Title: Time-resolved fluorescence assay for the detection of multimeric forms of A-beta 1-40
Abstract: The present invention provides for a polypeptide aggregation screening assay for the purpose of detecting modulators of polypeptide aggregation, which can provide for new therapies in pathologic states associated with polypeptide aggregation, especially considered is Alzheimer's Disease.
Patent Number: 6,846,640 Issued on 01/25/2005 to Peach, et al.
| Inventors:
|
Peach; Matthew L. (Portage, MI);
Laborde; Alice L. (Kalamazoo, MI)
|
| Assignee:
|
Pharmacia & Upjohn Company (Kalamazoo, MI)
|
| Appl. No.:
|
135592 |
| Filed:
|
April 30, 2002 |
| Current U.S. Class: |
435/7.1; 435/7.2 |
| Intern'l Class: |
G01N 033/53; G01N033/533 |
| Field of Search: |
435/7.1,7.2,500
|
References Cited [Referenced By]
U.S. Patent Documents
| 5721106 | Feb., 1998 | Maggio et al.
| |
| 5817626 | Oct., 1998 | Findeis et al.
| |
| 5837473 | Nov., 1998 | Maggio et al.
| |
| 5854215 | Dec., 1998 | Findeis et al.
| |
| 6319498 | Nov., 2001 | Findeis et al.
| |
| Foreign Patent Documents |
| WO 98/08868 | Mar., 1998 | WO.
| |
| WO 99/06545 | Feb., 1999 | WO.
| |
| WO 99/06838 | Feb., 1999 | WO.
| |
Other References
Ross (Aug. 29, 2002) "Polyglutamine pathogenesis: emergence of unifying
mechanisms for Huntington's diseases and related disorders." Neuron
35(5):819-822.*
Tobin & Signer (Dec. 2000) "Huntington's disease: the challenge for cell
biologists." Trends Cell Biol. 10(12):531-536.*
Sipe (1992) "Amyloidosis" Annu. Rev. Biochem. 61: 947-975.*
Akikusa et al., "Practical assay and molecular mechanism of aggregation
inhibitors of .beta.-amyloid," J. Pept. Res., 61:1-6 (2003).
Hetenyi et al., "Pentapeptide Amides Interfere with the Aggregation of
.beta.-Amyloid Peptide of Alzheimer's Disease," Biochemical and
Biophysical Research Communications, 292:931-936 (2002).
Howlett et al., "Inhibition of fibril formation in .beta.-amyloid peptide
by a novel series of benzofurans," Biochem. J., 340:283-280 (1999).
Hughes et al., ".beta.2-macroglobulin associates with .beta.-amyloid
peptide and prevents fibril formation," Proc. Natl. Acad. Sci. USA,
95:3275-3280 (1998).
Watanabe et al., "Inhibitors of Fibril Formation and Cytotoxicity of
.beta.-Amyloid Peptide Composed of KLVFF Recognition Element and Flexible
Hydrophilic Disrupting Element," Biochemical and Biophysical Research
Communications, 290:121-124 (2002).
Allsop et al., Biochem. Biophy. Res. Comm., 285:58-63 (2001).
Azriel et al., J. Biol. Chem., 276:34156-161 (2001).
Berthelier et al., Anal. Biochem., 295:227-236 (2001).
Cane et al., Science, 282:63-68 (1998).
Citron et al., Nature, 360:672-674 (1992).
Davies P., Annals of the N.Y. Acad. of Sci., 924:8-16 (2000).
Findeis et al., Biochemistry, 38:6791-6800 (1999).
Haas et al., Nature, 359:322-325 (1992).
Howlett et al., FEBS Letters, 417:249-251 (1997).
Iwatsubo et al., Neuron., 13:45 (1994).
Kang et al., Nature, 325:733-736 (1987).
Kitaguchi et al., Nature, 331:530-532 (1988).
Lannfelt et al., Neurosci. Lett., 153:85-87 (1993).
Lowe et al., Biochemistry, 40:7882-89 (2001).
Myers, Curr. Opin. Biotechnol., 8:701-707 (1997).
Perutz and Windle, Nature, 412:143-144 (2001).
Ponte et al., Nature, 331:525-527 (1988).
Schenk et al., Nature, 400:173-177 (1999).
Seubert et al., Nature, 359:325-327 (1992).
Tanzi et al., Nature, 331:528-530 (1988).
Yan et al., Nature, 402:533-537 (1999).
|
Primary Examiner: Kemmerer; Elizabeth
Assistant Examiner: Nichols; Christopher James
Attorney, Agent or Firm: Pharmacia & Upjohn Company, Wootton; Thomas A.
Claims
We claim:
1. A method for identifying modulators of .beta.-amyloid aggregation
comprising:
i) providing a substrate attached to streptavidin, wherein the streptavidin
binds with biotin;
ii) providing a polypeptide composition comprising .beta.-amyloid monomers,
wherein at least some of the .beta.-amyloid monomers are modified to
comprise biotin;
iii) incubating the polypeptide composition in the presence and absence of
a test agent, under conditions in which the .beta.-amyloid monomers
aggregate into multimers;
iv) contacting the polypeptide composition to the substrate attached to the
streptavidin under conditions wherein biotin binds to streptavidin;
whereby .beta.-amyloid or multimers that comprise biotin bind to the
streptavidin of the substrate;
v) contacting the substrate with a detecting agent comprising the
streptavidin, wherein the detecting agent binds to biotin on
.beta.-amyloid aggregates that are bound to the substrate; and
vi) measuring .beta.-amyloid aggregation by detecting the detecting agent
bound to .beta.-amyloid aggregates bound to the substrate, wherein
differential .beta.-amyloid aggregation measurements in the presence
versus the absence of a test agent identifies the test agent as a
modulator of .beta.-amyloid aggregation.
2. A method according to claim 1 wherein the monomers are selected from the
group consisting of .beta.-amyloid 1-40, .beta.-amyloid 1-42,
.beta.-amyloid 1-43, and mixtures thereof.
3. A method according to claim 1 wherein the .beta.-amyloid aggregation
correlates with a pathologic disease state.
4. A method according to claim 3 wherein the pathologic state is a member
of the group consisting of Alzheimer's Disease and Down's syndrome.
