U.S. patent application number 12/944558 was filed with the patent office on 2011-06-30 for neurodegenerative protein aggregation inhibition methods and compounds.
Invention is credited to Gerald R. Crabtree, Jason E. Gestwicki, Isabella A. Graef.
Application Number | 20110160246 12/944558 |
Document ID | / |
Family ID | 34115506 |
Filed Date | 2011-06-30 |
United States Patent
Application |
20110160246 |
Kind Code |
A1 |
Graef; Isabella A. ; et
al. |
June 30, 2011 |
Neurodegenerative Protein Aggregation Inhibition Methods and
Compounds
Abstract
Methods and compositions are provided for reducing aggregation
of neurodegenerative proteins associated with neurotoxicity or
other proteins. The compounds comprise a first domain or targeting
element for binding to the target proteins linked to a second
domain or recruiting element that binds to an aggregation
inhibiting protein, e.g. a prolyl isomerase. By associating the
aggregating forming proteins or neuronal cells under conditions
where aggregating proteins are produced with the compound and the
aggregation inhibiting protein, aggregation is reduced. The subject
agents can be used in assays, investigating the etiology of the
neuronal diseases and for prophylaxis and therapy.
Inventors: |
Graef; Isabella A.;
(Woodside, CA) ; Crabtree; Gerald R.; (Woodside,
CA) ; Gestwicki; Jason E.; (Ann Arbor, MI) |
Family ID: |
34115506 |
Appl. No.: |
12/944558 |
Filed: |
November 11, 2010 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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12050017 |
Mar 17, 2008 |
7923230 |
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12944558 |
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10901848 |
Jul 28, 2004 |
7485706 |
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12050017 |
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60491482 |
Jul 30, 2003 |
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Current U.S.
Class: |
514/319 ;
435/68.1; 546/206 |
Current CPC
Class: |
A61K 31/7076 20130101;
A61K 31/5415 20130101; A61P 25/00 20180101; A61P 25/16 20180101;
A61K 47/54 20170801; A61K 31/4745 20130101; A61P 25/28 20180101;
A61K 31/655 20130101 |
Class at
Publication: |
514/319 ;
546/206; 435/68.1 |
International
Class: |
A61K 31/445 20060101
A61K031/445; C07D 211/60 20060101 C07D211/60; C12P 21/00 20060101
C12P021/00; A61P 25/00 20060101 A61P025/00; A61P 25/28 20060101
A61P025/28; A61P 25/16 20060101 A61P025/16 |
Goverment Interests
[0002] This invention was made in part with government support
under Grant No. NS046789 awarded by the National Institutes of
Health. The government has certain rights in this invention. This
work was also supported by the Howard Hughes Medical Institute.
Claims
1. A method for diminishing the presence of protein aggregates in
an environment susceptible to the formation of a protein aggregate,
said method comprising: introducing into said environment a
compound having two domains, a first domain that binds to a protein
aggregation having at least two monomers, and a second domain
binding to an aggregation diminishing protein, whereby the presence
of aggregations is diminished.
2. A method according to claim 1, wherein said aggregation
diminishing protein sterically interferes with the formation of
aggregations.
3. A method according to claim 1, wherein said aggregation
diminishing protein enzymatically modifies said monomers to inhibit
aggregation.
4. A method for reducing aggregation of neurodegenerative proteins,
said method comprising: associating neurodegenerative proteins with
a compound comprising a first domain binding to said
neurodegenerative proteins linked to a second domain binding to an
aggregation formation reducing protein. whereby aggregations are
diminished.
5. A method according to claim 4, wherein said aggregation
formation reducing protein is a prolyl isomerase.
6-7. (canceled)
8. A method according to claim 4, wherein said first domain is a
polycyclic compound.
9. A method according to claim 5, wherein said polycyclic compound
comprises a biphenyl or stilbene.
10. A compound comprising a polycyclic domain binding to
neurodegenerative proteins linked to a prolyl isomerase-binding
domain.
11-17. (canceled)
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to provisional application
Ser. No. 60/491,482, filed Jul. 30, 2003.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The invention relates generally to modification of
neurodegenerative protein aggregations associated with neuronal
disease.
[0005] 2. Background Information
[0006] In an aging population there is an increasing incidence of
neurodegenerative diseases. These diseases that waste the mental
faculties while still leaving the physical capabilities
substantially intact are an emotional drain on those related to the
patient and a major financial drain on the patient, patient
families and societies. Many of these diseases have certain common
features. One of the features is the formation of pathological
conformations of proteins that can lead to protofibrils, fibrils
and amorphous aggregations resulting in neuronal cell death.
[0007] Among the diseases associated with such etiology are
Alzheimer's disease, Parkinson's disease, transmissible spongiform
encephalopathies, such as Creutzfeld-Jakob disease, polyglutamine
diseases, Huntington disease, and Lou Gehrig's disease, as well as
other neuropathologies. Efforts to treat these diseases have been
substantially unavailing. The efforts have been impeded by the
inability to diagnose the diseases at their early stages and
recognizing the existence of the disease until the patient becomes
symptomatic. Also, there are the problems of the blood brain
barrier, the identification of effective drugs and the incidence of
side effects of attempted treatments.
[0008] Originally, assay methods were developed that relied on
cells in culture, which have provided insights into the etiology of
these diseases. The effect of various treatments on neuronal
mortality has aided in understanding how one may reduce the level
of neuronal toxicity of the aggregates. Today, there are mouse
models, where the mice have been genetically modified to represent
the neuronal pathologies. In this way, experiments can be performed
that more readily approximate the human condition. The Kiessling
laboratory, as well and the Lee and Kung laboratories, among
others, have done extensive studies on methods of interfering with
protein aggregation in amyloid related diseases, e.g. Alzheimer's
disease. The Kiessling group has identified an amino acid sequence
in A.beta. that appears to be a domain involved with aggregation,
amino acids 16-20 (KLVFF). Further efforts are needed to provide
compounds that can be used in research to elucidate the mechanisms
of the neuropathies, the role played by the formation of fibrils
and plaques and the effect of interference of such formation. In
this way, compounds can be developed that will be used in the
treatment of the neuropathies.
[0009] Besides the neuropathies associated with aggregation, other
diseases may also involve aggregation, where there is an interest
in preventing or deterring the aggregation from inducing a diseased
state. In addition, there are other situations where the inhibition
of aggregate formation, such as in the analysis of naturally
occurring mixtures are of interest.
RELEVANT LITERATURE
[0010] Work by the Kiessling group may be found in Ghanta, et al.,
1996 J Biol Chem 271, 29525-28; Lowe, et al., 2001 Biochemistry 40,
7882-89 and Cairo, et al., 2002 Biochemistry 41, 8620-9, describing
the binding domain of A.beta. and assay methodology. Other articles
related to A.beta. include Korth, et al., 2001 PNAS 98, 9836-41,
Hardy and Selkoe 2002 Science 297, 353-6; Lee, 2002 Neurobiology of
Aging 23, 1039-42 and Kim and Lee, 2003, Biochem Biophys Res Comm
303, 576-9, where the latter reports that fullerene has an
aggregation inhibiting effect on A.beta.. Description of the prolyl
isomerase Pin 1 and its role with phosphorylated tau is described
in Lu, et al., 1999 Nature 399, 739-40; and Lu, et al., 1999
Nature, 399.
