U.S. patent application number 15/438949 was filed with the patent office on 2017-08-24 for high-throughput screening for compounds modulating expression of cellular macromolecules.
The applicant listed for this patent is The Scripps Research Institute. Invention is credited to Corinne Lasmezas, Charles Weissmann.
Application Number | 20170241984 15/438949 |
Document ID | / |
Family ID | 46969578 |
Filed Date | 2017-08-24 |
United States Patent
Application |
20170241984 |
Kind Code |
A1 |
Lasmezas; Corinne ; et
al. |
August 24, 2017 |
High-throughput screening for compounds modulating expression of
cellular macromolecules
Abstract
A method of screening for compounds that module expression of
specific macromolecules, the "target". The method is particularly
useful in that it does not require separation of target-bound and
excess ligand and therefore enables, but is not limited to, High
Throughput Screening for compounds that increase or decrease the
levels or amounts of a target present in a biological sample. The
method can also be used for high-throughput diagnosis of a
condition leading to an increase or decrease of a cellular
macromolecule.
Inventors: |
Lasmezas; Corinne; (Palm
Beach Gardens, FL) ; Weissmann; Charles; (Palm Beach,
FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Scripps Research Institute |
La Jolla |
CA |
US |
|
|
Family ID: |
46969578 |
Appl. No.: |
15/438949 |
Filed: |
February 22, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14046483 |
Oct 4, 2013 |
9612238 |
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15438949 |
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PCT/US2012/032587 |
Apr 6, 2012 |
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14046483 |
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61472962 |
Apr 7, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5041 20130101;
G01N 2500/04 20130101; G01N 33/5023 20130101; G01N 33/5058
20130101; A61K 31/35 20130101; A61K 31/14 20130101; G01N 33/542
20130101; A61K 31/351 20130101; G01N 2500/10 20130101; A61K 31/4745
20130101; A61K 31/454 20130101; A61K 31/436 20130101; G01N 33/543
20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; A61K 31/454 20060101 A61K031/454; A61K 31/14 20060101
A61K031/14; A61K 31/351 20060101 A61K031/351; A61K 31/35 20060101
A61K031/35; A61K 31/4745 20060101 A61K031/4745; G01N 33/542
20060101 G01N033/542; A61K 31/436 20060101 A61K031/436 |
Claims
1. A method of preventing or treating a neurological disease or
disorder in an individual comprising: administering a
therapeutically effective amount of one or more compounds
comprising Astemizole, Tacrolimus, Lasalocid sodium, Monensin
sodium, Emetine, Cetrimonium, analogs or combinations thereof.
2. The method of claim 1, wherein the compounds are administered in
a pharmaceutical composition.
3. The method of claim 1, wherein a neurological disease or
disorder comprises Creutzfeldt-Jakob disease; prion infection;
Alzheimer's disease; Parkinson's disease, synucleinopathies;
Multiple Sclerosis (MS); amyotrophic lateral sclerosis (ALS);
Huntington's disease or neurological disorders associated with a
virus infection.
4. A method of modulating PrP in vitro or in vivo comprising,
contacting a cell or administering to a subject an effective amount
of one or more compounds comprising: Astemizole, Tacrolimus,
Lasalocid sodium, Monensin sodium, Emetine, Cetrimonium, analogs or
combinations thereof.
5. The method of claim 4, wherein the amount of PrP is reduced as
compared to a control.
6. A method of modulating a protein kinase in vitro or in vivo,
comprising contacting a cell or administering to a subject an
effective amount of one or more compounds comprising: Astemizole,
Tacrolimus, Lasalocid sodium, Monensin sodium, Emetine,
Cetrimonium, analogs or combinations thereof.
7. A method of preventing or treating cancer comprising:
administering a therapeutically effective amount of one or more
compounds comprising Astemizole, Tacrolimus, Lasalocid sodium,
Monensin sodium, Emetine, Cetrimonium, analogs or combinations
thereof.
8. A pharmaceutical composition comprising a therapeutically
effective amount of one or more compounds comprising: Astemizole,
Tacrolimus, Lasalocid sodium, Monensin sodium, Emetine,
Cetrimonium, analogs or combinations thereof.
9. A compound identified by method of screening for a candidate
therapeutic compound comprising: screening a sample containing a
specific target molecule in a high-throughput screening assay
comprising the steps of: (i) contacting the sample with the
candidate therapeutic compound (ii) adding a first and second
ligand, wherein the first ligand comprises a first detectable label
and the second ligand comprises a second detectable label, (iii)
the first and second ligands each binding to separate and specific
sites on a specific target molecule, wherein the screening assay
does not require the step of (iv) washing; detecting an emission of
light when the first and second ligands specifically bind to the
specific target molecule; selecting the compound(s) that
modulate(s) the amount of the target molecule as compared to a
control; thereby, identifying the compound.
10. A compound identified by a method comprising the steps of:
placing the sample containing a specific target molecule into a
receptacle permitting irradiation of the sample at a wavelength
suitable for exciting the donor fluorophore and measurement of the
fluorescence of the acceptor fluorophore via a high-throughput
assay; contacting the sample with the candidate therapeutic
compound; adding a first ligand that binds to a specific site on
the target molecule wherein the first ligand is linked to a first
fluorophore (the "donor fluorophore"); adding a second ligand that
binds to a specific site on the same target molecule distinct from
that to which the first ligand binds wherein the second ligand is
linked to a second fluorophore (the "acceptor fluorophore");
irradiating the sample containing the target molecule linked to the
ligands at a wavelength optimal for exciting the donor fluorophore
and measuring the intensity of the light emitted by the acceptor
fluorophore; selecting the compound(s) that modulate(s) the amount
of the target molecule as compared to a control; thereby, screening
for a candidate compound.
11. The method of claim 9 or 10, wherein the compound modulates an
amount, a function, activity or expression of a target molecule as
measured by the emission of light.
12. A pharmaceutical composition comprising a compound identified
by the method of claim 9 or 10.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] In accordance with 37 C.F.R. 1.76, a claim of priority is
included in an Application Data Sheet filed concurrently herewith.
Accordingly, the present invention claims priority as a divisional
of U.S. patent application Ser. No. 14/046,483, filed Oct. 4, 2013,
which is a continuation-in-part of International Application No.
PCT/US2012/032587, filed Apr. 6, 2012, which claims priority to
U.S. Provisional Patent Application No. 61/472,962, entitled
"HIGH-THROUGHPUT SCREENING FOR COMPOUNDS MODULATING LEVELS OF
CELLULAR MACROMOLECULES" filed Apr. 7, 2011, the entire contents of
which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] Embodiments are directed to methods for the quantification
of specific cellular components ("targets") by a FRET-based assay
which does not require attachment of the targets to a solid phase
nor the separation of the target from excess reagents, making it
suitable for high-throughput screening.
