WO2013043669A1 - Compositions peptoïdes pour le traitement de la maladie d'alzheimer et d'un trouble de l'expansion de polyglutamine - Google Patents

Compositions peptoïdes pour le traitement de la maladie d'alzheimer et d'un trouble de l'expansion de polyglutamine Download PDF

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WO2013043669A1
WO2013043669A1 PCT/US2012/056025 US2012056025W WO2013043669A1 WO 2013043669 A1 WO2013043669 A1 WO 2013043669A1 US 2012056025 W US2012056025 W US 2012056025W WO 2013043669 A1 WO2013043669 A1 WO 2013043669A1
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compound
compound according
alkyl
substituted
hqp09
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PCT/US2012/056025
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Ilya Bezprozvanny
Yuan Luo
Xuesong Chen
Thomas Kodadek
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The Board Of Regents Of The University Of Texas System
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K14/575—Hormones

Definitions

  • the invention relates to the fields of biology and medicine. More particularly, the invention provides compositions and methods for the selective inhibition of polyglutamine expansion disorders, such as Huntington's disease and Spinocerebellar ataxias (SCAs). The invention also relates to compositions and methods for treatment and prevention of neurodegenerative diseases such as Alzheimer's Disease. More particularly, it concerns compositions and methods of treating memory deficiency in patients suffering from dementia.
  • polyglutamine expansion disorders such as Huntington's disease and Spinocerebellar ataxias (SCAs).
  • SCAs Spinocerebellar ataxias
  • the invention also relates to compositions and methods for treatment and prevention of neurodegenerative diseases such as Alzheimer's Disease. More particularly, it concerns compositions and methods of treating memory deficiency in patients suffering from dementia.
  • HD Huntington's Disease
  • HTT huntingtin
  • Antisense oligonucleotides or siRNAs that reduce HTT expression have been proposed as a therapeutic strategy (Hasholt et ah, 2003; Boado et ah, 2002; Harper et ah, 2005; Denovan-Wright and Davidson, 2006; DiFiglia et ah, 2007) but most oligomers inhibit the mutant and wild-type protein expression indiscriminately. HTT
  • ⁇ 00014010 ⁇ _ ⁇ _ is known to play an essential role in embryogenesis ( asir et al, 1995), neurogenesis (White et al, 1997), and normal adult function in heterozygotes (Nasir et al, 1995), suggesting that agents inhibiting both mutant and wild-type HTT will induce significant side-effects.
  • nucleic acid-based approaches do not address polyglutamine protein aggregates that may already be present in a subject. Thus, there remains a need to identify agents that selectively bind to and inhibit the function of polyglutamine expanded polypeptides.
  • AD Alzheimer's disease
  • AD drug discovery is to identify specific ⁇ 42 ligands or inhibitors of ⁇ 42 oligomerization.
  • Most drug candidates that work through such a mechanism and are currently in human clinical trials are anti- ⁇ antibodies. While antibodies against amyloid precursor protein (APP) improve memory in the SAMP8 mouse, a strain which overproduce ⁇ protein (Morley et al, 2002), they impair performance of passive avoidance tasks in rats (Huber et al, 1993) and chicks (Mileusnic et al, 2000).
  • BBB blood-brain-barrier
  • Ri and R2 are each, independently, -H, -OH, -F, -CI, -Br, -NH 2 , -CN, alkyl(c ⁇ 6), acyl(c ⁇ 6), alkoxy(c ⁇ 6), acyloxy( C ⁇ 6), alkylaminO( C ⁇ 6), dialkylaminO( C ⁇ 6), amidO( C ⁇ 6), or a substituted version of any of these groups; or Ri and R2 are both together alkanediyl(c ⁇ 6), alkenediyl(c ⁇ 6), alkoxydiyl(c ⁇ 5), -0-alkanediyl(c ⁇ 4)-0-, or a substituted version of any of these groups; R3 is aryl( C ⁇ i2) or substituted aryl( C ⁇ i2); R4 is -H, alkyl( C ⁇ 6), or substituted alkyl(c ⁇ 6); R5 and R6 are each independently alkyl((c
  • Ri and R 2 are both together -0-alkanediyl(c ⁇ 4 ) -0- or -OCH 2 0-
  • compounds of the embodiments comprise an R 3 wherein R 3 is aryl (C ⁇ i 2) , such as wherein R 3 phenyl.
  • a compound of the embodiments can comprise, for instance, an R4 wherein R4 is alkyl(c ⁇ 6), such as wherein R4 is methyl.
  • R5 is a substituted alkyl( C ⁇ 6), such as wherein R5 is 2-hydroxyethan-l-yl.
  • R6 is a substituted alkyl( C ⁇ 6), such as 2- methoxyethan- 1 -yl.
  • X of the compounds of the embodiments is -NH 2 , an affinity tag (e.g., biotin) or another compound of the embodiments.
  • a compound of the embodiments is defined as:
  • a compound of the embodiments is defined as:
  • a method of treating or preventing Huntington's disease, spinocerebellar ataxia type 3 (SCA3) or another polyglutamine expansion disorder in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound according the embodiments.
  • a method of the embodiments can comprise administering to a subject a therapeutically effective amount of a compound of the formula:
  • a compound of the embodiments is defined as a fluorescent probe.
  • a fluorescent probe compound can have the formula:
  • a method for detecting polyQ expansion proteins longer than 35 Q comprising contacting a fluorescent probe of the embodiments with a poly Q expansion protein longer than 35 Q.
  • kits for the detection of polyQ expansion proteins longer than 35 Q, comprising a compound according to the embodiments and a microbead or a glass slide, wherein the compound is covalently attached to the microbead or glass slide.
  • a method of detecting a polyQ expansion protein comprising: contacting a sample suspected of comprising a polyQ expansion protein longer than 35 Q with a compound according to the embodiments; and detecting binding of the compound to the polyQ expansion protein.
  • detecting the binding can comprise detecting binding to the compound by performing an enzyme-linked immunosorbent assay (ELISA)-like assay (e.g., on a streptavidin plate).
  • ELISA enzyme-linked immunosorbent assay
  • Ri is -H, alkyl( C ⁇ 6), substituted aryl( C ⁇ i2), aralkyl( C ⁇ i2), substituted aralkyl( C ⁇ i2), or benzo[i/][ l,3]dioxol-5-yl
  • R2 is -H, aryl( C ⁇ i2), substituted aryl( C ⁇ i2), aralkyl( C ⁇ i2), substituted aralkyl(c ⁇ i2), or benzo[ ⁇ i][ l,3]dioxol-5-yl
  • Ri and R2 are both together alkanediyl(c ⁇ 6), alkenediyl(c ⁇ 6), alkoxydiyl(c ⁇ 5), or a substituted version of any of these groups
  • n is 0 or 1
  • R 3 is -H, -OH, alkenyl(c ⁇ 6), alkoxy(c ⁇ 6), alky
  • Ri is -H, alkyl( C ⁇ 6). Methyl, aryl( C ⁇ i2) or phenyl.
  • R2 is aryl( C ⁇ i2) (e.g., phenyl), substituted aralkyl( C ⁇ i2) (e.g., 4- sulfamoylphenylmethyl) or benzo[ ⁇ i][l,3]dioxol-5-yl.
  • Ri and R2 are both together alkanediyl(c ⁇ 6) such as butanediyl.
  • R3 is -H, alkyl( C ⁇ 6) (e.g., methyl) or aryl( C ⁇ i2) (e.g., phenyl).
  • R4 is aryl( C ⁇ i2) (e.g., phenyl), substituted aralkyl( C ⁇ i2) (e.g., 4- sulfamoylphenylmethyl), alkyl( C ⁇ 6) (e.g., methyl), substituted alkyl( C ⁇ 6) (e.g., methoxymethyl or hydroxymethyl), or alkenyl( C ⁇ 6) (e.g., vinyl).
  • R 3 and R4 are both together alkanediyl(c ⁇ 6) such as butanediyl.
  • R5 is -H, alkyl( C ⁇ 6) (e.g., methyl) or aryl( C ⁇ i2) (e.g., phenyl).
  • R6 is aryl( C ⁇ i2) (e.g., phenyl), substituted aralkyl( C ⁇ i2) (e.g., 4- sulfamoylphenylmethyl), alkyl( C ⁇ 6) (e.g., methyl), substituted alkyl( C ⁇ 6) (e.g., methoxymethyl, hydroxymethyl or 3-aminopropan-l-yl).
  • R5 and Re are both together alkanediyl(c ⁇ 6), such as butanediyl.
  • R7 is -H, alkyl( C ⁇ 6) (e.g., methyl), aryl( C ⁇ i2), (e.g., phenyl).
  • Rs is aryl( C ⁇ i2) (e.g., phenyl), benzo[ ⁇ i][l,3]dioxol-5-yl, substituted aralkyl( C ⁇ i2) (e.g., 4-sulfamoylphenylmethyl), substituted alkyl( C ⁇ 6) (e.g., hydroxymethyl), alkenyl( C ⁇ 6) (e.g., vinyl).
  • R7 and Rs are both together alkanediyl(c ⁇ 6), such as butanediyl.
  • R9 is a substituted alkyl( C ⁇ 6) (e.g., 4-aminobutan-l-yl).
  • Rio is a substituted alkyl(c ⁇ 6) (e.g., 4-aminobutan-l-yl).
  • X is -NH 2 , an affinity tag (e.g., biotin) or another compound of the embodiments.
  • a compound of the embodiments is defined as:
  • a method of inhibiting oligomerization of ⁇ -amyloid peptide 42 comprising contacting at least one such peptide with a compound according to the embodiments.
  • the inhibition is selective for ⁇ -amyloid peptide 42. For example, inhibition can occur even in the presence of a higher concentration of ⁇ -amyloid peptide 40 relative to ⁇ -amyloid peptide 42.
  • a method for treating or preventing Alzheimer's disease or other forms of progressive dementia in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a compound according to the embodiments.
  • a method of detecting ⁇ -amyloid peptide 42 or ⁇ - amyloid peptide 40 comprising: contacting a sample suspected of comprising a ⁇ -amyloid peptide 42 or ⁇ -amyloid peptide 40 with a compound according to the embodiments; and detecting binding of the compound to the ⁇ -amyloid peptide 42 or ⁇ - amyloid peptide 40.
  • detecting binding of the compound can comprise performing an enzyme-linked immunosorbent assay (ELISA)-like assay.
  • ELISA enzyme-linked immunosorbent assay
  • detection is selective for ⁇ -amyloid peptide 42, such as wherein detection occurs even in the presence of a higher concentration of ⁇ -amyloid peptide 40.
  • 14010 ⁇ -13- performing an ELISA-like assay comprising performing the assay on a plate, such as a streptavidin plate.
  • a compound of the embodiments (or compounds used in the methods of the embodiments) comprise a label, such as a radioactive, fluorescent or enzymatic label or an affinity tag (e.g., biotin).
  • a label such as a radioactive, fluorescent or enzymatic label or an affinity tag (e.g., biotin).
  • kits for the detection of detecting ⁇ - amyloid peptide 42 or ⁇ -amyloid peptide 40, comprising a compound according to the embodiments and a microbead or a glass slide, wherein the compound is covalently attached to the microbead or plate (e.g., a streptavidin plate).
  • a microbead or a glass slide wherein the compound is covalently attached to the microbead or plate (e.g., a streptavidin plate).
  • FIGS. 1A-C Peptoid library used in the screen for specific mHtt ligands.
  • FIG. 1A General chemical structure of the 6-mer peptoid library containing a fixed proline14010 ⁇ -14- residue at the 4 position and 5 variable positions (R1-R5). The calculated diversity of the library is 59,049 diverse peptoids.
  • FIG. IB Chemical structures and names of 9 amines used in synthesizing the peptoid library.
  • FIG. 1C A fluorescence microscopic image showing a representative hit isolated from the peptoid library as a potential ligand for MBP-Htt-N-82Q protein. The bead containing a putative hit (shown by the red arrow) is identified by a red halo around the bead resulting from Qdot fluorescence. Scale bar: 170 ⁇ .
  • FIGS. 2A-C HOP09 peptoid specifically binds to polyO-expanded proteins.
  • FIG. 2A Chemical structure of HQP09 peptoid identified from the screen of the peptoid library.
  • RP01 is a random 6-mer peptoid used as a control.
  • FIG. 2B MBP-Htt-N-82Q or MBP-Htt-N-15Q recombinant proteins were used in pull-down experiments with bio- (QBP1)2, HQP09 or RP01 beads. The precipitated fractions were analyzed by Western blotting with an anti-MBP monoclonal antibodies.
  • FIG. 2A Chemical structure of HQP09 peptoid identified from the screen of the peptoid library.
  • RP01 is a random 6-mer peptoid used as a control.
  • FIG. 2B MBP-Htt-N-82Q or MBP-Htt-N-15Q recombinant proteins were
  • Atxn3-77Q or Atxn3-19Q recombinant proteins were used in pull-down experiments with bio- (QBP1)2, HQP09 or RP01 beads.
  • the precipitated fractions were analyzed by western blotting with an anti- Atxn3 monoclonal antibodies.
  • the input lane contained 1% of total protein used in pull-down experiments.
  • FIGS. 3A-B Competitive mHtt binding experiments with HQP09 peptoid and QBP 1 peptide.
  • FIG. 3A, 3B, MBP-Htt-N-82Q and MBP-Htt-N-15Q recombinant proteins were used in pull-down experiments with HQP09 beads (FIG. 3 A) or QBP1 beads (FIG. 3B).
  • the QBP1 peptide, HQP09 peptoid or RP01 peptoid were added in binding reaction as indicated.
  • the precipitated fractions were analyzed by Western blotting with an anti- MBP monoclonal antibody.
  • the input lane contained 1% of total protein used in pull-down experiments.
  • FIGS. 4A-C Fluorescence polarization assay for HQP09 association with mHtt.
  • FIG. 4A Chemical structure of HQP09 G2 and HQP09_G2_Fluo.
  • FIG. 4C Competitive fluorescence polarization assay.
  • HQP09 peptoid, RP01 peptoid or the seven14010 ⁇ i c sarcosine scan derivatives of HQP09 as indicated were added in a final concentration of 300 ⁇ to a binding assay between 5 nM HQP09_G2_Fluo and 5 ⁇ MBP-Htt-N-82Q.
  • the significant reduction in Amp values when compared to control is shown (*** p ⁇ 0.001, ** p ⁇ 0.01).
  • FIGS. 5A-B Identification of minimal HOP09 active analog.
  • FIG. 5A Chemical structures and molecular weights of four 4-mer HQP09 analogs.
