TAU proteomimetics

Abstract

A tau proteomimetic comprising a cyclic peptide comprising an amino acid sequence NIKHVPGGGSVQIVY (SEQ ID NO: 1) or VPGGGCVQIVYK (SEQ ID NO: 2), such that two amino acids are substituted with cysteine residues, or cysteine residues are positioned at both an amino terminus and a carboxy terminus of the peptide, a linker moiety covalently linking the sulfur atoms of the cysteine residues to form the cyclic peptide, and the N-terminus of the peptide is acetylated and the C-terminus of the peptide is amidated.

Description

[0001] TAU PROTEOMIMETICS RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No.63 / 551,707 filed February 9, 2024, which is incorporated herein by reference. GOVERNMENT SUPPORT This invention was made with government support under grant AG074570 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. BACKGROUND OF THE INVENTION A growing number of neurodegenerative diseases are characterized by the presence of neurofibrillary tangles (NFTs) composed of tau. Tau is an intrinsically disordered, microtubule- associated protein that plays a role in cellular signaling, mRNA translation, and maintenance of neuronal microtubule dynamics. Aberrant post-translational modifications, mutations in the encoding MAPT gene, and certain environmental stimuli can promote the transition of normal tau into cross-β amyloid assemblies and NFTs. Misfolded tau is self-perpetuating, with the capacity to propagate from neuron to neuron in a prion-like fashion. The ability of seed-competent forms of tau to recruit and convert intracellular tau into homotypic fibrils is strongly linked to cognitive decline. Recent cryo-electron microscopy (cryo-EM) studies using patient-derived extracts have revealed that the conformation of filamentous tau protomers varies depending on the disease. Pathological folds, or “strains,” of tau correlate with neurodegenerative phenotype even when fibrils are composed of the same isoform or primary sequence in the ordered core. Despite intense interest in anti-tau therapies, a lack of relevant in vitro and in vivo models of pathological tau is a major impediment to drug screening and conformation-specific antibody development. Given the scarcity, variability, and structural diversity of tau proteoforms from patient samples, efforts to recapitulate pathological tau folds in vitro are urgently needed. In addition, there remain key gaps in our understanding of the specific epitopes within tau that are responsible for templated fibril growth and aggressive prion-like spread. The conformational transition of tau from a disordered monomeric state to cross-b assembly is largely driven by exposure of aggregation-prone hexapeptide modules (tau306-311or PHF6, and tau275-280 or PHF6*) in the tau repeat domains. However, fibrils comprised of the PHF6 and PHF6* hexapeptides are unable to promote the aggregation of endogenous tau in cell-based assays. This has prompted efforts to identify larger fragments that adopt cross-b architecture and exhibit seeding activity in vitro and in vivo. While such efforts have begun to shed light on the structural characteristics of seed-competent fragments, they still fall short. Accordingly, there is a need for new and effective tools and methods to investigate tau seeding and their subsequent pathologies. The present disclosure satisfies these needs. SUMMARY OF THE INVENTION The present disclosure describes the design and synthesis of functional tau proteomimetics using an operationally simple peptide stapling approach. Diversity-oriented macrocyclization enables the identification of conformationally pre-organized β-arch peptides that self-assemble into amyloid filaments. A subset of these “mini-tau” fibrils potently seed endogenous tau in engineered biosensor cells and primary neurons. Structural elucidation of a mini-tau filament by cryo-EM reveals several conformational features congruent with those in pathological tau folds. These studies provide a framework for the minimization of pathological epitopes of tau and other amyloidogenic proteins. Accordingly, in some embodiments, the disclosure provides for a tau proteomimetic comprising a cyclic peptide comprising an amino acid sequence NIKHVPGGGSVQIVY (SEQ ID NO: 1) or VPGGGCVQIVYK (SEQ ID NO: 2), wherein two amino acids are substituted with cysteine residues, or cysteine residues are positioned at both an amino terminus and a carboxy terminus of the cyclic peptide; a linker moiety covalently linking the sulfur atoms of the cysteine residues to form the cyclic peptide; and wherein the N-terminus of the cyclic peptide is acetylated and the C-terminus of the peptide is amidated. In some embodiments, the cyclic peptide comprises DNIKHVPGGGSVQIVYK (SEQ ID NO: 3), DNIKHVPGGGSVQIVYKPV (SEQ ID NO: 4), KDNIKHVPGGGSVQIVYKPVD (SEQ ID NO: 5), or GSKDNIKHVPGGGSVQIVYKPVD (SEQ ID NO: 6), In some embodiments, a tau proteomimetic comprises a cyclic peptide comprising an amino acid sequence of: CNIKHVPGGGSVQIVYC (SEQ ID NO: 7), DNIKCVPGGGCVQIVYK (SEQ ID NO: 8), CDNIKHVPGGGSVQIVYKPVC (SEQ ID NO: 9), KDNIKCVPGGGSVQIVCKPVD (SEQ ID NO: 10), GSKDNIKCVPGGGSVCIVYKPV (SEQ ID NO: 11), GSKDNIKHVCGGGCVQIVYKPV (SEQ ID NO: 12), GSKDNIKHVCGGGSVQCVYKPV (SEQ ID NO: 13), KDNICHCPGGGSVQIVYKPVD (SEQ ID NO: 14), KDCICHVPGGGSVQIVYKPVD (SEQ ID NO: 15), or DNICVPGGGCVQIVYK (SEQ ID NO: 16), wherein a linker moiety covalently links the sulfur atoms of the cysteine residues to form the cyclic peptide; and wherein the N-terminus of the peptide is acetylated and the C-terminus of the peptide is amidated. In some embodiments, the di-cysteine linker moiety is