BACKGROUND
[0003] Quantification of a specific macromolecule (the "target"),
particularly in the presence of other components, such as, a
specific protein on the cell surface, in a protein mixture, in a
cell homogenate, is commonly performed with use of an antibody that
specifically recognizes the target. In general, the protein mixture
is first immobilized on a support, for example, by adsorption or
covalent linkage to a membrane or a plastic surface, or to a
support surface to which an antibody specific for the target
("immobilizing antibody") has been attached, and exposed to a
target-specific antibody ("primary antibody", different from the
immobilizing antibody, if one was used). After an appropriate
reaction time, excess primary antibody is removed by repeated
washes, and the amount of bound antibody is determined by one of
several methods. For example, the primary antibody may have been
covalently linked to a fluorescent tag, and may be detected by
measuring the intensity of fluorescence; alternatively, a
"secondary" antibody (tagged with a marker, such as a fluorescent
dye or an enzyme) directed against the primary antibody, may be
used for quantification. Many different approaches for the
DM2\2763089.1 quantification of the primary antibody are available,
but common to all is the requirement that any primary or secondary
antibody that is not bound to the target be completely removed,
because it would give rise to a signal indistinguishable from that
of specifically bound antibody. Because high-throughput screening
does not allow for washing steps, the above-mentioned approaches
are not applicable.
SUMMARY
[0004] This Summary is provided to present a summary of the
invention to briefly indicate the nature and substance of the
invention. It is submitted with the understanding that it will not
be used to interpret or limit the scope or meaning of the
claims.
[0005] Embodiments are directed to a method of screening, in
particular in a high throughput mode, in a homogeneous or
heterogeneous solution for compounds modulating expression of a
specific macromolecule, the "target", including but not restricted
to a specific protein or nucleic acid. In a preferred embodiment,
at least two target-specific, FRET-enabling ligands, are directed
against at least two specific, distinct sites on the macromolecule,
one ligand being linked to at least one donor fluorophore and the
other to at least one acceptor fluorophore. Examples of
FRET-enabling ligands, include but are not restricted to:
antibodies, antibody mimetics, peptoids, peptide or nucleic acid
aptamers. The embodiments enable high throughput screening of
compounds able to modify the amounts of a specific macromolecule in
a biological sample. Further, the embodiments enable high
throughput identification and quantification of a specific
macromolecule in a biological sample.
[0006] Other aspects are described infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIGS. 1A-1B are graphs showing PrP detection at the surface
of living LD9 cells using the PrP detection assay in the 96-well
format. PrP knock-out cells (KO) lacking expression of PrP are used
as a negative control. PrP levels are expressed as [Delta F %]
which is a value resulting from the ratiometric measurement of the
HTRF signal corresponding to the detection of PrP. FIG. 1A: PrP
detection as a function of cell number and effect of the treatment
with brefeldine A (BFA). Z' is a statistical parameter measuring
the quality of an assay (Z'>0.5 is considered an excellent
assay). Z' of the assay was 0.7 when 20K or 40K LD9 cells were
used, and 0.8 for 80K LD9 cells. FIG. 1B: PrP detection after
treatment of LD9 cells with increasing doses of BFA during 24
hours; 4 and 8 .mu.g/ml reduce the PrP signal to background.
Triplicates are shown. FIG. 1C shows that none of the tested doses
of BFA were toxic to LD9 cells. Cell viability was measured using
the CELLTITER-GLO.RTM. luminescent assay (Promega) in
singlicate.
[0008] FIG. 2 is a graph showing PrP detection at the surface of
living LD9 cells using the PrP detection assay described in the
384-well format. DMSO is the solvent used in most small molecules
screening libraries and will be used as a control in the screening
plates. Therefore the Z' is calculated using LD9+DMSO as control
for the highest PrP signal, LD9+BFA as control for the lowest PrP
signal. Z' of the assay was 0.4, 0.6, 0.8 and -2.7 for 10.sup.3,
5.times.10.sup.3, 10.sup.4 and 2.times.10.sup.4 of LD9 cells,
respectively. Each point was done in triplicate.
[0009] FIG. 3 is a graph showing results from the preliminary
screening of the US Drug Collection using the primary screening
assay. Only data from the candidate hits are shown (selected by
using 50% reduction of cell surface PrP expression as a threshold).
Upper panels show PrP expression at the surface of LD9 cells after
treatment during 24 hours with the compounds indicated in the
abscissa. PrP levels are expressed as a percentage of the DMSO
control. Each screening plate is shown as a separate panel. DMSO
control is shown in purple for each plate. BFA control reduced
cell-surface PrP to background (0% PrP expression). Z' was 0.7 for
all four plates. Lower panels show viability of LD9 cells treated
with the compounds for 24 hours at the screening dose. Cell
viability was measured by our counter-screening assay using the
CELLTITER-GLO kit. Nine compounds exhibited less than 10% toxicity
and were selected as hits.
[0010] FIGS. 4A to 4D show the reduction of cell surface PrP by one
of the hits, Tacrolimus, confirmed by secondary screening on N2a
cells. PrP was labeled at the surface of living cells at +4.degree.
C. with monoclonal antibody D18 and fixed with 4% PFA prior to the
addition of the Alexa-488 labeled secondary antibody. FIGS. 4A, 4B:
microscopic analysis of N2a cells treated with DMSO (left picture)
and Tacrolimus (right picture). Quantification was performed by
flow cytometry (FIG. 4C) and IN Cell analyzer 1000 with Developer
software (FIG. 4D). Key for FIG. 4C: red: PrP signal of the
negative control (secondary antibody alone); blue: PrP signal for
the positive control (DMSO treated cells); green and orange:
duplicate analysis of PrP signal for the cells treated with
Tacrolimus at three different doses indicated in the panels.
[0011] FIGS. 5A to 5B are graphs showing the reduction of cell
surface PrP by two other hits, Lasalocid sodium and Astemizole,
confirmed by secondary screening on N2a cells. PrP labeling and IN
Cell Analyzer quantification were performed as described in FIGS.
4A-4D.