[0011] Review articles of protein aggregation associated
neuropathies include Zoghbi and Orr, 2000 Ann Rev Neurosci 23,
217-47 (glutamine repeats); Lee, et al., 2000 Ann Rev Neurosci 24,
1121-59; Goedert 2001 Nature Reviews/Neuroscience 2, 482-501
(.alpha.-synuclein); Sacchettini, J. C. and Kelly, J. W. (2002)
Nature Rev. Drug Discovery 1:267-275--a good recent review of
inhibitor strategies; Voiles, M. J. and Lansbury, P. T. Jr (2003)
Biochemistry 42:7871-7878.--discussion of the latest models of
Parkinson's pathology; Koo, E. H. et al (1999) Proc. Natl. Acad.
Sci. USA 96:9989-9990--one of the most commonly cited reviews on
the subject of amyloid diseases; Kayed, R. et al (2003) Science
300:486-489.--suggests that various amyloidogenic peptides display
a common mechanism(s) of aggregation; and Serpell, L. C. (2000)
Biochimica et Biophysica Acta 1502:16-30--discusses current
thinking about the structure of A.beta. aggregates.
[0012] Other references of interest include references associated
with the experimental procedures: Gordon, D. J. and Meredith, S. C.
(2003) Biochemistry 42:475-485; and Graef, I. A. et al 1999 Nature
401:1703-1708 and other references associated with inhibitors:
Gordon, D. J. and Meredith, S. C. (2003) Biochemistry 42:475-485
--inhibitor peptides; Kim, Y-S. et al (2003) J. Biol. Chem.
278:10842-10850 --data shows that Congo red binds to unfolded
A.beta. peptide; Hammarstrom, P. et al (2003) Science
299:713-716.--inhibitors based on kinetics; Klettner and Henlegen
(2003) Curr Drug Target CNS Neurol Disord 2, 152-62 --FK506 for
treatment of neurological disorders; and Klunk, W. E. et al (1998)
Life Sciences 63:1807-1814 --first chrysamine G paper.
[0013] Spencer, et al., 1983 Science 262, 1019-24 describes the
construction of small molecules that are dimeric and bind two
different proteins created by DNA recombination. See also, U.S.
Pat. Nos. 5,830,462; 6,011,018 and 6,316,418. Briesewitz, et al.,
1999 PNAS USA 96, 1953-8 and U.S. Pat. No. 6,372,712 describe the
use of an endogenous protein to modulate the affinity of a small
molecule.
SUMMARY OF THE INVENTION
[0014] The presence of protein aggregations, particularly as
associated with neuropathies or other environments in which
aggregation is detrimental, are diminished by adding to an
environment in which such protein aggregations occurs a compound
having two domains, one that targets aggregations and a second that
recruits proteins to the site of aggregation, particularly prolyl
isomerases or other applicable proteins, by binding to the
aggregate forming protein. The compounds are characterized by
having a domain that preferentially binds to aggregations
comprising at least two monomeric proteins linked to a second
domain that recruits the aggregation modifying protein(s). In the
case of neuropathies, the presence of prolyl isomerases intra- and
extracellular ensure the availability of the aggregation modifying
protein, as well as the opportunity to modify the aggregate monomer
by isomerizing proline to reduce the propensity to form aggregates
as well as sterically hinder aggregate formation. Of particular
interest are domains of compounds that when combined are able to
cross the blood brain barrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a diagram of the current model for aggregation of
amyloidogenic proteins. Properly folded protein or peptide is
believed to progress through a series of conversions to form a
highly toxic protofibril (or seed aggregate). Protofibrils further
mature into progressively more extended structures, such as fibrils
and amorphous aggregates.
[0016] FIG. 2 is a description of two possible mechanisms by which
agents could influence toxicity and/or prevent fibril formation. In
the first model, the prolyl isomerase activity of FKBP, which has
been recruited to an unfolding amyloidogenic peptide by the
bifunctional agent, acts to lower the barrier to re-folding. In the
second model, the steric bulk of the recruited FKBP prevents
formation of a toxic fibrillar species.
[0017] FIG. 3 is a diagram of the agents of this invention.
[0018] FIG. 4 is a diagram of the synthesis of the agents of this
invention.
[0019] FIG. 5 is a graph of the observed fluorescence with the
Thioflavin T assay under the conditions outlined in the
Experimental section. The graph shows that fibril formation is
inhibited by addition of a combination of SLF-CR and FKBP12.
[0020] FIG. 6 is a graph showing the effect of varying the
concentration of an agent of this invention on fibril formation
using the Thioflavin T assay. The concentration of FKBP, which was
present during the fibrillization procedure, was held constant at 1
.mu.M.
[0021] FIG. 7 is a series of TEM images that demonstrate the
disruption of fibril formation by an agent of this invention when
FKBP is added. Representative fibrils have been outlined with
dotted lines and highlighted by arrows. The fibrils formed in the
presence of a combination of SLF-CR and FKBP are clearly less
abundant and generally smaller. TEM was performed as outlined in
the Experimental section.
[0022] FIG. 8 is a diagram and graph describing the luciferase
assay and the results obtained from this assay. Panel A is a
diagram of the assay procedure. Panel B is a graph that
demonstrates that an agent of this invention can prevent toxicity
of aggregated amyloidogenic peptide. Further, this inhibition of
toxicity is greatly enhanced by the presence of FKBP.
[0023] FIG. 9 is a graph of data from the luciferase assay showing
that addition of an agent of this invention can promote survival of
primary neurons transfected with the Q72 form of Huntingtin
amyloidogenic protein.
[0024] FIG. 10 illustrates: (A) Schematic representation of how
improvements in linker length or flexibility can allow bound FKBP
to sample a greater area around the A.beta. surface. (B) Chemical
structures of bifunctional inhibitors generated for the study, with
the variable linker region shown in blue and SLF-CR shown for
comparison. (C) Relative potency of the bifunctional linker series,
as measured by thioflavin T fluorescence. FKBP was added to each
aggregation reaction at a final concentration of 1 .mu.M. The
average IC.sub.50 (.+-.SEM) of two experiments performed in
triplicate is shown in the inset. (D) TEM images confirm the
presence of intermediate particles in A.beta. samples treated with
SLF-CR/FKBP. Particles of similar size and shape are observed in
samples treated with compounds from the linker series. Drug and
FKBP concentrations are 0.5 .mu.M and 1 .mu.M, respectively. An
untreated A.beta. sample is shown. (E) The survival of hippocampal
neurons subjected to A.beta. samples that had been treated with
compounds from the bifunctional linker series. Results are the
average (.+-.SEM) of 1 to 2 experiments performed in
triplicate.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Methods and compositions are provided for diminishing the
presence of aggregates in an environment susceptible to the
formation of aggregates, for example, by inhibiting protein
aggregation in an environment where the formation of such aggregate
is detrimental. Of particular interest is where the formation of
protein aggregation is associated with neuronal pathologies. The
method comprises introducing into an environment susceptible to
protein aggregation a compound having a first domain or agent that
functions as an aggregate binding element having an affinity for
incipient or existing protein aggregations and a second independent
domain or agent that serves as a recruiting element for an active
factor that impedes protein aggregation. In this manner the
compound recruits to the site of the protein aggregation a factor
that is able to inhibit the formation of the protein aggregation
and/or diminish aggregates. In the absence of the aggregation
occurring, the detrimental effect or injury resulting from
formation of the aggregation is avoided. Combinations of agents are
preferred that can penetrate a membrane.
