U.S. patent application number 16/986311 was filed with the patent office on 2021-01-14 for dyes for analysis of protein aggregation.
This patent application is currently assigned to Enzo Life Sciences, Inc.. The applicant listed for this patent is Enzo Life Sciences, Inc.. Invention is credited to Anatoliy Balanda, Jack Coleman, Lijun Dai, Vladyslava Kovalska, Mykhaylo Losytskyy, Anthony Ludlam, Praveen Pande, Wayne Forrest Patton, Dee Shen, Kateryna Volkova, Sergiy M. Yarmoluk.
Application Number | 20210009809 16/986311 |
Document ID | / |
Family ID | 1000005109589 |
Filed Date | 2021-01-14 |
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United States Patent
Application |
20210009809 |
Kind Code |
A1 |
Patton; Wayne Forrest ; et
al. |
January 14, 2021 |
DYES FOR ANALYSIS OF PROTEIN AGGREGATION
Abstract
Provided are dyes and compositions which are useful in a number
of applications, such as the detection and monitoring protein
aggregation, kinetic studies of protein aggregation,
neurofibrillary plaques analysis, evaluation of protein formulation
stability, and analysis of molecular chaperone activity.
Inventors: |
Patton; Wayne Forrest; (Dix
Hills, NY) ; Yarmoluk; Sergiy M.; (Kyiv, UA) ;
Pande; Praveen; (Holbrook, NY) ; Kovalska;
Vladyslava; (Kyiv, UA) ; Dai; Lijun;
(Farmingville, NY) ; Volkova; Kateryna; (Kyiv,
UA) ; Coleman; Jack; (East Northport, NY) ;
Losytskyy; Mykhaylo; (Kyiv, UA) ; Ludlam;
Anthony; (Ypsilanti, MI) ; Balanda; Anatoliy;
(Kyiv, UA) ; Shen; Dee; (Glen Head, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Enzo Life Sciences, Inc. |
Farmingdale |
NY |
US |
|
|
Assignee: |
Enzo Life Sciences, Inc.
Farmingdale
NY
|
Family ID: |
1000005109589 |
Appl. No.: |
16/986311 |
Filed: |
August 6, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15963441 |
Apr 26, 2018 |
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16986311 |
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15156565 |
May 17, 2016 |
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15963441 |
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13510976 |
Feb 12, 2013 |
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PCT/US10/03061 |
Nov 30, 2010 |
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15156565 |
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12592639 |
Nov 30, 2009 |
9133343 |
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13510976 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C09B 23/06 20130101;
C09B 23/102 20130101; G01N 2800/2835 20130101; C09B 23/0008
20130101; G01N 33/582 20130101; C09B 23/0066 20130101; G01N
2800/2821 20130101; C09B 23/0058 20130101; G01N 33/6845 20130101;
C09B 23/145 20130101; C09B 23/0025 20130101; C09B 23/141 20130101;
C09B 23/04 20130101; C07D 401/12 20130101; G01N 2458/30
20130101 |
International
Class: |
C09B 23/14 20060101
C09B023/14; C09B 23/01 20060101 C09B023/01; C09B 23/04 20060101
C09B023/04; C09B 23/06 20060101 C09B023/06; C09B 23/10 20060101
C09B023/10; G01N 33/58 20060101 G01N033/58; G01N 33/68 20060101
G01N033/68; C07D 401/12 20060101 C07D401/12 |
Claims
1. A compound comprising the structure ##STR00155## wherein m and n
are independently 1, 2 or 3; wherein L is a linker arm comprising
carbon, sulfur, oxygen, nitrogen, or any combination thereof;
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.9, R.sub.10,
R.sub.11, R.sub.12, R.sub.13, R.sub.14, R.sub.15, R.sub.16,
R.sub.19, R.sub.20, R.sub.21 and R.sub.22 are independently
hydrogen, halogen, amino, ammonium, nitro, sulfo, sulfonamide,
carboxy, ester, cyano, phenyl, benzyl, an alkyl group wherein the
alkyl group is saturated or unsaturated, linear or branched,
substituted or unsubstituted, an alkoxy group wherein the alkoxy
group is saturated or unsaturated, branched or linear, substituted
or unsubstituted, or when taken in combination R.sub.1 and R.sub.2,
or R.sub.3 and R.sub.4, or R.sub.9 and R.sub.10, or R.sub.11 and
R.sub.12, or R.sub.13 and R.sub.14, or R.sub.15 and R.sub.16, or
R.sub.19 and R.sub.20, or R.sub.21 and R.sub.22 form a five or six
membered ring wherein the ring is saturated or unsaturated,
substituted or unsubstituted, and wherein R.sub.9 and R.sub.10, or
R.sub.11 and R.sub.12, or R.sub.13 and R.sub.14, or R.sub.15 and
R.sub.16 can comprise alkyl chains that are joined together,
wherein a quinoline moiety can be formed; wherein R.sub.7, R.sub.8,
R.sub.17 and R.sub.18 are independently hydrogen, Z, an alkyl group
wherein the alkyl group is saturated or unsaturated, linear or
branched, substituted or unsubstituted, an alkoxy group wherein the
alkoxy group is saturated or unsaturated, branched or linear,
substituted or unsubstituted, or when taken together, R.sub.7 and
R.sub.8 and R.sub.17 and R.sub.18, may form a 5 or 6 membered ring
wherein the ring is saturated or unsaturated, substituted or
unsubstituted; wherein Z comprises a carboxyl group
(CO.sub.2.sup.-), a carbonate ester (COER.sub.25), a sulfonate
(SO.sub.3.sup.-), a sulfonate ester (SO.sub.2ER.sub.25), a
sulfoxide (SOR.sub.25), a sulfone
(SO.sub.2CR.sub.25R.sub.26R.sub.27), a sulfonamide
(SO2NR.sub.25R.sub.26), a phosphate (PO.sub.4.sup.=), a phosphate
monoester (PO.sub.3.sup.-ER.sub.25), a phosphate diester
(PO.sub.2ER.sub.25ER.sub.26), a phosphonate (PO.sub.3.sup.=) a
phosphonate monoester (PO.sub.2.sup.-ER.sub.25) a phosphonate
diester (POER.sub.25ER.sub.26), a thiophosphate (PSO.sub.3.sup.=),
a thiophosphate monoester (PSO.sub.2.sup.-ER.sub.25) a
thiophosphate diester (PSOER.sub.25ER.sub.26), a thiophosphonate
(PSO.sub.2.sup.=), a thiophosphonate monoester (PSO.sup.-ER.sub.25)
a thiophosphonate diester (PSER.sub.25ER.sub.26), a phosphonamide
(PONR.sub.25R.sub.26NR.sub.28R.sub.29), its thioanalogue
(PSNR.sub.25R.sub.26NR.sub.28R.sub.29), a phosphoramide
(PONR.sub.25R.sub.26NR.sub.27NR.sub.28R.sub.29), its thioanalogue
(PSNR.sub.25R.sub.26NR.sub.27NR.sub.28R.sub.29), a phosphoramidite
(PO.sub.2R.sub.25NR.sub.28R.sub.29) or its thioanalogue
(POSR.sub.25NR.sub.28R.sub.29) wherein E is independently O or S;
wherein R.sub.25, R.sub.26, R.sub.27, R.sub.28, and R.sub.29 are
independently a hydrogen, an unsubstituted straight-chain, branched
or cyclic alkyl, alkenyl or alkynyl group, a substituted
straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group
wherein one or more C, CH or CH.sub.2 groups are substituted with
an O atom, N atom, S atom, or NH group, or an unsubstituted or
substituted aromatic group; wherein Z is attached directly, or
indirectly through a second linker arm comprising carbon, sulfur,
oxygen, nitrogen, and any combinations thereof and wherein the
second linker arm may be saturated or unsaturated, linear or
branched, substituted or unsubstituted or any combinations thereof;
wherein R.sub.5, R.sub.6, R.sub.23 and R.sub.24 can independently
be hydrogen or an alkyl group wherein the alkyl group is saturated
or unsaturated, linear or branched, substituted or unsubstituted,
or when taken in combination R.sub.5 and R.sub.6 or R.sub.2 and
R.sub.5 or R.sub.3 and R.sub.6 or R.sub.23 and R.sub.24 or R.sub.22
and R.sub.23 or R.sub.20 and R.sub.24 form a five or six membered
ring wherein the ring is saturated or unsaturated, substituted or
unsubstituted; and wherein said compound is modified to comprise a
reactive group which is an isocyanate, isothiocyanate,
monochlorotriazine, dichlorotriazine, 4,6,-dichloro-1,3,5-triazine,
mono- or di-halogen substituted pyridine, mono- or di-halogen
substituted diazine, maleimide, haloacetamide, aziridine, sulfonyl
halide, acid halide, hydroxysuccinimide ester,
hydroxysulfosuccinimide ester, imido ester, hydrazine,
azidonitrophenol, azide, 3-(2-pyridyl dithio)-propionamide, glyoxal
or aldehyde group.
2. The compound of claim 1, wherein the compound exhibits increased
fluorescence in the presence of an aggregated form of a protein
when compared to the fluorescence exhibited when the compound is in
the presence of the unaggregated form of the protein.
3. The compound of claim 1, comprising the structure ##STR00156##
modified to comprise the reactive group, or ##STR00157## modified
to comprise the reactive group.
4. The compound of claim 4, wherein each of R.sub.5, R.sub.6,
R.sub.23 and R.sub.24 are a methyl or an ethyl moiety.
5. The compound of claim 1, wherein the compound is S25, S43, TOL3,
YAT2134, YAT2148, YAT2149, S13, YAT2135, YAT2324 or YAT2150,
modified to comprise the reactive group.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/963,441 filed Apr. 26, 2018 which is a divisional of
U.S. patent application Ser. No. 15/156,565, filed May 17, 2016,
now abandoned, which is a divisional of U.S. patent application
Ser. No. 13/510,976, filed Feb. 12, 2013, now abandoned, which is
the U.S. national stage application of PCT/US2010/03061, filed Nov.
30, 2010, which claims priority to U.S. patent application Ser. No.
12/592,639, filed Nov. 30, 2009 (now U.S. Pat. No. 9,133,343), all
of which are hereby incorporated by reference in its entirety.
SEQUENCE LISTING STATEMENT
[0002] This application contains a Sequence Listing which has been
submitted in ASCII format via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Aug. 5,
2020, is named ENZ-89-CIP-D1-D1-D1-SL.txt and is 955 bytes in
size.
BACKGROUND OF THE INVENTION
(1) Field of the Invention
[0003] The present application generally relates to dyes and
compositions comprising dyes. More particularly, provided are dyes
and compositions for identifying and quantifying protein
aggregation.
(2) Description of the Related Art
[0004] The deposition of insoluble protein aggregates, known as
amyloid fibrils, in various tissues and organs is associated with a
number of neurodegenerative diseases, including Alzheimer's,
Huntington's and Parkinson's diseases, senile systemic amyloidosis
and spongiform encephalopathies (Volkova et al., 2007; Stefani
& Dobson, 2003). Fibrillar deposits with characteristics of
amyloid are also formed by several other proteins unrelated to
disease, including the whey protein beta-lactoglobulin (BLG). All
amyloid fibers, independent of the protein from which they were
formed, have very similar morphology: long and unbranched, a few
nanometers in diameter, and they all exhibit a cross-beta X-ray
diffraction pattern. The ability to form amyloid fibrils of
structurally and functionally diverse proteins, some of which are
not associated with amyloid-deposition diseases, suggests that this
property is common to all polypeptides Such amyloid structures are
also known to possess a binding affinity for certain dyes, notably,
thioflavin T and congo red dyes.
[0005] Many proteins are known to be only marginally stable in
solution, undergoing conformational changes due to various stresses
during purification, processing and storage (Arakawa et al., 2007).
Such stresses may include elevated temperature, agitation and
exposure to extremes of pH, ionic strength, or various interfaces
(e.g., an air-liquid interface) and high protein concentration (as
observed for some monoclonal antibody formulations). A wide variety
of aggregates are encountered in biopharmaceutical samples, which
range in size and physiochemical characteristics (e.g., solubility,
reversibility). Protein aggregates span a broad size range, from
small oligomers that are only a couple nanometers in length to
insoluble micron-sized aggregates that extend to millions of
monomeric units. Structurally altered proteins have an especially
strong tendency to aggregate, often leading to their eventual
precipitation. Irreversible aggregation is a major problem for the
long-term storage and stability of therapeutic proteins and for
their shipment and handling.
Mechanisms of Protein Aggregation
[0006] Aggregation is a major degradation pathway that needs to be
characterized and controlled during the development of protein
pharmaceuticals. In the bioprocessing arena, the mechanisms of
protein aggregation are still not fully understood, despite the
fact that aggregation is a major problem in therapeutic protein
development (Arakawa et al., 2006). One plausible mechanism is that
aggregation is driven or catalyzed by the presence of a small
amount of a contaminant which serves as a nucleation site. That
contaminant could be a damaged form of the protein product itself,
host cell proteins, or even nonprotein materials, such as leachates
from the container or resin particles associated with purification
of the protein.
[0007] If the contaminant is the damaged protein itself, then its
aggregation may lead to soluble oligomers, which become larger
aggregates, visible particulates, or insoluble precipitates. Such
soluble oligomers, host-cell contaminants, or nonprotein materials
may serve as a nucleus onto which native proteins assemble and are
incorporated into larger aggregates. Damaged forms of a protein
product can also arise from chemical modification (such as
oxidation or deamidation) and from conformationally damaged forms
arising from thermal stress, shear, or surface-induced
denaturation. Minimizing protein aggregation thus requires ensuring
both chemical and physical homogeneity; that is, chemically
modified or conformationally altered proteins must be removed from
the final product.
[0008] A second mechanism that often leads to protein aggregation
is initiated by the partial unfolding of the native protein during
its storage. Protein conformation is not rigid--the structure
fluctuates around the time-averaged native structure to different
extents depending upon environmental conditions. Some partially or
fully unfolded protein molecules are always present at equilibrium
in all protein solutions, but most such molecules simply refold to
their native structure. These unfolded proteins may in some
instances, however, aggregate with other such molecules or may be
incorporated into an existing aggregate nucleus, eventually forming
larger aggregates, as described above. Factors such as elevated
temperature, shaking (shear and air-liquid interface stress),
surface adsorption, and other physical or chemical stresses may
facilitate partial unfolding of proteins, leading to the cascade of
events that cause aggregation.
[0009] A third aggregation mechanism is reversible self-association
of the native protein to form oligomers. According to the law of
mass action, the content of such reversible aggregates will change
with total protein concentration. The tendency of different
proteins to associate reversibly with one another is highly
variable, and the strength of that association typically varies
significantly with solvent conditions, such as pH and ionic
strength. In principle, these reversible oligomers will dissociate
completely as the protein becomes highly diluted, for example,
after delivery of a therapeutic protein in vivo. Consequently, this
class of aggregates is generally less of a concern than
irreversible aggregates. Such reversible oligomers can eventually
become irreversible aggregates, however. Preventing accumulation of
irreversible aggregates may thus require minimizing the reversible
association as well. Further, reversible self-association of
proteins can significantly alter overall pharmaceutical properties
of product solutions, such as solution viscosity.
[0010] Detection of reversible aggregates can be an especially
challenging task. As such, aggregates can dissociate after their
dilution during attempts to measure them. Additionally, the results
of any analysis method incorporating a separation process in the
workflow may depend very much upon the kinetic rates of the
reversible association-dissociation reactions as well as the
equilibrium constants.
[0011] One consequence of the complexities of monitoring aggregate
formation processes is the difficulty of linking the effect
(presence of aggregates) to its underlying cause, particularly
because the key damage may occur at a time or place quite separated
from the observed consequence. One example arises during the
large-scale production of therapeutic monoclonal antibodies (MAbs).
Acid stability plays a major role in the aggregation of MAbs
because the process for their purification usually involves both
low-pH elution from protein-A affinity columns and acid-treatment
for viral inactivation.
[0012] The exposure of MAbs to a low-pH environment can result in
small but significant conformational changes that can additionally
depend upon factors such as temperature, and solvent composition.
While such partially unfolded MAbs may not aggregate at low pH,
they may aggregate during subsequent manufacturing steps involving
changes in pH or ionic strength. A larger conformational change at
low pH generally leads to more aggregates upon increasing the pH.
Typically, protein aggregate formation from the low-pH structure is
not a fast process, but it does occur slowly from the association
of damaged monomers that have not returned to their fully native
structure. This and other types of protein aggregation phenomena
may not manifest themselves until months after manufacturing a
particular lot of protein or until later stages of the product
development process. Regardless of the mechanism of aggregation,
preventing aggregation problems requires sensitive and reliable
technologies for quantitative determination of aggregate content
and aggregate characteristics.
[0013] Since the earliest clinical applications of protein
pharmaceuticals in medicine, aggregation problems have been
implicated in adverse reactions in humans and other safety issues.
In order to minimize such risks from therapeutic proteins in the
clinic, formulations must be optimized to minimize aggregation
during storage, handling, and shipping.
Analysis of Protein Aggregation
[0014] The analysis of protein aggregation can be formally
classified into four experimental types (Arakawa et al., 2006,
2007; Krishnamurthy et al., 2008). The first type of protein
aggregation analysis is the most conventional approach, wherein a
small volume of sample is applied to a separation medium and forms
a band or zone. As the band migrates through the medium, the
proteins separate according to differences in size, electrophoretic
charge, or mass. Gel electrophoresis, size exclusion chromatography
(SEC), field flow fractionation (FFF), and the occasionally used
band sedimentation technique belong to this class of methods. The
movement of the band or zone in these methods is often monitored
using absorbance or refractive index detection.
[0015] In the second type of analysis, the sample initially and
uniformly fills a measurement cell. When an electrical or
centrifugal driving force is then applied, the protein moves along
the applied field, leaving a protein-depleted solvent, which
creates a boundary between protein-free and protein-containing
solution phases. The movement of this boundary over time is
measured. This mode of separation is used in analytical
ultracentrifugation-sedimentation velocity (AUC-SV) and
moving-boundary electrophoresis.
[0016] The third type of analysis is a measurement of particle size
with no physical separation. An example of this method is referred
to as correlation spectroscopy and it measures the fluctuation of
particles in solution due to Brownian motion (i.e., measures
protein diffusion coefficients). Fluctuations of scattered light
and of fluorescence intensity have been employed in this type of
measurement. One of the most widely employed methods in this
category is referred to as dynamic light scattering (DLS).
[0017] SEC is the most commonly implemented control method and has
become an industry benchmark for quantification of protein
aggregates. SEC is seen as a versatile technique for separation and
quantification of protein aggregates because of its high precision,
high throughput, ease of use, compatibility with a quality control
(QC) environment, and in most cases ability to accurately quantify
protein aggregates. In spite of these strengths, several concerns
exist with the technique including: a potential loss of aggregates
(especially multimers), interaction of samples with a column
matrix, the required change of a sample buffer matrix to an SEC
mobile phase, and the inherent requirement for dilution of samples.
Additionally, perturbation of the distribution of protein
aggregates under standard SEC methodological conditions is
possible.
[0018] AUC-SV relies on hydrodynamic separation of various species
in a heterogeneous protein mixture under strong centrifugal force.
AUC-SV complements SEC in resolving and quantifying low levels of
protein aggregates. The main advantages of AUC-SV are seen in its
ability to detect and measure higher order aggregates (which may
elute in the void volume of an SEC column) and to conduct these
measurements without exposing samples to a column resin or SEC
mobile phase. AUC-SV is considered an accurate method because it
does not require standards or dissociate aggregates; thus it can be
used as an orthogonal method to verify the accuracy of SEC results.
AUC-SV suffers from lower precision than SEC, however. The
practical aspects of AUC-SV that impact precision and accuracy are
beginning to be understood better, and several recent studies have
demonstrated the utility of AUC-SV to detect and quantify
aggregates present at relatively low (.about.1%) levels. Despite
its advantages, AUC-SV is not yet readily amenable for use as a
routine release test in the biotechnology industry because of
issues related to low throughput, the need for specialized
equipment, performance problems at high protein concentrations, the
need for skilled practitioners of the method, and difficulty in
validating data analysis software.
[0019] DLS uses the time-dependent fluctuations of a
scattered-light signal to calculate the hydrodynamic diameter of
protein aggregates and their relative proportions. This method is
highly sensitive to large aggregates because the intensity of
scattered light increases proportionally with molecular weight. As
a result, very large aggregates (e.g., a 1,000-mer) present at
trace levels (.ltoreq.0.1%) can be detected with high sensitivity.
If present, such aggregates would elute in the void volume of an
SEC column or they may be filtered out. Although this method is
ideal for detecting very low mass fractions of large aggregates, it
cannot resolve species that are similar in size. At least a three-
to five-fold difference in hydrodynamic diameter is required for
resolving different species. DLS is also not amenable to use as a
control method because it is semi-quantitative and very sensitive
to dust or other extraneous particles. Results also depend on the
algorithm used for data analysis, which is often proprietary to the
manufacturer of a particular instrument.
[0020] As an orthogonal technique to SEC and AUC-SV, analytical
field-flow fractionation (aFFF) has gained popularity in recent
years for its ability to fractionate protein aggregates without a
column. aFFF most commonly uses two fluid flows ("fields") in a
channel to achieve particle separation based upon molecular weight
and hydrodynamic size (diffusion coefficient). Injected
macromolecular species are held in place by a cross flow on a
semi-permeable membrane while a perpendicular channel flow carries
molecules forward based on their diffusion coefficient, thereby
providing size-based fractionation. Because aFFF involves no column
interactions, it is considered a gentler separation technique than
SEC. Concerns regarding the interaction of aggregates with the
membrane have yet to be completely addressed, however. aFFF can be
coupled with different detectors including light scattering,
refractive index, and ultraviolet (UV) detectors. When compared
with SEC, the precision and limit of detection of aFFF is inferior
in the high-molecular-weight range, because of increased baseline
noise. Experimental conditions (e.g., cross-flow rate) for
reasonable separations in one size range are also not generally
applicable to other size ranges, making the technique cumbersome,
especially when analyzing a broad range of masses. Along with other
limitations, such as the need for specialized equipment and a
skilled operator, and the difficulty in validating the method
prevents the use of aFFF in applications for release and stability
monitoring.
[0021] Resolution and the size range that can be evaluated in one
particular analysis vary widely among the above mentioned
techniques. SEC cannot handle a large range of sizes because the
pore size or degree of polymerization of the resin must be adjusted
to the size of the protein species. If a protein sample contains
widely different sizes, many techniques are unsuitable for
analyzing all sizes simultaneously. FFF and DLS can cover a very
large range of sizes, but in the case of DLS, resolution is
generally fairly poor, and FFF entails some trade-off between
resolution and dynamic range. SV-AUC is intermediate in capability
relative to FFF and DLS. The dynamic range of SV-AUC is fairly
good, generally a factor of 100 or more in molecular weight at any
particular rotor speed. The resolution of SV-AUC is generally not
ideal for separating monomer from dimer, compared with the best SEC
columns (especially for lower molecular weight proteins). SV-AUC is
often much better, however, than SEC for resolving moderate size
oligomers, (tetramers to decamers).
[0022] The cited analytical techniques also differ significantly
with respect to their overall sensitivity, in other words, their
ability to detect and quantify small percentages of irreversible
aggregates. SEC, FFF, and SV-AUC are all capable of detecting
aggregates at levels as low as .about.0.1% when they are well
separated from other species. The quantification of species that
elute from SEC or FFF is quite good, but aggregates can easily be
lost during the separation process. Thus, SEC and FFF may provide
good precision but poor accuracy. For SV-AUC, loss of protein
aggregates to surfaces is usually not a problem, but accurate
quantification of small oligomers (dimer-tetramer) at total levels
of .about.2% or less is quite difficult.
[0023] The sensitivity of DLS increases linearly with the
stoichiometry of the protein aggregate. DLS is for all practical
purposes useless for detecting oligomers smaller than an octamer,
because the technique cannot resolve such oligomers from monomeric
species, and for those protein aggregate species that are resolved,
the accuracy of the weight fractions is quite poor, typically plus
or minus factors of two to ten. DLS exhibits excellent sensitivity,
however, for very large aggregate species, which can often be
detected at levels far below 0.01% by weight.
[0024] Overall, no single analytical technique is ideal for every
protein or is optimal for analyzing the wide range of aggregation
problems that can arise with protein pharmaceutical formulation.
One important industry trend are recent requests from regulatory
agencies that the protein aggregation analytical method used for
lot release and/or formulation development. Typically, this means
SEC which is cross-checked through one or more orthogonal
approaches to ensure detection of all relevant protein aggregate
species. Comparison of protein aggregate content using various
technologies is thus an emerging topic of interest in biotechnology
research.