[0012] FIG. 6 shows a secondary assay used to prioritize hits
reducing cell surface PrP expression. The graphs represent the
number of infected cells (detected as spots by the scrapie cell
assay--SCA) as a function of cell number. Blue lines: untreated
cells; Red lines: cells treated with 1 .mu.g/ml PIPLC (for the time
indicated on each panel). The "RI.sub.200" value is defined as the
reciprocal of the cell number required to give 200 spots. The
RI.sub.200 for control PK1 [RML] at 26 hours is 3.3.times.10.sup.-3
and for PIPLC-treated PK1 [RML] it is 5.times.10.sup.-4. Therefore
PIPLC caused a 85% inhibition of infection
(1-[5.times.10.sup.-4/3.3.times.10.sup.-3].times.100).
[0013] FIGS. 7A and 7B illustrate that tacrolimus (Tac) and
astemizole (Ast), two compounds screened using the method described
herein that reduce cell surface PrP amounts as shown in FIGS. 4A-4D
and 5A-5B, block infection of PK1 neuroblastoma cells by RML and 22
L prions. PK1 cells were pretreated for 3 days with the indicated
doses of drugs and infected with RML (FIG. 7A) or 22 L (FIG. 7B)
prions using a 10.sup.-4 dilution of brain homogenate from an RML-
or 22 L-infected mouse. Treatment was continued for 12 days after
infection. Cells were analyzed by western blot for proteinase
K-resistant PrP.sup.Sc (a hallmark of prion infection) 9 and 18
days post-infection (i.e. 3 days before and 6 days after treatment
cessation). PPS (pentosan polysulfate), a drug that prevents prion
infection, was used at the dose of 10 .mu.g/ml as positive control
for treatment efficacy. CTRL: untreated cells. Both astemizole and
tracrolimus blocked prion infection, and there was no rebound of
infectivity after treatment cessation.
[0014] FIGS. 8A-8E show the toxicity of A.beta. oligomers for human
neuroblastoma cells restricted to those expressing PrP. FIGS. 8A,
8C: non-treated cells. FIGS. 8B, 8D: cells exposed to A.beta.42
oligomers for 4 days (100 .mu.g/m1). In FIG. 8B, cell vacuolation
and loss was observed. FIG. 8E is a Western-blot analysis in
triplicate showing PrP expression only in SK-NSH cells.
[0015] FIGS. 9A-9C are graphs showing detection of the tau protein
in human neuroblastoma cells using the tau detection assay in the
384-well format. Tau levels are expressed as [Delta F %] which is a
value resulting from the ratiometric measurement of the HTRF signal
corresponding to the detection of tau. FIG. 9A: Tau detection in
SH-SY5Y cells as a function of cell number. Z' is a statistical
parameter measuring the quality of an assay (Z'>0.5 is
considered an excellent assay). Z' of the assay was 0.7 when 18K or
24K SH-SY5Y cells were used. Triplicates are shown. Error bars
correspond to standard deviations. FIG. 9B: Tau knock-down in
SH-SY5Y cells using RNA interference leads to a specific decrease
in tau detection. Triplicates are shown. Error bars correspond to
standard deviations. FIG. 9C: Tau detection in SK-NSH cells after
treatment with increasing doses of staurosporine (in 0.75% DMSO
final) or 0.75% DMSO alone (CTRL) during 24 hours using the tau
detection assay in the 384-well format. [Delta F %] was calculated
using medium containing 0.75% DMSO as blank (average signal from 16
wells). Z' of the assay was 0.5. Bars correspond to the average
signal from 8 wells (each dose of staurosporine) or 96 wells
(CTRL). Error bars correspond to standard deviations.
DETAILED DESCRIPTION
[0016] Embodiments are directed to methods for the efficient
screening, identification and quantification of samples containing
target molecules for use in High-Throughput Screening (HTS) assays.
In particular, the assays described herein do not require washing
steps nor the attachment of the target molecules to solid supports.
Thus, the targets can be assayed under conditions where they retain
their natural, in vivo, conformation. Novel compounds or new uses
of known compounds that affect the level of a cellular
macromolecule can be identified.
[0017] The present invention is described with reference to the
attached figures, wherein like reference numerals are used
throughout the figures to designate similar or equivalent elements.
The figures are not drawn to scale and they are provided merely to
illustrate the instant invention. Several aspects of the invention
are described below with reference to example applications for
illustration. It should be understood that numerous specific
details, relationships, and methods are set forth to provide a full
understanding of the invention. One having ordinary skill in the
relevant art, however, will readily recognize that the invention
can be practiced without one or more of the specific details or
with other methods. The present invention is not limited by the
illustrated ordering of acts or events, as some acts may occur in
different orders and/or concurrently with other acts or events.
Furthermore, not all illustrated acts or events are required to
implement a methodology in accordance with the present
invention.
[0018] Embodiments of the invention may be practiced without the
theoretical aspects presented. Moreover, the theoretical aspects
are presented with the understanding that Applicants do not seek to
be bound by the theory presented.
[0019] Unless otherwise defined, all terms (including technical and
scientific terms) used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. It will be further understood that terms, such
as those defined in commonly used dictionaries, should be
interpreted as having a meaning that is consistent with their
meaning in the context of the relevant art and will not be
interpreted in an idealized or overly formal sense unless expressly
so defined herein.
Definitions
[0020] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting of
the invention. 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. Furthermore, to the extent
that the terms "including", "includes", "having", "has", "with", or
variants thereof are used in either the detailed description and/or
the claims, such terms are intended to be inclusive in a manner
similar to the term "comprising."
[0021] As used herein, the terms "comprising," "comprise" or
"comprised," and variations thereof, in reference to defined or
described elements of an item, composition, apparatus, method,
process, system, etc. are meant to be inclusive or open ended,
permitting additional elements, thereby indicating that the defined
or described item, composition, apparatus, method, process, system,
etc. includes those specified elements--or, as appropriate,
equivalents thereof--and that other elements can be included and
still fall within the scope/definition of the defined item,
composition, apparatus, method, process, system, etc.
[0022] The term "about" or "approximately" means within an
acceptable error range for the particular value as determined by
one of ordinary skill in the art, which will depend in part on how
the value is measured or determined, i.e., the limitations of the
measurement system. For example, "about" can mean within 1 or more
than 1 standard deviation, per the practice in the art.
Alternatively, "about" can mean a range of up to 20%, preferably up
to 10%, more preferably up to 5%, and more preferably still up to
1% of a given value. Alternatively, particularly with respect to
biological systems or processes, the term can mean within an order
of magnitude, preferably within 5-fold, and more preferably within
2-fold, of a value. Where particular values are described in the
application and claims, unless otherwise stated the term "about"
meaning within an acceptable error range for the particular value
should be assumed.