5. A method for identifying modulators of polyglutamine repeat containing
protein aggregation comprising:
i) providing a substrate attached to streptavidin, wherein the streptavidin
binds with biotin;
ii) providing a polypeptide composition comprising polyglutamine repeat
containing protein monomers, wherein at least some of the polyglutamine
repeat containing protein monomers are modified to comprise biotin;
iii) incubating the polypeptide composition in the presence and absence of
a test agent, under conditions in which the polyglutamine repeat
containing protein monomers aggregate into multimers;
iv) contacting the polypeptide composition to the substrate attached to the
streptavidin under conditions wherein biotin binds to streptavidin;
whereby polyglutamine repeat containing protein or multimers that comprise
biotin bind to the streptavidin of the substrate;
v) contacting the substrate with a detecting agent comprising the
streptavidin, wherein the detecting agent binds to biotin on polyglutamine
repeat containing protein aggregates that are bound to the substrate; and
vi) measuring polyglutamine repeat containing protein aggregation by
detecting the detecting agent bound to polyglutamine repeat containing
protein aggregates bound to the substrate, wherein differential
polyglutamine repeat containing protein aggregation measurements in the
presence versus the absence of a test agent identifies the test agent as a
modulator of polyglutamine repeat containing protein aggregation.
6. A method according to claim 5 wherein aggregation of the polyglutamine
containing polypeptide correlates with a pathologic disease state.
7. A method according to claim 6 wherein the pathologic state is a member
of the group consisting of Huntington's disease and spinal bulbar muscular
atrophy.
Description
FIELD OF THE INVENTION
The present invention relates to methods of detection of modulators of
polypeptide aggregation observed in many human disease states.
BACKGROUND OF THE INVENTION
A number of serious diseases affecting the human population can be closely
associated with the improper aggregation of polypeptide fragments and
characterized by aggregate deposition in lesions or plaques often
resulting in abnormal physiological function at the plaque site. For
instance, a stretch of polyglutamine repeats in a particular protein has
been proposed as the cause for many neurological disorders including
Huntington's disease and spinal bulbar muscular atrophy, while aggregation
of other unrelated proteins are cited as the causes of prion disease (PrP
aggregation), Parkinson's disease and amyotrophic lateral sclerosis (ALS)
(.alpha.-synuclein aggregation), dialysis-related amyloidosis (.beta.-2
microglobulin aggregation), corneal dystrophy (kerato-epithelial
deposits), and aggregation of islet amyloid polypeptide in more than 95%
of type II diabetes. Of particular interest is the aggregation of
.beta.-amyloid polypeptides in Alzheimer's Disease.
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by
the gradual decline of cognitive function and memory loss associated with
amyloid plaque formation, neurofibrillary tangles, and associated neuronal
toxicity. The initiation and onset of Alzheimer's disease is believed to
have both genetic origins as well as sporadic, environmentally influenced
onset (Davies P., Annals of the N.Y. Acad Of Sci. 924: 8-16.2000). Several
genetic mutations have been characterized to date that are associated with
familial onset AD. Mutations which are significantly involved in AD onset
have been found in the Amyloid Precursor Protein (APP) itself, and in
either the presenilin-1 or presenilin-2 genes. However, the common
denominator in all of these mutations is the formation of protein, or
senile plaques derived from a cleavage fragment, designated amyloid beta
(A.beta.), of the APP molecule which is deposited in the brain of affected
individuals, and results in toxicity and death of neuronal cells. This
phenomenon of plaque formation can also be detected in another genetic
defect not directly related to AD, the trisomy of chromosome 21 involved
in Down's syndrome.
Amyloid precusor protein (APP), a single transmembrane glycoprotein
possessing a large cytoplasmic domain and a short intracellular C-terminal
region, occurs naturally as several splice forms of either 751 amino acids
(Ponte et al., Nature 331: 525-527. 1988; Tanzi et al., Nature 331:
528-530. 1988), 770 amino acids (Kitaguchi et. al., Nature 331: 530-532.
1988), or 695 amino acids (Kang et. al., Nature 325: 733-736. 1987),
designated as "normal" APP. The APP695 variant is more widely expressed in
neurons.
Amyloid precursor protein cleavage takes place through a series of
enzymatic reactions mediated by the .alpha., .beta., and .gamma.
secretases. The .alpha.-secretase cleaves .about.12 residues N-terminal of
the transmembrane domain of APP, at approximately residue 687 of APP770,
generating the large soluble protein s-APP.alpha., which is nonpathogenic,
and a C-terminal fragment of 83 amino acids. The .gamma.-secretase cleaves
after amino acid 711 or 712 in the C-terminal end to create a free peptide
termed p3. Alternatively, APP can be cleaved N-terminal to the
transmembrane domain before aspartyl residue 672 by .beta.-secretase
enzyme, a member of the aspartyl protease family of enzymes (Yan et al.,
Nature 402: 533-537. 1999), forming a truncated verison of sAPP.alpha.,
referred to as sAPP.beta.. The remaining C-terminal fragment can also be
cleaved by a .gamma.-secretase, near residue 711, giving rise to soluble
A.beta. peptide. The .gamma.-secretase generates an A.beta. peptide of
either 1-39, 1-40, 1-42, or 1-43 amino acids (where position 1 immediately
follows the .beta.-secretase cleavage site) depending upon its cleavage
site. A.beta.1-40 is the more abundant cleavage product produced by most
cell types.
In addition to the formation of senile plaques by A.beta. fragments,
A.beta. deposition can also be detected in nonfibrillar, granular
associations termed diffuse plaques (Tagliavini et al., Neurosci. Lett.
93: 191). Diffuse A.beta. plaques are detectable in brains of normal,
healthy individuals, while very few senile, amydyloidogenic plaques are
detected in the brains of non-AD affected individuals. Antibody staining
against A.beta. peptide revealed that the diffuse plaques are composed
primarily of the highly amyloidogenic A.beta.1-42 (Iwatsubo et al., Neuron
13:45. 1994) while senile plaques contain a mixture of both A.beta.1-42
and A.beta.1-40 peptides.