[0002] These and other features and advantages of this invention will be more fully understood from the following detailed description of the invention taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description. BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention. Fig. 1A-C. (A) Solid-phase synthesis and diversity-oriented macrocyclization of tau epitope mimics. (B) Structures and isolated yields (see Table 1) for synthesized macrocycles. (C) End-point of the fluorescence of tau mimics (500 mM) incubated 48 h at 37oC. Fig. 2A-K. (A) Optimized protocol for seeding the endogenous tauRD[LM]-YFP in biosensor cells with synthetic macrocycle fibrils. Representative fluorescence microscopy images of biosensor cells (20× magnification under FITC channel) in the (B) absence or presence of (C- D) 5 mM KD-mxyl and (E-F) KD-pyr fibrils. (G) Screening of synthesized macrocycles for seed- competency in the biosensor cell assay at 1 mM. (H) Structures of synthesized linear and cyclic control analogues. (I) Biosensor cell seeding activity of KD-mxyl and KD-pyr relative to linear and sequence-scrambled controls. Dose-dependent seeding activity of (J) KD-mxyl and (K) KD-pyr. Fig. 3A-R. (A-B) Representative negative stain TEM images of dispersed KD-mxyl filaments prepared in aq TBS (pH 7.6). (C) 2D classes of filaments observed for KD-mxyl by cryo- EM. (D) Side and (E-F) top view 3D reconstructions of type 1 and type 2 KD-mxyl filaments. (G) Inter-monomer steric zipper formed from antiparallel cross-β association of VQIVYK segments in the type 1 KD-mxyl filament. (H) Parallel inter-monomer association of VQIVYK segments in the type 2 KD-mxyl filament. Macrocycle conformation in the (I) type 1 and (J) type 2 KD-mxyl filaments. (K) Sequence and structure map for KD-mxyl. (L) Overlay of the type 1 and type 2 KD-mxyl backbone conformations and calculated RMSD. (M) Inter-layer S305-G302 sidechain- to-backbone H-bonds in the type 1 KD-mxyl fold. (N) Cross-β sidechain-to-sidechain H-bond between S305 and H299 in the type 2 KD-mxyl fold. (O) Overlay of the VPGGG β-arc segments from the type 1 KD-mxyl filament and the CBD type 1 tau fold (pdb 6TJO) with calculated backbone RMSD. (P) Overlay of the VPGGG β-arc segments from the type 1 KD-mxyl filament and the AGD type 1 tau fold (pdb 7P6D) with calculated backbone RMSD. (Q) Inter-monomer H-bonding interaction between Y310 / K311 and D295 in KD-mxyl filaments. (R) Cartoon depicting the tripartite assembly of KD-mxyl macrocycles linked through Y310 / K311-D295 H- bonds. Fig. 4A-G. (A) Seeding and immunohistochemistry of human wt tau in primary neurons isolated from transchromosomal mice by KD-mxyl fibrils. Confocal microscopy images (20× magnification, DAPI staining, TRITC filter) of primary hTau+ / mTau- neurons treated for 5 d with (B-D) 500 nM KD-mxyl fibrils, (E) vehicle, (F) 500 nM linear-Ala, or (G) 500 nM scrm-mxyl, followed by AT8 antibody and DAPI staining for nuclei. Fig.5A-M. Screening of embodiment of synthesized macrocycles for seed-competency in the biosensor cell assay at 5 mM. DETAILED DESCRIPTION OF THE INVENTION Primary tauopathies are a class of neurodegenerative disorders whose predominant feature is tau protein deposits in the brain. Misfolded tau has the capacity to seed the aggregation of naïve tau, leading to the prion-like spread of neurofibrillary tangles. Tau protomers within fibrils always exhibit cross-b amyloid structure, but distinct conformations of misfolded tau correlate with specific pathology. An understanding of how seeding capacity is impacted by conformation remains elusive. Identification of the minimal epitopes required for transcellular propagation represents a key step toward more relevant models of disease progression. Here, we implement a peptide macrocyclization approach toward seed-competent miniature tau, or “mini-tau”, proteomimetics. Structural elucidation of a potent mini-tau macrocycle reveals several conformational features present in 4R tauopathic strains. The discovery of functional b-arch folds through diversity-oriented peptide stapling has broad-ranging implications for the mimicry of other proteopathic seeds. Additional information and data supporting the invention can be found in the following publication by the inventors: Angera, et al., Structure-based design of seed-competent proteomimetic macrocycles derived from 4R tauopathic folds. ChemRxiv. 2024; doi:10.26434 / chemrxiv-2024-l7zgc and its Supporting Information, which are incorporated herein by reference in its entirety. Definitions. The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2d ed., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary of Biology. Harper Perennial, N.Y. (1991). General laboratory techniques (DNA extraction, RNA extraction, cloning, cell culturing. etc.) are known in the art and described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., 4th edition, Cold Spring Harbor Laboratory Press, 2012. References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described. The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations. The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is disubstituted. As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect. The term "about" can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent, or as otherwise defined by a particular claim. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, composition, or embodiment. The term about can also modify the endpoints of a recited range as discussed above in this paragraph. As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units are also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually, and as part of a range. A recited range (e.g., weight percentages) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation. The term “substantially” as used herein, is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified. For example, the term could refer to a numerical value that may not be 100% the full numerical value. The full numerical value may be less by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%. Wherever the term “comprising” is used herein, options are contemplated wherein the terms “consisting of” or “consisting essentially of” are used instead. As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect. In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The disclosure illustratively described herein may be suitably practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC / GENE (Intelligenetics, Mountain View, Calif.). As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. The term “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. In certain embodiments, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, JMB, 48, 443 (1970)). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Thus, embodiment of the invention also provides nucleic acid molecules and peptides that are substantially identical to the nucleic acid molecules and peptides presented herein. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. An "effective amount" refers to an amount effective to treat a disease, disorder, and / or condition, or to bring about a recited effect. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host. Thus, an "effective amount" generally means an amount that provides the desired effect. An appropriate "effective" amount in any individual case may be determined using techniques, such as a dose escalation study. This disclosure provides methods of making the compounds and compositions of the invention. The compounds and compositions can be prepared by any of the applicable techniques described herein, optionally in combination with standard techniques of organic synthesis. Many techniques such as etherification and esterification are well known in the art. However, many of these techniques are elaborated in Compendium of Organic Synthetic Methods (John Wiley & Sons, New York), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol.2, Ian T. Harrison and Shuyen Harrison, 1974; Vol.3, Louis S. Hegedus and Leroy Wade, 1977; Vol.4, Leroy G. Wade, Jr., 1980; Vol.5, Leroy G. Wade, Jr., 1984; and Vol.6; as well as standard organic reference texts such as March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Ed., by M. B. Smith and J. March (John Wiley & Sons, New York, 2001); Comprehensive Organic Synthesis. Selectivity, Strategy & Efficiency in Modern Organic Chemistry. In 9 Volumes, Barry M. Trost, Editor-in-Chief (Pergamon Press, New York, 1993 printing); Advanced Organic Chemistry, Part B: Reactions and Synthesis, Second Edition, Cary and Sundberg (1983); for heterocyclic synthesis see Hermanson, Greg T., Bioconjugate Techniques, Third Edition, Academic Press, 2013. The formulas and compounds described herein can be modified using protecting groups. Suitable amino and carboxy protecting groups are known to those skilled in the art (see for example, Protecting Groups in Organic Synthesis, Second Edition, Greene, T. W., and Wutz, P. G. M., John Wiley & Sons, New York, and references cited therein; Philip J. Kocienski; Protecting Groups (Georg Thieme Verlag Stuttgart, New York, 1994), and references cited therein); and Comprehensive Organic Transformations, Larock, R. C., Second Edition, John Wiley & Sons, New York (1999), and referenced cited therein. The term amino acids described herein are among the 20 naturally occurring amino acids that have the L-configuration unless indicated otherwise. An amino acid that has the D- configuration is denoted symbolically with a superscript capital “D” preceding its name or its standard abbreviation. For example, the D-configuration of alanine with be abbreviated asDalanineDAla, orDA; and it may also be indicated as D-alanine or a lower case single letter abbreviation, e.g., “a” for D-alanine. As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprising amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides, and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof. The term “cyclic peptide” means the entire peptide sequence forms the macrocycle of the cyclic peptide. It can also mean only part of the peptide sequence forms the macrocycle of the cyclic peptide. It is also to be understood that the mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it is also to be understood that the mention of one or more components in a composition does not preclude the presence of additional components than those expressly identified. Embodiments of the Invention. Aggregation of the tau protein into toxic fibrils underlies several neurodegenerative pathologies. These fibrils spread throughout the brain (from neuron-to-neuron) leading to disease progression. We have developed miniaturized mimics of the tau protein based on a peptide macrocyclization platform. These peptides exhibit tau-like function and seed endogenous tau aggregation in vitro, in engineered cells, in primary neurons, and in a humanized tau mouse model. We have solved the high-resolution structure of our peptides and confirmed that they recapitulate the conformation of tau associated with disease. In some embodiments, a tau proteomimetic comprises a cyclic peptide comprising an amino acid sequence NIKHVPGGGSVQIVY (SEQ ID NO: 1) or VPGGGCVQIVYK (SEQ ID NO: 2), wherein at least two amino acids are substituted with cysteine residues, or cysteine residues are positioned at both an amino terminus and a carboxy terminus of the peptide; a di-cysteine linker moiety covalently links the sulfur atoms of the cysteine residues to form the cyclic peptide; and wherein the N-terminus of the cyclic peptide is acetylated and the C-terminus of the cyclic peptide is amidated. In some embodiments, the peptide is SEQ ID NO: 1 and further comprises an amino acid sequence DNIKHVPGGGSVQIVYK (SEQ ID NO: 3), DNIKHVPGGGSVQIVYKPV (SEQ ID NO: 4), KDNIKHVPGGGSVQIVYKPVD (SEQ ID NO: 5), or GSKDNIKHVPGGGSVQIVYKPVD (SEQ ID NO: 6), wherein at least two amino acids are substituted with cysteine residues, or cysteine residues are positioned at both an amino terminus and a carboxy terminus of the peptide; a di-cysteine linker moiety covalently links the sulfur atoms of the cysteine residues to form the cyclic peptide; and wherein the N-terminus of the cyclic peptide is acetylated and the C-terminus of the cyclic peptide is amidated. In some embodiments, the cyclic peptide is: CNIKHVPGGGSVQIVYC (SEQ ID NO: 7); DNIKCVPGGGCVQIVYK (SEQ ID NO: 8); CDNIKHVPGGGSVQIVYKPVC (SEQ ID NO: 9); KDNIKCVPGGGSVQIVCKPVD (SEQ ID NO: 10); GSKDNIKCVPGGGSVCIVYKPV (SEQ ID NO: 11); GSKDNIKHVCGGGCVQIVYKPV (SEQ ID NO: 12); GSKDNIKHVCGGGSVQCVYKPV (SEQ ID NO: 13); KDNICHCPGGGSVQIVYKPVD (SEQ ID NO: 14); or KDCICHVPGGGSVQIVYKPVD (SEQ ID NO: 15). In some embodiments, a tau proteomimetic comprises a cyclic peptide comprising an amino acid sequence VPGGGCVQIVYK (SEQ ID NO: 2), wherein two amino acids are substituted with cysteine residues, or cysteine residues are positioned at both an amino terminus and a carboxy terminus of the peptide; a di-cysteine linker moiety covalently links the sulfur atoms of the cysteine residues to form the cyclic peptide; and wherein the N-terminus of the cyclic peptide is acetylated and the C-terminus of the cyclic peptide is amidated. In some embodiments, the cyclic peptide is DNICVPGGGCVQIVYK (SEQ ID NO: 16). In some embodiments, the di-cysteine linker (DCL) is In some embodiments, the di-cysteine linker (DCL) is In some embodiments, a tau proteomimetic comprises a cyclic peptide comprising an amino acid sequence according to any one of SEQ ID NO: 1-6, wherein at least two amino acids of the peptide are substituted with cysteine residues; a di-cysteine linker moiety covalently links the sulfur atoms of the cysteine residues to form the cyclic peptide; wherein the N-terminus of the peptide is acetylated and the C-terminus of the peptide is amidated; and wherein the di-cysteine linker moiety comprises one of:

[0003] In some embodiments, a tau proteomimetic comprises a peptide comprising an amino acid sequence according to any one of SEQ ID NO: 1-6, wherein a cysteine residue is positioned at both an amino terminus and a carboxy terminus of the peptide; and a di-cysteine linker moiety covalently links the sulfur atoms of the cysteine residues to form the cyclic peptide wherein the di-cysteine linker moiety comprising one of: In some embodiments, the tau proteomimetic is one or more cyclic peptides having the structure:

[0004]

[0005]

[0006]

[0007]

[0008]

[0009]

[0010]

[0011]

[0012]

[0013] or a salt or solvate thereof. Methods of making the described tau proteomimetic cyclic peptides are known in the art, and described, for example, by U.S. Patent Publication No.2023 / 0399361 to Del Valle et al. The disclosure also provides for compositions comprising one or more tau peptidomimetic having an amino acid sequence according to any one of SEQ ID NO: 1-6, wherein at least two amino acids are substituted with cysteine residues, or cysteine residues are positioned at both an amino terminus and a carboxy terminus of the peptide; a di-cysteine linker moiety covalently links the cysteine residues; wherein the N-terminus of the peptide is acetylated and the C-terminus of the peptide is amidated, and wherein the di-cysteine linker moiety comprises one of: , , , ; and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, a composition comprises one or more tau proteomimetic comprising a cyclic peptide having a structure according to any one of structures 1-33. The compounds may be added to a carrier in the form of a salt or solvate. For example, in cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiologically acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, α-ketoglutarate, and β-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, halide, sulfate, nitrate, bicarbonate, and carbonate salts. Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable ionic compound. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example, calcium) salts of carboxylic acids can also be prepared by analogous methods. Results and Discussion. Design and synthesis of "mini-tau" macrocycles featuring sidechain-to-sidechain tethers. We examined available cryo-EM structures of pathological tau fibrils isolated from patients to identify potentially seed-competent core folds. We focused on primary 4R tauopathic structures given the presence of densely packed, multi-strand β-arch motifs encompassing the aggregation- driving PHF6 module. In addition to extensive cross-strand hydrophobic interactions, each of the pathological strains in features at least one sidechain-to-sidechain polar interaction in the core, which we hypothesized may be important for stabilizing a seed-competent conformation. Highlighted interactions in are shown in representative pathological folds but are not necessarily exclusive to a given strain. For example, the D295-K311 and H299-S305 H-bonds are observed in both AGD and CBD folds, while the K294-D314 interaction is found in GGT as well as GPT fibril structures. In contrast, the H299-Y310 H-bond is only observed in the cryo-EM structures of tau fibrils associated with PSP. We truncated the tau core sequences and replaced the interacting residues with Cys to allow for sidechain stapling. Macrocycles are classified according to the residue pairs that were replaced with Cys (DK, HS, KD, or HY). We expected that tethers with differing flexibility and geometry would enforce a variety of conformations, including some that are distinct from the parent fibrillar folds. Four di-Cys-containing linear substrates were prepared by conventional solid-peptide synthesis on Rink amide MBHA resin (Figure 1A). Following cleavage and global deprotection the crude peptides were subjected to bis-alkylation with dibromide electrophiles to afford 20 unique macrocycles. The identity and purity of all synthesized compounds were verified by HRMS and analytical HPLC (Figure 1B). The aggregation of purified macrocycles was monitored in a thioflavin T (ThT) fluorescence assay. Several of the wells exhibited high ThT fluorescence immediately upon initiation of the assay, suggesting that some macrocycles were already in an aggregated state following purification. Other macrocycles exhibited time-dependent aggregation with little to no discernable lag phase indicating rapid formation of amyloid nuclei. A comparison of endpoint fluorescence readings shows that 18 out of the 20 synthesized tau mimics elicited high (> 5 × 105RFU) ThT fluorescence after 48 h, with the majority reaching levels between 1−5 × 106RFU (Figure 1C). For comparison, heparin-induced 0N4R tauP301Lachieved a maximum ThT fluorescence of approximately 2 × 105RFU, whereas aggregates of PHF6 reached a level similar to that of several macrocycles (~ 1.2 × 106RFU). Interestingly, an unstapled control peptide corresponding to the longest synthesized sequence (KD), but with wild-type Lys and Asp residues at the termini, did not exhibit significant fluorescence under the same conditions. Mini-tau macrocycles seed endogenous tau in a sequence and staple-dependent manner. We next evaluated the ability of our synthetic macrocycles to template aggregation in HEK293 cells expressing a tau-yellow fluorescent protein fusion (tau-RD[LM]-YFP). Under normal conditions these cells exhibit only diffuse background fluorescence when observed by microscopy. However, exposure to recombinant or patient-derived tau fibril seeds in the presence of lipofectamine leads to the formation of fluorescent puncta indicative of endogenous tau inclusions. Initial screening of compounds pre-incubated for 4 days prior to cell treatment revealed reproducible albeit sparse punctate fluorescence in wells treated with KD-mxyl and KD-pyr. We then optimized the fibril preparation procedure to maximize the signal induced by these two hit compounds (Figure 2A). Cells treated with 5µM KD-mxyl and KD-pyr seeds prepared in this manner produced significant tau-RD[LM]-YFP inclusions relative to vehicle-treated cells (Figure 2B-F). We then tested all 20 of our macrocycles in the biosensor cell assay and quantified seeding activity using image-based brightness threshold analysis. We confirmed KD-mxyl and KD-pyr as the most effective seeds (Figure 2G ). Although less efficacious, HS-pyr and KD-pxyl also induced endogenous tau aggregation at 1 µM. Additional hits were identified when the assay was conducted at 5 µM. However, KD-pyr and KD-mxyl remained the most consistently active compounds in repeated experiments. The prion-like activity of amyloids is highly sensitive to oligomerization state, fibril maturity, and membrane permeation. We found fibril shearing to be necessary for seeding, as the activity of KD-mxyl and KD-pyr was completely abolished in the absence of a sonication step. KD-mxyl and KD-pyr incubated in unbuffered water also failed to induce endogenous tau inclusions, confirming the importance of PBS for self-assembly into seed-competent fibrils. Finally, we confirmed that lipofectamine is required for potent seeding of biosensor cells by both KD-mxyl and KD-pyr. These results show that both monomeric and mature fibrillar forms of KD- mxyl and KD-pyr lack seeding activity, whereas sonicated fibrils are capable of templating tau aggregation in cells. To test whether macrocyclic constraint is important for activity, we synthesized unstapled control peptides that harbor either Ala (linear-Ala) or the wild-type Lys and Asp (linear-wt) residues in place of the terminal Cys residues (Figure 2H). We also prepared sequence-scrambled macrocycles (scrm-mxyl and scrm-pyr) that retained the linker present in our lead compounds. When tested in same biosensor cell assay, we observed a dramatic reduction in seeding activity for linear-wt and scrm-mxyl, and complete loss of seeding activity in the case of linear-Ala and scrm- pyr (Figure 2I). The activity of linear-wt was weak (~500 puncta / well) but still significantly higher than that of linear-Ala (~30 puncta / well). This may be due to restoration of a key electrostatic interaction when the wild-type residues are positioned at the peptide termini. In addition to the scrm-mxyl