[0026] The compounds of this invention are complex organic
compounds, usually synthetic, having one or more rings, both
carbocyclic and heterocyclic, of from 10 to 120, usually of from 12
to 80 carbon atoms, more usually 12 to 60 carbon atoms, and having
at least two heteroatoms and not more than about 60, usually not
more than about 36 heteroatoms, more usually not more than about 30
heteroatoms, where the heteroatoms will be N, chalcogen (O or S),
P, B, halogen and as cations, alkali and alkaline earth metals. The
subject compounds will be in the higher molecular weight range when
naturally occurring or modified naturally occurring compounds, such
as the cyclosporins, are included in the molecule. Generally there
will be at least 2 rings and not more than about 12 rings, usually
not more than about 10 rings, where the rings will have from 3 to
8, usually 5 to 7 annular members. There may be from 1 to 4,
usually 1 to 3 heteroatoms in a ring, where fused rings will
usually have from 2 to 5, more usually 2 to 4 rings.
[0027] The compounds of the subject invention will for the most
part have a first domain R1 that serves to bind aggregating protein
molecules that are detrimental, particularly where the aggregates
from injurious entities leading to neuronal pathologies; a second
domain R2 that has specificity for a factor that impedes, prevents
or reverses protein aggregation formation, serving to recruit the
factor to an aggregate; and an entity L as a linker that serves to
connect R1 and R2 to keep them together. The factor can affect the
formation or dispersible of aggregates by steric hindrance to
aggregate formation, modification of the components of the
aggregate reducing the affinity of aggregation, or the like. R1, R2
and L will be described in seriatim.
[0028] Many compounds that bind the proteins associated with
aggregation and neuronal pathologies are well known. Therefore, R1
will be such compounds as they may be modified to be linked to L
and such other compounds that presently exist or may be developed
in the future. In many cases R1 will have one or more functional
groups that can serve as the site to bind to L. Where such
opportunity does not exist, such compounds may be modified by
introducing functional groups, such as --OH, --NH2 or
mono-substituted --NH2, --CO2H, --CH.dbd.CH--, --C(O)CH2X, where X
is halogen or pseudohalogen, etc. Such modifications will depend
upon the nature of the compound, the site that permits modification
without a significant adverse effect on the binding affinity of the
compound, and the synthetic protocol. All of these permutations can
be readily determined using combinatorial methods, if necessary.
See, for example, U.S. Pat. Nos. 5,968,736 and 6,503,759. R1 can
serve as a binding agent, but may also serve in disaggregation of
the aggregating monomeric proteins.
[0029] The domains of interest for R1 will for the most part be
cyclic compounds, having from 1 to 8, usually 1 to 6 rings, fused
or unfused, as a core structure, usually having from 2 to 6 rings,
more usually 2 to 4 rings, where other rings may be present as
substituents. The compounds serving as the domain will generally
have at least 8 carbon atoms, usually at least about 12 carbon
atoms, and at least about 1 heteroatom, usually at least about 2
heteroatoms, generally from 1 to 16, usually 1 to 12, more usually
2 to 8, heteroatoms, that are nitrogen, chalcogen (O and S),
phosphorous, halogen, boron, and with acids, anions, such as the
alkali and alkaline earth metals.
[0030] For the most part, the R1 domain will not exceed 60 atoms
other than hydrogen, usually not more than about 36 atoms other
than hydrogen and generally will not be more than 30 carbon atoms.
Substituents on the core rings may be aliphatic (including
alicyclic), aromatic and heterocyclic, and combinations thereof,
generally of from 1 to 16, usually from 1 to 12 carbon atoms, halo,
e.g. F, Cl, Br, and I, oxo-carbonyl of from 1 to 8, usually 1 to 6
carbon atoms, non-oxo-carbonyl of from 1 to 12, usually 1 to 8,
more usually 1 to 6 carbon atoms, e.g. acid, ester and amide, and
the nitrogen and sulfur analogs thereof, amino (including
substituted amino, particularly alkyl or oxyalkyl) of from 0 to 12,
usually 0 to 8, more usually 0 to 6 carbon atoms, oxy of from 0 to
8, usually 0 to 6 carbon atoms, thio of from 0 to 8, usually 0 to 6
carbon atoms, inorganic acids and their esters and amides of from 0
to 8, usually 0 to 6 carbon atoms, including sulfonic acid,
sulfate, sulfenic acid, boronic acid, phosphoric acid, phosphonic
acid, phosphinic acid and the nitrogen and sulfur analogs thereof,
azo, nitro, cyano, etc.
[0031] The rings will generally be of from 3 to 8 annular members,
more usually of from 5 to 7 annular members. Rings include benzene,
furan, thiophene, azole, pyrazole, oxazole, thiazole, imidazole,
pyrrole, pyrrolidine, pyrroline, piperidine, pyridine, pyrazine,
pyridazine, thiazine, cycloheptane, azacycloheptane, azulene,
diazacycloheptane, anthracene, etc. Various core structures include
dibenzazacycloheptane, phenylpiperidine, dibenzdiazacycloheptane,
dibenzdiazacyclohexane, dibenzthiazacyclohexane,
dibenzthiacyclohexane, biphenyl, benzpyrrole, naphtho[2,1-b]furan,
naphthacene, anthracene, stilbene, etc.
[0032] Compounds of interest include: 1-Br or
I-2,5-bis(3-hydroxycarbonyl-4-hydroxy)stilbene, thioflavin,
thioflavin T, chrysamine G, X-34, Congo red, IMPY (Kung, et al.,
2002 Brain Res 956, 201-10), imipramine, carbamazepine, phenazine,
phenothiazine, promazine, chlorpromazine, haloperidol, clozapine,
2-chlorophenothiazine, promethazine, chlorprothixen, acepromazine,
deoxydoxorubicin, rifamycin, acridone and acridone derivatives,
such as flavinoids and alkaloids, benzofurans, such as
5-(3-(4-(2-methoxyphenyl)-1-piperazinyl)propionyl)benzofuran (He,
et al., 2002 Chinese Chem J on Internet 4, 13), and benzofurane
(SKF74652), quinacrine and other 9-substituted acridines, e.g.
1-(3-dimethylaminopropyl-1-amino)4-nitro-7-methylthio-9-acridone,
pamaquine, chloroquine and amacrine, methylene blue, and the
like.
[0033] The disaggregation protein binding compound can be any
protein that interferes with aggregation formation by steric
hindrance, enzymatic activity or other mechanism. By virtue of an
incipient or formed aggregation having a plurality of members and
an extended surface, a plurality of the first domain may bind to
the aggregation. In this way, multiple target proteins may be
recruited to the site of the aggregation to act on the aggregation.
As discussed previously, the subject compounds allow for the
recruitment of various protein agents to inhibit or modify
incipient or formed aggregations. Of particular interest in vivo as
the second domain, R2, is an agent that serves to recruit a prolyl
isomerase. R2 may bind to any epitope of the recruited protein, for
an enzyme including the active site of the enzyme. The binding will
usually be reversible, particularly where the binding compound
binds at the active site.
