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WO2018005980A1 - Compositions et méthodes de diagnostique associées aux maladies transthyrétine amyloïdes - Google Patents

Compositions et méthodes de diagnostique associées aux maladies transthyrétine amyloïdes Download PDF

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WO2018005980A1
WO2018005980A1 PCT/US2017/040314 US2017040314W WO2018005980A1 WO 2018005980 A1 WO2018005980 A1 WO 2018005980A1 US 2017040314 W US2017040314 W US 2017040314W WO 2018005980 A1 WO2018005980 A1 WO 2018005980A1
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seq
ttr
probe
amino acid
compound
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Jeffery W. Kelly
Joseph D. SCHONHOFT
Cecilia MONTEIRO
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Scripps Research Institute
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Scripps Research Institute
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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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present disclosure is in the medical and biomedical field, specifically as it relates to amyloidosis.
  • Transthyretin is a 127-amino acid ⁇ -sheet-rich protein that folds and assembles into a tetramer in the endoplasmic reticulum of liver, choroid plexus, and retinal pigmented epithelial cells, from which TTR is secreted into the plasma, cerebrospinal fluid, and eye, respectively.
  • Rate-limiting TTR tetramer dissociation, and relatively fast monomer misfolding leads to TTR aggregation associated with a number of systemic amyloid diseases, which are autosomal dominant diseases collectively referred to as the TTR amyloidoses (ATTR).
  • Wild type TTR amyloidosis affects approximately 10-15% of individuals older than 65 years of age. ATTR-WT manifests predominantly as a cardiac disease, but the involvement of other organ systems, including the nervous system, is increasingly recognized in this malady that is being diagnosed with increasing frequency.
  • familial TTR amyloidoses caused by the dissociation of TTR tetramers comprising WT and/or mutant TTR subunits, followed by their misfolding and misassembly, the clinical heterogeneity is even more marked.
  • the familial TTR amyloidoses can manifest with different primary phenotypes, including heart failure with a preserved ejection fraction, in a disease called familial amyloid cardiomyopathy (FAC) caused by inherited TTR mutations like Vall22Ile (present in 4 % of individuals of African descent).
  • FAC familial amyloid cardiomyopathy
  • Individuals inheriting other mutations, e.g. Val30Met can present with a predominantly neuropathic disease, a long fiber neuropathy with autonomic nervous system involvement, in a disease called familial amyloid polyneuropathy (FAP).
  • FAP familial amyloid polyneuropathy
  • V30M FAP has high prevalence in endemic areas of Japan and Portugal. Variability in clinical presentation is common and can even occur within the same kindred.
  • V30M carriers e.g., present with marked involvement of other less common organs, such as the eye or the central nervous system and/or the kidneys.
  • TTR amyloidosis patients present first to different clinical specialties, including neurology, cardiology, nephrology, hematology, ophthalmology or gastroenterology. In part because this is still perceived to be a relatively rare disease, too many physicians have a low suspicion level for systemic amyloidosis. Even if the physician is aware that amyloidosis is a possible diagnosis, these patients' initial symptoms can mimic a number of other more common diseases and there is no single diagnostic method that is non-invasive and easy to apply currently.
  • amyloid fibril detection still considered the diagnostic gold standard, is generally combined with organ damage detected by echocardiographic and/or neurophysiological studies to make a diagnosis. As a direct result, diagnosis is often made later in the course of disease. This is problematic, as currently available therapies for the TTR amyloidoses - liver transplant and kinetic stabilizer (tafamidis and diflunisal) administration - have proven to be more effective when used early in the course of FAP.
  • Various embodiments disclosed herein include a compound comprising: a polypeptide comprising a sequence P1P2P3P4P5P6P7P8P9P10 or a pharmaceutically acceptable salt thereof, wherein Pi, P 3 and P 10 are each independently any amino acid residue, analog or absent; P2 is a ⁇ -branched amino acid residue, analog or absent; P 4i ⁇ and Pg are each independently a ⁇ -branched amino acid residue; and Ps , P7 and P9 are each independently any amino acid residue or analog.
  • the polypeptide is a synthetic or recombinant peptide.
  • the polypeptide consists of from 6 to about 20 amino acid residues.
  • the compound further comprises a diagnostic moiety.
  • the diagnostic moiety is an alkyne reporter group and/or an alkyl diazirine reactive group.
  • the diagnostic moiety is conjugated to the polypeptide.
  • the diagnostic moiety is conjugated to the N-terminal residue of the peptide.
  • the diagnostic moiety comprises an absorbent, fluorescent or luminescent label moiety.
  • the diagnostic moiety comprises a fluorophore, a coumarin moiety, or a rhodamine moiety.
  • P 4i ⁇ and Ps are each Val
  • P5 is a hydrophobic amino acid residue
  • P7 is a hydrophobic amino acid residue or His
  • P9 is a hydrophobic amino acid residue
  • P3 is an uncharged polar amino acid residue or Ala.
  • ⁇ 2 ⁇ P 4i ⁇ and Pg are each independently Val or He
  • P3 is Asn or Ala
  • P5 is Ala
  • P7 is His or Ala
  • P9 is Phe or Ala.
  • the polypeptide consists of an amino acid sequence INVAVHVF (SEQ ID NO: 21).
  • Pi is a hydrophobic amino acid residue or absent
  • P2 is a ⁇ -branched amino acid residue
  • P3 is an uncharged polar amino acid residue or Ala
  • P lo is Arg, Lys or absent.
  • ⁇ 2 ⁇ P 4i ⁇ and Pg are each independently Val or He
  • Pi is Ala or propargyl glycine
  • P3 is Asn or Ala
  • P5 is Ala
  • P7 is His or Ala
  • P9 is Phe or Ala
  • P lo is Arg.
  • the polypeptide consists of an amino acid sequence selected from the group consisting of VAVHVF (SEQ ID NO: 1), AINVAVHVFR (SEQ ID NO: 2), AINAAAHVFR (SEQ ID NO: 3), AINAAVHVFR (SEQ ID NO: 4), AINVAAHVFR (SEQ ID NO: 5), AINVAVHAFR (SEQ ID NO: 6), AANVAVHVFR (SEQ ID NO: 7), AINVAVHVAR (SEQ ID NO: 8), AINVAVAVFR (SEQ ID NO: 9), AIAVAVHVFR (SEQ ID NO: 10), INVAVHVFR (SEQ ID NO: 11), NVAVHVFR (SEQ ID NO: 12), VAVHVFR (SEQ ID NO: 13), AVHVFR (SEQ ID NO: 14), VAVHV (SEQ ID NO: 15), AINVAVHVF (SEQ ID NO: 16), AINVAVHV (SEQ ID NO:
  • the polypeptide consists of an amino acid sequence AINVAVHVFR (SEQ ID NO: 2), and the diagnostic moiety comprises fluorescein-5-carboxamido)hexanoic acid (5-FAM-X).
  • the polypeptide consists of an amino acid sequence propargyl glycine-INV AVHVFR (SEQ ID NO: 22), and the diagnostic moiety comprises a photoreactive diazirine group and a terminal alkyne group.
  • a composition comprising the compound of claim 1 and a pharmaceutically acceptable excipient.
  • Various embodiments disclosed herein also include a method for diagnosing TTR amyloidosis in a subject, comprising: providing a blood sample of the subject; contacting the blood sample with a compound comprising a polypeptide comprising a sequence P1P2P3P4P5P6P7P8P9P10 or a pharmaceutically acceptable salt thereof, wherein Pi, P 3 and P 10 are each independently any amino acid residue, analog or absent; P2 is a ⁇ -branched amino acid residue, analog or absent; P ⁇ ⁇ and Pg are each independently a ⁇ -branched amino acid residue; and Ps , P7 and P9 are each independently any amino acid residue or analog; and diagnosing TTR amyloidosis if there is binding between a misfolded TTR oligomer in the blood sample and the polypeptide compound.
  • the blood sample is a plasma sample or a serum sample.
  • the method further comprises treating the TTR amyloidosis in the subject by liver transplant and/or administering an effective amount of TTR amyloidosis medicine to the subject.
  • TTR amyloidosis is treated by administering an effective amount of a transthyretin kinetic stabilizer.
  • the transthyretin kinetic stabilizer is tafamidis and/or diflunisal.
  • the method further comprises a diagnostic moiety conjugated to the polypeptide compound.
  • the diagnostic moiety is an alkyne reporter group and/or an alkyl diazirine reactive group.
  • the diagnostic moiety comprises an absorbent, fluorescent, or luminescent label moiety. In one embodiment, the diagnostic moiety comprises a fluorophore, a coumarin moiety, or a rhodamine moiety. In one embodiment, the specific binding is detected via a fluorescence assay. In one embodiment, the specific binding is detected via photocrosslinking and ELISA assay.
  • the polypeptide consists of an amino acid sequence selected from the groups VAVHVF (SEQ ID NO: 1), AINV AVHVFR (SEQ ID NO: 2), AINAAAHVFR (SEQ ID NO: 3), AINAAVHVFR (SEQ ID NO: 4), AINVAAHVFR (SEQ ID NO: 5), AINVAVHAFR (SEQ ID NO: 6), AANV AVHVFR (SEQ ID NO: 7), AINVAVHVAR (SEQ ID NO: 8), AINVAVAVFR (SEQ ID NO: 9), AIAV AVHVFR (SEQ ID NO: 10), INV AVHVFR (SEQ ID NO: 11), NVAVHVFR (SEQ ID NO: 12), VAVHVFR (SEQ ID NO: 13), AVHVFR (SEQ ID NO: 14), VAVHV (SEQ ID NO: 15), AINVAVHVF (SEQ ID NO: 16), AINVAVHV (SEQ ID NO: 15),
  • Other embodiments disclosed herein include a method for monitoring treatment of a patient being treated for TTR amyloidosis, comprising: obtaining a first blood sample from the subject prior to treatment, detecting and quantifying misfolded TTR oligomer in the first blood sample by contacting the first blood sample with a compound comprising a polypeptide comprising a sequence P1P2P3P4P5P6P7P8P9P10 or a pharmaceutically acceptable salt thereof, wherein Pi, P 3 and P 10 are each independently any amino acid residue, analog or absent, P2 is a ⁇ -branched amino acid residue, analog or absent, P ⁇ ⁇ and Pg are each independently a ⁇ - branched amino acid residue, and Ps , P7 and P9 are each independently any amino acid residue or analog; obtaining a second blood sample from the subject during or subsequent to treatment, detecting and quantifying misfolded TTR oligomer in the second blood sample by contacting the second blood sample with the polypeptide compound, and monitoring
  • the blood sample is a plasma sample or a serum sample.
  • the method further comprises adjusting the treatment based on the misfolded TTR oligomer in the second blood sample.
  • the polypeptide in the compound consists of an amino acid sequence selected from the group consisting of VAVHVF (SEQ ID NO: 1), AINVAVHVFR (SEQ ID NO: 2), AINAAAHVFR (SEQ ID NO: 3), AINAAVHVFR (SEQ ID NO: 4), AINVAAHVFR (SEQ ID NO: 5), AINVAVHAFR (SEQ ID NO: 6), AANVAVHVFR (SEQ ID NO: 7), AINVAVHVAR (SEQ ID NO: 8), AINVAVAVFR (SEQ ID NO: 9), AIAVAVHVFR (SEQ ID NO: 10), INVAVHVFR (SEQ ID NO: 11), NVAVHVFR (SEQ ID NO: 12), VAVHVFR (SEQ ID NO:
  • FIG. 1 depicts, in accordance with embodiments herein, that the B ⁇ -strand of transthyretin (TTR) labels non-native TTR aggregates.
  • TTR transthyretin
  • FIG. 2 depicts, in accordance with embodiments herein, that the B ⁇ -strand (Bl) is a selective probe for recombinant TTR aggregates within buffer and human plasma.
  • Bl labels TTR aggregates and minimally natively folded tetramers. Native gel of Bl incubated with TTR tetramers with the indicated hereditary mutations.
  • Incorporation is measured by Native PAGE or Size Exclusion Chromatography (SEC). D) Native PAGE. E) SEC. F) Effect of alanine mutations on peptide incorporation into MTTR oligomers added into plasma from three healthy donors. The peptides are the same as in panel B (SEQ ID NOs: 2-10, respectively). Incorporation was measured by integrating the peak in the high MW fraction of the SEC chromatogram (shaded in panel E). All errors are ⁇ s.d. *p-value ⁇ 0.05.
  • FIG. 3 depicts, in accordance with embodiments herein, that the B peptide differentiates patients from controls.
  • FIG. 4 depicts, in accordance with embodiments herein, that the B peptide differentially labels TTR in patient plasma.
  • FIG. 5 depicts, in accordance with embodiments herein, that TTR photo- crosslinking by B2 correlates with Bl incorporation into the high MW fractions.
  • B) Labeling of TTR in the high MW fraction by B2 photo-crosslinking correlates with the high MW fluorescence signal from the SEC chromatograms (correlation coefficient 0.85).
  • FIG. 6 depicts, in accordance with embodiments herein, that Clusterin is a target of B-2.
  • Representative V30M patient and control plasma samples probed with B-2 and an anti-Clusterin antibody (A) and Streptavidin pull-down CuACC click conjugation of Biotin to B-2 after photo-crosslinking (B).
  • Figure 7 depicts, in accordance with embodiments herein, that probe B-1 detects disease-relevant structures in patient plasma.
  • Circulating misfolded transthyretin oligomers in plasma of familial amyloid polyneuropathy patients correlate with early clinical symptoms (r 2 0.5013), and B) decrease upon tafamidis treatment in a subset of patients (upper panel) or remain stable in other patient (lower panel).