  • FIGS. 6A-B PolyQ-binding peptoids inhibit mHtt aggregation.
  • FIG. 12B AFM images of Htt53Q aggregates formed following 24h incubation in the presence of 3% DMSO, QBP1 peptide, HQP09, HQP09 9, HQP09 1 or RP01 peptoids as indicated.
  • the particles corresponding to fibrils and oligomers formed by Htt53Q are clearly visible in DMSO control (top left) and in the presence of HQP09 1 (bottom center) or RP01 (bottom right).
  • the number of particles is reduced in the presence of QBP1 (top center), HQP09 (top right) and HQP09 9 (bottom left).
  • Scale bar 2 ⁇ for all panels. Quantification of the number of particles over 100 nm detected on these images is shown as FIG. 12B, below.
  • FIGS. 7A-E PolyQ-binding peptoids stabilize glutamate-induced Ca 2+ signals in HD-YAC128 MSN.
  • FIG. 7A Repetitive application of 20 ⁇ glutamate (shown as black bars) induces enhanced Ca 2+ signals in MSN cultures from HD-YAC128 mice. The intracellular Ca 2+ concentraton is measured by Fura-2 ratio imaging and presented as 340/380 Fura-2 ratio at each time point in the experiment.
  • FIG. 7b-d The same experiment14010 ⁇ as in panel (a) was performed after preincubation with 20 ⁇ HQP9 (FIG. 7B), HQP9 9 (FIG. 7C) or RP01 (FIG. 7D). On panels FIG.
  • FIG. 7A-D the average traces from all HD- YAC128 MSN in each experimental group are shown.
  • FIG. 7E The summary of Ca 2+ increase in WT (open column) and YAC128 (filled column) MSN following 20 pulses of glutamate. The average increase in Ca 2+ levels is shown by change in 340/380 ratio as mean ⁇ S.E. (n > 33 for the number of MSN analyzed in each group).
  • Preincubation with HQP09 or HQP09 9 significantly (***p ⁇ 0.001) attenuated glutamate-induced Ca 2+ responses in YAC128 MSN.
  • HQP09_9 peptoid had significantly (*p ⁇ 0.05) stronger effect than HQP09.
  • FIGS. 8A-B PolvQ-binding peptoids protect HD-YAC128 MSN from glutamate-induced apoptosis.
  • FIG. 8A Representative images of TUNEL staining of MSN cultures from WT (top) and HD-YAC128 (bottom) mice. The MSN cultures were exposed to 250 ⁇ glutamate for 8 h, fixed and analyzed by TUNEL staining with propidium iodide counterstaining. Scale bar: 40 ⁇ .
  • FIGS. 9A-C PolvQ-binding peptoid inhibits mHtt aggregates in YAC128 mice.
  • FIG. 9A Representative confocal images of the striatum region of brain slices showing mHtt aggregates stained with EM48 antibody and nuclei stained with Nissl stain from ACSF-treated (control, top) or HQP09-treated (HQP09, bottom) YAC128 mice.
  • ACSF 100 uL/mice
  • HQP09 2.5 mg/100 ⁇ /mice
  • FIG. 9C The total mHtt aggregates intensity was quantified 14010 ⁇ - 1 7- using MetaXpress from the striatum images of control (treated with ACSF) and HQP09- treated mice. The mean from 15 images per mouse were used. The data are presented as mean ⁇ S.E.
  • FIG. lOA-C Chemical structures of 10 initial peptoid hits from the primary screen of 60,000 peptoid library with MBP-Htt-N-82Q protein as a bait. Based on structural similarity the peptoids are grouped in group a, b, and c. The variable residues are highlighted for peptoids within groups b and c.
  • FIG. 11 Chemical structures of 7 analogs of HQP09 generated as a result of sarcosine scan. The position of sarcosine residue in each derivative is shown by grey shading.
  • FIG. 12A-B Effect of peptoids on mutant Huntingtin aggregation in vitro.
  • FIG. 12A Western blot analysis of aggregates formed by Htt53Q. The samples of Htt53Q prepared as described in the main text were resolved by SDS-PAGE electrophoresis and analyzed by Western blotting with MW8 antibodies.
  • FIG. 12B Quantification of Htt53Q particles detected by AFM. The AFM images of Htt53Q were collected as as described in the main text. The number of particles with effective diameter larger than 100 nm was calculated from AFM images.
  • FIG. 14 Histological evaluation of effects resulting from intracerebroventricular delivery of HQP09 in striatum of WT and YAC128 mice. Representative images of striatal sections from WT ICV-ACSF, WT ICV-HQP09, YAC128 ICV-ACSF and YAC128-HQP09 mice stained with NeuN (top panel), MAP2 14010 ⁇ -18- and Neurotrace (middle panel) and DARPP32 (bottom panel) are shown as indicated. Scale bars, 20 ⁇ .
  • FIGS. 15A-B BBB studies.
  • HQP09 9 400 ug was injected from the tail vein of 2 months old WT mouse; (20 mg/kg). Samples were collected from the blood (FIG. 15B) and brain (FIG. 15 A) at different time points and the concentration of HQP09_9 was determined by LC/MS/MS.
  • FIG. 16 A schematic showing an example AD progression by the amyloid cascade mechanism.
  • FIGS. 17A-B FIG. 17A, schematic depicting an example on-bead peptoid library and peptoid identification protocol.
  • FIG. 17B schematic depicting an example library screening protocol.
  • FIGS. 18A-E Candidate IAMl peptoid binds ⁇ 42.
  • FIG. 18A IAM1 or random peptoids (RP) were coated on resin beads and tested for binding with ⁇ 42 or ⁇ 40.
  • FIG. 18B-E IAMl binding to (FIG. 18B) ⁇ 42; (FIG. 18C) ⁇ 40; (FIG. 18D) ⁇ 38; (FIG. 18E) Scrambled ⁇ 42, as indicated was quantified by an ELISA-like assay.
  • FIGS. 19A-B IAMl inhibits ⁇ 42 oligomerization.
  • FIG. 19A shows the results of an anti- ⁇ (DE2B4) Western blot following separation of proteins by electrophoresis on a 15% Tris-glycine gel.
  • FIG. 19B shows the results of an anti- ⁇ (DE2B4) Western blot after separation of proteins by electrophoresis on a 10-20% Tricine gel.
  • FIGS. 20A-G IAMl inhibits ⁇ 42 aggregation.
  • FIGS. 20A-F, ⁇ 40 and ⁇ 42 aggregation was assessed by an in situ kinetic thioflavin T assay.
  • FIG. 20F Effects of Scyllo-inositol (Scl, 1 :500); RP peptoid (1 : 100), 6E10 antibodies are compared with DMSO control on aggregation.
  • FIG. 20G, ⁇ 42 aggregation was assessed by transmission electron microscopy (TEM) in the presence of IAMl or control preparations.
  • TEM transmission electron microscopy
  • FIGS. 21A-B IAMl rescues neurons from ⁇ 42 oligomer toxicity.
  • FIG. 21A shows concentration-dependent toxicity of ⁇ 42 in primary hippocampal neuronal cultures.
  • FIGS. 21B shows neuroprotection by IAMl of primary hippocampal neuronal cultures from ⁇ 42 oligomer-associated toxicity. 14010 ⁇ -19-
  • FIGS. 22A-B IAM1 pharmacokinetics.
  • FIG. 22A shows IAM1 pharmacokinetics in the brain.
  • FIG. 22B shows IAMl pharmacokinetics in the plasma. IAM1 concentrations were measured by LCMS/MS in each case.
  • FIGS. 23A-B IAMl reduces the amyloid load in the brain of APPPS1 mice.
  • Graphs and tissue staining show measurements of the amyloid load in brain slices prepared from APPPS 1 mice that were administered with IAMl subcutaneously with implanted ALZET ® osmotic pumps.
  • FIGS. 24A-E Evaluation of the dimeric derivative (IAM1)2.
  • FIG. 24A chemical structure of the dimeric derivative (IAM1)2.
  • FIG. 24B binding curves of (IAM1)2 with ⁇ 42 and ⁇ 40 using fluorescence solid phase binding assay.
  • FIGS. 24C- D time courses of the fluorescence of aggregate-bound ThT in the aggregation processes of ⁇ 42 or ⁇ 40 in the presence of (IAM1)2 at different concentrations.
  • ⁇ 42 and ⁇ 40 were used as monomers (18 ⁇ ) and mixed with 2 ⁇ stock solution of (IAM1)2 (1 mM, 2 mM, 5 mM, 10 mM).
  • the fluorescence (excitation at 440 nm and emission at 485 nm) was measured every 10 min up to 24h.
  • FIG. 24E the ThT max normalized for the aggregation processes of ⁇ 42 and ⁇ 40 at different concentrations of (IAM1)2.
  • FIGS. 25A-D Evaluation of ASR1.
  • FIG. 25A chemical structure of ASR1.
  • FIG. 25B binding curves of ASR1 with ⁇ 42 and ⁇ 40 using fluorescence solid phase binding assay.
  • ⁇ 42 and ⁇ 40 were used as monomers (18 ⁇ ) and mixed with 2 ⁇ stock solution of ASR1 (50 mM). The fluorescence (excitation at 440 nm and emission at 485 nm) was measured every 10 min up to 24 h.
  • FIGS. 26A-B Neuroprotective effects of (IAM1)2 in amyloid toxicity assay.
  • FIG. 26A MAP2 staining of neurons incubated in the control medium (upper panel) and in ⁇ -containing conditioned medium (lower panel). The images are shown for control (first column), 6E10 (second column) and (IAM1)2 (third column).
  • FIG. 26B percent viability of neurons treated with ⁇ -containing conditioned mediumin the presence of (IAMI)2 at increasing concentrations. The wells that received only the control medium were regarded as a control and with al00% viability.
  • the data presented as mean ⁇ SE (n 3), 14010 ⁇ _2Q_ DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
  • polyglutamin expansion disorders also known as CAG- repeat related diseases
  • CAG- repeat related diseases present significant obstacles with respect to the selective inhibition of disease-associate proteins versus normal proteins.
  • the instant application provides a novel approach for targeting polyglutamine expansion-related diseases with peptoid binding molecules.
  • Peptoids are the oligomers of N-substituted glycines, which have a number of advantages over peptide ligands: (i) peptoids cover a greater diversity of conformational states than peptides; (ii) the peptoid linkage is resistant to hydrolytic enzymes, resulting in improved stability in vivo; and (iii) short peptoids are membrane permeable.
  • HQP09 and HQP09 9 were able to prevented aggregation of mHtt in vitro (see, e.g., FIG. 6 and 12).
  • the binding specificity and the anti-aggregation activity of HQP09 was comparable to those of polyQ binding peptide (QBP1) (Nagai et ah, 2000) and HQP09 and QBP 1 appeared to bind to non-overlapping sites on expanded polyQ sequence (FIGS. 2, 3, 6 and 12).
  • QBP1 polyQ binding peptide
  • HQP09 and QBP 1 appeared to bind to non-overlapping sites on expanded polyQ sequence (FIGS. 2, 3, 6 and 12).
  • both HQP09 and HQP09 9 stabilized abnormal Ca 2+ signals in striatal medium spiny neuron culture systems (FIG.
  • the peptoid molecules of the embodiments provide a new class of therapeutics for treatment and/or prevention of polyglutamine expansion disorders, such as Huntington's Disease.
  • AD Alzheimer's disease
  • ⁇ 42 oligomers are major neurotoxic species contributing to the pathogenesis of AD.
  • a 42-binding ligands may provide a potential avenue for AD therapy and diagnosis.
  • Several approaches to targeting ⁇ 42 have been considered, such as using antibodies that bind to amyloid proteins, but a range of technical limitations has limited the effectiveness of such agents. 14010 ⁇ _2 ⁇ .
  • the instant application provides a novel approach for targeting ⁇ 42 with peptoid binding molecules.
  • Peptoids are the oligomers of N-substituted glycines, which have a number of advantages over peptide ligands: (i) peptoids cover a greater diversity of conformational states than peptides; (ii) the peptoid linkage is resistant to hydrolytic enzymes, resulting in improved stability in vivo; and (iii) short peptoids (up to 8 monomers) are membrane permeable.
  • a 42-binding peptoids using a novel peptoid library and screening protocol that allows for the rapid identification of AD- targeted molecules.
  • a peptoid library comprising 38,000 diverse 6-mer peptoids was screened for specific ⁇ 42 ligands.
  • An array of peptoids was identified that displayed the ability to selectively bind ⁇ 42 over ⁇ 40.
  • a representative of isolated molecules peptoid IAM1 inhibited ⁇ 42 oligomerization in vito, protected primary cultured hippocampal neurons from toxicity caused by ⁇ 42 oligomers and reduced total amyloid load in vivo in a murine AD model system.
  • ⁇ 42 -binding peptoids of the embodiments provide both AD therapeutic candidates and highly effective biological reagents.
  • the peptoids can be administered alone or in combination with other agents for the treatment or prevention of symptoms of AD.
  • peptoid compounds described here are highly specific for ⁇ 42, they can be used to detect the presence of ⁇ 42 in a sample.
  • a peptoid can be immobilized, contacted with a sample and binding of any protein to ⁇ 42 from sample detected (e.g., an ELISA-like assay).
  • peptoids can be labeled (e.g., with contrast agents) and used for in vivo detection of ⁇ 42.
  • embodiments of the application provide new approaches to AD therapy and diagnosis not previously available. I. PEPTOIDS
  • Peptoids are a type of small molecules that mimic peptides, comprising oligomers of N-substituted glycines. Such ligands provide better potential drug candidates than peptides because of: (i) peptoids cover a greater diversity of conformational states than peptides (ii) the peptoid linkage is resistant to hydrolytic enzymes, resulting in improved stability in vivo (iii) short peptoids (up to 8 monomers) are membrane permeable.
  • Peptoid compounds can comprise a broad array of side chains. Exemplary chemical groups for such side chains are detailed further below.
  • Peptoid libraries such as the on-bead library provided herein, can be rich sources of ligands for a variety of proteins (Zuckermann et al, 1994; Zuckerman and Kodadek, 2009; Kodadek and Bachihawat- Sikder, 2006; Yoo and Kirshenaum, 2008).
  • Compounds can be further modified and optimized using the principles and techniques of organic chemistry as applied by a person skilled in the art. Such principles and techniques are taught, for example, in March 's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (2007), which is incorporated by reference herein.
  • Compounds employed in methods of the invention may contain one or more asymmetrically-substituted carbon or nitrogen atoms, and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated.
  • Compounds may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained.
  • the chiral centers of the compounds of the present invention can have the S or the R configuration.
