WO2017004560A1 - Procédés et compositions pour des agrégats amyloïdes - Google Patents

Procédés et compositions pour des agrégats amyloïdes Download PDF

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WO2017004560A1
WO2017004560A1 PCT/US2016/040772 US2016040772W WO2017004560A1 WO 2017004560 A1 WO2017004560 A1 WO 2017004560A1 US 2016040772 W US2016040772 W US 2016040772W WO 2017004560 A1 WO2017004560 A1 WO 2017004560A1
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substituted
unsubstituted
amyloid
heteroaryl
heterocycloalkyl
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PCT/US2016/040772
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English (en)
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Jagesh SHAH
Dominic Walsh
Jerry Yang
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The Regents Of The University Of California
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Publication of WO2017004560A1 publication Critical patent/WO2017004560A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • novel amyloid ( ⁇ ) aggregate-binding probe aryl cyano amide (ARCAM), which exhibits a large enhancement in fluorescence properties ( ⁇ 5X) upon binding to aggregates (compared to free probe in solution) when assayed by Probe- Enabled Fluorescence Correlation Spectroscopy (PE-FCS).
  • ARCAM aryl cyano amide
  • P-FCS Probe- Enabled Fluorescence Correlation Spectroscopy
  • compositions and methods for the detection of amyloid aggregates for use in the detection, diagnosis, prognosis, and treatment of neurological diseases propagated by aggregated amyloid proteins.
  • a method for detecting an amyloid aggregate in a biological sample includes contacting a biological sample containing an amyloid aggregate with an amyloid-binding fluorophore thereby forming a fluorescent amyloid aggregate complex comprising said amyloid aggregate anon-covalently bound to said amyloid-binding fluorophore, and detecting said fluorescent amyloid aggregate complex using fluorescence correlation spectroscopy.
  • the method also includes determining the size of said amyloid aggregate.
  • determining the size of said amyloid aggregate comprises measuring a number of fluorescent burst events for a resident time in a confocal volume.
  • the size of the amyloid aggregate is less than 1000 nm.
  • amyloid-binding fluorophore includes the structure of Formula (I),
  • EDG is an electron donor group
  • 7lCE is a pi-conjugation element
  • WSG is a water soluble group.
  • EDG is R ⁇ -substituted or unsubstituted alkyl, R ⁇ substituted or unsubstituted cycloalkyl, R 1 - substituted or unsubstituted heteroalkyl, R 1 -substituted or unsubstituted heterocycloalkyl, R ⁇ substituted or unsubstituted aryl, R ⁇ substituted or unsubstituted heteroaryl, -OR 2 , -NR 4 C(0)R 3 , -NR 4 R 5 , -SR 6 , or -PRV.
  • R 1 is halogen, -OR 9 , -NR 10 R n , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.
  • R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen, R 12 -substituted or unsubstituted alkyl, R 12 -substituted or unsubstituted heteroalkyl, R 12 -substituted or unsubstituted cycloalkyl, R 12 -substituted or unsubstituted heterocycloalkyl, R 12 -substituted or unsubstituted aryl or R 12 -substituted or unsubstituted heteroaryl.
  • R 4 and R 5 are optionally joined together to form an R 12 -substituted or unsubstituted heterocycloalkyl, or R 12 -substituted or unsubstituted heteroaryl;
  • R 9 , R 10 and R 11 are
  • R 12 -substituted or unsubstituted alkyl independently hydrogen, R 12 -substituted or unsubstituted alkyl, R 12 -substituted or unsubstituted heteroalkyl, R 12 -substituted or unsubstituted cycloalkyl, R 12 -substituted or unsubstituted heterocycloalkyl, R 12 -substituted or unsubstituted aryl, or R 12 -substituted or unsubstituted heteroaryl.
  • R 10 and R 11 are optionally joined together to form an R 12 -substituted or unsubstituted heterocycloalkyl, or R 12 -substituted or unsubstituted heteroaryl;
  • R 12 is halogen, -OR 13 , -NR 14 R 15 , R 16 -substituted or unsubstituted alkyl, R 16 -substituted or unsubstituted heteroalkyl, R 16 - substituted or unsubstituted cycloalkyl, R 16 -substituted or unsubstituted heterocycloalkyl, R 16 - substituted or unsubstituted aryl, or R 16 -substituted or unsubstituted heteroaryl.
  • R 13 , R 14 and R 15 are independently hydrogen or unsubstituted alkyl.
  • R 16 is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.
  • the pi-conjugation element has the formula: or -L*-(A L 4 -A 3 -L 2 -(A 2 )r-L 3 -.
  • q and r are independently 0 or 1;
  • L 1 , L 2 , L and L are independently a bond or a linking group having the formula: V / ⁇ , x is an integer from 1 to 50.
  • a 1 , A2 and A 3 are independently R 17 -substituted or unsubstituted arylene, or
  • R 17 -substituted or unsubstituted heteroarylene.
  • R 17 is halogen, -OR 18 , -NR19 R 20 , R 21 -substituted or unsubstituted alkyl, R 21 -substituted or unsubstituted heteroalkyl, R 21 -substituted or unsubstituted cycloalkyl, R 21 -substituted or unsubstituted heterocycloalkyl, R 21 -substituted or unsubstituted aryl, or R 21 -substituted or unsubstituted heteroaryl.
  • R 18 , R 19 and R 20 are
  • R 21 -substituted or unsubstituted alkyl independently hydrogen, R 21 -substituted or unsubstituted alkyl, R 21 -substituted or unsubstituted heteroalkyl, R 21 -substituted or unsubstituted cycloalkyl, R 21 -substituted or unsubstituted heterocycloalkyl, R 21 -substituted or unsubstituted aryl, or R 21 -substituted or unsubstituted heteroaryl.
  • R 21 is halogen, -OR 22 , -NR 23 R 24 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.
  • R 22 , R 23 and R 24 are independently hydrogen or unsubstituted alkyl.
  • a 1 , A 2 and A 3 are independently R 21 -substituted or unsubstituted naphthylene, or R 21 -substituted or unsubstituted phenylene.
  • x is an integer from 1 to 10.
  • the water soluble group is R -substituted or unsubstituted alkyl, R - substituted or unsubstituted heteroalkyl, R 25 -substituted or unsubstituted cycloalkyl, R 25 - substituted or unsubstituted heterocycloalkyl, R 25 -substituted or unsubstituted aryl, R 25 - substituted or unsubstituted heteroaryl.
  • R 25 is halogen, -OR 26 , -NR 27 R 28 , R 29 -substituted or
  • R 26 , R 27 and R 28 are independently hydrogen,
  • R -substituted or unsubstituted alkyl R -substituted or unsubstituted heteroalkyl, R -
  • R 28 are optionally joined together to form an R 29 -substituted or unsubstituted heterocycloalkyl, or
  • R 29 -substituted or unsubstituted heteroaryl.
  • R 29 is halogen, -OR 30 , -NR 31 R 32 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.
  • R 30 , R 31 and R 32 are independently hydrogen or unsubstituted
  • the water soluble group is an ethylene glycol moiety having the formula: y is an integer from 1 to 50.
  • R is -OH.
  • amyloid-binding fluorophore comprises the structure:
  • the biological sample is a biofluid.
  • the biofluid is blood, urine, saliva, or cerebrospinal fluid.
  • the biological sample further comprises a buffer.
  • the invention includes a fluorescent amyloid aggregate complex including an amyloid aggregate non-covalently bound to said amyloid-binding fluorophore.
  • the complex is within a vessel.
  • the vessel further comprises a buffer.
  • the buffer has a pH of between 4.5 and 7.5.
  • the buffer has a pH of between 5.0 and 7.0.
  • FIGS. 1A-1C Structure and spectroscopic properties of fluorescent probe 1.
  • FIG. 1A Structures of fluorescent, aggregate-binding compound ARCAM 1. Excitation (FIG. IB) and emission (FIG. 1C) properties of 1 in the presence or absence of aggregated ⁇ (1-42).
  • FIGS. 2A-2B Comparison of aggregate size measured using ThT, ARCAM 1 and fluorescently labeled ⁇ peptide.
  • Dyes ThiT or ARCAM 1
  • TAMRA-labeled ⁇ (1-42) peptides were added to pre-aggregated ⁇ samples.