  • Figure 8 depicts, in accordance with embodiments herein, screen of fragment peptides with TTR derived aggregates or oligomers. Each fragment peptide was incubated overnight with the listed aggregates and incorporation was assessed by size exclusion chromatography.
  • Figure 9 depicts, in accordance with embodiments herein, that M-TTR oligomers and TTR52-127 actively aggregating more readily incorporate probe B-1 relative to oligomers that have been aged.
  • FIG. 10 depicts, in accordance with embodiments herein, that the B-peptide does not incorporate into ⁇ 1-42 ADDLs. After overnight incubation and size exclusion chromatography, the B-peptide signal shows minimal overlap with the ⁇ 1-42 signal from the monomer, dimer and trimer species. Overlapping signal is observed in the high MW region (5-6 minutes), although comprises -1% or less of the total B-peptide fluorescent signal.
  • Figure 11 depicts, in accordance with embodiments herein, a comparison of binding activities of peptide Bl and several shortened variants (SEQ ID NOs: 2, 11-14, 1 and 15-18, respectively).
  • the data suggest a minimal binding motif of VAVHVF (SEQ ID NO: l).
  • Relative peptide incorporation as measured by Native PAGE.
  • Each B-1 deletion mutant labeled with fluorescein was incubated with 1 week old MTTR oligomers. After fluorescence imaging of the PAGE gel the oligomer band density was normalized to the B-1 sequence (AINVAVHVFR) (SEQ ID NO: 2).
  • Figure 12 depicts, in accordance with embodiments herein, linear labeling of M- TTR oligomers in human healthy plasma by probe B-1.
  • Increasing concentrations of MTTR derived oligomers were incubated in healthy human plasma from three different donors and next these oligomers were selectively labeled using B-1 probe (20 ⁇ ).
  • Figure 13 depicts, in accordance with embodiments herein, that 4% of healthy controls show some labeling and the presence of cleaved TTR.
  • Figure 14 depicts, in accordance with embodiments herein, that the B-peptide does not cross-react with the Anti-TTR antibody (DAKOTM, #A0002).
  • MTTR or B-l was spotted onto the membrane and either directly imaged (fluorescein channel) or probed with the polyclonal Anti-TTR antibody from DAKOTM.
  • Figure 15 depicts, in accordance with embodiments herein, that B-2 does not label the haptoglobin alpha chain.
  • Figure 16 depicts, in accordance with embodiments herein, that probe B-l is selective for nascently formed soluble non-native oligomeric TTR.
  • B) Probe B-l is most selective for nascently formed MTTR oligomers ( ⁇ 3 days old) as measured by SEC. (Right) Native PAGE showing the molecular weight and labeling intensity differences between 1 -week-old MTTR oligomers and 2-month-old MTTR oligomers.
  • Figure 17 depicts, in accordance with embodiments herein, that probe B-l selectively differentiates FAP patient samples from controls.
  • Figure 18 depicts, in accordance with embodiments herein, that probe B-2 labels non-native TTR in patient plasma.
  • TTR labeling by B-2 is indicated by the magenta box.
  • Figure 19 depicts, in accordance with embodiments herein, that targeted mass spectrometry confirms non-native TTR as a target of the B-peptide.
  • Figure 20 depicts, in accordance with embodiments herein, labeling of high molecular weight non-native TTR oligomers decreases V30M FAP patients treated with tafamidis or liver transplantation.
  • Figure 21 depicts, in accordance with embodiments herein, non-native TTR is detected in additional ATTR mutations and not detected in cardiomyopathy-associated genotypes (V122I and WT).
  • V30M FAP data from Fig. 4G is presented for comparison.
  • Figure 22 depicts, in accordance with embodiments herein, that the B ⁇ -strand of TTR labels TTR50-127 oligomers.
  • A AFM image of TTR50-127 oligomers that were used to assay incorporation.
  • B Quantification of incorporation of each candidate TTR-derived peptide probe into TTR50-127 oligomers using size exclusion chromatography.
  • Figure 23 depicts, in accordance with embodiments herein, determination of probe stoichiometry in MTTR Oligomers.
  • B Method for determination of fluorescence quenching and stoichiometry of B-1 bound to recombinant oligomers.
  • Figure 24 depicts, in accordance with embodiments herein, additional microscopy images showing that the B-peptide does not label V30M FAP derived TTR amyloid in salivary gland biopsies.
  • FIG. 26 depicts, in accordance with embodiments herein, that a similar B-1 probe Structure Activity Relationship (SAR) is found when MTTR oligomers are incubated in healthy plasma.
  • SAR Structure Activity Relationship
  • MTTR oligomers were incubated in plasma of three healthy donors and the incorporation of the B-1 alanine mutants was measured by size exclusion chromatography (see Fig. 1G for similar results in buffer).
  • Each bar represents the mean of the three plasma samples for a specified mutant; all error bars represent ⁇ s.d.
  • Figure 27 depicts, in accordance with embodiments herein, that labeling of the high MW SEC fraction in FAP patients by disulfoCy5-B is fluorophore independent.
  • Figure 28 depicts, in accordance with embodiments herein, that alanine substitutions of probe B-1 have identical effects on incorporation of peptide into the HMW fraction of FAP V30M patient plasma.
  • the alanine mutants incorporation was measured in three separate patient plasma samples and three healthy control samples using the size exclusion chromatography method.
  • FIG. 29 depicts, in accordance with embodiments herein, that B-1 is the only peptide of the TTR ⁇ -strands that incorporates into the high MW fraction of patient plasma.
  • Each bar represents the mean of the three plasma samples for a specified mutant; all error bars represent ⁇ s.d.
  • Figure 30 depicts, in accordance with embodiments herein, that Diazirine- containing probe B-2 selectively labels oligomeric TTR.
  • FIG 31 depicts, in accordance with embodiments herein, the schematic of B-2 non-native TTR gel quantification method and representative data.
  • the samples were photocrosslinked, separated by SEC using the Agilent Bio SEC- 3 4.6 x 300 mm column and N3-Rhodamine was then clicked onto the B-2 alkyne handle.
  • the samples were then analyzed by reducing SDS PAGE and the bands were quantified by densitometry and then normalized to the total protein present.
  • each gel was loaded with a standard curve of retroaldolase labeled quantitatively with a single alkyne as described in the Methods.
  • the gel above depicts 5 plasma samples from V30M asymptomatic carriers (5 unique individuals) and 5 V30M FAP patients (5 unique individuals).
  • Figure 32 depicts, in accordance with embodiments herein, that probe B-1 does not cross-react with the anti-TTR antibody.
  • MTTR or probe B-1 was spotted onto the nitrocellulose membrane and either directly imaged (fluorescein channel) or probed with the polyclonal Anti-TTR antibody.
  • Figure 33 depicts, in accordance with embodiments herein, correlation of the spectral counts (MSI spectra) of the identified protein targets in the B-2/B-2-Mut treated samples from V30M FAP patients (average of 3 patients) with plasma concentration. Approximate human plasma concentrations were obtained from the Plasma Proteome Database (accessed October 1, 2016).
  • Figure 34 depicts, in accordance with embodiments herein, validation of N- terminally cleaved non-native TTR as a target of the B-peptide in V30M FAP patient plasma.
  • the inventors have developed novel peptide-based probes that allow detection of misfolded transthyretin oligomers in the plasma of patients with hereditary transthyretin amyloid polyneuropathy and these probes reveal that the levels of these oligomers are lowered upon treatment with disease-modifying therapies.
  • TTR amyloid polyneuropathy a chronic respiratory disease characterized by apoptosis.
  • oligomer levels decrease in TTR amyloid polyneuropathy patients treated with disease-modifying therapies (Tafamidis or liver transplant-mediated gene therapy). Quantification of plasma oligomer levels by such peptide probes could become an early diagnostic strategy, a response-to-therapy biomarker, and a useful tool for understanding structure-proteotoxicity relationships in the TTR amyloidoses.
  • a compound comprising a polypeptide comprising a sequence P1P2P3P4P5P6P7P8P9P10 or a pharmaceutically acceptable salt thereof, wherein Pi, P 3 and P 10 are each independently any amino acid residue, analog or absent; P2 is a ⁇ -branched amino acid residue, analog or absent; P 4i ⁇ and Pg are each independently a ⁇ - branched amino acid residue; and Ps , P7 and P9 are each independently any amino acid residue or analog.
  • the polypeptide is a synthetic or recombinant peptide.
  • the polypeptide consists of from 6 to about 20 amino acid residues.
  • the compound further comprises a diagnostic moiety.
  • the diagnostic moiety is an alkyne reporter group and/or an alkyl diazirine reactive group.
  • the diagnostic moiety is conjugated to the polypeptide.
  • the diagnostic moiety is conjugated to the N-terminal residue of the peptide.
  • the diagnostic moiety comprises an absorbent, fluorescent or luminescent label moiety.
  • the diagnostic moiety comprises a fluorophore, a coumarin moiety, or a rhodamine moiety.
  • P 4i ⁇ and Ps are each Val
  • P5 is a hydrophobic amino acid residue
  • P7 is a hydrophobic amino acid residue or His
  • P9 is a hydrophobic amino acid residue
  • P3 is an uncharged polar amino acid residue or Ala.
  • ⁇ 2 ⁇ P 4i ⁇ and Pg are each independently Val or He
  • P3 is Asn or Ala
  • P5 is Ala
  • P7 is His or Ala
  • P9 is Phe or Ala.
  • the polypeptide consists of an amino acid sequence INVAVHVF (SEQ ID NO: 21).
  • Pi is a hydrophobic amino acid residue or absent
  • P2 is a ⁇ -branched amino acid residue
  • P3 is an uncharged polar amino acid residue or Ala
  • P lo is Arg, Lys or absent.
  • ⁇ 2 ⁇ ⁇ 4 ⁇ ⁇ and Pg are each independently Val or He
  • Pi is Ala or propargyl glycine
  • P3 is Asn or Ala
  • P5 is Ala
  • P7 is His or Ala
  • P9 is Phe or Ala
  • P lo is Arg.
  • the polypeptide consists of an amino acid sequence selected from the group consisting of VAVHVF (SEQ ID NO: 1), AINVAVHVFR (SEQ ID NO: 2), AINAAAHVFR (SEQ ID NO: 3), AINAAVHVFR (SEQ ID NO: 4), AINVAAHVFR (SEQ ID NO: 5), AINVAVHAFR (SEQ ID NO: 6), AANVAVHVFR (SEQ ID NO: 7), AINVAVHVAR (SEQ ID NO: 8), AINVAVAVFR (SEQ ID NO: 9), AIAVAVHVFR (SEQ ID NO: 10), INVAVHVFR (SEQ ID NO: 11), NVAVHVFR (SEQ ID NO: 12), VAVHVFR (SEQ ID NO: 13), AVHVFR (SEQ ID NO: 14), VAVHV (SEQ ID NO: 15), AINVAVHVF (SEQ ID NO: 16), AINVAVHV (SEQ ID NO:
  • the polypeptide consists of an amino acid sequence AINVAVHVFR (SEQ ID NO: 2), and the diagnostic moiety comprises 6-(fluorescein-5-carboxamido)hexanoic acid (5- FAM-X).
  • the polypeptide consists of an amino acid sequence propargyl glycine-INVAVHVFR (SEQ ID NO: 22), and the diagnostic moiety comprises a photoreactive diazirine group and a terminal alkyne group.
  • a method for diagnosing TTR amyloidosis in a subject comprising: providing a blood sample of the subject; contacting the blood sample with a compound comprising a polypeptide comprising a sequence P1P2P3P4P5P6P7P8P9P10 or a pharmaceutically acceptable salt thereof, wherein Pi, P 3 and P 10 are each independently any amino acid residue, analog or absent; P2 is a ⁇ -branched amino acid residue, analog or absent; P ⁇ ⁇ and Pg are each independently a ⁇ -branched amino acid residue; and Ps , P7 and P9 are each independently any amino acid residue or analog; and diagnosing TTR amyloidosis if there is binding between a misfolded TTR oligomer in the blood sample and the polypeptide compound.
  • the blood sample is a plasma sample or a serum sample.
  • the method further comprises treating the TTR amyloidosis in the subject by administering an effective amount of TTR amyloidosis medicine to the subject.
  • TTR amyloidosis is treated by administering an effective amount of a transthyretin kinetic stabilizer.
  • the transthyretin kinetic stabilizer is tafamidis.
  • the method further comprises a diagnostic moiety conjugated to the polypeptide compound.
  • the diagnostic moiety is an alkyne reporter group and/or an alkyl diazirine reactive group.
  • the diagnostic moiety comprises an absorbent, fluorescent, or luminescent label moiety.
  • the diagnostic moiety comprises a fluorophore, a coumarin moiety, or a rhodamine moiety.
  • the specific binding is detected via a fluorescence assay. In one embodiment, the specific binding is detected via photocrosslinking and ELISA assay.