[0023] The terms "specific binding" or "specifically binding" when
used in reference to the interaction of a protein and an antibody
or alternative protein scaffold or peptoid or aptamers, means that
the interaction is dependent upon the presence of a particular
structure (i.e., the antigenic determinant or epitope) on the
protein; in other words the antibody is recognizing and binding to
a specific protein structure rather than to proteins in general.
Thus, an antibody that "specifically binds to" or is "specific for"
a particular polypeptide or an epitope on a particular polypeptide
is one that binds to that particular polypeptide or epitope on a
particular polypeptide without substantially binding to any other
polypeptide or polypeptide epitope.
[0024] The term "ligand," includes any compound, composition or
molecule capable of specifically or substantially specifically
(that is with limited cross-reactivity) binding another compound or
molecule, which, in the case of immune-recognition contains an
epitope. In many instances, the ligands are antibodies, such as
polyclonal or monoclonal antibodies. "Ligands" also include
derivatives or analogs of antibodies, including without limitation:
Fv fragments; single chain Fv (scFv) fragments; Fab' fragments;
F(ab').sub.2 fragments; humanized antibodies and antibody
fragments; camelized antibodies and antibody fragments; and
multivalent versions of the foregoing. Multivalent binding reagents
also may be used, as appropriate, including without limitation:
monospecific or bispecific antibodies, such as disulfide stabilized
Fv fragments, scFv tandems ((scFv)fragments), diabodies, tribodies
or tetrabodies, which typically are covalently linked or otherwise
stabilized (i.e., leucine zipper or helix stabilized) scFv
fragments. "Ligands" also include peptoids, peptide or nucleic acid
aptamers, or antibody mimetics such as DARPins, affibody molecules,
affilins, affitins, anticalins, avimers, fynomers, recombinant
probes, Kunitz domain peptides and monobodies.
[0025] A "label" or a "detectable label" is a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, chemical or any other means. For example, useful
labels include radio labeled molecules, fluorophores, luminescent
compounds, electron-dense reagents, enzymes (e.g., as commonly used
in an ELISA), biotin, digoxigenin, or haptens and proteins which
can be made detectable, e.g., by incorporating a label into the
peptide or used to detect antibodies specifically reactive with the
peptide.
[0026] The term "fluorophore" includes any compound, composition or
molecule capable of emitting light in response to irradiation. In
many instances, fluorophores emit light in the visible region of
light. In other instances, the fluorophores can emit light in the
non-visible regions of light, such as ultraviolet,
near-ultraviolet, near-infrared, and infrared. For example and
without limitation, examples of fluorophores include: quantum dots;
nanoparticles; fluorescent proteins, such as green fluorescent
protein and yellow fluorescent protein; heme-based proteins or
derivatives thereof; carbocyanine-based chromophores, such as IRDye
800CW, Cy 3, and Cy 5; coumarin-based chromophores, such as
(7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin) (CPM);
fluorine-based chromophores, such as fluorescein, fluorescein
isothiocyanate (FITC); and numerous ALEXA FLUOR.TM. chromophores
and ALEXA FLUOR.TM. bioconjugates, which absorb in the visible and
near-infrared spectra. The emission from the fluorophores can be
detected by any number of methods, including but not limited to,
fluorescence spectroscopy, fluorescence microscopy, fluorimeters,
fluorescent plate readers, infrared scanner analysis, laser
scanning confocal microscopy, automated confocal nanoscanning,
laser spectrophotometers, fluorescent-activated cell sorters
(FACS), image-based analyzers and fluorescent scanners (e.g.,
gel/membrane scanners).
[0027] As used herein, the term "chromophore" refers to a
substituent which, with another chromophore, can be used for energy
transfer (e.g., FRET assay).
[0028] The term "chemiluminescent compound" includes any compound,
composition or molecule capable of emitting light in response to a
chemical reaction. A "bioluminescent compound" refers to a
naturally occurring form of a chemiluminescent compound. Examples
of chemiluminescent compounds include: lucigenin, luminol. Examples
of bioluminescent compounds include: luciferins, coelenterazines.
The emission from chemiluminescent compounds can be detected by
luminometers or scanning spectrometers.
[0029] The term "luminescent component" or "luminescent compound"
as used herein refers to a component capable of absorbing energy,
such as electrical (e.g., electro-luminescence), chemical (e.g.,
chemi-luminescence) or acoustic energy and then emitting at least
some fraction of that energy as light over time. The term
"component" as used herein includes discrete compounds, molecules,
bioluminescent proteins and macro-molecular complexes or mixtures
of luminescent and non-luminescent compounds or molecules that act
to cause the emission of light.
[0030] "Sample" is used herein in its broadest sense. A sample
comprising polynucleotides, polypeptides, peptides, antibodies and
the like may comprise a bodily fluid; a soluble fraction of a cell
preparation, or media in which cells were grown, a cell culture; a
chromosome, an organelle, or membrane isolated or extracted from a
cell; genomic DNA, RNA, or cDNA, polypeptides, or peptides in
solution or bound to a substrate; a cell; a tissue; a tissue print;
a fingerprint, skin or hair; and the like.
[0031] As used herein, "biological samples" include solid and body
fluid samples. The biological samples used in the present invention
can include cells, cell cultures, protein or membrane extracts of
cells, blood or biological fluids such as ascites fluid or brain
fluid (e.g., cerebrospinal fluid). Examples of solid biological
samples include, but are not limited to, samples taken from tissues
of the central nervous system, bone, breast, kidney, cervix,
endometrium, head/neck, gallbladder, parotid gland, prostate,
pituitary gland, muscle, esophagus, stomach, small intestine,
colon, liver, spleen, pancreas, thyroid, heart, lung, bladder,
adipose, lymph node, uterus, ovary, adrenal gland, testes, tonsils,
thymus and skin, or samples taken from tumors. Examples of "body
fluid samples" include, but are not limited to blood, serum, semen,
prostate fluid, seminal fluid, urine, feces, saliva, sputum, mucus,
bone marrow, lymph, and tears.
[0032] The term "high-throughput screening" or "HTS" refers to a
method drawing on different technologies and disciplines, for
example, optics, chemistry, biology or image analysis to permit
rapid, highly parallel biological research and drug discovery. HTS
methods are known in the art and they are generally performed in
multiwell plates with automated liquid handling and detection
equipment; however it is envisioned that the methods of the
invention may be practiced on a microarray or in a microfluidic
system.
[0033] The term "library" or "drug library" as used herein refers
to a plurality of chemical molecules (test compound), a plurality
of nucleic acids, a plurality of peptides, or a plurality of
proteins, organic or inorganic compounds, synthetic molecules,
natural molecules, or combinations thereof.