  • Compounds of the invention may also have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and/or have a better pharmacokinetic profile (e.g., higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.
  • a better pharmacokinetic profile e.g., higher oral bioavailability and/or lower clearance
  • atoms making up the compounds of the present invention are intended to include all isotopic forms of such atoms.
  • Isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include tritium and deuterium
  • isotopes of carbon include 13 C and 14 C.
  • one or more carbon atom(s) of a compound of the present invention may be replaced by a 014010 ⁇ _2 _ silicon atom(s).
  • one or more oxygen atom(s) of a compound of the present invention may be replaced by a sulfur or selenium atom(s).
  • Compounds of the present invention may also exist in prodrug form. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.), the compounds employed in some methods of the invention may, if desired, be delivered in prodrug form. Thus, the invention contemplates prodrugs of compounds of the present invention as well as methods of delivering prodrugs. Prodrugs of the compounds employed in the invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound.
  • prodrugs include, for example, compounds described herein in which a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a subject, cleaves to form a hydroxy, amino, or carboxylic acid, respectively.
  • the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.
  • Linkers or cross-linking agents may be used to fuse peptoids to other proteinaceous sequences.
  • Bifunctional cross-linking reagents have been extensively used for a variety of purposes including preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural studies. Homobifunctional reagents that carry two identical functional groups proved to be highly efficient in inducing cross-linking between identical and different peptoids. Heterobifunctional reagents contain two different functional groups. By taking advantage of the differential reactivities of the two different functional groups, cross- linking can be controlled both selectively and sequentially.
  • the bifunctional cross-linking reagents can be divided according to the specificity of their functional groups, e.g., amino-, sulfhydryl-, guanidino-, indole-, or carboxyl-specific groups. Of these, reagents directed to free amino groups have become especially popular because of their commercial availability, ease of synthesis and the mild reaction conditions under which they can be14010 ⁇ _24_ applied.
  • a majority of heterobifunctional cross-linking reagents contains a primary amine- reactive group and a thiol-reactive group.
  • heterobifunctional cross-linking reagents and methods of using the cross-linking reagents are described in U.S. Patent 5,889, 155, specifically incorporated herein by reference in its entirety.
  • the cross-linking reagents combine a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing coupling in one example, of aldehydes to free thiols.
  • the cross-linking reagent can be modified to cross-link various functional groups and is thus useful for cross-linking peptoids. In instances where a particular peptide does not contain a residue amenable for a given cross- linking reagent in its native sequence, conservative genetic or synthetic amino acid changes in the primary sequence can be utilized.
  • Any undefined valency on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to the atom.
  • R may replace any hydrogen atom attached to any of the ring atoms, including a depicted, implied, or expressly defined hydrogen, so long as a stable structure is formed.
  • a group "R” is depicted as a "floating group” on a fused ring system, as for example in the formula:
  • R may replace any hydrogen attached to any of the ring atoms of either of the fused rings unless specified otherwise.
  • Replaceable hydrogens include depicted hydrogens (e.g., the hydrogen attached to the nitrogen in the formula above), implied hydrogens (e.g., a hydrogen of the formula above that is not shown but understood to be present), expressly defined hydrogens, and optional hydrogens whose presence depends on the identity of a ring atom (e.g., a hydrogen attached to group X, when X equals -CH-), so long as a stable structure is formed.
  • R may reside on either the 5-membered or the 6-membered ring of the fused ring system.
  • the subscript letter "y" immediately following the group "R” enclosed in parentheses represents a numeric variable. Unless specified otherwise, this variable can be 0, 1, 2, or any integer greater than 2, only limited by the maximum number of replaceable hydrogen atoms of the ring or ring system. 14010 ⁇ -26- [0075]
  • the following parenthetical subscripts further define the group/class as follows: "(Cn)” defines the exact number (n) of carbon atoms in the group/class.
  • (C ⁇ n) defines the maximum number (n) of carbon atoms that can be in the group/class, with the minimum number as small as possible for the group in question, e.g., it is understood that the minimum number of carbon atoms in the group “alkenyl(c ⁇ 8)” or the class “alkene(c ⁇ 8)” is two.
  • alkoxy(c ⁇ io) designates those alkoxy groups having from 1 to 10 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range derivable therein (e.g., 3 to 10 carbon atoms).
  • (Cn-n') defines both the minimum (n) and maximum number ( ⁇ ') of carbon atoms in the group.
  • alkyl(c 2- io) designates those alkyl groups having from 2 to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range derivable therein (e.g., 3 to 10 carbon atoms)).
  • saturated means the compound or group so modified has no carbon-carbon double and no carbon-carbon triple bonds, except as noted below.
  • the term does not preclude carbon-heteroatom multiple bonds, for example a carbon oxygen double bond or a carbon nitrogen double bond. Moreover, it does not preclude a carbon-carbon double bond that may occur as part of keto-enol tautomerism or imine/enamine tautomerism.
  • aliphatic when used without the "substituted” modifier signifies that the compound/group so modified is an acyclic or cyclic, but non-aromatic hydrocarbon compound or group.
  • the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic).
  • Aliphatic compounds/groups can be saturated, that is joined by single bonds (alkanes/alkyl), or unsaturated, with one or more double bonds (alkenes/alkenyl) or with one or more triple bonds (alkynes/alkynyl).
  • one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, - H 2 , -N0 2 , -C0 2 H, -C0 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , -C(0)NH 2 , -OC(0)CH 3 , or -S(0) 2 NH 2 .
  • alkyl when used without the "substituted” modifier refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, and no atoms other than carbon and hydrogen.
  • cycloalkyl is a subset of alkyl.
  • the groups -CH 3 (Me),014010 ⁇ _27_ -CH 2 CH 3 (Et), -CH 2 CH 2 CH 3 ( «-Pr), -CH(CH 3 ) 2 (iso-Pr), -CH(CH 2 ) 2 (cyclopropyl), -CH 2 CH 2 CH 2 CH 3 ( «-Bu), -CH(CH 3 )CH 2 CH 3 (sec-butyl), -CH 2 CH(CH 3 ) 2 (iso-bul l), -C(CH 3 ) 3 (tert-butyl), -CH 2 C(CH 3 ) 3 (weo-pentyl), cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl are non-limiting examples of alkyl groups.
  • alkanediyl when used without the "substituted” modifier refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched, cyclo, cyclic or acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen.
  • alkanediyl groups are non-limiting examples of alkanediyl groups.
  • one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, -NH 2 , -N0 2 , -C0 2 H, -C0 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , -C(0)NH 2 , - OC(0)CH 3 , or -S(0) 2 NH 2 .
  • the following groups are non-limiting examples of substituted alkyl groups: -CH 2 OH, -CH 2 C1, -CF 3 , -CH 2 CN, -CH 2 C(0)OH, -CH 2 C(0)OCH 3 , -CH 2 C(0)NH 2 , -CH 2 C(0)CH 3 , -CH 2 OCH 3 , -CH 2 OC(0)CH 3 , -CH 2 NH 2 , -CH 2 N(CH 3 ) 2 , and -CH 2 CH 2 C1.
  • fluoroalkyl is a subset of substituted alkyl, in which one or more hydrogen has been substituted with a fluoro group and no other atoms aside from carbon, hydrogen and fluorine are present.
  • alkane refers to the compound H-R, wherein R is alkyl.
  • alkenyl when used without the "substituted” modifier refers to an monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one nonaromatic carbon- carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen.
  • alkenediyl when used without the "substituted” modifier refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of 014010 ⁇ -28- attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen.
  • -CH CHCH 2 -, and are non-limiting examples of alkenediyl groups.
  • substituents one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, -NH 2 , -N0 2 , "C0 2 H, -C0 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , -C(0)NH 2 , -OC(0)CH 3 , or - S(0) 2 NH 2 .
  • alkene refers to the compound H-R, wherein R is alkenyl.
  • alkynyl when used without the "substituted” modifier refers to an monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen.
  • alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds.
  • the groups, -C ⁇ CH, -C ⁇ CCH 3 , and -CH 2 C ⁇ CCH 3 are non-limiting examples of alkynyl groups.
  • alkynediyl when used without the “substituted” modifier refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen.
  • aryl when used without the "substituted” modifier refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, said carbon atom forming part of a one or more six-membered aromatic ring structure, wherein the ring atoms are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl group (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present.
  • Non-limiting examples of aryl groups include phenyl14010 ⁇ _2 Q _ (Ph), methylphenyl, (dimethyl)phenyl, -C6H 4 CH 2 CH 3 (ethylphenyl), naphthyl, and the monovalent group derived from biphenyl.
  • the term "arenediyl” when used without the "substituted” modifier refers to a divalent aromatic group, with two aromatic carbon atoms as points of attachment, said carbon atoms forming part of one or more six-membered aromatic ring structure(s) wherein the ring atoms are all carbon, and wherein the monovalent group consists of no atoms other than carbon and hydrogen.
  • the term does not preclude the presence of one or more alkyl group (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused.
  • alkyl group carbon number limitation permitting
  • arenediyl groups include:
  • one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, -NH 2 , -N0 2 , "C0 2 H, -C0 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , -C(0)NH 2 , -OC(0)CH 3 , or - S(0) 2 NH 2 .
  • An "arene” refers to the compound H-R, wherein R is aryl.
  • aralkyl when used without the “substituted” modifier refers to the monovalent group -alkanediyl-aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above.
  • Non-limiting examples of aralkyls are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl.
  • substituted aralkyls are: (3- chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-l-yl.
  • heteroaryl when used without the "substituted” modifier refers to a monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of an aromatic ring structure wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. As used herein, the term does not preclude the presence of one or more
  • heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), methylpyridyl, oxazolyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, thienyl, and triazinyl.
  • heteroarenediyl when used without the "substituted” modifier refers to an divalent aromatic group, with two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as the two points of attachment, said atoms forming part of one or more aromatic ring structure(s) wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur.
  • the term does not preclude the presence of one or more alkyl group (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused.
  • Non-limiting examples of heteroarenediyl groups include:
  • one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, -NH 2 , -N0 2 , "C0 2 H, -C0 2 CH 3 , -CN, -SH, -OCH3, -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , -C(0)NH 2 , -OC(0)CH 3 , or - S(0) 2 NH 2 .
  • acyl when used without the "substituted” modifier refers to the group -C(0)R, in which R is a hydrogen, alkyl, aryl, aralkyl or heteroaryl, as those terms are defined above.
  • the groups, -CHO, -C(0)CH 3 (acetyl, Ac), -C(0)CH 2 CH 3 , -C(0)CH 2 CH 2 CH 3 , -C(0)CH(CH 3 ) 2 , -C(0)CH(CH 2 ) 2 , -C(0)C 6 H 5 , -C(0)C 6 H 4 CH 3 , -C(0)CH 2 C 6 H 5 , -C(0)(imidazolyl) are non-limiting examples of acyl groups.
  • a “thioacyl” is defined in an analogous manner, except that the oxygen atom of the group -C(0)R has been replaced with a sulfur atom, -C(S)R.
  • one or more hydrogen atom has been independently replaced by-OH, -F, -CI, -Br, -I, - H 2 , -N0 2 , -C0 2 H, -C0 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , -C(0)NH 2 , -OC(0)CH 3 , or -S(0) 2 NH 2 .
  • the groups, -C(0)CH 2 CF 3 , -C0 2 H (carboxyl), -C0 2 CH 3 (methylcarboxyl), -C0 2 CH 2 CH 3 , 01 4 010 ⁇ -31- -C(0)NH 2 (carbamoyl), and -CON(CH 3 ) 2 are non-limiting examples of substituted acyl groups.
  • alkoxy when used without the "substituted” modifier refers to the group -OR, in which R is an alkyl, as that term is defined above.
  • alkoxy groups include: -OCH 3 , -OCH 2 CH 3 , -OCH 2 CH 2 CH 3 , -OCH(CH 3 ) 2 , -OCH(CH 2 ) 2 , -O-cyclopentyl, and -O-cyclohexyl.
  • alkenyloxy when used without the “substituted” modifier, refers to groups, defined as -OR, in which R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl, and acyl, respectively.
  • alkylthio when used without the "substituted” modifier refers to the group -SR, in which R is an alkyl, as that term is defined above.
  • alkylamino when used without the "substituted” modifier refers to the group -NHR, in which R is an alkyl, as that term is defined above.
  • alkylamino groups include: -NHCH 3 and -NHCH 2 CH 3 .
  • dialkylamino when used without the "substituted” modifier refers to the group -NRR', in which R and R' can be the same or different alkyl groups, or R and R' can be taken together to represent an alkanediyl.
  • Non-limiting examples of dialkylamino groups include: -N(CH 3 ) 2 , -N(CH 3 )(CH 2 CH 3 ), and N-pyrrolidinyl.
  • dialkylamino groups include: -N(CH 3 ) 2 , -N(CH 3 )(CH 2 CH 3 ), and N-pyrrolidinyl.
  • alkoxyamino refers to groups, defined as -NHR, in which R is alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, and alkylsulfonyl, respectively.
  • a non- limiting example of an arylamino group is -NHC 6 H 5 .
  • a non-limiting example of an amido group is -NHC(0)CH 3 .
  • alkylphosphate when used without the "substituted” modifier refers to the group -OP(0)(OH)(OR), in which R is an alkyl, as that term is defined above.
  • alkylphosphate groups include: -OP(0)(OH)(OMe) and -OP(0)(OH)(OEt).
  • dialkylphosphate when used without the "substituted” modifier refers to the group -OP(0)(OR)(OR'), in which R and R' can be the same or different alkyl groups, or R and R' can be taken together to represent an alkanediyl.
  • Non- limiting examples of dialkylphosphate groups include: -OP(0)(OMe) 2 , -OP(0)(OEt)(OMe) and -OP(0)(OEt) 2 .
  • -OP(0)(OMe) 2 When any of these terms is used with the "substituted" modifier one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, - H 2 , -N0 2 , -C0 2 H, -C0 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , -C(0)NH 2 , -OC(0)CH 3 , or -S(0) 2 NH 2 .
  • alkylsulfonyl and “alkylsulfinyl” when used without the “substituted” modifier refers to the groups -S(0) 2 R and -S(0)R, respectively, in which R is an alkyl, as that term is defined above.
  • alkenylsulfonyl are defined in an analogous manner.
  • one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, -NH 2 , -N0 2 , -C0 2 H, -C0 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , -C(0)NH 2 , - OC(0)CH 3 , or -S(0) 2 NH 2 .
  • a "chiral auxiliary” refers to a removable chiral group that is capable of influencing the stereoselectivity of a reaction. Persons of skill in the art are familiar with such compounds, and many are commercially available.