Fluorescent Dyes and Protein Aggregation
[0025] In a fourth method of aggregate analysis, fluorescent dyes
have been used to stain amyloidogenic material in histology, while
insights into the prerequisites and kinetics of amyloid formation
have been obtained by the in vitro analysis of this process using
similar dyes (Volkova et al., 2007, 2008; 2009; Demeule et al.,
2007). The fluorescent probes, thioflavin T and Congo red, have
been the most frequently used dyes to detect the presence of
amyloid deposits. Both the benzothiazole dye thioflavin T and the
symmetrical sulfonated azo dye congo red have been adapted to study
the formation of amyloid fibrils in solution using the fluorescence
properties of these molecules. The amyloid aggregates cause large
enhancements in fluorescence of the dye thioflavin T, exhibit
green-gold birefringence upon binding the dye congo red, and cause
a red-shift in the absorbance spectrum of congo red. Amyloid fibril
detection assays have suffered from several drawbacks, however,
when using thioflavin T, Congo red and their derivatives. For
instance, congo red can bind to native .alpha.-proteins such as
citrate synthase and interleukin-2 (Khurana et al., 2001). As a
consequence of its poor optical properties, the congo red
derivative chrysamine-G only weakly stains neuritic plaques and
cerebrovascular amyloid in postmortem tissue (Klunk et al., 1998).
Furthermore, the binding of dyes can influence the stability of
amyloid aggregates, and the interplay with other components (for
example, during testing of potential amyloid inhibitors) is
unpredictable (Murakami et al., 2003). Importantly, there exists a
great variability among the different amyloid fibrils in their
ability to bind congo red and thioflavin T. Fluorescence intensity
using thioflavin T can vary depending upon the structure and
morphology of the amyloid fibrils (Murakami et al., 2003). Despite
the widespread use of thioflavin T, its application to amyloid
quantification often generates inconsistent and inaccurate results.
Variations in spectral properties caused by buffer conditions and
protein-dye ratios result in poor reproducibility, complicating the
use of thioflavin T for quantitative assessment of fibril
formation. In the absence of other more reliable assays,
investigators have relied heavily upon thioflavin T as a reporter
probe for amyloid protein aggregation. A reliable method for
amyloid quantification likely would be useful not only for
detecting mature amyloid fibrils, but also for monitoring the
kinetics of fibrillogenesis, which is essential for better
understanding of the underlying biophysics and mechanism of the
protein aggregation process. Furthermore, such an assay would be a
tool for discovery and development of therapeutic compounds capable
of blocking protein aggregation.
[0026] Thus the design of new dyes which can selectively interact
with fibrillar amyloidogenic proteins is of substantial importance
for basic research, and has a crucial practical significance for
biotechnology and medicine. Dialkylamino-substituted monomethine
cyanine T-284 and meso-ethyl-substituted trimethine cyanine SH-516
have demonstrated higher emission intensity and selectivity to
aggregated .alpha.-synuclein (ASN) than the classic amyloid stain
thioflavin T; while the trimethinecyanines T-49 and SH-516 exhibit
specifically increased fluorescence in the presence of fibrillar
.beta.-lactoglobulin (BLG) (Volkova et al., 2007). These dyes
demonstrated the same or higher emission intensity and selectivity
to aggregated BLG as thioflavin T. Recently, nile red dye has been
used to detect antibody A aggregate, but it did not stain all types
of protein aggregates, underscoring the need to several analytical
methods in order to assess protein aggregation (Demeule et al.,
2007).
Optimization of Protein Formulations
[0027] Another potential application of a fluorescence based
protein aggregate detection technique relates to pharmaceutical
protein formulations (U.S. Pat. Nos. 6,737,401; 5,192,737;
6,685,940; US Patent Application Publication 2008/0125361 A1). The
physical stability of pharmaceutical protein formulations is of
great importance because there is always a time delay between
production, protein formulation and its subsequent delivery to a
patient. The physical stability of a protein formulation becomes
even more critical when using drug delivery devices to dispense the
protein formulation, such as infusion pumps and the like. When the
delivery device is worn close to the body or implanted within the
body, a patient's own body heat and body motion, plus turbulence
generated in the delivery tubing and pump, impart a high level of
thermo-mechanical stress to a protein formulation. In addition,
infusion delivery devices expose the protein to hydrophobic
interfaces in the delivery syringes and catheters. These
interfacial interactions tend to destabilize the protein
formulation by inducing denaturation of the native structure of the
protein at these hydrophobic interfaces.
[0028] In an optimized protein formulation, the protein should
remain stable for several years, maintaining the active
conformation, even under unfavorable conditions that may occur
during transport or storage. Protein formulation screening needs to
be performed before the assessment of safety, toxicity, ADME
(absorption distribution metabolism excretion), pharmacology and
the testing of biological activity in animals. Currently, protein
formulation in the pharmaceutical industry is generally a slow
process and would benefit from fast formulation screening
approaches that do not require overly complicated instrumentation
techniques.
[0029] The formulation of protein drugs is a difficult and
time-consuming process, mainly due to the structural complexity of
proteins and the very specific physical and chemical properties
they possess. Most protein formulations contain excipients which
are added to stabilize protein structure, such as a particular
buffer system, isotonic substances, metal ions, preservatives and
one or more surfactants, with various concentration ranges to be
tested. The conventional analytical methods usually require a long
period of time to perform, typically twenty or more days, as well
as manual intervention during this period. The development of new
formulations is costly in terms of time and resources. Moreover,
even for a known protein formulation, batch to batch quality
control analysis is often less than optimal using the current state
of the art methods. Therefore, a versatile, reliable, rapid and
resource-efficient analytical method is desired for both developing
novel protein formulations and identifying protein stability in
quality control procedures. The ideal analytical method would be
sensitive, accurate, and linear over a broad range, resistant to
sample-matrix interference, capable of measuring all possible
structural variants of a protein, and compatible with high
throughput screening.
[0030] A high throughput screening (HTS) platform for optimization
of protein formulation has been proposed based upon the use of
multi-well microplates (Capelle Martinus et al., 2009). Basically,
such an HTS platform was envisioned to consist of two components:
(i) sample preparation and (ii) sample analysis. Sample preparation
involves automated systems for dispensing the drug and the
formulation ingredients in both liquid and powder form. The sample
analysis involves specific methods developed for each protein to
investigate physical and chemical properties of the formulations in
the microplates.
[0031] The techniques that could be coupled with such an HTS
platform include UV-Visible absorbance/turbidity, light scatter,
fluorescence intensity, resonance energy transfer, fluorescence
anisotropy, Raman spectroscopy, circular dichroism, Fourier
transform infrared spectroscopy (FTIR), surface plasmon resonance
and fluorescence lifetime. Ideally, however, the analysis technique
should be specific, quantitative, robust, cost-effective, easily
accessed, easy to use and informative. Capelle Martinus et al.
(2009) utilized several assays coupled with HTS to optimize a
salmon calcitonin formulation: turbidity (absorbance at 350 nm),
intrinsic tyrosine fluorescence, 1-anilino-naphthalene-8-sulfonate
(ANS) fluorescence and Nile red fluorescence. Addition of the dyes
(Nile red and ANS) were employed to examine protein conformational
changes. Their findings were in accordance with the salmon
calcitonin formulations that were patented and used commercially,
lending credence to the concept that fluorescent probe-based
approaches can be employed in protein formulation optimization
activities. The use of several complementary analytical methods
permits the selection of formulations using carefully designed
assay criteria. The investigators found that in some cases, an
increase in turbidity was observed without an increase in ANS or
Nile red fluorescence. In other formulations, an increase in
fluorescence was detected without an increase in turbidity. This
suggests that these dyes are not necessarily measuring the exact
same biophysical phenomenon as the turbidity measurements.
Measuring the fluorescence of at least two dyes in combination with
turbidity and intrinsic fluorescence was, therefore,
recommended.
[0032] Among these techniques, fluorescence detection from
externally added dyes, which enhances fluorescence intensity upon
interacting with misfolded or aggregated protein, is most
attractive, because this technique requires minimum protein
concentration due to its high sensitivity and simple implementation
on a microplate reader.
[0033] Real time stability testing of a particular formulation may
demonstrate no immediately apparent effect on physical or chemical
stability. Accelerated stability testing can help, therefore, in
facilitating the determination of the most suitable excipients and
concentrations. Storage at different target temperatures
(0-50.degree. C.), illumination of samples, mechanical stress
(i.e., agitation that simulates handling and transportation),
multiple freeze-thaw cycles (mimicking frozen storage, freeze
drying), oxygen purging, increased humidity and seeding are
different ways to accelerate protein degradation.
[0034] High throughput spectroscopy is a fast and versatile method
for initial screening of the physical stability of protein
formulations. The microplate well-based platform could be enhanced
with accelerated stress testing and methods to determine chemical
stability, e.g., electrophoresis, HPLC, mass spectrometry. For
instance, thioflavin T has been used to select and optimize
FDA-approved surfactant(s) in insulin formulations using
magnetically stirring to accelerate insulin aggregation (U.S. Pat.
No. 6,737,401).
Thermal Shift Assay
[0035] Fluorescent dyes have been used to monitor protein stability
by systematically varying the temperature of test samples, also
known as the Thermofluor.RTM. technique (U.S. Pat. No. 6,020,141;
Matulis et al., 2005; Mezzasalma et al., 2007; Volkova et al.,
2008; Ericsson et al., 2006; Todd et al., 2005). Protein stability
can be altered by various additives including but not limited to
excipients, salts, buffers, co-solvents, metal ions, preservatives,
surfactants, and ligands. Protein stability can be shifted by
various stresses, including elevated temperature, referred to as
thermal shift, or chemical denaturants, such as urea, guanidine
isocyanate or similar agents. A protein stability shift assay
offers a wide spectrum of applications in the investigation of
protein refolding conditions, optimization of recombinant protein
expression/purification conditions, protein crystallization
conditions, selection of ligand/drug/vaccine/diagnostic reagents
and protein formulations.
[0036] The classic thermal shift technology utilizes the dye
SYPRO.RTM. Orange and involves the use of a melting point device to
raise the temperature stepwise (Raibekas, 2008). Thermal shift
technology is coupled with aggregation detection technologies, such
as light scattering technology or internal fluorescence from
protein (such as tyrosine or tryptophan) to monitor protein
aggregation and unfolding respectively. This type of technology
usually requires a high protein concentration, therefore, it is not
cost-effective. In addition, thermal shift technology cannot work
effectively on formulations with low protein concentrations or
finalize protein formulations which require a very low detection
limit (typically .about.1-5% protein aggregates).
Fluorometric Screening Assay for Protein Disulfide Isomerase
(PDI)
[0037] Protein disulfide isomerase (PDI, EC5.3.4.1) is a 57-kDa
enzyme expressed at high levels in the endoplasmic reticulum (ER)
of eukaryotic cells (Ferrari and Soling, 1999). PDI was the first
enzyme known to possess the disulfide isomerase activity and has
been well characterized over the past three decades. In ER, PDI
catalyzes both the oxidation and isomerization of disulfides of
nascent polypeptides. Under the reducing condition of the
cytoplasm, endosomes and cell surface, PDI catalyzes the reduction
of protein disulfide bonds.
[0038] Folding catalysts such as PDI and peptidylprolyl isomerase
accelerate slow chemical steps that accompany folding. Disulfide
bond formation can occur quite rapidly, even before the completion
of synthesis, but for some proteins disulfide bond formation is
delayed and occurs post-translationally. PDI catalyzes disulfide
formation and rearrangement by thiol/disulfide exchange during
protein folding in the ER. As a member of the thioredoxin
superfamily, which also includes homologs such as ERp57, PDIp,
ERp72, PDIr and ERp5, PDI has two independent but non-equivalent
active sites, with one positioned close to the C-terminus and
another close to the N-terminus. Each site possesses two cysteine
residues (CGHC) that cycle between the dithiol and disulfide
oxidation states. The disulfide bond at the active site of PDI is a
good oxidant that directly introduces a disulfide bond into protein
substrates. The dithiol redox state is essential for catalyzing
disulfide rearrangements. The necessity of having oxidized and
reduced active sites for catalysis of different steps results in a
redox optimum. Besides its major role in the processing and
maturation of secretory proteins in ER, PDI and its homologs have
been implicated in other important cellular processes. For example,
cellular insulin degradation occurs in a sequential fashion with
several identified steps. The initial degradative step occurs in
endosomes with two or more cleavages in the B chain occurring. This
is followed by reduction of disulfide bonds by PDI, or a related
enzyme, generating an intact A chain and fragments of B chain. The
insulin fragments are further cleaved by multiple proteolytic
systems, such as the lysosomal degradation pathway.
[0039] PDI and its homologs also play roles in the processing and
maturation of various secretory and cell surface proteins in the ER
following their synthesis. Several in vitro studies have also
suggested a chaperone function of PDI, to assist in protein folding
or refolding. During ER stress, as for example during hypoxia in
endothelial cells and astrocytes in the cerebral cortex, PDI is
up-regulated. This indicates that PDI is involved in protecting
cells under pathological or stressful conditions.
[0040] Besides ER, PDI also exists on many cell surfaces, such as
endothelial cells, platelets, lymphocytes, hepatocytes, pancreatic
cells and fibroblasts. For the reductive activity of plasma
membrane, PDI is required for endocytosis of certain exogenous
macromolecules. The cytotoxicity of diphtheria toxin is blocked by
PDI inhibitors, which block the cleavage of the inter-chain
disulfide bonds in the toxin. PDI also mediates reduction of
disulfide bonds in human immunodeficiency virus envelope
glycoprotein 120, which is essential for infectivity. PDI
inhibitors can thus prevent virus entry into cells. Such functional
activities make PDI and its homologs attractive drug targets.
[0041] Biochemical assays related to measuring PDI activity have
been described:
[0042] (1) ScRNase assay: PDI converts scrambled (inactive) RNase
into native (active) RNase that further acts on its substrate. The
reported sensitivity of the assay is in the micromolar range (Lyles
& Gilbert, 1991).
[0043] (2) The Insulin Turbidity Assay: PDI breaks the two
disulfide bonds between the two insulin chains (A and B) that
results in precipitation of the B chain. This precipitation can be
monitored by measuring turbidity (absorbance at 620 nm), which in
turn indicates PDI activity. Sensitivity of this assay is in the
micromolar range (Lundstrom & Holmgren, 1990). Recently an
end-point, high throughput screening assay of PDI isomerase
activity based on enzyme-catalyzed reduction of insulin in the
presence of dithiothreitol using hydrogen peroxide as a stop
reagent has been developed (Smith et al., 2004; U.S. Pat. No.
6,977,142).
[0044] (3) The Di-E-GSSG assay: This is the fluorometric assay that
can detect picomolar quantities of PDI and is, therefore,
considered the most sensitive assay to date for detecting PDI
activity. Di-E-GSSG has two eosin molecules attached to oxidized
glutathione (GSSG). The proximity of eosin molecules leads to the
quenching of its fluorescence. Upon breakage of the disulfide bond
by PDI, however, fluorescence increases 70 fold (Raturi & Mutus
2007). Certain common excipients can cause signal generation as
well, such as 2-mercaptoethanol and dithiothreitol.
[0045] In view of the important functional activities of PDI and
homologous enzymes, sensitive, real-time, high throughput methods
that are time and cost-effective are highly desirable.
Chaperone/Anti-Chaperone Activity
[0046] A chaperone is a protein that can assist unfolded or
incorrectly folded proteins to attain their native state by
providing a microenvironment in which losses due to competing
folding and aggregation reactions are reduced (Puig & Gilbert,
1994). Chaperones also mediate the reversibility of pathways
leading to incorrectly folded structures. One of the major
complications encountered in both in vitro and in vivo protein
folding is aggregation resulting from the commonly encountered low
solubility of the unfolded protein or different folding
intermediates. The efficiency of folding depends upon how the
unfolded protein partitions between pathways leading to aggregation
and pathways leading to the native structure. In vivo, the
partitioning between productive and non-productive folding pathways
may be influenced by "foldases" and molecular chaperones. Foldases
accelerate folding by catalyzing the slow chemical steps, such as
disulfide bond formation and proline isomerization that may retard
folding. Molecular chaperones do not appreciably accelerate folding
but bind to nonnative proteins in a way that is thought to inhibit
non-productive aggregation and misfolding. In order to prevent
these improper interactions, chaperones must be present at
concentrations that are stoichiometric with the newly synthesized
proteins. Consequently, chaperones are often found at very high
concentrations in the cell.
[0047] PDI is a very abundant protein within cells. Although
primarily classified as a foldase, PDI has also been shown to
possess chaperone or anti-chaperone activity (Puig & Gilbert,
1994). PDI accelerates lysozyme folding, and at high concentration,
it displays a chaperone-like activity that prevents lysozyme
misfolding and aggregation. In addition, PDI also exhibits an
unusual "anti-chaperone" activity. Under conditions that favor
lysozyme aggregation, low concentrations of PDI greatly reduce the
yield of native lysozyme and facilitate the formation of aggregates
that are extensively cross-linked by intermolecular disulfide
bonds. Similarly, PDI breaks the two disulfide bonds between two
insulin chains (A and B) that results in precipitation of The B
chain, thus serving as an "anti-chaperone in this case." (Lundstrom
& Holmgren. 1990.
[0048] Alpha-crystallin, a major protein component of the mammalian
lens of the eye, belongs to the heat shock protein (Hsp) family and
acts as a molecular chaperone by preventing aggregation of target
proteins (e.g. beta and gama-crystallins) under stress conditions
through the formation of stable, soluble high-molecular mass
complexes with them. Aggregation of BLG (beta-lactoglobulin) occurs
mainly via intermolecular disulfide bond exchange. Upon heating,
BLG aggregates, which can be accelerated by subjecting the protein
to either an elevated pH or through the additional of DTT.
.alpha.-crystallin prevents heat-induced BLG aggregation, acting as
a chaperone in the absence of DTT; in the presence of DTT, however,
this chaperone activity is less efficient due to faster aggregation
of heated and reduced beta-lactoglobulin. Another Hsp protein, Hsp
27, protects myosin 51 from heat-induced aggregation, but not from
thermal denaturation and ATPase inactivation.
[0049] Highly sensitive fluorescent probes useful to monitoring
various protein functions relating to aggregation should assist in
formulation optimization. Preferably, these probes should be
applicable to a broad ranges of proteins and concentrations even in
the presence of excipients, salts and buffers, providing sensitive
limits of detection and excellent linear dynamic ranges.
BRIEF SUMMARY OF THE INVENTION
[0050] The present invention provides dyes, reagents and methods
useful for detection of protein aggregates.
[0051] In some embodiments, a compound is provided. The compound
comprises the structure
##STR00001##
[0052] wherein m and n are independently 1, 2 or 3;
[0053] wherein L is a linker arm comprising carbon, sulfur, oxygen,
nitrogen, or any combination thereof;
[0054] wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.9,
R.sub.10, R.sub.11, R.sub.12, R.sub.13, R.sub.14, R.sub.15,
R.sub.16, R.sub.19, R.sub.20, R.sub.21 and R.sub.22 are
independently hydrogen, halogen, amino, ammonium, nitro, sulfo,
sulfonamide, carboxy, ester, cyano, phenyl, benzyl, an alkyl group
wherein the alkyl group is saturated or unsaturated, linear or
branched, substituted or unsubstituted, an alkoxy group wherein the
alkoxy group is saturated or unsaturated, branched or linear,
substituted or unsubstituted, or when taken in combination R.sub.1
and R.sub.2, or R.sub.3 and R.sub.4, or R.sub.9 and R.sub.10, or
R.sub.11 and R.sub.12, or R.sub.13 and R.sub.14, or R.sub.15 and
R.sub.16, or R.sub.19 and R.sub.20, or R.sub.21 and R.sub.22 form a
five or six membered ring wherein the ring is saturated or
unsaturated, substituted or unsubstituted, and wherein R.sub.9 and
R.sub.10, or R.sub.11 and R.sub.12, or R.sub.13 and R.sub.14, or
R.sub.15 and R.sub.16 can comprise alkyl chains that are joined
together, wherein a quinoline moiety can be formed;
[0055] wherein R.sub.7, R.sub.8, R.sub.17 and R.sub.18 are
independently hydrogen, Z, an alkyl group wherein the alkyl group
is saturated or unsaturated, linear or branched, substituted or
unsubstituted, an alkoxy group wherein the alkoxy group is
saturated or unsaturated, branched or linear, substituted or
unsubstituted, or when taken together, R.sub.7 and R.sub.8 and
R.sub.17 and R.sub.18, may form a 5 or 6 membered ring wherein the
ring is saturated or unsaturated, substituted or unsubstituted;
[0056] wherein Z comprises a carboxyl group (CO.sub.2.sup.-), a
carbonate ester (COER.sub.25), a sulfonate (SO.sub.3.sup.-), a
sulfonate ester (SO.sub.2ER.sub.25), a sulfoxide (SOR.sub.25), a
sulfone (SO.sub.2CR.sub.25R.sub.26R.sub.27), a sulfonamide
(SO2NR.sub.25R.sub.26), a phosphate (PO.sub.4.sup.=), a phosphate
monoester (PO.sub.3.sup.-ER.sub.25), a phosphate diester
(PO.sub.2ER.sub.25ER.sub.26), a phosphonate (PO.sub.3.sup.=) a
phosphonate monoester (PO.sub.2.sup.-ER.sub.25) a phosphonate
diester (POER.sub.25ER.sub.26), a thiophosphate (PSO.sub.3.sup.=),
a thiophosphate monoester (PSO.sub.2.sup.-ER.sub.25) a
thiophosphate diester (PSOER.sub.25ER.sub.26), a thiophosphonate
(PSO.sub.2.sup.=), a thiophosphonate monoester (PSO.sup.-ER.sub.25)
a thiophosphonate diester (PSER.sub.25ER.sub.26), a phosphonamide
(PONR.sub.25R.sub.26NR.sub.28R.sub.29), its thioanalogue
(PSNR.sub.25R.sub.26NR.sub.28R.sub.29), a phosphoramide
(PONR.sub.25R.sub.26NR.sub.27NR.sub.28R.sub.29), its thioanalogue
(PSNR.sub.25R.sub.26NR.sub.27NR.sub.28R.sub.29), a phosphoramidite
(PO.sub.2R.sub.25NR.sub.28R.sub.29) or its thioanalogue
(POSR.sub.25NR.sub.28R.sub.29) where E can be independently O or S;
[0057] wherein R.sub.25, R.sub.26, R.sub.27, R.sub.28, and R.sub.29
are independently a hydrogen, an unsubstituted straight-chain,
branched or cyclic alkyl, alkenyl or alkynyl group, a substituted
straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group
wherein one or more C, CH or CH.sub.2 groups are substituted with
an O atom, N atom, S atom, or NH group, or an unsubstituted or
substituted aromatic group; [0058] wherein Z is attached directly,
or indirectly through a second linker arm comprising carbon,
sulfur, oxygen, nitrogen, and any combinations thereof and wherein
the second linker arm may be saturated or unsaturated, linear or
branched, substituted or unsubstituted or any combinations thereof;
and
[0059] wherein R.sub.5, R.sub.6, R.sub.23 and R.sub.24 can
independently be hydrogen or an alkyl group wherein the alkyl group
is saturated or unsaturated, linear or branched, substituted or
unsubstituted, or when taken in combination R.sub.5 and R.sub.6 or
R.sub.2 and R.sub.5 or R.sub.3 and R.sub.6 or R.sub.23 and R.sub.24
or R.sub.22 and R.sub.23 or R.sub.20 and R.sub.24 form a five or
six membered ring wherein the ring is saturated or unsaturated,
substituted or unsubstituted.
[0060] In other embodiments, a compound is provided that exhibits
at least three times increased fluorescence in the presence of an
aggregated form of a protein when compared to the fluorescence
exhibited when the compound is in the presence of the unaggregated
form of the protein. In some embodiments, the compound is D95, D97,
L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43,
S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7,
TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150,
YAT2213, YAT2214 or YAT2324.
[0061] A multi-dye composition comprising at least three dyes is
also provided. In this composition, each of the at least three dyes
exhibits increased fluorescence in the presence of an aggregated
form of a protein when compared to the fluorescence exhibited when
the compound is in the presence of the unaggregated form of the
protein.
[0062] Further provided is a multi-dye composition comprising two
or more dyes. In this composition, at least one of the two or more
dyes comprises Dye F, Dye Fm(b), D95, D97, L-30, L-33, Lu-1, Lu-2,
S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592,
Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3,
YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 or
YAT2324.
[0063] A reactive compound comprising at least one compound from
Table 1B or Table 2B is additionally provided. In these
embodiments, the compound is modified by the addition of a reactive
group.
[0064] Additionally, a labeled target molecule is provided. The
labeled target molecule comprises a target molecule attached to the
above-described reactive compound through the reactive group.