[0034] As used herein, the term "target" or "target molecule"
refers to any type of molecule, or structure to be detected or
characterized. The molecule can be an intracellular molecule, such
as for example, nucleic acid sequences, peptides, structures (e.g.
intracellular membranes, ribosomes, etc.), surface molecules (e.g.
receptors), extracellular molecules (e.g. cytokines, enzymes, viral
particles, organisms, biological samples and the like.
[0035] A "polynucleotide" means a single strand or parallel and
anti-parallel strands of a nucleic acid. Thus, a polynucleotide may
be either a single-stranded or a double-stranded nucleic acid.
[0036] Unless otherwise indicated, the terms "peptide",
"polypeptide" or "protein" are used interchangeably herein,
although typically they refer to peptide sequences of varying
sizes.
[0037] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a polynucleotide, such as a gene, a
cDNA, or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having either a
defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a
defined sequence of amino acids and the biological properties
resulting therefrom. Thus, a gene encodes a protein if
transcription and translation of mRNA corresponding to that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the
mRNA sequence and is usually provided in sequence listings, and the
non-coding strand, used as the template for transcription of a gene
or cDNA, can be referred to as "encoding" the protein or other
product of that gene or cDNA.
[0038] As used herein, the term "kit" refers to any delivery system
for delivering materials. In the context of reaction assays, such
delivery systems include systems that allow for the storage,
transport, or delivery of reaction reagents (e.g.,
oligonucleotides, enzymes, etc. in the appropriate containers)
and/or supporting materials (e.g., buffers, written instructions
for performing the assay etc.) from one location to another. For
example, kits include one or more enclosures (e.g., boxes)
containing the relevant reaction reagents and/or supporting
materials. As used herein, the term "fragmented kit" refers to a
delivery systems comprising two or more separate containers that
each contain a subportion of the total kit components. The
containers may be delivered to the intended recipient together or
separately. For example, a first container may contain an enzyme
for use in an assay, while a second container contains
oligonucleotides. The term "fragmented kit" is intended to
encompass kits containing Analyte specific reagents (ASR's)
regulated under section 520(e) of the Federal Food, Drug, and
Cosmetic Act, but are not limited thereto. Indeed, any delivery
system comprising two or more separate containers that each
contains a subportion of the total kit components are included in
the term "fragmented kit." In contrast, a "combined kit" refers to
a delivery system containing all of the components of a reaction
assay in a single container (e.g., in a single box housing each of
the desired components). The term "kit" includes both fragmented
and combined kits.
[0039] An "amount" or "quantity" of a molecule refers to the
concentration, volume, mass, weight, percentage or any other factor
that one of skill in the art would recognize as a means to measure
how much of the molecule is present as compared to a baseline
control. The amount can decrease or increase or remain the same as
compared to the control. An example of measuring the amount of a
molecule is shown in the examples section which follows.
[0040] By the term "modulate," it is meant that the amounts,
activity, function, expression, of a molecule, e.g. PrP, tau etc.,
are, e.g., increased, enhanced, agonized, promoted, decreased,
reduced, suppressed blocked, or antagonized. Modulation can
increase amounts, activity, function, expression of a molecule more
than 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, etc., over
baseline values. Modulation can also decrease its amounts,
activity, function, expression below baseline values. Modulation
can also normalize amounts, activity, function, expression to a
baseline value.
[0041] The terms "determining", "measuring", "evaluating",
"detecting", "assessing" and "assaying" are used interchangeably
herein to refer to any form of measurement, and include determining
if an element is present or not. These terms include both
quantitative and/or qualitative determinations. Assessing may be
relative or absolute. "Assessing the presence of" includes
determining the amount of something present, as well as determining
whether it is present or absent.
[0042] "Optional" or "optionally" means that the subsequently
described circumstance may or may not occur, such that the
description includes instances where the circumstance occurs and
instances where it does not.
[0043] The terms "patient", "subject" or "individual" are used
interchangeably herein, and refers to a mammalian subject to be
treated, with human patients being preferred. In some cases, the
methods of the invention find use in experimental animals, in
veterinary application, and in the development of animal models for
disease, including, but not limited to, rodents including mice,
rats, and hamsters; and primates.
[0044] "Diagnostic" or "diagnosed" means identifying the presence
or nature of a pathologic condition. Diagnostic methods differ in
their sensitivity and specificity. The "sensitivity" of a
diagnostic assay is the percentage of diseased individuals who test
positive (percent of "true positives"). Diseased individuals not
detected by the assay are "false negatives." Subjects who are not
diseased and who test negative in the assay, are termed "true
negatives." The "specificity" of a diagnostic assay is 1 minus the
false positive rate, where the "false positive" rate is defined as
the proportion of those without the disease who test positive.
While a particular diagnostic method may not provide a definitive
diagnosis of a condition, it suffices if the method provides a
positive indication that aids in diagnosis.
[0045] As used herein the phrase "diagnosing" refers to classifying
a disease or a symptom, determining a severity of the disease,
monitoring disease progression, forecasting an outcome of a disease
and/or prospects of recovery. The term "detecting" may also
optionally encompass any of the above. Diagnosis of a disease
according to the present invention can be effected by determining a
level of a polynucleotide or a polypeptide of the present invention
in a biological sample obtained from the subject, wherein the level
determined can be correlated with predisposition to, or presence or
absence of the disease. It should be noted that a "biological
sample obtained from the subject" may also optionally comprise a
sample that has not been physically removed from the subject.
[0046] "Treatment" is an intervention performed with the intention
of preventing the development or altering the pathology or symptoms
of a disorder. Accordingly, "treatment" refers to both therapeutic
treatment and prophylactic or preventative measures. Those in need
of treatment include those already with the disorder as well as
those in which the disorder is to be prevented. As used herein,
"ameliorated" or "treatment" refers to a symptom which approaches a
normalized value (for example a value obtained in a healthy patient
or individual), e.g., is less than 50% different from a normalized
value, preferably is less than about 25% different from a
normalized value, more preferably, is less than 10% different from
a normalized value, and still more preferably, is not significantly
different from a normalized value as determined using routine
statistical tests.
Assay Description.
[0047] In a preferred embodiment, the sample containing the protein
or the desired molecule to be screened for (the "target") is placed
into a receptacle. In one embodiment, the target is a known
molecule, for example when screening for a particular molecule
diagnostic of a disease or disorder or identifies subjects that may
be at risk of developing a disease or disorder, or when screening
for a compound that will modify the amount of a disease-associated
molecule. The assay is termed herein as FRET-enabled high
throughput assay (FEHTA). The assay specifically identifies and/or
quantifies specific proteins in a sample and includes
intra-cellular, extra-cellular or cell surface molecules. The
assays embodied herein do not require the step of washing. In some
embodiments, one or more washing steps can be omitted.
