WO2020232440A1 - Inhibiteurs à base de peptides qui bloquent l'agrégation, l'ensemencement et l'ensemencement croisé de protéine bêta-amyloïde et de protéine tau - Google Patents

Inhibiteurs à base de peptides qui bloquent l'agrégation, l'ensemencement et l'ensemencement croisé de protéine bêta-amyloïde et de protéine tau Download PDF

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WO2020232440A1
WO2020232440A1 PCT/US2020/033426 US2020033426W WO2020232440A1 WO 2020232440 A1 WO2020232440 A1 WO 2020232440A1 US 2020033426 W US2020033426 W US 2020033426W WO 2020232440 A1 WO2020232440 A1 WO 2020232440A1
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tau
seq
peptide
aggregation
seeding
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PCT/US2020/033426
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English (en)
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David S. Eisenberg
Paul M. SEIDLER
Sarah GRINER
Kevin Murray
David R. Boyer
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The Regents Of The University Of California
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • the invention relates to compositions and methods useful in inhibiting aggregation of Tau and amyloid b proteins.
  • AD Alzheimer’s disease
  • Ab amyloid beta
  • tau intracellular neurofibrillary tangles of tau (1, 2). While Ab aggregation is thought to occur at the early stages of AD, tau aggregation correlates better to disease progression, with characteristic spreading along linked brain areas, and severity of symptoms correlating to the number of observed inclusions (3–8). Structural information about the aggregated forms of Ab and tau is accumulating, but to date this knowledge has not led to successful chemical interventions (9).
  • AD tissue extracts 5, 22.
  • soluble complexes of Ab and tau have been found to promote aggregation of tau (22), while another study found that Ab fibrils can seed tau (23).
  • AD pathology is characterized by plaques of amyloid beta (Ab) and neurofibrillary tangles of tau.
  • Ab aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline.
  • peptide-based inhibitors that reduce Ab and/or tau aggregation and toxicity of already-aggregated species, and methods of making and using these peptide-based inhibitors.
  • Embodiments of the invention include compositions of matter comprising a peptide inhibitor of Ab aggregation disclosed herein.
  • the peptide inhibitor comprises an amino acid sequence LYIWVQ (SEQ ID NO: 3), LYIWIQMQ (SEQ ID NO: 4), LYIWIWRT (SEQ ID NO: 5), LYIWIWFS (SEQ ID NO: 6), LYIWIQKT (SEQ ID NO: 7), MYIWVQ, MYIWRQ (SEQ ID NO: 9) or MLIVRN (SEQ ID NO: 10), wherein at least one amino acid is a D-amino acid.
  • Embodiments of the invention also include a method of inhibiting aggregation of Ab polypeptides by combining Ab polypeptides with one or more peptide inhibitors of Ab aggregation disclosed herein; and allowing the peptide(s) to bind to the Ab polypeptides; so that Ab aggregation is inhibited.
  • a peptide used in the method is selected for an ability to inhibit cross- seeding of tau by Ab polypeptides; and/or inhibit tau homotypic seeding.
  • the peptide is combined with Ab and/or Tau in vivo.
  • Certain embodiments of the invention include therapeutic methods of inhibiting formation of Ab aggregates in vivo inhibits development or progression of a Ab plaque formation in an individual; and/or inhibiting homotypic seeding of Tau fibrils in vivo inhibits development or progression of a tauopathy in an individual.
  • Yet another embodiment of the invention is a composition of matter comprising a peptide inhibitor of tau aggregation that is disclosed herein.
  • the peptide inhibitor comprises an amino acid sequence: S-V-W-I-V-Y-E (SEQ ID NO: 11), S-V-Q-W-V-Y-E (SEQ ID NO: 12), S-V-Q-I-W-Y-E (SEQ ID NO: 13), S- V-W-I-W-Y-E (SEQ ID NO: 14), D-V-W-I-I-N-K-K-L-K (SEQ ID NO: 15), D-V-Q- M-I-N-K-K-L-K (SEQ ID NO: 16), D-V-Q-I-I-N-K-K-R-K (SEQ ID NO: 17) or D- V-W-M-I-N-K-K-R-K (SEQ ID NO: 18).
  • compositions further comprise a Tau polypeptide (SEQ ID NO: 1).
  • a related composition of matter comprises a polynucleotide encoding a peptide inhibitor of tau (or Ab) aggregation such as a peptide inhibitor comprising an amino acid sequence: S-V-W-I-V-Y-E (SEQ ID NO: 11), S-V-Q-W-V-Y-E (SEQ ID NO: 12), S-V-Q-I-W-Y-E (SEQ ID NO: 13), S-V-W-I-W-Y-E (SEQ ID NO: 14), D-V-W-I-I-N-K-K-L-K (SEQ ID NO: 15), D-V-Q-M-I-N-K-K-L-K (SEQ ID NO: 16), D-V-Q-I-I-N-K-K-R-K (SEQ ID NO: 17) or D-V-W-M-I-N-K-K-R-K
  • Yet another embodiment of the invention is a method of inhibiting formation of tau aggregation, the method comprising combining tau polypeptides with one or more peptide inhibitors of tau aggregation that are disclosed herein; and then allowing the peptide(s) to bind to the tau polypeptides, so that tau aggregation is inhibited.
  • the peptide(s) used in the method is selected for an ability to inhibit seeding of purified tau fibrils and/or inhibit seeding of tau fibrils present in unpurified or partially purified brain extracts.
  • the peptide is combined with Tau in vivo (e.g. in therapeutic methods designed to inhibit development or progression of a tauopathy in an individual).
  • an inhibitory peptide disclosed herein is coupled to a plurality of heterologous amino acids.
  • the peptide is fused, optionally via a linker sequence, to a plurality of heterologous amino acids comprising a cell penetrating peptide (CPP).
  • the plurality of heterologous amino acids e.g. a CPP
  • the CPP comprises a plurality of arginine residues, for example 4 to 16 contiguous arginine residues.
  • the peptide comprises a non-naturally occurring amino acid such as a D amino acid.
  • the composition comprises a cocktail of different peptide inhibitors such as a combination of the peptide inhibitors of Ab aggregation that are disclosed herein, a combination of the peptide inhibitors of tau aggregation that are disclosed herein, or a combination of the peptide inhibitors of Ab aggregation and the peptide inhibitors of tau aggregation that are disclosed herein.
  • a first and a second (and a third etc.) peptide within the composition are selected to effect the ability of the composition to inhibit one or more discreet phenomena observed in pathological protein aggregation (e.g. inhibition of homotypic seeding of protein fibrils).
  • Figures 1A-1C provide disclosure on the microED structure of segment Ab 16-26 D23N from microcrystals.
  • Figure 1A shows an electron micrograph of 3D crystals used for data collection, scale bar is 1mm.
  • Figure 1B provides a schematic showing the crystal structure reveals tightly mated pairs of anti-parallel b-sheets with opposing sheets in grey and cyan. The side-chains interdigitate to form a dry interface. Two neighboring sheets are viewed perpendicular to the b-sheets.
  • Figure 1C provides a view of 6 layers perpendicular to the fibril axis (black line). The b-sheets stack out of register along the fibril axis.
  • Figures 2A-2E provide disclosure on the development of inhibitors of Ab fibril formation using structure-based design against Ab 16-26 D23N.
  • Figure 2B and 2C provide schematics of the segment KLVFFAEN (SEQ ID NO: 25), derived from the Ab 16-26 D23N crystal structure, that was used as the design target.
  • Smaller hydrophobic residues of D1 mimic interactions with the fibril interface on one side of the peptide ( Figure 2B), whereas the other side of the peptide positions large aromatic residues between Ab residues, breaking possible further interactions (Figure 2C).
  • Figure 2D provides an overview of peptide inhibitors in D and L amino acid conformations, as indicated, used in this study and their sequences.
  • Peptide LC is the L-form cognate peptide of D-form peptide D1 and is the negative control for peptide inhibitor D1 and its derivatives D1b and D1d. IC50 values were determined using 4 parameter nonlinear fit. N.D., not determined.
  • Figures 3A-3C provide data showing that designed inhibitors reduce aggregation of Ab1-42.
  • Figure 3A provides data showing how peptide inhibitors D1, D1b, and D1d reduce fibril formation of Ab1-42, while negative control peptide LC does not. 10 mM of Ab1-42 was incubated alone or at a 1:10, 1:1, or 10:1 molar ratio to each inhibitor under quiescent conditions at 37 °C. Fibril formation was monitored using ThT fluorescence. Curves show the average of three technical replicates with one standard deviation below.
  • Figure 3B provides data showing a negative-stain TEM analysis that confirms the results of the ThT assays in Figure 3A. Samples were prepared as above and incubated for 72 hours before TEM analysis.
  • Figures 4A-4C provide data showing that inhibitors bind and block toxicity of aggregated Ab1-42.
  • Figures 4B and 4C provides data showing that inhibitors bind to Ab1-42 fibrils.
  • Figure 4B provides data showing that peptide inhibitors do not disaggregate Ab. 10 mM Ab1-42 was incubated alone for 72 hours at 37 °. Peptide inhibitors were added at 10-fold molar excess and incubated at RT for 24 hours before TEM analysis. Images were captured at 24,000; scale bars are 500 nm.
  • Figures 5A-5E provide data showing that tau aggregation is seeded by Ab and reduced by structure-based inhibitors.
  • Figure 5A shows disclosure where 20 mM tau40 was seeded with 10% monomer equivalent of pre-formed fibrils of Ab1-42 or tau-K18m under shaking conditions at 700 RPM at 37 °C in buffer containing 0.5mg/ml heparin. Fibril formation was monitored using ThT fluorescence. Error bars show the standard deviation of the average of three technical replicates.
  • Figure 5B shows disclosure where the number of intracellular aggregates present in tau-K18CY biosensor cells normalized to cell confluence seeded by the addition of 250 nM tau40 or 250 nM Ab1-42 fibrils.
  • Figure 5C shows representative images of seeded cells from B at 10x magnification, scale bar 100 mm.
  • Figures 5D and 5E show concentration dependent inhibition of Ab1-42 induced seeding of tau aggregation in tau-K18CY biosensor cells.
  • Figure 5D shows disclosure of an average by Ab as a function of indicated inhibitor concentration.
  • Figures 6A-6D provide disclosure showing that Ab inhibitors also reduce fibril formation and seeding by tau40.
  • Figure 6A shows disclosure on how peptide inhibitors D1, D1b, and D1d reduce fibril formation of tau40. 10 mM tau40 monomer was incubated at a 1:10, 1:1, or 10:1 molar ratio to each inhibitor with 0.5mg/ml heparin under shaking conditions at 700 RPM at 37 oC. Fibril formation was monitored using ThT fluorescence. Plots show the average of three technical replicates with one standard deviation below.
  • Figure 6B and 6C show disclosure on the effects of the inhibitors on seeding by tau40 fibrils in tau-K18CY biosensor cells.
  • Figure 6C shows representative images of effect of D1b on seeding.
  • Figures 7A and 7B show that peptide inhibitors reduce seeding by crude brain-extract from tauopathy donor tissue.
  • Brain lysate was prepared in TBS buffer from 3 brain regions of one AD patient, and from a one sample of a PSP patient lacking Ab plaques. Cells were seeded with a 1/400 dilution of brain tissue lysate; for samples with inhibitor, lysates were incubated with inhibitor overnight prior to addition to cells. A concentration of 10 mM peptide was used for all of the experiments shown.