  • FCS measurements after a thirty minute incubation, revealed larger species in the ThT labeled sample (FIG. 2A, p ⁇ 0.05 for 0.1, Is delay time).
  • ARCAM 1 revealed larger species in the pre-aggregated samples when compared to TAMRA- ⁇ (1-42) peptides (FIG. 2B, p ⁇ 0.05 for 10s delay time). Note that pre-aggregated samples were matched for measurements in (FIG. 2A) and (FIG. 2B) independently, resulting in the different ARCAM 1 FCS spectra.
  • FIGS. 3A-3B Monitoring the kinetics of aggregation of ⁇ (1-42) peptides by bulk fluorescence and FCS burst analysis.
  • FIG. 3A Increase in total fluorescence (blue squares) or fluorescent burst number (squares) in solutions containing ⁇ and fluorescent ARCAM 1.
  • FIG. 3B Detection of aggregates interacting with ARCAM 1 by monitoring the intensity and number of fluorescent bursts within a 120 second acquisition time window as a function of the concentration of total peptide.
  • the amplitude of autocorrelation curve G(0) increases along the reaction time point 0, 120 and 600 min, indicating a decrease in particle number for the dominant fluorescent species.
  • the autocorrelation functions show increasing contribution from long delay times indicating the increasing size of the particles.
  • FIG. 5 A plot of fluorescence intensity versus concentration of ARCAM 1 in the presence of aggregated A ?(l-42) peptide at pH 7.4.
  • FIG. 6 Hydrolytic stability studies of ARCAM 1 in pH 7.4 IX PBS over 24 hours.
  • FIG. 9. ⁇ (1-42) aggregation kinetics are not perturbed by the addition of ARCAM 1 or ThT.
  • FIG. 10 ⁇ monomer was incubated in the absence or presence of ThT and bulk fluorescence monitored at intervals. Maximal fluorescence was attained after 140 min and remained constant thereafter. Samples lacking ThT were collected after 240 min and used as the aggregate standard.
  • FIG. 11 The intensity trace of ⁇ peptide (5 ⁇ ) alone by FCS measurement.
  • FIG. 12A-12C Burst selection method.
  • FIG. 12A the histogram of intensity trace with 1 ms resolution at 240 min of ⁇ reaction.
  • the solid line indicates the modal value of the histogram. This value is treated as the average of intensity background due to the majority of intensity events.
  • the dashed line is the position at four times of the standard deviation of this intensity trace distribution above the average background.
  • FIG. 12B solid and dash lines are the same as FIG. 12A, showing the relative positions of cutoff.
  • FIG. 12C Selected bursts clearly shown with all other intensities set as zero.
  • FIG. 13 The diffusion coefficients of large species by two-component analysis at different time points.
  • FIGS. 14A-14H NMR spectra.
  • FIG. 14A 1H NMR spectrum of Cmpd A at 500 MHz in CDC1 3 ;
  • FIG. 14B 13 C NMR spectrum of Cmpd A at 125 MHz in CDCI3;
  • FIG. 14C 1H NMR spectrum of Cmpd B at 500 MHz in CDCI 3 ;
  • FIG. 14D 13 C NMR spectrum of Cmpd B at 125 MHz in CDCI 3 ;
  • FIG. 14E 1H NMR spectrum of Cmpd C at 500 MHz in CDC1 3 ;
  • FIG. 14F 13 C NMR spectrum of Cmpd C at 125 MHz in CDC1 3 ;
  • FIG. 14G 1H NMR spectrum of Cmpd 1 at 500 MHz in CDCI 3 ;
  • FIG. 14H 13 C NMR spectrum of Cmpd 1 at 125 MHz in CDCI 3 .
  • substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH 2 0- is equivalent to -OCH 2 -.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include mono-, di- and multivalent radicals, having the number of carbon atoms designated (i.e., C 1 -C 10 means one to ten carbons). Alkyl is an uncyclized chain.
  • saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl, n- hexyl, n-heptyl, n-octyl, and the like.
  • An unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-0-).
  • alkylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, - CH 2 CH 2 CH 2 CH 2 -.
  • an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred herein.
  • a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • alkenylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, and S), and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized.
  • the heteroatom(s) e.g., N, S, Si, or P
  • Heteroalkyl is an uncyclized chain.
  • a heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include two optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • heteroalkylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH 2 -CH 2 -S-CH 2 -CH 2 - and -CH 2 -S-CH 2 -CH 2 -NH-CH 2 -.
  • heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy,
  • heteroalkyl groups include those groups that are attached to the remainder of the molecule through a heteroatom, such as - C(0)R', -C(0)NR', -NR'R", -OR', -SR', and/or -S0 2 R'.
  • heteroalkyl is recited, followed by recitations of specific heteroalkyl groups, such as -NR'R” or the like, it will be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R" or the like.
  • cycloalkyl and heterocycloalkyl by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for
  • heterocycloalkyl a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule.
  • cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.
  • heterocycloalkyl examples include, but are not limited to, 1-(1,2,5,6- tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1- piperazinyl, 2-piperazinyl, and the like.
  • a "cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and
  • halo or halogen
  • haloalkyl by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • terms such as “haloalkyl” are meant to include monohaloalkyl and polyhaloalkyl.
  • halo(Ci-C 4 )alkyl includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
  • acyl means, unless otherwise stated, -C(0)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • aryl means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently.
  • a fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring.
  • heteroaryl refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized.
  • heteroaryl includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring).
  • a 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring.
  • a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring.
  • a 6,5- fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring.
  • a heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom.
  • Non- limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1- naphthyl, 2-naphthyl, 4-biphenyl, 1 -pyrrolyl, 2-pyrrolyl, 3 -pyrrolyl, 3 -pyrazolyl, 2-imidazolyl
  • arylene and heteroarylene are selected from the group of acceptable substituents described below.
  • a heteroaryl group substituent may be -O- bonded to a ring heteroatom nitrogen.
  • Spirocyclic rings are two or more rings wherein adjacent rings are attached through a single atom.
  • the individual rings within spirocyclic rings may be identical or different.
  • Individual rings in spirocyclic rings may be substituted or unsubstituted and may have different substituents from other individual rings within a set of spirocyclic rings. Possible substituents for individual rings within spirocyclic rings are the possible substituents for the same ring when not part of spirocyclic rings (e.g. substituents for cycloalkyl or heterocycloalkyl rings).
  • Spirocylic rings may be substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heterocycloalkylene and individual rings within a spirocyclic ring group may be any of the immediately previous list, including having all rings of one type (e.g. all rings being substituted heterocycloalkylene wherein each ring may be the same or different substituted heterocycloalkylene).
  • heterocyclic spirocyclic rings means a spirocyclic rings wherein at least one ring is a heterocyclic ring and wherein each ring may be a different ring.
  • substituted spirocyclic rings means that at least one ring is substituted and each substituent may optionally be different.
  • oxo means an oxygen that is double bonded to a carbon atom.
  • alkylarylene as an arylene moiety covalently bonded to an alkylene moiety (also referred to herein as an alkylene linker).
  • alkylarylene group has the formula:
  • An alkylarylene moiety may be substituted (e.g. with a substituent group) on the alkylene moiety or the arylene linker (e.g. at carbons 2, 3, 4, or 6) with halogen, oxo, -N 3 , -CF 3 , - CC1 3 , -CBr 3 , -CI 3 , -CN, -CHO, -OH, -NH 2 , -COOH, -CONH 2 , -N0 2 , -SH, -S0 2 CH 3 -S0 3 H, , - OSO 3 H, -SO 2 NH 2 , -NHNH 2 , -ONH 2 , -NHC(0)NHNH 2 , substituted or unsubstituted C 1 -C5 alkyl or substituted or unsubstituted 2 to 5 membered heteroalkyl).
  • the alkylarylene is unsubstituted.
  • R, R', R", R'", and R" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or
  • each of the R groups is independently selected as are each R', R", R'", and R"" group when more than one of these groups is present.
  • R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring.
  • -NR'R includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF 3 and -CH 2 CF 3 ) and acyl (e.g., -C(0)CH 3 , -C(0)CF 3 , -C(0)CH 2 OCH 3 , and the like).
  • haloalkyl e.g., -CF 3 and -CH 2 CF 3
  • acyl e.g., -C(0)CH 3 , -C(0)CF 3 , -C(0)CH 2 OCH 3 , and the like.