[0048] An example of the assay, which is meant to be illustrative
and should not be construed as limiting, is provided. For example,
the first ligand, which is linked to the donor fluorophore, and the
second ligand which is linked to the acceptor fluorophore are added
to the receptacle. Each of the ligands bind to a specific and
distinct site on the same target molecule. The sample containing
the target linked to the ligands is irradiated at a wavelength
optimal for exciting the donor fluorophore. The intensity of the
light emitted by the acceptor fluorophore as a result of its
excitation by the energy transferred from the donor fluorophore
(Forster Resonance Energy Transfer (FRET)) is measured. The
distance between the donor fluorophore and the acceptor fluorophore
is defined as being equal to or less than the distance defined by
the Forster radius.
[0049] The samples can be identified and/or quantified by any
useful fluorescence detection method, such as by fluorimeters,
time-resolved fluorimeters, fluorescent microscopy, fluorescent
plate readers, infrared scanner analysis, spectrophotometers,
fluorescent-activated cell sorters (FACS), and fluorescent scanners
(e.g., gel/membrane scanners). Although samples can be analyzed
with a laser scanning confocal microscope, an automated confocal
nannoscanner or a microplate spectrofluorimeter, the method can be
easily adapted to other devices (i.e. FACS cell sorter) for
applications in other fields. FRET can be detected directly or
indirectly. Direct detection of FRET is performed exciting the
donor (CPM) and detecting the signal emitted by the acceptor (FITC
or Alexa488). As used herein the term "signal" means any detectable
event (whether direct or indirect) indicative of FRET, and includes
without limitation, emission of a photon. FRET is detected
indirectly using the method described by Karpova and co-workers (T.
S. Karpova et al., J Microsc 209, 56-70, 2003).
[0050] The term "assay" used herein, whether in the singular or
plural shall not be misconstrued or limited as being directed to
only one assay with specific steps but shall also include, without
limitation any further steps, materials, various iterations,
alternatives etc., that can also be used. Thus, if the term "assay"
is used in the singular, it is merely for illustrative
purposes.
[0051] In a preferred embodiment, the assay optionally omits or
does not require washing between steps. In one aspect the washing
steps are omitted before and/or after adding the ligands to the
receptacles. The drawback with currently available assays is that
the requirement that the target be firmly bound to a support raises
two major problems: (a) binding may be incomplete, the efficiency
of binding may be different for distinct conformations or forms of
the target, or binding of the target to the support may mask to an
unknown degree the accessibility of the site to be recognized by
the primary antibody. (b) The requirement that excess antibody
(both secondary and/or primary) be removed as completely as
possible requires repeated washes, which is laborious and
time-consuming, and in some cases not doable. In particular in High
Throughput assays, where tens- or hundreds of thousands of samples
are screened in 384 or 1536 well-plates, washing procedures cannot
be implemented.
[0052] The assays (FEHTA) embodied herein, obviate the necessity of
attaching the target to a support and of a washing step. In one
embodiment, the assay employs Forster Resonance Energy Transfer or
FRET, a process in which a fluorophore ("donor") that can be
excited by light and can transfer the excitation to a second
fluorophore ("acceptor") if and only if they are sufficiently
close, that is, within a distance in the order of 100 .ANG. or
less, defined by the Forster radius. Further details regarding FRET
assays are provided below. Although FRET is used as an illustrative
example, the assays described herein are not limited to FRET based
assays. For example, an assay which uses a bioluminescent protein,
such as luciferase, to excite a proximal fluorophore (BRET),
typically a fluorescent protein (Xu et al. (1999) Proc. Natl. Acad.
Sci. USA 96(1), 151-6). Another assay alternative is a luminescent
oxygen-channeling chemistry (Ullman et al. (1994) Proc. Natl. Acad.
Sci. USA 91(12), 5426-30), wherein a light induced singlet oxygen
generating system transfers the singlet oxygen to a
chemiluminescent system in proximity.
[0053] In one embodiment, the donor and acceptor fluorophores
(detectable label/detectable molecules) are attached to two
distinct ligands, for example, antibodies that can bind
specifically to distinct sites of one and the same target. When the
ligands carrying the donor and the acceptor fluorophore,
respectively, bind to the same target molecule and in doing so
become sufficiently close to each other, inadiation of the sample
at a wavelength that allows excitation of the donor results in
emission of radiation by the acceptor. Ligands that are not bound
to the same target do not give rise to FRET and therefore need not
be removed prior to measurement of emitted radiation. Since the
target is not adsorbed or bound to a support, it is fully available
for interaction with the ligands.
[0054] In one embodiment, a method of identifying and quantifying a
specific target molecule in a sample comprises screening a sample
containing the specific target molecule in a high-throughput
screening assay comprising the steps of: (i) adding a first and
second ligand, each having a first and second detectable label,
(ii) the first and second ligands each binding to separate and
specific sites on a specific target molecule, wherein the screening
assay optionally omits or does not require the step of (iii)
washing, and detecting an emission of light when the first and
second ligands specifically bind to the specific target molecule.
Preferably, the detectable label comprises: fluorophores,
luminescent molecules, enzymes or radionuclides. In some
embodiments, the light comprises: fluorescence, chemiluminescence,
or bioluminescence.
[0055] In some embodiments, the assay is a high-throughput
screening assay wherein the high-throughput screening assay
comprises a Forster Resonance Energy Transfer (FRET),
Bioluminescence Resonance Energy Transfer (BRET), or fluorescence
polarization assay.
[0056] In one embodiment, the ligands comprise: polypeptides such
as antibodies or antibody fragments bearing epitope recognition
sites, such as Fab, Fab', F(ab').sub.2 fragments, Fv fragments,
single chain antibodies, antibody mimetics (such as DARPins,
affibody molecules, affilins, affitins, anticalins, avimers,
fynomers, Kunitz domain peptides and monobodies), recombinant
probes, peptoids, aptamers and the like. In one embodiment the
first and second ligands are the same type of molecule. In another
embodiment, the first and second ligands are different types of
molecules. In some embodiments, the first or second ligands
comprise: antibodies, antibody fragments, Fv fragments; single
chain Fv (scFv) fragments; Fab' fragments; F(ab')2 fragments,
humanized antibodies and antibody fragments; camelized antibodies
and antibody fragments, human antibodies and antibody fragments,
monospecific or bispecific antibodies, disulfide stabilized Fv
fragments, scFv tandems ((scFv) fragments), diabodies, tribodies or
tetrabodies, peptoids, peptide or nucleic acid aptamers, antibody
mimetics or combinations thereof. In other embodiments, the first
and second ligands comprise: a polypeptide, antibodies, antibody
fragments, antibody mimetics, single chain antibodies, nucleic
acids, an aptamer, a peptoid or a sugar moiety or combinations
thereof. In certain embodiments, the first and second ligands are
peptide or nucleic acid aptamers. In other embodiments, the first
and second ligands are sugar moieties comprising
glycosaminoglycans, heparan sulfates or chondroitin sulfates.