  • the polypeptide consists of an amino acid sequence selected from the groups VAVHVF (SEQ ID NO: 1), AINVAVHVFR (SEQ ID NO: 2), AINAAAHVFR (SEQ ID NO: 3), AINAAVHVFR (SEQ ID NO: 4), AINVAAHVFR (SEQ ID NO: 5), AINVAVHAFR (SEQ ID NO: 6), AANVAVHVFR (SEQ ID NO: 7), AINVAVHVAR (SEQ ID NO: 8), AINVAVAVFR (SEQ ID NO: 9), AIAVAVHVFR (SEQ ID NO: 10), INVAVHVFR (SEQ ID NO: 11), NVAVHVFR (SEQ ID NO: 12), VAVHVFR (SEQ ID NO: 13), AVHVFR (SEQ ID NO: 14), VAVHV (SEQ ID NO: 15), AINVAVHVF (SEQ ID NO: 16), AINVAVHV (SEQ ID NO: 17),
  • a method for monitoring treatment of a patient being treated for TTR amyloidosis comprising: obtaining a first blood sample from the subject prior to treatment, detecting and quantifying misfolded TTR oligomer in the first blood sample by contacting the first blood sample with a compound comprising a polypeptide comprising a sequence P1P2P3P4P5P6P7P8P9P10 or a pharmaceutically acceptable salt thereof, wherein Pi, P 3 and P 10 are each independently any amino acid residue, analog or absent, P2 is a ⁇ -branched amino acid residue, analog or absent, P ⁇ ⁇ and Pg are each independently a ⁇ - branched amino acid residue, and Ps , P7 and P9 are each independently any amino acid residue or analog; obtaining a second blood sample from the subject during or subsequent to treatment, detecting and quantifying misfolded TTR oligomer in the second blood sample by contacting the second blood sample with the polypeptide compound, and monitoring
  • the blood sample is a plasma sample or a serum sample.
  • the method further comprises adjusting the treatment based on the misfolded TTR oligomer in the second blood sample.
  • the polypeptide in the compound consists of an amino acid sequence selected from the group consisting of VAVHVF (SEQ ID NO: 1), AINVAVHVFR (SEQ ID NO: 2), AINAAAHVFR (SEQ ID NO: 3), AINAAVHVFR (SEQ ID NO: 4), AINVAAHVFR (SEQ ID NO: 5), AINVAVHAFR (SEQ ID NO: 6), AANVAVHVFR (SEQ ID NO: 7), AINVAVHVAR (SEQ ID NO: 8), AINVAVAVFR (SEQ ID NO: 9), AIAVAVHVFR (SEQ ID NO: 10), INVAVHVFR (SEQ ID NO: 11), NVAVHVFR (SEQ ID NO: 12), VAVHVFR (SEQ ID NO:
  • composition comprising a compound comprising a polypeptide having the sequence P1P2P3P4P5P6P7P8P9P10 or a pharmaceutically acceptable salt thereof, wherein Pi, P 3 and P lo are each independently any amino acid residue, analog or absent; P2 is a ⁇ -branched amino acid residue, analog or absent; P ⁇ ⁇ and Pg are each independently a ⁇ -branched amino acid residue; and Ps , P7 and P9 are each independently any amino acid residue or analog; and a pharmaceutically acceptable excipient.
  • “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration.
  • Route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral.
  • Parenteral refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.
  • the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.
  • compositions according to the invention can also contain any pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body.
  • the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof.
  • Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
  • compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration.
  • Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition.
  • Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water.
  • Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin.
  • the carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • the pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms.
  • a liquid carrier When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension.
  • Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
  • the pharmaceutical compositions according to the invention may be delivered in a therapeutically effective amount.
  • the precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration.
  • Typical dosages of an effective composition can be in the ranges recommended by the manufacturer where known therapeutic compounds are used, and also as indicated to the skilled artisan by the in vitro responses or responses in animal models. Such dosages typically can be reduced by up to about one order of magnitude in concentration or amount without losing the relevant biological activity. Thus, the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of the relevant primary cultured cells or histocultured tissue sample, such as biopsied malignant tumors, or the responses observed in the appropriate animal models.
  • TTR Transthyretin
  • TTR Transthyretin
  • RBP holo-retinol binding protein
  • TTR is one of greater than 20 nonhomologous amyloidogenic proteins that can be transformed into fibrils and other aggregates leading to disease pathology in humans (ATTR amyloidosis). These diseases do not appear to be caused by loss of function due to protein aggregation. Instead, aggregation appears to cause neuronal/cellular dysfunction by a mechanism that is not yet clear. Under denaturing conditions, rate limiting wild type TTR tetramer dissociation and rapid monomer misfolding enables misassembly into amyloid that causes wildtype TTR amyloidsis (WT-ATTR). Dissociation and misfolding of one of more than 120 TTR variants results in hereditary ATTR amyloidoses, including familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC).
  • FAP familial amyloid polyneuropathy
  • FAC familial amyloid cardiomyopathy
  • TTR amyloid diseases are diseases that are caused by amyloid deposits made up of transthyretin (TTR).
  • TTR amyloidosis There are a few distinct different types of ATTR amyloidosis, including (1) familial amyloid polyneuropathy (FAP) which is hereditary and can overlap with FAC, (2) familial amyloid cardiomyopathy (FAC) which is hereditary and can overlap with FAP), and (3) senile systemic amyloidosis or wild- type ATTR amyloidosis which is not hereditary and it mostly causes a cardiomyopathy.
  • FAP familial amyloid polyneuropathy
  • FAC familial amyloid cardiomyopathy
  • senile systemic amyloidosis or wild- type ATTR amyloidosis which is not hereditary and it mostly causes a cardiomyopathy.
  • the inventors synthesized peptide-based probes that selectively integrate into the structure(s) of the non-native TTR oligomers that can be prepared in vitro and integrate into apparently similar structures that circulate in TTR polyneuropathy and cardiomyopathy patient blood.
  • This approach of quantifying misfolded TTR oligomers in patient plasma could be useful not only for aiding physicians in point-of-care diagnosis and in following the response to particular therapies, but also for basic science purposes in terms of understanding the aggregate structure-proteotoxicity relationships driving the TTR amyloidoses.
  • the methods described herein enables detection of non-native TTR oligomers in patients suffering from TTR amyloidoses - both polyneuropathy and cardiomyopathy phenotypes.
  • An important aspect of the probes described herein is that, unlike other probes used for diagnostic purposes (i.e., Congo Red or thioflavin T), these probes preferentially recognize nascently misfolded and actively aggregating TTR, making them suitable for detection of the earliest possible misfolding events that lead to TTR related amyloid diseases.
  • TTR related systemic amyloid disease class and the probes disclosed herein provide the initial tools to understand the tissue tropism of the amyloid diseases. For example, why is the peripheral nervous system affected in V30M-FAP, versus the heart in VI 221 related cardiomyopathies?
  • probes developed here will certainly assist future studies directed at elucidating the structure-proteotoxicity relationship driving loss of postmitotic tissue, which is currently a major gap in understanding amyloid diseases mechanisms.
  • a specific probe described herein, probe B-2 can detect circulating TTR that is fragmented, which heretofore has only been detected in amyloid tissue biopsies, and may explain particular aspects of the tissue tropism associated with these diseases. It is possible that particular variants including the WT protein are or become susceptible to proteolysis, which leads to non-native TTR that preferentially targets the heart.
  • the non-native oligomeric TTR that probes B-l and B-2 detect in patient blood is not without other protein partners.
  • the peptidomimetic probes enable numerous diagnostic applications described herein. For example, both the probe B-l fluorescence assay and the probe B-2 photo-crosslinking and SDS-PAGE gel assay described in the Examples below can be applied to other amyloid diseases to detect the onset of these diseases at the earliest possible time-point.
  • the present disclosure provides novel peptides or peptidomimetic probes that can be used in diagnosing distinct TTR amyloid diseases or monitoring disease status. Also provided in the instant disclosure are various diagnostic uses and disease monitoring applications of such probes.
  • peptide and “polypeptide,” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length unless otherwise defined.
  • Peptides and polypeptides are composed of linearly arranged amino acids linked by peptide bonds, and may be produced biologically and isolated from the natural environment, produced using recombinant technology, or produced synthetically typically using naturally occurring amino acids.
  • the polypeptide is a "modified polypeptide" comprising non-naturally occurring amino acids.
  • the polypeptides comprise a combination of naturally occurring and non-naturally occurring amino acids, and in some embodiments, the peptides comprise only non-naturally occurring amino acids.
  • peptide is contemplated to encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to, N-terminus modification, C-terminus modification, peptide bond modification, backbone modification, and/or side chain modification.
  • amino acid of a peptide refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Unless otherwise noted, the TTR derived probe peptides of the present disclosure may encompass derivative or analogs which have be modified with non-naturally coding amino acids.
  • Natural amino acids that make up a polypeptide or protein can be grouped according to what their side chains are like. Based on the propensity of the side chain to be in contact with polar solvent like water, it may be classified as hydrophobic, polar or charged.
  • Hydrophobic amino acid refer to amino acids or residues that have hydrophobic side chains. These include glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (He), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp). These side chains are composed mostly of carbon and hydrogen, have very small dipole moments, and tend to be repelled from water.
  • Amyloidosis is a rare disease that results from the buildup of misfolded proteins into a spectrum of aggregates including oligomers and amyloid fibrils.
  • proteins that are normally dissolvable in water misassemble into amyloid fibrils they become insoluble and deposit in organs or tissues, disrupting normal function.
  • the type of protein that is misfolded and misassembled, and the organ or tissue in which the misfolded aggregated proteins are deposited determine the clinical manifestations of the specific amyloidosis.
  • the second most common is AA amyloidosis due to the accumulation of S amyloid A protein or SAA, which occurs in association with chronic infections - e.g. tuberculosis - or inflammatory illnesses such as rheumatoid arthritis.
  • SAA S amyloid A protein
  • the third and the fourth type are due to the deposition of a genetically defective or normal form of a protein called transthyretin respectively.
  • a "branched-chain amino acid” is an amino acid having aliphatic side-chains with a branch (a central carbon atom bound to three or more carbon atoms).
  • proteinogenic amino acids there are two ⁇ -branched amino acids (isoleucine and valine) and one ⁇ -branched amino acid (leucine).
  • non-proteinogenic branched- chain amino acids include norvaline and 2-aminoisobutyric acid.
  • the peptide probes of the instant disclosure may include non-proteinogenic branched-chain amino acids.
  • the term "consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • “conservatively modified variants” refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a "derivative" of a reference molecule is a molecule that is chemically modified relative to the reference molecule while substantially retaining the biological activity.
  • the modification can be, e.g., oligomerization or polymerization, modifications of amino acid residues or peptide backbone, cross-linking, cyclization, conjugation, fusion to additional heterologous amino acid sequences, or other modifications that substantially alter the stability, solubility, or other properties of the peptide.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same.
  • Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • a "diagnostic moiety” or “label moiety” is a functional group, compound, molecule, substituent, or the like, that can enable detection of a target molecule (e.g., a protein or peptide) to which it is conjugated, either via covalent bonding or non- covalent attachment. It can provide a detectable biological or physiochemical signal that allows detection via any means, e.g., fluorescence, phosphorescence, absorbance, luminescence, chemiluminescence, radioactivity, colorimetry, magnetic resonance, or the like.
  • the detectable signal provided by the diagnostic moiety or label moiety can be directly due to a biochemical or physiochemical property of the moiety (e.g., a fluorophore) or indirectly due to its interaction with another compound or agent.
  • a biochemical or physiochemical property of the moiety e.g., a fluorophore
  • the diagnostic moiety or label moiety does not encompass peptide sequences that are naturally present in the TTR protein.
  • the diagnostic moiety or label moiety contemplated in the instant disclosure is a small functional group or small organic compound.
  • the employed diagnostic moiety or label moiety has a molecular weight of less than about 1,000 Da, 750 Da, 500 Da or even smaller.
  • Non-native or misfolded TTR oligomer refers to soluble oligomers or other aggregate types of TTR which are formed as a result of the misfolding and misassembly of TTR monomers. Unlike normal circulating TTR which are tetrameric, these TTR aggregates results from misfolding and misassembly of monomeric TTR molecules released by dissociation of the normal tetrameric TTR.
  • photoactivatable crosslinker is used herein to indicate a reactive functional group capable of becoming covalently bound to another molecule upon irradiation by light, preferably ultraviolet light.
  • a diazirine ring is an example photoactivatable crosslinker that can be employed in the instant disclosure, and it typically involves the generation of a carbene.
  • a diazirine functional group has advantages in that it will only result in minimal structural changes within the target molecule to which it binds.
  • terminal alkyne group refers to reactive species in a number of important chemical reactions such as the 1,3-dipolar cycloadditionl between azides and alkynes to give 1,2,3-triazoles (click-chemistry), the Sonogashira reaction and its forerunner, the Stephens-Castro reaction. Furthermore, alkynes can undergo the Vollhardt cyclization, alkyne trimerization to form aromatic compounds, or can act as dienophiles in Diels-Alder reactions. A number of protecting groups have been developed for alkyne chemistry such as trialkylsilyl, benzyl- or phenyl-substituted alkylsilyl groups and propargylic alcohols.
  • a terminal alkyne group used in the present disclosure is capable of being covalently linked in a chemical reaction with a molecule containing an azide.
  • the terminal alkyne or azide can serve as a non-native and non-perturbing bioorthogonal chemical handle that can be derivatized employing a chemistry that is known as click chemistry.
  • photolysis is meant to indicate the light induced activation of a photoactivatable group (e.g., a diazirine functional group) resulting in a reactive species (e.g., carbene) that can form a covalent linkage with a molecule in close proximity.
  • a photoactivatable group e.g., a diazirine functional group
  • a reactive species e.g., carbene
  • the target molecule e.g., a TTR derived peptide
  • click chemistry refers to the copper(I)-catalyzed [3+2]- Huisgen 1,3-dipolar cyclo-addition of terminal alkynes and azides leading to 1,2,3-triazoles. It may also refer to a copper free variant of this reaction that might also be used. (J. M. Baskin, J. A. Prescher, S. T. Laughlin, N. J. Agard, P. V. Chang, I. A. Miller, A. Lo, J. A. Codelli, C. R. Bertozzi, Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16793.).
  • peptide mimetic refers to a derivative compound of a reference peptide (e.g., a TTR derived probe polypeptide disclosed herein) that biologically mimics the peptide's functions.