  • hydrate when used as a modifier to a compound means that the compound has less than one (e.g., hemihydrate), one (e.g., monohydrate), or more than one (e.g., dihydrate) water molecules associated with each compound molecule, such as in solid forms of the compound.
  • IC5 0 refers to an inhibitory dose which is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or chemical process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half.
  • An "isomer" of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the configuration of those atoms in three dimensions differs.
  • the term "patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof.
  • the patient or subject is a primate.
  • Non-limiting examples of human subjects are adults, juveniles, infants and fetuses.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily 014010 ⁇ -34- fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • “Pharmaceutically acceptable salts” means salts of compounds of the present invention which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity.
  • Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2 -hydroxy ethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-l-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene- 1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinna
  • Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases.
  • Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide.
  • Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002).
  • Prevention includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or 014010 ⁇ -35- patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.
  • Prodrug means a compound that is convertible in vivo metabolically into an inhibitor according to the present invention.
  • the prodrug itself may or may not also have activity with respect to a given target protein.
  • a compound comprising a hydroxy group may be administered as an ester that is converted by hydrolysis in vivo to the hydroxy compound.
  • Suitable esters that may be converted in vivo into hydroxy compounds include acetates, citrates, lactates, phosphates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene- bis-P-hydroxynaphthoate, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, -toluenesulfonates, cyclohexylsulfamates, quinates, esters of amino acids, and the like.
  • a compound comprising an amine group may be administered as an amide that is converted by hydrolysis in vivo to the amine compound.
  • a "repeat unit” is the simplest structural entity of certain materials, for example, frameworks and/or polymers, whether organic, inorganic or metal-organic. In the case of a polymer chain, repeat units are linked together successively along the chain, like the beads of a necklace. For example, in polyethylene, -[-CH 2 CH 2 -] n -, the repeat unit is -CH 2 CH 2 - The subscript "n" denotes the degree of polymerisation, that is, the number of repeat units linked together.
  • a “stereoisomer” or “optical isomer” is an isomer of a given compound in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs.
  • “Enantiomers” are stereoisomers of a given compound that are mirror images of each other, like left and right hands.
  • “Diastereomers” are stereoisomers of a given compound that are not enantiomers.
  • Chiral molecules contain a chiral center, also referred to as a stereocenter or stereogenic center, which is any point, though not necessarily an atom, in a molecule bearing groups such that an interchanging of any two groups leads to a stereoisomer.
  • the chiral center is14010 ⁇ t typically a carbon, phosphorus or sulfur atom, though it is also possible for other atoms to be stereocenters in organic and inorganic compounds.
  • a molecule can have multiple stereocenters, giving it many stereoisomers.
  • n is the number of tetrahedral stereocenters. Molecules with symmetry frequently have fewer than the maximum possible number of stereoisomers.
  • a 50:50 mixture of enantiomers is referred to as a racemic mixture.
  • a mixture of enantiomers can be enantiomerically enriched so that one enantiomer is present in an amount greater than 50%.
  • enantiomers and/or diasteromers can be resolved or separated using techniques known in the art.
  • stereocenter or axis of chirality for which stereochemistry has not been defined, that stereocenter or axis of chirality can be present in its R form, 5 * form, or as a mixture of the R and S forms, including racemic and non- racemic mixtures.
  • the phrase "substantially free from other stereoisomers” means that the composition contains ⁇ 15%, more preferably ⁇ 10%, even more preferably ⁇ 5%, or most preferably ⁇ 1% of another stereoisomer(s).
  • Substituent convertible to hydrogen in vivo means any group that is convertible to a hydrogen atom by enzymological or chemical means including, but not limited to, hydrolysis and hydrogenolysis.
  • Examples include hydro lyzable groups, such as acyl groups, groups having an oxycarbonyl group, amino acid residues, peptide residues, o-nitrophenylsulfenyl, trimethylsilyl, tetrahydropyranyl, diphenylphosphinyl, and the like.
  • Examples of acyl groups include formyl, acetyl, trifluoroacetyl, and the like.
  • groups having an oxycarbonyl group include ethoxycarbonyl, tert-butoxycarbonyl (-C(0)OC(CH 3 ) 3 ), benzyloxycarbonyl, / methoxybenzyloxycarbonyl, vinyloxycarbonyl, -(p-toluenesulfonyl)ethoxycarbonyl, and the like.
  • Suitable amino acid residues include, but are not limited to, residues of Gly (glycine), Ala (alanine), Arg (arginine), Asn (asparagine), Asp (aspartic acid), Cys (cysteine), Glu (glutamic acid), His (histidine), He (isoleucine), Leu (leucine), Lys (lysine), Met (methionine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine), Val (valine), Nva (norvaline), Hse (homoserine), 4-Hyp (4-hydroxyproline), 5-Hyl (5-hydroxylysine), Orn (ornithine) and ⁇ -Ala.
  • suitable amino acid residues also include amino acid residues that are protected with a protecting group.
  • suitable protecting groups include those typically employed in peptide synthesis, including acyl groups (such as 14010 ⁇ -37- formyl and acetyl), arylmethyloxycarbonyl groups (such as benzyloxycarbonyl and p- nitrobenzyloxycarbonyl), tert-butoxycarbonyl groups (-C(0)OC(CH 3 ) 3 ), and the like.
  • Suitable peptide residues include peptide residues comprising two to five amino acid residues. The residues of these amino acids or peptides can be present in stereochemical configurations of the D-form, the L-form or mixtures thereof.
  • amino acid or peptide residue may have an asymmetric carbon atom.
  • suitable amino acid residues having an asymmetric carbon atom include residues of Ala, Leu, Phe, Trp, Nva, Val, Met, Ser, Lys, Thr and Tyr.
  • Peptide residues having an asymmetric carbon atom include peptide residues having one or more constituent amino acid residues having an asymmetric carbon atom.
  • suitable amino acid protecting groups include those typically employed in peptide synthesis, including acyl groups (such as formyl and acetyl), arylmethyloxycarbonyl groups (such as benzyloxycarbonyl and p- nitrobenzyloxycarbonyl), tert-butoxycarbonyl groups (-C(0)OC(CH 3 ) 3 ), and the like.
  • acyl groups such as formyl and acetyl
  • arylmethyloxycarbonyl groups such as benzyloxycarbonyl and p- nitrobenzyloxycarbonyl
  • tert-butoxycarbonyl groups tert-butoxycarbonyl groups
  • Suitable reductively eliminable hydrogenolyzable groups include, but are not limited to, arylsulfonyl groups (such as o- toluenesulfonyl); methyl groups substituted with phenyl or benzyloxy (such as benzyl, trityl and benzyloxymethyl); arylmethoxycarbonyl groups (such as benzyloxycarbonyl and o-methoxy-benzyloxycarbonyl); and haloethoxycarbonyl groups (such as ⁇ , ⁇ , ⁇ - trichloroethoxycarbonyl and ⁇ -iodoethoxycarbonyl).
  • arylsulfonyl groups such as o- toluenesulfonyl
  • methyl groups substituted with phenyl or benzyloxy such as benzyl, trityl and benzyloxymethyl
  • arylmethoxycarbonyl groups such as benzyl
  • Effective amount means that amount which, when administered to a subject or patient for treating a disease, is sufficient to effect such treatment for the disease.
  • Treatment includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.
  • inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease e.g., arresting further development of the pathology and/or symptomatology
  • ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease e.g., reversing the pathology and/or symptomatology
  • water soluble means that the compound dissolves in water at least to the extent of 0.010 mole/liter or is classified as soluble according to literature precedence.
  • DMSO dimethyl sulfoxide
  • NO nitric oxide
  • iNOS inducible nitric oxide synthase
  • COX-2 cyclooxygenase-2
  • NGF nerve growth factor
  • IBMX isobutylmethylxanthine
  • FBS fetal bovine serum
  • GPDH glycerol 3-phosphate dehydrogenase
  • RXR retinoid X receptor
  • TGF- ⁇ transforming growth factor- ⁇
  • IFNy or IFN- ⁇ interferon- ⁇
  • LPS bacterial endotoxic lipopolysaccharide
  • TNFa or TNF-a tumor necrosis factor-a
  • IL- ⁇ ⁇ interleukin- ⁇
  • GAPDH glyceraldehyde-3 -phosphate dehydrogenase
  • MTT 3-[4,5-dimethylthiazol-2-yl]-2,5- dimethylthiazol-2-yl]-2,5- dimethylthiazol
  • Compounds of the present disclosure may be made using the methods described above and in Example 1 below. These methods can be further modified and optimized using the principles and techniques of organic chemistry as applied by a person skilled in the art. Such principles and techniques are taught, for example, in March 's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (2007), which is incorporated by reference herein.
  • Compounds employed in methods of the invention may contain one or more asymmetrically-substituted carbon or nitrogen atoms, and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated.
  • Compounds may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained.
  • the 014010 ⁇ -39- chiral centers of the compounds of the present invention can have the S or the R configuration.
  • Compounds of the invention may also have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and/or have a better pharmacokinetic profile (e.g., higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.
  • atoms making up the compounds of the present invention are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include tritium and deuterium
  • isotopes of carbon include 13 C and 14 C.
  • one or more carbon atom(s) of a compound of the present invention may be replaced by a silicon atom(s).
  • one or more oxygen atom(s) of a compound of the present invention may be replaced by a sulfur or selenium atom(s).
  • Compounds of the present invention may also exist in prodrug form. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.), the compounds employed in some methods of the invention may, if desired, be delivered in prodrug form. Thus, the invention contemplates prodrugs of compounds of the present invention as well as methods of delivering prodrugs. Prodrugs of the compounds employed in the invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound.
  • prodrugs include, for example, compounds described herein in which a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a subject, cleaves to form a hydroxy, amino, or carboxylic acid, respectively.
  • the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically 14010 ⁇ -40- acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.
  • the polyglutamine disorders include nine neurodegenerative disorders that are inherited gain-of-function diseases caused by expansion of a translated CAG repeat. Even though the disease-causing proteins are widely expressed, specific collections of neurons are more susceptible in each disease. There is substantial evidence linking the function of the polyglutamine disease-associated proteins with the regulation of gene transcription, and a variety of mechanisms have been suggested by which the polyglutamine proteins impact upon transcription, including altering the function of a very specific DNA-binding factor like the AR (SBMA), general DNA-binding proteins like TBP (SCA17), Spl, TFIID and TFIIF (HD), chromatin structure (SCA7), coregulators (HD, SCA1, and DRPLA), and possibly the ubiquitin-proteasome system (SCA3).
  • SBMA DNA-binding factor
  • SCA17 general DNA-binding proteins
  • Spl Spl
  • TFIID and TFIIF chromatin structure
  • SCA7 chromatin structure
  • coregulators HD, SCA1, and DRPLA
  • Huntington disease also called Huntington's chorea, chorea major, or HD
  • Huntington's chorea is a genetic neurological disorder characterized by abnormal body movements called chorea and a lack of coordination; it also affects a number of mental abilities and some aspects of behavior.
  • the accession number for Huntingtin is NM_002111.
  • the gene causing the disorder is dominant and may, therefore, be inherited from a single parent.
  • Global incidence varies, from 3 to 7 per 100,000 people of Western European descent, down to 1 per 1,000,000 of Asian and African descent.
  • the onset of physical symptoms in HD occur in a large range around a mean of a person's late forties to early fifties. If symptoms become noticeable before a person is the age of twenty, then their condition is known as Juvenile HD. 14010 ⁇ _41_ [00119]
  • a trinucleotide repeat expansion occurs in the Huntingtin gene, which produces mutant Huntingtin protein. The presence of this protein increases the rate of neuron cell death in select areas of the brain, affecting certain neurological functions. The loss of neurons isn't fatal, but complications caused by symptoms reduce life expectancy. There is currently no proven cure, so symptoms are managed with a range of medications and supportive services.
  • Symptoms increase in severity progressively, but are not often recognised until they reach certain stages. Physical symptoms are usually the first to cause problems and be noticed, but these are accompanied by cognitive and psychiatric ones which aren't often recognized. Almost everyone with HD eventually exhibits all physical symptoms, but cognitive symptoms vary, and so any psychopathological problems caused by these, also vary per individual. The symptoms of juvenile HD differ in that they generally progress faster and are more likely to exhibit rigidity and bradykinesia instead of chorea and often include seizures. [00121] The most characteristic symptoms are jerky, random, and uncontrollable movements called chorea, although sometimes very slow movement and stiffness (bradykinesia, dystonia) can occur instead or in later stages.
  • Cognitive cognitive abilities are progressively impaired, including executive function (planning, cognitive flexibility, abstract thinking, rule acquisition, initiating appropriate actions and inhibiting inappropriate actions), psychomotor function (slowing of thought processes to control muscles), perceptual and spatial skills of self and surrounding environment, selection of correct methods of remembering information (but not actual memory itself), short-term memory, and ability to learn new skills, depending on the pathology of the individual. 14010 ⁇ _42_ [00123]
  • Psychopathological symptoms vary more than cognitive and physical ones, and may include anxiety, depression, a reduced display of emotions (blunted affect) and decreased ability to recognize negative expressions like anger, disgust, fear or sadness in others, egocentrism, aggression, and compulsive behavior. The latter can cause, or worsen, hypersexuality and addictions such as alcoholism and gambling.
  • HD is autosomal dominant, needing only one affected allele from either parent to inherit the disease. Although this generally means there is a one in two chance of inheriting the disorder from an affected parent, the inheritance of HD is more complex due to potential dynamic mutations, where DNA replication does not produce an exact copy of itself. This can cause the number of repeats to change in successive generations. This can mean that a parent with a count close to the threshold, may pass on a gene with a count either side of the threshold. Repeat counts maternally inherited are usually similar, whereas paternally inherited ones tend to increase. This potential increase in repeats in successive generations is known as anticipation. In families where neither parent has HD, new mutations account for truly sporadic cases of the disease. The frequency of these de novo mutations is extremely low.
  • Huntingtin protein is variable in its structure as there are many polymorphisms of the gene which can lead to variable numbers of glutamine residues present in the protein. In its wild-type (normal) form, it contains 6-35 glutamine residues; however, in individuals affected by HD, it contains between 36-155 glutamine residues. Huntingtin has a predicted mass of ⁇ 350kDa, however, this varies and is largely dependent on the number of glutamine residues in the protein. Normal huntingtin is generally accepted to be 3144 amino acids in size. [00127] Two transcriptional pathways are more extensively implicated in HD - the CBP/p300 and Spl pathways - and these are transcription factors whose functions are vital 14010 ⁇ -43- for the expression of many genes.