[0065] A solid support attached to the above-described reactive
compound through the reactive group is also provided.
[0066] A kit for assaying aggregation of a protein is also
provided. The kit comprises in packaged combination: (a) the
above-described compound, and (b) instructions for using the
compound for assaying aggregation of a protein.
[0067] Another kit for assaying aggregation of a protein is
additionally provided. The kit comprises in packaged combination:
(a) two or more compounds, wherein each of compound exhibits
increased fluorescence in the presence of an aggregated form of a
protein when compared to the fluorescence produced when the
compound is in the presence of the unaggregated form of the
protein, and (b) instructions therefor.
[0068] Additionally provided is a method for detecting an aggregate
of a protein in a sample. The method comprises (a) combining the
sample with the above-described compound or multidye composition;
(b) measuring the amount of fluorescence in the mixture;
[0069] (c) comparing the amount of fluorescence determined in (b)
with the amount of fluorescence in [0070] (i) a mixture of the
compound or multidye composition with a control sample without
aggregated protein, or [0071] (ii) a mixture of the compound or
multidye composition with a known standard quantity of aggregated
protein; and
[0072] (d) determining the aggregation of the protein in the sample
based on the comparison in (c).
[0073] A method for separating aggregates of a protein from
monomeric forms of the protein in a sample is also provided. The
method comprises (a) combining the sample to the above-described
solid support under conditions where aggregates of the protein
preferentially bind to the compound; and (b) separating sample
protein bound to the solid support from unbound protein. In this
method, protein bound to the solid support are substantially
aggregates and unbound protein is substantially monomers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0074] FIG. 1, Panels A-D, are micrographs demonstrating IgG
stability in two different buffer formulations.
[0075] FIG. 2 is a graph showing the fluorescence of various dye
concentrations with 20 .mu.M of aggregated lysozyme.
[0076] FIGS. 3A-B are graphs showing the effect of pH on
fluorescent detection sensitivity and linearity for different
probes of the invention.
[0077] FIG. 4 is a graph showing the linear dynamic range of
lysozyme aggregate detection using a two dye combination ST (S25
and Tol3) compared with thioflavin T.
[0078] FIG. 5 is a graph showing the effective linear dynamic range
of antibody aggregate detection using a two dye combination ST (S25
and Tol3) compared with thioflavin T.
[0079] FIGS. 6A-D are graphs showing protein aggregate detection as
a function of various protein species with the dyes S25, Tol3 and
thioflavin T.
[0080] FIG. 7 is a graph showing the kinetics of lysozyme
aggregation monitored with dyes S25, Tol3, Thioflavin T and the two
dye combination ST (S25 and Tol3).
[0081] FIG. 8 is a graph showing the kinetics of IgG aggregation as
a function of temperature.
[0082] FIG. 9 is a graph showing IgG aggregation induced by
temperature (50.degree. C.) as a function of pH.
[0083] FIGS. 10A-C are graphs of a high-throughput protein
formulation optimization workflow using IgG and the two dye
combination ST (S25 and Tol3).
[0084] FIG. 11 is a graph showing measurement of the inhibition of
Lysozyme aggregation by Chitotriose.
[0085] FIG. 12 is a graph showing a thermal shift assay of BLG
aggregation using a dye of the present invention.
[0086] FIGS. 13A-B are graphs showing a thermal shift assay of
carbonic anhydrase II aggregation at two different pH values using
a dye of the present invention.
[0087] FIGS. 14A-B are graphs comparing the fluorescence response
between unfolded and aggregated forms of IgG.
[0088] FIGS. 15A-C are graphs showing PDI activity monitored by
turbidity and by a fluorometric assay using a dye of the present
invention.
[0089] FIG. 16 is a graph showing activity assay of Hsp 27 (heat
shock protein) as a chaperone preventing .beta.-lactoglobulin (BLG)
aggregation induced by heat.
[0090] FIG. 17 is a graph showing fluorescence of IgG aggregates
induced by stirring using a dye combination of the present
invention.
[0091] FIG. 18, Panels A-B, are fluorescence micrographs of control
cells (A) and cells treated with dye YAT2150 (B).
[0092] FIG. 19, Panels A-D are fluorescence micrographs of control
cells (A) and cells treated with proteasome inhibitors and dye
YAT2150.
[0093] FIGS. 20A-C are fluorescence micrographs of cells treated
with various dyes to show that dye YAT2150 co-localizes with
ubiquitin.
[0094] FIG. 21, Panels A-D, are fluorescence micrographs of control
cells (A) and cells treated with amyloid beta peptide 1-42 (B, C,
D) with (C, D) or without (B) treatment with SMER28, an inducer of
autophagy.
[0095] FIGS. 22A (Panels A and B) and 22B (Panels A and B) are
fluorescence micrographs of control cells of normal or Alzheimer's
disease brain tissue after staining with thioflavin T (A) or
YAT2150.
[0096] FIGS. 23A (Panels A and B) and 23B (Panels A and B) are
fluorescence micrographs showing that dye YAT2150 co-localized with
the Tau-13 protein in post-mortem brain tissue of Alzheimer's
disease patients.
[0097] FIGS. 24A-B are graphs comparing YAT2150 (ProteoStat.RTM.)
with fluorescein-p62 antibody for identifying aggresomes by flow
cytometry.
DETAILED DESCRIPTION OF THE INVENTION
[0098] As used herein, the singular forms "a", "an" and "the" are
intended to include the plural forms as well, unless the context
clearly indicates otherwise. Additionally, the use of "or" is
intended to include "and/or", unless the context clearly indicates
otherwise.
[0099] The present invention provides dyes, reagents and methods
that are useful for detecting protein aggregates. In some
embodiments, the invention provides a family of dimeric styryl dyes
containing either a picoline or lepidine ring and a dialkyl amino
or alkyloxy substituent. The dyes of the invention are useful for
generating fluorescence signals that depend upon the presence of an
aggregated form of a protein, while conveying minimal levels of
signals when only the native form of the protein is present. A
number of novel dimeric styryl dyes having these properties are
also disclosed. Other dyes have been described previously in the
context of binding to nucleic acids, but it has been discovered
that many of these dyes demonstrate a useful property where an
enhanced level of fluorescence is produced after binding to
aggregated forms of proteins compared to the level that is emitted
in the presence of the native forms. Some of these dyes also
exhibit large Stokes shifts between their absorption and emission
wavelength optima thereby increasing the ease of detection.
[0100] Thus, in some embodiments, a compound is provided. The
compound comprises the structure
##STR00002##
[0101] wherein m and n are independently 1, 2 or 3;
[0102] wherein L is a linker arm comprising carbon, sulfur, oxygen,
nitrogen, or any combination thereof;
[0103] wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.9,
R.sub.10, R.sub.11, R.sub.12, R.sub.13, R.sub.14, R.sub.15,
R.sub.16, R.sub.19, R.sub.20, R.sub.21 and R.sub.22 are
independently hydrogen, halogen, amino, ammonium, nitro, sulfo,
sulfonamide, carboxy, ester, cyano, phenyl, benzyl, an alkyl group
wherein the alkyl group is saturated or unsaturated, linear or
branched, substituted or unsubstituted, an alkoxy group wherein the
alkoxy group is saturated or unsaturated, branched or linear,
substituted or unsubstituted, or when taken in combination R.sub.1
and R.sub.2, or R.sub.3 and R.sub.4, or R.sub.9 and R.sub.10, or
R.sub.11 and R.sub.12, or R.sub.13 and R.sub.14, or R.sub.15 and
R.sub.16, or R.sub.19 and R.sub.20, or R.sub.21 and R.sub.22 form a
five or six membered ring wherein the ring is saturated or
unsaturated, substituted or unsubstituted, and wherein R.sub.9 and
R.sub.10, or R.sub.11 and R.sub.12, or R.sub.13 and R.sub.14, or
R.sub.15 and R.sub.16 can comprise alkyl chains that are joined
together, wherein a quinoline moiety can be formed;
[0104] wherein R.sub.7, R.sub.8, R.sub.17 and R.sub.18 are
independently hydrogen, Z, an alkyl group wherein the alkyl group
is saturated or unsaturated, linear or branched, substituted or
unsubstituted, an alkoxy group wherein the alkoxy group is
saturated or unsaturated, branched or linear, substituted or
unsubstituted, or when taken together, R.sub.7 and R.sub.8 and
R.sub.17 and R.sub.18, may form a 5 or 6 membered ring wherein the
ring is saturated or unsaturated, substituted or unsubstituted;
[0105] wherein Z comprises a carboxyl group (CO.sub.2.sup.-), a
carbonate ester (COER.sub.25), a sulfonate (SO.sub.3.sup.-), a
sulfonate ester (SO.sub.2ER.sub.25), a sulfoxide (SOR.sub.25), a
sulfone (SO.sub.2CR.sub.25R.sub.26R.sub.27), a sulfonamide
(SO2NR.sub.25R.sub.26), a phosphate (PO.sub.4.sup.=), a phosphate
monoester (PO.sub.3.sup.-ER.sub.25), a phosphate diester
(PO.sub.2ER.sub.25ER.sub.26), a phosphonate (PO.sub.3.sup.=) a
phosphonate monoester (PO.sub.2.sup.-ER.sub.25) a phosphonate
diester (POER.sub.25ER.sub.26), a thiophosphate (PSO.sub.3.sup.=),
a thiophosphate monoester (PSO.sub.2.sup.-ER.sub.25) a
thiophosphate diester (PSOER.sub.25ER.sub.26), a thiophosphonate
(PSO.sub.2.sup.=), a thiophosphonate monoester (PSO.sup.-ER.sub.25)
a thiophosphonate diester (PSER.sub.25ER.sub.26), a phosphonamide
(PONR.sub.25R.sub.26NR.sub.28R.sub.29), its thioanalogue
(PSNR.sub.25R.sub.26NR.sub.28R.sub.29), a phosphoramide
(PONR.sub.25R.sub.26NR.sub.27NR.sub.28R.sub.29), its thioanalogue
(PSNR.sub.25R.sub.26NR.sub.27NR.sub.28R.sub.29), a phosphoramidite
(PO.sub.2R.sub.25NR.sub.28R.sub.29) or its thioanalogue
(POSR.sub.25NR.sub.28R.sub.29) where E can be independently O or S;
[0106] wherein R.sub.25, R.sub.26, R.sub.27, R.sub.28, and R.sub.29
are independently a hydrogen, an unsubstituted straight-chain,
branched or cyclic alkyl, alkenyl or alkynyl group, a substituted
straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group
wherein one or more C, CH or CH.sub.2 groups are substituted with
an O atom, N atom, S atom, or NH group, or an unsubstituted or
substituted aromatic group; [0107] wherein Z is attached directly,
or indirectly through a second linker arm comprising carbon,
sulfur, oxygen, nitrogen, and any combinations thereof and wherein
the second linker arm may be saturated or unsaturated, linear or
branched, substituted or unsubstituted or any combinations thereof;
and
[0108] wherein R.sub.5, R.sub.6, R.sub.23 and R.sub.24 can
independently be hydrogen or an alkyl group wherein the alkyl group
is saturated or unsaturated, linear or branched, substituted or
unsubstituted, or when taken in combination R.sub.5 and R.sub.6 or
R.sub.2 and R.sub.5 or R.sub.3 and R.sub.6 or R.sub.23 and R.sub.24
or R.sub.22 and R.sub.23 or R.sub.20 and R.sub.24 form a five or
six membered ring wherein the ring is saturated or unsaturated,
substituted or unsubstituted.
[0109] In many of these embodiments, the compound exhibits
increased fluorescence in the presence of an aggregated form of a
protein when compared to the fluorescence exhibited when the
compound is in the presence of the unaggregated form of the
protein.
[0110] These compounds can be modified by the addition of charged
groups, as exemplified by sulfonates, phosphates, phosphonates and
their derivatives and/or polar groups as exemplified by sulfoxide,
sulfone and sulfonamide moieties.
[0111] It is also understood that when a dye comprises an anionic
group, there will also be a cationic counterion present. Any cation
may serve this purpose as long as it does not interfere with the
use of the dye. Examples of cations that may serve as counterions
can include but are not limited to hydrogen, sodium, potassium,
lithium, calcium, cesium, ammonium, alkyl ammonium, alkoxy ammonium
and pyridinium. It is also understood that when a dye comprises a
cationic group, there will also be an anionic counterion present.
Any anion may serve this purpose as long as it doesn't interfere
with the use of the dye. Examples of anions that may serve as
counterions can include but not be limited to perchlorate
(ClO.sub.4.sup.-), sulfate (SO.sub.4.sup.=), sulfonate, alkane
sulfonate, aryl sulfonate, phosphate, tosylate, mesylate and
tetrafluoroborate moieties and halides such as a bromide, chloride,
fluoride and iodide. In some cases the counterion or counterions
are provided by the dye being a salt where they exist as separate
ionic species. In other cases, the counterion or counterions may be
present as part of the compound (sometimes called inner salts). It
is understood that there may also be a combination of ions that are
provided by the compound and salts. With regard to acid moieties
that are shown in forms such as COOH it is also understood that
these compounds may be found in ionized forms such as
COO.sup.-.
[0112] It should also be appreciated by those skilled in the art
that the stoichiometric number of counterion or counterions which
balance the charge or charges on the compound can be the same or
they can be different provided that the counterions balance the
charge(s) on the compound. The combination of counterions can be
selected from any of the above mentioned anions. This applies for
the combination of cations also.
[0113] It should be further appreciated by those skilled in the art
that the foregoing descriptions of the anions and their
stoichiometric number and/or combination are applicable to the
compounds and dyes of the present invention, and to methods which
use these compounds and dyes.
[0114] Alkyl or alkoxy R groups in the above compounds may be
substituted or unsubstituted. Examples of substitutions can include
but are not limited to one or more fluorine, chlorine, bromine,
iodine, hydroxy, carboxy, carbonyl, amino, cyano, nitro or azido
groups as well as other alkyl or alkoxy groups. The length of the
alkoxy groups may be as desired. For instance, they may
independently comprise from 1 to 18 carbons in length. They may be
shorter as well, for instance they may be only 1 to 6 carbons in
length in a dye molecule of the present invention.
[0115] The polar groups, charged groups and other substituents may
be connected to the dye directly or they may be connected by a
linker arm comprising carbon, nitrogen, sulfur, oxygen or any
combination thereof. The linker arm may be saturated or
unsaturated, linear or branched, substituted or unsubstituted as
well as any combination of the foregoing.
[0116] As described above some of the R groups may be joined
together to form one or more fused 5 or 6 membered ring structures.
It is understood that the complex rings that are formed by closure
of R groups may be further substituted with any of the R groups
described previously. Examples of complex rings that may be formed
for the picoline or lepidine portion of the cyanine dyes of the
invention can comprise but not be limited to:
##STR00003##
[0117] Examples of rings and complex rings that may be part of the
styryl portion of the dye can comprise but not be limited to:
##STR00004##
[0118] In various embodiments, the compound comprises the
structure
##STR00005##
[0119] In some of these embodiments, each of R.sub.5, R.sub.6,
R.sub.23 and R.sub.24 are a methyl or an ethyl moiety.
[0120] As described in Example 1, numerous compounds having the
above structure, as well as other compounds, were tested for the
ability to exhibit increased fluorescence in the presence of an
aggregated form of a protein (human .alpha.-synuclein) when
compared to the fluorescence exhibited when the compound is in the
presence of the unaggregated form of the protein. The excitation
and emission wavelength in the presence and absence of the protein
aggregate was also determined. Results of these tests, and the
structures of the tested compounds, are provided in Tables 1 and 2.
Table 1 gives results where the compounds exhibited a ratio of 3 or
more for fluorescence from binding to protein aggregates compared
to being in the presence of monomeric protein; Table 2 gives
results with other compounds.
TABLE-US-00001 TABLE 1 Compounds tested that exhibit a ratio of 3
or more for fluorescence from binding to protein aggregates
compared to being in the presence of monomeric protein. A.
Properties of compounds. Dye Dye Dye with Dye with Alone: Alone:
Aggregate: Aggregate: Fluore- Fluore- Fluore- .lamda..sub.Ex
.lamda..sub.Em .lamda..sub.Ex .lamda..sub.Em cence cence cence
I.sub.Agg/ Dye (nm) (nm) (nm) (nm) I.sub.dye I.sub.Mono I.sub.Agg
I.sub.Mono S25 485 613 516 607 3.4 4.6 87.3 19.0 S43 527 637 550
623 0.35 0.58 27.3 47 TOL3 471 611 511 603 2.7 2.7 40.2 14.9 Yat
500 620 535 613 4.2 4.9 63.2 12.9 2134 Yat 520 632 553 625 1.2 3.4
53 15.6 2148 Yat 502 614 534 617 0.6 0.7 29.5 42 2149 Yat 485 612
515 610 6.7 9.7 42.3 4.4 2150 F 460 610 518 607 3.6 3.4 57.4 16.9
L-33 465 527 462 504 7.7 7.6 53 7.0 S49 501 584 524 576 2.2 2.2
20.1 9.1 S33 479 616 513 611 5.5 5.7 19.4 3.4 TOL- 389 539 554 603
10 6.5 22.5 3.5 11 SL- 491 578 516 578 7.4 7.5 30.3 4.1 2131 SL-
401 608 400 608 7.5 8 25.4 7.5 2592 Tio-1 494 578 526 578 2.8 2.8
19 6.8 S-13 568 662 580 670 0.2 0.2 3.1 15.5 L-30 457 515 478 512
1.5 1.8 8 4.4 YA-1 446 491 461 498 7.7 11 45 4.1 YA-3 460 514 456
537 6.4 7.9 24.2 3.1 (Diph40) TOL-2 527 595 566 600 0.5 0.5 3.5 7
[T-33] TOL-5 428 581 460 535 3.8 4.5 22.2 4.9 Dil-10 548 595 564
599 3.7 3.6 15.3 4.3 [TOL-7] S-39 540 599 577 605 3.1 3.3 11.3 3.4
Fm [b] 461 610 504 597 5.5 5.5 21 3.8 S-42 547 600 559 603 1.1 1.1
5 4.5 S-48 491 581 527 588 2.3 2.3 15.8 6.9 TOL-6 501 559 512 559
4.6 4.6 17.5 3.8 Lu-1 453 583 452 526 7.3 7.6 24 3.2 Lu-2 473 506
485 503 3.8 3 14 4.7 Yat2135 500 618 540 620 0.9 0.7 12.5 17.9
Yat2214 507 626 549 625 1.5 1.4 6.4 4.6 Yat2213 483 622 540 622 0.6
1.2 5.5 4.6 D-95 450 585 555 598 0.6 1 8.4 8.4 D-97 516 650 587 650
3.6 3.7 13.4 3.6 S-8 547 671 566 667 0.8 0.8 2.8 3.5 Yat2324 500
619 551 619 0.8 0.7 7.1 10.1 S-22 543 598 562 602 0.5 0.8 2.4 3.0
B. Structures of compounds. Dye Structure S25 ##STR00006## S43
##STR00007## TOL3 ##STR00008## Yat 2134 ##STR00009## Yat 2148
##STR00010## Yat 2149 ##STR00011## Yat 2150 ##STR00012## F
##STR00013## L-33 ##STR00014## S49 ##STR00015## S-33 ##STR00016##
TOL-11 ##STR00017## S-22 ##STR00018## SL-2131 ##STR00019## SL-2592
##STR00020## Tio-1 ##STR00021## S-13 ##STR00022## L-30 ##STR00023##
YA-1 ##STR00024## YA-3 (Diph40) ##STR00025## TOL-2 [T-33]
##STR00026## TOL-5 ##STR00027## Dil-10 [TOL-7] ##STR00028## S-39
##STR00029## Fm [b] ##STR00030## S-42 ##STR00031## S-48
##STR00032## TOL-6 ##STR00033## Lu-1 ##STR00034## Lu-2 ##STR00035##
Yat-2135 ##STR00036## Yat-2214 ##STR00037## Yat-2213 ##STR00038##
D-95 ##STR00039## D-97 ##STR00040## S-8 ##STR00041## Yat2324
##STR00042##
TABLE-US-00002 TABLE 2 Compounds tested that exhibit a ratio of
less than 3 or more for fluorescence from binding to protein
aggregates compared to being in the presence of monomeric protein.
A. Properties of compounds Dye with Dye with Dye alone: Dye alone:
aggregate: aggregate: Fluorescence Excitation Emission Excitation
Emission enhancement: wavelength wavelength wavelength wavelength
Aggregate/ Dye (nm) (nm) (nm) (nm) monomer S-11 531 594 560 600 2.6
S-12 539 597 553 599 2.2 SH-330 393 278 398 483 1.6 SH-654 370 443
359 434 0.91 SH-675 445 472 449 475 1.8 SH-975 471 631 471 630 1.8
SH-1036 478 611 464 605 2.7 Sl-2599 468 564 468 569 1.1 Sl-2600 518
535 518 536 1.9 S-7 460 612 465 609 1.1 L-28 460 654 572 577 1.4
L-31 450 527 462 534 2.7 TOL-4 488 665 458 654 1.4 TOL-10 394 544
397 539 1.4 S-26 532 593 562 602 2.7 S-29 543 597 554 600 1.6 S-44
498 586 525 582 2.8 S-45 534 596 558 600 2.3 Dbt-5 539 597 545 598
2.2 [TOL-9] S-30 530 598 570 600 1.9 Sip-7 397 576 397 576 1.2
[TOL-12] S-28 384 608 384 608 1 S-23 464 546 471 553 1.2 SH-1070
408 500 408 480 1.2 Yat2212 485 623 530 620 2.6 D-91 395 520 396
517 1.03 D-78 426 621 426 621 0.94 D-68 553 696 558 694 1.1 D-69
483 638 483 637 1.07 D-160 481 631 493 617 1.2 D-155 500 625 516
619 1.6 D-72 380 477 375 469 1.1 D-163 493 588 490 589 1.06 D-159
487 603 494 593 1.5 D-80 489 669 486 668 0.31 D-84 494 623 507 602
2.8 D-90 475 662 479 655 0.36 D-162 472 706 565 692 9.9 D-70 506
615 506 614 0.92 D-86 388 544 387 544 1 D-87 430 534 428 534 1.08
D-85 450 515 534 608 2.3 Tol-24 530 594 540 598 2.4 Yat2325 503 624
538 622 2.5 S-5 527 597 535 598 1.1 S-38 535 599 555 602 2.1 S-37
545 600 551 602 1.9 S-3 562 595 565 596 1.2 S-27 522 607 540 608
1.2 SIP-2 397 576 397 575 1.3 D-74 517 601 527 601 1 Sbt 520 592
551 594 2.8 D-75 494 554 494 555 1.5 D-71 482 585 498 587 1.4
Dbo-10 505 559 515 597 2.1 SI-1999 582 595 582 595 1 SL-42 555 567
555 567 0.9 Dimer- 431 577 440 580 1.12 NN SIP-3 398 579 408 582
0.98 SIP-10 404 582 440 590 0.79 Dst-NN-6 397 572 402 572 1 SIP-8
446 582 442 584 0.28 Dst-NN- 396 572 398 572 1.24 10 Dst-NN- 398
584 413 600 0.76 11 Dst-NN- 404 581 412 595 0.79 12 SI-1035 512 545
512 546 1 SI-1047 574 596 575 596 0.95 SI-1056 546 571 548 572 1
SL-1722 673 700 676 699 1 SL-2153 547 573 547 573 0.9 SI-2596 491
594 492 609 1.1 SI-2611 456 554 460 555 1 T-164 559 575 559 572 1
SH-0229 520 628 525 641 0.7 T-33 589 656 589 656 0.7 SH-0423 409
536 409 588 1 SH-0428 588 601 588 603 2.3 SH-0627 558 569 558 569
1.1 T-333 559 576 559 576 1 T-74 561 576 561 576 0.9 SH-0999 640
653 585 596 2 T-165 583 623 588 632 2.3 T-364 582 628 581 630 1.4
Dst-NN- 398 576 409 576 0.65 13 T-119 530 635 532 636 1 T-15 554
571 564 575 1.2 TOL-26 563 609 564 607 0.9 Dst-NN-8 366 474 374 472
1.26 SL-2057 589 603 591 605 1.26 SL-2059 582 608 582 607 0.97
SL-2132 532 604 558 609 1.46 B. Structures of compounds. Dye
Structure S-11 ##STR00043## S-12 ##STR00044## SH-330 ##STR00045##
SH-654 ##STR00046## SH-675 ##STR00047## SH-975 ##STR00048## SH-1036
##STR00049## SI-2599 ##STR00050## SI-2600 ##STR00051## S-7
##STR00052## L-28 ##STR00053## L-31 ##STR00054## L-28 ##STR00055##
TOL-4 ##STR00056## TOL-10 ##STR00057## S-26 ##STR00058## S-29
##STR00059## S-44 ##STR00060## S-45 ##STR00061## S-30 ##STR00062##
Dbt-5 [TOL-9] ##STR00063## Sip-7 [TOL-12] ##STR00064## S-28
##STR00065## S-23 ##STR00066## SH-1070 ##STR00067## Yat-2212
##STR00068## D-91 ##STR00069## D-78 ##STR00070## D-68 ##STR00071##
D-69 ##STR00072## D-160 ##STR00073## D-155 ##STR00074## D-72
##STR00075## D-163 ##STR00076## D-159 ##STR00077## D-80
##STR00078## D-84 ##STR00079## D-90 ##STR00080## D-162 ##STR00081##
D-70 ##STR00082## D-86 ##STR00083## D-87 ##STR00084## D-85
##STR00085## Tol-24 ##STR00086## Yat2325 ##STR00087## S-5
##STR00088## S-38 ##STR00089## S-37 ##STR00090## S-3 ##STR00091##
S-27 ##STR00092## SIP-2 ##STR00093## D-74 ##STR00094## Sbt
##STR00095## D-75 ##STR00096## D-71 ##STR00097## Dbo-10
##STR00098## SI-1999 ##STR00099## SL-42 ##STR00100## Dimer-NN
##STR00101## SIP-3 ##STR00102## SIP-10 ##STR00103## Dst-NN-6
##STR00104## SIP-8 ##STR00105## Dst-NN-10 ##STR00106## Dst-NN-11
##STR00107## Dst-NN-12 ##STR00108## SI-1035 ##STR00109##
SI-1047 ##STR00110## SI-1056 ##STR00111## SL-1722 ##STR00112##
SL-2153 ##STR00113## SI-2596 ##STR00114## SI-2611 ##STR00115##
T-164 ##STR00116## SH-0229 ##STR00117## T-33 ##STR00118## SH-0423
##STR00119## SH-0428 ##STR00120## SH-0627 ##STR00121## T-333
##STR00122## T-74 ##STR00123## SH-0999 ##STR00124## T-165
##STR00125## T-364 ##STR00126## Dst-NN-13 ##STR00127## T-119
##STR00128## T-15 ##STR00129## TOL-26 ##STR00130## Dst-NN-8
##STR00131## SL-2057 ##STR00132## SL-2059 ##STR00133## SL-2132
##STR00134##
[0121] Although the compounds in Tables 1 and 2 are shown with a
particular counterion, it should be understood that the compounds
can also utilize other counterions as described above. As such,
when the above compounds are identified by name herein, the named
compound includes the structure identified in Table 1 or 2 with any
counterion, unless the counterion is particularly specified.