[0057] In some embodiments, the methods are used to identify and
quantify a specific molecule in a sample. In such embodiments, a
method of quantifying a specific molecule, e.g. a protein in a
sample, the method comprises the steps of: placing the sample
containing the specific target molecule into a receptacle
permitting irradiation of the sample at a wavelength suitable for
exciting the donor fluorophore and measurement of the fluorescence
of the acceptor fluorophore via a high-throughput assay; adding a
first ligand that binds to a specific site on the target molecule
wherein the first ligand is linked to a first fluorophore (the
"donor fluorophore"); adding a second ligand that binds to a
specific site on the same target molecule distinct from that to
which the first ligand binds wherein the second ligand is linked to
a second fluorophore (the "acceptor fluorophore"); optionally,
omitting washing steps between each step; irradiating the sample
containing the target molecule linked to the ligands at a
wavelength optimal for exciting the donor fluorophore and measuring
the intensity of the light emitted by the acceptor fluorophore or
both the donor and acceptor fluorophores. In embodiments, the assay
is a high-throughput screening assay. In one embodiment the
specific molecule is a prion protein (PrP). In another embodiment,
the specific molecule is a Tau protein or peptides, or
hyperphosphorylated tau molecules.
[0058] In one embodiment, the intensity of the light emitted is
measured by time resolved fluorimetry. In embodiments, the
excitation is transferred to the acceptor fluorophore when the
acceptor fluorophore is at a distance from the donor fluorophore
that is equal to or less than the distance defined by the Forster
radius.
[0059] In embodiments, the target is present in a sample
comprising: a liquid, a semi-liquid, a gel, a biological sample, an
intact cell, a permeabilized cell, a disrupted cell, a cell
homogenate, a membrane, or a cellular organelle.
[0060] In other embodiments, the ligands are linked to a detectable
label (detectable molecule), either directly or linked via a
suitable linker. The present invention is not limited to any
particular linker group. Indeed, a variety of linker groups are
contemplated, suitable linkers could comprise, but are not limited
to, alkyl groups, ether, polyether, alkyl amide linker, a peptide
linker, a polypeptide linker, a modified peptide or polypeptide
linker, a peptide nucleic acid (PNA) a Poly(ethylene glycol) (PEG)
linker, a streptavidin-biotin or avidin-biotin linker,
polyaminoacids (e.g. polylysine), functionalized PEG,
polysaccharides, glycosaminoglycans, dendritic polymers PEG-chelant
polymers, oligonucleotide linker, phospholipid derivatives, alkenyl
chains, alkynyl chains, disulfide, or a combination thereof.
[0061] In another preferred embodiment, the detectable label is
linked to the ligand, through a chemical bond, or noncovalently,
through ionic, van der Waals, electrostatic, or hydrogen bonds.
[0062] There are various methods that one of skill in the art can
practice to identify various ligands or combinations of ligands. In
one example, a best ligand pair analysis was carried out as
described in the "Examples" section which follows. In addition, to
select the best compounds from the hits generated during the
primary screening, the following strategy can be used: (1).
Selection of the compounds exerting the highest effect. (2).
Selection of the compounds exerting the effect at the lowest
concentration and harboring the least toxicity on cells. To this
end, EC.sub.50 and TC.sub.50 can be determined by any of the assays
routinely used by those of ordinary skill in the art. (3).
Selection of the compounds exhibiting the highest specificity for a
molecule. Although complete specificity is not required for a
compound to achieve a good therapeutic index, this can be used as
criteria for compound selection. (4). Selection of the compounds
showing the highest of the desired property or therapeutic capacity
can be determined. The "therapeutic capacity" (or "treatment") is
dependent on the condition to be treated. For example, an
anti-viral would inhibit a viral infection by either inhibiting
replication, slowing growth of the virus, etc. Any desired output
parameter can be used. An "effect" would be the type of parameter
that one of skill in the art is screening the compounds for. This
is inclusive of, for example, amount, activity, function,
expression etc., of the target molecule. For example, the amounts
of the target molecule can be due to upstream or downstream
activities of other molecules which may modulate the amount of the
target molecule. Thus, for example, if one of skill in the art is
screening compounds for an inhibitory effect on a certain target
molecule, then the parameters used, can be expression profiles if
the target molecule is a nucleic acid peptide etc. In other cases
it can be the activity, e.g. if it is an enzyme. In other cases it
can be a receptor and the effect measured would be modulation of
signaling, or surface expression, or conformation change. In other
cases, the test compound or a candidate therapeutic agent, may have
an effect on the formation or properties (e.g., conformation or
binding affinity) between the target molecule and its binding
partner. In other cases, the compound or test agent may have an
effect on the secondary or tertiary structure of the target
molecule. In other cases, the test agent may inhibit the function
of the target molecule. Thus, the effects measured would be limited
only by the imagination of the user.
[0063] In certain embodiments, provided herein are methods for
identifying the effects of a compound that modulates amounts or
quantities of a target molecule, comprising: (a) providing a target
molecule labeled with a first chromophore at a first position; (b)
exciting the chromophore; and (c) measuring the fluorescence
lifetime of the first chromophore; wherein a difference between the
fluorescence lifetime in the presence of the test compound and the
fluorescence lifetime in the absence of the test compound indicates
that the test compound modulates the target molecule, such that the
fluorescence lifetime of the chromophore is altered. In one
embodiment, the target molecule is further labeled with a second
chromophore at a second position, wherein the second position is
different from the first position, and wherein the chromophores can
be used for energy transfer.
[0064] In certain embodiments, provided herein are methods for
identifying the effects of a compound that modulates (e.g. amounts
of) a target molecule, comprising: (a) providing a target molecule
labeled with a first chromophore at a first position and a second
chromophore at a second position, wherein the second position is
different from the first position, and wherein the first and the
second chromophores can be used for energy transfer; (b) exciting
either the first or the second chromophore; and (c) measuring FRET
between the chromophores; wherein a difference between FRET in the
presence of the test compound and FRET in the absence of the test
compound indicates that the test compound produces the desired
effect, such that the energy transfer between the two chromophores
is altered.