  • Mutant huntingtin was also shown to interact with the acetyltransferase domain of CBP and inhibit the acetyltransferase activity of CBP, p300, and the p300/CBP-associated factor P/CAF (Steffan et al, 2001). [00128] These observations prompted a hypothesis whereby the pathogenic process was linked to the state of histone acetylation; specifically, mutant huntingtin induced a state of decreased histone acetylation and thus altered gene expression. Support for this hypothesis was obtained in a Drosophila HD model expressing an N-terminal fragment of huntingtin with an expanded polyglutamine tract in the eye.
  • HDAC inhibitors have been reported for other polyglutamine disorders, prompting the concept that at least some of the observed effects in polyglutamine disorders are due to alterations in histone acetylation (Hughes 2002).
  • Studies published in 2002 revealed that the N-terminal fragment of huntingtin and intact huntingtin interact with Spl (Dunah et al, 2002; Li et al, 2002), a transcriptional activator that binds to upstream GC- rich elements in certain promoters.
  • Standard treatments to alleviate emotional symptoms include the use of antidepressants and sedatives, with antipsychotics (in low doses) for psychotic symptoms.
  • Speech therapy helps by improving speech and swallowing methods; this therapy is more effective if started early on, as the ability to learn is reduced as the disease progresses.
  • Nutrition is an important part of treatment; most third and fourth stage HD sufferers need two to three times the calories of the average person to maintain body weight. Healthier foods in pre-symptomatic and earlier stages may slow down the onset and progression of the disease. High calorie intake in pre-symptomatic and earlier stages has been shown to speed up the onset and reduce IQ level. Thickening agent can be added to drinks as swallowing becomes more difficult, as thicker fluids are easier and safer to swallow. The option of using a stomach PEG is available when eating becomes too hazardous or uncomfortable; this greatly reduces the chances of aspiration of food, and the subsequent increased risk of pneumonia, and increases the amount of nutrients and calories that can be ingested.
  • EPA an Omega-3 fatty acid
  • EPA may slow and possibly reverse the progression of the disease.
  • it is in FDA clinical trial as ethyl-EPA, (brand name Miraxion), for prescription use.
  • Clinical trials utilise 2 grams per day of EPA. In the United States, it is available over the counter in lower concentrations in Omega-3 and fish oil supplements.
  • SCA Spinocerebellar ataxia
  • SCA is one of a group of genetic disorders characterized by slowly progressive incoordination of gait and often associated with poor coordination of hands, speech, and eye movements. Frequently, atrophy of the cerebellum occurs.
  • SCA results in unsteady and clumsy motion of the body due to a failure of the fine coordination of muscle movements, along with other symptoms.
  • the symptoms of the condition vary with the specific type (there are several), 14010 ⁇ -45- and with the individual patient. Generally, a person with ataxia retains full mental capacity but may progressively lose physical control.
  • spinocerebellar ataxia is a progressive disease (it gets worse with time), although not all types cause equally severe disability. Treatments are generally limited to softening symptoms, not the disease itself. The condition can be irreversible. A person with this disease will usually end up needing to use a wheelchair, and eventually they may need assistance to perform daily tasks.
  • the treatment of incoordination or ataxia then mostly involves the use of adaptive devices to allow the ataxia individual to maintain as much independence as possible. Such devices may include a cane, crutches, walker, or wheelchair for those with impaired gait; devices to assist with writing, feeding, and self care if hand and arm coordination are impaired; and communication devices for those with impaired speech.
  • Ataxia Many patients with hereditary or idiopathic forms of ataxia have other symptoms in addition to ataxia. Medications or other therapies might be appropriate for some of these symptoms, which could include tremor, stiffness, depression, spasticity, and sleep disorders, among others. Both onset of initial symptoms and duration of disease can be subject to variation, and it can be easily misdiagnosed as another neurological condition, such as multiple sclerosis (MS).
  • MS multiple sclerosis
  • SCA1 Spinocerebellar ataxia type 1
  • SCA1 is an autosomal dominant ataxia that results in gait ataxia, dysarthria, and bulbar dysfunction, with death usually between 10 and 15 years after the onset of symptoms. The average age of onset is in the 4 th decade of life.
  • protein ataxin- 1 being widely expressed in the central nervous system, the most frequently seen and most severe pathological alterations are restricted to loss of Purkinje cells in the cerebellar cortex, as well as loss of neurons in the inferior olivary nuclei, the cerebellar dentate nuclei and the red nuclei.
  • Ataxin- 1 the product of the SCA1 gene, is prominently located in the nuclei of neurons (Servadio et ah, 1995). Indication that SCA1 pathogenesis was due to alterations in nuclear function began with the observation that for mutant ataxin- 1 to cause disease, it had to enter the nucleus of Purkinje cells (Klement et ah, 1998). Consequent studies revealed that wild-type ataxin- 1 has properties consistent with a role in the 14010 ⁇ -46- regulation of gene expression in the nucleus. These include the ability to bind RNA (Yue et ah, 2001) and to shuttle between the nucleus and cytoplasm (Irwin et ah, 2005).
  • SCA2 Spinocerebellar ataxia type 2
  • SCA2 is characterized by progressive cerebellar ataxia, including nystagmus, slow saccadic eye movements and, in some individuals, ophthalmoparesis. Pyramidal findings are present; deep tendon reflexes are brisk early on and are absent later in the course. Age of onset is typically in the 3 rd to 4 th decade with a 10- 15 -year disease duration.
  • SCA2 The diagnosis of SCA2 rests upon the use of molecular genetic testing to detect an abnormal CAG trinucleotide repeat expansion of the ATXN2 gene. Affected individuals have alleles with greater than 32 CAG trinucleotide repeats. Such testing detects nearly 100% of cases and is available in clinical laboratories.
  • SCA3 Spinocerebellar ataxia type 3
  • MJD Machado Joseph disease
  • Ataxin-3 contains an N-terminal Josephin domain (JD) with recently ascribed ubiquitin protease activity (Burnett et ah, 2003; Scheel et ah, 2003), two ubiquitin interacting motifs (UIMs) capable of binding ubiquitin (Chai et ah, 2004; Burnett et ah,14010 ⁇ _47_ 2003; Donaldson et al, 2003) followed by a polyglutamine stretch, and a C-terminal variable domain.
  • JD N-terminal Josephin domain
  • UAMs ubiquitin interacting motifs
  • the crystal structure of the ataxin-3 JD provided insight into the potential function of ataxin-3 as a polyubiquitin chain editing protein by demonstrating a tight connection between polyubiquitin binding and the deubiquitylating activity of ataxin-3 (Mao et al, 2005; Nicastro et al, 2005).
  • ataxin-3 has a role in the ubiquitin and/or the ubiquitin-proteasome system.
  • Ataxin-3 is unique from the other polyglutamine diseases in that wild-type ataxin-3 expression in Drosophila protects neurons from toxicity initiated by other polyglutamine- expanded proteins (Warrick et al, 2005). This protection afforded by wild-type ataxin-3 was dependent on active proteasomes and both the UIM and ubiquitin protease domains of ataxin-3.
  • accession numbers for these genes are as follows: Ataxinl ( M_000332), ataxin2 (NM_002973), and ataxin3 ( M_004993).
  • DPLA Dentatorubral-pallidoluysian atrophy
  • DRPLA can be juvenile-onset ( ⁇ 20 years), early adult-onset (20 ⁇ 10 years), or late adult-onset (> 40 years). Late adult-onset DRPLA is characterized by ataxia, choreoathetosis and dementia. Early adult-onset DRPLA also includes seizures and myoclonus. Juvenile-onset DRPLA presents with ataxia and symptoms consistent with progressive myoclonus epilepsy.
  • Atrophin-1 encodes a hydrophilic 1184 amino acid protein with several repetitive motifs including a serine-rich region, a variable length polyglutamine tract, a polyproline tract, and a region of alternating acidic and basic residues. It contains a putative nuclear localization signal in the N-terminus of the protein and a putative nuclear export signal in the C-terminus. ATN1 is ubiquitously expressed in all tissues, but proteolytically cleaved in neuronal cells. The function of ATN1 is not clear, however it is believed to be a transcriptional co-repressor.
  • ATN1 and atrophin-2 can be co-014010 ⁇ _A Q _ immunoprecipitated, indicating that they may carry out some functions together in a molecular complex.
  • Atrophin-1 may be a dispensable or redundant protein as mice bred with a null allele for atrophin-1 produce viable and fertile offspring and show no compensatory upregulation of atrophin-2.
  • the accession number for atrophinl is NM_001940.
  • DRPLA is characterized by marked, generalized brain atrophy and the accumulation of atrophin-1 with expanded glutamine stretches. Mutant atrophin-1 proteins have been found in neuronal intranuclear inclusions (Nil) and diffusely accumulated in the neuronal nuclei. While the role of Nils (pathologic or protective) is unclear, the diffuse accumulation of mutant protein is regarded as toxic.
  • Transgenic DRPLA mice demonstrated several neuronal abnormalities including a reduction in the number and size of dendritic spines as well as in the area of perikarya and diameter of dendrites. Spine morphology and density have been linked to learning and memory functions as well as epilepsy. The stubby-type spines seen in DRPLA mice are morphologically different from the thin and mushroom-type spines seen in Huntington's mice. [00150] Morphometric analysis of DRPLA mouse brains has shown a loss of normal inter-microtubule spacing in neuronal axons. The microtubules were relatively compacted, suggesting abnormalities in protein transport may play a role in neuronal degeneration.
  • Nils are not exclusive to DRPLA; they have been found in a variety of neurodegenerative disorders. In DRPLA, Nils have been demonstrated in both neurons and glial cells in the striatum, pontine nuclei, inferior olive, cerebellar cortex and dentate nucleus, though the incidence of neurons with Nils is low, roughly 1-3%. In DRPLA, the Nils are spherical, eosinophilic structures of various sizes. They are non-membrane-bound and are composed of both granular and filamentous structures. They are ubiquitinated and may be paired or in doublet form within the nucleus.
  • Nils have also been demonstrated to alter the distribution of the intranuclear structures, such as promyelocytic leukemia protein (PML) nuclear bodies.
  • PML bodies In neurons with Nil, PML bodies in DRPLA patients form a shell or ring around the ubiquitinated core. In similar polyQ diseases, the association of this PML shell has been shown to be size-dependent with larger Nils being PML negative. This has led to two models, one in which PML bodies represent sites for Nil formation and a second in which PML bodies are involved in degradation and proteolysis of Nils.
  • PML bodies represent sites for Nil formation and a second in which PML bodies are involved in degradation and proteolysis of Nils.
  • DRPLA diffuse accumulation of mutant ATN1 occurs far more extensively than Nil formation.
  • the extent and frequency of neurons showing the diffuse nuclear accumulations changes depending on CAG repeat length. It is believed that the diffuse nuclear accumulations contribute to the clinical features such as dementia and epilepsy.
  • ATN1 contains both a nuclear localization sequence and a nuclear export sequence.
  • ATN1 Cleavage of ATN1 to an N terminal fragment relieves ATN1 of its nuclear export signal and concentrates it in the nucleus. Increased nuclear concentrations have been demonstrated via transfection assay to enhance cellular toxicity.
  • regions in which more than 40% of neurons became immunoreactive to 1C2 included: the nucleus basalis of Meynert, large striatal neurons, globus pallidus, subthalamic nucleus, thalamic intralaminar nucleus, lateral geniculate body, oculomotor nucleus, red nucleus, substantia nigra, trigeminal motor nucleus, nucleus 014010 ⁇ _-: Q _ raphe pontis, pontine nuclei, vestibular nucleus, inferior olive and the cerebellar dentate nucleus.
  • the juvenile type also shows reactivity in the cerebral cortex, hippocampal CA1 area, and the reticular formation of the brainstem. Nuclei containing accumulations of mutant atrophin-1 are deformed with nuclear membrane indentations. [00156] Diagnosis of DRPLA rests of positive family history, clinical findings, and genetic testing. Family history can be difficult to obtain if a relative was misdiagnosed, died young, or experiences late onset of symptoms. Other diseases in the differential diagnosis of adult-onset DRPLA include Huntington's and the spinocerebellar ataxias.
  • FEME familial essential myoclonus and epilepsy
  • Lafora For juvenile-onset, familial essential myoclonus and epilepsy (FEME), Lafora, Unverricht- Lundborg, Neuroaxonal dystrophy, Gaucher's disease, Sialidosis, and Galactosialidosis.
  • MRI Magnetic resonance Imaging
  • EEG EEG
  • neuropsychological testing are recommended. Seizures are treated with anticonvulsants and psychiatric disturbances with psychotropic medications.
  • AD Alzheimer's disease
  • the disease usually begins after age 60, and risk goes up with age. While younger people also may get AD, it is much less common. About 3 percent of men and women ages 65 to 74 have AD, and nearly half of those age 85 and older may have the disease. While the subject of intensive research, the precise causes of AD are still unknown, and there is no cure. The course of the disease varies from person to person. Some people have the disease only for the last 5 years of life, while others may have it for as many as 20 years. The most common cause of death in AD patients is infection.
  • AD is a progressive, neurodegenerative disease characterized by memory loss, language deterioration, impaired visuospatial skills, poor judgment, indifferent attitude, but preserved motor function. AD usually begins after age 65, however, its onset may occur as early as age 40, appearing first as memory decline and, over several years, destroying cognition, personality, and ability to function. Confusion and restlessness may also occur. The type, severity, sequence, and progression of mental changes vary widely. The early symptoms of AD, which include forgetfulness and loss of concentration, can be missed easily because they resemble natural signs of aging. Similar symptoms can also 014010 ⁇ -51- result from fatigue, grief, depression, illness, vision or hearing loss, the use of alcohol or certain medications, or simply the burden of too many details to remember at once.
  • AD Alzheimer's disease
  • medication such as tacrine may alleviate some cognitive symptoms.
  • Aricept donepezil
  • Exelon rivastigmine
  • acetylcholinesterase inhibitors that are indicated for the treatment of mild to moderate dementia of the Alzheimer's type.
  • some medications may help control behavioral symptoms such as sleeplessness, agitation, wandering, anxiety, and depression. These treatments are aimed at making the patient more comfortable.
  • the molecular aspect of AD is complicated and not yet fully defined.
  • AD Alzheimer's disease
  • ⁇ amyloid ⁇ protein
  • PS presenilin
  • ApoE apolipoprotein E
  • Tau protein Tau protein
  • ⁇ contains approximately 40 amino acid residues.
  • the 42 and 43 residue forms are much more toxic than the 40 residue form.