[0122] Notable examples of compounds useful compounds from Table 1
include
##STR00135##
wherein X comprises an anion (compound S25).
##STR00136##
wherein X comprises an anion (TOL3).
##STR00137##
wherein X comprises an anion (S43).
##STR00138##
wherein X comprises an anion (YAT2134).
##STR00139##
wherein X comprises an anion (YAT2148).
##STR00140##
wherein X comprises an anion (YAT2149).
##STR00141##
wherein X comprises an anion (YAT2150).
[0123] Especially useful for many purposes are dyes that have
fluorescence emissions in the range of 600-650 nM since such dyes
can avoid interference of biological proteins for the application
in tissue staining, such as green fluorescent proteins (GFPs).
Excitation fluorescence for such dyes are preferred to be in the
range of 500-600 nM. It can be seen that the dyes in Table 1
fulfill these requirements where the maxima of the fluorescence
excitation spectra of these dyes in the presence of aggregates of
.alpha.-synuclein (ASN) are between 511 and 553 nm, and
fluorescence emission have their maxima between 603 and 625 nm. The
values of the fluorescence quantum yield (QY) of the dyes of the
invention in the presence of saturating concentrations of fibrillar
protein are situated in the range between 0.01 and 0.08, which
allow using relatively small amounts of dye for interaction with
protein aggregates, tissues or cell staining. Stokes shift of the
dyes of the invention are in the range of 73 to 95 nm and are much
larger than the classic amyloid detection dyes, such as thioflavin
T, which only has a 23 nm Stokes shift. The wider Stokes shift of
the dyes of the present invention ensures a much lower overlap
between excitation and emission, thus allowing more flexible filter
set selection, such as a wide excitation and or emission filter to
improve the brightness of the dye or increasing the exposure time
to enhance the fluorescence intensity. With these considerations,
particularly useful compounds from Table 1 include S25, S43, TOL3,
YAT2134, YAT2148, YAT2149, S13, YAT2135 and YAT2324.
[0124] It is to be understood that with any particular dye, the
excitation maximum, emission maximum, and/or ratio of fluorescence
intensity in the presence of aggregates vs. monomers can vary to
some extent with different proteins. Thus, the selection of a dye
to use for detection of the aggregates of any particular protein
could benefit from information of the fluorescence characteristics
of the dye with the particular protein. Such information can be
obtained for any protein-dye combination without undue
experimentation, for example by using the methods described in
Example 1. Nonlimiting examples of useful proteins whose
aggregation could be detected using the above compounds include
immunoglobulin, a DNA polymerase or a fragment thereof,
.alpha.-synuclein, synphilin-1, TCR.alpha., P23H mutant of
rhodopsin, .DELTA.F508 mutant of CFTR, amyloid-.beta., prion
protein, Tau, SOD1, Ig light chains, ataxin-1, ataxin-3, ataxin-7,
calcium channel, atrophin-1, androgen receptor, p62/sequestosomel
(SQSTM1), Pael receptor, serum amyloid A, transthyretin,
.beta.2-microglobulin, apolipoprotein A-1, gelsolin, atrial
natriuretic factor, lysozyme, insulin, fibrinogen, crystallin,
surfactant protein C, lactoferrin, .beta.ig-h3, PAPB2,
corneodesmosin, neuroserpin, cochlin, RET, myelin, protein 22/0,
SCAD, prolactin, lactadherin, p53, procalcitonin, cytokeratin,
GFAP, ATP7B, prolyl hydroxylase PHD3, presenilin, or
huntingtin.
[0125] A further consideration of the present invention, is that
detection and/or quantification of aggregates may also be improved
by a mixture of dyes where at least one of the dyes is one of the
compounds illustrated in Table 1. The additional dye or (dyes) may
also be from Table 1 or 2. The use of more than one dye may widen
the breadth of proteins that will successfully generate signals
after aggregation when these dyes become bound. The signal will
derive from the net amount of fluorescence enhancement derived from
each dye in the mixture. Particularly useful multi-dye compositions
comprise dyes where the emission maximum of each dye is within 150
nm of the emission maximum of each of the other dyes. For some
applications, multi-dye compositions may be even more useful where
the compositions comprise dyes where the emission maximum of each
dye is within 50 nm of the emission maximum of each of the other
dyes.
[0126] Thus, in some embodiments, a multi-dye composition is
provided. This multi-dye composition comprises at least three dyes,
where each of the at least three dyes exhibits increased
fluorescence in the presence of an aggregated form of a protein
when compared to the fluorescence exhibited when the compound is in
the presence of the unaggregated form of the protein. In some of
these embodiments, each of the three dyes is selected from the
group consisting of Dye F, Dye Fm(b), D95, D97, L-30, L-33, Lu-1,
Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131,
SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1,
YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214
and YAT2324. In other embodiments, at least one of the three dyes
is selected from the group consisting of S25, S43, TOL3, YAT2134,
YAT2148, YAT2149, S13, YAT2135, YAT2324 and YAT2150.
[0127] Another multi-dye composition is also provided herein. This
multi-dye composition comprises two or more dyes, where at least
one of the two or more dyes comprises Dye F, Dye Fm(b), D95, D97,
L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43,
S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7,
TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150,
YAT2213, YAT2214 or YAT2324. In some of these embodiments, at least
one of the two dyes is selected from the group consisting of S25,
S43, TOL3, YAT2134, YAT2148, YAT2149, S13, YAT2135, YAT2324 and
YAT2150. In other embodiments, both of the two dyes are selected
from the group consisting of S25, S43, TOL3, YAT2134, YAT2148,
YAT2149, S13, YAT2135, YAT2324 and YAT2150. In particular
embodiments, the two dyes are S25 and TOL3. See, e.g., Example
26.
[0128] In another embodiment of the present invention, any of the
above dyes further comprises a reactive group, thereby allowing
their attachment to targets of interest. Examples of reactive
groups that may find use in the present invention can include but
not be limited to a nucleophilic reactive group, an electrophilic
reactive group, a terminal alkene, a terminal alkyne, a platinum
coordinate group or an alkylating agent.
[0129] There are a number of different electrophilic reactive
groups that may find use with the present invention; examples can
include but not be limited to isocyanate, isothiocyanate,
monochlorotriazine, dichlorotriazine,
4,6,-dichloro-1,3,5-triazines, mono- or di-halogen substituted
pyridine, mono- or di-halogen substituted diazine, maleimide,
haloacetamide, aziridine, sulfonyl halide, acid halide,
hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido
ester, hydrazine, azidonitrophenol, azide, 3-(2-pyridyl
dithio)-propionamide, glyoxal and aldehyde groups. Nucleophilic
reactive groups can include but not be limited to reactive thiol,
amine and hydroxyl groups. For purposes of synthesis of dyes,
reactive thiol, amine or hydroxyl groups can be protected during
various synthetic steps and the reactive groups generated after
removal of the protective group. Use of a terminal alkene or alkyne
groups for attachment of markers has been previously described in
U.S. Patent Application Serial No. 2003/0225247, hereby
incorporated by reference. The use of platinum coordinate groups
for attachment of other dyes has been previously disclosed in U.S.
Pat. No. 5,580,990 and the use of alkyl groups has been previously
described in U.S. Pat. No. 6,593,465 B1, both of which patents are
hereby incorporated by reference. In some cases the molecules that
have been disclosed already have a suitable group that can be used
as a reactive group; in other cases standard chemical manipulations
can be used to modify a dye to comprise a desired reactive
group.
[0130] Thus, the present invention provides a composition
comprising any of the above-identified compounds, where such
compound or compounds have been modified by the addition of a
reactive group (Rx) for attachment of a target molecule thereto.
The reactive group (Rx) comprises an electrophilic reactive group
comprising isocyanate, isothiocyanate, monochlorotriazine,
dichlorotriazine, 4,6,-dichloro-1,3,5-triazines, mono- or
di-halogen substituted pyridine, mono- or di-halogen substituted
diazine, maleimide, haloacetamide, aziridine, sulfonyl halide, acid
halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester,
imido ester, hydrazine, azidonitrophenol, azide, 3-(2-pyridyl
dithio)-propionamide, glyoxal or aldehyde groups, and a combination
of any of the foregoing. In another embodiment, the reactive group
(Rx) comprises a nucleophilic reactive group comprising reactive
thiol, amine or hydroxyl, and a combination of the foregoing. In
other aspects, the reactive group (Rx) comprises a terminal alkene
group, a terminal alkyne group, a nickel coordinate group or a
platinum coordinate group for attachment. The reactive group (Rx)
can be attached to the compound through a linker arm.
[0131] Another aspect of the present invention is a labeled target
molecule comprising a target molecule attached to any of the
above-described reactive compounds through the reactive group. The
target molecule is not narrowly limited to any particular type of
molecule, and can comprise any molecule that can be attached to the
above-described reactive compounds. Nonlimiting examples of target
molecules include a nucleoside, a nucleotide, an oligonucleotide, a
polynucleotide, a peptide nucleic acid, a protein, a peptide, an
enzyme, an antigen, an antibody, a hormone, a hormone receptor, a
cellular receptor, a lymphokine, a cytokine, a hapten, a lectin,
avidin, streptavidin, digoxigenin, a carbohydrate, an
oligosaccharide, a polysaccharide, a lipid, a liposomes, a
glycolipid, a viral particle, a viral component, a bacterial cell,
a bacterial component, a eukaryotic cell, a eukaryotic cell
component, a natural drug or synthetic drug, and any combination
thereof.
[0132] Examples of useful target molecules and solid-phase supports
can include but are not limited to a nucleoside, nucleotide,
oligonucleotide, polynucleotide, peptide nucleic acid, protein,
peptide, enzyme, antigen, antibody, hormone, hormone receptor,
cellular receptor, lymphokine, cytokine, hapten, lectin, avidin,
streptavidin, digoxigenin, carbohydrate, oligosaccharide,
polysaccharide, lipid, liposomes, glycolipid, viral particle, viral
component, bacterial cell, bacterial component, eukaryotic cell,
eukaryotic cell component, natural drug, synthetic drug, glass
particle, glass surface, natural polymers, synthetic polymers,
plastic particle, plastic surface, silicaceous particle,
silicaceous surface, organic molecule, dyes and derivatives
thereof.
[0133] The nucleoside, nucleotide, oligonucleotide, or
polynucleotide can comprise one or more ribonucleoside moieties,
ribonucleotide moieties, deoxyribonucleoside moieties,
deoxyribonucleotide moieties, modified ribonucleosides, modified
ribonucleotides, modified deoxyribonucleosides, modified
deoxyribonucleotides, ribonucleotide analogues, deoxyribonucleotide
analogues or any combination thereof.
[0134] As indicated above, the target molecule of these embodiments
may have dyes as targets thereby creating composite dyes. By
joining the dyes of the present invention to another dye, unique
properties may be enjoyed that are not present in either dye alone.
For instance, if one of the dyes of the present invention is joined
to another dye such that it creates an extended conjugation system,
the spectral characteristics of the dye may be different than
either dye component.
[0135] Another example of this method is where the conjugation
systems do not overlap but the proximity allows an internal energy
transfer to take place thereby extending the Stokes shift, a system
that is commonly referred to as FRET (Fluorescent Resonance Energy
Transfer) or Energy Transfer in short. For an example of this, see
U.S. Pat. Nos. 5,401,847; 6,008,373; 5,800,996, all three of which
are hereby incorporated by reference.
[0136] Other properties may also be enhanced by this joining; for
example, it has been previously described that the joining together
of two ethidium bromide molecules generates a dye that has enhanced
binding to nucleic acids and novel fluorescent properties that are
different from the monomeric forms (U.S. Patent Application
Publication No. 2003/0225247, hereby incorporated by reference).
Other composite dyes have been described that simultaneously enjoy
both properties, i.e., enhanced binding and energy transfer (U.S.
Pat. No. 5,646,264, hereby incorporated by reference). Furthermore,
these composites dyes are not limited to binary constructs of only
two dyes, but may comprise oligomeric or polymeric dyes. These
composite dyes may be comprised of the same dye or different dyes
may be joined together depending upon the properties desired.
[0137] Utility may also be achieved by attaching a dye of the
present invention to a target specific moiety. Thus, binding
between the target specific moiety and its corresponding target may
be monitored by essentially determining the presence or amount of
dye that is bound to the target. Well-known examples of such assays
are hybridizations between complementary nucleic acids as well as
binding that take place between antibodies and their corresponding
antigens.
[0138] Other binding pairs that may be of interest can include but
not be limited to ligand/receptor, hormone/hormone receptor,
carbohydrate/lectin and enzyme/substrate. Assays may be carried out
where one component is fixed to a solid-phase support and a
corresponding partner is in solution. By binding to the component
fixed to the support, the partner now becomes attached to the
support as well. A well-known example of this method is the
microarray assays where labeled analytes become bound to discrete
sites on the microarray.
[0139] Homogeneous probe dependent assays are also well known in
the art and may take advantage of the present invention. Examples
of such methods are energy transfer between adjacent probes (U.S.
Pat. No. 4,868,103), the Taqman exonuclease assay (U.S. Pat. Nos.
5,538,848 and 5,210,015), Molecular Beacons (U.S. Pat. Nos.
5,118,801 and 5,925,517) and various real time assays (US Patent
Application Publication 2005/0137388), all of which are
incorporated by reference.
[0140] Antibodies labeled with dyes of the present invention may be
used in various formats. For example, an antibody with one of the
dyes of the present invention may be used in an immunofluorescent
plate assay or in situ analysis of the cellular location and
quantity of various antigenic targets. Antibodies labeled with dyes
may also be used free in solution in cell counting or cell sorting
methods that use a flow cytometer or for in-vitro and in-vivo
imaging of animal models.
[0141] The presence or absence of a signal may then be used to
indicate the presence or absence of the target itself. An example
of this is a test where it is sufficient to know whether a
particular pathogen is present in a clinical specimen. On the other
hand, quantitative assays may also be carried out where it is not
so much the intention of evaluating if a target is present but
rather the particular amount of target that is present. An example
of this is the previously cited microarray assay where the
particular rise or fall in the amount of particular mRNA species
may be of interest.
[0142] In another embodiment of the present invention, dyes that
have been disclosed above as well as dyes described previously in
the literature may be attached to a carrier with a more general
affinity. Dyes may be attached to intercalators that in themselves
do not provide signal generation but by virtue of their binding may
bring a dye in proximity to a nucleic acid. A further example is
attachment of dyes to SDS molecules thereby allowing dyes to be
brought into proximity to proteins. Thus this embodiment describes
the adaptation of a dye or dyes that lack affinity to a general
class of molecules may be adapted by linking them to non-dye
molecules or macromolecules that can convey such properties.
[0143] Various applications may enjoy the benefits of binding the
dyes of the present invention to appropriate targets. As described
above, staining of macromolecules in a gel is a methodology that
has a long history of use. More recent applications that also may
find use are real time detection of amplification (U.S. Pat. Nos.
5,994,056, 6,174,670 and US Patent Application Publication
2005/0137388, all of which are hereby incorporated by reference),
and binding of nucleic acids to microarrays. In situ assays may
also find use where the binding of dyes of the present invention is
used to identify the location or quantity of appropriate
targets.
[0144] In other aspects, this invention provides a composition
comprising a solid support to which is attached any of the
above-described reactive compounds. In some embodiments, the solid
support comprises glass particle, glass surface, natural polymers,
synthetic polymers, plastic particle, plastic surface, silicaceous
particle, silicaceous surface, glass, plastic or latex beads,
controlled pore glass, metal particle, metal oxide particle,
microplate or microarray, or any combination thereof. The
aforementioned reactive group for attachment comprises or may have
comprised an electrophilic reactive group comprising isocyanate,
isothiocyanate, monochlorotriazine, dichlorotriazine,
4,6,-dichloro-1,3,5-triazines, mono- or di-halogen substituted
pyridine, mono- or di-halogen substituted diazine, maleimide,
haloacetamide, aziridine, sulfonyl halide, acid halide,
hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido
ester, hydrazine, azidonitrophenol, azide, 3-(2-pyridyl
dithio)-propionamide, glyoxal or aldehyde groups, a nucleophilic
reactive group comprising reactive thiol, amine or hydroxyl, a
nickel coordinate group, a platinum coordinate group, a terminal
alkene or a terminal alkyne, and any combination of the foregoing.
As in the case of other embodiments previously described above, a
linker arm can be usefully positioned between the compound and the
reactive group, or between the solid support and the reactive
group.
Reagent Kits
[0145] Commercial kits are valuable because they eliminate the need
for individual laboratories to optimize procedures, saving both
time and resources. They also allow better cross-comparison of
results generated from different laboratories. The present
invention thus additionally provides reagent kits, i.e., reagent
combinations or means, comprising all of the essential elements
required to conduct a desired assay method. The reagent system is
presented in a commercially packaged form, as a composition or
admixture where the compatibility of the reagents will allow, in a
test kit, i.e., a packaged combination of one or more containers,
devices or the like holding the necessary reagents, and usually
written instructions for the performance of the assays. Reagent
systems of the present invention include all configurations and
compositions for performing the various labeling and staining
formats described herein.
[0146] The reagent system will generally comprise (1) one or more
dye of the present invention preferably in the form of concentrated
stock solutions in an aprotic dipolar solvent, for example, DMSO
designed to target specific protein aggregate structures; (2) a
buffer, such as Tris-HCl or phosphate buffer; (3) a positive
control comprising both protein aggregates and protein monomers in
the state of solution or lyophilized powder; and (4) instructions
for usage of the included reagents. Generic instruction, as well as
specific instructions for the use of the reagents on particular
instruments, such as a wide-field microscope, confocal microscope,
flow cytometer, high content screening instrument, microplate-based
detection platform, RT-PCR instrument or standard fluorometer may
be provided. Recommendations regarding filter sets and/or
illumination sources for optimal performance of the reagents for a
particular application may be provided.
[0147] The dyes, compounds and compositions of the present
invention are fluorescently detectable or localized. Techniques and
fluorescence methods are well known in the art. A compilation of
such techniques and methods are set forth below in Table 3 which
was obtained from Hawe et al., 2008.
TABLE-US-00003 TABLE 3 Fluorescence methods and their application
with extrinsic fluorescent dyes for protein characterization.
Application with Noncovalent Method Information Extrinsic Dyes
Steady-state Spectral information Detection of protein fluorescence
(emission spectrum and structural changes by fluorescence intensity
dye-protein interactions Time-resolved Fluorescence lifetime
Detection of protein fluorescence structural changes by dye-protein
interactions Anisotrophy Rotational motions Study of rotational
(steady-state and dynamics Determination of time-resolved size of
dye-protein complexes Fluorescence Translational Determination of
size of correlation motions/diffusion dye-protein complexes
spectroscopy (FCS) Fluorescence Visualization of Detection of large
microscopy particles dye-protein complexes Determination of size
and morphology of large aggregates, fibrils, etc.
For an expert review on such fluorescence methods, see the entire
above cited publication by Hawe et al., 2008, pp. 1487-1499, the
contents of which are incorporated herein by reference.
Protein Aggregation Detection and Analysis
[0148] Fluorescence microscopy allows an early detection of changes
in protein solutions, while minimizing alterations to the observed
sample after staining with appropriate dyes. In protein
formulations, the ability to detect protein aggregates at early
time points with the dyes of the present invention can accelerate
stability testing and reduce number of samples in long term
stability studies. Fluorescence microscopy provides the possibility
of studying subtle changes in the aggregation state of the
proteins, which is also of interest in medicine and biology,
whenever protein characterization is needed. Also, fluorescence
microscopy allows the characterization of high-concentration
protein formulations without dilution and with minimal impact on
the protein's local environment. Furthermore, high-content
screening fluorescence-based imaging methods allow quantification
of populations of protein aggregates including number of branches,
mean fiber length, mean fiber width, size distribution,
polydispersity, kinetics of formation and kinetics of
disassembly.
[0149] The present invention includes an example of IgG aggregate
detection using dyes of the invention by fluorescence microscopy
(Examples 2 and 10; FIG. 1). The aggregate formation is barely
visible before staining, but clearly becomes visible after
staining.
[0150] The dyes of the invention are also capable of detecting a
broader range of protein aggregates than the conventional amyloid
detecting dyes, such as thioflavin T (Thio-T) or congo red. These
styryl dyes are able to sensitively detect protein aggregates,
ranging in size (nanometers to visually observable turbid solution
to precipitates) and physicochemical characteristics (e.g., soluble
or insoluble, covalent or non-covalent, reversible or
irreversible). Structurally altered proteins have a strong tendency
to aggregate, often leading to their precipitation. Irreversible
aggregation is a major concern for long-term storage stability of
therapeutic proteins and for their shipping and handling.
[0151] The styryl dyes of the present invention are also able to
detect aggregates at different stages of formation induced by
various stresses, such as elevated temperature, agitation and
exposure to extremes of pH, ionic strength, or various interfaces
(e.g., air-liquid interface) and high protein concentration (as in
the case of some monoclonal antibody formulations), chemicals and
protein-protein interactions (i.e., PDI-insulin interaction). These
fluorescent probes are able to detect broad types and concentration
ranges of proteins, in the presence of excipients, at different pH
values (2.about.10) and in the presence of salts and buffers,
exhibiting desirable detection limits and dynamic range, excellent
sensitivity as well as linear response. This is exemplified by the
broad categories of proteins/peptides system in the present
invention, including lysozyme, insulin, and IgG molecules, as well
as serum proteins, such as .beta.-lactoglobulin (BLG) and BSA.
Therefore, these novel dyes are capable of providing quantitative
analysis of protein aggregates in a robust, high throughput
fashion.
[0152] Thus, the present invention provides a method for detecting
the presence of aggregates of a protein in a sample. The method
comprises
[0153] (a) combining the sample with any of the above-described
compounds or multidye compositions to form a dye-sample
mixture;
[0154] (b) measuring the amount of fluorescence in the dye-sample
mixture;
[0155] (c) comparing the amount of fluorescence determined in (b)
with the amount of fluorescence in [0156] (i) a mixture of the
compound or multidye composition with a control sample without
aggregated protein, or [0157] (ii) a mixture of the compound or
multidye composition with a known standard quantity of aggregated
protein; and
[0158] (d) determining the aggregation of the protein in the sample
based on the comparison in (c).