Several mutations of amyloid precursor protein that result in increased
cleavage of APP into A.beta. peptides have been characterized. The
"Swedish" mutation, at amino acid residues KM 670/671.fwdarw.NL (of
APP770), enhances the production of both A.beta.1-40 and A.beta.1-42
(Citron et al., Nature 360:6724. 1992; Lannfelt et al., Neurosci Lett 153:
85-7. 1993). The "London" mutation at residue 717, V.fwdarw.I, G, or F,
also results in increased production of A.beta. peptide fragments (Schenk
et al., Nature 400: 173-177. 1999). Several other mutations have been
identified, all of which cluster around one of the three secretase
cleavage sites in APP, all leading to increased A.beta. cleavage.
Many of the therapies contemplated for the treatment of AD target the
formation of A.beta. peptides by secretase enzyme activity, particularly
.beta. and .gamma. secretases involved in the cleavage of APP into A.beta.
peptides. Cleavage of APP into A.beta. is a natural enzymatic reaction
that generates A.beta. peptides in areas not associated with the neuronal
damage such as basement membrane and arterioles and venules, and areas of
the brain not associated with AD pathology. These deposits of A.beta. are
generally diffuse in nature rather than fibrillary, and A.beta. is
constitutively secreted by cells throughout life and is found in the
cerebrospinal fluid and plasma of all normal individuals (Haas et al.,
Nature 359: 322-5. 1992; Seubert et al., Nature 359: 325-7. 1992). These
and other data suggest that A.beta. aggregation (as opposed to A.beta.
formation) represents another target for therapeutic intervention.
One recent approach for therapeutic intervention into Alzheimer's Disease
and other diseases associated with polypeptide aggregation is treatment
with agents that inhibit the nucleation/aggregation of polypeptides.
Several screening assays are currently available for the detection of
aggregating proteins or aggregating polypeptides involved in various
debilitating human diseases.
A unique method for detecting aggregation of proteins is termed Time
Resolved Anisotrpy Measurements (TRAMS) (Allsop et al, Biochem. Biophy.
Res. Comm. 285: 58-63), which measures the movement of fluorescent
particles in solution. TRAMS require a mixture of a fluorescently-labeled
peptide and non-labeled peptide which are then mixed to the desired
concentration, and anisotropy measurements taken over a course of time
points. For this particular assay a modified single photon counter with a
light emitting diode with a repetition rate of 1 MHz is used for measuring
the light emission spectra. Slower movement of the fluorescing particles
over time correlates with an increasing number of aggregates. While the
TRAMS assay may measure the initial steps in protein aggregation, it is a
very complex method of detecting peptide complexes which requires
equipment not readily available and involves difficult interpretation of
the data.
A scintillation proximity assay (SPA) can also be used to assess
aggregation of .beta.-amyloid polypeptides. In the SPA method, three
species of .beta.-amyloid.sub.1-40 are employed, an unlabelled
.beta.-amyloid, biotinylated-.beta.-amyloid, and [.sup.125 I]-labeled
.beta.-amyloid. A mixture of the three types of peptides are allowed to
aggregate at 37.degree. C. for 4 hrs. At this time, 1 mg of streptavidin
coated SPA beads (Amersham) are added to the mixture and allowed to
incubate for several hours at 37.degree. C, with measurement of .sup.125 I
incorporation (into the beads) taken at varying timepoints. To carry out
this protocol, large amounts of .beta.1-40 are needed per assay and each
assay requires a large amount of time to complete. Thus, this type of
assay does not have the high throughput ability needed in the
pharmaceutical industry, as well as using potentially hazardous reagents
to carry out the protocol.
In the standard Enzyme-Linked Immunosorbant Assay (ELISA) protocol outlined
for the detection of .alpha.-amyloid aggregation (Howlett et al., FEBS
Letters, 417: 249-251. 1997), a polystyrene microtitre plate is coated
with a monoclonal antibody to the .beta.-amyloid peptide (e.g. antibody
6E10, Senetek, Napa, Calif.). In a separate microtitre plate,
.beta.-amyloid is diluted to a desired concentration in an appropriate
buffer and allowed to aggregate overnight in the presence or absence of
test compound. After the 24 hr incubation, the aggregation mixture is
transferred from the microtitre plate to the p-amyloid antibody coated
plate and allowed to bind to antibody. A second, biotinylated 6E10
antibody is then added to the assay plate to bind .beta.-amyloid
aggregates. The secondary 6E10 will only be bound if there are
.beta.-amyloid molecules present in the assay well bound to other
p-amyloid peptides but not to the primary antibody. For detection of the
bound biotinylated antibody, Eu3+ labeled streptavidin is added to the
wells and detected by excitation at the appropriate wavelength. The amount
of Eu3+fluorescence detected will decrease with inhibition of
.beta.-amyloid aggregation by the test compound.
While this method is useful in detecting aggregation of .beta.-amyloid
peptides, the requirement for peptide-specific monoclonal antibodies
limits this assay to availability of the particular antibody and the
specificity and binding affinity of the antibody for the peptide.
Berthelier et al, in Anal. Biochem. 295: 227-36. 2001, describe an assay
for the detection of polyglutamine aggregate extension where microtiter
plate wells are coated with pre-formed polyglutamine aggregates to which
are added additional biotinylated-polyglutamine peptides. The rate of
incorporation of these newly added peptides is measured using
Eu3+labeled-streptavidin to detect bound biotin molecules, corresponding
to integrated polyglutamine. This assay, however, does not address whether
a test compound affects the prevention of aggregation, or the nucleation
event, but rather only provides compounds which modulate continuing
polypeptide aggregation.
To this end, Perutz and Windle state in Nature 412: 143-44. 2001, "For any
process that occurs on a timescale of years, the controlling step will be
nucleation, not growth, and it will occur at random intervals of time."
Thus, there exists a need in the art for improved materials and methods
that address the drawbacks of existing protocols designed to detect
polypeptide aggregation, and in doing so expedites development of new
therapies and reduces the cost of development.