  • a peptidomimetic derivative of a TTR derived probe peptide may have at least 25%, at least 50%, at least 75% or at least 90% of the misfolded TTR oligomer-binding activity of the reference peptide.
  • the term "subject” includes human and non-human animals.
  • Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, and reptiles. Except when noted, the terms “patient” or “subject” are used herein interchangeably.
  • the term “variant” refers to a molecule (e.g., a polypeptide or peptide) that contains a sequence that is substantially identical to the sequence of a reference molecule. In some other embodiments, the variant differs from the reference molecule by having one or more conservative amino acid substitutions but substantially retains the biological activity of the reference molecule.
  • the instant disclosure provides synthetic or recombinantly produced peptides that are capable of specifically binding to soluble non-native TTR oligomers.
  • these probe peptides or peptide probes can selectively integrate into both the structure of non-native TTR oligomers prepared in vitro and similar structures found in the blood of TTR amyloidosis patients.
  • the peptide probes disclosed herein typically contain from 6 amino acid residues to about 20 amino acid residues.
  • the peptide probes can contain 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues.
  • the peptides contain from 6 amino acid residues to about 12 amino acid residues.
  • some of these peptides contain about 8 amino acid residues.
  • Some other peptides contain about 9 amino acid residues.
  • Some other peptides contain about 10 amino acid residues.
  • peptides of the present disclosure also contain a 6-aa sequence motif P4P5P6P7P8P9.
  • residues ⁇ 4 ⁇ ⁇ and Pg are each independently a branched amino acid residue such as ⁇ -branched residues, and the other residues each independently may be any amino acid residue or analog.
  • residue P5 is a hydrophobic amino acid residue
  • residue P7 is a hydrophobic amino acid residue
  • His or Lys residue P9 is a hydrophobic amino acid residue.
  • residues P 4i ⁇ and Pg are each independently Val or He, residue P5 is Ala, residue P7 is His or Ala, and residue P9 is an aromatic amino acid residue or Ala.
  • residues P 4i ⁇ and Pg are each Val, residue P5 is Ala, residue P7 is His or Ala, and residue P9 is Phe, Tyr or Ala.
  • the peptides contain a 7-aa sequence motif. Relative to the 6-aa sequence motif, the motif additionally contains an extra ⁇ - branched amino acid residue at its C-terminus.
  • the 6-aa or 7-aa sequence motif is VAVHVF (SEQ ID NO: 1), INVAVH (SEQ ID NO: 19), or INVAVHV (SEQ ID NO: 20).
  • VAVHVF SEQ ID NO: 1
  • INVAVH SEQ ID NO: 19
  • INVAVHV SEQ ID NO: 20
  • a number of peptides of varying length that harbor one of these sequence motifs are able to bind to misfolded TTR oligomers. These include 6-aa peptide VAVHVF (SEQ ID NO: 1), 7-aa peptide VAVHVFR (SEQ ID NO: 13), 8-aa peptides NVAVHVFR (SEQ ID NO: 12) and AINVAVHV (SEQ ID NO: 17).
  • the peptides contain an 8-aa sequence motif P2P 3 P4P5P 6 P7- PgP i.
  • residue P2 in this 8-aa sequence is a branched amino acid residue such as ⁇ -branched residue Val or He
  • residue P3 is any amino acid residue or analog.
  • residue P3 is an uncharged polar amino acid residue or Ala.
  • residues P 2i P 4i ⁇ and Pg are each independently Val or He
  • residue P3 is Asn or Ala
  • residue P5 is Ala
  • residue P7 is His or Ala
  • residue P9 is Phe or Ala.
  • Examples of such peptides that have shown activities in binding to misfolded TTR oligomer include, e.g., 8-aa peptide INVAVHVF (SEQ ID NO: 21), and 9-aa peptides INVAVHVFR (SEQ ID NO: 11) and AINVAVHVF (SEQ ID NO: 16).
  • the peptides contain a sequence motif P1P2P3P4P5P6P7- P 8 P 9 P1 0 .
  • residue Pi in this sequence motif can be any amino acid residue, analog or absent
  • residue P2 is a ⁇ -branched amino acid residue
  • residue P3 is any amino acid residue or analog
  • residue P 10 is any amino acid residue, analog or absent.
  • residue Pi is a hydrophobic amino acid residue or absent
  • residue P2 is a ⁇ - branched amino acid residue
  • residue P3 is an uncharged polar amino acid residue or Ala
  • residue P lo is Arg, Lys or absent.
  • residues ⁇ 2 ⁇ ⁇ 4 ⁇ ⁇ and Pg are each independently Val or He
  • residue Pi is Ala
  • residue P3 is Asn or Ala
  • residue P5 is Ala
  • residue P7 is His or Ala
  • residue P9 is Phe or Ala
  • residue P lo is Arg.
  • Exemplary peptides harboring such a sequence motif that have demonstrated binding activity for misfolded TTR oligomer include AINVAVHVFR (SEQ ID NO: 2), propargyl glycine-INVAVHVFR (SEQ ID NO: 22), AIAVAVHVFR (SEQ ID NO: 10), AINVAVAVFR (SEQ ID NO: 9), and AINVAVHVAR (SEQ ID NO: 8).
  • the present disclosure provides peptide probe compounds that contain a synthetic or recombinant peptide and a diagnostic moiety.
  • the present disclosure provides modifying the peptides of SEQ ID Nos: 1-22 by incorporating a diazirine functional group and an alkyne handle.
  • probe B-2 As illustrated below.
  • probe B-2 it may be advantageous to use probe B-2 over probe B-l because after photocrosslinking, probe B-2 is covalently attached to the proteins it initially binds to, thus denaturing and reducing SDS PAGE can be used to identify the proteins bound by probe B-2, including initially aggregated non-native TTR (Fig. 18B).
  • non-denaturing separation methods like SEC must be used to preserve the non-covalent probe B-l interactions.
  • probe B-2 displays a high degree of conformational selectivity for non-native TTR oligomers, as does probe B-l .
  • an important advantage of the covalent peptide-based probe B- 2 introduced herein is that, unlike other amyloid-selective probes used for diagnostic purposes (e.g., Congo Red or thioflavin T), these probes preferentially recognize soluble, misfolded and actively aggregating TTR oligomers that adopt a non-amyloid conformation, rendering these probes suitable for detection of early misfolding events that may lead to degenerative phenotypes characteristic of the TTR amyloidoses.
  • probes detect non-native TTR in neuropathic TTR amyloidosis patients that are very early on in the course of pathology (NIS-LL ⁇ 10), although with this small cohort, and a correlation with the NIS-LL was not observed.
  • the ability of probe B-2 to detect circulating fragmented TTR in a subset of polyneuropathy patients allows a skilled artisan in the art to understand whether particular variants are more susceptible to proteolysis, which in turn could change the type of TTR aggregates that are formed, leading to unique proteotoxicity mechanisms and potentially explaining why there is early onset vs. late onset V30M FAP disease or why men typically progress faster than women in FAP.
  • probe B-2 also provides the data for understanding the tissue tropism of the TTR amyloid diseases.
  • a one amino acid change in the TTR sequence determines whether non-native TTR oligomers are detected in patient plasma, suggesting that distinct aggregate structures influence whether the peripheral nervous system or the heart is compromised by a given TTR sequence.
  • Probe B-2 is not able to detect circulating non-native TTR oligomers in the plasma of patients harboring cardiomyopathy associated mutations, suggesting that a unique aggregate structure and rapid heart deposition may contribute to the distinct disease etiology in cardiomyopathy.
  • Evidence from other amyloid diseases indicates that conformational differences within amyloid fibrils themselves may be linked with different disease phenotypes.
  • Peptide-based probes for soluble ⁇ oligomers have been developed and exhibit oligomer selectivity.
  • the selectivity of the covalent peptide probe B-2 for non- native oligomeric TTR may be useful as an early diagnostic strategy for FAP or could be used as a response-to-therapy biomarker in polyneuropathy, as indicated within for tafamidis treatment / liver transplant-mediated gene therapy, that both slow the progression of FAP.
  • the probe B-l fluorescence- based assay or the probe B-2 photo-crosslinking approach can be elaborated beyond the methods disclosed herein, and such methods are contemplated by the present disclosure.
  • the probes disclosed herein may be useful in an ELISA format, commonly used by clinical laboratories, rendering these peptide probes generally useful.
  • the probe peptides also encompass variants, analogs, peptidomimetics or other derivative compounds that can be generated from the specific peptide sequences exemplified herein. These derivative compounds can be subject to the assays described herein to ascertain their binding activity for misfolded TTR oligomer. As noted above, the probe peptides typically contain from 6 amino acid residues to about 20 amino acid residues. Thus, in addition to the specific peptide sequences or motifs described herein, peptides that are suitable for probing misfolded TTR oligomer can also include one or more additional residues at the N-terminus and/or the C-terminus.
  • the derivative peptides are modified versions of the exemplified peptides which are generated by conservative amino acid substitutions. In some other embodiments, the derivative peptides are variants produced by non-conservative substitutions to the extent that that they substantially retain the binding activity of the exemplified peptides.
  • the analogs or derivative peptides of an exemplified probe peptide e.g., SEQ ID NOs: 2, 10, 11 or 21
  • polypeptides may also include amino acids that are not linked by polypeptide bonds. Similarly, they can also be cyclic polypeptides and other conformationally constrained structures. Methods for modifying a polypeptide to generate analogs and derivatives are well known in the art, e.g., Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Eds. Gross and Meinhofer, Vol. 5, p. 341, Academic Press, Inc., New York, N.Y. (1983); and Burger 's Medicinal Chemistry and Drug Discovery, Ed. Manfred E. Wolff, Ch. 15, pp. 619-620, John Wiley & Sons Inc., New York, N.Y. (1995).
  • the disclosure provides probe peptidomimetics that are derived from the exemplified peptide sequences.
  • Peptidomimetics based on an exemplified probe peptide substantially retain the activities of the reference peptide. They include chemically modified peptides, peptide-like molecules containing non-naturally occurring amino acids, peptoids and the like, have a structure substantially the same as the reference peptide upon which the peptidomimetic is derived (see, for example, Burger's Medicinal Chemistry and Drug Discovery, 1995, supra).
  • the peptidomimetics can have one or more residues chemically derivatized by reaction of a functional side group.
  • a chemical derivative can have one or more backbone modifications including alpha-amino substitutions such as N-methyl, N-ethyl, N-propyl and the like, and alpha-carbonyl substitutions such as thioester, thioamide, guanidino and the like.
  • alpha-amino substitutions such as N-methyl, N-ethyl, N-propyl and the like
  • alpha-carbonyl substitutions such as thioester, thioamide, guanidino and the like.
  • a peptidomimetic shows a considerable degree of structural identity when compared to the reference peptide or polypeptide, and exhibits characteristics which are recognizable or known as being derived from or related to the reference polypeptide.
  • Peptidomimetics include, for example, organic structures which exhibit similar properties such as charge and charge spacing characteristics of the reference polypeptide. Peptidomimetics also can include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid functional groups.
  • the peptides described herein can include modifications within the sequence, such as, modification by terminal -NH 2 acylation, e.g., acetylation, or thiogly colic acid amidation, by terminal-carboxylamidation, e.g., with ammonia, methylamine, and the like terminal modifications.
  • Terminal modifications are useful to reduce susceptibility by proteinase digestion, and therefore can serve to prolong half-life of the polypeptides in solution, particularly in biological fluids where proteases may be present.
  • Amino terminus modifications include methylation (e.g., -NHCH 3 or -N(CH 3 ) 2 ), acetylation (e.g., with acetic acid or a halogenated derivative thereof such as a-chloroacetic acid, a-bromoacetic acid, or a-iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group, or blocking the amino terminus with any blocking group containing a carboxylate functionality defined by RCOO- or sulfonyl functionality defined by R-SO2-, where R is selected from the group consisting of alkyl, aryl, heteroaryl, alkyl aryl, and the like, and similar groups.
  • the N-terminus is acetylated with acetic acid or acetic anhydride.
  • Carboxy terminus modifications include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints.
  • Other functional groups to modify the C-terminus include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.
  • the probe peptides described herein, including variants and derivatives thereof, can be chemically synthesized and purified by standard chemical or biochemical methods that are well known in the art. Some of the methods are described in the Examples herein. Thus, in some embodiments, solid phase peptide synthesis can be employed for producing the probe peptides and their derivative compounds. In some embodiments, the peptides may be synthesized using t-Boc (tert-butyloxycarbonyl) or FMOC (9-flourenylmethloxycarbonyl) protection group described in the art. See, e.g., "Peptide synthesis and applications" in Methods in molecular biology Vol. 298, Ed.
  • the probe peptides and derivatives thereof may also be synthesized and purified by recombinant methods that are well known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3 rd ed., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003).
  • the probe compound typically contains one or more detectable labels (label moieties or diagnostic moieties).
  • a detectable label is a molecule or functional group that can itself be detected or can produce a detectable signal upon reacting with another molecule under appropriate conditions. It can be detected by a variety of methods including fluorescence, electrical conductivity, radioactivity, size, and the like.
  • the label may be directly or indirectly detectable. For example, the label can be detected directly by its ability to emit and/or absorb light of a particular wavelength.
  • a label can be detected indirectly by its ability to bind, recruit and, in some cases, cleave (or be cleaved by) another compound, thereby emitting or absorbing energy.
  • indirect detection is the use of a reporter function group (e.g., an alkyne group) which can react with and become attached to a reporter molecule (e.g., rhodamine-azide).
  • a reporter function group e.g., an alkyne group
  • a reporter molecule e.g., rhodamine-azide
  • Another example of indirect detection is the use of an enzyme substrate that can be cleaved to produce detectable products.