[0034] The prolyl isomerase enzyme will be classified as EC.5.2.1.8
and may be one of the human genes related to FKBP or cyclophilin,
Pin1, hParv14, etc. The binding domain will bind to at least the
human enzyme and may bind to enzymes of other species. These
domains will in many situations be based on naturally occurring
compounds and may therefore be relatively large. The domains will
generally have from about 10 to 80 carbon atoms and from about 2 to
30, usually 6 to 24 heteroatoms, where the heteroatoms will be N,
chalcogen (O or S), P, B, halogen and as cations, alkali and
alkaline earth metals. The functional groups listed for R1 will
also be applicable for R2. Compounds of interest to serve as
domains include FK506, rapamycin and derivatives thereof,
cyclosporinA and modified cyclosporine (See, U.S. Pat. No.
6,444,643), 18-OH-ascomycin (Mollison, et al., 1997 J Pharmacol Exp
Ther 283, 1509-19), sanglifehrin A derivatives (Zenke, et al., 2001
J Immunol 166, 7165-71), KLVFF, and the like. Other proteins may be
recruited that will discourage aggregation, particularly of the
individual neurodegenerative proteins. These proteins include the
heat shock proteins, e.g. hsp70 and hsp90 that serve as
chaperonins, or other chaperonins, where the heat shock proteins
can be recruited by geldenamycin.
[0035] The two domains are joined by a linking group L that may
only be a bond or have not more than about 20 atoms in the chain,
usually not more than about 12 atoms in the chain. It should be
understood that the functional group associated with the domain for
binding to the linker is not included in the counting of the atoms
of the chain. For cyclic compounds the shortest path will be
counted. Depending on the nature of the two domains, different
linking groups may provide for optimization of the activity of the
product. The linkers can provide spacing between the two domains,
rigidity or flexibility between the two domains, hydrophilicity or
hydrophobicity, hydrogen bonding, etc. Thus, the examples given in
the Experimental section provide guidance as to the effect of the
linking group with the particular domains that are employed.
[0036] The total number of atoms other than hydrogen for the
linking group will not exceed about 36 atoms, usually not more than
20 atoms, more usually not more than 16 atoms, frequently not more
than about 12 atoms. The chain may be aliphatic (including
alicyclic), aromatic or heterocyclic, or combinations thereof,
saturated or unsaturated, where heteroatoms will be N, chalcogen,
or P, including such functionalities as oxy, ester, amide and the
nitrogen and sulfur analogs thereof, amino, substituted amino,
inorganic acids, particularly phosphorous acids. e.g. phosphate,
thio, oxo, halo, etc. Where rigidity of the linking group is
desired, of particular interest is the inclusion of an aromatic
group in the chain, particularly a carbocyclic aromatic group, of
from 5, usually 6 to 12 carbon atoms, where heteroannular atoms
will for the most part be N, O and S. Alternatively, one may use
ethylenic or acetylenic unsaturation to provide for the rigidity.
The linking group may serve solely to join R1 and R2 without
significantly affecting the characteristics of the compound or may
provide for solubility, enhanced membrane penetration, stability,
pharmacological distribution, excretion, and reduction in toxicity
or other desired characteristic.
[0037] The linking group will be selected in light of the two
domains being linked, the available functionalities for linking,
the presence of functionalities that must be protected and
deprotected, the effect on the properties of the final product, and
the like. Illustrative linking groups include a bond, amino,
methylamino, succinoyl, maleoyl, pimeloyl, ethylenediamino,
glycine, 1,3-dihydroxypropylene, phenylenediamine, p-aminobenzoic
acid, glycinamido 4-aminobutyric acid,
bis(2-hydroxyethyl)phosphate, di(amino acids), such as
glycylglycine, tryptophanylalanine, methionylvaline, etc.,
.omega.-amino-aliphatic acids of from 3 to 10, usually 3 to 6,
carbon atoms, oxyalkylamino substituted aliphatic acids of from 2
to 10, usually 2 to 6, carbon atoms, such as
2'-aminoethoxy-3-propionic acid, 3'-aminopropoxyethoxycrotonic
acid, etc. See, FIG. 3, where a number of compounds are indicated,
with a targeting element for binding to the neurodegenerative
proteins and a recruitment element for binding to the prolyl
isomerase. The domains can be derived from compounds that are known
and have been shown to be physiologically benign. Using these known
compounds to provide the domains allows for the ready preparation
of compounds that will have physiologically acceptable
properties.
[0038] The compounds will be synthesized in accordance with known
protocols and reactions. Methods for modifying the various domain
compounds are well known in the literature and can be used
successfully, if necessary, to introduce functionalities, when
required. See, for example, U.S. Pat. No. 6,372,712.
[0039] A number of in vitro (including cultures) and in vivo tests
are known for evaluating activity of compounds in reversing
aggregation of the neurodegenerative proteins. In vitro studies
include transient transfection of cells (Dou, et al., 2003 PNAS
100, 721-6) with tau gene, use of anti-phosphorylated tau in an
immunoassay (Lu, et al., supra), surface plasmon resonance assay
(Cairo, et al., supra), cellular toxicity with PC-12 cells (Lowe,
et al., supra), and ThT fluorescence spectroscopy (Ghanta, et al.,
supra). Mouse models are described in Lee, supra, references cited
in Goedert, supra, references cited in Lee, et al., supra, and
references cited in Zoghbi and Orr, supra.
[0040] The subject compounds find use in investigating the etiology
of the different neurodegenerative diseases, the response of the
diseases to various compounds in conjunction with the subject
compounds, that substantially reduce the level of aggregation of
the individual proteins, and the response of the neurodegenerative
proteins to various proteins that are recruited by the subject
compounds to the aggregate. The subject compounds may also be used
with naturally occurring or synthetic compounds to evaluate their
propensity to induce aggregation formation. Where a prolyl
isomerase is recruited, one can investigate the effect of the
modification of the geometry of the proline on the stability of the
aggregates and the response to other drugs at a reduced level of
aggregation.
[0041] By using the subject protocols with conjugates having a
first domain binding to the neurodegenerative proteins,
particularly in their .beta.-sheet conformation, and a second
domain that recruits proteins, such as prolyl isomerases, one can
evaluate other drugs that affect the formation of the aggregates
and the neurotoxicity of such aggregates. One can also evaluate the
effect on the aggregate formation, as to time, fibril formation and
aggregate formation. In addition, one can use the subject compounds
to screen for other compounds that bind the aggregates, evaluating
the effect of the binding of such compounds on aggregate formation
and neurotoxicity. By using standardized tests, one can determine
the binding affinity of test compounds and their effect on
aggregation by providing a competition between an agent of the
subject invention, which can also serve as a standard, and such
test compound. By testing in the presence and absence of a prolyl
isomerase, and measuring the effect on aggregation and
neurotoxicity, the effect of the test compound on these two
indications can be determined.