  • Figure 7A shows disclosure on the average number of aggregates seeded by lysate from each respective brain region, with or without addition of inhibitors.
  • Figure 7B shows representative images of seeded biosensor cells from Figure 7A shown at 10X magnification, scale bar 100 mm.
  • Figure 8 provides a schematic showing the crystal packing of the Ab 16-26 D23N atomic structure. View down the‘a’ axis of the unit cell, outlined in red.
  • Figures 9A-9C show extended toxicity data from peptide inhibitors.
  • Figures 9A and 9B provide data showing that peptide inhibitors are not toxic. 100 mM of each peptide inhibitor was incubated for 12 hours at 37 °C and then diluted 1:10 with pre-plated N2a cells. Cytotoxicity was quantified using MTT dye reduction. Bars represent mean with individual technical replicates.
  • Figures 10A-10C provide data showing that peptide inhibitors D1a, D1c, D1e, and D1f are less effective ate reducing fibril formation of Ab1-42.
  • Figure 10A shows data from studies where 10 mM of Ab1-42 was incubated alone or at a 1:10, 1:1, or 10:1 molar ratio to each inhibitor under quiescent conditions at 37 °C. Fibril formation was monitored using ThT fluorescence. Lines show the average of three technical replicates with one standard deviation below. Inhibitors D1a and D1c are not shown at (1:10) ratio due to high ThT signal from peptide self-association.
  • Figure 10B shows disclosure where inhibitors D1b and D1d self-associate at high concentrations.
  • Peptide inhibitors were incubated at 10 mM and 100 mM under quiescent conditions at 37 °C. Fibril formation was monitored using ThT fluorescence. Lines show the average of three technical replicates with one standard deviation.
  • Figure 10C shows unspliced dot blots from Figure 4C. Column key displayed at lower right is representative of all displayed blots.
  • Figure 11 shows a representative sensorgram obtained when D1d solutions at the indicated concentrations were flowed across the Ab1-42 sensor chip.
  • Figures 12A-12C shows disclosure from tau seeding experiments.
  • Figure 12A shows extended ThT data of Tau seeding experiment. K18 and Ab1-42 seeds have some baseline ThT signal.
  • Figure 12B shows disclosure where 10 mM Ab1-42 was seeded with 10% monomer equivalent of pre-formed seed of Ab1-42 or K18, under quiescent conditions at 37 °C. Fibril formation was monitored using ThT fluorescence. Lines show average of three technical replicates.
  • Figure 12C shows disclosure examining the relative seeding efficiency of tau40 and other amyloid fibrils into the HEK293 biosensor that stably expresses K18 yellow fluorescent protein (YFP) fusion. The cells were seeded with 250 nM final concentration of amyloid fibril.
  • YFP yellow fluorescent protein
  • Amyloid fibrils were confirmed by endpoint ThT or Electron Microscopy.
  • Figures 13A-13F shows disclosure from tau fibril experiments.
  • Figure 13A shows that a control peptide inhibitor LC does not reduce fibril formation of tau40. 10 mM of tau40 was incubated at a 1:10 molar ratio to LC with 0.5mg/ml heparin under shaking conditions at 700 RPM at 37 °C. Fibril formation was monitored using ThT fluorescence. Lines show the average of three technical replicates with one standard deviation below. esigned inhibitors are not general amyloid inhibitors.
  • Figure 13B shows data where 50 mM of a-synuclein was incubated at a 1:5 molar ratio to peptide inhibitors under shaking conditions at 700 RPM at 37 °C.
  • Figures 13C-13F show extended data of seeding by tau interface mutation fibrils in tau-K18CY biosensor cells. The cells were seeded with 250 nM tau40 fiber (final concentration); in samples with inhibitor, tau40 fibers were incubated with indicated final concentrations of peptide inhibitor for one hour prior to addition to cells.
  • Figures 14A-14C show schematics of peptide inhibitor interactions.
  • the spines of Ab 16-26 D23N and tau are structurally similar.
  • Figures 14A-14B show Ab 16-26 D23N overlay with tau 274-283 in parallel and antiparallel orientations.
  • Figure 14A shows 32 backbone atoms differ from each other by 0.53 ⁇ RMSD. RMSD values were calculated using LSQ in Coot.
  • Figure 14B shows the C a atoms differ from each other by 0.56 ⁇ RMSD.
  • Backbone and side chain rotomers were optimized with Foldit over 2000 iterations to minimize energy to -603 REU.
  • Figure 14C shows Ab 16-22 overlay with tau 304-310 in parallel, backbone atoms differ from each other by 0.54 ⁇ RMSD.
  • FIGS 15A-15B show schematics of tau.
  • the VQIVYK (SEQ ID NO: 27) segment of tau harbors two aggregation-prone surfaces.
  • Figure 15A shows (Top) Schematic of full-length Tau showing the positions of the VQIINK (SEQ ID NO: 28) and VQIVYK (SEQ ID NO: 27) aggregation segments in the microtubule binding domain which contains four repeats (R1-4), together termed K18. Also shown are the domain boundaries of the cryoEM AD fibril core (13), and a modified K18 construct used in this work called K18+, which includes an 8 additional amino acids at the C- terminus to match the AD fibril core.
  • the vertical axis plots the energy of a steric zipper formed by a hexapeptide beginning with the residue above, and extending 5 additional amino acids to the right (N-to-C). Residues exceeding a predetermined threshold of - 23 kcal/mol are predicted to form steric zippers.
  • Figure 15B shows the crystal structure of the SVQIVY (SEQ ID NO: 29) steric zipper segment determined in this work.
  • the structure contains two different steric zippers formed on opposite faces of the peptide, a Class 1 interface that is created by the top two strands colored in tan and orange, and a Class 3 interface that is formed by the bottom two strands colored grey and orange.
  • a Class 1 interface that is created by the top two strands colored in tan and orange
  • a Class 3 interface that is formed by the bottom two strands colored grey and orange.
  • the buried surface area (Ab) and shape complementarity (Sc) are shown in inset boxes.
  • Figures 16A-16D show disclosure that provides evidence that the Class 3 steric zipper interface is involved in the formation of disease-relevant tau fibrils.
  • the Class 3 steric zipper interface maps to a region of unmodeled density in the tau PHF and SF.
  • Residues VQIVY from a single protomer chain of the steric zipper were aligned to the same sequence of the respective cryoEM structures, and show that the mated strand that forms the Class 3 interface lays in a patch of unmodeled density present in both the SF and PHF cryoEM maps. Numbering along the backbone corresponds to residue positions that were modified in the panel of capping inhibitors of Table 1.
  • Figure 16C shows seeding inhibition by VQIVYK (SEQ ID NO: 27) and VQIINK (SEQ ID NO: 28) capping inhibitors. Unlabeled fibrils of Tau40 were transfected into HEK293 biosensor cells that stably express P301S tau-K18 YFP.
  • FIG. 16D shows seeding following transient transfection of a PiggyBac vector encoding the WIW capping inhibitor peptide, or a scrambled peptide as a control, into tau biosensor cells.
  • Figures 17A-17E show disclosure from inhibition of seeding by AD- derived tau fibrils using the VQIVYK (SEQ ID NO: 27) and VQIINK (SEQ ID NO: 28) panel of capping inhibitors. Seeding in tau biosensor cells was induced by ( Figure 17A and Figure 17B) crude brain extract or (Figure 17C) fibrils purified by size exclusion chromatography from donors with AD. Seeding inhibition was measured by counting the number of fluorescent puncta as a function of inhibitor.
  • Figure 17D shows negative-stain electron micrograph of fibrils used for seeding in 17C.
  • Figure 17E shows representative images from seeding inhibition experiments from 17B (seeded with crude brain extract), and 17C (seeded with purified AD fibrils). Red arrows indicate representative cells containing seeded tau aggregates, and white arrows point to representative cells lacking aggregated tau.
  • Figures 18A-18F show disclosure from inhibitor profiling studies.
  • Figure 18(A) shows CTE-derived tau seeds from the temporal cortex
  • Figure 18(B) shows recombinant tau K18+ that was seeded with CTE-derived tau seeds
  • Figure 18(C) shows recombinant tau K18+ fibrils aggregated with heparin. Seeding inhibition measurements for the seeded recombinant fibrils from Figure 18B were carried out in tau biosensor cells after 3 sequential rounds of in vitro seeding. Seeding inhibition was measured by counting the number of fluorescent puncta as a function of inhibitor.
  • Figures 18A-C red arrows were used to mark capping inhibitors that were effective at blocking seeding by CTE-derived tau from crude brain extracts, blue arrows mark inhibitors effective at blocking seeding by recombinant tau fibrils, and a purple arrow marking IN-W3 in Figure 18B emphasizes that it is the only of the different inhibitors that blocks seeding by both CTE-derived tau and recombinant tau fibrils.
  • Figures 18D-18F show representative images from Figures 18A, 18B and 18C, respectively showing seeding and inhibition in tau biosensor cells. Red arrows indicate representative cells containing seeded tau aggregates, and white arrows point to representative cells lacking aggregated tau.
  • Figures 19A-19K show disclosure from inhibitor profiling studies. Inhibitor profiling in biosensor cells seeded by brain extract from 4 different PSP donors.
  • Figure 19A shows tissue sections from donors 1 and 2 were harvested from the midbrain, and from the locus coeruleus for donor 3. Seeding inhibition was measured by counting the number of fluorescent puncta as a function of inhibitor.
  • VQIINK (SEQ ID NO: 28) inhibitors showing greater than 70% inhibition are highlighted on bar graphs with a red outline.
  • Figure 19B shows seeding by extracts from PSP donors 1, 2 and 3 after treatment with the capping peptide W4.
  • Figure 19C shows representative images from a showing seeding and inhibition in tau biosensor cells.
  • Figure 19D and 19E show that, as in Figure 19A, except tissue sections came for a two different brain regions, the cerebellum (Figure 19D) or frontal cortex (Figure 19E), of a 4 th PSP donor.
  • Figures 19F-19H show representative images from Figure 19D.
  • Figures 19I- 19K show representative images from 19E.
  • Figure 20 provides a schematic summary of sensitivities to panel of capping peptides measured by seeding inhibition using extracts from donors with different tauopathies.
  • the top panel of cartoons shows locations of segments in tau targeted by different inhibitors of the panel, and crystal structures of corresponding interfaces.
  • the Table shows efficacies of respective inhibitors for each donor tested in this study. For this analysis, inhibitors were scored as effective (filled box) if seeding was inhibited by 50% or more. Otherwise inhibitors were scored ineffective (open box).
  • Figures 21A-21C provide photos and schematics of tau.
  • Figure 21A Left panel– microcrystals of SVQIVY (SEQ ID NO: 29) in hanging drop crystallization screens.
  • Middle panel Electron micrograph of SVQIVY (SEQ ID NO: 29) microcrystals (Scale bar 1 micron).
  • Right panel representative electron diffraction image from SVQIVY (SEQ ID NO: 29) microcrystals.
  • Figure 21B Refined atomic model for SVQIVY (SEQ ID NO: 29) shown with 2Fo-Fc map (grey) and Fo-Fc map (green and red).
  • Figure 21C Alignment of VQIVYK (SEQ ID NO: 27) (2ON9) and Class 1 interface of SVQIVY (SEQ ID NO: 29).
  • Figures 22A-22D provide schematics of tau.