  • the type of label moieties or diagnostic moieties that may be used are typically sterically and chemically compatible with the peptide component of the probe compound. In general, the label should not interfere with the folding or other activity of the target TTR oligomer protein.
  • the detectable label does not include a peptide sequence that is present in the natural TTR protein.
  • the label in the probe compounds can be a fluorescent molecule, a chemiluminescent molecule (e.g., chemiluminescent substrates), a phosphorescent molecule, a radioisotope, an enzyme substrate, an affinity molecule, a ligand, an antigen, a hapten, an antibody, an antibody fragment, a chromogenic substrate, a contrast agent, an MRI contrast agent, a positron emission tomography (PET) label (e.g., Technetium-99m and fludeoxy glucose), a phosphorescent label, and the like.
  • PET positron emission tomography
  • the detectable label is preferably a small moiety such as a detectable atom (e.g., a radioactive isotope), a small organic molecule, or a small reactive chemical moiety or functional group, as opposed to bigger molecules such as enzymes or other polypeptides.
  • the detectable label in the probe compounds can be a fluorescent group or moiety as exemplified herein.
  • the detectable label can be a photoactivatable cross-linker.
  • the label moiety in the probe compounds can be radioactive isotopes such as 2P or H.
  • the detectable label in the probe compounds can be haptens such as digoxigenin and dintrophenyl.
  • the detectable label can also be an analyte-binding group such as but not limited to a metal chelator (e.g., a copper chelator).
  • the label may also be a heavy atom carrier such as iodine, Au, Pt and Hg.
  • the detectable labels or label moieties in the probe compounds are fluorescent, luminescent, or absorbent label moieties.
  • label moieties include fluorophores, rhodamine moieties, and coumarin moieties (e.g., such as 7- amino-4-carbamoylcoumarin, 7-amino-3-carbamoylmethyl-4-methylcoumarin, or 7-amino-4- methylcoumarin).
  • fluorophores examples include, e.g., fluorescein, fluorescein analogs, BODIPY-fluorescein, arginine, rhodamine-B, rhodamine-A, rhodamine derivatives, and the like.
  • fluorescent label moieties and fluorescence techniques see, e.g., Handbook of Fluorescent Probes and Research Chemicals, by Richard P. Haugland, Sixth Edition, Molecular Probes, (1996).
  • the detectable label is a long wavelength fluorophore such as fluorescent dyes in the Alexa Fluor family (Thermo Fisher Scientific Inc.).
  • a fluorescein derivative such as 5-FAM-X-SE (6 - (Fluorescein - 5 - carboxamido)hexanoic acid, succinimidyl ester) is used to label the probe compounds.
  • This will result in the probe peptide being conjugated to a fluorophore, 6-(fluorescein-5-carboxamido)hexanoic acid (5- FAM-X).
  • An example of such peptide probes is Probe Bl as exemplified herein.
  • Probe Bl contains Peptide AINVAVHVFR (SEQ ID NO: 2) conjugated to a fluorophore diagnostic moiety 5-FAM-X (X refers to the hexanoic acid linker).
  • the detectable label can be attached to the probe peptide at any position.
  • the detectable label is attached to the N-terminal residue of the probe peptide.
  • the attachment can be either covalent or non-covalent.
  • some probe compounds have a detectable label that is covalently bonded to the N- terminus of the probe peptide.
  • Preparation of fluorophore-labeled peptide probes can be readily performed via protocols exemplified herein and/or method well known in the art. See, e.g., Weder et al., J. Chromatogr. 698, 181, 1995; Cavrois et al, Nat. Biotechnol.
  • labeled peptide probes can be prepared by either modifying isolated peptides or by incorporating the label during solid-phase synthesis.
  • fluorophores can be conjugated to the N-terminus of a resin-bound peptide before other protecting groups are removed and the labeled peptide is released from the resin. Labeling of the peptide probes can also be achieved indirectly by using a biotinylated amino acid.
  • biotin group allows specific binding of streptavidin or avidin-conjugate to that site.
  • fluorophores are available as (strept)avidin conjugates.
  • quantum dots can be employed as label moieties.
  • semiconductor nanocrystals or quantum dots such as cadmium selenide and cadmium sulfide can be used as fluorescent probes. See, e.g., Bruchez et al, Science 281 :2013-2016, (1998).
  • the label moieties in the probe compounds are electroactive species for electrochemical detection or chemiluminescent moieties for chemiluminescent detection.
  • UV absorption is also an optional detection method, for which UV absorbers are optionally used.
  • Phosphorescent, colorimetric, e.g., dyes, and radioactive labels can also be optionally attached to the probe peptides.
  • the probe compounds can contain a diagnostic moiety or label moiety that is a photoactivatable cross-linker.
  • a photoactivable cross linker is a photo-affinity group that becomes reactive upon exposure to radiation (e.g., a ultraviolet radiation, visible light, etc.). Examples include diazirine functional groups, benzophenones, aziridines, a photoprobe analog of geranylgeranyl diphosphate (2-diazo- 3,3,3-trifluoropropionyloxy-farnesyl diphosphate or DATFP-FPP) (Quellhorst et al. J Biol. Chem. 2001 Nov.
  • Non-native misfolded TTR oligomers can be readily detected via photo-affinity labeling of the probe compounds with such photo-reactive functional groups.
  • Photo-affinity labeling has been well known for target identification in complex protein mixtures.
  • PAL utilizes a biologically active small-molecular photo-affinity probe that bears photo-reactive and reporter functional groups to identify macromolecular binding partners.
  • the photo- affinity probe is designed and synthesized based on SAR (structure-activity relationships) of a parent small-molecule having known biological activity.
  • SAR structure-activity relationships
  • Photo-crosslinked protein targets are then visualized by the reporter group (e.g. fluorophore, biotin, or radioactive label). Covalent bond formation between the probe and targets enable the subsequent purification and identification of the targets using techniques such as SDS-PAGE, immunoprecipitation, biotin-streptavidin affinity purification and mass spectrometry.
  • a highly reactive chemical species e.g. carbene, nitrene, or radical
  • the reporter group e.g. fluorophore, biotin, or radioactive label
  • the probe peptide is labeled with an alkyl diazirine photo- reactive group.
  • X N, S, O
  • C-H bonds C-H bonds
  • the alkyl carbene intermediate undergoes rapid quenching by solvent or internal rearrangement to a stable olefin side-product.
  • the alkyl diazirine is also stable toward acidic and basic conditions and toward ambient light encountered during routine chemical synthesis.
  • heterobifunctional amine-reactive alkyl diazirine crosslinkers as well as alkyl diazirine- containing amino acid analogs can be obtained commercially, e.g., from Pierce or Thermo Scientific. Labeling a peptide with a diazirine functional group can be readily performed in accordance with the protocols described herein or other methods routinely practiced in the art. See, e.g., MacKinnon et al, Curr. Protoc. Chem. Biol. 1 : 55-73, 2009; Bond et al., Nat. Protoc. 4: 1044-63, 2009; and MacKinnon et al., J. Am. Chem. Soc. 129: 14560-14561, 2007.
  • the probe compound can additionally include a diagnostic moiety that is a reporter function group for conjugation to a detectable reporter compound, e.g., an alkyne reporter group as exemplified herein for Probe B2.
  • a detectable reporter compound e.g., an alkyne reporter group as exemplified herein for Probe B2.
  • detection of a target protein with a photo-affinity labeled probe compound can be carried out via, e.g., Cu(I)-catalyzed click chemistry (see, e.g., Wang et al. J. Am. Chem. Soc. 125: 11164-11165, 2003). This method allows bio-conjugation of probe- labeled proteins with reporter groups.
  • the click reaction catalyzes a highly selective, bio-orthogonal 1,3 dipolar cycloaddition reaction between a terminal alkyne group and an azide group. This results in the formation of a stable triazole product.
  • the terminal alkyne is typically present in the labeled probe compound, while the azide is present in a fluorescent or biotinylated reporter compound, e.g., Rhodamine-azide or biotin-azide.
  • the azide can be incorporated into the probe and the alkyne incorporated into the reporter.
  • the diazirine and azide labeled probe compound covalently labels the TTR oligomer protein upon irradiation, which is then further conjugated to the azi de-bearing reporter under click chemistry conditions.
  • the alkyne group for labeling the probe compound that Rhodamine- azide reporter exemplified herein
  • many other azide function group and alkyne-reporters that are known in the art for bio-conjugate click reactions may also be utilized. See, e.g., Speers et al, Chem Biol. 11 :535-46, 2004.
  • the probe compounds can contain a photolabile protecting group or a photoswitch label.
  • Photolabile protecting groups are useful for photocaging reactive functional groups. Examples of photolabile protecting group include a nitrobenzyl group, a dimethoxy nitrobenzyl group, nitroveratryloxycarbonyl (NVOC), 2- (dimethylamino)-5-nitrophenyl (DANP), Bis(o-nitrophenyl)ethanediol, brominated hydroxy quinoline, and coumarin-4-ylmethyl derivative.
  • a photoswitch label is a molecule that undergoes a conformational change in response to radiation.
  • the molecule may change its conformation from cis to trans and back again in response to radiation.
  • the wavelength required to induce the conformational switch will depend upon the particular photoswitch label.
  • photoswitch labels include azobenzene, 3-nitro-2- naphthalenemethanol.
  • photoswitches are also described in van Delden et al. Chemistry. 2004 January 5; 10(l):61-70; van Delden et al. Chemistry. 2003 June 16; 9(12):2845-53; Zhang et al. Bioconjug Chem. 2003 July-August; 14(4):824-9; Irie et al. Nature. 2002 December 19-26; 420(6917):759-60; as well as many others.
  • binding of the probe compounds to target misfolded TTR oligomer proteins can be either non-covalent or covalent.
  • the binding can be detected via various detection systems. Detection systems may be selected from a number of detection systems known in the art. These include a fluorescent detection system, a photographic film detection system, a chemiluminescent detection system, an enzyme detection system, an atomic force microscopy (AFM) detection system, a scanning tunneling microscopy (STM) detection system, an optical detection system, a nuclear magnetic resonance (NMR) detection system, a near field detection system, and a total internal reflection (TIR) detection system.
  • AFM atomic force microscopy
  • STM scanning tunneling microscopy
  • optical detection system a nuclear magnetic resonance (NMR) detection system
  • NMR nuclear magnetic resonance
  • TIR total internal reflection
  • binding to a target protein by a probe compound containing a photo-affinity label and a reporter function group can be first subject to photo-crosslinking. This is followed by performing appropriate assays to isolate (e.g., via size exclusion chromatography) and detect the protein complexes (e.g., via electrophoresis and fluorescence imaging, or Streptavi din-based precipitation and Western blot).
  • a photo-affinity label and a reporter function group e.g., rhodamine- or biotin-conjugated azide
  • the detection system can be based on an ELISA-like assay.
  • the biological sample can be first contacted with a probe compound that can pull down the misfolded TTR oligomers.
  • the isolated probe-TTR complexes can then be analyzed and quantified in an ELISA-like plate format.
  • this detection system can be used to examine any other molecules that specifically interact with the non-native TTR oligomer protein.
  • one such TTR interacting protein partner is the extracellular chaperone Clusterin.
  • Some of these embodiments can employ probe B-2 exemplified herein which is labeled with a photoreactive group and a terminal alkyne reporter group. Following irradiation to crosslink the probe to the TTR oligomer and reacting with a biotin-azide reporter molecule via click chemistry, the complexes can be affinity purified via streptavidin- biotin interaction. The isolated non-native TTR oligomers from the biological sample can then be further analyzed on a plate with antibodies to TTR, Clusterin and other related molecules. With this assay system, multiple biological samples can be analyzed simultaneously for the presence of different target molecules of interest. By analyzing the presence of the different target molecules, this assay format also enables the generation of a risk score for the subject whose biological sample was tested.
  • binding of fluorescein-labeled probes to the misfolded TTR oligomer can be detected via monitoring fluorescein fluorescence following sample separation by gel electrophoresis or gel filtration chromatography analysis.
  • binding of the probe compounds to the target TTR protein may be determined by a number of other methods, e.g., quenching and other intensity measurements, donor or acceptor depletion kinetics, and fluorescence lifetime or emission anisotropy measurements.
  • TTR amyloid diseases encompass transthyretin-related hereditary amyloidoses (ATTR).
  • TTR amyloid diseases encompass transthyretin-related hereditary amyloidoses (ATTR).
  • TTR amyloid diseases include transthyretin-related hereditary amyloidoses (ATTR).
  • GTR transthyretin-related hereditary amyloidoses
  • these hereditary disorders include conditions with predominant degeneration of the heart (cardiomyopathy), the peripheral nervous system (polyneuropathy), the central nervous system (meningocerebrovascular amyloidosis), and the eye (ocular amyloidosis).
  • TTR amyloid diseases suitable for the diagnostic methods disclosed herein further include wild type TTR amyloidosis (ATTR-WT), aka senile systemic amyloidosis (SSA), which is not inherited and has aggregation formed from wild type TTR protein.
  • FAP familial amyloid polyneuropathy
  • FAC familiar amyloid cardiomyopathy
  • TTR amyloid diseases suitable for the diagnostic methods disclosed herein further include wild type TTR amyloidosis (ATTR-WT), aka senile systemic amyloidosis (SSA), which is not inherited and has aggregation formed from wild type TTR protein.
  • TTR-WT wild type TTR amyloidosis
  • SSA senile systemic amyloidosis
  • TTR normal "wild-type" TTR functions as a transporter of thyroid hormones and vitamin A (retinol) within the bloodstream.