[0042] In addition, the subject compounds can find use in the
treatment of neurodegenerative disorders, based on protein
aggregation. The subject compounds will generally have an IC50 of
less than about 10 uM, preferably less than about 1 uM and more
preferably less than about 50 nM. The subject compounds are capable
of being administered orally, by injection, parenterally,
intravascularly, intraperitoneally, intracranially, subcutaneously,
etc. Dosages will vary widely depending upon the mode of
administration, the activity of the subject compound, the status of
the patient, whether the subject compound is being administered
prophylactically or therapeutically, the frequency of
administration, the response of the patient to the drug and dosage,
the rate of distribution, excretion, metabolism and ability to
cross the blood brain barrier.
[0043] Administration may be by continuous infusion, slow release
formulations, a pump, bolus, etc. The administration may be daily
or more or less frequently, generally not less than about 1 time a
week, when the patient is under therapy. The dosage will be an
effective dosage administered in accordance with its effectiveness
to achieve the level of response desired from the patient, while
protecting the patient from detrimental side effects. Generally the
daily dosage will be less than 500 mg, usually less than about 100
mg, and preferably less than about 1 mg. Usually, the daily dosage
will be greater than about 0.01 mg. The dose will be regulated to
provide a blood level at a prophylactically or therapeutically
effective amount.
[0044] The subject compounds can be formulated in accordance with
known formulation techniques and compositions. These compositions
will include a prophylactically or therapeutically effective amount
of the active compound and a pharmaceutically acceptable carrier,
which may include a variety of additives as part of the carrier. As
used herein, the phrase "pharmaceutically acceptable carrier"
intends a non-toxic, usually inert, solid, semi-solid, or liquid
filler, diluent, encapsulating material, formulation, auxiliary of
any type, including a sterile aqueous medium, saline, etc.
[0045] Exemplary of such materials are sugars, such as lactose,
glucose and sucrose, starches, such as corn starch and potato
starch, cellulose and modified cellulose, such as sodium
carboxymethylcellulose, hydroxyethylcellulose, ethyl cellulose,
cellulose acetate, etc., powdered tragacanth, malt, gelatin, talc;
excipients, such as cocoa butter and suppository waxes, oils, such
as peanut oil, cottonseed oil, safflower oil, sesame oil, olive
oil, corn oil, and soybean oil, polyols, such as propylene glycol,
glycerin, sorbitol, mannitol and polyethylene glycol, esters such
as ethyl oleate and ethyl laurate, agar, buffering agents, such as
magnesium and aluminum hydroxide, and phosphate buffered saline,
alginic acid, Ringer's solution, ethyl alcohol, etc.
[0046] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
releasing agents, coating agents, sweetening agents, flavoring
agents, perfuming agents, preservatives and antioxidants may also
be included in the formulations. Examples of pharmaceutically
acceptable antioxidants include, but are not limited to, water
soluble antioxidants, such as ascorbic acid, cysteine
hydrochloride, sodium bisulfite, sodium metabisulfite, sodium
sulfite, etc.; oil soluble antioxidants, such as ascorbyl
palmitate, butylated hydroxyanisole, butylated hydroxytoluene,
lecithin, propyl gallate, alpha-tocopherol, etc.; and the metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid, sorbitol, tartaric acid, phosphoric acid, etc.
[0047] Liquid formulations for oral administration will include
pharmaceutically acceptable solutions, emulsions, microemulsions,
suspensions, syrups and elixirs, employing the agents described
previously.
[0048] Injectable formulations may be solutions or suspensions,
employing media described previously, such as water, Ringer's
solution, isotonic saline, bland fixed oils, such as mono- or
diglycerides, and may include fatty acids, e.g. oleic acid.
[0049] Depots of the drug may be employed with subcutaneous or
intramuscular injection. The injection will usually be a slow
release suspension of crystalline or amorphous material, where the
rate of dissolution of the suspension and/or transport of the drug
to the surface of the particles will determine the release rate of
the drug. Alternatively microencapsulation may be employed
employing biodegradable polymers, e.g. polylactide-polyglycolide,
polyorthoesters, and polyanhydrides.
[0050] Solid dosage forms for oral administration may include
capsules, tablets, pills, powders, gelcaps, and granules. The
active ingredient is mixed with at least one inert diluent, such as
sucrose, lactose or starch. Other components may include tableting
lubricants, extenders, release controlling coatings, etc. Soft- or
hard-filled gelatin capsules may be employed, using such excipients
as lactose or milk sugar, high molecular weight polyethylene
glycols, and the like.
[0051] In some instances transdermal administration may be
effective. Appropriate additives are provided for transferring the
subject compounds across the skin and into the blood stream.
Inhalation may also be used, where an aerosol of the active
ingredient is employed and the active ingredient crosses the lung
membrane.
[0052] The compositions of the subject invention may be
administered as the only active ingredient or in combination with
other compounds, such as dopamine, antiinflammatory agents, enzyme
inhibitors, blood brain barrier permeabilizing agents, and the
like.
[0053] The subject compounds can be used in vivo or in vitro to
inhibit aggregation in environments or compositions that result in
aggregation by enhancing localized concentrations of enzymes that
interact with the aggregation. As described above, in in vivo
situations, one can administer a compound according to this
invention that will bind to an aggregate and recruit the enzymes to
the site. In lysates or other complex mixture, particularly having
a variety of proteins, one can determine what domains may be used
to prevent one or more components of the mixture to form an
aggregate. Where the binding of the target domain is known, one can
enjoy the benefit of an endogenous protein or add the protein, if
necessary. In this way, where aggregation of components of a
mixture to be analyzed will interfere with the analysis, by adding
a compound according to this invention, the binding of the compound
to aggregate forming components at an early stage of aggregation
and recruitment of a target protein to the site of aggregation can
inhibit the formation of the aggregate.
[0054] For recruiting enzymes, one may use substrate or co-enzyme
mimics, compounds that bind to epitopes or other feature of the
enzyme, etc. Mimics may include for kinases AMP, .gamma.-SATP,
etc., for phosphatases, phosphoramide, thiophosphate,
thiophosphoramide, etc., for proteases, amine substitutes for
carboxamide, and compounds that bind to enzymes, such as SLF to
prolyl isomerases, etc. By recruiting enzymes that can modify the
monomers of the aggregate, aggregation may be impeded or the
monomers dispersed.
[0055] The following examples are intended to illustrate but not
limit the invention.
EXPERIMENTAL
[0056] Synthesis of C8-Congo Red. Agents of this invention were
synthesized using standard chemical procedures. EDC (13.7 mg;
0.0885 mmole; 5 eq), sulfa-NHS (20.6 mg; 0.0885 mmole; 5 eq), and
octanoic acid (2.55 uL; 0.0177 mmole; 1 equivalent) were dissolved
in 100 uL dimethyl furane (DMF). After stirring for 10 minutes at
room temperature, Congo red (16.8 mg; 0.0345 mmole; 2 equivalents)
was added in 100 uL of DMF. This mixture was stirred at room
temperature and monitored by thin layer chromatography (TLC) on
silica gel plates. After 60 minutes, the reaction was brought up in
methanol:ethyl acetate (10:90) and subjected to purification by
silica gel chromatography. Fractions were analyzed by TLC and
UV-visible spectroscopy. Pooled fractions were dried and a sample
analyzed by mass spectrometry.