[0159] In these methods, the standard quantity of aggregated
protein recited in (c)(ii) can be prepared by any means known in
the art. Examples include the provision of a previously determined
quantity of aggregated protein, or the preparation of a standard
curve derived from measurements of protein aggregates and protein
monomers in selected proportions. When a standard curve is
utilized, the protein for the standard curve can be the same or
different protein as the protein in the sample.
[0160] The sample for this method is not limited to any particular
composition. The sample can be from any prokaryote, archaea, or
eukaryote, or from an environmental sample. In some embodiments,
the sample is from a mammal, for example a bodily fluid of the
mammal (e.g., blood [e.g., serum, plasma], bile, sputum, urine, or
perspiration).
[0161] In other embodiments, the sample comprises a cell from the
mammal. In some of these embodiments, the cell is intact. Such an
intact cell, either fixed (see, e.g., Example 28) or living (e.g.,
Example 29), can be combined with the compound or multidye
composition and the fluorescence is measured histologically. Here,
the fluorescence can be measured by visual observation or by
quantifying the amount of fluorescent light emitted from the cell,
by known methods.
[0162] Example 29 exemplifies embodiments utilizing living cells
where a compound can be tested for an effect on the aggregation of
proteins. In these embodiments, the cell is treated with a protein,
for example amyloid beta peptide (e.g., amyloid beta peptide 1-42),
known to aggregate in the cell. Such cells treated with the
compound can be compared with cells not treated with the compound
(the control composition of (c)(ii) in the above-described methods)
to determine the effect of the compound on the aggregation of the
protein in the cell.
[0163] The sample of these methods can also comprise homogenized
cells from a mammal that is part of a tissue from a mammal. In some
embodiments, the mammal has a disorder characterized by altered
protein aggregation, e.g., Alzheimer's disease, Huntington's
disease, Parkinson's disease, senile systemic amyloidosis, or a
spongiform encephalopathy.
[0164] These methods can be utilized to detect any known form of
aggregated protein, including but not limited to aggresomes,
aggresome-like structures, inclusion bodies, Lewy bodies, Mallory
bodies, neurofibriliary tangles, or any combination thereof.
Protein Aggregation Kinetic Studies
[0165] Protein aggregation is an important phenomenon that
alternatively is part of the normal functioning of nature or has
negative consequences via its hypothesized central role in
neurodegenerative diseases. A key in controlling protein
aggregation is to understand the mechanism(s) of protein
aggregation. Kinetic studies, including data curve-fitting, and
analysis are, in turn, keys to performing rigorous mechanistic
studies.
[0166] The many approaches in the literature striving to determine
the kinetics and mechanism of protein aggregation can be broadly
divided into three categories: (i) kinetic and thermodynamic, (ii)
empirical, and (iii) other approaches. The large literature of
protein aggregation can be distilled down to five classes of
postulated mechanisms: i) the subsequent monomer addition
mechanism, ii) the reversible association mechanism, iii) prion
aggregation mechanisms, iv) an "Ockham's razor"/minimalistic model,
and v) quantitative structure activity relationship (QSAR) models
(Morris et al., 2009). Corresponding equations derived from
aggregation kinetic data can enlighten which proposed mechanism is
applicable to the specific protein.
[0167] Detection of aggregates at their nascent stages, such as
intermediates consisting of a couple of monomers, is key in
determining critical nucleus size and aggregate growth mechanism.
In addition, kinetic studies are also very helpful in screening
excipients or inhibitors that can stop or suppress protein
aggregation and in assessing enzyme activity in various clinical
and research settings. Hence, a sensitive kinetic assay in a
robust, high-throughput manner is highly desirable in mechanism
determination studies and in drug discovery. Most of the current
aggregate analysis technologies, unfortunately, are neither
sensitive nor accurate enough to quantify nascent aggregates.
Various factors affecting aggregation can be studied by these
means; a number of these are described by Bondos and Bicknell
(2003) and in addition, Table 4 below is reproduced from this
article (Table 1 therein) showing components (including recommended
concentrations) that might be used for decreasing aggregation:
TABLE-US-00004 TABLE 4 Agents that may promote protein solubility
Recommended concentration Additive range Kosmotropes MgSO.sub.4
0-0.4M (NH.sub.4).sub.2SO.sub.4 0-0.3M Na.sub.2SO.sub.4 0-0.2M
Ca.sub.2SO.sub.4 0-0.2M Weak kosmotropes NaCl 0-1M KCl 0-1M
Chaotropes CaCl.sub.2 0-0.2M MgCl.sub.2 0-0.2M LiCl 0-0.8M RbCl
0-0.8M NaSCN 0-0.2M NaI 0-0.4M NaClO.sub.4 0-0.4M NaBr 0-0.4M Urea
0-1.5M Amino acids Glycine 0.5-2% L-arginine 0-5M Sugars and
Sucrose 0-1M polyhydric Glucose 0-2M alcohols Lactose 0.1-0.5M
Ethylene glycol 0-60% v/v Xylitol 0-30% w/v Mannitol 0-15% w/v
Inositol 0-10% w/v Sorbitol 0-40% w/v Glycerol 5-40% v/v Detergents
Tween 80 0-0.2% w/v Tween 20 0-120 .mu.M Nonidet P-40 0-1%
[0168] The method described above can be adapted to measure the
kinetics of protein aggregation, e.g., by measuring fluorescence of
the protein-dye mixture at various time points while aggregation is
occurring. Thus, in some embodiments, the amount of fluorescence of
the above-described method is measured at preselected time
intervals to detect formation of protein aggregates, wherein
increasing fluorescence over time indicates formation of protein
aggregates.
[0169] These embodiments encompass two methods of applying the
above-described dyes into a kinetics study of protein aggregation,
such as lysozyme and IgG aggregation, induced by various types of
stress, including pH, shaking and temperature shift and in the
presence or absence of excipient(s). The first method comprises the
following steps: (1) apply a stress to a protein formulation for a
certain period of time; (2) release stress by switching off the
stress, such as heat or harsh pH to freeze or trap the aggregate
formation; (3) fluorescence reading of these formulations by
addition of selected dyes of the invention; (4) plot the relative
fluorescence unit (RFU) vs. time curve and further process the
kinetic curve to extract more desired information. This method is
beneficial for some proteins whose aggregation can be significantly
interfered with by probing dye binding (especially for nascent or
intermediate aggregates, characterized by a much smaller surface
area than those more matured aggregates) at stressed condition,
which is minimized after the release of the stress.
[0170] The second method is more convenient compared to the first
method. First, mix the dye with the protein formulation prior to
the application of the stress; second, apply the stress and start
recording the fluorescence response at various points of time;
finally, plot a relative fluorescence unit (RFU) vs. time curve and
possibly perform further processing of the curve to extract more
desired information. This method, though labor saving, much more
robust and accurate in time, may not be applicable for some
proteins if the dye blocks, promotes or interferes with the
addition of monomers to the aggregate intermediates or
polymerization of aggregate intermediates. However, notwithstanding
the mentioned caveats, the second method is generally preferred,
since it allows for a simpler high throughput assay.
[0171] The measurement of fluorescence in these methods can be
conducted using any appropriate time interval between measurements,
determined by a determination of the time expected for the
aggregation to occur in the particular system being investigated.
In some embodiments, the preselected time intervals are less than 2
minutes. In other embodiments, the preselected time intervals are
less than 10 minutes. In still other embodiments, the preselected
time intervals are less than 1 hour. In additional embodiments, the
preselected time intervals are more than 1 hour.
Methods of Evaluating Protein Formulation Stability Using
Accelerated Stability Testing
[0172] Embodiments of the present invention are directed to
reliable, time and cost-efficient methods for evaluating the
relative chemical and physical stability of a particular protein
formulation. Thus, embodiments of the invention are useful
analytical tools for developing new protein formulations with
increased stability, as well as for use in evaluating the stability
of newly prepared batches of known protein formulations in quality
control procedures, or the like.
[0173] Embodiments of the present invention encompass a fully
automated assay of protein stability that generally requires less
than one week for evaluating protein formulations. The present
invention method comprises preparing two series of formulations,
one formed before stress test (pre-stress formulations), another
formed after stress test (post-stress formulations), followed by an
adding aggregate detection reagent that include one or more dyes of
the present invention. The dye or dyes of the present invention may
be used alone for this purpose oror they may be used in conjunction
with other commercial dyes, such as Nile red, thioflavin-T, ANS or
Congo red. This is followed by comparing the fluorescence response
of different formulations to rank the amount of aggregates existing
within individual formulations.
[0174] In one exemplification of this method, the following 6 steps
may be carried out:
[0175] Step (1). A selected group of components, including, but not
limited to excipients, salts, buffers, co-solvents, metal ions,
preservatives, surfactants, and ligands are collected and their
stock solutions are prepared.
[0176] Step (2). Preliminary formulations comprising one or more
components following a standard design of experiment procedure
aimed at generating relevant information are designed and the
protein formulations, preferably containing the same concentration
of protein are prepared.
[0177] Step (3). A stress such as heat, agitation, rotation, harsh
pH, ultrasound, shearing or the like, is simultaneously applied
externally to multiple protein formulations under evaluation, which
are held in individual containers, preferably in separate wells of
one microplate (s), which is preferably sealed, each with zero, one
or more components of interests; meanwhile, the formulation with
zero component of interests, but the same protein concentration as
the formulations with component (s) of interests can be prepared in
a separately sealed container in bulk quantity.
[0178] Step (4). After stress is released, the bulk protein
formulation that has zero components of interests is split and
mixed with one or more components of interests to make up similar
formulations as those subjected to the stress test, preferably in
wells of another microplate. Note that the later added components
of interest solutions dilute the resulted non-stressed
formulations, making them less concentrated as their stressed
counterpart; this can be adjusted later in the step where the
probing dyes are added. These control formulations which have not
experienced the stress test allow accurate evaluation of the
functions of the components of interests during the stress test
since components of interests themselves can affect the
fluorescence response of protein aggregates to some extent.
[0179] Step (5). A solution of the dye or dyes of the present
invention (and the buffer in which the dyes are dissolved) are
added into the protein formulations such that post-stress
formulations are more concentrated than that added to the stressed
formulations to result in the same concentration of protein,
components of interests and dye(s) for both pre-stress formulation
and post-stress counterpart. After an incubation period, the
microplates are read in a conventional plate reader by, for
example, fluorescence intensity or fluorescence polarization
measurement.
[0180] Step (6). The formulations can be first evaluated within the
group (i.e. either pre-stress or post-stress formulations), which
are preferably tested in one microplate, by comparing formulations
containing one or more components of interests with that containing
no components of interests. This method can eliminate the errors
produced during the preparation of different plates (the sample
formulation plate(s) and the control formulation plate(s), which
can take 10.about.60 minutes. Then fluorescence ratio of each
stress tested formulation to its corresponding control without
stress application can be further calculated. The function of
components of interests during stress is evaluated by using the
fluorescence ratio of components of interests added before
application of stress vs. after application of stress using zero
components of interests as a reference. Therefore, the present
invention is further directed to a method to evaluate components of
interests that can stabilize or destabilize protein in order to
optimize protein formulations.
[0181] The properties of the dyes of the invention allow their wide
application in the protein/peptide formulation field, especially on
a high-throughput technology platform. Compared with other
fluorescent probes, such as intrinsic tyrosine or externally added
probes, such as 1-anilino-naphthalene-8-sulfonate (ANS), Nile red
or thioflavin-T, the dyes of the present invention are better
capable of providing quantitative analysis of protein aggregates in
a robust, high throughput fashion and are applicable to more
categories of proteins under various conditions. In some instances
two or more dyes of the present invention are applied to a sample.
This facilitates detection of the broadest range of protein
aggregates since these means provide that if one dye does not bind
a particular aggregate, another can compensate for this
deficiency.
Protein Stability Shift Assay Based on Fluorescent Detection of
Protein Aggregation Using Exogenously Added Fluorophores
[0182] Protein stability can be altered by various components
discussed in protein formulation embodiments, including, but not
limited to excipients, salts, buffers, co-solvents, metal ions,
preservatives, surfactants, and ligands. Protein stability can be
shifted by various stresses, including elevated temperature, which
is often referred to as a thermal shift or by addition of chemical
denaturants, such as urea, guanidinium isocyanate or the like. A
protein stability shift assay has a wide spectrum of applications
in, but not limited to investigation of protein refolding
conditions, optimization of recombinant protein
expression/purification conditions, protein crystallization
conditions, selection of ligand/drug/vaccine/diagnostic reagents
and protein formulations.
[0183] The classic thermal shift technologies based on protein
aggregate detection utilize a melting point device to raise the
temperature stepwise, coupled with aggregation detection
technologies, such as light scattering technology (an example
includes but is not limited to differential static light scattering
(DSLS)) to monitor protein aggregation. This type of technology
usually requires a high protein concentration, therefore, it is not
cost effective. In addition, it cannot work effectively on
formulations with low protein concentrations or finalize protein
formulations which require a very low detection limit for
aggregates (typically .about.1-5%), which is usually beyond the
detection limit of these classic technologies.
[0184] Thermofluor.RTM. (J&J, 3-Dimensional Pharmaceuticals,
Inc, Exton, Pa., U.S. Pat. No. 6,020,141 ["the '141 patent"]) is a
biophysical technique used to study (relative) protein stabilities.
The solution of protein is heated up stepwise from room temperature
to .about.95.degree. C. and the fluorescence is monitored at each
step. The rising temperature causes protein unfolding and the
fluorophore (SYPRO Orange.RTM. [Invitrogen] or ANS) partitions
itself into the melted protein and hence the overall effect is an
increase in fluorescence with increasing temperature. If a drug or
ligand is included which binds to the protein, the mid-point of the
curve can shift, arising from stabilizing or destabilizing effects
(e.g., ligand binding). Thermofluor.RTM. can rank binding affinity
in a rapid, HTS manner and help setup structure-activity
relationship. However, this particular methodology is related to
both denaturation of proteins as well as subsequent aggregations of
the denatured proteins and the patent clearly indicates that the
focus is on the unfolding and denaturation of proteins and as
described in column 16, lines 25-56, the fluorescent probes chosen
for application of this method are drawn from compounds that are
"capable of binding to an unfolded or denatured receptor". However,
some of the compounds that are listed (ANS, bis-ANS and JCVJ) are
known to bind to aggregates (Lindgren et al., 2005) and as such no
particular emphasis is laid upon distinguishing between
denaturation and aggregation events. In contrast, the present
invention is specifically directed towards aggregation
detection.
[0185] As such, one of the embodiments of the present invention
encompasses a novel thermal shift assay in which protein is heated
up stepwise from room temperature to .about.95.degree. C. using a
device, including, but not limited to, a microplate reader, a
thermocycler, a melting device or similar equipment, preferably
with a heating stage that can raise temperature stepwise and record
fluorescence change simultaneously, and the fluorescence of
externally added dyes of the present invention is monitored at each
heating step. Since the dyes that are used in the present invention
selectively interact with protein aggregates and not hydrophobic
domains exposed by protein unfolding, the increase in fluorescence
with increasing temperature is not due to protein unfolding as seen
in the technique described in the '141 patent, but rather is due to
protein aggregation. Therefore, this particular embodiment of the
present invention can be applied to directly targeting at ranking
components, including, but not limited to, excipients, salts,
buffers, co-solvents, metal ions, preservatives, surfactants, and
ligands in protein stabilization by preventing protein aggregation
to improve formulations, or to screening drugs (inhibitors)
preventing protein aggregates found in some diseases, including,
but not limited to, organic synthetic compounds, peptides and
proteins (recombinant or natural source). For most proteins,
unfolding directly precedes their aggregation. Hence, similar to
the unfolding-based Thermofluor.RTM. technique, the
aggregation-based thermal shift assay technology embodied in this
present invention also has the potential to being applied to
ranking the effect of additives on protein stability. Its
application can thus be expanded to more broad fields, including,
but not limited to, investigation protein refolding conditions,
optimization of recombinant protein expression/purification
conditions, protein crystallization conditions, and selection of
ligands, drug, vaccine and diagnostic reagents.
[0186] Thus, fluorescence can be measured at one or more different
temperatures after forming the first mixture and the second
mixture. Such different temperatures can be selected from
temperatures ranging from about 4.degree. C. to about 100.degree.
C. Further, fluorescence measurements can be carried out as a
series of discrete temperatures, where the measuring steps are
carried out after incubation at each of the different discrete
temperatures, or while changing temperatures.
[0187] Another useful method of the present invention is a method
for determining whether a test compound decreases aggregation of a
protein. The above-described method can be utilized, where a test
compound is added to a portion of the dye-sample mixture of (a) and
the fluorescence of the portion with the test compound is compared
to the fluorescence of the portion without the test compound to
determine whether the test compound decreases aggregation of the
protein, wherein decreased fluorescence in the portion with the
test compound indicates that the test compound decreases
aggregation of the protein.
[0188] The test compound is not limited to any particular class of
compound. Nonlimiting examples include a kosmotrope, a chaotrope,
an amino acid, a peptide, a reducing agent, a carbohydrate, a
detergent, a surfactant, a zwitterion or a polyhydric alcohol, or
any combination thereof. Any of these test compounds can have a
range of concentrations from about 0 molar to about 2 molar, a
range of pH values from about 4 to about 10. The test compound can
also comprise a storage buffer for said protein. Such storage
buffer can comprise a set of buffer formulations with a range of
concentrations from about 0 molar to about 2 molar, a range of pH
values from about 4 to about 10, and any combinations thereof.
Chaperone/Anti-Chaperone Activity Assays
[0189] Chaperone and anti-chaperone function oppositely in the
sense that one helps prevent aggregates and the other helps induce
aggregate formation. To assay activity of the opposite functions,
one needs to quantitatively analyze the substrate aggregate change
with time. The present invention uses methods described above in
the PDI/thioredoxin activity assay to analyze
chaperone/anti-chaperone activity, which has similar advantages
over methods based on other aggregate detection technologies,
particularly turbidity and back-scatter methods. The present
invention also encompasses a kit or kits comprising similar
components as the PDI isomerase activity kit (s) included in the
present invention. Assays can be devised to monitor assembly or
disassembly of protein aggregates or both.
[0190] Thus, in some embodiments of the above-described method,
[0191] (A) the protein is a substrate for a chaperone;
[0192] (B) the dye-sample mixture of step (a) is subjected to a
stress for a time and under conditions sufficient to induce
aggregation of the protein; and
[0193] (C) the amount of fluorescence determined in (b) is compared
to the amount of fluorescence from the protein with the compound or
multidye composition subjected to the same stress without the
sample. In these embodiments, a decrease in fluorescence of the
stressed dye-sample mixture with the sample when compared to the
fluorescence from the protein with the compound or multidye
composition but without the sample indicates that the sample
comprises the chaperone.
[0194] This method can utilize any chaperone now known or later
discovered, including chaperones that are small heat-shock proteins
(sHSPs), as they are known in the art. Examples of chaperones
include HSP33, HSP60, HSP70, HSP90 or HSP100, GRP94, GRP170,
calnexin, calreticulin, HSP 40, HSP47 and ERp29, GroEL, GroES,
HSP60, Cpn10, DnaK, DnaJ, Hsp70, Hsp71, Hsp72, Grp78 (BiP), PDI,
Erp72, Hsx70, Hdj1, Hdj2, Mortalin, Hsc70, Hsp70-A1, fHtpG, C62.5,
Hsp90 alpha, Hsp90 beta, Grp94, ClpB, ClpA, ClpX, Hsp100, Hsp104,
Hsp110, TRiC, alpha crystallin, HspB1, Hsp 25, Hsp27, clusterin,
GrpE, Trigger Factor, and Survival of Motor Neuron (SMN1, SMN2), or
any combination thereof. The substrate can comprise any chaperone
substrate now known or later discovered, including but not limited
to .beta.-lactoglobulin, citrate synthase, lysozyme,
immunoglobulin, CRYBB2, HSPB8, CRYAA, TGFB1I1, HNRPD or CRYAB, or
any combination thereof. The reaction mixture can be incubated for
a period of time from about 15 to about 60 minutes. The stress can
be an elevated temperature, preferably, from about 37.degree. C. to
about 95.degree. C. Alternatively, the stress can be a chaotropic
agent, such as guanidine-HCl or urea, or both. The concentration of
the chaotropic agent can be from about 4 to 8 M. Moreover, a
plurality of these methods can be performed in parallel.
[0195] Analogously, the invention methods can be utilized to
identify anti-chaperone activity. Here, the methods described above
are utilized, where
[0196] (A) the protein is a substrate for an anti-chaperone;
and
[0197] (B) the amount of fluorescence determined in (b) is compared
to the amount of fluorescence from the protein with the compound or
multidye composition without the sample. In these methods, an
increase in fluorescence of the dye-sample mixture when compared to
the fluorescence from the protein with the compound or multidye
composition but without the sample indicates that the sample
comprises the anti-chaperone.
High-Throughput Fluorometric Assay for Measuring Aggregates Formed
by Members of the Thioredoxin Superfamily
[0198] Thioredoxins and related proteins act as antioxidants by
facilitating the reduction of other proteins by cysteine
thiol-disulfide exchange. Such exchanges can lead to intermolecular
bridges being formed, thereby forming covalently linked aggregates.
Thioredoxins are characterized at the level of their amino acid
sequence by the presence of two vicinal cysteines in a CXXC motif.
These two cysteines are the key to the ability of thioredoxin to
reduce other proteins. A number of different families
(thioredoxins, protein disulfide isomerases [PDI's] and
glutaredoxins) form what can be considered the thioredoxin
superfamily. With regard to the glutaredoxins, they share many of
the functions of thioredoxins, but are reduced by glutathione
rather than a specific reductase and may be assayed by the
described methods of the present invention.
[0199] Thus, methods are disclosed to measure the activity of
thioredoxin-like enzymes by detecting the induction of aggregates
formation by utilizing any of the dyes described above. The
above-described method for detecting a protein aggregate can be
utilized, where
[0200] (A) the protein is a substrate for a member of the
thioredoxin superfamily;
[0201] (B) a reducing agent is included in the dye-sample mixture
of (a); and
[0202] (C) the dye-sample mixture of step (a) is incubated for a
period of time sufficient to reduce disulfide bonds in the protein.
In these methods, an increase in fluorescence of the dye-sample
mixture when compared to the fluorescence from the protein with the
compound or multidye composition without the sample indicates that
the sample comprises the member of the thioredoxin superfamily.
[0203] Substrates here include, but are not limited to, insulin,
hypoxia-inducible factor, prolyl 4-hydroxylase, HIV gp120, TXNIP,
ASK1, collagen type I, alpha 1 and glucocorticoid receptor. In some
embodiments, insulin is used as a substrate at a concentration of
less than 0.2 mM. This method can be used to measure the amount of
activity in a sample, identify the suitability of proteins as
substrates for such activity, and to screen for inhibitors of these
enzymes. This method may also be used to test the ability of a
particular protein to be used as a substrate by a member of the
thioredoxin superfamily to form aggregates. This method also allows
an accurate assay of multiple samples, such as samples from
patients, or therapeutic agents for drug discovery. The method can
be used in a high throughput manner using a microplate, as
reflected in the insulin aggregate detection example included in
the present invention.
[0204] The reducing reagent concentration should be optimized to
reduce the substrate disulfide bonds without minimizing the
competing chemical reaction. The reducing reagents may include, but
are not limited to glutathione, dithiothreitol (DTT),
dithioerythritol, .beta.-mercaptoethanol, thioglycolate, and
cysteine, with DTT being a preferred embodiment. A preferred DTT
concentration is less than 10 mM, and more preferably less than 1
mM. The assay buffer can include those buffers that stabilize
thioredoxin superfamily members and their substrates, with
optimized pH, salts, chelating agents (e.g. EDTA, and the like),
dyes, and potentially organic solvents such as DMSO.
[0205] When testing for the presence or amount of a particular
member of the thioredoxin superfamily in a sample (or for overall
activity), a variety of sources may be used that include biological
tissues, biological fluids and cells. Thus for instance, samples
may include cells up-regulating PDI during hypoxia or cells with
surface expressed PDI, including endothelial cells, platelets,
lymphocytes, hepatocytes, pancreatic cells and fibroblasts. The
sample may also include a thioredoxin superfamily member complexed
with other proteins, such as PDI complexed with hypoxia-inducible
transcription factor HIFa. Samples may also include fragments of a
member of the thioredoxin superfamily as well as recombinant forms
of these members, and in vitro protein synthesis reactions that are
presumed to have generated such proteins.
[0206] These methods may also find utility in identifying
modulators of thioredoxin superfamily activity; such modulators can
comprise enzyme mimetics, interacting proteins, competitive
inhibitors, small molecular inhibitors, and the like.