  • People with mutations in the TTR gene produce abnormal, amyloidogenic, "variant” TTR throughout their lives.
  • Amyloid deposits consisting of abnormal "variant” TTR may cause familial amyloid polyneuropathy (FAP).
  • FAP familial amyloid polyneuropathy
  • This disease affects the peripheral nervous system, often the heart, and sometimes the kidneys and eyes. More than 120 variants of TTR have been observed to be associated with the amyloidoses, with the most common mutation worldwide being TTR Val30Met (V30M TTR) and TTR Vall22Ile (VI 221 TTR).
  • the most common type of FAP is associated with the Val30Met mutation in the TTR protein. It is thought to affect about 10,000 people in the world. Patients with this mutation often start to experience symptoms in their 30s. Sensory and autonomic neuropathies are the main symptoms; heart, kidneys and eye involvement
  • FAC familial amyloid cardiomyopathy
  • wild type TTR may also be amyloidogenic, causing wild type ATTR amyloidosis (aka “senile systemic amyloidosis” or "SSA").
  • SSA systemic amyloidosis
  • These types of amyloid deposits are found at autopsy in 1 in 4 people over age 80, but in most cases they do not appear to cause any symptoms. Almost 50% of patients with wild type ATTR amyloidosis experience carpal tunnel syndrome - tingling and pain in the wrists, pins and needles in the hands. Carpal tunnel syndrome often appears 3-5 years before the symptoms of heart disease.
  • TTR amyloid diseases may be diagnosed by tissue biopsy, genetic testing and imaging studies, these currently available tests are often invasive and/or ineffective in early diagnosis of patients with very few symptoms, pre-symptomatic (immediately before developing any symptoms) patients or patients with wildtype ATTR amyloidosis.
  • the probe compounds can detect non-native TTR oligomers in patients with TTR aggregation associated amyloidoses - both polyneuropathy and cardiomyopathy phenotypes.
  • An important aspect of the probe compounds is that, unlike other probes used for diagnostic purposes (i.e.
  • Congo Red or thioflavin T they preferentially recognize nascently unfolded and actively aggregating TTR.
  • the diagnostic methods described herein are suitable for detection of the earliest possible misfolding events that lead to TTR related amyloid diseases.
  • a suspected biological sample e.g., a blood sample
  • a probe compound e.g., a probe compound
  • the complex can be readily detected and examined via routinely practiced assays.
  • the exact assay to be employed in the detection is dependent on the nature of the diagnostic moiety present in the probe compound.
  • suitable biological samples include, but are not limited to, blood samples, cerebrospinal fluid, vitreous fluid, tears, ascites, sweat, urine, saliva, buccal sample, cavity or organ rinse.
  • detection of misfolded TTR oligomer in candidate subjects having or suspected of having a TTR amyloidosis is performed with a blood sample from the candidate subjects.
  • blood samples including whole blood, blood plasma or blood serum can be readily employed for detection of misfolded TTR oligomer. If misfolded TTR oligomer is positively detected in the biological sample relative to a control as described below, the candidate subject is identified as one who likely has or is likely to develop a TTR amyloidosis disease or condition.
  • the diagnostic methods disclosed herein can be employed in conjunction with other diagnostic methods known in the art.
  • the diagnostic test disclosed herein can be administered to the candidate patients before or after the patients are examined with the known diagnostic methods.
  • suspected patients can be first screened with the non-invasive diagnostic methods disclosed herein.
  • the patients with positive test results can then be further subject to, e.g., genetic testing to confirm the existence of a known disease causing TTR mutant (e.g., V30M or V122I).
  • the patients can also be additionally examined via an imaging test, e.g., echocardiogram, DPD scanning or cardiac MR scanning.
  • the probe compounds can also be employed in assessing disease status or progression, and monitoring effect of treatments that TTR amyloidosis patients are undergoing.
  • Subjects suitable for these methods can be TTR amyloidosis patients that are receiving any therapeutic treatment or intervention. Treatment of all types of amyloidosis is currently based on the following principles: reducing the supply of amyloid forming precursor proteins, and supporting the function of organs containing amyloid. All the TTR in the blood, which forms the amyloid deposits everywhere except in the eye and the blood vessels around the brain, is made in the liver.
  • liver transplantation may be helpful for some patients with hereditary, variant ATTR amyloidosis, mainly for patients with FAP associated with the V30M mutation.
  • heart transplantation may also be an option, esp. for younger, otherwise healthy patients.
  • Another proven treatment is kinetic stabilizers, e.g., tafamidis, that bind to the TTR tetramer slowing its dissociation, which is the rate-limiting step associated with TTR aggregation.
  • treatment may also involve supporting the function of organs containing amyloid.
  • this may include treatment for heart disease, treatment of peripheral neuropathy symptoms, and treatment of autonomic neuropathy symptoms. Many medications can be used for these treatments.
  • medications that may help to alleviate neuropathic pain include gabapentin, pregabalin and duloxetine.
  • Other therapeutic regimens that may be available for treating TTR amyloid diseases include, e.g., drugs such as diflunisal and tafamidis, genetic based therapies, and antibody based therapies.
  • the probe compounds can be employed to assess disease status or monitor treatment effect in patients undergoing any of these treatments.
  • misfolded TTR oligomer via a chosen assay is based on a detected signal level indicative of the presence of non-native TTR oligomer relative to a background or control signal level determined via the same assay.
  • the background or control signal level is determined with the same type of biological sample that is known not to contain misfolded TTR oligomer (e.g., biological sample from healthy subject).
  • the control or background may be determined via a control probe peptide that does not bind to misfolded TTR oligomer.
  • the assays can further include comparing the detected signal level in the candidate subject with a positive standard or control signal level. The latter is determined with biological samples from a group of subjects known to be affected by a specific type of TTR amyloidosis.
  • the controls are age- matched subject, e.g., within 0-10 years of age as the candidate subject to be tested.
  • a positive diagnosis is obtained if there is no significant difference between the detected signal level and the positive control signal level.
  • a positive diagnosis can be established by a detected signal level that is comparable to or falls within the range of positive control signal levels determined from a population of subjects affected by a TTR amyloidosis.
  • a departure is considered significant or substantial if the detected signal level falls outside the range typically observed in unaffected subjects due to inherent variation between subjects and experimental error. For example, in some methods, a departure can be considered significant if a detected signal level does not fall within the mean plus one standard deviation of levels in a control population. Typically, a significant departure occurs if the difference between the detected signal level and background or control levels is at least 20%, 30%, or 40%. Preferably, the difference is by at least 50% or 60%. More preferably, the difference is more than at least 70% or 80%. Most preferably, the difference is by at least 90%. The extent of departure between a detected signal value and a background or control value in a control population also provides an indicator of the probable accuracy of the diagnosis, and/or of the severity of the disease being suffered by the subject.
  • signal levels via the same assay are detected with the same type of biological sample (e.g., blood plasma) that is obtained from the candidate subject at multiple time points.
  • the time points can be every month, every other month, every 6 months, every year, or every other year.
  • the monitoring period can last for a few years or for the remaining life of the candidate subject.
  • an increase of over 10%, 20%, 30%, 40%, 50%, 100%, 200%, 500% or more of a detected signal level over time in the same subject is indicative of an increased likelihood of developing a TTR amyloid disease or an increased severity of disease.
  • the time points can be, e.g., prior to treatment, during and/or after treatment.
  • a decrease of a detected signal level e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or more
  • a later time point relative to a signal level determined at a previous time point with the same type of sample from the same subject would suggest an improvement of disease symptoms or a positive treatment result.
  • Peptide synthesis and purification Peptides were synthesized by solid-phase peptide synthesis using a standard Fmoc a-amine protecting group strategy (Applied Biosystems Model 433A). Resin and amino acids used for peptide synthesis were purchased from Novabiochem Corp (San Diego, CA, USA).
  • HOBt (1-hydroxy-benzotriazole; Advanced Chem Tech)
  • HBTU N,N,N',N'-Tetramethyl-0-(lH-benzotriazol-l-yl)uronium hexafiuorophosphate, 0-(Benzotriazol- 1 -yl)-N,N,N'N'-tetramethyluronium hexafluorophosphate; Sigma Aldrich
  • DIEA ⁇ , ⁇ -diisopropylethylamine; Sigma Aldrich
  • piperidine Sigma Aldrich
  • the cleaved peptide was precipitated using cold diethylether, recovered by centrifugation, reconstituted in anhydrous DMF or DMSO and incubated with 5-FAM-X-SE (6 - (Fluorescein - 5 - carboxamido)hexanoic acid, succinimidyl ester) (Anaspec Inc.) or SE-Diazirine (succinimidyl 4,4'-azipentanoate) for two hours in the presence of DIEA (1 : 100 DIEA/DMF).
  • fluorophore coupling was done on the resin prior to deprotection and cleavage.
  • TTR5 0 -127 and/or TTR52-127 were expressed and purified as follows.
  • the protein coding sequence was cloned into a plasmid upstream of an IPTG inducible T7 promoter (pMMHa).
  • coli BL21 containing the TTR5 0 -127 and/or TTR52-127 plasmid were grown to an OD of 0.6 and induced at 37 °C with 1 mM IPTG and the protein was allowed to express overnight. After induction and protein expression the majority of the protein was found within inclusion bodies.
  • the E. coli cells were lysed by sonication and centrifuged at 15,000 x g. The supernatant was discarded and the insoluble fraction was resuspended in 10 mL 6M guanididium-HCl and sonicated for 35 minutes in a water bath, followed by vigorous stirring for 2 hrs. The mixture was then centrifuged again at 15,000 xg for 15 minutes.
  • the supernatant was then taken and dialyzed against 4L of 25 mM Tris pH 8.8, followed by injection onto a Sourcel5Q strong anion exchange column (GE Healthcare ).
  • a linear gradient was then run to elute the protein (Buffer A 25 mM Tris, 2M Urea pH 8.8, Buffer B: 25mM Tris pH 8.8, 2M Urea, 1M NaCl).
  • the fractions containing TTR50-127 and/or TTR52-127 were then concentrated and injected onto a sephadex 75 gel filtration column, run in 25 mM Tris pH 8.8, 2M Urea.
  • the purified protein was then dialyzed into 5 mM NH4HCO4, frozen on liquid N 2 and lyophilized.
  • TTR and MTTR readily form stable oligomers at neutral pH. Briefly, the proteins were concentrated to 2 mg/mL in lOmM sodium phosphate pH 7.6, lOOmM KC1, lmM EDTA and incubated at 37 °C for 7 days ("young") or 6 weeks ("old"). To make aggregates of TTR52-127, 0.5 mg of the protein powder was dissolved in 10 mM sodium phosphate pH 7.6, 100 mM KC1, 1 mM EDTA to a final concentration of 70 ⁇ . the solution was placed in sonicating water bath for 5 minutes to fully dissolve the peptide an then passed through a 0.2 ⁇ filter and incubated at room temperature for 4 hours. Soluble aggregates were found to form within hours (Fig. 9).
  • each candidate probe (2 ⁇ ) was incubated overnight with 50 ⁇ oligomeric TTR followed by injection onto an Agilent SEC-3 or SEC-5 column and monitored for fluorescence (495 ex., 520 em.).
  • fluorescein standard curve was used to compare results from day to day. The results represent the area of the fluorescently labeled high MW peak.
  • Plasma was carefully removed and centrifuged for an additional 20 min to remove any remaining cells. Aliquots of the clarified plasma were stored at -20 °C.
  • B-2 was synthesized and purified as mentioned above. Lyophillized B-2 powder was weighed and dissolved in anhydrous DMSO to a final stock concentration of 1 mM. This stock was aliquoted and stored at -80 °C. For each experiment a fresh aliquot of B-2 was taken and added directly to the sample of interest (plasma, buffer etc.) to a final concentration of 50 ⁇ and incubated at 37 °C overnight. Time course experiments with plasma from patients and MTTR within plasma from healthy donors, showed that the signal plateaued within 5-8 hours and did not change thereafter.
  • each sample was MeOH/CHC13 and resuspended in 500 uL of 8M Urea, IX PBS, 5 mM EDTA, followed by the addition of 140 uL of 10% SDS. Following resuspension the samples were then reduced by adding TCEP (10 mM final concentration) and incubated for 30 min at room temperature. The free sulfhydryls were then blocked by adding lodoacetamide to a final concentration of 12 mM and incubated at room temperature for 30 minutes in the dark.
  • the samples were then added to 6 mL of PBS containing 100 uL of Streptavidin conjugated agarose beads (Novagen) and incubated at room temperature with gentle agitation overnight.
  • the biotin beads were then washed 6 x with 1 x PBS, 0.5% SDS, 5 mM EDTA and eluted with 100 uL of dithionite containing solution by incubation for 30 min at room temperature.
  • the samples were then run in a denaturing gel for western blot analysis.
  • the gel was transferred to nitrocellulose, blocked with Odyssey blocking buffer and probed with the anti-TTR antibody (rabbit, DAKOTM).
  • the membrane was then imaged using a LicorTM infrared imager by the addition of an anti-rabbit alexa680 conjugated secondary antibody.
  • Salivary gland biopsies staining with Thioflavin T Anti-TTR antibody and B-l- Biotin: Deparaffinization and re-hydration was performed according to the following protocol: 2 x 5 min washes with 100% xylene, followed by 3 minutes wash with 50 : 50 xylene : ethanol, followed by 3 minutes washes with ethanol 100%, 95%, 70%, 50%, and 2 x 3 min with water.
  • Thioflavin T staining slices were incubated for 10 minutes at room temperature with 1% aqueous Thioflavin-T (AnaSpec. Inc Ultra Pure Grade, Cat # 88306; filtered before each use with a 0.22 ⁇ syringe filter).