  • Class 1 steric zipper structures formed by the VQIVYK (SEQ ID NO: 27) segment from ( Figure 22A) peptide crystal structure SVQIVY (SEQ ID NO: 29), and ( Figure 22C - Figure 22D) cryoEM fibril structures from PiD (6GX5) and AD (5O3L and 5O3T).
  • the VQIVYK (SEQ ID NO: 27) strand is colored yellow, and the mated sheet, which varies in sequence in the different structures, is colored magenta.
  • Figure 23 shows seeding inhibition in tau biosensor cells by titration of the D-peptide TLKIVW (SEQ ID NO: 30) into recombinant fibrils of tau40 prepared using heparin. No inhibition of seeding was observed up to a concentration of 50 mM peptide.
  • Figure 24 provides data from seeding inhibition studies with crude brain extract from two different donors (left and right panels) with AD. Seeding was measured by counting the number of fluorescent puncta in tau biosensor cells after transfecting with crude brain lysate, and seeding inhibition was determined by plotting the number of counted aggregates when crude brain lysate was pre-treated with the indicated capping inhibitor at a final concentration of 10 mM.
  • Figure 25 shows an inhibitor profile of tau biosensor cells seeded by crude brain extract from a donor with CBD. Seeding inhibition was measured by counting the number of fluorescent puncta as a function of inhibitor.
  • Figures 26A-26F provide data showing that seeding by soluble oligomers of tau are inhibited by SVQIVY (SEQ ID NO: 29)- and VQIINK (SEQ ID NO: 28)- based capping inhibitors.
  • Figure 26A shows shaking recombinant tau-K18 with ionic liquid 15 (Hampton Research) allows for slower aggregation compared to heparin.
  • Figure 26B and Figure 26C show endpoints of solutions aggregated with IL15 produce fibrils, as shown for the IL15 endpoint of (Figure 26B) Tau40 and (Figure 26C) tau-K18.
  • Figure 26D shows data from studies of aliquots removed between 16-20 hours for tau-K18 with IL15 were subjected to size exclusion chromatography on a superdex 200 column and result in 2 peaks.
  • Figure 26E shows the early eluting species has immunoreactivity to the oligomer antibody A11, whereas the late eluting species corresponding to the tau monomer lacks A11 immunoreactivity.
  • AD pathology is characterized by plaques of amyloid beta (Ab) and neurofibrillary tangles of tau.
  • Ab aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline.
  • peptide-based inhibitors that reduce Ab and/or tau aggregation and toxicity of already-aggregated species, and methods of using these peptide-based inhibitors.
  • Embodiments of the invention include, for example, a composition of matter comprising at least one peptide inhibitor of Ab aggregation wherein the at least one peptide inhibitor comprises an amino acid sequence LYIWVQ (SEQ ID NO: 3), LYIWIQMQ (SEQ ID NO: 4), LYIWIWRT (SEQ ID NO: 5), LYIWIWFS (SEQ ID NO: 6), LYIWIQKT (SEQ ID NO: 7), MYIWVQ, MYIWRQ (SEQ ID NO: 9) or MLIVRN (SEQ ID NO: 10); and the at least one peptide inhibitor comprises at least one D-amino acid.
  • the at least one peptide inhibitor comprises an amino acid sequence LYIWVQ (SEQ ID NO: 3), LYIWIQMQ (SEQ ID NO: 4), LYIWIWRT (SEQ ID NO: 5), LYIWIWFS (SEQ ID NO: 6), LYIWIQKT (SEQ ID NO
  • the composition comprises a plurality of peptide inhibitors of Ab aggregation.
  • the peptide inhibitor sequence comprises less than 15, 14, 13, 12, 11, 10, 9, 8 or 7 amino acids.
  • the peptide inhibitor sequence is coupled to a plurality of heterologous amino acids, for example a linker amino acid sequence and/or a cell penetrating peptide (CPP) amino acid sequence.
  • the composition further comprises an Ab polypeptide (SEQ ID NO: 2).
  • compositions of matter comprising a polynucleotide encoding at least one peptide inhibitor of Ab aggregation wherein the at least one peptide inhibitor comprises an amino acid sequence LYIWVQ (SEQ ID NO: 3), LYIWIQMQ (SEQ ID NO: 4), LYIWIWRT (SEQ ID NO: 5), LYIWIWFS (SEQ ID NO: 6), LYIWIQKT (SEQ ID NO: 7), MYIWVQ, MYIWRQ (SEQ ID NO: 9) or MLIVRN (SEQ ID NO: 10).
  • these polynucleotides are disposed within a vector selected for its ability to express the peptide inhibitor in a mammalian cell.
  • the composition comprises a vector disposed within a mammalian cell.
  • Another embodiment of the invention is a method of inhibiting aggregation of Ab polypeptides, the method comprising combining Ab polypeptides with at least one peptide inhibitor of Ab aggregation that is disclosed herein; and then allowing the at least one peptide inhibitor of Ab aggregation to bind to the Ab polypeptides, such that that Ab aggregation is inhibited.
  • the at least one peptide inhibitor of Ab aggregation used in the method is selected for an ability to inhibit cross-seeding of tau by Ab polypeptides; and/or to inhibit tau homotypic seeding.
  • the method uses a plurality of peptide inhibitors of Ab aggregation.
  • the at least one peptide inhibitor of Ab aggregation is combined with Ab and/or Tau in vivo.
  • inhibiting formation of Ab aggregates in vivo inhibits development or progression of a Ab plaque formation in an individual; and/or inhibiting homotypic seeding of Tau fibrils in vivo inhibits development or progression of a tauopathy in an individual.
  • composition of matter comprising at least one peptide inhibitor of tau aggregation wherein the at least one peptide inhibitor comprises an amino acid sequence: S-V-W-I-V-Y-E (SEQ ID NO: 11), S-V-Q-W-V-Y-E (SEQ ID NO: 12), S-V-Q-I-W-Y-E (SEQ ID NO: 13), S-V-W- I-W-Y-E (SEQ ID NO: 14), D-V-W-I-I-N-K-K-L-K (SEQ ID NO: 15), D-V-Q-M-I- N-K-K-L-K (SEQ ID NO: 16), D-V-Q-I-I-N-K-K-R-K (SEQ ID NO: 17) or D-V-W- M-I-N-K-K-R-K (SEQ ID NO: 18).
  • the composition comprises a plurality of peptide inhibitors of tau aggregation; and/or the composition comprises a pharmaceutical excipient selected from the group consisting of a preservative, a tonicity adjusting agent, a detergent, a viscosity adjusting agent, a sugar or a pH adjusting agent.
  • the peptide inhibitor sequence comprises less than 15, 14, 13, 12, 11, 10, 9, 8 or 7 amino acids.
  • the peptide inhibitor sequence is coupled to a plurality of heterologous amino acids, for example a linker and/or a cell penetrating peptide (CPP).
  • the composition further comprises a tau polypeptide (SEQ ID NO: 1).
  • Another embodiment of the invention is a composition of matter comprising a polynucleotide encoding a peptide inhibitor of tau aggregation wherein the peptide inhibitor comprises an amino acid sequence: S-V-W-I-V-Y-E (SEQ ID NO: 11), S-V- Q-W-V-Y-E (SEQ ID NO: 12), S-V-Q-I-W-Y-E (SEQ ID NO: 13), S-V-W-I-W-Y-E (SEQ ID NO: 14), D-V-W-I-I-N-K-K-L-K (SEQ ID NO: 15), D-V-Q-M-I-N-K-K-L- K (SEQ ID NO: 16), D-V-Q-I-I-N-K-K-R-K (SEQ ID NO: 17) or D-V-W-M-I-N-K- K-R-K (SEQ ID NO: 18).
  • the polynucleot SEQ
  • Yet another embodiment of the invention is a method of inhibiting aggregation of tau polypeptides comprising combining tau polypeptides with a composition comprising at least one peptide inhibitor of tau aggregation that is disclosed herein; and then allowing the at least one peptide inhibitor of tau aggregation to bind to the tau polypeptides, such that tau aggregation is inhibited.
  • the at least one peptide inhibitor of tau aggregation used in the method is selected for an ability to inhibit seeding of purified tau fibrils and/or to inhibit seeding of tau fibrils present in unpurified brain extracts.
  • the at least one peptide inhibitor of tau aggregation is combined with Tau in vivo, for example in methods designed to inhibit seeding of Tau fibrils in vivo so as to inhibit development or progression of a tauopathy in an individual.
  • the method uses a plurality of peptide inhibitors of tau aggregation.
  • the at least one peptide inhibitor of tau aggregation is coupled to a plurality of heterologous amino acids.
  • a tau and/or Ab inhibitory peptide disclosed herein is coupled to heterologous amino acids such as a cell penetrating peptide (CPP) amino acid sequence, typically one less than 30 amino acids in length.
  • CPP cell penetrating peptide
  • the peptide is coupled to the heterologous amino acids by a peptide linker comprising 1-7 amino acids.
  • the heterologous amino acids forms a polycationic structure.
  • the heterologous amino acids forms an amphipathic structure.
  • Embodiments of the invention include peptides wherein the peptide comprises at least one D amino acid (e.g. a peptide comprising all D-amino acids).
  • Embodiments of the invention can further compare metabolic stability and efficacy of L- and D form peptide inhibitors.
  • Embodiments of the invention include coupling of any of a tau and/or Ab inhibitory peptides disclosed herein by chemical or biological (genetic) means to proteins or peptides to carry the peptide inhibitor across the blood-brain-barrier (BBB), as a therapy for neurodegeneration or movement disorder.
  • embodiments of the invention include coupling of any of a tau and/or Ab inhibitory peptides disclosed herein to a cell penetrating peptide and then coupling the resulting peptide by chemical or biological (genetic) means to proteins or peptides to carry the inhibitor across the blood-brain-barrier (BBB), as a therapy for neurodegeneration or movement disorder.
  • Embodiments of the invention also include coupling of any of a tau and/or Ab inhibitory peptides disclosed herein to small molecules that aid in the ability of the peptide inhibitors to cross the BBB and/or cell membranes.
  • Embodiments of the invention also include coupling of any of a tau and/or Ab inhibitory peptides disclosed herein to enzymes or small molecules (e.g. fluorophores) to aid in the diagnosis of pathology (ante and/or postmortum).
  • Embodiments of the invention also include coupling of any of a tau and/or Ab inhibitory peptides disclosed herein to small molecules that aid in the ability of the peptide inhibitors to degrade amyloid aggregates.
  • Embodiments of the invention also include coupling of any of a tau and/or Ab inhibitory peptides disclosed herein by chemical means to a nano-particle capable of crossing the BBB and possibly also entering into cells, for example for use as a therapy for neurodegenation or movement disorder.
  • Embodiments of the invention also include coupling of any of a tau and/or Ab inhibitory peptides disclosed herein by chemical means to a metal-containing nano-particle capable of crossing the BBB to create a diagnostic/biomarker for MRI or PET diagnosis of neurodegenerative disease or movement disorders (e.g. Parkinson's disease).
  • Embodiments of the invention also include insertion of any of a tau and/or Ab inhibitory peptides disclosed herein into the CDRs of an antibody to produce inhibitors of greater potency and/or specificity.
  • the antibody can be a full antibody, and Fab domain, or a single-chain antibody. Coupling of the resulting antibody to a nano-particle provides embodiments of the invention useful for therapy or diagnosis as noted above.