SUMMARY OF THE INVENTION
The present invention relates to materials and methods for determining
causes for the initial aggregation, or nucleation, of .beta.-amyloid and
other disease inducing aggregating polypeptides, and for delineation of
compounds and additional therapies that specifically target polypeptide
aggregation and plaque formation in several human disease states. The
present invention provides an improved method for identifying compounds
which modulate the aggregation of polypeptides, such compounds will be
useful in developing therapies for many human disease states characterized
by the deleterious aggregation of polypeptides.
For example, the present invention provides a method for identifying
modulators of polypeptide aggregation wherein the method comprises: i)
providing a substrate attached to a first binding partner, wherein the
first binding partner binds with a second binding partner, ii) providing a
polypeptide composition comprising polypeptide monomers, wherein at least
some of the monomers are modified to comprise to the second binding
partner, iii) incubating the polypeptide composition in the presence and
absence of a test agent, under conditions in which the polypeptide
monomers aggregate into multimers, iv) contacting the polypeptide
composition the substrate attached to the first binding partner and v)
measuring polypeptide aggregation by detecting polypeptide aggregates
bound to the substrate, wherein the detecting comprises measuring the
second binding partner, and wherein differential polypeptide aggregation
in the presence versus the absence of a test agent identifies the test
agent as a modulator compound.
The method can be carried out wherein the incubating step is done either
prior to or concurrent with the contacting step.
The first and second binding partners of the invention are molecular pairs
that show high binding affinity and specificity for each other and bind to
each other very rapidly (relative to the rate at which polypeptide
aggregation occurs). At least one must be attachable to a solid substrate
and at least one must be attachable to the polypeptide. Exemplary classes
of binding partners include high affinity binding small molecules; peptide
tags and monoclonal antibodies thereto; fluorophores and monoclonal
antibodies thereto; and enzymes and substrates. Exemplary pairs include
strepavidin/biotin, anti-Histidine.sub.6 (His.sub.6 -tag)
antibodies/His.sub.6 peptide tags, anti-biotin/biotin molecules, and
anti-glutathione/glutathione tag. In a preferred embodiment, the first and
second binding partner pair of the invention are comprised of
strepavidin/biotin molecules.
The "first" binding partner is so designated because it is attached to a
substrate. The substrate may be any solid support suitable for use in a
quantitative assay, such as polymer beads or a solid container such as a
polystyrene culture dish. To permit running the assays in a high
throughput format, specifically contemplated is a microtiter plate,
preferably a 96-well, 384-well or a 1536-well polystyrene microtiter
plate, and more preferably a 384-well polystyrene microtiter plate. The
first binding partner is attached to the substrate using any suitable
techniques such as pre-coating the plate with the first binding partner in
an appropriate buffer such as phosphate buffered saline (PBS),
magnesium/calcium-free PBS, sodium carbonate, or similar.
The method as described above includes a step of providing a polypeptide
composition comprising polypeptide monomers. The polypeptide monomers are
unaggregated peptides or polypeptide that can coalesce into aggregates
with like polypeptide under physiological conditions. The method is
particularly useful when practiced with polypeptides whose aggregates are
associated with disease pathogenesis, such as those polypeptides seen to
aggregate in Alzheimer's Disease, prion disease, Parkinson's disease,
amyotrophic lateral sclerosis (ALS), dialysis-related amyloidosis, corneal
dystrophy, and diabetes. Exemplary polypeptides include .alpha.-amyloid,
Prion protein, .alpha.-synuclein, .beta.-2 microglobulin and islet amyloid
polypeptide.
The polypeptide composition preferably is comprised of polypeptides whose
aggregation correlates a pathologic disease state. Exemplary polypeptide
monomers include, but are not limited to, .beta.-amyloid 1-40,
.beta.-amyloid 1-42, .beta.-amyloid 1-43, polyglutamine repeats, prion
protein (PrP), .alpha.-synuclein, or pancreatic amyloid. In a preferred
embodiment, the polypeptide of the invention is chosen from amyloid 1-40,
.beta.-amyloid 1-42, .beta.-amyloid 1-43, and most preferably is
.beta.-amyloid 1-40. Mixtures of different A.beta. forms can also be
employed. Aggregation of the foregoing polypeptides has been correlated
with pathologic states such as Alzheimer's Disease, Down's syndrome,
Huntington's chorea, Parkinson's disease, prion disease, Amyotrophic
Lateral Sclerosis, Lewy Bodies, or type II diabetes. In a preferred
embodiment the pathologic state is chosen from Alzheimer's Disease, Down's
syndrome, Lewy Bodies, or type II diabetes. Specifically contemplated by
the invention is the assessment of polypeptide aggregation in Alzheimer's
disease.
The "polypeptide composition" is formed by mixing polypeptide monomers
together in a solution and at a concentration at which aggregation can
occur (in the absence of inhibitors). Such solutions and concentrations
are easily determined from experience and literature, or alternatively by
simple dose-response type analyses to select a concentration at which
measurable aggregation occurs at a desirable rate (e.g. in a matter of
minutes or hours). Exemplary solutions and concentrations for A.beta.
range from 2 mg/ml beta-amyloid peptide stock solution in 0.1% acetic
acid, to 20-50 .mu.m/ml diluted A.beta. peptide in appropriate assay or
reaction buffer, containing any or all of the following: Bovine Serum
Albumin, milk protein, or similar high molecular weight protein, and mild
detergent such as Tween 20, Tween 40, or similar.
In order to perform the method, at least some of the polypeptide monomers
are "modified" or chemically coupled to comprise the second binding
partner. In other words, the second binding partner is not an epitope or
other natural feature of the polypeptide. Instead, the polypeptide is
modified to attach the second binding partner. In one variation, the
modification comprises expressing a chimeric peptide in which the
polypeptide amino acid sequence is modified by attaching an epitope tag
such as a His.sub.6 tag or Hemagluttinin (HA) tag. In a preferred
variation, the polypeptide is modified by chemical reaction e.g., by
attaching a biotin molecule to the peptide by conventional techniques. In
a preferred embodiment, at least some of the monomers lack the second
binding partner.