[0057] Synthesis of SLF-Congo Red. EDC (13.7 mg; 0.0885 mmole; 5
eq), sulfo-NHS (20.6 mg; 0.0885 mmole; 5 eq), and SLF (10 mg;
0.0177 mmole; 8.8 mM final) were dissolved in 100 uL DMF. After
stirring for 10 minutes at room temperature, Congo red (16.8 mg;
0.0345 mmole; 2 equivalents) was added in 100 uL of DMF. This
mixture was stirred at room temperature and monitored by TLC on
silica gel plates. After 60 minutes, the reaction was brought up in
methanol:ethyl acetate (10:90) and subjected to purification by
silica gel chromatography. Fractions were analyzed by TLC and
UV-visible spectroscopy. Pooled fractions were dried and a sample
analyzed by mass spectrometry.
[0058] Fibrillization Procedure. Purified amyloid beta (1-42) was
purchased from AnaSpec and used without further purification.
Fibrillization conditions are based on those previously described
by Gordon and Meredith (Gordon, D. J. and Meredith, S. C. (2003)
Biochemistry 42:475-485). Briefly, peptide was dissolved in PBS to
a final concentration of 100 uM. To this solution, was added either
FKBP12 (to a final concentration of 1 uM) or an equivalent volume
of buffer alone (100 mM NaCl, 10 mM Tris-HCl pH 7.0). Additionally,
either agent (dissolved in DMF) or DMF alone was added. These
mixtures were placed at room temperature for at least 4 days
without disruption.
[0059] Thioflavin T assay. Assay conditions were based on those
previously described (Gordon, D. I. and Meredith, S. C. (2003)
Biochemistry 42:475-485). Briefly, a 5 uL solution, which was
derived from the fibrillization procedure above, was added to 200
uL of 5 uM thioflavin T in 50 mM glycine buffer pH 8.5. The mixture
was vortexed briefly and the fluorescence emission maximum
measured. For the dose dependence experiment, the emission
wavelength was recorded at 490 nm. Excitation was set at 446
nm.
[0060] Transmission Electron Microscopy (TEM). Electron microscopy
was performed essentially as previously described (Gordon, D. J.
and Meredith, S. C. (2003) Biochemistry 42:475-485). Briefly,
solutions subjected to the fibrillization conditions described
above were applied to Formvar coated 400-mesh glow-discharged
copper grids and contrasted with 1% uranyl acetate solution. Grids
were analyzed at 80 kV and a magnification of 100,000.times..
[0061] Luciferase assay. Neuronal culturing, transfection with
plasmid DNA, and luciferase assays were performed as previously
described (Graef et al 1999, supra).
[0062] Surface Plasmon Resonance. Amide coupling was used to
generate a carboxymethyldextran (CMS; BIAcore, Uppsala, Sweden)
sensor chip bearing a mockimmobilized surface on flow chamber 1
(Fc1), 950 RU of A.beta.(1-40) (AnaSpec, San Jose, Calif.) on Fc2,
and 2800 RU of bacterially-expressed recombinant FKBP12 on Fc3. The
regeneration conditions were as follows: for removing analyte from
A.beta. surfaces (4 M guanidine HCl, 10 mM Tris buffer pH 8.0) and
for removing analyte from the FKBP surface (750 mM NaCl, 250 mM
NaOH). To calculate K.sub.D1 and K.sub.D2 (.+-.SEM) for A.beta.
binding (in part C), the control signal was subtracted and data was
fit according to the method of Cairo et al, (Biochemistry 41,
8620-8629 (2002).
[0063] In each case, eight concentrations of analyte distributed
around the K.sub.D were injected in duplicate in the running buffer
(HBS-EP; BIAcore). Congo red is not uniquely specific for A.beta.
and, consistent with this, it also binds FKBP, but at a K.sub.D
20-fold higher than SLF-CR.
[0064] Light Scattering. Fresh A.beta.(1-42) (Anaspec, San Jose,
Calif.) was prepared by resuspending the peptide in 100 mM NaOH pH
9.5 to 10, sonicating for 5 minutes, and passing the resulting
solutions through a 10 kDa filter (YM-10; Millipore, Bedford,
Mass.) (31). Stock solutions (100 .mu.M in 2 mM NaOH) were stored
at -80.degree. C. and diluted to 25 uM with PBS pH 7.2 to initiate
fibrillogenesis. Identical methods were used to prepare the
A.beta.(1-42) solutions utilized for TEM, AFM, MTT, Tunel and
thioflavin T experiments. Turbidity was measured at 30.degree. C.
with orbital shaking in half area, nonbinding surface 96-well
plates (Corning) on a TECAN GENios plate reader. The total volume
was 50 uL and the concentration of FKBP was 1 uM. The results are
representative of two independent experiments each performed in
triplicate.
[0065] Thioflavin T. Compounds (CR or SLF-CR), FKBP (1 uM), and
A.beta. (25 uM) were incubated in a total volume of 10 .mu.L PBS pH
7.2 in black Corning 96-well plates. After a 96 hour incubation in
the dark at 22.degree. C., thioflavin T (200 uL of 5 .mu.M
thioflavin T in 50 mM glycine pH 8.5) was added. Fluorescence was
measured on a SpectraMax Gemini (Molecular Devices, Sunnyvale,
Calif.), using an excitation wavelength of 446 nm (.+-.5 nm) and an
emission of 490 nm (.+-.5 nm). The data was fit to sigmoidal curves
using DeltaGraph (DeltaPoint, Inc) and the IC.sub.50 for each
treatment recorded. The percent aggregation is arbitrarily defined
as 0% by the fluorescence of a solution of thioflavin T to which no
A.beta. has been added (Em.sub.490nm=3.3) and for 100% by the
fluorescence of a solution containing A.beta. but no inhibitors
(Em.sub.490nm=42). A linear relationship between thioflavin T
fluorescence and percent aggregation was used for simplicity.
[0066] Transmission Electron Microscopy. TEM was conducted using
300 mesh formvar-coated copper grids and 1% uranyl acetate stain on
a JEOL TEM1230 and imaged with a Gatan 967 slow-scan. CCD at 80 kV.
Samples were incubated for 4 days at 22.degree. C. in PBS pH 7.2
containing 25 uM A.beta.(1-42), 1 .mu.M FKBP and 1 uM drug, where
appropriate. The protocol used to prepare A.beta. can influence
fibrillogenesis and the reproducibility of the assays. Therefore,
it can be useful to examine A.beta. aggregation under a variety of
conditions. In addition to the hydroxide method used above, stock
solutions of A.beta. were also prepared using a trifluoroacetic
acid (TFA) protocol (Crystal, et al., J. Neurochem. 86, 1359-1368
(2003). TEM on samples originated from these stock solutions
yielded similar results, although the typical size of the fibrils
was considerably shorter.
[0067] Atomic Force Microscopy. Samples for AFM were prepared as
for TEM, above, applied to a clean silicon surface, washed twice
with dH.sub.2O, dried, and imaged at a scan rate of 1 Hz using a
silicon probe (TESP) on an AFM Nanoscope Dimension 3000 (Digital
Instruments) in Tapping Mode (C. Goldsbury, et al, J. Mol. Biol.
285, 33-39 (1999). The dimensions of the globular aggregates in the
samples treated with SLF-CR/FKBP were approximated using NIH
Image.