  • An inhibitory peptide or CPP inhibitor of the invention can be synthesized (e.g., chemically or by recombinant expression in a suitable host cell) by any of a variety of art-recognized methods.
  • a practitioner can, for example, using conventional techniques, generate nucleic acid (e.g., DNA) encoding the peptide and insert it into an expression vector, in which the sequence is under the control of an expression control sequence such as a promoter or an enhancer, which can then direct the synthesis of the peptide.
  • Suitable expression vectors e.g., plasmid vectors, viral, including phage, vectors, artificial vectors, yeast vectors, eukaryotic vectors, etc.
  • capping inhibitor peptides are loaded into adeno-associated virus (AAV) capsids and hydrogel-based polymers to mediate delivery across the blood brain barrier
  • One aspect of the invention is a method for reducing or inhibiting Tau aggregation, comprising contacting Tau amyloid protofilaments with an effective amount of one or more of the inhibitory peptides or CPP inhibitors of the invention. Such a method can be carried out in vitro (in solution) or in vivo (e.g. cells in culture or in a subject).
  • Another aspect of the invention is a method for restoring the conformation of a Tau protein molecule having an aberrant conformation.
  • An “aberrant conformation,” as used herein, refers to a conformation which is different from the wild type conformation, and which results in a loss of function of the molecule. Such aberrant conformation is sometimes referred to herein as pathological conformation.
  • the aberrant conformation can take the form of amyloid aggregates or fibers (fibrils) of Tau molecules with other Tau molecules or with other proteins.
  • the aberrant conformation can take the form of misfolding (e.g., partial or complete unfolding) of the Tau protein due to mutations or other factors.
  • the Tau molecule having the aberrant conformation is contacted with an effective amount of an inhibitory peptide or a CPP inhibitor of the invention.
  • the contacted Tau molecule has a restored conformation, and exhibits a restored or reactivated biological or biochemical activity.
  • Another aspect of the invention is a method for reducing or inhibiting Ab aggregation, comprising contacting Ab proteins with an effective amount of one or more of the inhibitory peptides or CPP inhibitors of the invention. Such a method can be carried out in vitro (in solution) or in vivo (e.g. cells in culture or in a subject).
  • Another aspect of the invention is a method for restoring the conformation of a Ab protein molecule having an aberrant conformation.
  • An“aberrant conformation,” as used herein, refers to a conformation which is different from the wild type conformation, and which results in a loss of function of the molecule. Such aberrant conformation is sometimes referred to herein as pathological conformation.
  • the aberrant conformation can take the form of amyloid aggregates or fibers (fibrils) of Ab molecules with other Ab molecules or with other proteins.
  • the aberrant conformation can take the form of misfolding (e.g., partial or complete unfolding) of the Ab protein due to mutations or other factors.
  • the Ab molecule having the aberrant conformation is contacted with an effective amount of an inhibitory peptide or a CPP inhibitor of the invention.
  • the contacted Ab molecule has a restored conformation, and exhibits a restored or reactivated biological or biochemical activity.
  • Another aspect of the invention is a method for reactivating or restoring a biological or biochemical activity (function) of Tau and/or Ab which results from aberrant conformation of the Tau and/or Ab proteins.
  • the method comprises contacting the Tau and/or Ab protein molecule having an aberrant conformation with an effective amount of an inhibitor peptide or CPP inhibitor of the invention.
  • an inhibitor peptide or CPP inhibitor of the invention As a result of contacting the Tau and/or Ab protein having the aberrant conformation, the lost biological or biochemical activity of the Tau and/or Ab molecule is reactivated or restored.
  • Another aspect of the invention is a method for inhibiting or preventing a loss of a biological or biochemical activity (function), of a Tau and/or Ab protein which results from aberrant conformation of the Tau and/or Ab protein.
  • the method comprises contacting the Tau and/or Ab protein molecule having an aberrant conformation with an effective amount of an inhibitor peptide or CPP inhibitor of the invention.
  • an inhibitor peptide or CPP inhibitor of the invention As a result of contacting the Tau and/or Ab protein having the aberrant conformation, the loss of activity of the Tau and/or Ab molecule is inhibited or prevented.
  • Another aspect of the invention is a method for treating a subject having a disease or condition which is mediated by loss of function of Tau and/or Ab, such as a pathological syndrome in which Tau and/or Ab has an abnormal conformation (e.g. is aggregated or misfolded). That is, the pathological syndrome is associated with Tau and/or Ab having an aberrant conformation.
  • the method comprises administering to the subject an effective amount of one or more peptides of the invention.
  • a cocktail of two of more of the peptides or CPP inhibitor peptides is used.
  • Yet another embodiment of the invention is a method of observing the presence or absence of Tau amyloid fibrils and/or Ab in a biological sample comprising combining a biological sample with a peptide disclosed herein that binds to Tau or Ab, allowing the peptide to bind to Tau amyloid fibrils and/or Ab that may be present in the biological sample, and then monitoring this combination for the presence of complexes formed between Tau amyloid fibrils and/or Ab and the peptide; wherein the presence of said complexes show the presence of Tau amyloid fibrils and/or Ab in the biological sample.
  • the presence of complexes formed between Tau amyloid fibrils and/or Ab and the peptide is monitored using a detectable label that is coupled to the peptide (e.g. a heterologous peptide tag).
  • a detectable label that is coupled to the peptide (e.g. a heterologous peptide tag).
  • the method is performed on a biological sample obtained from an individual suspected of suffering from a tauopathy.
  • Such embodiments of the invention can be used, for example, in diagnostic methods designed to observe the presence or status of Alzheimer’s disease, for example to detect disease beginnings before clinical symptoms, and to follow the effectiveness (or lack of effectiveness), of a therapeutic treatment.
  • Peptide inhibitors of the invention bind specifically (selectively, preferentially) to Tau and/or Ab rather than to unintended proteins.
  • the protein to which the peptide inhibitor binds may be, e.g., a monomer, small aggregate, oligomer, or fibril.
  • the binding can be 2 times, 5 times, 10 times, 100 times or 200 times stronger, or no binding at all can be detected to an unintended target.
  • Conventional methods can be used to determine the specificity of binding, such as e.g. competitive binding assays or other suitable analytic methods.
  • An“active variant” is a variant which retains at least one of the properties of the inhibitory peptides described herein (e.g., the ability to bind to Tau and/or Ab and/or to block, inhibit or prevent Ab or Tau fibrillation (aggregation) and/or Tau cytotoxicity).
  • Fibrilization refers to the formation of fiber or fibrils, such as amyloid fibrils.
  • Suitable active variants include peptidomimetic compounds (any compound containing non-peptidic structural elements that is capable of mimicking the biochemical and/or biological action(s) of a natural mimicked peptide), including, for example, those designed to mimic the structure and/or binding activity (such as, for example, hydrogen bonds and hydrophobic packing interactions) of the peptides according to the methods disclosed herein).
  • Inhibitory peptides of the invention, including active variants thereof, are sometimes referred to herein as“peptidic compounds” or“compounds.”
  • active variants of the inhibitory peptides are shortened by 1-3 (e.g., 1, 2 or 3) amino acids at either the N-terminus, the C-terminus, or both of the starting inhibitory peptide.
  • the active variants are lengthened (extended) by 1, 2, 3 or 4 amino acids at the C-terminal end of the starting inhibitory peptide, e.g. with amino acid residues at the position in which they occur in Tau and/or Ab.
  • amino acids other than the ones noted above are substituted. These amino acids can help protect the peptide inhibitors against proteolysis or otherwise stabilize the peptides, and/or contribute to desirable pharmacodynamic properties in other ways.
  • the non-natural amino acids allow an inhibitor to bind more tightly to the target because the side chains optimize hydrogen bonding and/or apolar interactions with it.
  • non- natural amino acids offer the opportunity of introducing detectable markers, such as strongly fluorescent markers which can be used, e.g., to measure values such as inhibition constants.
  • peptide mimetics such as, e.g., peptoids, beta amino acids, N-ethylated amino acids, and small molecule mimetics.
  • non-natural amino acids are substituted for amino acids in the sequence. More than 100 non-natural amino acids are commercially available. These include, for example,
  • Non-natural amino acids which can substitute for LEU are N-natural amino acids which can substitute for LEU:
  • Non-natural amino acids which can substitute for THR:
  • Non-natural amino acids which can substitute for ILE allo-Ile-OH 251316-98-0
  • Non-natural amino acids which can substitute for ARG:
  • Non-natural amino acids which can substitute for TYR:
  • Non-natural amino acids which can substitute for LYS:
  • an inhibitory peptide of the invention can comprise, e.g., L-amino acids, D- amino acids, other non-natural amino acids, or combinations thereof.
  • Active variants include molecules comprising various tags at the N-terminus or the C-terminus of the peptide (e.g. tags comprising a stretch of heterologous amino acids).
  • an inhibitory peptide of the invention can comprise as tags at its N-terminus and/or at its C-terminus: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more Lysine residues; 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more Arginine residues; 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more Glutamate residues; 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more Aspartate residues; combinations of these amino acid residues; or other polar tags that will be evident to a skilled worker.
  • Other active variants include mutations of the Tau and/or Ab sequence which increase affinity of the inhibitory peptides for Tau and/or Ab.
  • an inhibitory peptide of the invention is isolated or purified, using conventional techniques such as the methods described herein.
  • isolated is meant separated from components with which it is normally associated, e.g., components present after the peptide is synthesized.
  • An isolated peptide can be a cleavage product of a protein which contains the peptide sequence.
  • a “purified” inhibitory peptide can be, e.g., greater than 90%, 95%, 98% or 99% pure.
  • the peptide is fused to any of a variety of cell penetrating peptides (CPPs).
  • CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non- polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively.
  • a third class of CPP’s are the hydrophobic peptides, containing only apolar residues, with low net charge or have hydrophobic amino acid groups that are crucial for cellular uptake.
  • TatP59W GRKKRRQRRRPWQ (SEQ ID NO:25)
  • BMVGag(7-25) KMTRAQRRAAARRNRWTAR SEQ ID NO:24
  • Other represntative CPPs useful in embodiments of the invention are found, for example in WO 2018/005867, the contents of which are incorporated herein by refernce.
  • the CPP comprises a plurality of arginine residues (e.g. R1-16).
  • the length of the CPP is rather short, e.g. less than about 30 amino acids, in order to improve stability and pharmacodynamic properties once the molecule enters a cell.
  • the CPP is directly attached (fused) to a peptide of the invention.
  • Any of a variety of linkers can be used. The size of the linker can range, e.g., from 1-7 or even more amino acids (e.g., 1, 2, 3, 4, 5, 6 or 7 amino acids).
  • the inhibitory peptide can be detectably labeled.
  • Labeled peptides can be used, e.g., to better understand the mechanism of action and/or the cellular location of the inhibitory peptide.
  • Suitable labels which enable detection e.g., provide a detectable signal, or can be detected
  • Suitable detectable labels include, e.g., radioactive active agents, fluorescent labels, and the like. Methods for attaching such labels to a protein, or assays for detecting their presence and/or amount, are conventional and well-known.
  • An inhibitory peptide of the invention can be synthesized (e.g., chemically or by recombinant expression in a suitable host cell) by any of a variety of art- recognized methods.
  • a practitioner can, for example, using conventional techniques, generate nucleic acid (e.g., DNA) encoding the peptide and insert it into an expression vector, in which the sequence is under the control of an expression control sequence such as a promoter or an enhancer, which can then direct the synthesis of the peptide.