"Mixing" here may be defined as the incubation in the same plate/well of a
solution or suspension of polypeptide monomers that comprise the second
binding partner of the invention, optionally with polypeptide monomers
which lack the second binding partner. The ratio of monomers having and
lacking the second binding partners empirically selected to improve the
signal:background measurements of the assay. With respect to A.beta.
solutions described in the preceding paragraph, a preferred ratio is
within the range of 5:1 to 1:1 monomers lacking a second binding partner
to monomers comprising the second binding partner, and more preferably
wherein the ratio is 2:1 monomers lacking the second binding partner to
monomers comprising the second binding partner. This mixing of monomers to
form the polypeptide composition may be done in the presence of a test
agent or in the absence of a test agent.
The contacting is preferably performed in a liquid environment such as an
aqueous buffer, such as magnesium/calcium-free PBS. In one embodiment the
polypeptide composition is contacted with the substrate for a time period
within the range of 15 minutes to 2 hours, in a preferred embodiment the
polypeptide composition is contacted with the substrate for 30 minutes.
The method optionally includes a washing step, wherein the substrate is
washed prior to measuring polypeptide aggregation to reduce background
measurements caused by unbound polypeptides. Washing of the substrate
after incubation of the polypeptide composition and before detection of
aggregation is performed in appropriate buffer plus detergent. For
example, in a preferred embodiment the wash is performed in
magnesium/calcium-free PBS containing mild detergent, and especially
contemplated is the use of the mild detergent Tween 20.
The measuring step entails a determination (preferably quantitative) of
whether and how much polypeptide aggregate has bound to the substrate, and
it involves detecting (measuring) the second binding partner bound
(indirectly) to the substrate. In a preferred embodiment the measuring is
achieved by contacting the substrate with labeled first binding partner
under conditions where the first binding partner will bind to the second
binding partner washing the substrate to remove unbound labeled second
binding partner, and then measuring the amount of label that remains bound
to the substrate. According to this design of the assay, the substrate
will bind to polypeptide monomers and/or polypeptide aggregates during the
contacting step, due to the affinity of the first binding partner on the
substrate for the second binding partner that is present on at least some
of the polypeptides. However, unaggregated polypeptide monomers that are
bound to the substrate will not be detected during the measuring step
because the labeled first binding partner will fail to attach to such
monomers (the second binding partner being occupied by the substrate
attachment).
In contrast, polypeptide aggregates that include at least two monomers
containing second binding partners are detected during the measuring step
because only one of the second binding partners is attached to the
substrate comprising the first binding partner and at least one of the
second binding partners in the aggregate will be unattached and exposed,
and thus free to bind the labeled first binding partner in the detecting
step. Measuring label that is bound to the substrate thus provides a
measurement of aggregation.
The label preferably is detectable and quantifiable at very low
concentrations. In preferred embodiments the label attached to the first
binding partner is a radionuclide, a fluorophore, a chromophore, and the
like. In highly preferred embodiments, the label is chosen from the group
consisting of .sup.35 S, .sup.125 I, .sup.32 P, .sup.3 H, Europium
(Eu.sup.3+) molecules, fluorescein, phycoerythrin, horseradish peroxidase,
alkaline phosphotase, and like molecules that demonstrate predictable
excitation in detection methods commonly used in the art. Embodiments in
which the first binding partner comprises a Europium label are especially
preferred.
The method of detection of the label is dependent on the type of label
linked to the first binding partner: in one embodiment a radionuclide is
detected using scintillation counting, a fluorophore such as fluorescein
isothiocyanate (FITC) is detected using the appropriate excitation
wavelength, and a chromophore is detected using substrate/enzyme complexes
that change color upon mixing and are detected at the appropriate
wavelength. In a preferred embodiment a Europium detectable label is
detected by excitation at the required wavelength of light and quantitated
using methods commonly used in the art.
In one variation, the method of the invention further comprises a step of
manufacturing a modulator composition comprising a modulator compound
identified as outlined above in a pharmaceutically acceptable carrier.
Such a manufacturing step is useful because it permits introducing the
modulator into a cell-based assay and/or into a living animal model or
human being to evaluate its modulating effects in systems more relevant to
efficacy of disease treatment. By "pharmaceutically acceptable carrier" is
contemplated any excipient(s), diluent(s), adjuvant(s) or carrier(s)
compatible with the modulator composition of the invention and suitable
for administration to human or animals. Typically, the carriers, which
will be sterile and pyrogen free, will be water, saline, phosphate
buffered saline, glucose, or other carriers commonly used in the art to
deliver therapeutics.
The method optionally further comprises a step of administering the
modulator composition to a mammal, most preferably a human, susceptible to
polypeptide aggregation or demonstrating a pathologic state as a result of
polypeptide aggregation and determining an improvement in polypeptide
aggregation or pathologic state as a result of administration of the
modulator composition.
By "administering" is contemplated the introduction of the modulator
composition of the invention by any medically-accepted means known in the
art to a subject exhibiting an aggregation-induced pathologic state. The
modulator composition may be administered via several routes, including
but not limited to intravenous injection, subcutaneous injection,
intranasal ingestion, oral ingestion, topical administration, and the
like. Routes that achieve delivery of the agent to sites of polypeptide
aggregation are preferred. Practice of methods of the invention in other
mammalian subjects, especially mammals that are conventionally used as
models for demonstrating therapeutic efficacy in humans (e.g. primate,
porcine, canine, or small rodents such as rabbit, rat and mouse animals),
also is contemplated.
By "improvement" in aggregation or pathologic state is contemplated any
lessening of detectable polypeptide aggregation, lessening of symptoms of
the pathologic state as a result of polypeptide aggregation, such as
dementia in Alzheimer's patients, as a result of the administration of the
modulator compound. A slowing or arresting of disease progression also is
considered an improvement with respect to disease such as Alzheimer's
which are known to progress and worsen with time.
In a related aspect the invention provides a kit comprising the materials
packaged together in a manner which facilitates their use to practice
methods of the invention. In a simplest embodiment, such a kit includes a
composition (e.g. substrate, first binding partner, second binding
partner, polypeptide monomers, detectable label) packaged in a container
such as a sealed bottle or vessel with a label affixed to the container or
included in the package that describes use of the composition to practice
the method of the invention. In another embodiment, the kit comprises the
substrate attached to the first binding partner packaged with the
aggregating polypeptide attached to a second binding partner. The kit
preferably also includes the first binding partner attached to a
detectable label.