control macrocycle, the KD-pxyl analogue from our initial library represents a compound with identical chemical composition to that of KD-mxyl. Among these 3 compounds, KD-mxyl exhibited significantly higher seeding activity than either of its constitutional isomers. While KD-pxyl retained moderate cellular seeding capacity at 1 µM (~1200 puncta / well), scrambling of the native core sequence, as in scrm-mxyl, led to > 90% reduction in activity. Similarly, scrm-pyr exhibited no discernable activity at 1 µM despite the potency of its constitutional isomer KD-pyr. These results establish that covalent tethering, linker geometry, and sequence are indeed critical for potentiating the seeding capacity of KD-mxyl and KD-pyr. The minimum effective seeding concentrations of KD-mxyl and KD-pyr were examined by dose-response experiments in the biosensor cell assay. As shown in Figure 2J-K, both macrocycles exhibited clear dose-dependent seeding activity and maintained efficacy even at concentrations below 500 nM. Although the lowest concentration tested (1 nM) resulted in only sparse fluorescent tau inclusions (~250-350 puncta / well), seeding activity remained significant relative to vehicle-treated control wells. Pre-assembled and sonicated fibrils of KD-mxyl and KD- pyr are thus remarkably potent seeds of tau-RD[LM]-YFP in cells. Structural characterization of KD-mxyl and KD-pyr macrocycles. We next investigated the self-assembly and associated conformational transitions of KD-mxyl and KD-pyr in vitro. Maximum ThT fluorescence induced by KD-mxyl and KD-pyr correlated with macrocycle concentration and did not exhibit a lag phase. With both macrocycles, we observed large numbers of densely-packed fibrillar or proto-fibrillar structures by TEM (Figure 4C-D). Mature mini-tau fibrils were soluble in unbuffered water after pelleting, allowing us to obtain circular dichroic (CD) spectra of pre-fibrillar and post fibrillar states. Pre-fibrillar samples of both macrocycles exhibited no positive Cotton effect and a minimum molar ellipticity near 198 nm indicative of random coil conformation. In contrast, the self-assembled, pelleted, and re-dissolved samples of KD-mxyl and KD-pyr exhibited intense positive CD bands near 200 nm and pronounced negative ellipticity near 220 nm, which are characteristic of parallel β-sheet structure. Variable temperature CD measurements with KD-mxyl and KD-pyr revealed well-defined isodichroic points and cooperative melting transitions. Although KD-mxyl did not appear to reach a fully unfolded state even at 90oC, we calculated a melting temperature of ~69oC for KD-pyr. Neither of the denatured samples returned to a folded state upon re-cooling in unbuffered water. X-ray fiber diffraction of the more thermally stable KD-mxyl exhibited reflections near 4.6 Å attributable to repeating β-strands along the fiber meridian and weaker equatorial reflections at ~10 Å representing face-to-face cross-β- sheet interactions through the fibril axis. We next optimized fibril growth and dispersion conditions to obtain a high-resolution structure of KD-mxyl using cryo-electron microscopy (cryo-EM). Although images of KD-mxyl fibrils seemed to show only one morphology, two-dimensional class averaging showed 3 distinct classes due to readily discernible features and cross-over distances. We solved the structures of the first class (type 1) at 3.20 Å resolution and the second class (type 2) at 2.95 Å based on the 0.143 Fourier shell correlation criterion, respectively. Both type 1 and type 2 KD-mxyl filaments are composed of 6 macrocycles per layer, with a 3-fold symmetrical core whose structure could be fully assigned (Figure 3D-E). Partial assignment of macrocycles surrounding the trimeric core was only possible for the PHF6 motif that abuts its complementary strand. We observed a rise of 4.8 Å for both filament types, consistent with the protomer spacing characteristic of amyloids in a parallel β-sheet assembly. Successive rungs of KD-mxyl type 1 and type 2 macrocycles exhibit a twist of −2.9oand −1.2o, respectively. The manner in which the 3 peripheral macrocycles pack against the PHF6 segments in the trimeric core differs significantly between type 1 and type 2 filaments. The antiparallel cross-β arrangement in the type 1 structure results in an intermolecular class 1 steric zipper like that observed in solid- state structures of PHF6 hexapeptides (Figure 3F). In contrast, the type 2 trimeric core engages the peripheral PHF6 segments in a parallel arrangement that precludes tight hydrophobic packing (Figure 3G). Macrocycles in both the type 1 and type 2 filaments feature β-arch folds in which two β- strands are joined by a five-residue β-arc (Figure 3H-I). The di-Cys aromatic staples are buried within the macrocyclic cores and do not engage other monomers within their fibril layers. Both KD-mxyl polymorphs exhibit a cross-sectional conformation reminiscent of a T-type solenoid in which one of the β-strand modules is replaced by an unnatural staple. Most of the deviation between the structures originates from residues 300-304 (Figure 3K), with unique polar interactions at S305 that appear to govern β-arc conformation. In the KD-mxyl type 1 polymorph the S305 sidechain acts as a donor in an inter-layer H-bond with the carbonyl group of G302 (Figure 3L). This carbonyl O does not participate in an H-bond in the type 2 structure and S305 instead engages H299 in a cross-β H-bond that is also observed in the CBD tau fold (Figure 3M). This interaction is attended by N296 and Q307 amide ladders that are only present in the type 2 structure. We carried out RMSD calculations for each KD-mxyl β-arc in comparison to the pathological folds. Good agreement was found between the type 1 KD-mxyl arc and that observed in CBD tau fibrils. The type 2 KD-mxyl arc exhibits remarkably close overlap with that of the AGD tau fold (backbone RMSD = 0.62 Å). Both KD-mxyl polymorphs also feature a complex network of intra- and inter-layer H-bonds involving the sidechains of Y310 and K311 from one monomer and D295 from another (Figure 3P). These polar interactions appear to mediate lateral arrangement into a trimeric assembly (Figure 3Q). Of the 4R tauopathic strains, only the GGT fold contains Y310 and K311 sidechains that project in the same direction rather than the alternating pattern typical of β-strands. KD-mxyl induces the aggregation of wild-type human tau in primary hippocampal neurons. The recipient tau fusion in the HEK293 biosensor cells used for seeding experiments harbors P301L and V337M mutations associated with inherited frontotemporal dementia (FTD). These mutations are known to facilitate tau aggregation and render the cells more sensitive to fibril seeds. We next sought to determine if KD-mxyl could induce inclusions of wild-type tau in neurons. Hippocampi were dissected from the embryos of MAPT transchromosomal mice expressing wild-type human tau and the isolated neurons were treated with KD-mxyl, scrm-mxyl, or linear-Ala. After incubation for 5 days, the cells were fixed and treated with AT8 anti-tau antibody followed by a fluorescently conjugated secondary antibody (Figure 4A). As shown in Figure 4B-D, cells treated with 500 nM KD-mxyl displayed strong and clustered fluorescence throughout the cell body, indicating accumulation of misfolded wild-type tau into aggregates. In contrast, neurons treated with vehicle exhibited very low levels of background fluorescence distributed evenly throughout the cell, including the axon (Figure 4E). Both linear-Ala and scrm-mxyl control peptides failed to induce AT8-positive fluorescent tau inclusions (Figure 4F-G). These data show that fibrils of KD-mxyl can seed wild-type human tau in primary hippocampal neurons, and that effective seeding of these cells is also dependent on sequence and macrocyclic tethering. The structural diversity of pathological tau filaments has raised important questions on the potential link between conformational strain and seed infectivity. Considerable effort has been devoted to recapitulating the tauopathic folds observed in patient extracts, delineating the minimal tau fragments required for seeding, and elucidating the structure of functional tau