[0207] The method may also comprise the use of appropriate controls
for the sample, including controls that do not include any
thioredoxin superfamily member activity as well as controls that do
not include any reducing reagents. These controls can be used as
background to be subtracted from gross signal to gain net signal
induced by the enzyme activity.
[0208] A preferred addition sequence of the present invention is:
(1) Substrate and related buffers; (2) Dye(s) dissolved in organic
solvent(s), (3) PDI or similar thioredoxin-like enzyme (s) and
related buffers; (4) Reducing reagent (s). The enzyme(s) and
reducing reagents are preferred to be added with a multi-channel
addition device that can simultaneously add reducing agent into the
multiple assay containers, such as wells of a microplate to
minimize the time interval between the addition of enzyme and the
reducing reagent. This may be important for kinetic assays under
some circumstances since PDI and similar thioredoxin-like enzymes
can induce enzymatic reaction in the absence of reducing agent,
especially with a high concentration of enzyme or substrate or
both. This can minimize the background levels. The multi-channel
addition device can minimize the background levels derived from the
foregoing effects it may also minimize timing errors among the
multiple samples to be analyzed, which can minimize statistical
deviation among the samples.
[0209] In addition to the methods described above, the thioredoxin
superfamily aggregation assays can be formulated into kits
comprising one or more thioredoxin superfamily members, appropriate
substrates, buffers, reducing agents and one or more dyes of the
described in FIG. 1 as well as instructions for their use. These
kits may be used for any of the applications described above.
[0210] Such member of the thioredoxin superfamily (a) can comprise
a protein disulfide isomerase, a thioredoxin or a glutaredoxin, and
combinations thereof. The substrate (b) in this method can comprise
insulin ribonuclease, choriogonadotropin, coagulation factor,
glucocorticoid receptor or HIV gp 120, and combinations thereof.
The reducing agent (c) can be selected from the group comprising
dithiothreitol (DTT), Tris(2-carboxyethyl)phosphine hydrochloride
(TCEP HCl) or dithioerythritol (DTE), and combinations thereof. The
reaction mixture can be preferably incubated for a period of time
from about 15 to about 60 minutes. The protein disulfide isomerase
can comprise PDI, ERp57, PDIp, ERp72, P5, PDIr, ERp28/29, ERp44,
ERjd5/JPDI or ERp18, and combinations thereof.
[0211] This method can further comprise the step of terminating the
reaction prior to the measuring step (iii) by adding hydrogen
peroxide to the incubating reaction mixture. As in the case of
earlier described embodiments of this invention, a plurality of
such methods can be performed in parallel.
Assaying Various Enzymatic Activities and Post-Translational
Modifications by Monitoring Protein Aggregation Status
[0212] With respect to various pathological disorders, abnormal
protein aggregates are often sequestered into intracellular protein
deposits such as aggresomes, aggresome-like structures, inclusion
bodies. Lewy bodies or Mallory bodies (Stefani, 2004; Garcia-Mata
et al., 2002). These may trigger in turn the expression of
inflammatory mediators, such as cyclooxygenase 2 (COX-2) (Li et
al., 2004). Disruption of the ubiquitin-proteasome pathway, as for
example, thru impairment of ubiquitin hydrolase activity, triggered
by modulators such as .DELTA.12-PGJ2, lactacystin .beta.-lactone or
MG-132 can readily be analyzed directly in cells using the
disclosed methods to detect intracellular protein deposits as well
as in either cell-based or biochemical assays for screening of
other selective inhibitors of the ubiquitin-proteasome pathway that
lead to protein aggregation.
[0213] The principle advantages of the delineated approach relative
to use of conventional substrates of ubiquitin hydrolase activity,
such as ubiquitin-7-amino-4-methylcoumarin (ubiquitin-AMC), include
employment of a natural protein substrate in the assay as well as
an inherent signal amplification, arising from the formation of
polymerized amyloid fibrils as reporters. Examples of potential
protein substrates useful in this regard include, but are not
limited to, immunoglobulin, .alpha.-synuclein, synphilin-1,
TCR.alpha., P23H mutant of rhodopsin, .DELTA.F508 mutant of CFTR,
amyloid-.beta., prion protein, Tau, SOD1, Ig light chains,
ataxin-1, ataxin-3, ataxin-7, calcium channel, atrophin-1, androgen
receptor, p62/sequestosomel (SQSTM1), Pael receptor, serum amyloid
A, transthyretin, .beta.2-microglobulin, apolipoprotein A-1,
gelsolin, atrial natriuretic factor, lysozyme, insulin, fibrinogen,
crystallins, surfactant protein C, lactoferrin, .beta.ig-h3, PAPB2,
corneodesmosin, neuroserpin, cochlin, RET, myelin, protein 22/0,
SCAD, prolactin, lactadherin, p53, procalcitonin, cytokeratins,
GFAP, ATP7B, prolyl hydroxylase PHD3, presenilin, and huntingtin.
Additionally, proteins specifically engineered to be unstable or
highly prone to self-association into aggregates may be employed as
substrates using the disclosed assay methods.
[0214] With respect to coupled enzyme reactions the product of one
reaction is used as the substrate of another, more
easily-detectable reaction. The cited compositions and methods are
especially advantageous in the development of biochemical assays
involving coupled reactions leading to the formation of protein
aggregates. In this instance no meaningful physiological
relationship between the activity being monitored and the
generation of the aggregated protein-dye reporter is explicitly
required. The protein aggregate-dye complex is simply serving as an
indicator to establish the amount of product formed in a particular
catalytic reaction. For example, a protein substrate may be
employed that is marginally stable under the specified solution
conditions employed in the assay. When this substrate is acted upon
by a histone acetyltransferase, a particular lysine residue becomes
acetylated, the overall protein structure is destabilized and the
protein undergoes a conformational change resulting in its
aggregation. The dyes described in this disclosure are then able to
bind to the aggregates, generating a fluorescent signal. While
illustrated with histone acetyltransferase, a wide range of
activities that could potentially modify a substrate protein,
leading to its structural destabilization under the assay
conditions employed, could be performed by similar approaches. In
addition activities that do not directly modify the substrate
protein can also be considered. For instance, an enzyme activity
that leads to the acidification of the assay buffer could in turn
lead to destabilization of the substrate protein structure and its
aggregation.
Separation of Protein Aggregates from Protein Monomers
[0215] Those skilled in the art will appreciate that the present
invention is applicable to the separation or isolation of protein
aggregates from other protein forms, notably protein monomers. The
dyes described above are useful in subtraction of protein
aggregates from protein monomers.
[0216] Thus, the present invention provides a method for separating
aggregates of a protein from monomeric forms of the protein. The
method comprises
[0217] (a) combining the sample to the solid support of claim 25
under conditions where aggregates of the protein preferentially
bind to the compound;
[0218] (b) separating sample protein bound to the solid support
from unbound protein. In these methods, protein bound to the solid
support is substantially aggregates and unbound protein is
substantially monomers.
[0219] In carrying out the above isolation method, the solid
support can comprise glass particle, glass surface, natural
polymers, synthetic polymers, plastic particle, plastic surface,
silicaceous particle, silicaceous surface, glass, plastic or latex
beads, controlled pore glass, metal particle, metal oxide particle,
microplate or microarray, and combinations of any of the
foregoing.
[0220] Preferred embodiments are described in the following
examples. Other embodiments within the scope of the claims herein
will be apparent to one skilled in the art from consideration of
the specification or practice of the invention as disclosed herein.
It is intended that the specification, together with the examples,
be considered exemplary only, with the scope and spirit of the
invention being indicated by the claims, which follow the
examples.
Example 1. Testing Compounds for Ability to Sense Protein
Aggregation
[0221] Fluorescent readings were carried out in 50 mM Tris-HCl, pH
7.8 using 10 .mu.M dye. When present, 1 .mu.M recombinant human
.alpha.-synuclein (ASN, Sigma-Aldrich, St. Louis, Mo.) as monomers,
or aggregated as described in van Raaij et al. (2006) was included.
Fluorescence excitation and emission spectra were collected on a
Cary Eclipse fluorescence spectrophotometer (Varian, Australia).
Fluorescence spectra were measured with excitation and emission
slit widths set to 5 nm, and at a constant PMT voltage.
Spectroscopic measurements were performed in standard quartz cells.
All measurements were made at the respective excitation maxima of
each dye. All measurements were carried out at room temperature.
Results are summarized in Tables 1 and 2.
Example 2. Fluorescence Sensitivity of Different Protein
Aggregate-Sensing Dyes in the Presence of Excipients
[0222] IgG aggregate was prepared by adjusting 5.83 mg/ml of
purified goat-anti-mouse IgG (H&L, Pel Freez, Rogers, Ark.) to
pH 2.7 using HCl and incubating at 22.degree. C. for 24 hours. The
assay was performed using 2.8 .mu.M IgG, either native or
aggregated, and a dye concentration of 0.625 .mu.M. The protein and
dye were mixed together for 15 minutes at 22.degree. C., then
further incubated in the presence of the excipients shown in Table
5. The fluorescence intensity of S-25, Tol3 and Y2150 were
determined with a FLUOstar OPTIMA plate reader (BMG LABTECH) at
excitation wavelength of 550 nm and emission wavelength of 610 nm;
while the fluorescence intensity for thioflavin-T was determined
using a SpectraMAX GeminiXS (Molecular device, with Softmax Pro
7.0) using an excitation wavelength of 435 nm and emission
wavelength of 495 nm. The fluorescence enhancement
(aggregate/native IgG) is shown.
TABLE-US-00005 TABLE 5 Effect of various concentrations of various
excipients on fluorescence of four dyes in the presence of
aggregated IgG. Excipients & Concentrations S25 TOL3 Y2150
Thio-T Sodium Chloride, 10 mM 14.0 16.0 14.4 1.6 Sodium Chloride,
100 mM 13.6 16.2 11.3 1.3 Sodium Chloride, 1000 mM 11.7 17.4 15.0
2.7 Calcium Chloride, 10 mM 9.7 14.9 12.4 3.1 Calcium Chloride, 50
mM 9.6 13.9 14.7 1.5 Calcium Chloride, 200 mM 6.7 14.8 13.9 1.7
Ammonium Sulfate, 10 mM 15.4 15.6 12.4 2.8 Ammonium Sulfate, 100 mM
14.6 13.4 12.5 2.6 Ammonium Sulfate, 300 mM 13.3 16.9 14.6 1.4
Sorbitol, 100 mM 16.4 20.0 17.3 3.0 Sorbitol, 300 mM 21.0 19.2 15.6
1.9 Sorbitol, 600 mM 25.4 29.3 18.7 3.6 Mannitol, 100 mM 16.7 17.5
11.2 3.1 Mannitol, 300 mM 15.2 25.2 13.8 3.7 Mannitol, 600 mM 20.9
27.5 17.7 1.8 Trehalose, 100 mM 17.8 18.9 14.0 2.2 Trehalose, 300
mM 32.1 20.1 19.4 0.2 Trehalose, 600 mM 30.1 30.4 18.9 4.8 Lactose,
100 mM 23.0 19.9 17.5 1.2 Lactose, 300 mM 38.9 34.6 31.0 1.4
Ascoric Acid, 1 mM 13.9 15.4 14.5 1.5 X100, 0.01% 19.2 6.2 4.1 5.3
X100, 0.2% 7.3 3.4 2.6 6.6 X100, 1% 2.9 1.9 1.7 3.4 Arginine, 200
mM 14.8 18.6 14.4 1.4 Arginine, 500 mM 13.5 17.6 14.3 2.0 Glycine,
0.5% 14.1 16.3 12.5 3.1 Glycine, 2% 15.1 15.5 19.0 3.2 Tween 20,
0.01% 70.8 8.5 5.5 4.6 Tween 20, 0.2% 26.7 3.4 2.6 2.6 DTT, 1 mM
13.2 13.8 11.3 1.6 Average 19.3 17.6 14.8 2.9
Example 3. Synthesis of S25
(a) Preparation of 6-methylsulfonyloxyhexyl methylsulfonate
(Compound 1)
[0223] A solution of 1,6-hexanediol (13.15 g, 111.3 mmol) in 70 mL
of anhydrous pyridine was cooled to 0.degree. C. using ice bath. To
this methanesulfonyl chloride (27 g, 235.7 mmol) was slowly added
under mixing such that the temperature was maintained at
5-6.degree. C. The combined mixture was stirred overnight at the
temperature below 10.degree. C. and the precipitate formed was
filtered off, washed with 20% HCl (3.times.), water (3.times.), 5%
solution of sodium bicarbonate (3.times.), and then again with
water (3.times.). Product was dried under vacuum to obtain Compound
1 as a white solid (yield 32.8%). The structure of Compound 1 is
given below:
##STR00142##
(b) Preparation of Compound 2
[0224] A mixture of 4-methylpyridine (3.06 g, 32.9 mmol) and
Compound 1 (4.11 g, 15 mmol) was heated at 120.degree. C. for 3
hours. The reaction mixture was cooled and then 4 mL of isopropyl
alcohol was added and the combined mixture was refluxed for an
hour. After cooling the precipitate was collected by
centrifugation, washed with isopropyl alcohol (40 mL, 3.times.),
followed by diethyl ether (40 ml, 3.times.) and dried under vacuum
overnight to provide Compound 2 (yield 85%) which was then used
without any further purification. The structure of Compound 2 is
given below:
##STR00143##
(c) Preparation of S-25
[0225] To a suspension of Compound 2 (1.38 g) in n-butanol (15 mL),
p-dimethylaminobenzaldehyde (0.9 g) was added and the combined
mixture was stirred until it became homogeneous. To this mixture
.about.24 drops of piperidine was added and it was refluxed for 4.5
hours. Upon cooling, the precipitate formed was collected by
centrifugation, washed with isopropyl alcohol (40 ml, 3.times.),
diethyl ether (40 ml, 2.times.) and dried under vacuum to provide
dye S25 in a yield of about 68%. Abs=485 nm, Em=613 nm. The
structure of S25 is given below:
##STR00144##
Example 4. Synthesis of Tol3
(a) Preparation of Compound 3
[0226] A mixture of 3,4-dimethylpyridine (1.18 g, 11 mmol) and
1,10-diiododecane (1.97 g, 5 mmol) was alloyed during 3 hours at
120.degree. C. To the reaction mixture 5 mL of isopropyl alcohol
was added and the mixture was refluxed for an hour. Upon cooling,
the solvent was decanted, and the residue thus obtained was washed
with cold diethyl ether (40 ml, 2.times.), followed by
centrifugation to remove residual solvents. The solid obtained was
then dissolved in methanol (.about.4 mL) and dropwise added to cold
diethyl ether. Precipitated product was collected by
centrifugation, washed with diethyl ether (40 ml, 3.times.) and
dried under vacuum to provide Compound 3 in 88% yield. This product
was used without any further purification. The structure of
Compound 3 is given below:
##STR00145##
(b) Preparation of Tol3
[0227] A mixture of Compound 3 (0.61 g),
p-dimethylaminobenzaldehyde (0.3 g) and 6.about.8 drops of
piperidine in 5 mL of n-butanol was refluxed for 4 hours. After
cooling the precipitated solid was collected by centrifugation,
washed first with isopropyl alcohol (40 ml, 3.times.), diethyl
ether (40 ml, 2.times.) and then again isopropyl alcohol (40 ml,
1.times.) and diethyl ether (40 ml, 3.times.). The product was
dried under vacuum to provide dye Tol3 in 82% yield. Abs=471 nm,
Em=611 nm. The structure of Tol3 is given below:
##STR00146##
Example 5. Synthesis of S43
(a) Preparation of 1,1'-(1,2-phenylenebis(methylene))bis(4-methyl
pyridinium) bromide (Compound 4)
[0228] A mixture of 4-methylpyridine (1.02 g) and
1,2-bis-bromomethyl-benzene (1.32 g) was heated during 2.5 hours at
120.degree. C. To the reaction mixture 5 mL of isopropyl alcohol
was added and the mixture was refluxed for 2 hours. After cooling
the product was filtered, washed with diethyl ether and dried under
vacuum to provide Compound 4 in 87% yield. The structure of
Compound 4 is given below:
##STR00147##
(b) Preparation of S43
[0229] A mixture of Compound 4 (0.45 g),
p-dimethylaminobenzaldehyde (0.3 g) and 6 drops of piperidine in 5
mL of n-butanol were refluxed for 4 hours. After cooling the
product was filtered and washed with isopropyl alcohol and diethyl
ether. The residue obtained was recrystallized from the
DMF-methanol mixture to provide S43 in 72% yield. Abs=527 nm,
Em=637 nm. The structure of S43 is given below:
##STR00148##
Example 6. Synthesis of Yat 2134
(a) Preparation of 1,1'-(butane-1,4-diyl)bis(4-methylpyridinium)
iodide (Compound 5)
[0230] A mixture of 4-methylpyridine (1.02 g) and 1,4-diiodobutane
(1.55 g) in 5 mL of dioxane was refluxed for 8 hours. The obtained
salt was precipitated with diethyl ether and filtered. The
precipitate was washed with ether and dried under vacuum to provide
Compound 5 in 91% yield. This product was used without any further
purification. The structure of Compound 5 is given below:
##STR00149##
(b) Preparation of Yat 2134
[0231] This procedure was carried out as described previously in
step (b) of Example 3 with Compound 5 (0.5 g), piperidine (.about.6
drops), p-diethylamino benzaldehyde (0.36 g) and n-butanol (5 mL).
Purification was carried out by recrystallization from DMF-methanol
mixture to provide Yat 2134 in 70% yield. Abs=500 nm, Em=620 nm.
The structure of Yat 2134 is given below:
##STR00150##
Example 7. Synthesis of Yat 2148
[0232] A mixture of Compound 4 [0.45 g, obtained in step (a) of
Example 3], p-diethylaminobenzaldehyde (0.36 g) and 6 drops of
piperidine in 5 mL of n-butanol was refluxed for 4 hours. Upon
cooling the product was filtered and washed with isopropyl alcohol
and diethyl ether. The crude dye obtained was recrystallized from
the DMF-methanol mixture to provide Yat 2148 in 69% yield. Abs=520
nm, Em=632 nm. The structure of Yat 2148 is given below:
##STR00151##
Example 8. Synthesis of Yat 2149
(a) Preparation of
1,1'-(2,2'-oxybis(ethane-2,1-diyl))bis(4-methylpyridinium) chloride
(Compound 6)
[0233] A mixture of 4-methylpyridine (1.02 g) and 0.72 g of
1-Chloro-2-(2-chloro-ethoxy)-ethane (0.72 g) was heated at
120-130.degree. C. for 3-4 hours. To the reaction mixture 5 mL of
isopropyl alcohol was added and the mixture was refluxed for an
hour. Upon cooling the product was filtered and washed with diethyl
ether to provide Compound 6 in 81% yield. This product was used
without any further purification. The structure of Compound 6 is
given below:
##STR00152##
(b) Preparation of Yat 2149
[0234] This procedure was carried out as described previously in
step (b) of Example 3 with Compound 6 (0.33 g), piperidine
(.about.6 drops), p-diethylamino benzaldehyde (0.36 g) and
n-butanol (5 mL). After cooling the dye was precipitated with
isopropyl alcohol or diethyl ether. In order to obtain the iodide
salt, a saturated aqueous solution of KI (0.34 g) was added to the
dye solution in methanol. After cooling, the product was filtered,
washed with isopropyl alcohol, diethyl ether and dried under vacuum
to provide Yat 2149 in 65% yield. Abs=502 nm, Em=614 nm. The
structure of Yat 2149 is given below:
##STR00153##
Example 9. Synthesis of Yat 2150
[0235] This procedure was carried out as described previously in
step (b) of Example 2 with Compound 3 (0.61 g), piperidine
(.about.5 drops), p-diethylamino benzaldehyde (0.36 g) and
n-butanol (5 mL). Purification was carried out by recrystallization
from DMF-methanol mixture to provide Yat 2150 in 71% yield. Abs=485
nm, Em=612 nm. The structure of Yat 2150 is given below:
##STR00154##
Example 10 Monitoring Protein Stability in Two Different Buffer
Formulations
[0236] Goat anti-mouse IgG from Vector Labs (1.5 mg) was
resuspended in 150 .mu.l deionized water (dH.sub.2O). Phosphate was
removed from the IgG using an Ambion NucAway spin column, following
the manufacturer's instructions, briefly the column was resuspended
in 700 .mu.l dH.sub.2O and allowed to hydrate for 60 minutes.
Excess liquid was removed by centrifugation at 700.times.g for 2
minutes. The column was placed in a fresh collection tube and the
sample was carefully loaded on the center of the column. The IgG
was eluted by centrifugation at 700.times.g for 2 minutes. The
purified IgG was diluted 10 fold in either 100 mM HCl or 12 mM
phosphate pH 7.4, 150 mM sodium chloride. The samples were
incubated for 18 hours at 37.degree. C. The solutions were stained
with a final concentration of 100 mM MES, pH 6, 0.25 mg/ml IgG, 3
.mu.M S-25 and 3 .mu.M Tol3 (1:1 ratio) for at least 15 minutes.
The stained protein was spotted on the surface of a glass
microscope slide and overlaid with a cover slip, sealed with nail
polish and observed using a BX51 microscope (Olympus, Tokyo,
Japan). Images were acquired with a 40.times. objective lens
(Olympus). Fluorescent images were acquired using a Texas Red
filter set (Chroma Technoloogy Corp., Rockingham, Vt.). FIG. 1
shows that fibrils were formed in HCl solution, but not in the
neutral phosphate buffer. The fibrils formed exhibited fluorescence
that was bright and specific to the fibers using the S-25 and Tol3
dye mixture. There was little or no fluorescence when fibrils had
not formed.
Example 11. Binding Curve of Different Fluorescent Probes to 20
.mu.M of Aggregated Lysozyme Protein
[0237] Lysozyme aggregates were formed by dissolving Lysozyme in 10
mM HCl to make a 1 mM Lysozyme solution (14.8 mg/ml). The Lysozyme
solution was heated to 65.degree. C. with shaking at 750 rpm in an
Eppendorf thermomixer for 90 hours. The lysozyme was diluted to 20
.mu.M in a 50 mM potassium phosphate solution containing different
concentrations of a mixture of the dyes S-25 and Tol3. The
aggregate was incubated for 15 minutes prior to measuring the
fluorescence using a BioTek SynergyMx plate scanner, with
excitation set at 515 nm and emission set to 603 nm, both with a 9
nm slit-width. Readings were taken in at least triplicate in a
Greiner .mu.Clear black, clear bottom 96-well microplate. As can be
seen in FIG. 2, there is little or no signal generated with up to 1
nM of each of the dyes. Above 1 nM, the signal increases until
about 1 .mu.M each of the dyes, at which point no further signal
increase is observed. This indicates that above 1 .mu.M S-25 and 1
.mu.M Tol3, the fluorescence of 20 .mu.M aggregated Lysozyme is
dependent on aggregate concentration, and not dye
concentration.
Example 12. pH Sensitivity of Fluorescence Response to Aggregated
Lysozyme
[0238] Chicken egg white lysozyme (Sigma-Aldrich) was dissolved at
1 mM in 10 mM HCl. This monomer solution was stored at 4.degree. C.
Lysozyme aggregate was formed by shaking the protein solution at
750 rpm in a Thermomixer (Eppendorf) at 65.degree. C. for 90 hours.
The aggregation process was monitored by thioflavin T binding and
after saturation of the fluorescence signal (for lysozyme after 90
hrs), the aggregate solution was also stored at 4.degree. C.
[0239] FIG. 3A shows the effect of pH on the aggregation-specific
fluorescence of the dyes S-25 and Tol3, as determined by incubation
of 4 .mu.M aggregated lysozyme, 4 .mu.M monomer lysozyme (or 8
.mu.M lysozyme monomer alone) with 0.5 .mu.M S-25 and 0.5 .mu.M
Tol3 in buffers with a pH ranging from 3.about.10. The buffers used
were: 8 mM glycine-HCl, pH 3; 8 mM sodium acetate, pH 4.4; 8 mM
ammonium acetate, pH 6.0; 8 mM Tris-HCl, pH 7.4; 40 mM Tris-HCl, pH
7.8; 8 mM Tris-HCl, pH 8.5; and 8 mM sodium carbonate, pH 10. The
dye-protein mixture was incubated at room temperature (22.degree.
C.) for at least 15 minutes. Four replicates for either the 50%
aggregate or monomer at each pH were prepared and the plate was
scanned on a FLUOstar OPTIMA plate reader using an excitation
wavelength of 550 nm and an emission of 610 nm.