Additional features and variations of the invention will be apparent to
those skilled in the art from the entirety of this application, and all
such features are intended as aspects of the invention.
Likewise, features of the invention described herein can be re-combined
into additional embodiments that also are intended as aspects of the
invention, irrespective of whether the combination of features is
specifically mentioned above as an aspect or embodiment of the invention.
Also, only such limitations which are described herein as critical to the
invention should be viewed as such; variations of the invention lacking
limitations which have not been described herein as critical are intended
as aspects of the invention.
In addition to the foregoing, the invention includes, as an additional
aspect, all embodiments of the invention narrower in scope in any way than
the variations specifically mentioned above. Although the applicant(s)
invented the full scope of the claims appended hereto, the claims appended
hereto are not intended to encompass within their scope the prior art work
of others. Therefore, in the event that statutory prior art within the
scope of a claim is brought to the attention of the applicants by a Patent
Office or other entity or individual, the applicant(s) reserve the right
to exercise amendment rights under applicable patent laws to redefine the
subject matter of such a claim to specifically exclude such statutory
prior art or obvious variations of statutory prior art from the scope of
such a claim. Variations of the invention defined by such amended claims
also are intended as aspects of the invention.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a schematic flow diagram illustrating an aggregation assay
according to the invention.
FIG. 2 represents the aggregation assay as performed using test compounds
to modulate aggregation.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for identifying compounds useful in
modulating the aggregation of polypeptides involved in the onset of many
human disease states. Such compounds are useful in developing therapies
for diseases characterized by the aggregation of polypeptides. For
instance, a stretch of polyglutamine repeats in a particular protein has
been proposed as the cause for many neurological disorders including
Huntington's disease and spinal bulbar muscular atrophy, while aggregation
of other unrelated proteins are cited as the causes of prion disease (PrP
aggregation), Parkinson's disease and amyotrophic lateral sclerosis (ALS)
(.beta.-synuclein aggregation), dialysis-related amyloidosis (.beta.-2
microglobulin aggregation), corneal dystrophy (kerato-epithelial
deposits), aggregation of islet amyloid polypeptide in more than 95% of
type II diabetes and .beta.-amyloid polypeptide aggregation in Alzheimer's
Disease.
One advantage of the methods of the present invention is that the rapid
method of detecting polypeptide aggregation allows for the rapid
assessment of numerous potential modulator compounds. Another advantage of
the present invention is that the decreased amount of reagent required per
assay reduces cost of the assay per sample.
An exemplary method of the invention may be carried out as follows and as
schematically illustrated in FIG. 1. As depicted in Panel 10, a 96 well
polystyrene dish substrate 2 is coated with a first binding partner 4,
such as streptavidin, which is provided by incubating the substrate 2 with
a solution containing the first binding partner 4, thereby resulting in
the coated plate of Panel 10.
As shown in Panel 20, a composition of polypeptide monomers 8, e.g.
.beta.-amyloid 1-40 monomers, is provided, e.g. by making a solution in
which some of the monomers 16 are linked to a second binding partner 12,
an example of which is biotin, which binds to streptavidin. The monomer
composition is first incubated 24 in order to allow the monomers to form
polypeptide aggregates 28,44 as shown in Panel 30. The incubation 24 is
carried out in the presence or absence of a test compound which may
modulate, i.e. inhibit or promote, monomer aggregation.
As shown in Panel 30, after the incubation, the polypeptide composition may
comprise a variety of species such as polypeptide monomers 8,
biotin-linked polypeptide monomers 16 and polypeptide aggregates 28,44.
This incubated polypeptide composition is then contacted 32 with the
coated plate depicted in 10 comprising the substrate 2 containing the
streptavidin first binding partner 4. As depicted in Panel 40, polypeptide
species that include at least one biotin molecule will bind to the coated
plate of 10 during the contacting step. Molecules from the polypeptide
composition bound to the coated plate from Panel 10 may comprise several
species of molecules: single polypeptide monomers linked only to the
biotin second binding partner 34 and comprising only one streptavidin
binding site; aggregated molecules comprising biotinylated polypeptide
monomers in association with monomers lacking the second binding partner
44, and therefore also comprising only one stretpavidin binding site; and
aggregated monomers comprising biotinylated polypeptide monomers in
association with other biotinylated monomers 28, and thus comprising two
or more sites for streptavidin 4 binding. Multiple streptavidin binding
sites in an aggregated molecule 28 allows for at least one .beta.-amyloid
1-40-biotin to attach to the substrate coated plate 10, while the
unoccupied biotin 12 on an associated .beta.-amyloid 1-40-biotin molecule
allows for detection of aggregated monomers 28 using a streptavidin first
binding partner 4 linked to a detectable label 54, such as
Europium.sup.3+. Monomers 34 that attach to the substrate lack any free
biotin.
Polypeptide aggregation is measured by detecting polypeptide aggregates 28
that have bound to the coated plate of 10. The bound aggregated molecules
are detected by contacting 45 a detecting agent in 50 (containing the
first binding partner 4 linked to a detectable label 54) to the substrate
coated plate of 10. As shown in Panel 60, the detecting agent 50 only
attaches to substrate-bound, aggregated polypeptides which comprise two or
more biotinylated polypeptide monomers 28. Using techniques common in the
art, the substrate-bound streptavidin-Eu.sup.3+ 50 is detected by
excitation of the Eu.sup.3+ label 54 at an appropriate wavelength 64 and
subsequent measurement with a detector 65 of Eu.sup.3+ fluorescence
emitted 66, in units of light absorbance. The degree of Europium.sup.3+ 54
excitation measured reflects the amount of detecting agent 50 bound, and
thereby correlates with the amount of monomer aggregation occurring during
the incubation step 24. Test compounds which result in altered levels of
Eu.sup.3+ 54 detection when compared to control compounds are considered
modulators of polypeptide monomer aggregation.