  • Substituents for rings may be depicted as substituents on the ring rather than on a specific atom of a ring (commonly referred to as a floating substituent).
  • the substituent may be attached to any of the ring atoms (obeying the rules of chemical valency) and in the case of fused rings or spirocyclic rings, a substituent depicted as associated with one member of the fused rings or spirocyclic rings (a floating substituent on a single ring), may be a substituent on any of the fused rings or spirocyclic rings (a floating substituent on multiple rings).
  • the multiple substituents may be on the same atom, same ring, different atoms, different fused rings, different spirocyclic rings, and each substituent may optionally be different.
  • a point of attachment of a ring to the remainder of a molecule is not limited to a single atom (a floating substituent)
  • the attachment point may be any atom of the ring and in the case of a fused ring or spirocyclic ring, any atom of any of the fused rings or spirocyclic rings while obeying the rules of chemical valency.
  • a ring, fused rings, or spirocyclic rings contain one or more ring heteroatoms and the ring, fused rings, or spirocyclic rings are shown with one more floating substituents (including, but not limited to, points of attachment to the remainder of the molecule), the floating substituents may be bonded to the heteroatoms.
  • the ring heteroatoms are shown bound to one or more hydrogens (e.g. a ring nitrogen with two bonds to ring atoms and a third bond to a hydrogen) in the structure or formula with the floating substituent, when the heteroatom is bonded to the floating substituent, the substituent will be understood to replace the hydrogen, while obeying the rules of chemical valency.
  • Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups.
  • Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure.
  • the ring-forming substituents are attached to adjacent members of the base structure.
  • two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure.
  • the ring-forming substituents are attached to a single member of the base structure.
  • two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure.
  • the ring- forming substituents are attached to non-adjacent members of the base structure.
  • Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -T-C(0)-(CRR') q -U-, wherein T and U are independently -NR-, -0-, - CRR'-, or a single bond, and q is an integer of from 0 to 3.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH 2 ) r -B-, wherein A and B are independently -CRR'-, -0-, -NR-, -S-, -S(O) -, - S(0) 2 -, -S(0) 2 NR'-, or a single bond, and r is an integer of from 1 to 4.
  • One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR')s-X'- (C"R"R"')d-, where s and d are independently integers of from 0 to 3, and X' is -0-, -NR'-, -S-, -S(O)-, -S(0) 2 -, or -S(0) 2 NR'-.
  • R, R', R", and R' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
  • heteroatom or "ring heteroatom” are meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).
  • a “substituent group,” as used herein, means a group selected from the following moieties:
  • a "size-limited substituent” or " size-limited substituent group,” as used herein, means a group selected from all of the substituents described above for a "substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-C 2 o alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 3 -C 8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C 6 -Cio aryl, and each substituted or unsubstituted hetero
  • a "lower substituent” or " lower substituent group,” as used herein, means a group selected from all of the substituents described above for a "substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-C 8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 3 -C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C 6 -Cio aryl, and each substituted or unsubstituted heteroaryl is a
  • each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent group.
  • each substituted or unsubstituted alkyl may be a substituted or unsubstituted C 1 -C 20 alkyl
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 3 -C 8 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl
  • each substituted or unsubstituted aryl is a substituted or unsubstituted C 6 -Cio aryl
  • each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl.
  • each substituted or unsubstituted alkylene is a substituted or unsubstituted C 1 -C 20 alkylene
  • each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20 membered heteroalkylene
  • each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C 3 -C 8 cycloalkylene
  • each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 8 membered heterocycloalkylene
  • each substituted or unsubstituted arylene is a substituted or unsubstituted C 6 -Cio arylene
  • each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 10 membered heteroarylene.
  • each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-C 8 alkyl
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 3 -C 7 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl
  • each substituted or unsubstituted aryl is a substituted or unsubstituted C 6 -Cio aryl
  • each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl.
  • each substituted or unsubstituted alkylene is a substituted or unsubstituted Q-Cg alkylene
  • each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8 membered heteroalkylene
  • each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C 3 -C 7 cycloalkylene
  • each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 7 membered heterocycloalkylene
  • each substituted or unsubstituted arylene is a substituted or unsubstituted C 6 -Cio arylene
  • each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered heteroarylene.
  • the compound is a chemical species set forth in the Examples section, figures, or tables below
  • Certain compounds of the present invention possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute
  • stereochemistry as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers are encompassed within the scope of the present invention.
  • the compounds of the present invention do not include those that are known in art to be too unstable to synthesize and/or isolate.
  • the present invention is meant to include compounds in racemic and optically pure forms.
  • Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
  • the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.
  • the term “isomers” refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.
  • tautomer refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.
  • structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention.
  • compounds which differ only in the presence of one or more isotopically enriched atoms are within the scope of this invention.
  • compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by 13 C- or 14 C-enriched carbon are within the scope of this invention.
  • compounds which differ only in the presence of one or more isotopically enriched atoms are within the scope of this invention.
  • compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by 13 C- or 14 C-enriched carbon are within the scope of this invention.
  • the compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds.
  • the compounds may be radiolabeled with radioactive isotopes, such as for example tritium ( H), iodine-125 ( 125 I), or carbon-14 ( 14 C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.
  • each amino acid position that contains more than one possible amino acid. It is specifically contemplated that each member of the Markush group should be considered separately, thereby comprising another embodiment, and the Markush group is not to be read as a single unit.
  • an analog is used in accordance with its plain ordinary meaning within Chemistry and Biology and refers to a chemical compound that is structurally similar to another compound (i.e., a so-called “reference” compound) but differs in composition, e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Accordingly, an analog is a compound that is similar or comparable in function and appearance but not in structure or origin to a reference compound.
  • a or “an,” as used in herein means one or more.
  • substituted with a[n] means the specified group may be substituted with one or more of any or all of the named substituents.
  • a group such as an alkyl or heteroaryl group
  • the group may contain one or more unsubstituted C 1 -C 20 alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls.
  • R-substituted where a moiety is substituted with an R substituent, the group may be referred to as "R-substituted.” Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different. Where a particular R group is present in the description of a chemical genus (such as Formula (I)), a Roman alphabetic symbol may be used to distinguish each appearance of that particular R group. For example, where multiple R 13 substituents are present, each R 13 substituent may be distinguished as R 13A , R 13B , R 13C , R 13D , etc., wherein each of R 13A , R 13B , R 13C , R 13D , etc. is defined within the scope of the definition of R 13 and optionally differently.
  • a “covalent cysteine modifier moiety” as used herein refers to a subtituent that is capable of reacting with the sulfhydryl functional group of a cysteine amino acid (e.g. cysteine YYY of the XXX (e.g., human XXX)) to form a covalent bond.
  • a cysteine amino acid e.g. cysteine YYY of the XXX (e.g., human XXX)
  • the covalent cysteine modifier moiety is typically electrophilic.
  • a “detectable moiety” as used herein refers to a moiety that can be covalently or noncovalently attached to a compound or biomolecule that can be detected for instance, using techniques known in the art. In embodiments, the detectable moiety is covalently attached.
  • the detectable moiety may provide for imaging of the attached compound or biomolecule.
  • the detectable moiety may indicate the contacting between two compounds.
  • Exemplary detectable moieties are fluorophores, antibodies, reactive dies, radio-labeled moieties, magnetic contrast agents, and quantum dots.
  • Exemplary fluorophores include fluorescein, rhodamine, GFP, coumarin, FITC, Alexa fluor, Cy3, Cy5, BODIPY, and cyanine dyes.
  • Exemplary radionuclides include Fluorine- 18, Gallium-68, and Copper-64.
  • Exemplary magnetic contrast agents include gadolinium, iron oxide and iron platinum, and manganese.
  • FCS Fluorescence correlation spectroscopy
  • PE-FCS can enable significantly improved sensitivity for monitoring the aggregation of amyloidogenic proteins vs. current methods which measure the fluorescence of an amyloid-binding dye, e.g., Thioflavin T (ThT) or Thioflavin S (ThS), using bulk fluorescence measurements.
  • an amyloid-binding dye e.g., Thioflavin T (ThT) or Thioflavin S (ThS)
  • ThT Thioflavin T
  • ThS Thioflavin S
  • amyloid is used herein according to its customary meaning in the art.