  • Plasma 45 ⁇ was incubated with probe B-l (20 ⁇ ; 3.2 of probe B-l from a 300 ⁇ stock solution in DMSO) or 3.2 ⁇ . of DMSO for 24 hours at 37°C.
  • probe B-l 20 ⁇ ; 3.2 of probe B-l from a 300 ⁇ stock solution in DMSO
  • DMSO 3.2 ⁇ . of DMSO
  • Diazirine photocrosslinking and pulldown Lyophilized probe B-2 powder was weighed and dissolved in DMSO to a final stock concentration of 1 mM and added to the plasma samples to give a final concentration of 50 ⁇ and incubated at 37 °C overnight. Time course experiments with plasma from patients and MTTR oligomers within plasma from healthy donors, showed that the signal plateaued within 5-8 hours and did not change thereafter. After incubation, plasma was pipetted into a 96-well plate and irradiated at 355 nm in a Stratalinker® UV Crosslinker 1800 for 1 hour.
  • the proteins were precipitated with methanol/chloroform to remove any unconjugated rhodamine dye.
  • the resulting protein pellet was resuspended in 50 ⁇ of Laemmli buffer containing DTT and boiled for 5 minutes. 20 nL of the sample was then analyzed by SDS PAGE (4-12% BOLT Gel, InvitrogenTM, run at 200 V for 30 minutes). To compare band intensities between multiple gels, a standard curve was loaded in each gel.
  • the inventors used a 28 kDa recombinant protein (retroaldolase) labeled quantitatively at a single cysteine with an alkyne using a maleimide derivative (retroaldolase-alkyne).
  • the inventors separately conjugated rhodamine azide from an identical fluorophore stock to retroaldolase- alkyne and loaded varying concentrations of the rhodamine-labeled retroaldolase. This procedure is depicted in fig. 31. All gels were imaged on a Biorad ChemiDocTM MP system and bands were quantified in ImageLabTM (Biorad).
  • samples were first subjected to the CuAAC click reaction with N 3 -Diazo-Biotin. After biotin conjugation, each sample was precipitated with methanol/chloroform and resuspended in 500 ⁇ of 8 M Urea, 1 X PBS, 5 mM EDTA, followed by the addition of 140 ⁇ . of 10% SDS. Following resuspension, the samples were then reduced by adding TCEP (10 mM final concentration) and incubated for 30 min at room temperature. The free sulfhydryls were then blocked by adding iodoacetamide to a final concentration of 12 mM and incubated at room temperature for 30 minutes in the dark.
  • the samples were then added to 6 mL of PBS containing 100 of Streptavidin-conjugated agarose beads and incubated at room temperature with gentle rocking overnight.
  • the biotin beads were then washed once with IX PBS, 0.5% SDS, 5 mM EDTA, 3 times with 2 M Urea, IX PBS, 5 mM EDTA, and 3 times with IX PBS.
  • the samples were then eluted from the beads by incubation with 100 of dithionite containing solution (42 mM sodium dithionite, 0.5% SDS, IX PBS, pH ⁇ 7) for 30 min at room temperature.
  • the samples were then run in a denaturing gel for western blot analysis or prepared for mass spectrometry analysis as described below.
  • the gel was transferred to nitrocellulose, blocked with Odyssey blocking buffer (LicorTM), and probed with the anti-TTR antibody (rabbit anti- TTR, DAKOTM) or a C-terminal TTR specific antibody made by immunizing mice against TTR5 0 -127.
  • the membrane was then imaged using a LicorTM infared imager following the addition of an anti-rabbit or anti-mouse IR-800 secondary antibody (LicorTM).
  • Plasma samples from 3 unique individuals were run in parallel using the following scheme. Plasma (95 ⁇ ) was mixed with 5 ⁇ . of either probe B-2 or probe B-2-Mut and incubated and crosslinked as described above. The plasma samples were then passed through a Bio-Spin® P-30 gel- filtration column equilibrated in 10 mM phosphate buffer pH 7.6, 100 mM KC1 and subjected to conjugation of N 3 -Diazo-Biotin as described above. Probe-crosslinked proteins were enriched using streptavidin agarose as described above.
  • the biotin-enriched eluted protein samples were then precipitated in methanol/chloroform and washed twice with 100% methanol.
  • the air-dried protein pellets were then resuspended, reduced, acetylated and trypsin digested, and labeled with respective TMT-NHS isobaric reagents (Thermo Fisher) as described previously.
  • the 3 plasma samples treated with either probe B2 or probe B2-mut were pooled, resulting in a total of 6 channels per run.
  • MudPIT columns were prepared as described previously and LC-MS/MS analysis was performed using a Q-Exactive mass spectrometer with an EASY nLC 1000 (Thermo) LC pump.
  • MudPIT experiments consisted of 5 min sequential injections of 0, 20, 50, 80, 100 % buffer C (500 mM ammonium acetate in buffer A) followed by a final step of 10 % buffer B (20% water, 80% acetonitrile, 0.1% formic acid v/v/v)/90% buffer C (95% water, 5% acetonitrile, 0.1% formic acid, v/v/v). Each injection was followed by a linear gradient from buffer A (95% water, 5% acetonitrile, 0.1% formic acid, v/v/v) to buffer B. Electrospray was carried out directly from the analytical CI 8 columns by applying a voltage of 2.5kV using an inlet capillary temperature of 275°C.
  • Transthyretin aggregation appears to form a spectrum of structures, including amyloid fibrils in buffers.
  • the aggregation process is associated with a rough free energy landscape ( Figure 1A, B), especially in vivo, probably due to the presence of extracellular holdase chaperones that kinetically stabilize misfolded and misassembled TTR structures, thus certain aggregate structures can be kinetically trapped.
  • TTR aggregation in particular is very fast in vitro, although amyloid fibrils are not readily formed under aggregation reactions in buffers.
  • misfolded oligomeric TTR structures would be less densely packed than native TTR or amyloid fibrils, allowing certain complementary peptides to integrate into misfolded TTR oligomer structures by docking and integrating into the structure at imperfection or defect sites ( Figures 1A and IB).
  • This hypothesis was tested by synthesizing peptides derived from the TTR primary sequence as candidate probes for detecting misfolded TTR oligomer structures.
  • TTR oligomers made from TTR sequences other than full-length TTR Since the B ⁇ -strand is located near the N-terminus, the inventors tested TTR oligomers made from TTR5 0 -127, which lacks the B ⁇ -strand. This TTR fragment is deposited in cardiomyopathy and some polyneuropathy patients. TTR5 0 -127 is highly oligomerization prone, affording TTR oligomers based on native gel analysis and atomic force microscopy (fig. 22). Despite differences in oligomer size and sequence, again probe B-l is the only peptide of 18 that incorporates significantly into these oligomers (fig. 23).
  • Probe B-l is a selective for identifying misfolded TTR oligomers
  • probe B-l is selective for binding to and integrating into the structure of misfolded TTR oligomers.
  • native WT TTR tetramers or freshly purified V30M or T119M or L55P or T60A or A25T TTR homotetramers comprising disease-associated TTR subunits, no significant labeling was observed, indicating that probe B-l is selective for non-native aggregate forms of TTR over natively folded TTR ( Figure 2A, 16A).
  • probe B-l was used on salivary gland biopsy from a FAP patient and a sectioned human heart from a TTR FAC patient, revealing that unlike Congo red, probe B-l does not bind to or integrate into amyloid.
  • salivary gland biopsies of a V30M FAP patient and a control were stained with a biotin labeled version of probe B-l (B-l-Biotin).
  • B-l-Biotin biotin labeled version of probe B-l
  • the localization of probe B- 1 -Biotin was compared with i) Thiofiavin T fluorescence (an amyloid selective fiuorophore) and ii) anti-TTR antibody localization.
  • the amyloid fibrils in the salivary gland were found to be localized around glandular acini as revealed by Thioflavin T fluorescence (Fig. 16C (top left panel; white arrowheads) and fig. 24, and the anti-TTR antibody confirmed these are TTR amyloid (Fig. 16C; third panel from top left).
  • probe B-l-Biotin was found inside the glandular acini of both patient and control (Fig. 16C and fig. 24, white asterisks), revealing that the peptide does not bind to or integrate into the amyloid fibrils present within tissue, and that the probe B-l-Biotin fluorescence signal results from non-specific binding to other proteins inside the glandular cells.
  • TTR52-127 a highly aggregation prone fragment that has been implicated in TTR related cardiomyopathies and polyneuropathies, notably a sequence lacking the ⁇ - ⁇ -strand.
  • the entire TTR peptide probe library was screened against the TTR52-127 oligomeric aggregates, and only probe B-l incorporated into TTR52-127 aggregates.
  • the TTR52-127 fragment is highly aggregation prone and forms soluble aggregates that are morphologically distinct from M-TTR oligomers.
  • probe B-l was the only peptide that incorporated significantly into these aggregates (Fig. 8). These results suggested that there exists a fundamental structural commonality between all of these non-native TTR oligomeric forms, which allowed probe B-1 to recognize these aggregates while excluding probe B-1 incorporation into native TTR. This structural motif appeared to involve residues 52-127 common to all aggregates.
  • probe B-1 may integrate into WT TTR aggregates made at pH 4.4.
  • probe B-l- misfolded TTR oligomer complex is resistant to dilution by gel filtration ( Figure IE) qualitatively characterizes probe B-1 as a slow, very tight, and selective binder of non-native TTR among other conformations-the off-rate appears to be very slow based on the chromatography experiments.
  • the probe B-1 was screened against Abetal-42 ADDL oligomers. The probe B-1 exhibited no binding to the monomer, dimer and trimer species present, and minimal binding to higher order aggregates which is consistent with prior reports (Fig. 10).
  • Probe B-1 structure-activity relationship suggests that probe B-1 is integrating
  • truncation of the peptide probe B-l from the N- and C-termini identified the sequence VAVHVF (SEQ ID NO: l) as a minimal binding/integration competent sequence, although the truncated peptide incorporates to a lesser extent (-90% less) in comparison with the original probe B-l sequence itself (Fig. 11).
  • a major goal of this work was to develop peptide-based probes that are able to detect misfolded forms of TTR in relevant biological contexts, such as in the blood, plasma or cerebrospinal fluid.
  • relevant biological contexts such as in the blood, plasma or cerebrospinal fluid.
  • the inventors have demonstrated that circulating tetrameric TTR dissociates, and monomeric TTR misfolds and misassembles into soluble oligomers or other aggregate types.
  • the inventors tested whether these aggregates are present in plasma of patients with symptoms of disease and whether they can be used as early diagnostic markers.
  • the development of these types of probes also allowed them to determine if the lowering of non- native TTR oligomers may correlate with clinical outcome upon disease modifying therapy treatment.
  • probe B-l could label M-TTR oligomers that were added to the human plasma of a healthy blood donor ( Figure 2C).
  • probe B-l selectively labels the recombinant non-native M-TTR oligomers ( Figure 2D & E).
  • a standard curve exhibits a linear probe B-l response with misfolded oligomeric TTR concentration (Fig. 12).
  • Other proteins are labeled to a lesser degree, including serum albumin, which is not unexpected, as albumin is known to bind to short, relatively hydrophobic peptides.
  • a similar SAR trend was observed when probe B-l alanine mutants were added to plasma containing non-native M-TTR oligomers ( Figure 2F).
  • Probe B-l was incubated overnight with plasma samples from ten symptomatic V30M FAP patients, ten asymptomatic V30M mutation carriers, and two patients with wild-type TTR cardiomyopathy. All 12 patients (10 V30M FAP and 2 WT cardiomyopathy) had symptoms defining the onset of disease at the time the blood samples were collected.
  • Probe B-l is the only TTR-derived peptide probe that integrates into the TTR oligomers in patient plasma ( Figure 3D). These results also demonstrate the potential utility of probe B-l as a diagnostic probe. It is reasonable to expect that the soluble misfolded TTR oligomer peak in plasma could contain holdase chaperones like ERdJ3, or Clusterin, and possibly glycosaminoglycans and other components.
  • Photo-crosslinker identifies non-native oligomeric TTR as the target of probe
  • probe B-1 binding to naked non-native TTR oligomers in FAP patient plasma versus non-native TTR oligomers interacting with holdase chaperones and potentially other plasma protein binding partners
  • a diazirine functional group and an alkyne handle was incorporated into probe B-1, affording probe B-2 (Fig. 18 A).
  • the diazirine forms a highly reactive short-lived (-100 ns) carbene that inserts into proximal bonds, resulting in covalent conjugates with the target protein(s) and potentially other macromolecules.
  • probe B-2 After incubation of probe B-2 with plasma samples, the samples were irradiated and rhodamine-azide was covalently attached to the alkyne handle using a denaturing copper catalyzed alkyne-azide cycloaddition or 'click' reaction (CuAAC) to render the conjugates fluorescent.
  • CuAAC denaturing copper catalyzed alkyne-azide cycloaddition or 'click' reaction
  • the advantage of probe B-2 over probe B-1 is that after photocrosslinking, probe B-2 is covalently attached to the proteins it initially binds to, thus denaturing and reducing SDS PAGE can be used to identify the proteins bound by probe B-2, including initially aggregated non-native TTR (Fig. 18B). In contrast, non-denaturing separation methods like SEC must be used to preserve the non-covalent probe B-1 interactions.
  • Probe B-2 selectively labels MTTR oligomers, both in buffer (fig. 30) and when oligomers were added to the plasma of healthy donors, but does not label natively folded tetrameric TTR that is also present in ⁇ concentrations within plasma (Fig. 18C, middle panel, magenta box) indicated by the intense rhodamine fluorescence signal at the TTR monomer MW, and absence in the control lanes (cf. lanes 1 and 2 of the B-2 Rhodamine data).