[0240] FIG. 3B shows the effect of pH on the linearity of
aggregation specific fluorescence, as determined using 1.25 .mu.M
S-25 and 1.25 .mu.M Tol3 in 50 mM of the following buffers:
succinic acid-HCl, pH 5.0; histidine-HCl, pH 7.0; and tris-HCl, pH
8.0. The total concentration of lysozyme was kept constant at 20
but the percent of the total that was aggregated as opposed to
monomeric was varied from 0% to 100% aggregate. At least three
replicates of each sample was prepared, incubated at 22.degree. in
the dark for 15 minutes, then scanned on a FLUOstar OPTIMA plate
reader using an excitation wavelength of 550 nm and an emission of
610 nm.
Example 13. Linear Dynamic Range of Lysozyme Aggregate Detection
Using a Two Dye Combination ST (525& Tol3) Compared with
Thioflavin T
[0241] Hen egg white lysozyme was solubilized in 10 mM HCl and
heated to 65.degree. C. for 90 hours to form aggregates. The signal
from the aggregate was determined after mixing aggregated lysozyme
with monomeric lysozyme at different ratios such that the total
lysozyme concentration remained at 20 .mu.M protein. The readings
were taken in 50 mM potassium phosphate, pH 7, containing either ST
(3 .mu.M S-25 and 3 .mu.M Tol3) or 5 .mu.M thioflavin T. Protein
was incubated with dye for 15 minutes prior to determining the
fluorescence using a BioTek Synergy Mx plate scanner, with
excitation setting at 515 nm and emission setting at 603 nm, both
with a 9 nm slit-width for S-25 and Tol3, and Thioflavin T was
detected with excitation setting at 435 nm and emission setting at
495 nm, both with a 9 nm slit-width. Readings were taken in at
least triplicate in a Greiner .mu.Clear black, clear bottom 96-well
microplate. As seen in FIG. 4, the concentration curve is more
linear with S25/Tol3 as compared to Thioflavin T.
Example 14. Effective Linear Dynamic Range of Antibody Aggregate
Detection Using a Two Dye Combination ST (525& Tol3), Compared
with Thioflavin T
[0242] Purified Rabbit anti-Goat IgG (4.26 mg/ml) was incubated in
HCl, pH 2.7 at 80.degree. for 90 minutes to form aggregates. The
signal from the aggregate was determined after mixing aggregate
with monomer at different ratios such that the total IgG
concentration remained at 240 .mu.g/ml protein. The readings were
taken in 50 mM potassium phosphate, pH 7, containing either ST (3
.mu.M S-25 and 3 .mu.M Tol3) or 5 .mu.M thioflavin T. Protein was
incubated with dye for 15 minutes prior to determining the
fluorescence using a BioTek SynergyMx plate scanner, with
excitation setting at 515 nm and emission setting at 603 nm, both
with a 9 nm slit-width for S-25 and Tol3, and thioflavin T was
detected with excitation setting at 435 nm and with emission
setting at 495 nm, both with a 9 nm slit-width. Readings were taken
in at least triplicate in a Greiner .mu.Clear black, clear bottom
96-well microplate. As can be seen in FIG. 5, the signal from ST is
more than 10 times higher than the signal from thioflavin T under
these conditions. Also the concentration curve is more linear with
S-25/Tol3 as compared to thioflavin T.
Example 15. Protein Aggregate Detection as a Function of Protein
Species
[0243] The linearity of aggregation induced fluorescence of S-25,
Tol3 and Thioflavin T (Thio-T) for four different proteins was
determined. The proteins were hen egg white lysozyme (results shown
in FIG. 6A), rabbit anti-goat IgG (FIG. 6B), bovine insulin (FIG.
6C) and .beta.-lactoglobulin (FIG. 6D)).
[0244] Chicken egg white lysozyme aggregate solution and monomer
solution as well as their mixtures were prepared as described in
Example 12. The protein concentration was maintained at 20 .mu.M,
and the dye concentration was 2.5 .mu.M in 50 mM Tris-HCl, pH 8.
The ratio of aggregated protein to native protein was varied from 0
to 100% aggregate. Each sample was analyzed in at least 3
replicates. The mixtures were incubated in the dark at 22.degree.
C. for 15 minutes, then the fluorescence intensity was determined
with a FLUOstar OPTIMA plate reader (BMG LABTECH) with excitation
setting at 550 nm and emission setting of 610 nm; while the
fluorescence intensity for Thioflavin-T was determined using a
SpectraMAX GeminiXS (Molecular Devices, with Softmax Pro 7.0) using
an excitation wavelength of 435 nm and emission wavelength of 495
nm.
[0245] Rabbit-anti-goat IgG (H&L, Pel-Freez.RTM., formulated in
the same manner as goat-anti-mouse IgG, described in Example 2) was
diluted to 29.4 .mu.M with double deionized water adjusted to pH
2.7 using HCl. Then IgG aggregate was prepared by shaking the
protein solutions at 750 rpm in a Thermomixer (Eppendorf) at
80.degree. C. for 2 hours. Using a final protein concentration of 3
.mu.M, the linearity of aggregation induced fluorescence was
determined as described above for lysozyme.
[0246] Insulin aggregate was prepared by dissolving bovine pancreas
insulin (Sigma-Aldrich) at 170 .mu.M in 100 mM HCl, which was
subsequently transferred to a Thermomixer (Eppendorf), set at 750
rpm continuous shaking at 65.degree. C. for 150 min. Using a final
protein concentration of 20 .mu.M, the linearity of aggregation
induced fluorescence was determined as described above for
lysozyme.
[0247] .beta.-Lactoglobulin (BLG, Sigma-Aldrich) was dissolved at 1
mM in double deionized water. The aggregate was prepared by shaking
the protein solutions at 750 rpm in a Thermomixer (Eppendorf) at
80.degree. C., which was stopped after 24 hours. Using a final
protein concentration of 50 .mu.M, the linearity of aggregation
induced fluorescence was determined as described above for
lysozyme.
Example 16. Kinetics of Lysozyme Aggregation
[0248] A 1 mM solution of hen egg white lysozyme in 10 mM HCl was
incubated at 65.degree. C. in an Eppendorf thermomixer shaking at
750 rpm. At the indicated times, aliquots of the lysozyme were
removed, diluted to 30 .mu.M in 100 mM Tris-HCl, pH 8.0, and
incubated with 5 .mu.M of the indicated dye. After 15 minutes
incubation, fluorescence intensity was determined with a FLUOstar
OPTIMA plate reader (BMG LABTECH) at excitation wavelength of 550
nm and emission wavelength of 610 nm; while the fluorescence
intensity for thioflavin-T was determined using a SpectraMAX
GeminiXS (Molecular Devices, with Softmax Pro 7.0) using an
excitation wavelength of 435 nm and emission wavelength of 495 nm.
Every sample was evaluated in 4 replicates. As can be seen in FIG.
7, Tol3, S-25 and Thioflavin T all detect similar kinetics for
protein aggregate formation.
Example 17. Protein Aggregation as a Function of Temperature
[0249] A 0.9 mg/ml solution of goat-anti-mouse IgG (Pel Freeze) was
prepared in 73 mM sodium acetate, pH 4.5. This solution was
incubated at 21.degree. C. or 50.degree. C. for various times.
After incubation, the solution was diluted further to create a
solution that was 50 mM histidine, pH 7, 0.45 mg/ml IgG, 2.5 .mu.M
S-25 and 2.5 .mu.M Tol3. After 15 minutes further incubation, the
fluorescence intensity was determined with a FLUOstar OPTIMA plate
reader (BMG LABTECH) at an excitation wavelength of 550 nm and
emission wavelength of 610 nm. As seen in FIG. 8, aggregation is
much more rapid at 50.degree. C. than at 21.degree. C.
Example 18. Protein Aggregation as a Function of pH
[0250] Goat-anti-mouse IgG was diluted to 40 .mu.M at either pH 7.6
in sodium phosphate buffer, or adjusted to pH 2.46 using HCl. Both
solutions were then kept at 21.degree. C. After various times,
aliquots were removed and diluted to a final concentration of 2
.mu.M in 100 mM histidine buffer, pH 7 with 2.5 .mu.M S-25 and 2.5
.mu.M Tol3. After 15 minutes further incubation at 21.degree. C.,
the fluorescence intensity was recorded. As seen in FIG. 9,
aggregation is observed to be much more rapid under acidic pH
conditions.
Example 19. Illustration of High-Throughput Protein Formulation
Optimization
[0251] (A). Goat anti-mouse IgG was diluted in sodium acetate, pH
4.5, then mixed with the excipients shown in FIG. 10A giving a
final concentration of 400 mM sodium acetate, 18 .mu.M IgG and the
excipient concentration shown in FIG. 10A. This mixture was heated
to 50.degree. C. for 6 hours. After this incubation, the protein
solution was diluted two-fold to give a final concentration of 50
mM histidine buffer, originally pH 7, 2.5 .mu.M S-25, 2.5 .mu.M
Tol3 and 9 .mu.M IgG. After 30 minutes of incubation on the shaker,
the fluorescence intensity was recorded on the plate reader
(FLUOstar Optima). The fluorescence intensity from each individual
excipient was then compared with that without any excipient (value
set as 1.0) as shown on the top of the corresponding excipient bar
in FIG. 10A.
[0252] (B). In the control plate, the IgG was added to the plate at
the same volume and concentration as in A. above, in 400 mM sodium
acetate. This mixture was heated to 50.degree. C. for 6 hours, as
described above. After 6 hours, the excipient was added followed by
S-25 and Tol3 to give all the final concentrations as in A. above.
Similar to the sample plate, the fluorescence intensity from
individual excipients was also compared with that from water
without any excipient (values set as 1.0) to obtain the relative
fluorescent intensity as shown on the top of the corresponding
excipient bar in FIG. 10B.
[0253] (C). A ratio between the fluorescent intensity of the
protein aggregated with the excipient versus the intensity derived
from the protein aggregated without excipient is a good measure of
the effect of the given excipient on aggregation. FIG. 10C shows
the ratio of fluorescence intensity in the sample plate (A. above)
divided by the fluorescence intensity of the control plate (B.
above). Those compounds with a value of 1 (dotted line) do not
significantly affect aggregation of IgG. Those compounds
substantially higher than 1, such as 0.2% Triton X-100 induce
aggregation of IgG. Those compounds with a value substantially
lower than 1, such as 100 mM trehalose, inhibit or slow down
aggregation of IgG.
Example 20. Inhibition of Lysozyme Aggregation by Chitotriose
[0254] Hen egg white lysozyme (300 .mu.M) was incubated with or
without N,N',N''-triacetyl-chitotriose ("Chitotriose", 510 .mu.M)
in 10 mM potassium phosphate, pH 7.3 for 16 hours. Aggregation was
induced by 3.5 fold dilution into 50 mM potassium phosphate, pH
12.2. Aggregation was followed by removing an aliquot of the
protein and diluting such that the final composition was 20 .mu.M
protein, 50 mM potassium phosphate, pH 7, 3 .mu.M S-25 and 3 .mu.M
Tol3. Protein was incubated with dye for 15 minutes prior to
determining the fluorescence using a BioTek Synergy Mx plate
scanner, with excitation setting at 515 nm and emission setting at
603 nm, both with a 9 nm slit-width. The zero time point was taken
before dilution to pH 12.2. Readings were taken in at least
triplicate in a Greiner .mu.Clear black, clear bottom 96-well
microplate. Aggregation was followed for several weeks at room
temperature (19.degree.-23.degree. C.). As seen in FIG. 11, S-25
and Tol3 easily demonstrate that Chitotriose inhibits lysozyme
aggregation, as previously demonstrated by Kumar et al. (2009).
Example 21. Thermal Shift Assays of BLG Aggregation
[0255] A solution containing 4 or 16 mg/mL of .beta.-lactoglobulin
(BLG) and 2.times.SYPRO.RTM. Orange dye (Molecular Probes, supplied
as 5000.times. with unknown concentration) or 4 .mu.M TOL3 or 4
.mu.M S25 was prepared using 1.times.PBS, pH 7.4 as the dilution
buffer. This solution was then loaded into LightCycler.RTM.
capillaries (20 .mu.L, Roche Diagnostics GmbH). These capillaries
were then mounted on the sample holder of a LightCycler.RTM. 480
Real-Time PCR System (Roche), programmed to heat from 28.degree. C.
to 90.degree. C. at 3.degree. C./min, followed by cooling down to
28.degree. C. at the same rate. The thermal shift curves were
achieved by plotting fluorescence intensity vs. temperature. After
the heating cycle, protein aggregates were visually apparent.
However, SYPRO.RTM. Orange dye, known to detect protein, failed to
show a melting peak, probably because of a low binding affinity to
the aggregated BLG; but both TOL3 and S25 were able to detect BLG
thermal shift peaks due to the aggregation, as shown in FIG. 12.
The temperature of aggregation detected by TOL3 or S25 both showed
a protein concentration dependence, down-shifting from
81.about.83.degree. C. to 71.about.73.degree. C. when the BLG
concentration was increased from 4 mg/mL to 16 mg/mL, a
characteristic of protein aggregation, as opposed to protein
unfolding. This demonstrates that both TOl3 and S25 are detecting
aggregation thermal shift peaks of BLG, not transitions do to
unfolding of the protein.
Example 22. Thermal Shift Assays of Carbonic Anhydrase II
Aggregation
[0256] Carbonic anhydrase II (Sigma, 10 .mu.M) containing
5.times.SYPRO.RTM. Orange or 10 .mu.M TOL3 or S25 or Yat 2150 was
prepared using either 50 mM sodium acetate, pH 4.5 or 25 mM PIPES,
pH 7.0 buffer containing 100 mM NaCl and 0.5 mM EDTA. Sample
preparation and the thermal shift assay were then performed using
the same conditions as described in Example 21. As shown in FIG.
13, although SYPRO.RTM. orange and dyes of the invention all show
thermal shift peaks, there is a .about.5.degree. C. up-shift for
peaks from dyes of the invention, between pH 4.5 and pH 7.0. This
also highlights the fundamentally different detection mechanism
between SYPRO.RTM. Orange dye and the dyes described in this
invention; the former detects protein unfolding, while the later
detects protein aggregation.
Example 23. Comparison of Fluorescence Response Between Unfolded
and Aggregated Form of IgG Using Dyes of the Present Invention
[0257] (A) Chemical shift assay based on internal tryptophan
fluorescence: Rabbit-anti-goat IgG (Pel Freeze) in 1.times.PBS
buffer of pH 7.4 was mixed with urea in 1.times.PBS to achieve a
final IgG concentration of 0.25 mg/ml. After mixing on ice for 10
minutes, the fluorescence emission intensity at 330 nm was recorded
by exciting at 280 nm using a MD-5020 fluorimeter (Phototechnology
International). A chemical shift curve was plotted based on
internal tryptophan fluorescence intensity at each given urea
concentration. Results are shown in FIG. 14A. Urea denatures
proteins but prevents them from aggregating.
[0258] (B) A solution containing aggregated IgG (formed as in
Example 15) or monomeric IgG at 0.033 mg/mL, 4.55 M urea and 6.67
.mu.M Tol3 was prepared and transferred into a microplate. After
incubating at 4.degree. C. degree for about 10 minutes, the
fluorescence was recorded. Two control solutions without IgG but
with the same concentration of TOl3 were included, one including
4.55M urea, another without urea. From the previous chemical shift
curve generated (FIG. 14A), 4.55 M urea is known to unfold
approximately 60% of the IgG. The results shown in FIG. 14B
indicate that TOL3 is sensitive to IgG aggregates, which shows
significant fluorescence enhancement relative to controls without
IgG, but it is not sensitive to unfolded IgG monomer, which shows
insignificant fluorescence enhancement relative to controls without
IgG.
Example 24. PDI Isomerase Activity Assay by Monitoring Insulin
Aggregation Kinetics
[0259] (A) Turbidity assay: Protein disulfide isomerase (PDI, Assay
Designs) was diluted with 0.5M of sodium phosphate, pH 6.8. A
mixture was made with insulin to give a final solution comprising
188 mM sodium phosphate, pH 6.8, 5 mM Tris-HCl, 2 mM EDTA, 1 mM
DTT, 1 mg/mL insulin and PDI at the desired concentrations (0, 5,
10, 15, 20, 25 .mu.g/mL). The optical density (OD) at 630 nm was
recorded immediately after the addition of DTT in a 96-well
microplate reader at 2 minute-intervals, with every well containing
300 .mu.L solution. The OD from 0 .mu.g/mL of PDI at any time point
was used as a background value and was subtracted from the OD value
of samples with PDI at the same time point. Results are seen in
FIG. 15A.
[0260] (B) Fluorometric assay: PDI and insulin solutions were
prepared as in the turbidity assay described in (A) above. S25 and
TOL3 were mixed with the insulin solution and placed into a black
Greiner flat bottom 96-well plate. PDI solutions containing various
amount of PDI were then added. Just prior to fluorescence
recording, DTT was added. The final solution was 188 mM sodium
phosphate pH 6.8, 5 mM Tris-HCl, 2 mM EDTA, 1 mM DTT, 0.225 mg/mL
insulin and PDI at 0, 5, 10, and 20 .mu.g/ml. A FLUOstar Optima
plate reader was used to record the fluorescence change after 5
seconds' shaking with excitation set at 550 nm and emission set at
610 nm. The fluorescence intensity from 0 .mu.g/mL of PDI solution
at the corresponding time point was used as a background value and
was subtracted from the corresponding reading in the presence of
enzyme. Results are seen in FIGS. 15B and C. The turbidity assay
and fluorometric assay, though of significantly different
sensitivities, are orthogonal to each other, further supporting
that dyes of the present invention monitor aggregation status and
not unfolding status.
Example 25. Inhibition of .beta.-Lactoglobulin Aggregation by HSP
27
[0261] Aggregation of .beta.-lactoglobulin was monitored in the
presence or absence of the chaperone HSP 27. Aggregation of 8 mg/ml
.beta.-lactoglobulin was monitored using 1.25 .mu.M Tol3 and 1.25
.mu.M S25 in PBS, pH 7.4 with 2.5 mM EDTA and 0.05% sodium azide.
When the chaperone HSP 27 was added it was added to a final
concentration of 0.4 mg/ml. HSP 27 was also run in the absence of
.beta.-lactoglobulin as a control. Aggregation was initiated by
heating the protein solution to 68.degree. C. in a 96 well
half-volume clear plate (Biomol International, Inc). The
fluorescence intensity was then recorded every 2 minutes, with
shaking between reads. The excitation wavelength was set to 550 nm
and the emission was set to 610 nm on a BMG Fluorstar plate reader.
The fluorescence intensity of the starting point was subtracted
from the remaining points. The results (FIG. 16) indicate that Hsp
27 can significantly prevent the aggregation of BLG at a mass ratio
as low as 1:20. Since Hsp 27 is binding with unfolded BLG
intermediate, thus preventing protein aggregation, the dyes are
detecting protein aggregation, as opposed to unfolding.
[0262] Other chaperone activity assays can be configured using
.beta.-lactoglobulin or other substrates, such as citrate synthase
(CS). Table 6 shows suggestions for chaperone-to-CS ratios that
should find application for the disclosed assay methods.
TABLE-US-00006 TABLE 6 Chaperone:CS ratios. Chaperone system
Members ADI catalog #s Chaperone:CS DnaK/DnaJ/GrpE DnaK SPP-630 1:1
or less DnaJ SPP-640 GrpE SPP-650 Hsp70/Hsp40 Hsp70 NSP-555,
ESP-555, 1:1 or less SPP-758 Hdj1 SPP-400 Hdj2 SPP-405 Mortalin
SPP-828 Hsc70 SPP-751 Hsp70-A1 SPP-502, ESP-502 Hsp90 Hsp90 alpha
SPP-776 Depends on Hsp90 beta SPP-777 cochaperones Chaperonins
Hsp60/Cpn10 NSP-540, ESP-540 1:1 or less (human) Cpn10 SPP-110
Chaperonins GroEL SPP-610 1:1 or less (bacterial) GroES SPP-620
Small heat shock Hsp25 SPP-510 20:01 .sup. proteins Hsp27 SPP-715,
SPP-716 Crystallins SPP-225, SPP-226, SPP-235, SPP-236 ER
chaperones Grp78 SPP-765 5:1 .sup. PDI SPP-891 10:1 .sup. Erp72
H00009601-Q01 20:1 .sup. (abnova) Grp94 SPP-766 Depends on (ER
Hsp90) cochaperones Nascent chain NAC none 20:1 .sup. chaperones
Trigger Factor none 20:1 .sup.
[0263] Chaperone: CS ratios are based upon the known biology of the
individual systems. Active folders are likely to show significant
signal at less than 1:1 molar ratio to substrate, as each chaperone
complement will be able to inhibit aggregation while it actively
folds. Aggregate inhibitors like the small heat shocks and trigger
factor require substantially more, as they need to saturate the
solution to prevent aggregation. Pairs of holders and folders
(e.g., crystalline with low Hsp70 complex) may provide synergistic
effects.
Example 26. Monitoring Protein Stability in an Agitated
Solution
[0264] One method of creating aggregated proteins is by agitation
of the protein solution. Goat-anti-mouse IgG (12.8 mg/mL,
Pel-freeze Biologicals) was supplied in 10 mM sodium phosphate, 150
mM NaCl, 0.05% sodium azide, pH 7.2, filtered through 0.2 .mu.m
filter. The stirring experiment was performed by stirring 200 .mu.L
of IgG solution as supplied at 22.degree. C. in a 4 mL amber glass
vial with flat bottom at 990 rpm using Variomag.RTM. Poly
electronic stirrers. The control (without stirring) was also kept
at 22.degree. C. The stirring bar was 1.times.0.4.times.0.2
cm.sup.3.
[0265] A BioTek plate reader with a filter set as 550
(excitation)/603 nm (emission) and 9 nm filter band on both
excitation and emission was used to scan from the bottom of the
plate. 5 .mu.L of the IgG solution at various time points (stirred
or non-stirred) was added into 95 .mu.L of 2.5 .mu.M TOL3, 2.5
.mu.M of S25 and 50 mM potassium phosphate, pH 7.0 and incubated
for 20 minutes. Every time point was replicated twice. The
fluorescence of free dye was subtracted from that of the IgG/dye
mixing solution for both the stirred sample and non-stirred
control. The results (FIG. 17) indicate that the TOL3/S25 dye mix
can detect agitation induced aggregation.
Example 27. Thermal Shift Assay to Find Thermally Stabilizing
Buffers for DNA Polymerase I Klenow Fragment
[0266] In molecular biology, enzymes are often required that
function at elevated temperatures. Enzymes produced by mesophilic
organisms usually denature at elevated temperatures, followed by
aggregate formation. A rapid fluorescence-based assay was developed
for assessing a range of parameters impacting the thermal stability
of an enzyme. Overall, protein stability was monitored by a
fluorogenic dye that selectively detects aggregated protein.
Stability can be measured in the presence of different buffers,
cryoprotectants and excipients. By systematically raising the
temperature of the protein in solution, the temperature at which
the protein aggregates (T.sub.agg) can be determined. Using this
method with Klenow DNA polymerase, it was determined that trehalose
significantly increases T.sub.agg. The DNA polymerase activity of
the enzyme is significantly enhanced at 50.degree. C. in the
presence of trehalose under the same conditions. Low amounts of the
detergents Tween20.RTM. and Triton X-100.RTM. (0.05%) decreased
T.sub.agg and also compromised enzyme activity, especially at
elevated temperatures. The assay facilitates screening for buffers
and additives that structurally stabilize a protein of interest at
elevated temperatures.
[0267] Fifty units of DNA polymerase I Klenow fragment (New England
Biolabs, Ipswich, Mass.) was incubated in several different buffers
and excipients in the presence of 2.5 .mu.M YAT2150 dye. The
temperature was slowly raised and the fluorescence was determined
using a Qiagen (Valencia, Calif.) Rotorgene real-time thermocycler
with an excitation filter at 530 nm and an emission filter of 610
nm. Table 7 shows the detected aggregation temperatures in Buffer 1
(10 mM bis-Tris propane, 10 mM MgCl.sub.2, 1 mM dithiothreitol, pH
7), Buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl.sub.2, 1 mM
dithiothreitol, pH 7.9), Buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10
mM MgCl.sub.2, 1 mM dithiothreitol, pH 7.9), Buffer 4 (50 mM
potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1
mM dithiothreitol, pH 7.9), Buffer 2 with 0.5 M trehalose, Buffer 2
with 0.05% Tween20.RTM. and Buffer 2 with 0.05% Triton
X-100.RTM..
TABLE-US-00007 TABLE 7 Effect of various buffers on aggregation
temperature of DNA polymerase I Klenow fragment. Buffer T.sub.agg
Buffer #1 59.1.degree. +/-0.3.degree. Buffer #2 59.7.degree.
+/-0.2.degree. Buffer #3 60.2.degree. +/-0.3.degree. Buffer #4
59.8.degree. +/-0.4.degree. Buffer #2 Trehalose 62.6.degree.