  • Amyloids contain a plurality of associated amyloid peptides, such as aggregates of amyloid peptides.
  • amyloids include an amyloid peptide aggregated with one or more amyloid peptides.
  • amyloids include "amyloid plaques,” amyloid deposits,” “amyloid aggregates” or “aggregates of amyloid peptides.”
  • the compounds described herein can associate with (e.g., bind) an amyloid peptide and/or an amyloid. In certain embodiments, the compounds described herein can associate with an amyloid by hydrophobic interactions.
  • fluorescent amyloid aggregate complex is used herein to indicate an amyloid binding fluorophore in complex with an amyloid aggregate.
  • the amyloid aggregate can vary in size.
  • the amyloid aggregate can be a native amyloid aggregate.
  • the amyloid aggregate can be a non-native amyloid aggregate.
  • the amyloid binding fluorophore is non-covalently bound to the amyloid aggregate.
  • biofluid is used herein to describe a biological or bodily fluid that can be obtained from a subject, such as a mammalian subject (e.g. a human).
  • a biofluid is uring, blood, semen, saliva or cerebrospinal fluid.
  • a biofluid may be contained within a suitable buffer.
  • fluorescent burst event or "fluorescence burst event” is used herein to describe fluorescence measured by FCS that is detectably above background levels. Fluorescence can be measured via FCS across a set period of time. Fluorescence events can be measured for time course and intensity. Fluorescence burst events can be recorded in both their numerical quantity for a given time or by calculating a normalized bulk fluorescence correlating the number of burst events with the intensity of each event. II. Amyloid Binding Fluorophores
  • Amyloid binding fluorophores useful in the methods and compositions provided herein are compounds that are capable of binding an amyloid, amyloid peptide or amyloid aggregate and are fluorescent.
  • the amyloid binding fluorophore is not a fluorescently labeled peptide.
  • the amyloid binding fluorophore does not include an amino acid sequence.
  • the amyloid binding fluorophore is not a biomolecule (i.e. a molecule found in nature).
  • the amyloid binding fluorophore does not include a biomolecule or biomolecule moiety.
  • the amyloid binding fluorophore is less than 900 daltons.
  • the amyloid binding fluorophore is less than 800 daltons. In embodiments, the amyloid binding fluorophore is less than 700 daltons. In embodiments, the amyloid binding fluorophore is less than 600 daltons. In embodiments, the amyloid binding fluorophore is less than 500 daltons.
  • amyloid binding fluorophore has the structure of Formula (I),
  • EDG is an electron donor group.
  • 7rCE is a ⁇ -conjugation element.
  • WSG is a water soluble group.
  • EDG is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -OR ,
  • EDG is substituted alkyl, substituted cycloalkyl, substituted heteroalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, -OR 2 , -NR 4 C(0)R 3 , -CONR 4 R 5 , -NR 4 R 5 , -SR 6 , or -PR 7 R 8 .
  • EDG is R ⁇ substituted or unsubstituted alkyl, R ⁇ substituted or unsubstituted cycloalkyl, R ⁇ substituted or unsubstituted heteroalkyl, R ⁇ substituted or unsubstituted heterocycloalkyl, R ⁇ substituted or unsubstituted aryl, R ⁇ substituted or unsubstituted heteroaryl,
  • EDG is R 1 - substituted alkyl, R ⁇ substituted cycloalkyl, R ⁇ substituted heteroalkyl, R ⁇ substituted heterocycloalkyl, R ⁇ substituted aryl, R ⁇ substituted heteroaryl, -OR 2 , -NR 4 C(0)R 3 , -CONR 4 R 5 , -NR 4 R 5 , -SR 6 , or -PR 7 R 8 .
  • R 1 is halogen, -CN, -OR 9 , -CONR 10 R n , -NR 10 R n , -SR 9 , -SOR 9 , -S0 2 R 9 ,-COR 9 , -COOR 9 , -NR 10 COR 9 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 1 is halogen, -CN, -OR 9 , -CONR 10 R n , -NR 10 R n , -SR 9 , -SOR 9 ,
  • R 1 is halogen, -OR 9 , -NR 10 R n , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.
  • R 1 is -OR 9 , -NR 10 R n , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted
  • R 1 when R 1 is attached to alkyl, cycloalkyl, or aryl, R 1 includes at least one heteroatom. In some embodiments, R 1 includes at least one heteroatom. In some embodiments, R 1 is -OR 9 or -NR 10 R n . In some embodiments, R 1 is -NR 10 R n .
  • R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
  • R 5 J , R 6, R 7' and R 8° are independently hydrogen, R 12 -substituted or unsubstituted alkyl, R 12 - substituted or unsubstituted heteroalkyl, R 12 -substituted or unsubstituted cycloalkyl, R 12 - substituted or unsubstituted heterocycloalkyl, R 12 -substituted or unsubstituted aryl or R 12 - substituted or unsubstituted heteroaryl.
  • R 4 and R 5 are optionally joined together to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl.
  • R 4 and R 5 are optionally joined together to form R 12 - substituted or unsubstituted heterocycloalkyl, or R 12 -substituted or unsubstituted heteroaryl.
  • R 9 , R 10 and R 11 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 9 , R 10 and R 11 are independently hydrogen, R 12 -substituted or unsubstituted alkyl, R 12 -substituted or unsubstituted heteroalkyl,
  • R 12 -substituted or unsubstituted cycloalkyl, R 12 -substituted or unsubstituted heterocycloalkyl,
  • R 12 -substituted or unsubstituted aryl, or R 12 -substituted or unsubstituted heteroaryl.
  • R 10 and R 11 are optionally joined together to form an substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl.
  • R 10 and R 11 are optionally joined together to form an R 12 -substituted or unsubstituted heterocycloalkyl, or
  • R 12 -substituted or unsubstituted heteroaryl.
  • R 12 and R 12a are independently halogen, -CN, -SR 13 , -SOR 13 , -S0 2 R 13 ,-OR 13 , -NR 14 R 15 , -COR 15 , -COOR 15 , CONR 14 R 15 , -NR 14 COR 15 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 12 and R 12 ⁇ are independently halogen, -CN, -SR 13 , -SOR 13 ,
  • R 12 is -OR 13 , -NR 14 R 15 , R 16 -substituted or unsubstituted alkyl, R 16 -substituted or unsubstituted heteroalkyl, R 16 -substituted or unsubstituted cycloalkyl, R 16 - substituted or unsubstituted heterocycloalkyl, R 16 -substituted or unsubstituted aryl, or R 16 -substituted or unsubstituted heteroaryl.
  • R 13 , R 14 and R 15 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 13 , R 14 and R 15 are independently hydrogen R 16 -substituted or unsubstituted alkyl, R 16 -substituted or unsubstituted heteroalkyl, R 16 -substituted or unsubstituted cycloalkyl, R 16 -substituted or unsubstituted heterocycloalkyl, R 16 -substituted or unsubstituted aryl, or R 16 - substituted or unsubstituted heteroaryl.
  • R 13 , R 14 and R 15 are independently hydrogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted aryl or unsubstituted heteroaryl. In some embodiments, R 13 , R 14 and R 15 are independently hydrogen or unsubstituted alkyl.
  • R 16 is halogen, -NH 2 , -OH, -SH, -COOH, -COH, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.
  • R 12 is -OR 13 or -NR 14 R 15 .
  • R 12a is -OR 13 or -NR 14 R 15 .
  • R 12a forms part of an R 1 substituent (e.g. where R 1 is an alkyl, cycloalkyl or aryl)
  • R 12a includes a heteroatom.
  • R 12a forms part of an R 1 substituent (e.g. where R 1 is an alkyl, cycloalkyl or aryl)
  • R 12a is -OR 13 or -NR 14 R 15 .
  • R 4 and R 5 are independently hydrogen or R 12 -substituted or unsubstituted alkyl. In some embodiments, R 4 and R 5 are independently hydrogen, R 12 -
  • R 4 and R 5 are optionally joined together to form an R 12 - substituted or unsubstituted heterocycloalkyl.
  • heterocycloalkyl can be R 12 -substituted or unsubstituted piperidinyl, R 12 -substituted or unsubstituted morpholinyl, R 12 -substituted or unsubstituted tetrahydrofuranyl, R 12 -substituted or unsubstituted tetrahydrothienyl, or R 12 - substituted or unsubstituted piperazinyl.