  • Suitable expression vectors e.g., plasmid vectors, viral, including phage, vectors, artificial vectors, yeast vectors, eukaryotic vectors, etc.
  • compositions comprising one or more of the inhibitory peptides and a pharmaceutically acceptable carrier.
  • the components of the pharmaceutical composition can be detectably labeled, e.g. with a radioactive or fluorescent label, or with a label, for example one that is suitable for detection by positron emission spectroscopy (PET) or magnetic resonance imaging (MRI).
  • PET positron emission spectroscopy
  • MRI magnetic resonance imaging
  • peptides of the invention can be coupled to a detectable label selected from the group consisting of a radioactive label, a radio- opaque label, a fluorescent dye, a fluorescent protein, a colorimetric label, and the like.
  • the inhibitory peptide is present in an effective amount for the desired purpose.
  • the compositions may contain preservatives and/or antimicrobial agents as well as pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like.
  • “Pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use.
  • “pharmaceutically acceptable salts” of a compound means salts that are pharmaceutically acceptable, as defined herein, and that possess the desired pharmacological activity of the parent compound.
  • polynucleotide encoding an inhibitory peptide of the invention.
  • the polynucleotide is operably linked to a regulatory control sequence (e.g., a promoter or an enhancer) to facilitate production of the encoded protein following introduction (e.g. by transfection) into a suitable cell.
  • a regulatory control sequence e.g., a promoter or an enhancer
  • Other embodiments include a cell comprising the expression vector; and a method of making an inhibitory peptide of the invention comprising cultivating the cell and harvesting the peptide thus generated.
  • kits for carrying out any of the methods described herein may comprise a suitable amount of an inhibitory peptide of the invention; reagents for generating the peptide; reagents for assays to measure their functions or activities; or the like.
  • Kits of the invention may comprise instructions for performing a method.
  • Other optional elements of a kit of the invention include suitable buffers, media components, or the like; a computer or computer-readable medium providing the structural representation of a crystal structure described herein; containers; or packaging materials.
  • Reagents for performing suitable controls may also be included.
  • the reagents of the kit can be in containers in which the reagents are stable, e.g., in lyophilized form or stabilized liquids.
  • the reagents may also be in single use form, e.g., in single reaction form for administering to a subject.
  • Characterization of candidate inhibitory peptides of the invention can be carried out by any of a variety of conventional methods.
  • the peptides can be assayed for the ability to reduce or inhibit Tau and/or Ab aggregation or cytotoxicity or cell-to-cell spread.
  • the assays can be carried out in vitro or in vivo. Suitable assays will be evident to a skilled worker; some suitable assays are described herein.
  • One aspect of the invention is a method for reducing or inhibiting Tau and/or Ab aggregation, comprising contacting Tau and/or Ab proteins with an effective amount of one or more of the inhibitory peptides of the invention.
  • Such a method can be carried out in solution or in a cell (e.g. cells in culture or in a subject).
  • Another aspect of the invention is a method for treating a subject having a disease or condition which is mediated by the presence of fibrillated Tau (sometimes referred to herein as a Tauopathy or a Tau-mediated disease or condition), comprising administering to the subject an effective amount of an inhibitory peptide or pharmaceutical composition of the invention.
  • diseases or conditions are, e.g., Alzheimer’s disease.
  • Another aspect of the invention is a method to prevent the onset of such diseases or conditions (e.g., Alzheimer’s disease), or to treat a subject in the early stages of such diseases or conditions, or that is developing such a disease or condition, in order to prevent or inhibit development of the condition or disease.
  • An inhibitory peptide or pharmaceutical composition of the invention is sometimes referred to herein as an“inhibitor.”
  • An“effective amount” of an inhibitor of the invention is an amount that can elicit a measurable amount of a desired outcome, e.g. inhibition of Tau and/or Ab aggregation or cytotoxicity; for a diagnostic assay, an amount that can detect a target of interest, such as an Tau and/or Ab aggregate; or in a method of treatment, an amount that can reduce or ameliorate, by a measurable amount, a symptom of the disease or condition that is being treated.
  • A“subject” can be any subject (patient) having aggregated (fibrillated) Tau and/or Ab molecules associated with a condition or disease which can be treated by a method of the present invention.
  • the subject has Alzheimer’s disease.
  • Typical subjects include vertebrates, such as mammals, including laboratory animals, dogs, cats, non-human primates and humans.
  • the inhibitors of the invention can be formulated as pharmaceutical compositions in a variety of forms adapted to the chosen route of administration, for example, orally, nasally, intraperitoneally, or parenterally, by intravenous, intramuscular, topical or subcutaneous routes, or by injection into tissue.
  • Suitable oral forms for administering the inhibitors include lozenges, troches, tablets, capsules, effervescent tablets, orally disintegrating tablets, floating tablets designed to increase gastric retention times, buccal patches, and sublingual tablets.
  • the inhibitors of the invention can be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier, or by inhalation or insufflation. They can be enclosed in coated or uncoated hard or soft shell gelatin capsules, can be compressed into tablets, or can be incorporated directly with the food of the patient's diet.
  • a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier, or by inhalation or insufflation.
  • the compounds can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • compositions suitable for administration to humans are meant to include, but is not limited to, those ingredients described in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed. (2006) (hereinafter Remington's).
  • the inhibitors can be combined with a fine inert powdered carrier and inhaled by the subject or insufflated.
  • Such compositions and preparations should contain at least 0.1% compounds.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of a given unit dosage form.
  • the tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring can be added.
  • a liquid carrier such as a vegetable oil or a polyethylene glycol.
  • a syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.
  • any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • the inhibitors can be incorporated into sustained-release preparations and devices.
  • the inhibitors can be incorporated into time release capsules, time release tablets, and time release pills.
  • the composition is administered using a dosage form selected from the group consisting of effervescent tablets, orally disintegrating tablets, floating tablets designed to increase gastric retention times, buccal patches, and sublingual tablets.
  • the inhibitors may also be administered intravenously or intraperitoneally by infusion or injection.
  • Solutions of the inhibitors can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the compounds which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • Sterile injectable solutions are prepared by incorporating the compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Other solid carriers include conventional nontoxic polymeric nanoparticles or microparticles.
  • Useful liquid carriers include water, alcohols or glycols or water/alcohol/glycol blends, in which the compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
  • the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
  • Useful dosages of the peptides or pharmaceutical compositions of the invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.
  • concentration of the compounds in a liquid composition such as a lotion
  • concentration in a semi-solid or solid composition such as a gel or a powder can be about 0.1-5% by weight, or about 0.5- 2.5% by weight.
  • Effective dosages and routes of administration of agents of the invention are conventional.
  • the exact amount (effective dose) of the agent will vary from subject to subject, depending on, for example, the species, age, weight and general or clinical condition of the subject, the severity or mechanism of any disorder being treated, the particular agent or vehicle used, the method and scheduling of administration, and the like.
  • a therapeutically effective dose can be determined empirically, by conventional procedures known to those of skill in the art. See, e.g., The Pharmacological Basis of Therapeutics, Goodman and Gilman, eds., Macmillan Publishing Co., New York.
  • an, effective dose can be estimated initially either in cell culture assays or in suitable animal models. The animal model may also be used to determine the appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutic dose can also be selected by analogy to dosages for comparable therapeutic agents.
  • Treatment may involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years.
  • a suitable dose will be in the range of from about 0.001 to about 100 mg/kg, e.g., from about 0.01 to about 100 mg/kg of body weight per day, such as above about 0.1 mg per kilogram, or in a range of from about 1 to about 10 mg per kilogram body weight of the recipient per day.
  • a suitable dose can be about 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 30 mg/kg of body weight per day.
  • the inhibitors are conveniently administered in unit dosage form; for example, containing 0.05 to 10000 mg, 0.5 to 10000 mg, 5 to 1000 mg, or about 100 mg of active ingredient per unit dosage form.
  • the dosage unit contains about 0.1 mg, about 0.5 mg, about 1 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, or about 100 mg, of active ingredient.
  • EXAMPLE 1 STRUCTURE BASED INHIBITORS OF AMYLOID BETA CORE SUGGEST A COMMON INTERFACE WITH TAU
  • AD pathology is characterized by plaques of amyloid beta (Ab) and neurofibrillary tangles of tau.
  • Ab aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline.
  • Ab core segment determined by MicroED and in it, note characteristics of both fibrillar and oligomeric structure.
  • peptide-based inhibitors that reduce Ab aggregation and toxicity of already-aggregated species.
  • these inhibitors reduce the efficiency of Ab-mediated tau aggregation, and moreover reduce aggregation and self-seeding of tau fibrils.
  • the ability of these inhibitors to interfere with both Ab and tau seeds suggests these fibrils share a common epitope, and supports the hypothesis that cross-seeding is one mechanism by which amyloid is linked to tau aggregation and could promote cognitive decline.
  • AD Alzheimer’s disease
  • Ab amyloid beta
  • tau intracellular neurofibrillary tangles of tau (1, 2). While Ab aggregation is thought to occur at the early stages of AD, tau aggregation correlates better to disease progression, with characteristic spreading along linked brain areas, and severity of symptoms correlating to the number of observed inclusions (3–8) . Structural information about the aggregated forms of Ab and tau is accumulating, but to date this knowledge has not led to successful chemical interventions (9)
  • AD tissue extracts 5, 22.
  • soluble complexes of Ab and tau have been found to promote aggregation of tau(22), while another study found that Ab fibrils can seed tau (23). Taking the evidence together, we hypothesize that cross-seeding of tau by Ab promotes tangle formation in AD, which could be prevented not only by inhibiting Ab aggregation, but also by disrupting the binding site of Ab with tau.
  • micro-electron diffraction MocroED
  • Figure 1A The structure revealed pairs of anti-parallel b-sheets each composed of ⁇ 4000 strands, stacked into a fibril that spans the entire length of the crystal. Neighboring sheets are oriented face to back ( Figure 1B, Table 1) defining a Class 7 steric zipper motif.
  • Figure 8 the three C-terminal residues adopt an extended, non-b conformation which stabilizes the packing between steric zippers.
  • the sheet-sheet interface is strengthened by interdigitating side chains, Lys 16, Val18, Phe20, Glu22 from the face of one strand, and Leu17, Phe19, and the N-terminus from the back of the other.
  • the zipper has an extensive interface with a high shape complementarity of 0.76 and a total buried solvent accessible surface area of 258 ⁇ 2.
  • Inhibitors bind and reduce toxicity of A ⁇ aggregates
  • inhibitors D1b and D1d not only prevent aggregation of monomeric Ab, but also bind aggregated states.
  • Inhibitors reduce seeding of tau by aggregated A ⁇ 1-42
  • tau- K18 (P301S) EYFP which stably expresses the microtubule binding domain of tau P301S mutant.
  • This cell line referred to hereafter as tau-K18 biosensor cells, has been used to demonstrate prion like seeding from transfected tau fibrils to cells and has been used as a model system to test tau inhibitors (44, 53).
  • tau-K18 biosensor cells has been used to demonstrate prion like seeding from transfected tau fibrils to cells and has been used as a model system to test tau inhibitors (44, 53).
  • tau40 or Ab fibrils was used to demonstrate prion like seeding from transfected tau fibrils to cells and has been used as a model system to test tau inhibitors (44, 53).