+/-0.3.degree. Buffer #2 Tween 20 58.4.degree. +/-0.3.degree.
Buffer #2 Triton X-100 57.8.degree. +/-0.2.degree.
[0268] This data demonstrates that trehalose significantly raises
the aggregation temperature in Buffer 2. To show if this extends to
enzyme activity, the DNA polymerase was incubated in Buffer 2 with
400 .mu.M dATP, dCTP, dGTP and fluorescein-12-dUTP at 37.degree.
C., 42.degree. C. and 50.degree. C. After 15 minutes at the given
temperature, the DNA template and primer were added for an
additional 15 minutes at the given temperature. The template used
was 5-ACTTCTTACT TCTTACTTCT TACTTCTTAC TTCTTACTTC TTACTTCTTA
CTTCTTACTT CTTCATTGGT CATCTCGATC CATGACCTCA GC-3' and the primer
was 5'-TTGCTGAGGT CATGGATCGA GA-3'. The amount of oligo extended
full length is shown in Table 8, as measured by relative
fluorescence of the incorporated fluorescein.
TABLE-US-00008 TABLE 8 Oligonucleotide extension by DNA polymerase
I Klenow fragment using various buffer additives during incubation
at three temperatures. Additive in Buffer 2 37.degree. 42.degree.
50.degree. None 0.507 0.303 0.189 0.5M Trehalose 0.567 0.449 0.308
0.05% Tween20 0.317 0.154 0.180 0.05% Triton X-100 0.257 0.159
0.170
This data shows a correlation with the increased aggregation
temperature in the presence of 0.5 M trehalose and the enzyme
activity at elevated temperatures.
Example 28. YAT2150 Dye Detecting Aggregated Proteins within Fixed
and Permeabilized Cells
[0269] In mammalian cells, aggregated proteins may be concentrated
by microtubule-dependent retrograde transport to perinuclear sites
of aggregate deposition, referred to as aggresomes. Aggresomes are
inclusion bodies that form when the ubiquitin-proteasome machinery
is overwhelmed with aggregation-prone proteins. Typically, an
aggresome forms in response to some cellular stress, such as
hyperthermia, viral infection, or exposure to reactive oxygen
species. Aggresomes appear to provide a cytoprotective function by
sequestering the toxic, aggregated proteins and may also facilitate
their ultimate elimination from cells by autophagy. Certain
cellular inclusion bodies associated with human disease are thought
to arise from an aggresomal response, including Lewy bodies
associated with neurons in Parkinson's disease, Mallory bodies
associated with liver cells in alcoholic liver disease, and hyaline
inclusion bodies associated with astrocytes in amyotrophic lateral
sclerosis.
[0270] The ability of YAT2150 to detect aggregated proteins within
fixed and permeabilized cells was evaluated. Human cervical
adenocarcinoma epithelial cell line HeLa was obtained from American
Type Culture Collection (ATCC, Manassas, Va.). HeLa cells were
routinely cultured in Eagle's Minimum Essential Medium (ATCC) with
low glucose, supplemented with 10% fetal bovine serum (FBS) (ATCC)
and 100 U/ml penicillin with 100 .mu.g/ml streptomycin
(Sigma-Aldrich). Cells were maintained in a saturated, humidified
atmosphere at 37.degree. C., 5% CO.sub.2 and 95% air. HeLa cells
were grown on glass slides or polystyrene tissue culture dishes
until .about.80% confluent. The cells were treated with various
modulators or vehicle at various concentrations and time intervals,
as detailed in Table 9. Proteasome inhibitors MG-132 (Enzo Life
Sciences Inc.), lactacystin (Enzo Life Sciences Inc.), bortezomib
(Velcade.RTM.) (Selleck Chemicals LLC, Houston, Tex.) and
epoxomicin (Enzo Life Sciences Inc.) were employed in the studies.
The histone deacetylase 6 inhibitor
N-hydroxy-7-[5-(4-tertbutoxycarbonylaminophenyl)-3-isoxazolecarboxamido]
heptamide (BML-281) was also obtained from Enzo Life Sciences Inc.
Negative control cells were treated with a vehicle (DMSO, media or
other solvent used to reconstitute or dilute the inducer or
inhibitor) for an equal length of time under similar conditions.
The cells were subsequently washed with PBS, and fixed in 4%
formaldehyde in PBS for 30 min at room temperature, then
permeabilized with 0.5% Triton X-100, 3 mM EDTA in PBS on ice, for
30 minutes. The cells were washed with PBS, and then 500 nM of
YAT2150 dye was added. The samples were incubated for 30 minutes at
room temperature, protected from light. The cells were washed with
PBS, covered with glass coverslips and observed using a
fluorescence microscope (Carl Zeiss MicroImaging GmbH, Jena,
Germany) equipped with a Texas Red filter set. Images were acquired
with a 63.times. objective lens (Carl Zeiss, Inc).
TABLE-US-00009 .mu.M Induction Cell Aggresome Treatment Target
Effect used Time (hrs) Line Formation Starvation Inhibits mammalian
Activates N/A 1~4 HeLa, No target of rapamycin autophagy Jurkat
(mTOR) Rapamycin Inhibits mammalian Activates 0.2 6~18 HeLa, No
target of rapamycin autophagy Jurkat (mTOR) PP242 ATP-competitive
Activates 1 18 Hela No inhibitor of mTOR autophagy Lithium Inhibits
IMPase and Activates 10,000 18 HeLa, No reduce inositol and
autophagy Jurkat IP.sub.3 levels; mTOR- independent Trehalose
Unknown, mTOR- Activates 50,000 6 HeLa, No independent autophagy
Jurkat Bafilomycin A1 Inhibits Vacuolar- Inhibits 6~9 18 HeLa, Yes
ATPase autophagy .times.10.sup.-3 Jurkat Chloroquine Alkalinizes
Inhibits 10~50 18 HeLa, Yes Lysosomal pH autophagy Jurkat Tamoxifen
Increases the Activates 4~10 6~18 HeLa, Yes intracellular level of
autophagy Jurkat ceramide and abolishes the inhibitory effect of
PI3K Verapamil Ca.sup.2+ channel blocker; Activates 40-100 18 HeLa,
Yes reducesintracytosolic autophagy Jurkat Ca.sup.2+ levels; mTOR-
independent Hydroxy- Alkalinizes Inhibits 10 18 HeLa, Yes
chloroquine Lysosomal pH autophagy Jurkat Loperamide Ca.sup.2+
channel Activates 5 18 HeLa No blocker;reduces intra- autophagy
cytosolic Ca.sup.2+ levels; mTOR-independent Clonidine
Imidazoline-1 Activates 100 18 HeLa No receptor agonist; autophagy
reduces cAMP levels; mTOR-independent MG-132 Selective proteasome
Induce 2~5 18 HeLa, Yes inhibitor aggresome Jurkat Epoxomicin
Selective proteasome Induce 0.5 18 HeLa Yes inhibitor aggresome
Lactacystin Selective proteasome Induce 4 Yes inhibitor aggresome
Velcade .RTM. Selective proteasome Induce 0.5 18 HeLa Yes inhibitor
aggresome amyloid beta Induce oxidative Induce 25 18 SK-N- Yes
peptide 1-42 stress aggresome SH Norclomipramine Alkalinizes
Inhibits 5~20 18 HeLa Yes Lysosomal pH autophagy
[0271] MG-132, a relatively nonspecific proteasome inhibitor, has
also been shown to perturb protein homeostasis, inducing both the
unfolded protein response (UPR) and the heat shock response (HSR)
(Mu et al., 2008; Murakawa et al., 2007). MG-132 is known to
accelerate the formation of perinuclear aggresomes as well as
inclusion bodies within cells (Beaudoin S et al., 2008). After
treating cells with MG-132, YAT2150 dye was found to readily
highlight aggregated protein cargo accumulating within vacuolar
cytoplasmic structures, as observed by fluorescence microscopy
(FIG. 18). Examination of the distribution of the fluorescent dye
within cells treated with MG-132, revealed a punctate pattern of
cytoplasmic staining, as well as staining of certain inclusion
bodies within or immediately adjacent to the nucleus itself.
Multiple cytoplasmic inclusion bodies were readily discerned using
the fluorescent dye. It should be noted that true aggresomes are
characterized by a single large protein aggregate in cells that
co-localizes with the centrosome in a microtubule-dependent
fashion. The formation of the multiplicity of inclusion bodies
observed upon MG-132 treatment was not sensitive to the histone
deacetylase 6 inhibitor,
N-hydroxy-7-[5-(4-tertbutoxycarbonylaminophenyl)-3-isoxazolecarboxamido]h-
eptamide (BML-281) (Kawaguchi et al., 2003) or nocodazole (data not
shown). This suggests that the generated inclusion bodies do not
meet the strictest definition for aggresomes. The described probe
appears to detect aggregated protein cargo within a variety of
inclusion bodies, regardless of whether they are co-localized with
the centrosome or formed in a microtubule-dependent manner.
[0272] The ability to detect aggresomes and related inclusion
bodies was further demonstrated using various potent, cell
permeable, and selective proteasome inhibitors: lactacystin,
epoxomicin and bortezomib (Velcade.RTM.), as shown in FIG. 19.
Previous studies have shown that bortezomib-mediated proteasomal
inhibition results in the accumulation of large quantities of
ubiquitin-conjugated proteins organized into perinuclear structures
termed "aggresomes" (Nawrocki et al., 2006). All of the tested
proteasome inhibitors induced the accumulation of cytoplasmic
inclusion bodies within the cells, as demonstrated with the YAT2150
dye. Efforts to stain inclusion bodies in living cells were not met
with success, however. Instead of discrete punctuate staining of
aggregated cargo, a weak, diffuse cytoplasmic staining was observed
that differed little between control cells and cells treated with a
proteasome inhibitor. This could possibly be due to poor access of
the dye to the contents of membrane-bound vacuolar structures
within the cells.
Example 29. Co-Localization of Aggregated Protein with
Ubiquitinylation and Various Pathway Proteins Implicated in
Autophagy
[0273] Antibodies were obtained from the following commercial
sources: fluorescein-labeled p62 and LC3 reactive rabbit polyclonal
antibodies and ubiquitin-reactive mouse monoclonal antibody (clone
EX-9) were obtained from Enzo Life Sciences, Ltd. (Exeter, UK).
These labeled conjugates were produced by direct labeling of
antibodies raised to p62-derived, LC3-derived, and
ubiquitin-derived peptides, respectively. A mouse monoclonal
antibody reactive with human tau (clone tau-13) (Covance Inc,
Emeryville, Calif.) is able to stain brain tissue early in
Alzheimer's disease. It was used in conjunction with Alexa
Fluor.RTM. 488 dye-labeled goat anti-mouse secondary antibody from
Life Technologies (Carlsbad, Calif.). Alexa Fluor.RTM. 488
dye-labeled beta amyloid reactive mouse monoclonal antibody (clone
6E10), which is specifically reactive to amino acid residues 1-16
of the human .beta.-amyloid peptide, was obtained from Covance
Inc.
[0274] For antibody co-localization studies: cells were treated
overnight with 5 .mu.M MG-132, then fixed and permeabilized using
the protocol in Example 28. The cells were then incubated in PBS
containing 3% bovine serum albumin (blocking buffer).
Fluorescein-labeled p62, LC3 and ubiquitin (clone EX-9) reactive
antibodies were diluted to a concentration of 1 .mu.g/mL in
blocking buffer and incubated for 1 h at room temperature. Cells
were then washed in PBS containing 0.1% Tween-20 for 15 min. Next,
the cells were stained with YAT2150 dye for 30 minutes at room
temperature and washed with PBS, covered with glass cover slip,
sealed with nail polish, and observed by fluorescence microscopy
using a Texas Red filter set for the YAT2150 dye, and an FITC
filter set for fluorescein-labeled antibodies, respectively. All
images were acquired with a 63.times. objective lens (Carl Zeiss,
Inc).
[0275] Co-localization of fluorescently-labeled ubiquitin antibody
conjugate with YAT2150 dye is shown in FIG. 20A, highlighting
interactions between aggregated protein cargo and ubiquitinylation
status. The ubiquitin signal is observed to be co-localized
exclusively with the aggregated protein cargo, but it should be
remembered that cells were fixed and permeabilized, which likely
removed any free ubiquitin and ubiquitinylated substrates present
in the cytosol. FIG. 20B demonstrates that a fluorescein-conjugated
antibody directed towards p62 (a ubiquitin-binding scaffold protein
that co-localizes with ubiquitinylated protein aggregates) also
co-localizes with the YAT2150 dye within cells treated with MG-132.
Furthermore, co-localization between fluorescein-labeled antibodies
reactive with LC3 (a ubiquitin-like autophagy cascade protein
residing in the phagophore membrane) and aggregated protein cargo
was demonstrated, as shown in FIG. 20C. The described
co-localization studies demonstrate the capability of the YAT2150
dye to be used to analyze the interactions between aggregated
protein cargo, protein post-translational modifications, and
various autophagy pathway proteins. The long wavelength red
emission of the fluorescent probe is especially suitable for
studies using green fluorescent dye conjugates, such as
fluorescein, Alexa Fluor.RTM. 488, Oregon Green.RTM. 488,
BODIPY.RTM.-FL, HiLyte Fluor.TM. 488 and DyLight.RTM. 488.
Example 30. Cell Culture-Based Model Mimicking Elements of
Alzheimer's Disease Pathology
[0276] The human SK-N-SH neuroblastoma cell line was obtained from
American Type Culture Collection (ATCC, Manassas, Va.). SK-N-SH
cells were routinely cultured in Eagle's Minimum Essential Medium
(ATCC) with low glucose, supplemented with 10% fetal bovine serum
(FBS) (ATCC) and 100 U/ml penicillin, 100 .mu.g/ml streptomycin
(Sigma-Aldrich). Amyloid beta peptide 1-42 (21.sup.st Century
Biochemicals, Marlboro, Mass.) was added to the culture medium and
SKN-SH cells were incubated overnight to induce aggresome
formation. SMER28 (Enzo Life Sciences Inc.), an inducer of
autophagy, was employed to block this accumulation A cell
culture-based assay mimicking the accumulation of .beta.-amyloid,
as observed in Alzheimer's disease, was established.
[0277] FIG. 21B shows YAT2150 dye is able to detect amyloid fibrils
within an SK-N-SH human neuroblastoma cell line induced to form
inclusion bodies by overnight incubation with exogenously added
amyloid beta 1-42 peptide. Furthermore, SMER28, a small molecule
inducer of autophagy was evaluated with respect to its effect on
.beta.-amyloid peptide accumulation within the cells. SMER28 has
previously been shown to act via an mTOR-independent mechanism to
increase autophagosome synthesis and enhance the clearance of model
autophagy substrates, such as [A53T].alpha.-synuclein and mutant
huntingtin fragments (Sarkar et al., 2007; Renna et al., 2010). It
has also been demonstrated that SMER28 attenuates mutant
huntingtin-fragment toxicity in Drosophila models, suggesting
therapeutic potential. As shown in FIGS. 21C & D, SMER28 was
able to substantially reduce accumulation of .beta.-amyloid peptide
in SK-N-SH human neuroblastoma cells, suggesting this assay could
potentially enable screening of aggregation inhibitors relevant to
neurodegenerative disease, in an authentic cellular context.
Example 31. Detecting Protein Aggregates in Post-Mortem Brain
Tissue Sections from Patients with Alzheimer's Disease
[0278] Post-mortem brain tissue (cerebellum) from patients with
Alzheimer's disease and human adult normal brain tissue
(cerebellum) were obtained from BioChain Institute, Inc. (Hayward,
Calif.). All tissue samples were received from certified tissue
vendors who guarantee that they were collected with informed
consent from the donors and their relatives, all samples were
excised by licensed physicians, all normal and diseased tissues
were determined by the donor's clinical reports and all collections
were made with the relevant requirements for ethics committee/IRB
approvals. The frozen tissue sections were 5-10 .mu.m in thickness,
mounted on positively charged glass slides, and fixed with cold
acetone by the manufacturer. The embedded tissue sections were
fixed in formalin immediately after excision, and embedded in
paraffin. Tissue sections were .about.5 .mu.m in thickness, and
mounted on positively charged glass slides by the manufacturer.
[0279] Paraffin-embedded tissue sections were deparaffinized prior
to staining. Briefly, the microscope slide-mounted specimen was
immersed in a xylene substitute bath until the paraffin was
solubilized. The deparaffinized specimens were then washed with a
series of alcohol solutions of decreasing alcohol concentration, to
remove xylene, before a final wash with water. The tissue sections
were then fixed with 4% formaldehyde in PBS for 15 min at
37.degree. C. Following washing in deionized water, tissue sections
were stained with either 1 .mu.M thioflavin T in PBS or 500 nM
YAT2150 dye for 3 min, rinsed in water and destained in 1% acetic
acid for 20 min. Finally the tissues sections were washed
thoroughly in water, dehydrated, covered with glass coverslips,
mounted in anti-fade mounting medium and observed using a
fluorescence microscope (Carl Zeiss, Inc.) with an FITC filter set
for thioflavin T and a Texas Red filter set for YAT2150 dye,
respectively. All images were acquired with a 63.times. objective
lens (Carl Zeiss, Inc).
[0280] For the antibody co-localization studies, tissue sections
were stained with YAT2150 dye as described above. The tissue
sections were then blocked in PBS containing 3% bovine serum
albumin (blocking buffer). Tau-reactive monoclonal antibody (clone
tau-13) and Alexa Fluor.RTM. 488 labeled beta amyloid reactive
monoclonal antibody (clone 6E10) were diluted to a concentration of
2 .mu.g/mL in blocking buffer and incubated for 1 h at room
temperature. Tissues were then washed in PBS containing 0.1%
Tween-20 for 15 min. For tissues incubated with Tau-13 antibody,
the slides were subsequently incubated with Alexa Fluor.RTM. 488
goat anti-mouse secondary antibody for 30 min at room temperature.
Finally the tissue sections were washed with PBS, covered with
glass coverslips, mounted in anti-fade mounting medium and observed
using a fluorescence microscope (Carl Zeiss, Inc.) with a Texas Red
filter set for YAT2150 dye and FITC filter set for labeled
antibodies, respectively. All images were acquired with a 63.times.
objective lens (Carl Zeiss, Inc.).
[0281] Thioflavin T (ThT) is a widely employed histological probe
for detecting the formation of amyloid fibrils in brain tissue
(Gunilla et al., 1999). However, this dye is not an ideal predictor
of the degree of fibrillization because its fluorescence varies
substantially depending upon the structure and morphology of the
amyloid fibrils. It was found that the dye generates fairly high
background and weak fluorescent signal in brain tissue sections, as
shown in FIG. 22A. Thus, optimized protocols for the detection of
amyloid plaques in frozen and paraffin-embedded tissue sections of
human brain were developed using the YAT2150 dye. Relative to ThT,
this novel probe demonstrates significantly higher fluorescence
emission intensity enhancement in the presence of amyloid protein
fibrils and low non-specific background (shown in FIG. 22B). In
addition, use of antibodies directed against .beta.-amyloid and tau
protein, in conjunction with the YAT2150 dye, confirm the
selectivity of the probe for detection of amyloid plaques in
post-mortem brain tissue of patients with Alzheimer's disease
(FIGS. 22A & B).
Example 32. Utilizing Flow Cytometry to Quantify the Accumulation
of Protein Aggregates within Cells
[0282] Human leukemic Jurkat cells were obtained from ATCC. Jurkat
cells were grown in suspension in RPMI medium supplemented with 10%
(v/v) FBS, penicillin (100 U/ml), streptomycin (100 .mu.g/ml), and
glutamine (200 mM). Jurkat cells were maintained in a saturated,
humidified atmosphere at 37.degree. C., 5% CO.sub.2 and 95%
air.
[0283] Jurkat cells were grown to log phase, and treated with 5
.mu.M MG-132 or with vehicle for 16 hours. At the end of the
treatment, adherent cells were trypsinized; while Jurkat cells were
simply collected by centrifugation (400.times.g for 5 min). Samples
were resuspended at 1.times.10.sup.6 to 2.times.10.sup.6 cells per
ml. For each group, triplicate samples were prepared. The cells
were washed with PBS, fixed in 4% formaldehyde in PBS for 30 min
and then permeabilized with 0.5% Triton X-100, 3 mM EDTA, pH 8 on
ice, for 30 minutes. The cells were then washed, and resuspended in
500 .mu.L of 200 nM YAT2150 dye. The samples were incubated for 30
minutes at room temperature, protected from light. Experiments were
performed using a FACS Calibur benchtop flow cytometer (BD
Biosciences, San Jose, Calif.) equipped with a blue (488 nm) laser.
YAT2150 dye fluorescence was measured in the FL3 channel. No
washing was required prior to the flow cytometric analysis.
[0284] For the immunocytochemistry study, after fixing and
permeabilizing the cells, the cells were blocked in PBS containing
3% bovine serum albumin for one hour. Fluorescein-labeled p62
antibody was diluted to a concentration of 2 .mu.g/mL in blocking
buffer and incubated with the cells for 1 h at room temperature.
Cells were then washed in PBS containing 0.1% Tween-20 for 15 min.
Data was acquired by FACS Calibur benchtop flow cytometer (BD
Biosciences, San Jose, Calif.) equipped with a blue (488 nm) laser,
with the antibody signal measured in the FL1 channel.
[0285] All of the experiments were performed at least three times.
Flow cytometry data were analyzed by comparison of mean
fluorescence, through calculation of a term we refer to as the
Aggregation Propensity Factor (APF), having the following
definition.
APF=100.times.((MFI.sub.treated-MFI.sub.control)/MFI.sub.treated),
wherein MFI.sub.treated and MFI.sub.control are the mean
fluorescence intensity values from control and treated samples.
This metric is based upon a similar approach that is commonly
employed in the assessment of fluorescent signal between control
and treated groups in multidrug resistance experiments, using a
term referred to as Multidrug Resistance Activity Factor (MAF)
(Hollo et al., 1994). APF is a unitless term measured as the
difference between the amount of the YAT2150 dye accumulated within
cells in the presence and absence of a proteasome inhibitor or
other inducer of aggresome or inclusion body formation or protein
aggregation. The fluorescence measurement in the presence of the
proteasome inhibitor constitutes the maximal potential fluorescence
for the given cell population when aggregated protein cargo has
been generated. This represents a standardization method, which
eliminates unknown cell type-specific variables that might
influence dye accumulation, such as cell size, shape and volume,
allowing the potential for intra- and inter-laboratory comparison
of test results and APF values.
[0286] A flow cytometry cell-based assay was next developed using
the YAT2150 dye. FIG. 24A demonstrates typical results of flow
cytometry-based analysis of cell populations using the YAT2150 dye.
Uninduced control and 5 .mu.M MG-132-treated Jurkat cells were
employed in the investigation. After 16 hours treatment, fixed and
permeabilized cells were stained with the YAT2150 dye and then
analyzed without washing by flow cytometry. Results are presented
using histogram overlay graphs. Control cells displayed minimal
fluorescence staining with the dye. The YAT2150 dye signal
increased about three-fold in the MG-132 treated cells, readily
demonstrating that MG-132 induced protein aggregate formation in
Jurkat cells. An APF value of approximately 72, as defined above,
demonstrates that the control and treated cell populations were
readily distinguishable by flow cytometry. For comparison, an MAF
cut-off value of about 20-25 is routinely employed in flow
cytometry assays of multi-drug resistance (Hollo et al., 1994).
Protein aggregate accumulation in the Jurkat cells was confirmed by
flow cytometry analysis using fluorescein-conjugated p62-reactive
antibody (FIG. 24B). Thus, the described assay allows, for the
first time, easy quantification of aggresome accumulation by flow
cytometry. The advantage of the dye-based approach relative to the
antibody one is that staining and analysis are much more rapid.
Simultaneous staining with the fluorescein conjugated antibody and
the red fluorescent dye was also feasible (data not shown).
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[0350] In view of the above, it will be seen that several
objectives of the invention are achieved and other advantages
attained.
[0351] As various changes could be made in the above methods and
compositions without departing from the scope of the invention, it
is intended that all matter contained in the above description and
shown in the accompanying drawings shall be interpreted as
illustrative and not in a limiting sense.
[0352] All references cited in this specification are hereby
incorporated by reference. The discussion of the references herein
is intended merely to summarize the assertions made by the authors
and no admission is made that any reference constitutes prior art.
Applicants reserve the right to challenge the accuracy and
pertinence of the cited references.
Sequence CWU 1
1
2192DNAArtificial SequenceTemplate used in primer extension
experiment 1acttcttact tcttacttct tacttcttac ttcttacttc ttacttctta
cttcttactt 60cttcattggt catctcgatc catgacctca gc 92222DNAArtificial
SequencePrimer used in primer extension experiment 2ttgctgaggt
catggatcga ga 22
* * * * *