  • R 12 is R 16 -substituted or unsubstituted Ci-C 2 o (e-g-, Ci-Cio) alkyl or R 16 - substituted or unsubstituted heteroalkyl.
  • R 16 can be unsubstituted C 4 -C 8 heterocycloalkyl.
  • R 4 and R 5 are joined together to form R 12 -substituted or unsubstituted heteroaryl.
  • the R 12 -substituted or unsubstituted heteroaryl can be R 12 -substituted or unsubstituted purinyl, R 12 -substituted or unsubstituted pyrimidinyl, R 12 -substituted or unsubstituted imidazolyl, R 12 -substituted or unsubstituted pyrrolopyridinyl (e.g., lH-pyrrolo[2,3- b]pyridinyl), R 12 -substituted or unsubstituted pyrimidinyl, R 12 -substituted or unsubstituted indazolyl (e.g., lH-indazolyl), or R 12 -substituted or unsubstituted or unsubstitute
  • R 4 and R 5 are joined together to form R 12 - substituted or unsubstituted pyrrolopyrimidinyl, R 12 -substituted or unsubstituted indolyl, R 12 - substituted or unsubstituted pyrazolyl, R 12 -substituted or unsubstituted indazolyl, R 12 -substituted or unsubstituted imidazolyl, R 12 -substituted or unsubstituted thiazolyl, R 12 -substituted or unsubstituted benzothiazolyl, R 12 -substituted or unsubstituted oxazolyl, R 12 -substituted or unsubstituted benzimidazolyl, R 12 -substituted or unsubstituted benzoxazolyl, R 12 -substituted or unsubstituted is
  • R 4 and R 5 are joined together to form R 12 - substituted or unsubstituted 6,5 fused ring heteroaryl, R 12 -substituted or unsubstituted 5,6 fused ring heteroaryl, R 12 -substituted or unsubstituted 5,5 fused ring heteroaryl, or R 12 -substituted or unsubstituted 6,6 fused ring heteroaryl.
  • R 4 and R 5 are joined together to form a R 12 -substituted or unsubstituted 5 or 6 membered heteroaryl having at least 2 (e.g. 2 to 4) ring nitrogens.
  • the pi-conjugation element has the formula: -L 1 -(A1 ) q -L 2 -(A 2 ) r - L 3 - or -L 1 -(A 1 ) q -L 4 -A 3 -L 2 -(A 2 ) r -L 3 -.
  • L 1 , L 2 , L 3 and L 4 are independently a bond or a linking group having the formula:
  • x is an integer from 1 to 50. In some embodiments, x is an integer from 1 to 10, from 1 to 20, from 1 to 30, or from 1 to 40. In some embodiments, x is an integer from 1 to 3. In some embodiments, x is an integer of 1.
  • R 17a and R 17b are independently hydrogen, halogen, -CN, -OR 18 , -CONR 19 R 20 , -NR 19 R 20 , -SR 18 , -SOR 18 , -S0 2 R 18 ,-COR 18 ,
  • R 17a and R 17b are independently hydrogen, halogen, -CN, -OR 18 ,
  • a 1 , A2 and A 3 are independently substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene.
  • a 1 , A2 and A 3 are independently R 17 - substituted or unsubstituted arylene or R 17 -substituted or unsubstituted heteroarylene.
  • the symbols q and r are independently 0 or 1.
  • the pi-conjugation element has the formula: -L 1 -(A1 ) q -L 2 -(A 2 ) r -
  • L 1 and L3 are bonds
  • L 2 is a linking group (as defined above or below)
  • a 1 and A 2 are substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene
  • q and r are 1.
  • L 1 and L3 are bonds
  • L 2 is a linking group (as defined above or below)
  • a 1 and A 2 are R 17 -substituted or unsubstituted arylene, or
  • R 17 -substituted or unsubstituted heteroarylene and q and r are 1.
  • L 1 , L2 and L 3 are bonds
  • a 1 and A 2 are R 17 -substituted or unsubstituted arylene or R 17 -substituted or unsubstituted heteroarylene
  • q is 1 and r is 0.
  • the pi-conjugation element has the formula: -L 1 -(A 1 ) q -L 4 -A 3 -L 2 -
  • L 1 and L 3 are bonds
  • L 2 and L 4 are linking groups (as defined above or below)
  • a 1 , A2 and A 3 are substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene
  • q and r are 1.
  • L 1 and L3 are bonds
  • L 2 and L 4 are linking groups (as defined above or below)
  • a 1 , A2 and A 3 are R 17 -substituted or unsubstituted arylene or R 17 -substituted or unsubstituted heteroarylene
  • q and r are 1.
  • the pi-conjugation element is substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene.
  • the pi-conjugation element is
  • the pi-conjugation element is substituted or unsubstituted phenylene or substituted or unsubstituted naphthylene. In certain embodiments, the pi-conjugation element is
  • R 17 -substituted or unsubstituted phenylene or R 17 -substituted or unsubstituted naphthylene.
  • a linking group (L . , L2 , L3 and L 4 ) has the formula:
  • x is an integer from 1 to 50. In some embodiments, x is an integer from 1 to 10, from 1 to 20, from 1 to 30, or from 1 to 40. In some embodiments, x is an integer from 1 to 5, 1 to 3, 2 or 1. In some embodiments, x is an integer from 1 to 3. In some embodiments, x is an integer of 1. In some embodiments, L 1 , L2 , L 3 and L 4 are independently a bond.
  • a 1 , A2 and A 3 are independently substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene. In some embodiments, A 1 , A2 and A 3 are independently R 17 -substituted or unsubstituted arylene, or R 17 -substituted or unsubstituted heteroarylene. In certain embodiments, q and r are independently 0 or 1. In some embodiments, q is 1 and r is 0. In some embodiments, q is 0 and r is 1.
  • a 1 , A2 and A 3 are independently substituted or unsubstituted phenylene, or substituted or unsubstituted naphthylene. In some embodiments, A 1 , A2 and A 3 are independently R 17 -substituted or unsubstituted phenylene, or R 17 -substituted or unsubstituted naphthylene. In some embodiments,
  • a 1 , A2 and A 3 are independently R 17 -substituted or unsubstituted phenylene.
  • a 1 , A2 and A 3 are independently substituted or unsubstituted phenylene. In some embodiments, A 1 , A2 and A 3 are independently substituted or unsubstituted naphthylene. In some embodiments, A 1 , A2 and A 3 are independently R 17 -substituted or unsubstituted
  • R 17 is independently halogen, -CN, -OR 18 , -CONR 19 R 20 , -NR 19 R 20 , -SR 18 , -SOR 18 , -S0 2 R 18 ,-COR 18 , -COOR 18 , -NR 19 COR 20 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 17 is independently halogen, -CN, -OR 18 , -CONR 19 R 20 , -NR 19 R 20 , -SR 18 , -SOR 18 , -S0 2 R 18 ,-COR 18 , -COOR 18 , -NR 19 COR 20 , R 21 - substituted or unsubstituted alkyl, R- 21 substituted or unsubstituted heteroalkyl, R 21 -substituted or unsubstituted cycloalkyl, R 21 -substituted or unsubstituted heterocycloalkyl, R 21 -substituted or unsubstituted aryl, or R 21 -substituted or unsubstituted heteroaryl. In some embodiments, R 17 is independently halogen, -CN, -OR 18 , -CONR 19 R 20 , -NR 19 R 20 , -SR 18 , -SOR 18 ,
  • R is R -substituted or unsubstituted C1-C20 (e-g-, Ci-Cio) alkyl, or R 21 -substituted or unsubstituted heteroalkyl.
  • R 18 , R 1 and R 20 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 18 , R 19 and R 20 are independently hydrogen, R 21 -substituted or unsubstituted alkyl,
  • R 21 -substituted or unsubstituted heteroalkyl R 21 -substituted or unsubstituted cycloalkyl, R 21 - substituted or unsubstituted heterocycloalkyl, R 21 -substituted or unsubstituted aryl, or R 21 - substituted or unsubstituted heteroaryl.
