can estimate the fimbriae length of Htt to be between 240 nm and 280 nm. In ThT assays ( Figure 1 (A, E) We observed that the ThT signal of TauRD was almost completely suppressed, while that of HttEx1Q44 was not, although the plateau decreased. It is evident that the suppressed state of Htt involves more monomers than that of Tau. This may be because, in the presence of Fibrilpaint1, TauRD cannot assemble the amyloid structure recognized by ThT, while HttEx1Q44 can.

[0139] FibrilPaint1 for monitoring fibrils of patient origin FibrilPaint 1, 5, 6, 7, and 8 have the potential to serve as lead compounds for the diagnostic tracking of proteofibrosis in patients. This requires FibrilPaint to identify patient-derived proteofibrosis. Interestingly, cryo-electron microscopy of Tau proteofibrosis structures shows that their shapes differ for various tau proteopathies and heparin-induced recombinant proteofibrosis. Figure 5 B). Due to its potential as a Tau tracer, it is important to evaluate the ability of FibrilPaint to identify patient-derived fibrils in several Tau protein disorders.

[0140] Tau aggregation is a characteristic feature of the progression of various Tau protein disorders. Monomeric Tau can undergo post-translational selective splicing, producing six distinct subtypes with four (4R) or three (3R) repeating microtubule-binding domains. Depending on the subtype introduced into the fibrils, Tau protein disorders can be classified as 4R type (corticobasal degeneration, CBD), 3R type (frontotemporal disorder, FTD), or 4R / 3R type (AD). Size and morphology can reveal information about fibril formation, interface, and fibril stability. Therefore, characterizing the size of fibrils from various patient origins is important.

[0141] We set out to measure the size of Tau fibrils derived from patients with three different Tau diseases. To test whether FibrilPaint1 was also applicable to patient-derived fibrils, we purified fibrils from patients diagnosed with CBD, FTD, and AD. We imaged the purified fibrils using negative staining electron microscopy to confirm typical disease-specific fibril morphologies. Figure 5 C). Double-stranded helical filaments (PHF) and straight filaments (SF) with typical twisting can be observed.

[0142] Next, we performed FIDA analysis to determine the size of patient-derived Tau fibrils ( Figure 5 A). Our parallel-synthesized recombinant TauRD fibrils had an apparent mean Rh of 54 nm. Fibrils measured from patients diagnosed with AD had an apparent mean Rh of 49 nm, with minimal difference between measurements. Figure 5 A). For CBD, the observed Rh was 95 nm ( Figure 5 A). The fibrils from FTD exhibit a homogeneous population with an average Rh of 69 nm ( Figure 5 A). These data reveal that FibrilPaint1 is suitable for interacting with and characterizing protein fibrils derived from the original material.

[0143] Finally, we generated a model that correlates Rh with fibril length. In the computer simulation, we stacked different layers of fibrils on top of each other and calculated the predicted dimensions using the FIDAbio Rh prediction tool. A complete torsion of the PHF is composed of 342 stacked layers. Figure 5 D). The length of one layer is 4.7 Å, giving a full-turn twist length of approximately 160 nm (Fitzpatrick et al., 2017). For AD fibrils, we measured Rh at 49 nm, which corresponds to 1100 layers of fibrils ( Figure 5The F), yielded a fibril length of approximately 510 nm. In nsEM data obtained from fibrils purified from Alzheimer's patients, we evaluated a total of 103 fibrils, of which 48 exhibited typical PHF structures. We used repeating elements within the PHF fibrils to determine their average length. The average PHF in our samples had 2.7 turns, corresponding to 430 nm (F). Figure 5 E). This matches very well with the average length calculated based on the Rh value determined by FIDA measurements of Tau fibrils combined with FibrilPaint1.

[0144] Various Fibril Paints target amyloid substances Next, we set out to find FP1 derivatives that could maintain protofibril binding properties. Modifications included shortening the sequence to exclude the linker and E3 binding motif (FP5 and FP6), altering the net charge (FP5-FP13), sequence randomization (FP8), and using D-amino acids to modify the 3D structure (FP12 and FP13).