epitopes. Recently, a 95-residue tau fragment (dGAE) was induced to adopt an AD core fold in vitro upon fibrillization in the presence of MgCl2. However, the ability of these fibrils to seed endogenous tau was unsuccessful in our hands and has yet to be demonstrated in a cell-based system. Fibrils generated from shorter tau fragments including tau306-378, tau306-336, [P301L]tau295-311 exhibit seed- competency in cellular assays but their high-resolution structures have thus far not been determined. The diversity-oriented macrocyclization platform described here represents a simple yet powerful approach for identifying conformational "hot-spots" that may be critical for transcellular propagation. While covalent constraint is a broadly useful tool for stabilizing functional peptide conformations, its application to fibrillar β-arch topology remains limited. Using short tau-derived peptides, we demonstrate that macrocyclic tethering is important for enforcing β-arch conformation and imparting seed competency. Aggregation of most of our macrocycles was devoid of a lag phase in ThT assays, suggesting that di-Cys stapling dramatically reduces the barrier to primary nucleation. However, the poor correlation between high ThT fluorescence and seeding in biosensor cells highlights the limitations of this assay as a screening tool. Truncated tau analogues such as PHF6 that lack arc / loop residues but exhibit high affinity for ThT have thus far not been shown to seed endogenous tau. Our results suggest that conformational ordering of residues adjacent to PHF6 is critical for function. Structural elucidation of KD-mxyl polymorphs reveals β-arc conformations that are remarkably congruent with those in pathological tau folds. Although the presence of two filament types for KD-mxyl precludes a definitive assignment of the more active conformation, both structures share several other features with 4R tauopathic strains, including the bl conformation of the outward-facing310YK311dipeptide sidechains (observed only in GGT tau), the extended conformation of the 297IKH299 segment (shared by AGD, CBD, and PSP tau), and a cross-β H-bond between H299 and S305 (observed in CBD tau). These studies set the stage for further minimization of functional tau epitopes through structure-guided optimization or mimicry of other β-arch motifs. The resulting mini-tau macrocycles also represent attractive candidates for the development of anti-tau vaccines and conformation-specific antibodies. Coupled with fibril structure elucidation, the described approach has the potential to afford unprecedented insights into the misfolding and cell-to-cell transmission of tau and other amyloidogenic proteins. The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention. EXAMPLES Example 1. Material and Methods. Solid-phase peptide synthesis. Automated solid-phase peptide synthesis was carried out on NovaPEG Rink amide MBHA resin (35-100 mesh, 0.45 mmol / g) using a CEM Liberty Blue peptide synthesizer. Linear peptides were synthesized at 0.05 mmol scale. The following amino acid derivatives suitable for Fmoc SPPS were used: Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, Fmoc-His(Boc)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, and Fmoc-Ile-OH. Fmoc deprotection steps were carried out using a solution of 20% piperidine / DMF (5 min at rt, then 2 min at 75 °C). After Fmoc deprotection the resin was washed 4 × with DMF. Coupling of Fmoc-protected amino acids was achieved using 5 equiv HCTU (0.25 M in DMF), 10 equiv NMM (1 M in DMF), and 5 equiv of Fmoc-protected amino acid (0.2 M in DMF) at 50 °C (10 min × 2). Deprotection and coupling steps were repeated until sequence elongation was complete. The resin was transferred to a suitable vessel, washed with DCM (5 mL × 4) and dried under vacuum. Peptides were N-terminally acetylated using 5% acetic anhydride and 10% pyridine in DCM (15 min at rt), washed with DCM (5 mL × 4), and dried under vacuum. Cleavage and global deprotection was achieved by incubating the dried resin in 5 mL of TFA:TIPS:H2O:DODT (92.5:2.5:2.5:2.5) for 2.5 h. The resin was filtered, and the filtrate was collected in a 50 mL centrifuge tube. The resin was washed with DCM (10 mL), filtered, and crude peptides were precipitated from the combined filtrate by the addition of cold Et2O (40 mL). The mixture was centrifuged and the supernatant decanted. The pellet was washed with Et2O (25 mL × 2) and dried thoroughly under vacuum. Table 1. Macrocycle yield. Macrocyclization and characterization of mini-tau peptides. Crude Cys-containing linear peptides were dissolved in 1:1 MeCN:20 mM aq NH4HCO3 to a concentration of 1 mM and treated with 1.5 equiv of 1,3-bis(bromomethyl)benzene, 1,4- bis(bromomethyl)benzene, 2,6- bis(bromomethyl)pyridine, 4,4’-bis(bromomethyl)biphenyl, or E- 1,4- dibromo-2-butene. The pH was adjusted to 8.0 using 2 M aq NaOH. The reaction was stirred for 2 h before evaporating the MeCN under a stream of N2, freezing, and lyophilization. All peptides were purified by preparative RP-HPLC (C12, 250 mm × 21.2 mm, 4 μm, 90 Å) using linear gradients of MeCN in H2O (mobile phases modified with 0.1% formic acid) over 30 min. Analytical RP-HPLC (C12, 150 mm × 4.6 mm, 4 μm, 90 Å) spectra for all purified peptides were acquired using linear gradients of MeCN in H2O (mobile phases modified with 0.1% formic acid) over 20 min. HRMS of purified peptides were acquired using a Bruker Impact II ESI-QTOF. Circular dichroism. Peptides were dissolved from powder to 500 μM in 20 mM aq PBS and incubated with shaking at 37 °C for 3 d to allow for aggregation. Peptide aggregates were pelleted, the supernatant was discarded, and the pellet was redissolved in unbuffered water. CD spectra were acquired using a JASCO J-1700 CD spectrometer in a 1 mm path length quartz cell with 2 s DIT, 1 nm bandwidth, 0.5 nm datapitch, and a scan speed of 100 nm / min at 25 °C. The CD spectra of monomeric (pre- fibrillar) mini-tau macrocycles were obtained from 50 μM solutions of lyophilized peptides in unbuffered water. For variable temperature CD experiments, sample preparation and collection of spectra were carried out using the same conditions and parameters described above at intervals of 10 °C from 10-90 °C. The melting temperature was determined by plotting the change in ellipticity at 221 nm as a function of temperature and applying 4-parameter sigmoidal regression. Negative stain transmission electron microscopy. Lyophilized peptides were dissolved in DMSO to 5 mM and diluted to 500 μM in 20 mM aq PBS. Samples were incubated in a microcentrifuge tube for 4 d at 37 °C with a mixing speed of 150 rpm. A 10 μL aliquot of the sample was then applied to 400-meshed formvar- / carbon-coated copper grids and negative-stained with 2% uranyl acetate. Micrographs were obtained on a JEOL 2011 or Talos F200i transmission electron microscope at 200 kV. ThT fluorescence assay. Lyophilized peptides were dissolved in DMSO at 5 mM. Peptides were then diluted to 500, 200, or 100 µM with 20 mM aq PBS and 10 μM ThT in a 96-well clear bottom black plate to a final reaction volume of 200 μL. The plate was then sealed with a clear sealing film and allowed to incubate at 37 °C with 5 mm orbital shaking (205 cpm) in a Biotek Synergy H1 microplate reader. Error bars represent standard deviation from technical replicates. An automated method was used to carry out ThT fluorescence measurements at an excitation wavelength of 444 nm and an emission wavelength of 485 nm at an interval of every 5 min for 48 h. The same assay conditions were used to measure aggregation of PHF6 and linear wt control peptides. Recombinant tauP301L aggregation was monitored at 10 μM final protein concentration (20 mM aq PBS, 2 mM DTT, pH 7.4) in the presence of 10 μM ThT and 5 μM high-mw heparin sulfate. Cellular seeding of biosensor cells expressing tau-RD(LM)-YFP. HEK293 cells stably expressing tau-RD (LM)-YFP were cultured in DMEM media containing 10% FBS, 1% antibiotic (penicillin + streptomycin), antimycotic (amphotericin B), and 1% GlutaMAX in a 75 cm2 cell culture flask under 5% CO2 at 37 °C. For each experiment, cells were plated at a density of about 15000 cells / well into a 96-well tissue culture plate. Mini-tau macrocycles or control peptides were pre-incubated for 24 h in an aggregation buffer containing either 20 mM aq PBS pH 7.4 or 20 mM aq Tris, 150 mM NaCl, pH 7.6 at 37 °C with a mixing speed of 150 rpm. Following incubation, the reaction mixture was agitated using sonication for 10 min, diluted in DMEM complete media and mixed with Lipofectamine 2000 in 20:1 ratio (complex:Lipofectamine) and allowed to incubate for an additional 20 min at rt. A final concentration of either 5µM, 1µM, 0.5µM, 0.1 µM, 0.01 µM, or 0.001 µM of mini-tau macrocycles or control peptide was added to the cells. Cells were incubated for additional 48 h before taking measurements on a BioTek Cytation 5 cell imager and microplate reader. 