  • R 21 is halogen, -OR 22 , -NR 23 R 24 , halogen, -CN, -OR 22 , -CONR 23 R 24 , -NR 23 R 24 , -SR 22 , -SOR 22 , -S0 2 R 22 ,-COR 22 , -COOR 22 ,
  • R 21 can be halogen, -OR 22 , -NR 23 R 24 , halogen, -CN, -OR 22 , -CONR 23 R 24 , -NR 23 R 24 , -SR 22 , -SOR 22 , -S0 2 R 22 ,-COR 22 , -COOR 22 , -NR 23 COR 24 , R 21a - substituted or unsubstituted alkyl, R 21a -substituted or unsubstituted heteroalkyl, R 21a - substituted or unsubstituted cycloalkyl, R 21a -substituted or unsubstituted heterocycloalkyl, R 21a - substituted or unsubstituted aryl, or R 21a -substituted or unsubstituted heteroaryl.
  • R 21a is halogen, -NH 2 , -OH, -SH, -COOH, -COH, unsubstituted alkyl, unsubstituted, heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or unsubstituted heteroaryl.
  • R 22 , R 23 and R 24 are
  • R 22 , R 23 and R 24 are independently hydrogen or unsubstituted alkyl.
  • the water soluble group is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • the water soluble group is substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, or substituted heteroaryl.
  • the water soluble group is R 25 -substituted or unsubstituted alkyl, R 25 -substituted or unsubstituted heteroalkyl, R 25 -substituted or unsubstituted cycloalkyl,
  • R 25 -substituted or unsubstituted heterocycloalkyl R 25 -substituted or unsubstituted aryl, R 25 - substituted or unsubstituted heteroaryl.
  • the water soluble group is R 25 - substituted alkyl, R 25 -substituted heteroalkyl, R 25 -substituted cycloalkyl, R 25 -substituted heterocycloalkyl, R 25 -substituted aryl, R 25 -substituted heteroaryl.
  • R 25 is halogen, -CN, -OR 26 , -CONR 27 R 28 , -NR 27 R 28 , -SR 26 , -SOR 26 , -S0 2 R 26 ,-COR 26 ,
  • R 25 is halogen, -CN, -OR 26 , -CONR 27 R 28 , -NR 27 R 28 , -SR 26 , -SOR 26 , -S0 2 R 26 ,-COR 26 , -COOR 26 , -NR 27 COR 28 , R 29 - substituted or unsubstituted alkyl, R 29 -substituted
  • R 26 , R 27 and R 28 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 26 , R 27 and R 28 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 26 , R 27 and R 28 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cyclo
  • R 28 are independently hydrogen, R 29 -substituted or unsubstituted alkyl, R 29 -substituted or
  • R 27 and R 28 are optionally joined together to form a substituted or unsubstituted heterocycloalkyl, or a substituted or unsubstituted heteroaryl.
  • R 27 and R 28 are optionally joined together to form a R 29 -substituted or
  • R 29 is halogen, -CN, -OR 30 , -CONR 31 R 32 , -NR 31 R 32 , -SR 30 , -SOR 30 , -SO 2 R 30 ,-COR 30 ,
  • heterocycloalkyl substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.
  • R 30 , R 31 and R 32 are independently hydrogen or substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 30 , R 31 and R 32 are independently hydrogen or unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or unsubstituted heteroaryl. In some embodiments, R 30 , R 31 and R 32 are independently hydrogen or unsubstituted alkyl.
  • the water soluble group can include a moiety that increases the water solubility of a molecule.
  • the water soluble group can include a moiety containing a heteroatom (e.g., oxygen).
  • the heteroatom can be oxygen or nitrogen.
  • the water soluble group is an ethylene glycol moiety having the some embodiments, y is an integer from 1 to 50. In some embodiments, y is an integer from 1 to 10, from 1 to 20, from 1 to 30, or from 1 to 40. In some
  • R is -OMe
  • the water soluble group is R -substituted or unsubstituted C ⁇ -
  • C 2 o (e-g-, C 1 -C 10 ) alkyl or R -substituted or unsubstituted heteroalkyl.
  • C 2 o e-g-, C 1 -C 10 alkyl or R -substituted or unsubstituted heteroalkyl.
  • R ⁇ is -OH.
  • the water soluble group can be -(CH 2 ) b -(CH 2 0H)-CH 2 0H, and b is an integer from 0 to 20, or from 0-10.
  • the compound has the structure:
  • q and r are independently 0 or 1
  • y is an integer from 1 to 10.
  • R 4", R 5 J and R 29 are as defined above.
  • the compound has the structure:
  • q and r are independently 0 or 1
  • y is an integer from 1 to 10.
  • L 1 , L2 , L 3 , L 4 , A 1 , A 2 , A 3 , R 4 , R 5 and R 29 are as defined above.
  • the compound has the structure:
  • m is an integer from 0 to 4
  • z is an integer from 0 to 4
  • y is an integer from 1 to 10.
  • L 1 , L 2 , L 3 , R 4 , R 5 , R 17 and R 29 are as defined above.
  • the compound has the structure:
  • m is an integer from 0 to 6
  • z is an integer from 0 to 6
  • y is an integer from 1 to 10.
  • L 1 , V 2, If 3, R 4", R 5 J , R 17 and R 29 are as defined above.
  • m is 0.
  • z is 0.
  • m is 1.
  • z is 1.
  • the compound has the structure:
  • y is an integer from 1 to 10
  • z is an integer from 0 to 4.
  • R is -OMe.
  • the compound has the structure:
  • y is an integer from 1 to 10
  • z is an integer from 0 to 6.
  • R is -OMe.
  • m is 0.
  • z is 0.
  • m is 1.
  • z is 1.
  • the compound has the structure:
  • m is an integer from 0 to 4
  • x is an integer from 1 to 10
  • y is an integer from 1 to 10
  • z is an integer from 0 to 4.
  • x is 1, and m and z are 0.
  • each substituted group described above in the compounds of the Formulae provided herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or
  • unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene described above in the compounds of the Formulae provided herein is substituted with at least one substituent group.
  • at least one or all of these groups are substituted with at least one size-limited substituent group.
  • at least one or all of these groups are substituted with at least one lower substituent group.
  • each substituted or unsubstituted alkyl is a substituted or unsubstituted C ⁇ C ⁇ alkyl
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 4 -C 8 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 4 to 8 membered heterocycloalkyl
  • each substituted or unsubstituted alkylene is a substituted or unsubstituted C j -
  • each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20 membered heteroalkylene
  • each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 4 to 8 membered heterocycloalkylene.
  • each substituted or unsubstituted alkyl is a substituted or unsubstituted C j -C 8 alkyl
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 5 -C 6 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7 membered heterocycloalkyl
  • each substituted or unsubstituted alkylene is a substituted or unsubstituted C j -C 8 alkylene
  • each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8 membered heteroalkylene
  • compositions disclosed herein can include a compound described herein in combination with a pharmaceutically acceptable excipient (e.g., carrier).
  • a pharmaceutically acceptable excipient e.g., carrier
  • the pharmaceutical compositions include optical isomers
  • the pharmaceutical compositions include a compound disclosed herein and citrate as a pharmaceutically acceptable salt.
  • the compound included in the pharmaceutical composition may be covalently attached to a carrier moiety, as described above.
  • the compound included in the pharmaceutical composition is not covalently linked to a carrier moiety.
  • a "pharmaceutically acceptable carrier,” as used herein refers to pharmaceutical excipients, for example, pharmaceutically, physiologically, acceptable organic or inorganic carrier substances suitable for enteral or parenteral application that do not deleteriously react with the active agent.
  • Suitable pharmaceutically acceptable carriers include water, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, and polyvinyl pyrrolidine.
  • Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds disclosed herein.
  • the compounds disclosed herein can be administered alone or can be coadministered to the subject or biological sample from a subject. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound).
  • the preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation).
  • FCS Fluorescence Correlation Spectroscopy
  • Fluorescence correlation spectroscopy is a time-resolved spectroscopic technique that can measure the concentration and size of fluorescently labeled particles[6]. This method may be used to study aggregation phenomenon such as protein oligomerization (e.g. p53 [7,8]) or formation of large aggregates such as prions [9,10]. FCS has also been used to study ⁇ aggregation using fluorescently labeled peptides [11-13]. However, the effect of the fluorescent label covalently attached to the peptide on assembly dynamics in these prior studies remains unclear.