  • probe B-2 displays a high degree of conformational selectivity for non-native TTR oligomers, as does probe B-1.
  • Probe B-2 was incubated with FAP patient plasma. These samples exhibited the high MW SEC fluorescence signal upon incubation with probe B-1. After photocrosslinking and clicking on rhodamine, a band that migrates equally to that of monomeric TTR (13.5 kDa) is covalently labeled by probe B-2 in the denaturing SDS-PAGE of FAP patient plasma, but minimally in the plasma derived from a healthy donor (Fig. 18D, middle panel, magenta box), again demonstrating that probe B-2 does not form a complex with the natively folded TTR tetramer prior to photocrosslinking. Notably, other higher MW proteins are labeled by probe B-2 differentially between patients and controls, as revealed by the SDS-PAGE readout (Fig. 18D, middle panel).
  • TTR observed as a monomer in SDS PAGE in the rhodamine channel was in a more than 200 kDa MW complex, because only TTR in the "high MW fractions" (i.e., 3-5) reacted with covalent probe B-2.
  • a healthy donor plasma sample incubated with probe B-2 and irradiated does not crosslink with native WT TTR (Fig. 18F; right side middle panel), despite the fact that TTR is present in nearly all fractions, as ascertained by using an anti-TTR antibody (Fig. 18F, bottom panel, dark green box).
  • probe B-2 only labels high MW non-native TTR oligomers in the patient plasma and not native TTR in healthy donor plasma, demonstrating the selectivity of probe B2 for non-native oligomeric TTR in plasma.
  • the intensity of the B-2-TTR conjugate band was quantified from SDS-PAGE of the high MW plasma fraction by densitometry in a larger subset of V30M FAP patients, asymptomatic V30M carriers, and healthy donor controls. In the control groups, only minimal labeling of TTR is observed, whereas in the V30M FAP patient group, significantly more non-native TTR is labeled (Fig. 18G & fig. 31; Table 1).
  • Probe B-2 cross-linked plasma proteins were subjected to a click reaction with biotin, and subsequently the probe B-2 crosslinked proteins were affinity purified in patient vs. control plasma.
  • TTR is clearly identified in the eluted fractions by SDS-PAGE visualized by anti-TTR western blot, and is more prominent in the patient samples (Fig. 18H).
  • Control experiments show that the B-peptide sequence itself does not cross-react with the anti-TTR antibody used (Dako Inc., #A0002) ( Figure 13).
  • linear epitope mapping identifies the C-terminus of TTR as the primary epitope that the Dako TTR anibody targets.
  • the anti-TTR antibody is recognizing an antigen other than the B-peptide sequence (fig. 32).
  • Table 1 Sample age and demographics for the non-native TTR detection by the B-2 SDS PAGE assay presented in Fig. 18H, Fig. 20, and Fig. 21.
  • TTR labeling bv B-2 correlates with high MW SEC signal
  • TTR labeling by B-2 strongly correlates with the high MW SEC signal.
  • the results indicate that both probe B-2 photo-crosslinking and probe B-1 fluorescence chromatography can be used as general diagnostic for ATTR pathologies. Specifically, a standardized procedure was used where patient or control plasma was incubated with probe B-2. This was followed by collection of the high MW fraction using size exclusion chromatography, rhodamine labeling, and separation by reducing SDS PAGE. The labeling of a subset of plasma samples by probe B-2 was then quantified by densitometry (Figure 5A).
  • Probe B-1 selectively differentiates FAP patient samples from controls
  • probe B-1 selectively labels the non-native MTTR oligomers and exhibits a linear response with misfolded oligomeric TTR concentration based on SEC analysis (fig. 25).
  • SAR Ala-scan structure-activity relationship
  • FIG. 26 an Ala-scan structure-activity relationship with probe B-1 analogs added to plasma containing non-native MTTR oligomers was similar to that observed with non-native MTTR oligomers in buffer (Fig. 1), addressing selectivity even in this complex biological fluid.
  • Probe B-1 was incubated with plasma from symptomatic Portuguese and Japanese V30M FAP patients, asymptomatic Portuguese V30M mutation carriers, and healthy controls. All patients had symptoms defining disease onset at the time the blood samples were collected and most patients had a Neurological Impairment Score of the lower limbs (NIS-LL) of less than 20 points, reflecting early stage FAP.
  • NIS-LL Neurological Impairment Score of the lower limbs
  • probe B-1 labeled the high MW fraction in the FAP patients (Fig. 17A and B). In contrast, only minimal labeling was observed in the control groups.
  • SEC analysis of plasma afforded a statistically significant increase in the high MW fluorescence peak eluting between 800-1200 in the FAP patients relative to the control groups (Fig. 17C). Identical results were obtained using a B-peptide wherein the fluorescein substructure is replaced by disulfoCy5, showing that use of a less environmentally sensitive fluorophore makes no difference in this context (fig. 27
  • probe B-1 recognizes circulating misfolded TTR oligomers in patients that have not been detected previously. It is likely that the "TTR Oligomer-probe B-1" peak in plasma could also comprise holdase chaperones like ERdJ3 and clusterin, and possibly glycosaminoglycans and other macromolecular components.
  • Quantitative proteomics identifies the targets of the B-2 probe beyond non- native TTR oligomers
  • Clusterin is a multifunctional homodimeric 37 kDa sulfated glycoprotein that has been found to be co- deposited with amyloidogenic proteins in histopathology studies. Its variants have notably been positively linked in GWAS studies to neurodegenerative pathologies - most notably in Alzheimer's.
  • Haptoglobin is also a multifunctional high abundance plasma protein that has been shown to have chaperone activity and whose alpha subunit is similar in MW weight to TTR (9 and/or 18 kDa depending on the haplotype).
  • Rhodamine gel labeling experiments confirmed that probe B-2-Mut does not label non-native TTR oligomers in FAP patients (Fig. 19B, rightmost panel, magenta box).
  • each sample was digested with trypsin, and the tryptic fragments were labeled by one of the six unique isobaric mass tags (TMT tags, amine reactive).
  • TMT tags isobaric mass tags
  • the 6 samples e.g. the 3 unique V30M FAP patients treated with B-2 and B-2-Mut
  • MudPIT LC-MS/MS analysis were then combined and subjected to MudPIT LC-MS/MS analysis and the relative abundances of the tryptic peptides in each of the six samples were quantified by the intensity of the unique fragments in the MS2 spectra.
  • Proteins of interest are expected to be labeled by probe B-2, but not the probe B-2-Mut.
  • the intensity of the identified peptides in the MS2 spectra will be higher in the probe B-2 treated TMT channels, relative to the probe B-2-Mut TMT channels.
  • the protein list is sorted by the intensity ratio of probe B-2 to probe B-2-Mut
  • the most "B-2 enriched" proteins are TTR, several apolipoproteins including ApoE (36 kDa), clusterin (37 kDa), and alpha-2-macroglobin (-180 kDa), i.e., they have the highest intensity ratio of probe B-2 to probe B-2-Mut (Fig. 19D and E).
  • ApoE 36 kDa
  • clusterin 37 kDa
  • alpha-2-macroglobin i.e., they have the highest intensity ratio of probe B-2 to probe B-2-Mut (Fig. 19D and E).
  • the majority of the proteins that are most confidently identified by proteomics herein have been previously found in amyloid tissue biopsies from FAP patients, suggesting that these proteins may circulate with non-native TTR.
  • a clear proteomic signature emerges that distinguishes FAP patients from healthy donors and asymptomatic carriers.
  • the maj ority of probe B-2 targets identified in the FAP patients are also identified, including TTR (Fig. 19C).
  • the enrichment ratio for TTR was greatest for FAP patients followed by asymptomatic carriers, followed by healthy controls exhibiting no enrichment (probe B-2 : probe B-2-Mut ⁇ 1) (Fig. 19F).
  • the ratios for the circulating interacting proteins namely clusterin (a holdase chaperone), ApoE (transports lipoproteins, fat soluble vitamins and cholesterol in blood), and vitronectin (functions in hemostasis and modulating cell adhesion) display an inverse trend, i.e., the probe B-2 : probe B-2-Mut ratio is lowest for the FAP patients (Fig. 19F).
  • probe B-2-Mut ratio is lowest for the FAP patients (Fig. 19F).
  • non-native TTR oligomers may be interacting directly with these proteins, displacing probe B-2.
  • non-native TTR oligomers when present, could out-compete the available probe B-2 from binding to these other protein targets. Nonetheless, the biochemical and proteomics experiments described thus far fully validate non-native oligomeric TTR as a clear target of the B-2 probe, along with several TTR amyloid associated proteins.
  • probe B-l did not detect oligomers in plasma of mutation carriers that are asymptomatic, i.e., no symptoms or signs suggestive of polyneuropathy or any other organ involvement.
  • samples from patients with very few neurological symptoms defined as NIS ⁇ 10.
  • Tafamidis is a transthyretin kinetic stabilizer that is regulatory agency approved in Europe and Japan for the treatment of FAP.
  • the levels of non-native oligomers present in the plasma of tafamidis treated patients were investigated using probe B-l. These patients have been treated with tafamidis for at least 12 months and are considered clinical responders, i.e., with progression of the neurological impairment score scale less than 2 points (Suanprasert et al, supra).
  • Figure 7B shows the chromatograms of one of these typical responder patients (before treatment and after 12 months of tafamidis treatment), labeled with B-l .
  • the amount of non-native TTR labeled by B-l drops by about 50% after 12 months of treatment.
  • Transthyretin is a 127-amino-acid ⁇ -sheet-rich tetrameric protein that is predominantly secreted into the blood by the liver. Local production of TTR by the choroid plexus and the retinal epithelium accounts for the smaller quantities of TTR in the cerebrospinal fluid (CSF) and the eye, respectively. Natively folded tetrameric TTR circulating in blood, CSF, and in the eye of healthy individuals is known to function mainly as a transporter of vitamin A and thyroxine.
  • This natively folded TTR tetramer can slowly dissociate into monomers, that can subsequently misfold, enabling TTR aggregation, a process driving the dysfunction and ultimately the loss of post-mitotic tissue in a heterogeneous group of diseases collectively known as the TTR amyloidoses.
  • TTR amyloidosis-associated TTR mutations Approximately 120 amyloidosis-associated TTR mutations have been described so far; the autosomal dominant inheritance of one of these mutations leads to the incorporation of mutant subunits into a TTR tetramer otherwise composed of wild-type subunits, causing heterotetramer destabilization, i.e., faster TTR tetramer dissociation kinetics and/or the accumulation of higher quantities of misfolded aggregation-prone monomers.
  • the hereditary TTR amyloidoses are systemic amyloid diseases that can present with a variety of clinical phenotypes.
  • V122I a cardiomyopathy
  • V30M a peripheral nervous system associated with Familial Amyloid Polyneuropathy or FAP
  • FAP Familial Amyloid Polyneuropathy
  • the initial disease phenotype depends partially on the inherited TTR sequences, variability in clinical presentation is seen between patients with the same mutation and even within the same kindred, and some patients present with marked involvement of other less commonly involved organs, such as the eye (e.g., vitreous opacities and glaucoma), the central nervous system (e.g., stroke and dementia) or the kidney (e.g., nephrotic syndrome and chronic renal insufficiency).
  • the eye e.g., vitreous opacities and glaucoma
  • the central nervous system e.g., stroke and dementia
  • the kidney e.g., nephrotic syndrome and chronic renal insufficiency
  • the inventors developed reliable probes for each structure in the spectrum of aggregate structures that exists in patients. Determining which structures correlate with symptom development by studying asymptomatic mutation carriers and monitoring changes in the concentration of aggregate structures in patients in response to drug treatments that clinically halt amyloid disease progression provided an insight into structure-proteotoxicity relationships that drive amyloid diseases. In one embodiment, the goal of such an activity was to eliminate structures that do not correlate with symptom development and/or with clinical response to therapy, so as to produce a list of misfolded species that may drive loss of post-mitotic tissue (proteotoxicity) in human amyloid diseases.
  • the present disclosure provides probes that can selectively and/or specifically detect circulating non-native TTR structures that form in addition to amyloid fibrils.
  • the present disclosure provides peptide-based probes that selectively integrate into the structure(s) of non-native TTR oligomers prepared in vitro and notably integrate into apparently similar structures circulating in the blood of hereditary TTR amyloidosis patients, specifically those with predominant neurological or mixed peripheral nervous system and cardiac phenotypes, but not in patients with primarily cardiac phenotypes.
  • Proteolyzed TTR is identified in some FAP patient plasma samples.
  • Non-native TTR is detected in 3 additional polyneuropathy genotypes and not
  • the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

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Abstract

La présente invention concerne des peptides de sonde qui sont capables de se lier spécifiquement à des oligomères transthyrétines non natives (TTR) et des composés de sonde contenant de tels peptides et des marqueurs détectables. L'invention concerne également des procédés d'utilisation de composés de sonde pour détecter la présence d'oligomères mal repliés TTR dans des échantillons biologiques, des procédés pour diagnostiquer des amyloidases TTR chez un sujet humain, et des procédés pour surveiller la progression d'une maladie ou un effet de traitement chez des patients souffrant d'amyloidse TTR .
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WO2023245057A3 (fr) * 2022-06-15 2024-01-18 The Board Of Regents Of The University Of Texas System Sonde basée sur structure pour détection de fibrilles et d'agrégats amyloïdes de transthyrétine
WO2024240562A1 (fr) * 2023-05-19 2024-11-28 Neurimmune Ag Nouvelle immunothérapie pour troubles et états musculo-squelettiques

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