[0068] Cell Viability Assay. Rat hippocampal (E19.5 and P0) neurons
were prepared as previously described (Graef, et al., Nature 401,
703-708 (1999). Fresh A.beta.(1-42) (100 uM) was prepared under
basic conditions and incubated with inhibitors for two days at
22.degree. C. in PBS pH 7.2 and then diluted 1:4 with culture media
and applied to neurons. MTT assays were conducted as per
manufacturer's specifications after two additional days of
incubation at 37.degree. C. and 5% CO.sub.2. The inhibitor
concentrations are the in vitro incubation values.
[0069] Immunofluorescence Microscopy. Rat hippocampal neurons (P0)
were cultured 10 days on Matrigel-coated glass coverslips
(Deckglaser, Germany) in 24-well plates (Corning) at 37.degree. C.
and 5% CO.sub.2. A.beta. solutions were prepared and applied as
above. The final concentration of A.beta. was 25 uM and drug and
FKBP were present at 1 uM. Following a two-day incubation, cells
were prepared for microscopy as described (R. Kayed, et al.,
Science 300, 486-489 (2003).
[0070] The results from the assays indicated above are as
follows:
RESULTS
[0071] As a first measure of SLF-CR function, we examined the
binding of the compound to immobilized A.beta.(1-40) by surface
plasmon resonance. We found that the affinity of SLF-CR for A.beta.
is not significantly different from that of CR, which suggests that
the conjugation of SLF to Congo red does not dramatically interfere
with binding. Similarly, unmodified SLF and SLF-CR bind immobilized
FKBP with comparable affinity. These results confirmed that the
targeting and recruiting domains of SLF-CR retain affinity for
their respective targets.
[0072] To test the capacity of SLF-CR to simultaneously bind
A.beta. and FKBP, the bifunctional compound was pre-incubated with
FKBP and the resulting solutions passed over immobilized amyloid.
In surface plasmon resonance, the response (in arbitrary units,
RUs) is proportional to the mass of material assembled on the
surface. We used this characteristic to determine whether SLF-CR
could form a ternary complex with FKBP and immobilized A.beta..
Consistent with this model, the combination of SLF-CR and FKBP gave
a greater response (.about.230 RUs) than SLF-CR alone (.about.140
RUs). While interpretations of the efficiency of complex formation
are prevented by a lack of detailed information regarding the
binding site(s) of Congo red on A.beta., this result suggests that
SLF-CR can trigger formation of the FKBP/drug/A.beta. ternary
complex. Recruitment of FKBP to A.beta. by SLF-CR, but not CR, was
also confirmed by electron microscopy.
[0073] To assess the impact of chaperone recruitment on the potency
of aggregation inhibitors, the aggregation-prone 42-amino acid
version of A.beta.(A.beta.(1-42)) was incubated in the presence of
drug and FKBP. A.beta. fibrils scatter light; therefore, the
turbidity of A.beta. solutions can be used to follow
fibrillogenesis in real time. These experiments revealed that
SLF-CR/FKBP at 10 uM completely blocked the formation of A.beta.
aggregates capable of scattering light. Full inhibition by SLF-CR
required FKBP, but FKBP alone did not interfere with A.beta.
aggregation. CR was a modest inhibitor of turbidity and FKBP was
unable to significantly alter its potency. Even at nanomolar
concentrations, SLF-CR/FKBP (IC.sub.50SLF-CR 0.43 uM) delayed
amyloid aggregation.
[0074] As a second measure of inhibition, we explored the
aggregation of A.beta. by the well-established thioflavin T assay.
The fluorescence of thioflavin T at 490 nm dramatically increases
in the presence of A.beta. aggregates, which makes this an
attractive assay for screening inhibitors. Similar to the results
obtained by light scattering measurements, we found that the
IC.sub.50 of SLF-CR/FKBP was approximately 5-fold lower than that
of CR/FKBP for lowering the concentration of thioflavin-T-binding
species. Moreover, at 10 uM drug, only the combination of SLF-CR
and FKBP was capable of completely preventing thioflavin T
reactivity (i.e. the fluorescence of the sample is equivalent to
that of A.beta.-free solutions of thioflavin T). Between 20 and 35%
of aggregates remained after similar treatment (10 uM) with other
combinations and, even at the highest drug concentrations, CR
failed to fully block thioflavin T fluorescence. Importantly, FKBP
was required for enhanced potency of SLF-CR; in the absence of
FKBP, SLF-CR and CR had similar activity. The efficacy of CR was
not, however, influenced by the addition of FKBP. Increasing the
molar equivalents of FKBP further enhanced the potency of SLF-CR.
These results support that drug-mediated recruitment of FKBP to
A.beta. is required for enhanced potency.
[0075] To independently assess fibril formation, we explored the
ultrastructure of A.beta. aggregates after drug treatments by
direct imaging methods, both transmission electron microscopy (TEM)
and atomic force microscopy (AFM). Using TEM, we confirmed that
FKBP does not influence the formation or architecture of A.beta.
fibrils. At a concentration of 1 uM, CR, SLF-CR, and CR/FKBP were
ineffective at reducing the size or abundance of A.beta. fibrils.
However, consistent with the thioflavin T and turbidity
experiments, SLF-CR/FKBP almost fully blocked fibril formation.
Occasional fibrils remained in these samples and these tended to be
structurally similar to those seen in untreated samples. These rare
fibrils suggest that some A.beta. escapes inhibition and that this
peptide aggregates normally.
[0076] Recent evidence has strongly suggested a role for
nonfibrillar A.beta. aggregates in the pathology of Alzheimer's
disease. Blocking fibril formation at a stage that leads to
accumulation of these highly neurotoxic intermediates would likely
have deleterious effects. Preventing formation of these
intermediates is therefore an important measure of inhibitory
potency. To gain a more detailed understanding of the function of
SLF-CR/FKBP, we examined drug-treated mixtures by AFM, which
provides three-dimensional information about A.beta. aggregates.
Consistent with the results from TEM, the samples containing only
A.beta.(1-42) and FKBP contained a meshwork of elongated fibrils.
In contrast, the samples that were additionally treated with SLF-CR
failed to form fibrils. Rather, smaller and less elongated species
were observed. It appears that these likely represent an
intermediate that is trapped prior to formation of A.beta. fibrils.
The particles are approximately uniform in size (28.+-.5 nm.sup.2;
N=50), which suggests that SLFCR/FKBP interrupts fibril formation
by acting at a discrete step in the aggregation pathway. Because
these species are not observed in samples treated with CR/FKBP, it
appears that they represent a qualitative difference between the
mechanism of action of CR and SLF-CR/FKBP.
[0077] While a strategy that interrupts the aggregation process
provides mechanistic insight, the most important criterion of
therapeutic potential is the capacity to prevent the formation of
neurotoxic A.beta. aggregates. Hence, we examined whether the
bifunctional drugs could inhibit neurotoxicity of in
vitro-aggregated A.beta. on primary neurons. Cell viability of P0
rat hippocampal neurons was assessed with the
3-(4,5-dimethyl-2-thiazoyl)-2,5 diphenyltetrazoliumbromide (MTT)
reduction assay. SLF-CR/FKBP-treated A.beta. samples were
substantially less toxic than untreated- or CR/FKBP-treated
samples. Specifically, SLF-CR/FKBP displayed an EC.sub.50
approximately 4-fold better than CR/FKBP (0.9.+-.1.3 uM and
4.2.+-.1.4 uM, respectively). SLF-CR's ability to prevent A.beta.
induced neuronal death was dependent on the concentration of FKBP.