  • FCS Fluorescence correlation spectroscopy
  • the photons are recorded in a time resolved manner by a highly sensitive single-photon detection device. All signals resulting from the diffusion of a series of molecules through the confocal volume are recorded. The quanta belonging to particular fluorescing molecules are identified using autocorrelation software. The number of molecules in the illuminated volume, as well as their characteristic translational diffusion times, can be determined.
  • the spontaneous fluorescence fluctuating quantity is the number of observed molecules in a defined unit volume, and the diffusion coefficient and the kinetic coefficients of the system are two quantities that are generally measured.
  • FCS detects the time-dependent spontaneous intensity fluctuations in the fluorescence signal which may derive from Brownian motion, flow, and chemical reactions, such as binding.
  • fluorescence correlation spectroscopy FCS and a novel amyloid- binding fluorescent probe, ARCAM 1
  • FCS fluorescence correlation spectroscopy
  • ARCAM 1 a novel amyloid- binding fluorescent probe, can be used to monitor the aggregation of the Alzheimer's disease-associated amyloid- ⁇ peptide ( ⁇ ).
  • ⁇ Alzheimer's disease-associated amyloid- ⁇ peptide
  • ARCAM 1 exhibits a large increase in fluorescence emission upon binding to ⁇ assemblies, making it an excellent candidate for probe
  • an amyloid aggregate is a non-native molecule (e.g. a biological sample containing amyloid aggregates has been subjected to physical or chemical denaturing forces.) In embodiments, an amyloid aggregate is native.
  • PE-FCS/ARCAM 1 based assays can detect and provide some characterization of small ⁇ aggregation intermediates during the assembly process, which enables monitoring and study of such aggregates that transiently accumulate in biofluids of patients with Alzheimer's and other neurodegenerative diseases.
  • biofluids include urine, blood, semen, saliva, cerebrospinal fluids, or others.
  • the biofluid is mixed with a biological buffer (e.g.
  • a buffer has a pH of about 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or more.
  • a buffer has a pH of about 2.5 to about 8.5, about 3.0 to about 8.0, about 3.5 to about 7.5, about 4.0 to about 7.5, about 4.5 to about 6.5, about 5.0 to about 7.0, about 5.5 to about 6.5, about 5.5 to about 7.5, about 6.0 to about 7.0, or about 5.6 to about 6.5.
  • detection of an aggregate includes contacting a biological sample (e.g. a biofluid) containing an amyloid aggregate with an amyloid-binding fluorophore thereby forming a fluorescent amyloid aggregate complex comprising said amyloid aggregate anon- covalently bound to said amyloid-binding fluorophore.
  • detecting said fluorescent amyloid aggregate complex using fluorescence correlation spectroscopy.
  • detection and/or characterization of an amyloid aggregate can be used in the diagnosis, prognosis or treatment determination of diseases.
  • the disease can include Alzheimer's disease, bovine spongiform encephalopathy (BSE), Parkinson's disease, Huntington's disease, Down's Syndrome, Dementia with Lewy Body, or Amyotrophic Lateral Sclerosis (ALS).
  • BSE bovine spongiform encephalopathy
  • ALS Amyotrophic Lateral Sclerosis
  • the amyloid peptide is ⁇ peptide and the disease is Alzheimer's disease.
  • the methods of treating or diagnosing described herein include a method of treating Alzheimer's disease.
  • the methods of treating or diagnosing described herein include a method of treating Parkinson's disease.
  • characteristics of the amyloid aggregate are indicative of disease progression (e.g. smaller aggregates correlate to a less advanced disease, larger aggregates correlate to a more advanced disease state.)
  • the size of an amyloid aggregate is less than about 5,000nm, about 4,000nm, about 3,000nm, about 2,000nm, about 1000 nm, about
  • the size of an amyloid aggregate is about lOOnm to about 5,000nm, about 500nm, to about 4,000nm, about 500nm to about 3,000nm, about 500nm to about 2,000nm, about 500nm to about ⁇ , ⁇ , or about ⁇ , ⁇ to about 2,000.
  • a size of a fluorescent compound is possible using FCS as fluorescence is monitored in a defined confocal volume. Molecular resident time in the confocal volume is proportional to rates of diffusion and molecular size.
  • a size of an amyloid aggregate can be determined by measuring a number of fluorescent burst events for a given molecule while it resides within the confocal volume.
  • autocorrelation analysis is used in determining the size of the amyloid aggregates.
  • Methoxyethoxy)ethoxy ethanol (20.0 g, 0.122 mol) was added to a solution of dry pyridine (49.0 mL) and CH 2 C1 2 (152 mL). The solution was cooled to 0 °C and p-toluenesulfonyl chloride (27.9 g, 0.146 mol) was added in one portion with stirring. The reaction was allowed to come to room temperature and stir for 24 hours. The reaction was then concentrated in vacuo and the solution was filtered to remove solids. The filtrate was purified by flash silica column chromatography (0-3% MeOH/EtOAc) to give A (20.5 g, 53%) as a clear pale yellow oil.
  • ThT (20 ⁇ ) and ARCAM 1 (2.5 ⁇ ) were added into pre-aggregated ⁇ (5 ⁇ ) for comparison or TAMRA- ⁇ (2.5 ⁇ , AnaSpec, Fremont, CA) and ARCAM 1 (2.5 ⁇ ) were added into pre-aggregated ⁇ for comparison in FCS measurement.
  • the fluorescence of ARCAM 1 was measured every 20 minutes using a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA) with excitation and emission of 410 nm and 570 nm, respectively. Data are presented as normalized bulk fluorescence plotted vs. time.
  • ⁇ (1-42) monomers were diluted to 10 ⁇ with 20 mM ammonium bicarbonate pH 8.2 and incubated in the presence of fluorescence probe at a final concentration of 2.5 ⁇ . Aliquots (120 ⁇ ) of the peptide solutions were then dispensed into the wells of an ice-cold 96-well black microtiter plate (Nunc, Roskilde, Denmark) and read immediately. Plates were then sealed with an adhesive plastic cover (WVR, Radnor, PA) and incubated at room temperature with shaking at 500 rpm in a VorTemp 56
  • FCS setup and data analysis were described as before 7 ' 10.
  • the raw intensity traces were analyzed in the following four steps.
  • the histogram of intensity trace (l ms binning time windows) was plotted and the maximum peak (mode of the intensity trace) was used to designate the background signal (see Fig. 12A). The standard deviation (or width) of this histogram was also determined.
  • any signals from the trace with intensities greater than four times the width of the histogram above the background baseline (mode) were selected as burst candidates.
  • burst candidates due to the diffusion properties, there may be bursts in rapid succession without any intervening time. These bursts are merged as single burst event.
  • a final burst trace was generated that was used to calculate the burst number.
  • a solution of ARCAM 1 was prepared at 100 ⁇ in pH 7.4 IX PBS with 5% DMSO (by volume) and incubated quiescently at room temperature. Aliquots from this solution were removed at selected time points and flash frozen at -78°C. Aliquots were then warmed to room temperature and probe stability monitored by LC-UV-MS equipped with a CapCell MGIII C18 column with a 3 ⁇ particle size. The absorption was measured at 254 nm, 280 nm, and 480 nm using a solvent gradient of 2.5% to 100% MeCN in deionized H 2 0 with 0.1% formic acid at a flow rate of 0.3 mL/min. Relative probe stability was determined by integrating the peak area of the probe at different time intervals relative to the peak area at time zero.
  • ARC AM 1 was prepared to a final concentration of 4 ⁇ in 5% DMSO in IX PBS at various pH values both as free probe in solution and in the presence of 5 ⁇ aggregated ⁇ (1- 42) (t max ). Sample preparations were incubated at room temperature for 10 minutes prior to measurement.
  • ⁇ (1-42) was synthesized, purified, and characterized.
  • the peptide mass and purity (98%) were determined by electro spray/ion trap mass spectrometry and purified by reverse phase HPLC, respectively.
  • the fluorescence of ARC AM 1 was measured at 20 minute intervals using a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA) with excitation and emission of 410 nm and 570 nm, respectively. Data are presented as normalized bulk fluorescence plotted vs. time.
  • ThT fluorescence was measured at 20 minute intervals using a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA) with excitation and emission of 435 nm and 485 nm, respectively. Aggregation was allowed to proceed until the maximal fluorescence reached a plateau, then a portion of the same SEC-isolated monomer sample that had been held on ice was used for a repeat experiment exactly as described above, but adding an equal volume of MQ water in place of ThT.