  • ⁇ is generated from an amyloid precursor protein (APP) by sequential proteolysis.
  • One of the enzymes lacks sequence specificity and thus can generate ⁇ of varying (39-43) lengths.
  • the toxic forms of ⁇ cause abnormal events such as apoptosis, free radical formation, aggregation and inflammation.
  • Presenilin encodes the protease responsible for cleaving APP into ⁇ .
  • PS1 and PS2 There are two forms - PS1 and PS2. Mutations in PS 1, causing production of ⁇ 42 , are the typical cause of early onset AD.
  • PHFs paired helical filaments
  • the present invention also involves the treatment of polyglutamine neurodegenerative diseases and Alzheimer's Disease, discussed above.
  • treatment it is not necessary that all symptoms of the disease be addressed, or that any degree of "cure” be achieved. Rather, to accomplish a meaningful treatment, all that is required is that one or more symptoms of the disease be ameliorated to some degree, an advantageous effect be provided in combination with another therapy, or that the disease progression be slowed.
  • compositions of the present invention comprise an effective amount of the oligonucleotide to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or medium.
  • phrases "pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, liposomes, cationic lipid formulations, microbubble nanoparticles, and the like.
  • the use of such media and agents for pharmaceutically active substances is well-known in the art. I'm not so sure this is true. Delivery is a major issue in the field. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
  • compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the 14010 ⁇ -53- present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, or topical. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or introduction into the CNS, such as into spinal fluid. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • pharmaceutically acceptable carrier includes any and all solvents, lipids, nanoparticles, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the peptoids of the present invention may be incorporated with excipients.
  • the compositions of the present invention may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • the solution For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human 14010 ⁇ cc_ administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • Lipid vehicles encompass micelles, microemulsions, macroemulsions, liposomes, and similar carriers.
  • the term micelles refers to colloidal aggregates of amphipathic (surfactant) molecules that are formed at a well-defined concentration known as the critical micelle concentration. Micelles are oriented with the nonpolar portions at the interior and the polar portions at the exterior surface, exposed to water. The typical number of aggregated molecules in a micelle (aggregation number) is 50 to 100. Microemulsions are essentially swollen micelles, although not all micellar solutions can be swollen to form microemulsions.
  • Microemulsions are thermodynamically stable, are formed spontaneously, and contain particles that are extremely small. Droplet diameters in microemulsions typically range from 10 100 nm. In contrast, the term macroemulsions refers to droplets with diameters greater than 100 nm. Liposomes are closed lipid vesicles comprising lipid bilayers that encircle aqueous interiors. Liposomes typically have diameters of 25 nm to 1 ⁇ (see, e.g., Shah, 1998; Janoff, 1999).
  • the principal lipid of the vehicle may be phosphatidylcholine.
  • Other useful lipids include various natural (e.g., tissue derived L-a-phosphatidyl: egg yolk, heart, brain, liver, soybean) and/or synthetic (e.g., saturated and unsaturated l,2-diacyl-SN-glycero-3-phosphocholines, l-acyl-2-acyl-SN- glycero-3-phosphocholines, l,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of the same.
  • tissue derived L-a-phosphatidyl egg yolk, heart, brain, liver, soybean
  • synthetic e.g., saturated and unsaturated l,2-diacyl-SN-glycero-3-phosphocholines, l-acyl-2-acyl-SN- glycero-3-phosphocholines, l,2-diheptanoyl-SN-glycero-3-phosphocholine
  • Such secondary helper lipids may be non-ionic or uncharged at physiological pH, including non- ionic lipids such as cholesterol and DOPE (1,2-dioleolylglyceryl phosphatidylethanolamine).
  • the molar ratio of a phospholipid to helper lipid can range from about 3 : 1 to about 1 : 1, from about 1.5 : 1 to about 1 : 1, and about 1 : 1.
  • Another specific lipid formulation comprises the SNALP formulation, containing the lipids 3- ⁇ -[( ⁇ methoxypoly(ethylene glycol) 200 o)carbamoyl]-l,2- dimyristyloxy-propylamine(PEG-C-DMA), 1 ,2-dilinoleyloxy- N , N -dimethyl-3 - aminopropane (DLinDMA), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a 2:40: 10:48 molar % ratio. See Zimmerman et al. (2006).
  • a liposome is, in simplest form, composed of two lipid layers.
  • the lipid layer may be a monolayer, or may be multilamellar and include multiple layers.
  • Constituents of the liposome may include, for example, phosphatidylcholine, cholesterol, phosphatidylethanolamine, etc.
  • Phosphatidic acid which imparts an electric charge, may also be added.
  • Exemplary amounts of these constituents used for the production of the liposome include, for instance, 0.3 to 1 mol, 0.4 to 0.6 mol of cholesterol; 0.01 to 0.2 mol, 0.02 to 0.1 mol of phosphatidylethanolamine; 0.0 to 0.4 mol, or 0-0.15 mol of phosphatidic acid per 1 mol of phosphatidylcholine.
  • Liposomes can be constructed by well-known techniques (see, e.g., Gregoriadis (1993). Lipids are typically dissolved in chloroform and spread in a thin film over the surface of a tube or flask by rotary evaporation. If liposomes comprised of a mixture of lipids are desired, the individual components are mixed in the original chloroform solution. After the organic solvent has been eliminated, a phase consisting of water optionally containing buffer and/or electrolyte is added and the vessel agitated to suspend the lipid. Optionally, the suspension is then subjected to ultrasound, either in an ultrasonic bath or with a probe sonicator, until the particles are reduced in size and the suspension is of the desired clarity.
  • the aqueous phase is typically distilled water and the suspension is sonicated until nearly clear, which requires several minutes depending upon conditions, kind, and quality of the sonicator.
  • lipid concentrations are 1 mg/ml of aqueous phase, but could be higher or lower by about a factor of ten.
  • the treatment or prevention of AD may be implemented with a peptoid therapeutic of the present embodiments along with other AD therapeutic agents.
  • Secondary AD therapeutic agents include, but are not limited to, antibodies and small molecules. Antibodies developed by companies such as Elan and others, including antibodies that have been modified to improve transport across the BBB.
  • Small molecules have also been developed that inhibit amyloid aggregation. These molecules include Alzhemed (Neurochem Inc) and AZD-103 (Elan). It is also contemplated that gamma-secretase inhibitors can be used in combination with the compositions of the embodiments.
  • Treatment with the peptoid may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
  • the other agent and the peptoids are applied separately to the subject, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and the peptoid would still be able to exert an advantageously combined effect on the subject.
  • a peptoid composition is administered in conjunction with an anti-inflammatory agent.
  • a peptoid composition may precede or follow the anti-inflammatory agent treatment by intervals ranging from minutes to weeks.
  • the anti-inflammatory agent is administered immediately before the peptoid composition and immediately after the peptoid composition.
  • the anti-inflammatory agent may be given less than a day before and less than a day after the therapy.
  • the peptoid composition is "A” and the secondary agent (e.g., anti-inflammatory agent or AD therapeutic), is "B": A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B/B
  • administration of the peptoid composition of the present embodiments to a patient will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, of the vector. It is expected that the treatment cycles would be repeated as necessary.
  • a bead-displayed peptoid library was generated composed of 60,000 diverse 6-mer peptoids on TentaGelTM macrobeads (see Example 2, below).
  • a proline was fixed at the fourth position, while the other five positions were randomized using nine different primary amines (FIG. lb).
  • the fixed proline residue was chosen because proline has been reported to be essential for the association of QBP 1 peptide with polyQexpanded proteins(Hamuro et ah, 2007).
  • a residue containing a hydroxyethyl side group was added between the 6-mer peptoid and the resin in order to provide a consistent residue to aid in mass spectrometry-based characterization and to increase solubility.
  • Mutant Htt proteins that harbor more than 35 glutamine residues easily aggregate in vitro.
  • a fusion protein between Htt-82Q amino-terminal and a maltose-binding protein (MBP-Htt-N-82Q) was generated.
  • MBP-Htt-N-82Q maltose-binding protein
  • the wild type MBP-Htt-N-15Q protein was also expressed and purified. Both MBP-Htt-N-82Q and MBP-Htt-N-15Q proteins were monomeric and stable in solution.
  • the 60K peptoid library was screened with MBP-Htt-N-82Q protein as a bait using a recently developed beads-based screening method (Xiao et ah, 2007).
  • the beads were initially incubated with MBP-Htt-N-82Q protein and then the beads with the bound protein were visualized using anti-MBP mouse monoclonal antibodies and goat anti-mouse antibodies-coated red quantum dots (Qdots) (see Example 2 for details).
  • Qdots goat anti-mouse antibodies-coated red quantum dots
  • the beads displaying potential hits were identified by the red halo visible on the bead surface under a fluorescence microscope at excitation wavelength of 410 nm (FIG. lc). Twenty such beads were picked using a micropipette.
  • HQP09 only five representative peptoids HQP02, HQP04, HQP06, HQP07, and HQP09 were re-synthesized on TentaGelTM beads and evaluated in Qdot binding experiments with MBP-Htt-N-82Q protein. No binding was detected for HQP02, HQP04, HQP06 or HQP07 beads. However strong binding was detected for HQP09 beads. The HQP09 beads also displayed significantly stronger binding to MBP-Htt-N-82Q than to MBP-Htt-N-15Q. Thus, HQP09 (FIG. 2a) was selected as a specific mHtt ligand for further evaluation. HQP09 specifically binds to polyQ-expanded proteins in vitro
  • the next series of studies took advantage of FP-based binding assays in order to identify the structural determinants responsible for association of HQP09 with mHtt.
  • the minimum pharmacophore responsible for the HQP09 binding activity was first determined.
  • sarcosine scanning a method which was recently applied successfully to identifying the minimum pharmacophore of a peptoid antagonist of VEGFR2 (Udugamasooriya et ah, 2008), was used.
  • a series of sarcosine scanning derivatives was obtained by replacing one of the seven residues in HQP09 (FIG. 4a, positions 1-7) with a sarcosine.
  • the corresponding seven peptoids (HQP09_1 to HQP09 7, FIG. 11) were synthesized and used at concentration of 300 ⁇ in competitive FP binding assays with HQP09_G2_Fluo and MBP-Htt-N-82Q. It was found that HQP09 1 and HQP09 2 had no effect on FP signal and that HQP09 4 reduced FP signal by 50% (FIG. 4c). HQP09 3, HQP09_5, HQP09_6 and HQP09_7 were as potent as HQP09 itself and reduced FP signal by more than 75% (FIG. 4c).
  • HQP09 9 inhibited FP signal by more than 50%, HQP09 8 was modestly effective, and HQP09 10 and HQP09 1 1 were ineffective (FIG. 5b).
  • HQP09_9 was less effective than HQP09 (Fig. 5b)
  • HQP09 9 was selected as the lead HQP09 derivative for cell-based assay evaluation.
  • Htt-53Q protein The first exon of Htt-53Q protein was expressed in bacteria as glutathione S-transferase (GST) fusion protein and purified previously described (Wacker et ah, 2004). After purification, GST-Htt53Q appeared nonaggregated by the atomic force microscopy (AFM) analysis and the size-exclusion chromatography. Cleavage of a unique peptide sequence between the GST moiety and Htt53Q with a site- specific protease releases the Htt53Q fragment, initiating aggregation in a time-dependent manner (Wacker et ah, 2004).
  • GST glutathione S-transferase
  • Htt53Q aggregation To measure the degree of Htt53Q aggregation a filter trap assay was used in which detergent-insoluble mHtt aggregates were detected by MW8 antibody (Wacker et ah, 2004). Large amounts of trapped Htt53Q aggregates were detected in samples collected 24 h after cleavage from GST (FIG. 6a). Addition of 10 ⁇ QBP 1 peptide inhibited Htt53Q aggregation by 40% (FIG. 6a), consistent with the published results (Nagai et ah, 2007). When tested in the same concentration (10 ⁇ ), HQP09 peptoid inhibited Htt53Q aggregation by 80%, significantly better than QBP1 peptide (FIG. 6a).
  • the HQP09 9 derivative inhibited Htt53Q aggregation by 30 %, not significantly different from the QBP1 peptide (FIG. 6a).
  • the control peptoids RPOl and HQP09_1 derivative were not effective in preventing Htt53Q aggregation (FIG. 6a).
  • the formation of Htt53Q aggregates was also evaluated by SDS-PAGE gel followed by Western blotting with MW8 antibody.
  • PolyQ-binding peptoids stabilize glutamate-induced Ca signaling in YAC128 MSN
  • Random peptoid RPOl was used in the same concentration (20 ⁇ ) as a control for specificity of observed effects.
  • the peptoids were added to the culture media at DIV1 and DIV13 to allow sufficient time for binding mHtt.
  • Ca 2+ imaging experiments were performed at DIV13 as previously described (Tang et al, 2005).
  • the intracellular neuronal Ca 2+ concentration in these experiments was continuously monitored by Fura-2 imaging and the 340/380 ratio was used to quantify Ca 2+ levels.
  • the increase in Ca 2+ levels following 20 pulses of 20 ⁇ glutamate was significantly higher in YAC128 MSN than in WT MSN (FIG. 7a, e).
  • HQP09_9 appeared to be a more effective "Ca 2+ stabilizer" than HQP09 (FIG. 7e), most likely because of the better membrane permeability of the small derivative.
  • PolyQ-binding peptoids are neuroprotective in cellular HD model
  • the polyQ-binding peptoids prevent mHtt aggregation (FIG. 6, 12) and stabilize abnormal Ca 2+ signals in YAC128 MSN (FIG. 7). These results suggested that polyQ-binding peptoids could sequester mHtt and potentially reduce its toxicity.
  • a glutamate toxicity assay with YAC128 MSN cultures was used that was previously developed (Hu et al, 2009; Tang et al, 2005; Tang et al, 2007; Wu et al, 2006; Tang et al, 2009).
  • HQP09, HQP09 9 or RPOl (20 ⁇ each) were added to WT and YAC128 MSN cultures on DIVl and DIVl 3.
  • the apoptotic cell death of cultured MSN neurons was scored by TUNEL staining as previously described (Tang et al, 2005). In basal conditions approximately 5% of MSNs were apoptotic (TUNEL-positive) in all experimental groups (FIG. 8b), indicating that peptoids were not toxic for the cultured neurons at the concentrations tested.
  • HQP09 and HQP09 9 exerted significant and specific neuroprotective effects on the excitotoxic cell death of YAC128 MSNs. Consistent with Ca 2+ imaging studies (FIG. 7), HQP09_9 appeared to be more effective neuroprotector than HQP09 (FIG. 8b), presumably because of its better membrane permeability.