Using the same symbols and Panels depicted and defined in FIG. 1, FIG. 2
demonstrates three possible outcomes of the aggregation assay dependent
upon the test compound added during the incubation step 24. FIG. 2B
represents the aggregation shown in FIG. 1, as carried out using control
compounds (or buffers only) that do not modulate the natural process of
monomer aggregation. FIG. 2A represents aggregation that occurs when a
test compound added during the incubation step 24 is an inhibitor of
polypeptide aggregation. FIG. 2A, Panel 40 illustrates that inhibition of
polypeptide aggregation results in fewer aggregated monomers 28 bound to
the substrate coated plate 10. The decrease in aggregated monomers results
in less opportunity for the labeled first binding partner 50 to bind to
the substrate, as shown in FIG. 2A, Panel 60, thereby lowering the amount
of label 54 that is detectable by measurement of Eu.sup.3+ fluorescence.
FIG. 2C outlines a condition wherein the test agent administered during the
incubation step 24 enhances polypeptide aggregation. FIG. 2C, Panel 40
demonstrates that promotion of polypeptide monomer aggregation results in
an increase in aggregated monomers 28 bound to the substrate coated plate
10. The enhancement of polypeptide aggregation results in a greater amount
of labeled first binding partner 50 attached to the free second binding
partner on the aggregated monomers 28, as represented in FIG. 2C, Panel
60. Increased aggregation and subsequent increased binding of the labeled
first binding partner 50 leads to augmented detectable label 54 bound to
the substrate, resulting in amplification of Eu.sup.3+ signal emitted
compared to control compounds, thus providing a quantitative measure of
the capacity of a test compound to modulate polypeptide monomer
aggregation.
The aggregating polypeptides used in the invention, wherein "aggregating"
is defined as the binding of polypeptide monomers to each other forming a
higher order molecular structure, include polypeptides known to be
associated with human disease states. In a preferred invention the
polypeptides can be chosen from the group consisting of: .beta.-amyloid
polypeptides comprised of any peptide resulting from beta-secretase
cleavage of APP, especially considered are .beta.-amyloid 1-39,
.beta.-amyloid 1-40, .beta.-amyloid 1-41, .beta.-amyloid 1-42, and
.beta.-amyloid 1-43; polyglutamine repeats of at least 27 glutamine
residues to as many as 45 glutamine repeats, and most preferably of 38
glutamine residues; .alpha.-synuclein peptides of at least 11 amino acids
comprised of the common 11 residue repeat found in .alpha.-synuclein;
prion protein, and pancreatic amyliod peptides (ranging from 6 to 37 amino
acids in length) wherein the peptide includes the sequence NFGAIL (Azriel
et al., J. Biol. Chem. 276: 34156-161. 2001).
The first binding partner of the invention and the second binding partner
of the invention, selected from molecular pairs that show high affinity of
binding to each other and are easily applicable to many different
polypeptides, may be chosen from the group including but not limited to
strepavidin/biotin, anti-Histidine.sub.6 (His.sub.6 -tag)
antibodies/His.sub.6 peptide tags, anti-biotin/biotin molecules. The first
binding partner can be chosen from, but is not limited to, the group
consisting of streptavidin, anti-His.sub.6 antibodies, anti-myc tag,
anti-hemagluttin, anti-fluorophore (e.g. anti-fluorescein isothiocyanate),
anti-biotin, and the like.
The second binding partner of the invention can be chosen from the group
consisting of but not limited to biotin molecules, alkaline phosphatase,
fluorophores, genetically engineered peptide tags such as a histadine
His.sub.6 tag linked to the aggregating polypeptide, a myc-tag,
Hemagluttinin tags, and the like. Biotin, fluorophores, and other
contemplated small molecules comprising the second binding partner can be
linked to the polypeptide of the invention by means well-known in the art
such as a commercially produced Biotinylation kit (Sigma, St. Louis, Mo.),
or alternative methods commonly used in organic chemistry to chemically
attach a small molecule to a peptide or protein (see e.g., Current
Protocols in Protein Chemistry, John Wiley & Sons, 2001). Genetically
engineered tags, e.g., His.sub.6 and myc-tags, are operably linked to the
polypeptide of the invention using standard recombinant DNA methods well
known in the art (see e.g., Current Protocols in Molecular Biology, John
Wiley & Sons, 2001), or using conventional peptide synthesis techniques.
In a preferred embodiment, the first and second binding partner pair of the
invention are comprised of strepavidin/biotin molecules.
The first binding partner of the invention is attached to a substrate of
the invention. The substrate of the invention may be any material or
substance appropriate for performing the present method, such as polymer
beads or a solid container such as a polystyrene culture dish,
specifically contemplated is a polystyrene microtiter plate, preferably a
96-well, 384-well or 1536-well polystyrene microtiter plate, and more
preferably a 384-well polystyrene microtiter plate. The first binding
partner is attached to the substrate in an appropriate buffer such as
phosphate buffered saline (PBS), magnesium/calcium-free PBS, sodium
carbonate, or similar.
For measuring polypeptide aggregation, the first binding partner can be
operably linked to a label, wherein the label is a detectable label, and
preferably is detectable and quantifiable at very low concentrations. In a
preferred embodiment the label attached to the first binding partner can
be either a radionuclide, a fluorophore, a chromophore, and the like. In a
more preferred embodiment, the label is chosen from the group consisting
of .sup.35 S, .sup.125 I, .sup.32 H, europium (Eu.sup.3+) molecules,
fluorescein, phycoerythrin, horseradish peroxidase, alkaline phosphotase,
and like molecules that demonstrate predictable excitation in detection
methods commonly used in the art.
The test agent employed in the method of the invention can be any organic
or inorganic chemical or biological molecule that one would want to test
for the ability to modulate polypeptide aggregation. Since the most
preferred test agents are those that can be administered as therapeutics,
it will be apparent that molecules with limited toxicity are preferred.
However, toxicity can be screened in subsequent assays and often "designed
out" of molecules by pharmaceutical chemists. Screening of chemical
libraries such as those customarily kept by pharmaceutical companies,
consisting of both chemically synthesized and natural compounds, and
combinatorial libraries, is specifically contemplated.
Chemical libraries may contain known compounds, proprietary structural
analogs of known compounds, or compounds that are identified from natural
product screening.