[0145] We used TauRD and HttEx1Q44 fibrils and measured their size in FIDA. In the presence of TauRD and HttEx1Q44 fibrils grown for 20 h and 5 h respectively, the size of the five FP peptides increased (Figure 11). Specifically, the Rh of TauRD fibrils measured together with the five active FPs (FP1, FP5, FP6, FP7, and FP8) increased to an average size of 37 nm. In the case of HttEx1Q44, we observed an Rh of 188 nm (FP1, FP5, FP6, and FP7; the value for FP8 was not determined). The fibril size variation within measurements using different FPs was minimal and independent of the FP. Given that the aggregation process is a seed event, some variation in the measured Rh is expected. In summary, these results indicate that FP1, FP5, FP6, FP7, and FP8 are effective fibril binders.

[0146] The identification of four novel FP peptides (FP5, FP6, FP7, and FP8) binding to amyloid fibrils demonstrates that the sequence of FP1 can be modified while maintaining specific recognition of amyloid fibrils. First, charge changes do not impair binding ability. FP1 has a net charge of -1, while FP5, FP6, FP7, and FP8 have net charges of +3, +2, +2, and 0, respectively. FP5 and FP6 lack the GSGS and EEVD motifs, while in FP7, EEVD is replaced by RRVD, indicating that these motifs are not essential for the detection of amyloid fibrils. In FP8, the amino acid sequence order is altered, suggesting the potential for changing the residue order.

[0147] FibrilPaint as a PROTAC strategy for ubiquitination of amyloid fibrils PROTACs are small molecules that inhibit the function of their target proteins by targeting them for degradation by the ubiquitin-proteasome system. We set out to apply Firbrilpaint as a PROTAC for the ubiquitination of amyloid fibrils (see [link to article]). Figure 8 (Exemplary mode of action in A). First, we determined that we can use the FIDA system to track ubiquitin transfer by tracking Rh (data not shown). If ubiquitination occurs, the size of Rh increases significantly due to the formation of a larger complex ( Figure 8 B). We designed FibrilPaint1 as a PROTAC, utilizing the C-terminal portion of the Protac that recognizes amyloid and the EEVD motif contained in FibrilPaint1 to form a ternary complex with the target amyloid and the E3 ligase CHIP. Then, CHIP was titrated into FibrilPaint1, and the Rh value was measured. Figure 8 As shown in Figure C, FibrilPaint1 showed that Rh increased depending on the concentration of CHIP, indicating that FibrilPaint1 can indeed ubiquitinate amyloid fibrils.

[0148] To employ FibrilPaint1 as the PROTAC strategy, we further validated the transfer of ubiquitin to amyloid fibrils. To this end, we added four different recombinant fibrils (TauRD, HttEX1Q44, asyn, and Abeta fibrils) to the ubiquitination reaction with and without ATP, and measured the reactions using the FIDA system. ATP is essential for the formation of a high-energy thioester bond between ubiquitin and E1; therefore, ubiquitination does not occur in the absence of ATP.

[0149] The results are presented in Figure 9 In AD. In the presence of ATP, all Rh exceeds the maximum measurable size of ubiquitin bound by the ubiquitin transfer system ( Figure 9 The Rh size (BE) confirmed that ubiquitin was bound to the protofibrils. In the absence of ATP, the Rh size indicated that ubiquitination had not occurred.

[0150] Next, we investigated whether the protofibrils were covalently ubiquitinated. To do this, we added urea to the mixture. Urea denatures the ubiquitination mechanism. Therefore, if ubiquitin had already covalently bound to the protofibrils, the measured complex would remain intact. However, if the mechanism did not transfer ubiquitin, only the ubiquitin complex would remain. The results are presented in… Figure 9 In AD, it can be observed that the Rh value does not change after the addition of urea, indicating that the protofibrils are covalently ubiquitinated.

[0151] FibrilPaint can ubiquitinate fibrils derived from patients.

[0152] We wanted to test whether FibrilPaint could also ubiquitinate patient-derived fibrils. To this end, we extracted fibrils from patients diagnosed with AD, FTD, and CBD (Falcon et al., 2018; Fitzpatrick et al., 2017; Zhang et al., 2020) and repeated ubiquitination reactions with these patient-derived fibrils. The results confirmed that FibrilPaint1 also allows for the ubiquitination of patient-derived fibrils. Figure 10 ).

[0153] discuss We designed fibril paint peptides to recognize and bind to amyloid fibrils. The selected fibril paints can label fibrils from patients diagnosed with three different Tau protein diseases, which have different folding structures (Falcon et al., 2018; Fitzpatrick et al., 2017; Goedert et al., 2019; Schers et al., 2020; Zhang et al., 2020). Combined with FIDA, these fibril paints have proven to be valuable tools for studying the aggregation pathways of TauRD and HttEx1Q44, two sequence- and structurally unrelated aggregates. Importantly, fibril paints 1, 5, 6, 7, and 8 are specific for amyloid fibrils but not for monomeric precursors or amorphous aggregates. Therefore, fibril paints 1, 5, 6, 7, and 8 are specific tools that can be used to determine the presence of fibrils at all stages of the aggregation process.