  • PolyQ-binding peptoids inhibit the mHtt aggregates in the brain of YAC128 mice
  • the peptoid derivative HQP09_9 (20 mg/200 ⁇ , in saline) was delivered subcutaneously to YAC128 and WT mice using an Alzet osmotic pump for 8 months, beginning at the age of 3 months and replacing the pump every month.
  • HQP09 9 a beneficial effect of HQP09 9 on the motor coordination of YAC128 mice was not observed compared to the control group and a decrease in the staining of mHtt aggregates in the striatum was not observed.
  • Most likely these negative results were related to poor blood-brain-barrier permeability of HPQ09 9, so that therapeutic concentrations of HPQ09 9 have not been reached in striatum of YAC128 mice with subcutaneous delivery.
  • HQP09 9 was tested in BBB experiments. First, HQP09 9 (400 ug) was injected from the tail vein of 2 months old WT mouse; (20mg/kg). Samples were collected from the blood and brain at different time points and the concentration of HQP09 9 was determined by LC/MS/MS. Results are sghown in FIG. 15 and Table 2.
  • QBP 1 polyQ binding peptide
  • peptoid libraries constitute rich sources of protein-binding ligands that have in vitro and in vivo biological functions (Yoo et al, 2008).
  • both HQP09 and HQP09 9 prevented aggregation of mHtt in 14010 ⁇ -68- vitro (FIG. 6, FIG. 12).
  • the binding specificity and the anti-aggregation activity of HQP09 were comparable to those of QBPl peptide (FIGs. 2, 6 12).
  • HQP09 and QBP 1 appeared to bind to non-overlapping sites on expanded polyQ sequence and did not compete with each other (FIG. 3).
  • both HQP09 and HQP09 9 stabilized abnormal Ca 2+ signals in HD-YAC128 MSN cultures (FIG. 7) and protected HD-YAC128 MSN from glutamate-induced apoptosis (FIG. 8).
  • HQP09_9 displayed more potent Ca 2+ stabilizing and neuroprotectin activities than HQP09, presumably because of its smaller size and better membrane permeability.
  • HQP09 9 constituted a promising lead for developing HD therapy, especially in view of the fact that peptoids share favorable pharmacological characteristics such as resistance to degradation in vivo, good cell membrane and blood brain barrier permeability (Yoo et ah, 2008).
  • peptoid technology has had very limited application in the field of neuroscience and neurodegeneration. Only one study was performed previously, in which two 3-mer peptoids were isolated as potential neuroprotective agents in a cell-based screen for inhibitors of glutamate-induced excitotoxicity (Montoliu et ah, 2002).
  • TentaGelTM macrobeads (140-170 ⁇ ; capacity: 0.51 mmol/g) were obtained from Rapp Polymere.
  • Knorr Amide MBHA resin (0.60 - 0.90 mmol/g) was purchased from Novabiochem.
  • Preparative HPLC was performed on a Waters system with a C18 reversed-phase Peptide HPLC column (Vydac, 4.6 mm i.d. 250 mm).
  • MALDI-TOF MS was performed on a Voyager-DE PRO biospectrometry workstation (Applied Biosystems) using a-hydroxyl cinnamic acid as the matrix.
  • a Brunswick Scientific Innova 440014010 ⁇ £Q_ incubator shaker was used to perform the peptoid syntheses at 37°C.
  • Edman sequencing of peptoids was performed on an ABI 476A Protein Sequencer (Applied Biosystems).
  • On- bead fluorescence assays were visualized with an Olympus 1X70 fluorescence microscope equipped with a DAPI filter (410 nm excitation and 650 to 700 nm for emission) set and a CCD camera. All the other chemical reagents were purchased from commercial sources without further purification.
  • Bio-QBPl biotin-SNWKWWPGIFD; SEQ ID NO: l
  • Bio-(QBPl)2 and QBP1 SNWKWWPGIFD; SEQ ID NO:2
  • each peptoid residue was achieved through two steps including an acylation reaction and a SN2 substitution.
  • the acylation step was carried out in a standard 25-mL glass peptide synthesis reaction vessel (Chemglass) in an incubator shaker at 37°C for 10 min, in which all beads were pooled together and incubated in a mixture of 1.5 mL of 2.0 M chloroacetic acid in anhydrous DMF and 1.5 mL of 2.0 M diisopropylcarbodiimide in anhydrous DMF.
  • the beads were randomly split into nine aliquots.
  • each portion of the beads was incubated with one of the amines (2 M solution in 1-methyl- 2-pyrrolidinone) in a reaction vessel, and the mixture was shaken at 37°C for 90 min. Repeating the above two steps resulted in the extension of peptoid chains.
  • the coupling of a proline residue was done by mixing all of the beads with 400 mM proline, 400 mM HBTU and 800 mM DIPEA in DMF with shaking at room temperature for 1.5 h with one repetition. After the F-moc protective group was cleaved with 20% piperidine, the rest two residues were incorporated in the same way as described earlier. After this, beads from all the nine vessels were combined and randomized with bubbling argon.
  • a first exon (EX1) fragment of human Huntingtin protein (The Huntington's Disease Collaborative Research Group, 1993) (Metl-Gln66) containing 17 CAG repeats or 82 CAG repeats was amplified by PCR and cloned into Notl and Pstl sites of the pMAL vector with modified 3A-linker (Center et al, 1998).
  • the resulting MBP-Htt-N-15Q and MBP-Htt-N-82Q proteins were expressed in BL21 E. Coli strain by IPTG induction (100 ⁇ ) at 15°C with shaking for 16-18 h.
  • MBP fusion proteins were purified from bacterial lysates using amylose resin (New England Biolabs).
  • the MBP fusion proteins were eluted with 10 mM maltose (in lysis buffer) with shaking at 4°C and dialyzed against PBS buffer at 4°C overnight.
  • the final concentration of MBP fusion protein in PBS was 1.5 mg/ml (Bio-Rad Protein Assay Dye Reagent).
  • the MBP-Htt-N-82Q fusion protein was labeled with biotin following the protocol from the Pierce Biotinylation Kit (Pierce, # 21435). Briefly, this protein was dialyzed in PBS buffer (Sigma, P3813) overnight. Concentration of the protein in PBS was determined to be 2.3 mg/mL using the Bradford assay with BSA as a standard. 0.5 mL of the protein in PBS and 48.6 of 10 mM Sulfo-NHS-LC-Biotin in H 2 0 were mixed and incubated on ice for 2 h.
  • E. coli (BL21-RIL strain) cells were grown overnight at 37 °C in Luria broth medium. Cells were harvested using low-speed centrifugation (4,500 rpm, 15min), washed, and re-suspended in sonication buffer (Imidazole 20 mM, pH 6.8, NaCl 100 mM,14010 ⁇ _7
  • E. coli lysates from BL21-RTL strain were prepared as described above.
  • TentaGelTM beads 120 mg; approximately 60,000 beads) of the peptoids library were swollen in TBST buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 1 h.
  • the beads were incubated with the Binding Buffer [10 mg/mL E. coli lysate and 0.5% BSA in TBST for 1 h.
  • the beads were incubated with MBP-Htt-N-82Q (200 nM) in the Binding Buffer overnight. The unbound proteins were removed by washing twice with TBST.
  • the beads were incubated with anti-MBP mouse IgG (1 : 1000) in the Binding Buffer for 2 h.
  • the unbound antibody was removed by washes and 500 ⁇ of 5 nM Qdot 655 goat anti-mouse IgG in the Binding Buffer was added to the beads for 1.5 h. All of the above steps were done in cold room. Finally, after removing unbound Qdots, the beads were visualized under an Olympus 1X70 fluorescence microscope using an excitation wavelength of 410 nm.
  • the blue beads with red Qdot circles were manually picked out by using microloading tips, stripped off the bound target and re-probed with biotinylated bio- MBP-Htt-N-82Q protein and streptavidin-Qdot.
  • the peptoids were synthesized on the Knorr Amide MBHA resin (Novabiochem, #01-64-0459, 0.60 - 0.90 mmol/g) using the same protocol for synthesizing the peptoid library. After completion of the synthesis procedure, the beads were washed with DCM and peptoids were cleaved from the MBHA resin using 2 mL solution of 95% TFA, 2.5% water and 2.5% TIPS at room temperature for 1.5 h. Under slowly stream of nitrogen for 20 min, TFA was evaporated and the peptoids were purified by HPLC using 220 Absorbance Detector and Peptide preparation column.
  • MBP-Htt-N-82Q or MBP-Htt-N-15Q fusion proteins (4 ⁇ g each) were incubated overnight with the beads in 500 ⁇ of 0.5% BSA (in TBST buffer). The beads were washed twice with TBST, heated at 100 °C for 5 min in the gel loading buffer and analyzed by SDS-PAGE and Western blotting with an anti-MBP monoclonal antibody (New England Biolabs, E8032). Full- length Atxn3-19Q and Atxn3-77Q proteins were expressed in Sf9 cells by baculovirus infection as previously described31.
  • microsomes prepared from infected Sf9 cells were solubilized in 1% CHAPS, dialyzed in TBST buffer for 2 h and used in pull-down14010 ⁇ _74_ experiments.
  • the precipitated samples were analyzed by western blotting with an anti- Atxn3 monoclonal antibody (Millipore MAB5360).
  • the fluorescienated HQP09 peptoid was synthesized by reacting HQP09 G2 (Fig. 5a) with fluorescein-5-maleimide (Thermo Scientific, #46130).
  • the fluorescienated HQP09 (5 nM) was incubated with indicated concentrations of BSA or MBP-Htt-N-82Q in the presence of 1.0 ⁇ BSA in TBST (pH 7.4) in a final volume of 100 ⁇ on ice for 0.5 h in the dark.
  • the fluorescence polarization values were measured using Panvera Beacon 2000 instrument (Invitrogen). In competition experiments HQP09 and all analogs were included in the binding reaction in a final concentration of 300 ⁇ .
  • Htt53Q The filter trap and western blotting assays were performed as previously described (Wacker et ah, 2004).
  • the aggregation of Htt53Q was initiated by the addition of PreScission Protease (4 unites/100 ⁇ g fusion protein, Amersham Biosciences) to purified GST-Htt53Q protein.
  • PreScission Protease 4 unites/100 ⁇ g fusion protein, Amersham Biosciences
  • the Htt53Q proteins (6 ⁇ ) were incubated in 3% DMSO, QBP1 peptide or peptoids (10 ⁇ ) in 20 mM Tris-HCl pH7.5, 150 mM KC1, 1 mM DTT at 37°C with shaking at 800 rpm.
  • Htt53Q The 2 ⁇ g aliquots of Htt53Q were removed from the reaction, boiled in SDS-loading buffer and applied to a nitrocellulose membrane (0.2 ⁇ pore size, Schleicher & Schuell) through a slot-blot manifold. After washing with SDS-Buffer by vacuum filtering, the membranes were incubated in blocking buffer and probed with the MW8 monoclonal antibody (gift from Paul Patterson) raised against the last eight amino acids of Htt exon 1 (AEEPLHRP)49. The density of trapped Htt53Q aggregates was quantified by using Image J. For Western blotting analysis the proteins and aggregates were separated by SDS-PAGE and analyzed by Western blotting with the MW8 antibody.
  • the tapping mode AFM images were collected on an Asylum Research MFP- 3DTM atomic force microscope (Asylum Research, Santa Barbara, CA), with 120 ⁇ long oxide-sharpened silicon nitride V-shaped NP-S cantilevers (Veeco Instruments, Santa Barbara, CA). The samples were prepared and incubated for 24h as described for
  • YAC128 mice (FVBN/NJ background strain) were obtained from Jackson Labs (stock number 004938). The male YAC128 mice were crossed to wild type (WT) female FVBN/NJ mice and P1-P2 pups were collected and genotyped by PCR. The primary cultures of striatal medium spiny neurons (MSN) were established from YAC128 P1-P2 pups and control wild type pups as previously described (Tang et ah, 2005; Wu et ah, 2006; Tang et ah, 2007; Zhang et ah, 2008).
  • MSN striatal medium spiny neurons
  • neurons were plated on poly-L-lysine (Sigma) coated 12mm round coverslips (Assistent) in Neurobasal-A medium supplemented with 5% FBS (Fetal Bovine Serum), 2% B27, ImM glutamine and penicillin-streptomycin (all from Invitrogen) and kept at 37°C in a 5% CO 2 environment.
  • YAC128 mice (FVBN/NJ background strain) were obtained from Jackson Labs (stock number 004938). The male YAC128 mice were crossed to wild type (WT) female FVBN/NJ mice and P1-P2 pups were collected and genotyped by PCR. The primary cultures of striatal medium spiny neurons (MSN) were established from YAC128 P1-P2 pups (see above). The detailed procedures for Ca 2+ imaging and apoptosis experiments are provided below. HQP09, HQP09 9 and RP01 peptoids were added on DIV1 and DIV 13 at final concentration of 20 ⁇ in both Ca 2+ imaging and apoptosis experiments.
  • the MSN were loaded 14010 ⁇ with 5 ⁇ Fura-2 AM (Molecular Probes) for 45 min at 37°C in artificial cerebrospinal fluid (ACSF, containing the following: 140 mM NaCl, 5 mM KC1, 1 mM MgCl 2 , 2mM Ca(3 ⁇ 4 10 mM Hepes, pH7.3).
  • Coverslips were mounted onto a recording/perfusion chamber (RC-26G, Warner Instruments) and positioned on the movable stage of an Olympus (Melville) IX-70 inverted microscope. The cells were maintained in ACSF at 37°C during experiments (PHI heater, Warner Instruments).
  • Apoptosis experiments with 13 DIV (days in vitro) MSN cultures were performed as previously described (Tang et ah, 2005; Wu et ah, 2006).
  • Tested peptoids in final concentration 20 ⁇ were added to the WT and YAC 128 MSN culture medium on DIV 1 and DIV 13 from the aqueous stock solution (30 mM).
  • neurons were exposed to 250 ⁇ glutamate for 8 h, fixed, permeabilized and analyzed by propidium iodide (PI) and fluorescent terminal deoxynucleotidyltransferase-mediated dUTP-FITC labeling (TUNEL) staining as previously described.
  • PI propidium iodide
  • TUNEL fluorescent terminal deoxynucleotidyltransferase-mediated dUTP-FITC labeling
  • TUNEL-positive neuronal nuclei green was calculated as a fraction of Pi-positive neuronal nuclei (red) in each microscopic field.
  • the fractions of TUNEL-positive nuclei determined for each microscopic field were averaged and the results are presented as means ⁇ s.e. (n: number of fields count).