Natural product libraries are collections of materials isolated from
natural sources, typically, microorganisms, animals, plants, or marine
organisms. Natural products are isolated from their sources by
fermentation of microorganisms followed by isolation and extraction of the
fermentation broths or by direct extraction from the microorganisms or
tissues (plants or animal) themselves. Natural product libraries include
polyketides, non-ribosomal peptides, and variants (including non-naturally
occurring variants) thereof. For a review, see Cane et al., Science, 282,
63-68 (1998).
Combinatorial libraries are composed of large numbers of related compounds,
such as peptides, oligonucleotides, or other organic compounds as a
mixture. Such compounds are relatively straightforward to design and
prepare by traditional automated synthesis protocols, PCR, cloning or
proprietary synthetic methods. Of particular interest are peptide and
oligonucleotide combinatorial libraries.
Still other libraries of interest include peptide, protein, peptidomimetic,
multiparallel synthetic collection, recombinatorial, and polypeptide
libraries. For a review of combinatorial chemistry and libraries created
thereby, see Myers, Curr. Opin. Biotechnol., 8, 701-707 (1997).
Modulator compounds identified by assessment of the test agents may be
formulated into compositions which include pharmaceutically acceptable
(i.e., sterile and non-toxic) liquid, semisolid, or solid diluents that
serve as pharmaceutical vehicles, excipients, or media. Modulators
formulated in this manner can be further screened for modulating activity
in vivo, e.g., in animal models for disease; or can be administered to
humans in clinical trials; or can be made and sold as pharmaceuticals.
Modulator compositions according to the invention may be administered in
any suitable manner using an appropriate pharmaceutically-acceptable
vehicle, e.g., a pharmaceutically-acceptable diluent, adjuvant, excipient
or carrier. The composition to be administered according to methods of the
invention preferably comprises a pharmaceutically-acceptable carrier
solution such as water, saline, phosphate-buffered saline, glucose, or
other carriers conventionally used to deliver therapeutics.
The modulator compositions can be packaged in forms convenient for
delivery. The compositions can be enclosed within a capsule, caplet,
sachet, cachet, gelatin, paper, or other container. The dosage units can
be packaged, e.g., in tablets, capsules, suppositories or cachets.
The modulator compositions identified by the invention may also be
introduced into the subject being treated for a pathological disease state
as a result of polypeptide aggregation by any conventional method
including, e.g., by intravenous, intradermal, intramuscular,
intracerebral, intraperitoneal, or subcutaneous injection; by oral,
transdermal, sublingual, intranasal, anal, or vaginal, delivery. The
treatment may consist of a single dose or a plurality of doses over a
period of time. Dosing will be determined by standard dose-response
studies, first in animal models and then in clinical testing, revealing
optimal dosages for particular disease states and patient populations. The
amounts of modulator composition in a given dose will vary according to
the size of the individual to whom the therapy is being administered,
generally on a mg/kg basis, as well as the characteristics of the disorder
being treated.
The method of the invention will be more readily understood by reference to
the following examples, which are provided as a manner of illustration and
are not intended to be limiting.
EXAMPLE 1
Time Resolved Fluorescence Assay For .beta.-Amyloid Polypeptide Aggregation
The following example is an embodiment of the invention which demonstrates
a method for identifying modulators of .beta.-amyloid polypeptide
aggregation. .beta.-amyloid polypeptide monomers in solution in vitro are
pre-disposed toward aggregation of the peptide monomers into larger,
multimeric molecules. Aggregation of the .beta.-amyloid monomers in vivo
is a primary contributor to neuronal damage in Alzheimer's Disease. The
present invention allows for a sensitive in vitro analysis of
.beta.-amyloid aggregation and the effects of outside compounds or test
agents on the extent of .beta.-amyloid peptide aggregate formation.
Beta-amyloid 1-40 (A.beta.) and biotin-labeled .beta.-amyloid 1-40 peptides
were purchased from Biosource (Camarillo, Calif.). .beta.-amyloid and
biotinylated amyloid stock solutions, diluted in 0.1% acetic acid and
corrected for 80% peptide content, were diluted to 83 .mu.g/ml and 42
.mu.g/ml, respectively, in reaction buffer (Dulbecco's PBS pH 7.4, 0.02%
Tween 20). An equal mixture of the two .beta.-amyloid products was then
brought to a final concentration of 62.5 .mu.g/ml total .beta.-amyloid,
giving a final 2:1 ratio of unbiotinylated to biotinylated A.beta.
polypeptide. When analyzing aggregation in the presence of test compound,
5 ul of diluted test compound (120 uM in 10% DMSO) was added to each well
being analyzed in a 384-well polystyrene plate (Nunc). Test compounds are
added over a range of concentrations. Twenty microliters of the
.beta.-amyloid reaction mixture was dispensed into each well of the 384
well plate, giving the final assay conditions as 24 uM test compound, 2%
DMSO, and 50 ug/ml .beta.-amyloid in a final volume of 25 uL. Plates were
then sealed with foil sealers (Corning), and incubated at 37.degree. C.
for 24 hrs.
After 24 hrs, 35 uL of reaction buffer was added to a streptavidin coated
plate using a Multidrop pipettor. Using a multipette, 14 uL of the
aggregation assay plate media, now containing either aggregated or
monomeric A.beta., resulting from incubation with the test agent, was
added to the streptavidin coated plate containing 35 uL of buffer. Plates
were incubated 30 min at room temperature, washed three times with
reaction buffer (90 uL/wash), and 50 uL/well of Eu3+labeled streptavidin
(250 ng/ml, Wallac,/Perkin Elmer Life Sciences, Boston, Mass.) in DELFIA
assay buffer (Wallac) added to assay wells and incubated 2 hr at room
temperature. Plates were washed with reaction buffer and 75 uL of DELFIA
Enhancement solution (Wallac) was added to each well and the plates read
on TRF capable plate reader (excitation at 337 run and emission at 620
nm).
Results of the TRF aggregation assay demonstrate that the addition of test
compounds such as known A.beta. aggregation inhibitors ApoE3 or QKLVFF
peptide (Lowe et al., Biochemistry 40: 7882-89. 2001)(Bachem Bioscience,
Philadelphia, Pa.) prevents A.beta. aggregation by 75% and 44%,
respectively. Further screening with a priority compound library shows
A.beta. aggregation inhibited by greater than 90% compared to control
wells lacking test compound. The background of this TRF assay is