[0154] FibrilPaint bypasses the need for labeling protein protofibrils in modern fluorescence-based detection methods. As shown, FibrilPaint1 binds nonvalently to protofibrils without altering their structure. Figure 1 , 4 This proves useful in handling valuable patient-derived materials, avoiding material loss or artifacts caused by labeling procedures. It also allows for the detection of amyloid fibrils in the presence of cell lysates. Figure 2 A). Therefore, FibrilPaint1 has been shown to be a selective compound for the detection of amyloid and has the potential to be used as a tracer for the detection of a variety of aggregation diseases. Since amyloidosis is also present in other diseases such as diabetes and Parkinson's disease, FibrilPaint1 could be an interesting tool for the detection of pathological fibrils outside the brain (Brundin et al., 2017; Cao et al., 2020).

[0155] Developing tracer drugs for the early diagnosis of neurodegenerative diseases remains crucial. To date, Tauvid is the only FDA-approved Tau tracer for Alzheimer's disease (AD) (Commissioner, 2020; Jie et al., 2021; Mohammadi et al., 2023). Cryo-electron microscopy data revealed that Tauvid does not bind to AD fibrils in a conventional stoichiometric manner, but it does bind to CTE type I filaments in this manner (Shi et al., 2022). Regarding Htt tracers, those tracers have only been tested in ex vivo samples (Delva et al., 2022; Herrmann et al., 2021). We demonstrate that FibrilPaint can specifically bind to fibrils in patients diagnosed with AD, CBD, and FTD at substoichiometric concentrations (up to 1:1000; per monomer). Figure 5 A), and recombinant HttEx1Q44 fibrils and Htt brain supernatant (A), as well as recombinant HttEx1Q44 fibrils and Htt brain supernatant (A) Figure 1 J, Figure 7 C). This makes FibrilPaint 1, 5, 6, 7, and 8 potential tracers specific to amyloid fibrils of different properties, capable of recognizing repeating structures of fibrils (Figure 11). The peptide properties of FibrilPaint 1, 5, 6, 7, and 8 are biocompatible and can be further functionalized as lead compounds to develop tracers for early neurodegenerative diseases.

[0156] The combination of FibrilPaint 1, 5, 6, 7, and 8 with FIDA provides valuable research tools. FibrilPaint 1 with FIDA not only identifies the presence of amyloid material but also provides the structural parameter Rh (Jensen and Østergaard, 2010). The mean Rh of fibrils stained with FibrilPaint 1 is determined without bias towards larger or smaller particles, which can be used to assess disease stages (Lobanova, 2022; Nirmalraj et al., 2023). With recent advances in cryo-electron microscopy, many fibril structures are available as PDB files (Scheres et al., 2020). The mean Rh can then be directly compared with the fibril structure. Therefore, combining FibrilPaint 1, 5, 6, 7, and 8 with FIDA measurements allows for monitoring the mean length of fibrils. This is particularly important for monitoring disease progression, as fibril length is an indicator of disease stage (Nirmalra et al., 2023).

[0157] FibrilPaint 1, 5, 6, 7, and 8 are suitable tools for further development into tracers for neurodegenerative diseases and other protofibrotic diseases, as well as disease-modifying compounds such as those targeting protein degradation. Figure 12 Its ability to specifically bind to multiple amyloid fibrils opens the door to the diagnosis of various neurodegenerative diseases. Its ability to recognize early aggregates provides an opportunity to identify the first stage of the disease. This could allow for intervention before serious brain damage occurs.

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Claims

1. A polypeptide comprising... i) Amino acid sequence: P-W-W-X 1 -X 2 -P-W-W-P-W-H-H-P-X 3 Where X 1 It is R or K, preferably R, X 2 It is R or K, preferably R, and X 3 It is H or W, preferably H; or ii) Amino acid sequence: PWWRRPWWPWHHPH The amino acid sequence contains no more than 4, 3, 2, or 1 amino acid substitutions, deletions, or insertions. The amino acid sequences of i) and ii) are as follows: - It binds to aggregates or oligomeric precursors of amyloid fibrils; and - It does not bind to the non-aggregate monomeric form of amyloid protoplasts.