  • ⁇ (1-42) monomers were diluted to 10 ⁇ with 20 mM ammonium bicarbonate pH 8.2 and incubated in the presence of fluorescence probe at a final concentration of 2.5 ⁇ . Aliquots (120 ⁇ ) of the peptide solutions were then dispensed into the wells of an ice-cold 96-well black microtiter plate (Nunc, Roskilde, Denmark) and read immediately. Plates were then sealed with an adhesive plastic cover (WVR, Radnor,
  • FCS setup 0.1% BSA solution to reduce the non-specific adsorption
  • Two-photon FCS was performed on a customized setup based on an inverted Nikon TE2000 microscope.
  • the back aperture of the objective was slightly overfilled, creating a diffraction-limited focal spot.
  • the laser power was set to lOmW (at entrance to microscope) to reduce photobleaching of the fluorescent probe.
  • the emission fluorescence (collected via epifluorescence) was passed through an emission filter (HQ525/50m-2p for ARCAM1,
  • the raw intensity trace has 1 ⁇ 8 time resolution. By binning the time windows new time traces can be generated with 1 ms time resolution, which was analyzed by the burst analysis method.
  • T D is the residence time of species within the sampling volume
  • D is the diffusion coefficient of the species
  • ⁇ co z /co X y is the aspect ratio of the sampling volume
  • co z is the axial size of the excitation volume
  • co X y is its radius.
  • Brightness (Q) was calculated by dividing the average fluorescence intensity by average particle number (TV).
  • G(T) — (i + _L r > (i + -J—)- 1 - 2 + i— (i + _L)-' (i +
  • N and N 2 are the average particle number of small species and large species in the sampling volume, individually. and 3 ⁇ 4 are the particle brightness for small species and large species.
  • Example 2 Real-time monitoring of Alzheimer's-related amyloid aggregation via Probe Enhancement - Fluorescence Correlation Spectroscopy (PE-FCS).
  • PE-FCS Probe Enhancement - Fluorescence Correlation Spectroscopy
  • ThT Thioflavin T
  • ThT Thioflavin T
  • its emission intensity increases with increasing population of aggregates.
  • a major limitation of amyloid aggregation assays that use ThT is that the bulk fluorescence intensity increases above background only once protofibril and fibril structures have become abundant in solution, precluding the capability to detect small, transient intermediates [4,5].
  • ThT has a significant fluorescence as an unbound dye, decreasing the signal to noise ratio for sensitive measurements of small assemblies.
  • a family of fluorescent probes that bind ⁇ assemblies in solution and in tissue are described herein and previously [16-18]. These probes exhibit a large enhancement in fluorescence properties upon binding to aggregates compared to the weaker fluorescence of the free compounds in solution.
  • ARCAM aryl cyano amide
  • An important advantage of ARCAM 1 for aggregation studies is its stability in aqueous solutions (FIG. 6) and broad insensitivity of fluorescence as a function of pH (FIG. 7A-7D). For instance, the half-life of ARCAM 1 in phosphate buffered saline (PBS) at room temperature was -150 hours. Importantly, there is negligible change in the effective concentration of ARCAM 1 over the aggregation time-courses. In addition, ARCAM 1 shows similar multi-photon excitation (used in our FCS setup) to that of ThT (FIG. 8).
  • Probe-Enhancement FCS relies on the increase in fluorescence that occurs as a result of the probe binding to its target.
  • FCS curves were measured for solutions containing diluted, pre-aggregated ⁇ and the probes. Either ThT or ARCAM 1 was added to pre-aggregated ⁇ samples (FIG. 10).
  • ThT or ARCAM 1 was added to pre-aggregated ⁇ samples (FIG. 10).
  • TAMRA- labeled ⁇ (1-42) peptides or ARCAM 1 was added to another set of matched samples. FCS measurements of the probe- ⁇ solutions were taken after a 30 minutes incubation at room temperature to permit probe binding (for ThT and ARCAM 1) or monomer incorporation (for TAMRA- ⁇ ).
  • the size of the species at the onset of increased burst activity was also estimated (i.e., during the first 120 min of the aggregation of ⁇ monomers, FIG. 3A).
  • intensity time traces e.g. FIG. 3B
  • the result is a correlation function G(x) that is proportional to the number of burst events while a molecule is resident in the excitation volume for the delay time, ⁇ (FIG. 4, and below).
  • the residence time is directly related to the translational diffusion constant and, therefore, its size.
  • FCS can only provide an average diffusion constant that is biased towards the more fluorescent species.
  • G(0) point is inversely proportional to the number of particles in the solution.
  • ARC AM 1- ⁇ (1-42) solutions there were two diffusing species: unbound ARCAM 1 and bound to ⁇ .
  • hydrodynamic radius and proposed rod length are consistent with protofibrils, an early assembly intermediate in ⁇ aggregation [4] .
  • This PE-FCS method can detect low concentrations of early amyloid assembly intermediates that were consistent in size with a previous FCS study that used covalently labeled ⁇ peptides [11].
  • a major advantage of the method reported in this work is that using an exogenously added fluorescence reporter (as opposed to fluorescently labeled peptides) may make it possible to analyze patient samples containing a large mixture of native aggregated species.
  • the combination of a fluorescent reporter and PE-FCS -based detection of aggregated ⁇ represents a potentially important step towards establishing a reliable method for studying aggregate intermediates that may be present in human CSF [23].
  • Alzheimer's disease an emperor in need of clothes, Nature neuroscience 15, 349-357;

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Abstract

L'invention concerne entre autres des composés et une méthode de spectroscopie à corrélation de fluorescence, utiles pour la détection d'agrégats amyloïdes dans un échantillon biologique.
PCT/US2016/040772 2015-07-02 2016-07-01 Procédés et compositions pour des agrégats amyloïdes WO2017004560A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021525875A (ja) * 2018-05-31 2021-09-27 アミディス， インコーポレイテッド 外傷性脳損傷を検出するための組成物および方法
WO2024069193A1 (fr) * 2022-09-30 2024-04-04 Imperial College Innovations Limited Biomolécules dans une maladie

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998022494A2 (fr) * 1996-11-22 1998-05-28 Elan Pharmaceuticals, Inc. METHODES ET COMPOSES DESTINES A INHIBER LA LIBERATION ET/OU LA SYNTHESE DU PEPTIDE β-AMYLOIDE
US20040224365A1 (en) * 1997-08-14 2004-11-11 Charles Glabe Fluorescent amyloid Abeta peptides and uses thereof
US20080118938A1 (en) * 2006-09-06 2008-05-22 Lisbell Estrada Methods and Compositions for the Detection of Protein Folding Disorders

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998022494A2 (fr) * 1996-11-22 1998-05-28 Elan Pharmaceuticals, Inc. METHODES ET COMPOSES DESTINES A INHIBER LA LIBERATION ET/OU LA SYNTHESE DU PEPTIDE β-AMYLOIDE
US20040224365A1 (en) * 1997-08-14 2004-11-11 Charles Glabe Fluorescent amyloid Abeta peptides and uses thereof
US20080118938A1 (en) * 2006-09-06 2008-05-22 Lisbell Estrada Methods and Compositions for the Detection of Protein Folding Disorders

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KHURANA ET AL.: "Mechanism of thioflavin T binding to amyloid fibrils.", JOURNAL OF STRUCTURAL BIOLOGY, vol. 151, no. 3, 2005, pages 229 - 238, XP027216412, Retrieved from the Internet <URL:http://www.sciencedirect.com/ science /article/pii/S1047847705001309> [retrieved on 20160809] *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021525875A (ja) * 2018-05-31 2021-09-27 アミディス， インコーポレイテッド 外傷性脳損傷を検出するための組成物および方法
US20210369873A1 (en) * 2018-05-31 2021-12-02 Amydis, Inc. Compositions and methods for detection of traumatic brain injury
JP7381500B2 (ja) 2018-05-31 2023-11-15 アミディス， インコーポレイテッド 外傷性脳損傷を検出するための組成物および方法
WO2024069193A1 (fr) * 2022-09-30 2024-04-04 Imperial College Innovations Limited Biomolécules dans une maladie

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