10 × 10 pictures / well were taken at 20× magnification under FITC channel. Punctate counting was carried out using an automated procedure selecting for 5-100 µm punctate with GFP fluorescence readings above 20,000. Each data set was collected from technical replicates on at least two different days. Every experiment included control wells (vehicle and cells treated with fibrillar recombinant tauP301L). Error bars represent standard deviation from technical replicates. Cellular seeding by mini-tau macrocycles was limited by removing initial incubation in aggregation buffer, sonication, or coincubation with Lipofectamine. X-ray fiber diffraction. Lyophilized peptides were dissolved in DMSO to 10 mM and allowed to incubate in a microcentrifuge tube for 4 d at 37 °C with a mixing speed of 150 rpm. Peptides solutions were diluted 25-fold with unbuffered water, frozen, and lyophilized. Dried peptide was suspended in 10 µL of unbuffered water. An 8 μL aliquot of peptide was applied to a 30 x 300 µm MicroLoops E (MiTeGen) and dried overnight on a wax-tipped capillary system.2 Diffraction data were recorded on a Bruker Venture equipped with a CuDiamond microfocus X-ray source and Photon-III CPAD area detector. Frames were measured for 60 s at 70 mm and analyzed with the Bruker APEX-4 software suite. Primary murine neuron seeding with mini-tau macrocycles. Primary cultures and immunocytochemistry were carried out as described previously. (Hallinan et al., The Journal of Neuroscience 39, 9623-9632 (2019)) Briefly, hippocampi were dissected from embryonic day 15 embryos of MAPT(H2.1)-GR mice (JAX stock #033668). Dissociated neurons were plated in neurobasal medium supplemented with 2% B27 and 0.5 mM GlutaMAX (Invitrogen) on 12 mm glass coverslips that were pre-treated with 0.1 mg / mL poly-D- lysine. On day in vitro (DIV) 7, 500 nM of KD-mxyl, linear-Ala, or scrm-mxyl (pre-incubated as described for biosensor cell seeding assays) was mixed with lipofectamine 2000 and added to the neurons. Neurons were incubated with peptides until DIV12, at which point the neurons were fixed in 4% paraformaldehyde in 20% sucrose in PBS. Neurons were blocked in 10% goat serum, incubated overnight at 4 °C in anti-phosphoTau (Ser202 / Thr205) antibody (AT8, Thermo Fisher, 1:1000), followed by incubation in a fluorescently-conjugated secondary antibody (AlexaFluor 555, Invitrogen, 1:5000). Neurons were imaged on a BioTek Cytation C10 Confocal Imaging Reader (Agilent). Cryo-EM fibril preparation. KD-mxyl was incubated for 24 h in an aggregation buffer containing 20 mM aq Tris, 150 mM NaCl, pH 7.6 at 37 °C with a mixing speed of 150 rpm resulting in a 20mM KD-mxyl aggregated solution used for all cryo-EM studies. For negative staining, Lacey grid coated with continuous ultrathin carbon film (TedPella) were glow discharged with a PELCO easiGlow instrument for 60 s, then 3 µl of aggregated KD-mxyl was applied to the grid and incubated for 2 min. The excess solution was blotted away with filter paper and the grid was washed 3 times with 10 µL water and stained 2 times with 5 µL 2% (w / v) uranyl acetate. The excess uranyl acetate was blotted away, and the grid was dried in air for 4–5 min. The grid was examined in a Tecnai-12120 kV electron microscope. Cryo-EM data collection and image processing. KD-mxyl fibrils prepared as described above were diluted to 2 mM with 20 mM HEPES pH 7.5 before preparing grids. 3 μL of 20 mM KD- mxyl was applied to glow-discharged UltrAuFoil® grid in FEI Vitrobot Mark IV, then the excess sample was blotted with filter paper, and the grid was plunged into liquid ethane. All datasets were collected with a Titan Krios G4300 kV transmission electron microscope equipped with a post- column Gatan Quantum GIF energy filter and a Gatan K3 direct electron detector. Movie stacks were recorded using the EPU software at a nominal magnification of 105K in super-resolution mode but binned by a factor of 2 to have a calibrated pixel size of 0.822 Å at the specimen level. The slit width of energy filter was set to 20 eV. A total dose of 59.495 e / Å was fractionated into 50 frames with a defocus range set between -0.6 µm and -2 µm. All data processing was performed within the RELION 4.0. (Zivanov et al., A Bayesian approach to single-particle electron cryo-tomography in RELION-4.0. Elife 11 (2022).) The movie frames were aligned using MotionCor246 algorithm re-implemented in RELION and CTF parameters were fit using CTFFIND-4.1. (Rohou et al., J Struct Biol 192, 216-221 (2015)) Particles were picked manually using RELION helical picker as end-to-end line segments, then the filament segments were extracted using a box size of 768 pixels and an inter-box distance of 14.25 Å. The particle box was down scaled to 192 pixels to speed up analysis. 3 rounds of reference-free 2D classification were performed using a regularization value of T=2. Bad classes were discarded and the classes with different morphologies were selected and processed separately. The crossovers of the classes were measured using e2display.py in EMAN2. (Ludtke et al., J Struct Biol 128, 82-97 (1999)). Initial models for 3D classification were reconstructed from 2D class averages of segments.3D classification was performed with a regularization parameter of T= 8. The 3D classes with the clearest structural features, e.g. strands separation along the helical axis (Z-axis) and peptide backbone in X-Y plane, were selected. The particles of these classes were re-extracted using a box size of 320 pixels without rescaling. Another round of 3D classification with the re- extracted particles was used to sort out the classes with the clearest β-sheets (x−y plane) and peptide backbone structural features. The final selected segments were used for 3D auto-refinement. The first round of 3D auto-refinement was performed without reference mask or local searches of symmetry. The second round of 3D auto-refinement optimized the helical twist and rise. The FSC curves using half maps were calculated with trueFSC.py available in JSPR software. (Sun et al., Prog Biophys Mol Biol 160, 37-42 (2021)). The sharpened and masked 3D maps were used to manually build the atomic models using Coot. (Emsley et al., Acta Crystallogr D Biol Crystallogr 66, 486-501 (2010)). A stack of five layers was assembled and refined using phenix.real_space_refine in PHENIX. The non-crystallographic symmetry (NCS) restraints based on the helical symmetry were used on all the chains. The steric clashes, Ramachandran and rotamer outliers were iteratively corrected manually in Coot followed by further refinement using PHENIX (Adams et al., Acta Crystallogr D Biol Crystallogr 66, 213-221 (2010)). All publications, patents, and patent documents cited herein are incorporated by reference as though individually incorporated by reference, and in particular, Angera et al. Structure-based design of seed-competent proteomimetic macrocycles derived from 4R tauopathic folds. ChemRxiv. 2024; doi:10.26434 / chemrxiv-2024-l7zgc; Makawana et al., ACS Chem. Neurosci. 2021, 12, 20, 3928–3938; Rajewski et al., J Am Chem Soc. 2023 Oct 25;145(42):23131-23142. doi: 10.1021 / jacs.3c06830; U.S. Patent Publication No. 2023 / 0399361 to Del Valle et al., and U.S. Patent Publication No. 2020 / 0079867 to Watkins et al. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, many variations and modifications may be made while remaining within the spirit and scope of the invention. While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