2. The polypeptide of claim 1, wherein the polypeptide comprises at least one detectable marker.

3. The polypeptide according to claim 1 or 2, wherein the polypeptide comprises a second label.

4. The polypeptide according to any one of claims 1-3, wherein at least one of the detectable marker and the second marker is linked to the amino acid sequence or the polypeptide via a flexible linker, preferably a flexible amino acid sequence.

5. The polypeptide according to any one of claims 1-4, wherein the detectable label and the second label are attached to opposite ends of the polypeptide.

6. The polypeptide according to any one of claims 1-5, wherein the detectable label is selected from the group consisting of: radionuclides, isotopes, optical tags, magnetic materials, affinity labels, and any combination thereof.

7. The polypeptide according to claim 6, wherein at least one of the following: a) The radionuclide is selected from the following group: 124 I, 18 F, 11 C 99m Tc, 123 I and any combination thereof; b) The optical tag is selected from the group consisting of: fluorescent dyes, fluorescent proteins, chemiluminescent dyes, quantum dots, and any combination thereof; c) The magnetic material is selected from the group consisting of: nanoparticles containing nanocomposite materials, superparamagnetic iron oxide nanoparticles, and any combination thereof; d) The affinity marker is an epitope, streptavidin, or avidin-binding peptide, biotin, or an oligohistidine sequence.

8. The polypeptide according to any one of claims 3-6, wherein the second label is a negatively charged amino acid sequence, preferably wherein the negatively charged amino acid sequence comprises SEQ ID NO:

5.

9. A nucleic acid sequence encoding a polypeptide according to any one of the preceding claims.

10. A pharmaceutical composition comprising a polypeptide according to any one of the preceding claims, optionally further comprising a pharmaceutical excipient.

11. A method for diagnosing the progression of a pathological condition caused by amyloid fibrillation or identifying a disease state or predisposition to a pathological condition caused by amyloid fibrillation in a subject, the method comprising: a) Provide a sample from the subject; b) Contacting the sample with the polypeptide according to any one of claims 1 to 8, the polypeptide specifically binding to aggregated amyloid fibrils; and c) Detecting the presence of aggregated amyloid fibrils bound to the polypeptide, wherein the detection of aggregated amyloid fibrils bound to the polypeptide indicates the presence of aggregated amyloid fibrils.

12. A method for monitoring the progression of a pathological condition caused by amyloid fibrillation in subjects or for identifying the disease state of a pathological condition caused by amyloid fibrillation, the method comprising: a) Provide a sample from the subject; b) Contact the sample with a polypeptide as described herein, which specifically binds to aggregated amyloid fibrils; and c) Determine at least one of the following: the length of the amyloid fibrils bound to the polypeptide, the hydrodynamic radius, and the location of the fibrils. The length of the fibril, its hydrodynamic radius, and / or its location provide an indication of progression or identification of a disease state.

13. The method according to claim 11 or 12, wherein the sample is at least one of brain tissue or other tissue, plasma, cerebrospinal fluid (CSF), urine, or tears.

14. The method according to any one of claims 11 to 13, wherein the detected amyloid aggregates are derived from amyloid-forming proteins such as Tau, IAPP, β-amyloid, α-synuclein, huntingtin-tau protein, and / or α-synuclein.

15. The polypeptide according to any one of claims 1 to 8, the nucleic acid according to claim 9, or the pharmaceutical composition according to claim 10, for use as a medicine.

16. The polypeptide according to any one of claims 1 to 8, the nucleic acid according to claim 9, or the pharmaceutical composition according to claim 10, for use as a medicament for treating a pathological condition caused by amyloid fibrillary aggregation.

17. The method according to any one of claims 11-14 or the use according to claim 15 or 16, wherein the pathological condition is amyloidosis, such as systemic lysozyme, insulin, or hemodialysis amyloidosis; or a neurodegenerative disease, including tau protein diseases such as Alzheimer's disease (AD), corticobasal degeneration (CBD), and Pick's disease (PiD); and conucleoprotein diseases such as Parkinson's disease (PD) and Lewy body disease; or other neurodegenerative diseases such as prions, auricidal granulomas (AGD), progressive supranuclear palsy (PSP), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS).

18. Use of the polypeptide according to any one of claims 1 to 8, the nucleic acid according to claim 9, or the pharmaceutical composition according to claim 10 for the detection of aggregated amyloid fibrils.

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