  • tau40 or Ab fibrils to a final concentration of 250 nM.
  • We found that Ab was able to produce intracellular aggregates significantly greater than the vehicle alone, but only at around 2.5% efficiency of tau40.
  • the peptide inhibitors are not able to block aggregation of the amyloid forming proteins hIAPP or alpha synuclein, indicating that these inhibitors are specific for Abeta and tau, and are not general amyloid inhibitors (Figure 13).
  • the first 3 mutants were engineered to block the VQIVYK (SEQ ID NO: 27) aggregation interfaces in addition to all but 1 of the 3 different known VQIINK (SEQ ID NO: 28) interfaces.
  • Mutant 1 Q276W, L282R, I308P leaves only interface A of VQIINK (SEQ ID NO: 28) available for aggregation
  • mutant 2 Q276W, I277M, I308P
  • mutant 3 I277M, L282R, I308P leaves only interface C accessible for aggregation.
  • Constructs 4 and 5 were designed to test the effect of blocking VQIINK (SEQ ID NO: 28) and all but 1 of the VQIVYK (SEQ ID NO: 27) surfaces. Mutant 4 (Q276W, I277M, L282R, Q307W, V309W) leaves only the dry interface of VQIVYK (SEQ ID NO: 27) available for aggregation and mutant 5 (Q276W, I277M, L282R, I308W) leaves only the solvent accessible surface for aggregation.
  • Amyloid polymorphs may differ depending on whether they were aggregated in vitro or extracted from human brain tissue (56). We sought to determine if our inhibitors are capable of blocking pathological forms of either tau, or Ab. As suggested previously in our conformational antibody assay and structural alignment (Figure 3C), we hypothesized that our inhibitors would block disease-relevant amyloid polymorphs. Since we also found that our inhibitors blocked both homotypic and heterotypic tau seeding by aggregated tau and Ab, we tested our inhibitor series on crude lysate from AD donor patient brain tissue.
  • AD Alzheimer's disease
  • Ab the apparent initiator of the disease
  • aggregates into a wide variety of species from soluble oligomers ranging from dimers to those that contain dozens of copies, to polymorphic fibril deposits.
  • toxic assemblies targeting a specific sequence or structure of a toxic motif that is present in a variety of these assemblies could be an effective strategy for designing pharmaceuticals.
  • Both the R2 and R3 amyloid-prone regions of tau contain a D1b sensitive interface.
  • the surface of R2 blocked by D1b contains residue I277, which previously has been shown to be critical for tau aggregation (60).
  • These models support the hypothesis that Ab and tau may interact with favorable energy to form a stable heterozipper.
  • Ab overlays well with these regions of tau in both parallel and anti-parallel orientations suggesting that either fiber or smaller oligomers could be capable of cross seeding.
  • the effective dose to reduce toxicity of aggregated species is higher than to delay aggregation of monomeric species. This is emphasized by the differing efficacies of our related inhibitor series, where some inhibitors were able to prevent initial aggregation, but not toxicity or seeding from various assemblies. It appears that inhibitors to prevent an aggregation nucleus are much more promiscuous than those that ameliorate toxicity by binding to a distinct structure. This trend was observed in both Ab and tau, suggesting a common inhibitory mechanism for both proteins, and highlights the need for multiple experimental measures to validate inhibitor efficacy.
  • Recombinant Amyloid Beta Peptide purification- Ab was purified as described in Krotee et. Al (47) After purification, the protein was lyophilized. Dried peptide powders were stored in desiccant jars at -20 oC.
  • Peptide Preparation- Candidate inhibitors were custom made and purchased from Genscript (Piscataway, NJ). Lyophilized candidate inhibitors were dissolved at 10mM in 100% DMSO. 10mM stocks were diluted as necessary. All stocks were stored frozen at -20°C.
  • Amyloid Beta was prepared by dissolving lyophilized peptide in 100% DMSO or 100mM NaOH. Next, the sample was spin-filtered and the concentration was assessed by BCA assay (Thermo Scientific, Grand Island, NY). The DMSO or NaOH peptide stocks were diluted 100-fold in filter-sterilized Dulbecco’s PBS (Cat. # 14200-075, Life Technologies, Carlsbad, CA).
  • Crystallization- 16-Ac-KLVFFAENVGS-NH 3 -26 (SEQ ID NO: 26) (Ab 16- 26 D23N) was dissolved at 4.5 mg/ml in 20% DMSO. Micro crystals were grown in batch in 0.2M magnesium formate, 0.1M Tris base pH 8.0, and 15% isopropanol at room temperature under quiescent conditions. Crystals grew within 4 days to a maximum of 2 weeks.
  • MicroED data collection The procedures for MicroED data collection and processing largely follow published procedures (62, 63). Briefly, a 2-3 ml drop of crystals in suspension was deposited onto a Quantifoil holey-carbon EM grid then blotted and vitrified by plunging into liquid ethane using a Vitrobot Mark IV (FEI, Hillsboro, OR). Blotting times and forces were optimized to keep a desired concentration of crystals on the grid and to avoid damaging the crystals. Frozen grids were then either immediately transferred to liquid nitrogen for storage or placed into a Gatan 626 cryo-holder for imaging.
  • Crystals that appeared visually undistorted produced the best diffraction.
  • Datasets from individual crystals were merged to improve completeness and redundancy.
  • Each crystal dataset spanned a wedge of reciprocal space ranging from 40-80°.
  • the geometry detailed above equates to an electron dose rate of less than 0.01 e-/ ⁇ 2 per second being deposited onto our crystals.
  • Measured diffraction images were converted from TIFF format into SMV crystallographic format, using publicly available software (available for download at http://cryoem.janelia.org/downloads). We used XDS to index the diffraction images and XSCALE (65) for merging and scaling together datasets originating from thirteen different crystals.
  • the optimal set of rotamers was identified as those that minimize an energy function containing a Lennard-Jones potential, an orientation-dependent hydrogen bond potential, a solvation term, amino acid-dependent reference energies, and a statistical torsional potential that depends on the backbone and side-chain dihedral angles.
  • Area buried and shape complementarity calculations were performed with areaimol and Sc, respectively, from the CCP4 suite of crystallographic programs (69).
  • the solubility of each peptide was evaluated by hydropathy index (75).
  • the designed peptides were selected based on calculated binding energy of top or bottom binding mode, shape complementarity and peptide solubility.
  • Thioflavin-T (ThT) kinetic assays were performed in black 96-well plates (Nunc, Rochester, NY) sealed with UV optical tape. The total reaction volume was 150 mL per well. Ab1-42 was prepared as described. Inhibitors were added at indicted concentrations, with a final concentration of 1%DMSO. ThT fluorescence was recorded with excitation and emission of 444 nm and 482 nm, respectively, using a Varioskan Flash (Thermo Fisher Scientific, Grand Island, NY). Experiments were performed at 37°C without shaking in triplicate and readings were recorded every 5 minutes. ThT assays with tau40 were prepared as above with the following exceptions.
  • heparin Sigma cat. no. H3393
  • seeding assay included 10% monomer equivalent of preformed fibrils, sonicated for 10 minutes prior to addition.
  • N2a Cell culture- Neuro2a cells were a gift from the Pop Wongpalee in the laboratory of Douglas Black at UCLA. Cells were cultured in MEM media (Cat. # 11095-080, Life Technologies) plus 10% fetal bovine serum and 1% pen-strep (Life Technologies). Cells were cultured at 37°C in 5% CO2 incubator.
  • 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay for cell viability- N2a cells were plated at 5,000 cells per well in 90 mL of culture media, in clear 96-well plates (Cat. # 3596, Costar, Tewksbury, MA). Cells were allowed to adhere to the plate for 20-24 hours. Ab1-42 samples were incubated at 10mM with or without inhibitors at varying ratios for 12 hours at 37°C and then applied to N2a cells. 10 mL of sample was added to cells. By doing this, samples were diluted 1/10 from in vitro stocks. Experiments were done in triplicate.
  • Dot Blot Assay- Ab1-42 samples were incubated at 10 mM with or without inhibitors for 6, 24, and 72 hours at 37°C, and spotted onto a nitrocellulose membrane (Cat. # 162–0146, BioRad, Hercules, CA). 20mL was loaded for each condition; 2mL was spotted at a time and allowed to dry between application. The membranes were blotted as previously described (76), with the exception of the primary antibodies used. The antibodies used in the assay were previously generated and characterized (51). Surface Plasmon Resonance (SPR)- SPR experiments were performed using BiacoreT200 instrument (GE Healthcare). Ab42 fibrils/tau K18 fibrils were immobilized on a CM5 sensor chip.
  • SPR Surface Plasmon Resonance
  • the fibrils of Ab42 were prepared by placing a sample of 50 mM Ab42 in PBS pH 7.4 in two wells of a Nunc 96-well optical bottom plate (Thermo Scientific), 150 ml/well and incubating the plate in a microplate reader (FLUOstar Omega, BMG Labtech) at 37 °C with double orbital shaking at 600 rpm overnight. Sample from the two wells were pooled together and Ab42 fibrils were isolated from the incubation mixture by centrifuging it at 13,000 xg, 4°C for 45 minutes. The supernatant was removed and the pellet was re-dissolved in an equal volume of PBS as that of supernatant.
  • the isolated fibrils were sonicated using a probe sonicator for 1-2 minutes at 18% amplitude with 2 sec on, 5 sec off pulses.
  • the sonicated fibrils were filtered through a 0.22 m filter to remove large aggregates.
  • the sonicated and filtered fibrils were diluted to 60 mg/ml in 10 mM NaAc, pH 3 and then, immobilized immediately on a CM5 sensor chip using standard amine coupling chemistry. Briefly, the carboxyl groups on the sensor surface were activated by injecting 100 ul of 0.2 M EDC and 0.05 M NHS mixture over flow cells 1–2. The fibrils were then injected at a flow rate of 5 ml/min over flow cell 2 of the activated sensor surface for 900 seconds.
  • each peptide inhibitor was dissolved in 100 % DMSO at a concentration of 1 mM and diluted in PBS pH 7.4+1.2% DMSO to concentrations ranging from 5 mM to 260 mM.
  • Each peptide was injected at a flow rate of 30 ml/min over both flow cells (1 and 2) at increasing concentrations (in running buffer, PBS, pH 7.4+1.2% DMSO) at 25°C.
  • the contact time and dissociation time were 120 seconds and 160 seconds, respectively. 3 M NaCl was used as regeneration buffer.
  • the data were processed and analyzed using Biacore T200 evaluation software 3.1.
  • the data of flow cell 1 (blank control) was subtracted from the data of flow cell 2 (with immobilized fibrils/monomers).
  • the equilibrium dissociation constant (Kd) was calculated by fitting the plot of steady-state peptide binding levels (Req) against peptide concentration (C) with 1:1 binding model (Eq 1).
  • Cells were induced with 0.5 mM IPTG for 3 hours at 37°C and lysed by sonication in 50 mM Tris (pH 8.0) with 500 mM NaCl, 20 mM imidazole, 1 mM beta- mercaptoethanol, and HALT protease inhibitor. Cells were lysed by sonication, clarified by centrifugation at 15,000 rpm for 15 minutes, and passed over a 5 ml HisTrap affinity column. The column was washed with lysis buffer and eluted over a gradient of imidazole from 20 to 300 mM.