Claims

What is claimed is:

1. A tau proteomimetic comprising: a cyclic peptide comprising an amino acid sequence NIKHVPGGGSVQIVY (SEQ ID NO: 1) or VPGGGCVQIVYK (SEQ ID NO: 2), wherein two amino acids are substituted with cysteine residues, or cysteine residues are positioned at both an amino terminus and a carboxy terminus of the cyclic peptide; a di-cysteine linker moiety covalently linking sulfur atoms of the cysteine residues to form the cyclic peptide; and wherein the N-terminus of the cyclic peptide is acetylated and the C-terminus of the cyclic peptide is amidated.

2. The tau proteomimetic of claim 1, wherein the amino acid sequence comprises VPGGGCVQIVYK (SEQ ID NO: 2).

3. The tau proteomimetic of claim 2, wherein the cyclic peptide is DNICVPGGGCVQIVYK (SEQ ID NO: 16). 4, The tau proteomimetic of claim 1, wherein the amino acid sequence comprises NIKHVPGGGSVQIVY (SEQ ID NO: 1).

5. The tau proteomimetic of claim 4, wherein the amino acid sequence comprises DNIKHVPGGGSVQIVYK (SEQ ID NO: 3).

6. The tau proteomimetic of claim 4, wherein the amino acid sequence comprises DNIKHVPGGGSVQIVYKPV (SEQ ID NO: 4).

7. The tau proteomimetic of claim 4, wherein the amino acid sequence comprises KDNIKHVPGGGSVQIVYKPVD (SEQ ID NO: 5).

8. The tau proteomimetic of claim 4, wherein the amino acid sequence comprises GSKDNIKHVPGGGSVQIVYKPVD (SEQ ID NO: 6).

9. The tau proteomimetic of claim 4, wherein the cyclic peptide is: CNIKHVPGGGSVQIVYC (SEQ ID NO: 7); DNIKCVPGGGCVQIVYK (SEQ ID NO: 8);CDNIKHVPGGGSVQIVYKPVC (SEQ ID NO: 9); KDNIKCVPGGGSVQIVCKPVD (SEQ ID NO: 10); GSKDNIKCVPGGGSVCIVYKPV (SEQ ID NO: 11); GSKDNIKHVCGGGCVQIVYKPV (SEQ ID NO: 12); GSKDNIKHVCGGGSVQCVYKPV (SEQ ID NO: 13); KDNICHCPGGGSVQIVYKPVD (SEQ ID NO: 14); or KDCICHVPGGGSVQIVYKPVD (SEQ ID NO: 15).

10. The tau proteomimetic of claim 1, wherein the di-cysteine linker moiety is ,11. The tau proteomimetic of claim 10, wherein the di-cysteine linker moiety is12. The tau proteomimetic of claim 1, wherein the cyclic peptide is:45or a salt or solvate thereof.

13. A peptide comprising an amino acid sequence of: CNIKHVPGGGSVQIVYC (SEQ ID NO: 7); DNIKCVPGGGCVQIVYK (SEQ ID NO: 8); CDNIKHVPGGGSVQIVYKPVC (SEQ ID NO: 9); KDNIKCVPGGGSVQIVCKPVD (SEQ ID NO: 10); GSKDNIKCVPGGGSVCIVYKPV (SEQ ID NO: 11); GSKDNIKHVCGGGCVQIVYKPV (SEQ ID NO: 12); GSKDNIKHVCGGGSVQCVYKPV (SEQ ID NO: 13) KDNICHCPGGGSVQIVYKPVD (SEQ ID NO: 14); KDCICHVPGGGSVQIVYKPVD (SEQ ID NO: 15); or DNICVPGGGCVQIVYK (SEQ ID NO: 16), or a salt or solvate thereof; wherein an N-terminus of the peptide is acetylated and a C-terminus of the peptide is amidated.

14. The peptide of claim 13, wherein the peptide further comprises a di-cysteine linker moiety covalently linking sulfur atoms of the cysteine residues to form a cyclic peptide.

15. The peptide of claim 14, wherein the di-cysteine linker moiety is

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