These findings support the conclusion that drug mediated
recruitment of FKBP not only blocks aggregation but also inhibits
A.beta. toxicity.
[0078] To examine the morphology of the treated neurons, we applied
A.beta. and drug/protein combinations to cultured hippocampal
neurons. Cell death was measured after two days by counting the
Tunel positive nuclei. We assessed cell morphology by staining with
anti-.beta.-III tubulin antibody. As expected, the potent toxin,
camptothecin, induced cell death (36% of the remaining cells were
positive for Tunel) as well as abnormal cell morphology. Treatment
with A.beta. yielded a similar result; neurites became severely
dystrophic, some neurons detached from the slides, and those
remaining demonstrated signs of nuclear fragmentation and membrane
blebbing. Incubation of A.beta. with FKBP, CR, CR/FKBP, or SLF-CR
prior to addition to the cells failed to substantially reduce the
number of Tunel positive cells or prevent changes in cell
morphology. The SLF-CR/FKBP combination, however, markedly blocked
toxicity. Moreover, cells that were protected by SLFCR/FKBP
treatment display mostly normal nuclear and axonal morphology.
These studies support the MTT results and demonstrate that FKBP
recruitment can protect cultured neurons from cell death triggered
by toxic aggregates of A.beta..
[0079] The subject strategy is designed to allow A.beta.-bound
bifunctional drugs to wield the steric bulk of FKBP and, thus,
prevent nearby A.beta. from joining the nascent fibril. A
prediction of this hypothesis is that improved coverage of the
A.beta. surface might provide superior inhibition. This might be
achieved by altering the orientation or steric arrangement of FKBP
relative to the A.beta. surface. Our synthetic strategy permits the
assembly of bifunctional compounds from a collection of modular
targeting domains, recruiting domains, and interchangeable linkers.
Therefore, we envisioned that, by installing linkers that vary in
length and flexibility, we might identify compounds that permit
FKBP to scan the A.beta. surface for favorable arrangements (FIG.
10A).
[0080] The methods used to generate the compounds were similar to
those used to create SLF-CR by EDC/NHS methods and purified by
silica gel chromatography. The resulting SLFs with installed
linkers were coupled to CR, purified and characterized as above. In
a search for more potent inhibitors, we generated a series of
bifunctional compounds that vary in the linker employed (FIG. 10B).
These molecules are named according to the reagent used to create
the linker (i.e. the amino acid glycine was used to generate
SLF-Gly-CR). When we tested these compounds in conjunction with
FKBP in the thioflavin T assay, we found that SLF-But-CR/FKBP and
SLF-Benz-CR/FKBP are potent inhibitors (FIG. 10C). The most active
compound, SLFBenz-CR/FKBP, has an IC.sub.50 of approximately 50 nM.
This value is 40-fold better than CR/FKBP and a 6-fold improvement
over the parent combination, SLF-CR/FKBP. Like SLF-CR, the potency
of the compounds was dependent on the availability of FKBP.
Interestingly, TEM revealed that the size and shape of the
intermediates formed in A.beta. samples treated with the
bifunctional molecules were similar (FIG. 10D). This result
suggests that, regardless of the properties of the linker, a common
FKBP/drug/A.beta. complex is formed.
[0081] To explore the role of the linker in reduction of A.beta.
toxicity, we measured primary neuron viability in response to
pre-treated A.beta. samples. Consistent with the rank-order as
measured by thioflavin T fluorescence, SLF-Benz-CR/FKBP and
SLF-But-CR/FKBP were, at nanomolar concentrations, significantly
more potent than CR/FKBP or SLF-CR/FKBP (FIG. 10E). Even at the
high molar concentrations of A.beta. used in these experiments, in
which most neurons are killed and CR is unable to rescue them,
SLF-Benz-CR remained active at 100 nM. Despite the modest number of
compounds in the linker series, these results indicate that potent
inhibitors of toxicity can be uncovered by the combinatorial
assembly of modular components.
[0082] Our results indicate that recruitment of chaperones can
block A.beta. fibril formation and substantially reduce A.beta.
toxicity. While other inhibitors of A.beta. aggregation, such as CR
and short peptides, are active in the 2000 to 10000 nM range, our
best compound is potent at 50 nM. The advantage of therapeutic
intervention at the aggregation step is that it targets a purely
pathological event in disease development. Thus, directly
inhibiting A.beta. aggregation with the recruited chaperone
approach can provide a viable complement to recent efforts to
reduce the rate of A.beta. release (Citron, Neurobiol Aging 2002
23, 1017-22; Michaelis, J Pharmacol Exp Therapeutics 2003, 304,
897-904), enhance its clearance (Janus, et al., Biochim Biophys
Acta 2000, 1502, 63-75; Kayed, et al., Science 2003, 300, 486-9)
and/or template non-toxic aggregates (Ghanta, et al., J Biol Chem
1996, 271, 29525-8; Sacchettini and Kelly, Nature Rev Drug
Discovery 2002, 1. 267-75; Cohen and Kelly, Nature 2003, 426,
905-9).
[0083] It is evident from the above results that the subject
compounds and methods provide for a robust ability to investigate
the manner of aggregation and toxicity of neurodegenerative
proteins. One can also use the subject compounds as standards in
competitive assays, using the conjugate in the presence and absence
of the disaggregating element, to evaluate test compounds as to
their competitiveness in binding and disassociating aggregates, the
effect on the formation of aggregation and their effect on
neurotoxicity. The subject agents can be prepared from known
compounds whose physiology are known and can serve in test animals
as to their effect, as well as providing a basis for therapies for
the various neurodegenerative diseases.
[0084] In addition, the subject strategy provides for compounds
that can be used in various situations or contexts where
aggregation formation is detrimental to the situation. With complex
mixtures, such as lysates, aggregation formation can interfere with
assaying the mixture for one or more components, obscure the signal
from the mixture or the like. By using the subject strategy, small
molecules can be added to the mixture and either endogenous or
exogenous proteins recruited to impede or diminish the presence of
aggregates. The subject strategy also provides for the recruitment
of enzymes to an aggregate, where one is interested in modifying
the aggregation as a result of enzymatic activity. As demonstrated
with the A.beta. aggregates, recruitment of prolyl isomerase can
act to modify the prolyl groups of the members of the aggregate
with apparent modification of the presence of aggregates.
[0085] All references referred to in the text are incorporated
herein by reference as if fully set forth herein. The relevant
portions associated with this document will be evident to those of
skill in the art. Any discrepancies between this application and
such reference will be resolved in favor of the view set forth in
this application.
[0086] Although the invention has been described with reference to
the above examples, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
315PRTArtificial Sequencesynthetic peptide 1Lys Leu Val Phe Phe1
5242PRTArtificial Sequencesynthetic peptide 2Asp Ala Glu Phe Arg
His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe
Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met
Val Gly Gly Val Val Ile Ala 35 40340PRTArtificial Sequencesynthetic
peptide 3Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His
Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly
Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val 35 40
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