  • Inhibitors dissolved in DMSO were added to 20 ml of diluted fibrils at a concentration 20-fold greater than the final desired concentration. Fibrils were incubated for ⁇ 16 h with the inhibitor, and subsequently were sonicated in a Cup Horn water bath for 3 min before seeding the cells. The resulting‘pre-capped fibrils’ were mixed with one volume of Lipofectamine 2000 (Life Technologies, cat. no. 11668027) prepared by diluting 1 ml of Lipofectamine in 19 ml of OptiMEM. After 20 min, 10 ml of fibrils were added to 90 ml of the tau-K18CY biosensor cells to achieve the final indicated ligand concentration.
  • Lipofectamine 2000 Life Technologies, cat. no. 11668027
  • Quantification of seeding was determined by imaging the entire well of a 96-well plate seeded in triplicate and imaged using a Celigo Image Cytometer (Nexcelom) in the YFP channel. Aggregates were counted using ImageJ (77) by subtracting the background fluorescence from unseeded cells and then counting the number of peaks with fluorescence above background using the built-in Particle Analyzer. Dose-response curves were constructed for inhibitor peptides exhibiting concentration dependence by fitting to a nonlinear regression model in Graphpad Prism. High resolution images were acquired using a ZEISS Axio Observer D1 fluorescence microscope.
  • Brain lysate Human brain tissue was obtained from the Neuropathology Laboratory at UCLA Medical Center. AD and PSP cases were confirmed by the Neuropathology Laboratory by immunostaining autopsied brain tissue sections, and the PSP donor was confirmed to be free of amyloid immunoreactivity. Tissue sections from the indicated brain regions were manually homogenized using a disposable ultra-tissue grinder (Thermo Fisher) in TBS (pH 7.4) supplemented with 1X HALT protease inhibitor. Homoginzed tissue was aliquoted to several PCR tubes and prepared for seeding in biosensor cells by sonication as described by Kaufman et al.
  • tissue sections were sonicated twice as long, for a total of 2 h, in an ice cooled circulating water bath with individual sample tubes stirring to ensure each tube received the same sonication energy. Subsequently, seeding was measured by transfection into biosensor cells and quantified as described above.
  • Aggregation Inhibition Assay with a-synuclein- a-synuclein was expressed and purified as described previously in Rodriguez, et al. with the following exceptions to the expression protocol.
  • An overnight starter culture was grown in 15 mL instead of 100 mL, 7 mL of which was used to inoculate 1 L. After induction, cells were allowed to grown for 3-4 hours at 34 °C (instead of 4-6 hours at 30 °C). Cells were then harvested by centrifuging at 5,000 x g.
  • ThT assays with a-synuclein were performed in black 96-well plates (Nunc, Rochester, NY) sealed with UV optical tape. The total reaction volume was 180 mL per well.
  • ThT fluorescence was recorded with excitation and emission of 444 nm and 482 nm, respectively, using a Varioskan Flash (Thermo Fisher Scientific, Grand Island, NY). Experiments were performed at 37°C, shaking at 600 rpm with a teflon bead, in triplicate and readings were recorded every 15 minutes.
  • Alpha synuclein at 105 mM in PBS was diluted to a final concentration of 50 mM in 25 mM Thioflavin-T and PBS.
  • Inhibitors were added at the specified concentration by diluting 10 mM stocks in 100% DMSO 1 to 40 in the same manner. Thus, inhibitors were tested at 5:1 molar excess of a-synuclein.
  • IAPP- Human IAPP1-37NH 2 was purchased for Innopep (San Diego, CA). Peptides were prepared by dissolving lyophilized peptide in 100% 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) at 250mM for 2 hours. Next, the sample was spin-filtered and then HFIP was removed with a CentriVap Concentrator (Labconco, Kansas City, MO). After removal of the HFIP, the peptide was dissolved at 1mM or 10mM in 100% DMSO (IAPP alone) or 100% DMSO solutions containing 1mM or 10mM inhibitor.
  • HFIP 1,1,1,3,3,3-Hexafluoro-2-propanol
  • DMSO peptide stocks were diluted 100-fold in filter-sterilized Dulbecco’s PBS (Cat. # 14200-075, Life Technologies, Carlsbad, CA).
  • Thioflavin-T (ThT) assays with hIAPP were performed in black 384-well plates (Nunc, Rochester, NY) sealed with UV optical tape.
  • hIAPP1- 37NH2 and mIAPP1-37NH2 were prepared as described.
  • the total reaction volume was 150 mL per well.
  • ThT fluorescence was recorded with excitation and emission of 444 nm and 482 nm, respectively, using a Varioskan Flash (Thermo Fisher Scientific, Grand Island, NY). Experiments were performed at 25°C without shaking in triplicate and readings were recorded every 5 minutes.
  • Atomic structure overlay- A structural superposition of Ab 16-26 and tau (5V5B, 6HRE) was performed using LSQ from coot (Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr. D. Biol. Crystallogr. 60, 2126–32).
  • LSQ root mean square deviation
  • Anti-paprallel LSQ computation of Ab 16-22 and tau 275-281 (5V5B) of C a atoms was calculated, and side chain rotomers optimized with foldit (Kleffner, R., Cooper, S., Khatib, F., Flatten, J., Leaver-Fay, A., Baker, D., and Siegel, J. B. (2017) Foldit Standalone: a video game-derived protein structure manipulation interface using Rosetta. Bioinformatics. 33, 2765–2767) over 2000 iterations to minimize energy to -603 REU. Supplemental Table 1 (Table S1). Computed binding properties of designed inhibitors to amyloid-beta 16 KLVFFAEN 23 (SEQ ID NO: 25)
  • Amyloid b-protein Monomer structure and early aggregation states of Ab42 and its Pro19alloform. J. Am. Chem. Soc.127, 2075–2084
  • Aggregated tau is a histological hallmark of Alzheimer’s disease (AD) and a group of neurological disorders called tauopathies.
  • AD Alzheimer’s disease
  • tauopathies Tau pathology accompanies progressive neurodegeneration, and aggregated tau is thought to spread to adjacent neurons and anatomically connected brain regions by prion-like seeding.
  • inhibitors of seeding offer one possible route to therapy.
  • SEQ ID NO: 29 sequence SVQIVY
  • Our structure illuminates a previously unappreciated surface of SVQIVY (SEQ ID NO: 29) that contributes to proteopathic seeding by patient-derived fibrils.
  • tau pathology is a marker of neurodegeneration in AD and related tauopathies (1, 2).
  • amyloid core of tau is formed by steric zippers with rich b character, tightly interdigitated sidechains and strong shape complementarity (6, 7).
  • Amyloid structures are particularly stable relative to other protein folds owing to steric zipper interactions, and an extensive network of hydrogen bonds that form along the fibril axis (8, 9), potentially explaining their ability to spread transcellularly to distant, anatomically connected brain regions.
  • CryoEM models recapitulate all of the structural features, and additionally reveal novel protein folds and polymorphs that are unique to amyloids and contribute to their unusual stability and diversity.
  • crystal structures of steric zippers determined from short, aggregation-prone peptide segments reveal the exact molecular interfaces that are seen in cryoEM structures of fibrils containing the same aggregation-prone segments.
  • cryoEM structures of two different fibril polymorphs revealed homomeric interfaces, each formed by different homo-steric zippers (10). Both were identical to steric zipper interfaces determined from crystal structures of the same respective peptide segments (11), demonstrating that peptide structures recapitulate subsets of the interactions that are also seen in the fibril core.
  • amyloid proteins can involve different surfaces of peptides with the same sequence, leading to different structural polymorphs that are associated with different neurodegenerative diseases.
  • tau this is exemplified by 4 cryoEM structures of patient-derived fibrils from AD (12, 13) and Pick’s disease (14): two polymorphs from AD (referred to as PHFs and SFs, which are ultrastructural polymorphs of each other) and two from Pick’s disease (NPFs and WPFs).
  • PHFs and SFs two polymorphs from AD
  • NPFs and WPFs Pick’s disease
  • capping inhibitors from crystal structures of the steric zipper segments of tau with sequences 275 VQIINK 280 (SEQ ID NO: 28) and 306 VQIVYK 311 (SEQ ID NO: 27) (15, 16).
  • a capping inhibitor TLKIVW SEQ ID NO: 30
  • the SVQIVY (SEQ ID NO: 29) structure contains two different steric zipper interfaces that belong to two different classes.
  • a Class 1 steric zipper is formed by two copies of the peptide that are related by crystallographic symmetry (Fig. 15B), and is nearly identical to the Class 1 steric zippers formed by the VQIVYK (SEQ ID NO: 27) segment determined previously (7).
  • the opposite face of SVQIVY (SEQ ID NO: 29) forms a second steric zipper that is a Class 3 interface created by two molecules in the asymmetric unit.
  • the Class 3 interface is more interdigitated than the Class 1, having a shape complementarity (S c ) of 0.87 and solvent-accessible surface area buried (A b ) of 110.9 ⁇ 2 (compared to 0.79 and 106.1 ⁇ 2 , respectively, for the Class 1 zipper).
  • S c shape complementarity
  • a b solvent-accessible surface area buried
  • a Class 3 steric zipper interface was also observed previously in the cryoEM structure of an 11 residue segment from TDP43 (18), but has not been observed, or at least recognized, in any longer fibril structures determined by cryoEM to date.
  • SVQIVY SEQ ID NO: 29
  • the Class 3 interface formed by Ser305, Gln307, and Val309 of the SVQIVY (SEQ ID NO: 29) crystal structure maps to a solvent exposed surface of patient-derived fibrils from AD (Fig. 16A and B), and Pick’s disease.
  • the Class 1 steric zipper from the peptide crystal structure is formed by a homomeric steric zipper with a mated sheet of identical sequence, while the Class 1 interface from patient-derived fibrils forms through heteromeric steric zipper interactions. Nevertheless the features of the interface from the peptide crystal structure, mainly the zipper class and solvent-accessible surface area buried, is reminiscent of the cryoEM fibril structures (Fig. 22). Similarly, we suggest the Class 3 interface of SVQIVY (SEQ ID NO: 29) highlights a bona fide aggregation surface that could contribute to fibril stability and/or seeding, even though the sequences of the mated strands in the fibrillar and crystalline states are likely to differ.
  • the Class 1 and 3 steric zipper interfaces are not mutually exclusive as they are both observed simultaneously in the SVQIVY (SEQ ID NO: 29) crystal structure.
  • aligning the SVQIVY (SEQ ID NO: 29) crystal structure with AD-derived filaments reveals that unmodeled electron density overlaps with the same interface that forms the Class 3 steric zipper in both the PHF and SF, suggesting that similar interactions could form on the surfaces of these patient-derived fibrils.
  • the surface that forms the Class 3 interface is accessible to only one of the two protomers in the SF, as the other is occluded by a key intermolecular contact that forms with the mated protomer.
  • a second steric zipper interface can be formed by the aggregation-prone SVQIVY (SEQ ID NO: 29) segment, and the same surface of the PHF and SF harbors unmodeled, contiguous electron density.
  • a D-peptide inhibitor TLKIVW (SEQ ID NO: 30), which was previously designed using the Class 1 VQIVYK (SEQ ID NO: 27) structure and potently inhibited the primary aggregation of the 3R tau (15), likewise had no measurable inhibition of seeding by full length tau fibrils at concentrations up to 50 mM (Fig.23).
  • Table A Amino acid sequences of Tau VQIINK (SEQ ID NO: 28) and VQIVYK (SEQ ID NO: 27) capping inhibitors.