  • AD Alzheimer's disease 2019
  • APP ⁇ -amyloid precursor protein gene
  • PS1 presenilin-1 gene
  • PS2 presenilin-2 gene
  • On-bead peptoid libraries were designed and synthesized such that each peptoid in the library had four variable monomers and two constant monomers (Nlys) which served as a spacer (FIG. 17A).
  • Nlys constant monomers
  • a set of aromatic primary amines were chosen as submonomers. These amines were chosen also because aromatic rings commonly exist in CNS-active drug molecules.
  • a non-aromatic hydrophobic set provided additional amines for hydrophobic interactions with ⁇ 42. Amines in the hydrophilic set have functional groups that could interact with ⁇ 42 through electrostatic interactions or hydrogen bonding.
  • four additional amines were included to add more chemical diversity to the library. Overall, 14 amines were applied to form side chains at four variable positions, which resulted in a diversity of 38,416 (14 4 ) peptoids.
  • the generated peptoid library was screened using biotinylated ⁇ 42 as bait in the presence of an excess of ⁇ 40.
  • the design of the screen was aimed at isolating peptoids that specifically recognize ⁇ 42.
  • the library Prior to the screen the library was "prescreened” (FIG. 17B) to remove peptoids that bind to streptavidin nonspecifically. The rest of the beads were carried on through the “screening" (FIG. 2B). After step (8), the beads were visualized and fluorescent beads were identified as "hits".
  • the ratio of biotin-Ap42/Ap40 used in the screens decreased, the number of isolated hits also decreased correspondingly (Table 4). Because the 4th screen had the lowest ratio of biotin-Ap42/Ap40, it was reasoned that the 37 hits isolated in this screen are most selective ⁇ 42 ligands.
  • N-(tert-butoxycarbonyl)-l,4-diaminobutane (282 mg, 1.5 mmol, 0.5 M in 3 ml NMP) was added to the beads and the reaction mixture was shaken at 37°C for 2 h. The beads were again washed with DMF. The above two steps 14010 ⁇ -83- were repeated once.
  • Rink Amide AM resin 100 mg, 0.071 mmol; 200-400 mesh, capacity: 0.71 mmol/g was incubated in DMF at room temperature for 1 h. The resin was then incubated with 4 ml 20 % piperidine in DMF with shaking at room temperature for 20 min. This reaction step was repeated once and the resin was washed with DMF. Then the resin was mixed with Fmoc-Lys(Dde)-OH (0.355 mmol, 189 mg), HBTU (0.355 mmol, 135 mg), HOBt (0.355 mmol, 48 mg) and N-methylmorpholine (0.71 mmol, 72 mg) in 4 ml DMF at room temperature for 3h.
  • the resin was washed with DMF and then incubated with 4 ml 20 % piperidine in DMF with shaking at room temperature for 20 min. This reaction step was repeated once and the resin was washed with DMF. Then the resin was mixed with Fmoc-p-Ala-OH (0.355 mmol, 1 11 mg; AnaSpec), HBTU (0.355 mmol, 135 mg), HOBt (0.355 mmol, 48 mg) and N-methylmorpholine (0.71 mmol, 72 mg) in 4 ml DMF at room temperature overnight. The resin was washed with DMF and then incubated with 4 ml 20 % piperidine in DMF at room temperature for 20 min.
  • Binding affinity of IAMs for ⁇ 42 can also be quantitatively determined using a fluorescence solid-phase binding assay (Chauhan, 1999; LeVine, 2006; Matsubara et ah, 1995; Udugamasooriya, 2008).
  • biotin-tagged IAM1 can be coated on a 96- well NeutrAvidin-coated plate through the biotin-neutravidin interaction. Then ⁇ 42 of increasing concentrations is incubated in corresponding IAMl -coated wells for 1 h and unbound ⁇ 42 is decanted and washed away.
  • each well is incubated with a primary anti- ⁇ antibody 6E10 and a secondary antibody IgG-horseradish peroxidase (HRP) sequentially.
  • HRP fluorogenic peroxidase substrate QuantaBluTM
  • acquired fluorescence data can be plotted (representative binding curves were shown in FIGS. 18B-E) and subjected to nonlinear regression curve fit analysis using one site binding equations.
  • IAMs inhibit ⁇ 42 oligomerization. As shown in FIGS. 19A-B, IAMl inhibits high order ⁇ 42 oligomerization detectable by gel electrophoresis/Western blot when incubated with ⁇ 42 as compared to random peptoids or the Al l antibody.
  • ThT aggregation assays can likewise be used to evaluate effects of other IAMs on ⁇ 40 and ⁇ 42 aggregation.
  • direct binding and NMR measurements can be used to validate the most potent and specific ⁇ 42 peptoid ligand(s).
  • FIGS.14010 ⁇ 21A-B Results of these studies are shown in FIGS.14010 ⁇ 21A-B. Specifically, FIG. 21A shows concentration-dependent toxicity of ⁇ 42 in primary hippocampal neuronal cultures. FIG. 2 IB, shows neuroprotection by IAM1 of primary hippocampal neuronal cultures from ⁇ 42 oligomer-associated toxicity.
  • IAMl was tested for effect in the APPPSl mouse model (Thyl- APPKM670/671NL; Thyl-PS1L166P) (Radde et al, 2006).
  • the APPPS l mouse model displays early and robust onset of amyloid pathology, making evaluations of therapeutic strategies more cost-effective.
  • APPPSl mice that were administered with IAMl subcutaneously with implanted ALZET ® osmotic pumps and tissue staining from brain slices used to assess the amyloid load in brain of mice the indicated time points.
  • FIG. 23A IAMl reduces the amyloid load in the brain of APPPSl mice.
  • FIG. 23B shows a similar reduction in amyloid load in the brains of mice administered with IAMl at 2, 5 and 9 month time points.
  • a dimerized version of IAMl has improved activity
  • the K d values for the binding of ASRl-biotin to ⁇ 42 and ⁇ 40 were 1.50 ⁇ 0.34 ⁇ and 3.61 ⁇ 1.16 ⁇ , respectively, which reflected that ASR1 had much lower binding affinity toward ⁇ 42 and ⁇ 40 than both IAM1 and (IAM1)2.
  • Table 5 contains a summary of the binding affinities.
  • the inhibitory ability of (IAM 1)2 toward the aggregation of ⁇ 42 and ⁇ 40 was also measured by in situ kinetic ThT assay as for IAM1.
  • the time courses of the ThT fluorescence reflecting the aggregation processes of ⁇ 42 and ⁇ 40 in the presence of (IAM 1)2 are shown in FIGS. 24C-D.
  • the inhibition of ⁇ aggregation by (IAM 1)2 was concentration dependent as by IAM1, however, (IAM 1)2 showed stronger inhibitory potency than its parent compound.
  • the ThT max at the highest (IAM 1)2 concentrations in ⁇ 42 and ⁇ 40 aggregation was -34 % and -68% of that with the solvent control, respectively (FIG. 24E).
  • ThT max at the same concentration of IAM 1 in ⁇ 42 aggregation was -60%, which suggested that (LAM 1)2 demonstrated about 1.7-fold higher potency than IAM1 in inhibiting the aggregation of ⁇ 42.
  • (IAM 1)2 showed minimum inhibition of the aggregation of ⁇ 40.
  • the peptoid ASRI was also evaluated in its ability to inhibit the aggregation of ⁇ 42 or ⁇ 40.
  • Time courses of the ThT fluorescence of the aggregation processed of ⁇ 42 and ⁇ 40 in the presence of ASRI demonstrated that even14010 ⁇ OQ. at 50-fold molar excess of ASR1, there was no detectable inhibition of the aggregation of ⁇ 42 or ⁇ 40 (FIGS. 25C-D).
  • (IAM1)2 strongly binds to ⁇ 42 with a nanomolar affinity and effectively prevents its aggregation.
  • (IAM1)2 protects hippocampal neurons in amyloid-toxicity assay
  • ⁇ -containing conditioned media was added with different concentrations of (IAM1)2 to cultured hippocampal neurons.
  • the hippocampal neurons were fixed and stained with anti-MAP2 antibody specific for the neuronal dendrites and a fluorophore-tethered secondary antibody.
  • MAP2-positive neurons were quantified using Isocyte laser scanner system. The percentage of those neurons in the total number of neurons is taken as neuronal viability and the decrease in the MAP2 staining indicates the loss of neurons which reflects the neurodegeneration.
  • compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be14010 ⁇ _Q Q _ applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Abstract

La présente invention concerne des méthodes et des compositions pour le traitement et la prévention d'une maladie associée à une expansion de protéine polyglutamine. Par exemple, l'invention concerne des composés peptoïdes qui se lient sélectivement à des polypeptides étendus de polyglutamine. L'invention concerne également des procédés d'utilisation de tels composés pour le traitement, la prévention et la détection de troubles de l'expansion de polyglutamine, tels que la chorée de Huntington. De plus, l'invention concerne des méthodes et des compositions pour le traitement et la prévention de troubles neuronaux liés à une agrégation protéique pathogène. Par exemple, l'invention concerne des composés peptoïdes qui se lient sélectivement à Aβ42. L'invention concerne également des procédés d'utilisation de tels composés pour le traitement et la prévention de symptômes de la maladie d'Alzheimer.
PCT/US2012/056025 2011-09-21 2012-09-19 Compositions peptoïdes pour le traitement de la maladie d'alzheimer et d'un trouble de l'expansion de polyglutamine WO2013043669A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014193632A3 (fr) * 2013-05-29 2015-04-02 Vybion, Inc. Intracorps à chaîne unique modifiant la dégradation de la protéine mutante huntingtin
WO2015109107A1 (fr) * 2014-01-15 2015-07-23 Cal Poly Pomona Foundation, Inc. Traitement et diagnostic de la maladie de huntington
WO2017165438A1 (fr) * 2016-03-25 2017-09-28 Muralidhar Reddy Moola Synthèse combinatoire et développement de biomarqueurs
WO2017209805A1 (fr) * 2016-06-01 2017-12-07 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Polypeptoïdes modifiés de manière hydrophobe et leurs utilisations
US9986733B2 (en) 2015-10-14 2018-06-05 X-Therma, Inc. Compositions and methods for reducing ice crystal formation
JP2019512541A (ja) * 2016-03-09 2019-05-16 マイク−アン、エルエルシーＭｉｋｅ−Ａｎｎ，Ｌｌｃ ペプトイド親和性リガンド
CN113929744A (zh) * 2021-09-03 2022-01-14 国家纳米科学中心 一种靶向Aβ42纤维体的类肽及其制备方法与应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5700779A (en) * 1994-03-09 1997-12-23 Cortech, Inc. Bradykinin antagonist peptides incorporating N-substituted glycines
WO2007030804A1 (fr) * 2005-09-09 2007-03-15 Novartis Ag Reactifs peptoides specifiques des prions
US20080233137A1 (en) * 2004-04-16 2008-09-25 Johannes Antonius Gerardus Van Strijp Compounds that Block the C5a Receptor and Their Use in Therapy

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5700779A (en) * 1994-03-09 1997-12-23 Cortech, Inc. Bradykinin antagonist peptides incorporating N-substituted glycines
US20080233137A1 (en) * 2004-04-16 2008-09-25 Johannes Antonius Gerardus Van Strijp Compounds that Block the C5a Receptor and Their Use in Therapy
WO2007030804A1 (fr) * 2005-09-09 2007-03-15 Novartis Ag Reactifs peptoides specifiques des prions

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEN ET AL.: "Expanded Polyglutamine-Binding Peptoid as a Novel Therapeutic Agent for Treatment of Huntington?s Disease", CHEMISTRY & BIOLOGY, vol. 18, 22 September 2011 (2011-09-22), pages 1113 - 1125, XP028298159, DOI: doi:10.1016/j.chembiol.2011.06.010 *

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* Cited by examiner, † Cited by third party
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US11072650B2 (en) 2013-05-29 2021-07-27 Vybion, Inc. Single chain intrabodies that alter Huntingtin mutant degradation
AU2014272024B2 (en) * 2013-05-29 2018-03-15 Vybion, Inc. Single chain intrabodies that alter huntingtin mutant degradation
US9932394B2 (en) 2013-05-29 2018-04-03 Vybion, Inc. Single chain intrabodies that alter huntingtin mutant degradation
AU2018204243B2 (en) * 2013-05-29 2020-10-15 Vybion, Inc. Single chain intrabodies that alter huntingtin mutant degradation
WO2014193632A3 (fr) * 2013-05-29 2015-04-02 Vybion, Inc. Intracorps à chaîne unique modifiant la dégradation de la protéine mutante huntingtin
WO2015109107A1 (fr) * 2014-01-15 2015-07-23 Cal Poly Pomona Foundation, Inc. Traitement et diagnostic de la maladie de huntington
US10694739B2 (en) 2015-10-14 2020-06-30 X-Therma, Inc. Compositions and methods for reducing ice crystal formation
US11510407B2 (en) 2015-10-14 2022-11-29 X-Therma, Inc. Compositions and methods for reducing ice crystal formation
US9986733B2 (en) 2015-10-14 2018-06-05 X-Therma, Inc. Compositions and methods for reducing ice crystal formation
EP3426673A4 (fr) * 2016-03-09 2019-10-09 Mike-Ann, LLC Ligands à affinité peptoïdiques
JP2019512541A (ja) * 2016-03-09 2019-05-16 マイク−アン、エルエルシーＭｉｋｅ−Ａｎｎ，Ｌｌｃ ペプトイド親和性リガンド
US11053281B2 (en) 2016-03-09 2021-07-06 Mike-Ann, Llc Peptoid affinity ligands
US11053282B2 (en) 2016-03-09 2021-07-06 Mike-Ann, Llc Peptoid affinity ligands
JP7026644B2 (ja) 2016-03-09 2022-02-28 マイク－アン、エルエルシー ペプトイド親和性リガンド
US10458995B2 (en) 2016-03-25 2019-10-29 Muralidhar Reddy Moola Combinatorial synthesis and biomarker development
WO2017165438A1 (fr) * 2016-03-25 2017-09-28 Muralidhar Reddy Moola Synthèse combinatoire et développement de biomarqueurs
WO2017209805A1 (fr) * 2016-06-01 2017-12-07 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Polypeptoïdes modifiés de manière hydrophobe et leurs utilisations
CN113929744A (zh) * 2021-09-03 2022-01-14 国家纳米科学中心 一种靶向Aβ42纤维体的类肽及其制备方法与应用
CN113929744B (zh) * 2021-09-03 2024-05-10 国家纳米科学中心 一种靶向Aβ42纤维体的类肽及其制备方法与应用

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