  • the top row contains the residue position number for the referenced inhibitor peptide.
  • the second row is the native sequence from wild-type human Tau40 from which the respective capping inhibitor peptides were derived, and subsequent rows are inhibitor peptide sequences tested in this paper. Residues modified from the wild- type sequence for each given capping inhibitor are highlighted in yellow and are listed in red font. Because both Class 3 inhibitors WIV and QIW exhibited modest inhibition of seeding, we combined the two tryptophan substitutions at positions 3 and 5 from WIV and QIW into a single capping inhibitor peptide referred to as WIW.
  • the IC50 of WIW determined using recombinant fibrils of tau40 improved to 4 mM (Fig. 16C).
  • the series of VQIINK (SEQ ID NO: 28) capping inhibitors: WMINK (SEQ ID NO: 32), W3, M4 and R9 have similar IC50’s of about 1 mM. Since capping inhibitors are composed of L-peptides, we knew whether they could be delivered to cells by transfecting DNA that encoded the inhibitor sequence.
  • VQIINK SEQ ID NO: 28
  • VQIVYK SEQ ID NO: 27
  • the daughter fibrils also exhibited an unexplained sensitivity to WIW.
  • heparin induced fibrils of recombinant K18+ likewise exhibit sensitivity to WIW (Fig. 18C), suggesting that daughter fibrils inherited the inhibitor sensitivities of both the parent and recombinant fibrils.
  • heparin was not used for in vitro seeding to amplify the CTE-derived polymorph.
  • VQIVYK (SEQ ID NO: 27) inhibitors WIV, QIW and WIW produced variable effects generally ranging from no inhibition, to mild inhibition (Fig. 19A), whereas VQIINK (SEQ ID NO: 28) inhibitors IN-W3, IN-M4 and IN-R9 were stronger inhibitors, and interestingly inhibited seeding with different efficacies for the different PSP donors tested.
  • donors 1 and 3 shared similar profiles of inhibition in spite of deriving from two different brain regions, and were particularly sensitive to inhibition by IN-M4 and WIW, whereas little-to-no response was seen for IN-W3 and the other two VQIVYK (SEQ ID NO: 27) inhibitors, WIV and QIW.
  • PSP donors 1-3 appeared to show variable profiles of inhibitor sensitivities, we tested the susceptibility of a fourth PSP donor using tissue sections from two different brain regions, the frontal cortex and cerebellum using a subset of the inhibitors including IN-W3, IN-M4 and IN-R9, which proved to be the greatest discriminators observed for donors 1-3.
  • the inhibitor profile from the cerebellum of PSP donor 4 closely matched the profiles we observed for PSP donors 1 and 3, with strong inhibitor sensitivity limited mainly to the VQIINK (SEQ ID NO: 28)-based inhibitor IN-M4 (Fig. 19D and F-H).
  • the inhibitor profile we observed from a tissue section from the frontal cortex of the same donor more closely matched the profile that we observed from PSP donor 2, with the VQIINK (SEQ ID NO: 28)-based inhibitors IN-W3, IN-M4 and IN-R9 all showing strong inhibition (Fig. 19E and I-K).
  • extract from the frontal cortex of donor 4 additionally exhibited a strong sensitivity to WIV.
  • inhibitors targeting the solvent exposed surface blocked seeding, whereas inhibitors targeting the buried surface poorly inhibited seeding by AD fibrils and crude brain extracts.
  • inhibitors of seeding can be designed by targeting solvent exposed surfaces, and suggest that these surfaces contribute to seeding by pathological fibrils, perhaps by acting as scaffolds to allow for nucleation and growth of new fibrils by offering an adhesive interface on which tau monomers can accumulate.
  • these interactions are transient and dynamic, and that nucleated fibrils eventually break off to become independent fibrils.
  • structures of patient- derived fibrils from AD harbor unmodeled electron density that overlaps with the position of the Class 3 interface observed in our SVQIVY (SEQ ID NO: 29) crystal structure.
  • the Class 3 interface that is targeted by our inhibitors might contribute to fibril stability through intra- or interprotomer contacts that are poorly resolved in the cryoEM maps due to heterogeneity and/or partial occupancy.
  • capping inhibitor peptides could be functionally synthesized by ribosomes following delivery to cells in a DNA vector.
  • transfection of plasmid DNA encoding the WIW sequence into tau biosensor cells allowed cells to resist seeding with recombinant fibrils.
  • amyloid aggregates The prion-like nature of amyloid aggregates is thought to perpetuate specific disease-specific polymorphs, and thus, it is thought that seeding produces daughter fibrils that are identical to parent fibrils. Contrary to this, we found that daughter fibrils seeded using the sarkosyl insoluble fraction from a donor with CTE possessed a different profile of inhibitor sensitivity compared to the parent fibril polymorph. Parent fibrils exhibited a broad response to our panel of inhibitors, whereas daughter fibrils exhibited characteristics of both the parent and recombinant fibrils.
  • 305SVQIVY310 (SEQ ID NO: 29) synthetic peptide was purchased from GenScript and microcrystals were grown in batch at 3.3 mg/mL in 0.667 M DL-Malic acid pH 7.0, 8% w/v PEG 3350 at 18C.
  • Crystal solution was applied to a glow discharged Quantifoil R1/4 cryo-EM grid, and plunge frozen using a Vitrobot Mark 4.
  • Micro-ED data was collected in a manner similar to previous studies(19). Briefly, plunge-frozen grids were transferred to an FEI Technai F20 electron microscope operating at 200 kV and diffraction data were collected using a TVIPS F416 CMOS camera with a sensor size of 4,096 x 4,096 pixels, each 15.6 x 15.6 mm. Diffraction data was indexed using XDS, and XSCALE was used for merging and scaling together data sets from different crystals(20). XXX diffraction movies were merged using XSCALE to produce the final data set. Structure Determination
  • the SHELX macromolecular structure determination suite was used for phasing the measured intensities(21).
  • a combination of REFMAC, Phenix, and Buster refinement programs were used with electron scattering factors to refine the atomic coordinates determined by the direct-methods protocol. Recombinant protein expression and purification
  • Lysate was boiled for 20 minutes and the clarified by centrifugation at 15,000 rpm for 15 minutes and dialyzed to 20 mM MES buffer (pH 6.8) with 50 mM NaCl and 5 mM DTT. Dialyzed lysate was purified on a 5 ml HighTrap SP ion exchange column and eluted over a gradient of NaCl from 50 to 550 mM. Protein was polished on a HiLoad 16/600 Superdex 75 pg in 10 mM Tris (pH 7.6) with 100 mM NaCl and 1 mM DTT, and concentrated to ⁇ 20-60 mg/ml by ultrafiltration using a 3 kDa cutoff.
  • Cells were induced with 0.5 mM IPTG for 3 hours at 37 oC and lysed by sonication in 50 mM Tris (pH 8.0) with 500 mM NaCl, 20 mM imidazole, 1 mM beta-mercaptoethanol, and HALT protease inhibitor. Cells were lysed by sonication, clarified by centrifugation at 15,000 rpm for 15 minutes, and passed over a 5 ml HisTrap affinity column.
  • Tissue was cut into a 0.2-0.3 g section on a block of dry ice, and then manually homogenized in a 15 ml disposable tube in 1 ml of 50mM Tris, pH 7.4 with 150mM NaCl containing 1X HALT protease. Samples were then aliquoted to PCR tubes and sonicated in a cuphorn bath for 120 min under 30% power at 4 °C in a recirculating ice water bath. For purification of PHFs and SFs from AD brain tissue, extractions were performed according to the previously published protocol (Fitzpatrick AWP, et al. (2017) Cryo-EM structures of tau filaments from Alzheimer’s disease. Nature 547(7662):185–190) without any modifications. Inhibitor peptides
  • Inhibitor peptides were designed using the native crystal structure as a starting point. Bulky sidechains were modeled at sites in the VQIINK (SEQ ID NO: 28) structure that were in close contact with residues in the mated sheet of the steric zipper interface. Capping residues were chosen by modelling all possible rotamers to find sidechains without any compatible conformer with the steric zipper interface (that is, sidechains that clashed with the mated beta sheet at every rotamer conformer were selected). All of the inhibitor peptides shown in Table A were synthesized by Genscript with minimum purities of 90% and dissolved in deionized water or DMSO to a working concentration of 1.4 mM. Tau biosensor cell line maintenance and seeding
  • HEK293 cell lines stably expressing tau-K18 P301S-eYFP , referred to as“tau biosensor cells” were engineered by Marc Diamond’s lab at UTSW (5) and used without further characterization or authentication.
  • Cells were maintained in DMEM (Life Technologies, cat. 11965092) supplemented with 10% (vol/vol) FBS (Life Technologies, cat. A3160401), 1% penicillin/streptomycin (Life Technologies, cat. 15140122), and 1% Glutamax (Life Technologies, cat. 35050061) at 37 °C, 5% CO2 in a humidified incubator.
  • Fibrils and patient-derived seeds were incubated for 16 hours with inhibitor to yield a final inhibitor concentration of 10 mM (on the biosensor cells), except for IC50 determinations, which instead used adjustments to achieve the final indicated inhibitor concentration.
  • inhibitor-treated seeds were sonicated in a cuphorn water bath for 3 minutes, and then mixed with 1 volume of Lipofectamine 3000 (Life Technologies, cat. 11668027) prepared by diluting 1 ml of Lipofectamine in 19 ml of OptiMEM. After twenty minutes, 10 ml of fibrils were added to 90 ml of tau biosensor cells.
  • the number of seeded aggregates was determined by imaging the entire well of a 96-well plate in triplicate using a Celigo Image Cytometer (Nexcelom) in the YFP channel. Aggregates were counted using ImageJ by subtracting the background fluorescence from unseeded cells and then counting the number of peaks with fluorescence above background using the built-in Particle Analyzer. The number of aggregates was normalized to the confluence of each well, and dose-response plots were generated by calculating the average and standard deviations from triplicate measurements. For IC50 calculations, does- response curves were fit by nonlinear regression in Graphpad Prism. For high quality images, cells were photographed on a ZEISS Axio Observer D1 fluorescence microscope using the YFP fluorescence channel. Data Availability

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Abstract

La pathologie de la maladie d'Alzheimer (AD) est caractérisée par des plaques de protéine bêta-amyloïde (Aβ) et des enchevêtrements neurofibrillaires de protéine tau. L'agrégation de protéine Aβ est supposée se produire à des stades précoces de la maladie, et donne finalement lieu à la formation d'enchevêtrements de protéine tau qui vont de pair avec un déclin cognitif. L'invention concerne des inhibiteurs à base de peptides qui réduisent l'agrégation de protéine Aβ et de protéine tau et la toxicité d'espèces déjà agrégées.
PCT/US2020/033426 2019-05-16 2020-05-18 Inhibiteurs à base de peptides qui bloquent l'agrégation, l'ensemencement et l'ensemencement croisé de protéine bêta-amyloïde et de protéine tau WO2020232440A1 (fr)

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EP4186918A1 (fr) * 2021-11-30 2023-05-31 Université de Rennes Peptides inhibiteurs pour le diagnostic et/ou le traitement de tauopathies
WO2023099560A1 (fr) * 2021-11-30 2023-06-08 Universite De Rennes Peptides inhibiteurs pour le diagnostic et/ou le traitement de tauopathies

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