[0065] In another preferred embodiment, the ligands are covalently
bound, linked, attached fused or otherwise in contact with a
suitable donor or acceptor fluorophore.
[0066] In another preferred embodiment, the type of target molecule
that can be assayed is not limited by its form, structure, and the
medium it is assayed in. For example, the target molecule can be:
free in solution, part of an intact cell, a permeabilized cell, a
disrupted cell, a cell homogenate, a membrane, a cellular
organelle, attached to beads, attached or bound to nanoparticles,
lipids, columns, polymers, plastics, glass and the like. In some
aspects, the sample is free floating and not attached to the
surface of the cuvette or receptacle. Examples of types of
molecules include without limitation: a protein, a peptide, a
polypeptide, a nucleic acid, a polynucleotide, an oligonucleotide,
a peptide nucleic acid, a glycoprotein, a carbohydrate, an organic
or inorganic molecule, an isolated natural molecule, a synthetic
molecule, small molecules, or combinations thereof.
[0067] In other embodiments, the target molecule can be attached to
a lipid or cell membrane. Lipids suitable for methods and kits
provided herein may be any lipids or a combination thereof in
various ratios capable of forming a membrane known in the art. In
certain embodiments, the lipids are naturally occurring. In certain
embodiments, the lipids are synthetic. In certain embodiments, the
lipids are one or more of fatty acyls, glycerolipids,
glycerophospholipids, sphingolipids, saccharolipids, polyketides,
sterol lipids, prenol lipids and a derivative thereof. In certain
embodiments, the lipids are one or more of choline-based lipids
(e.g., phosphatidylcholine (PC)), ethanolamine-based lipids (e.g.,
phosphatidylethanolamine (PE)), serine-based lipids (e.g.,
phosphatidylserine), glycerol-based lipids (e.g.,
phosphatidylglycerol), cholesterol-based lipids, dolichols,
sphingolipids (e.g., sphingosine, gangliosides, or
phytosphingosine), inositol-based lipids (e.g.,
phosphatidylinositol), cardiolipin, phosphatidic acid,
lysophosphatides (e.g., lysophosphatides), hydrogenated
phospholipids and a derivative thereof.
[0068] In certain embodiments, the lipids are one or more of PC,
dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine
(DMPC), dipalmitoylphosphatidylcholine (DPPC),
distearoylphosphatidylcholine (DSPC), palmitoyl
oleoylphosphatidylcholine (POPC), 2-dioleoyl-3-succinyl-sn-glycerol
choline ester (DOSC), PE, dioleoylphosphatidylethanolamine (DOPE),
dimyristoylphosphatidylethanolamine (DMPE),
dipalmitoylphisphatidylethanolamine (DPPE),
dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylserine
(DPPS), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG), sphingomyelin (SM), sodium
dodecyl sulphate (SDS), cholesterol (CHOL), cholesterol
hemisuccinate (CHEMS), cholesterol-(3-imidazol-1-yl
propyl)carbamate (CHIM), diacylglycerol hemisuccinate (DG-Succ),
cholesterol sulphate (Chol-SO.sub.4), dimethyldioctadecylammonium
bromide (DDAB), dioleoylphosphatidic acid (DOPA),
1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride
(DORI), 11,2-dioleoyl-3-trimethylammonium propane (DOTAP),
N-(1-(2,3-dioleoyloxy)-propyl)-N,N,N-triethylammonium chloride
(DOTMA), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium
bromide (DMRIE), 1,2-dioleoyl-3-dimethylammonium propane (DODAP),
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),
1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORIE),
N-(1-(2,3-dioleyloxy)-propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimeth-
-ylammonium trifluoroacetate (DOSPA),
1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazolinium
chloride (DOTIM), N-(trimethylammonioacetyl)-didodecyl-D-glutamate
chloride (TMAG), N,N-di-n-hexadecyl-N,N
dihydroxyethylammoniumbromide (DHMHAC),
N,N-di-n-hexadecyl-N-methyl-N-(2-hydroxyethyl)ammonium chloride
(DHDEAB),
N,N-myristoyl-N-(1-hydroxyprop-2-yl)-N-methylammoniumchloride
(DMHMAC), 1,2-dioleoyl-3-(4'-trimethylammonio)butanoyl-sn-glycerol
(DOTB), Synthetic Amphiphiles Interdisciplinary (SAINT lipids),
4,(2,3-bis-acyloxy-propyl)-1-methyl-1H-imidazole (DOIM),
2,3-bis-palmitoyl-propyl-pyridin-4-yl-amine (DPAPy),
3.beta.-(N-(N9,N9-dimethylaminoethane)carbamoyl)cholesterol
(DC-Chol),
3.beta.-(N-(N9,N9-trimethylaminoethane)carbamoyecholesterol
(TC-Chol), 3.beta.(N-(N,N-Dimethylaminoethan)-carbamoyl)cholesterol
(DAC-Chol), cetyltrimethylammonium bromide (CTAB), cationic
cardiolipins (e.g.
(1,3-bis-(1,2-bis-tetradecyloxy-propyl-3-dimethylethoxyammoniumbromide)-p-
ropane-2-ol) (NEOPHECTIN.TM.), N-histidinyl-cholesterol
hemisuccinate (HistChol), 4-(2-aminoethyl)-morpholino-cholesterol
hemisuccinate (MoChol), histaminyl-cholesterol hemisuccinate
(HisChol), and a derivative thereof.
[0069] Solvents suitable for methods and kits provided herein may
be any solvent capable of facilitating lipid solubilization known
in the art. In certain embodiments, the solvent is one or more of
methanol, ethanol, acetonitrile and chloroform. In one embodiment,
the solvent is methanol. In another embodiment, the solvent is
ethanol. In yet another embodiment, the solvent is acetonitrile. In
yet another embodiment, the solvent is chloroform. In certain
embodiments, the solvent is an aqueous solution comprising one or
more amphiphilic detergents. Examples of such detergents include,
but are not limited to, oxylglucoside, octaethylene glycol
monododecyl ether (C.sub.12E.sub.8), dodecylphosphocholine and
deoxycholate.
[0070] Target Molecules: Below is a non-exhaustive list of cellular
macromolecules that represent therapeutic targets (also referred to
herein as "target molecules") for HTS drug discovery using the
methods described herein. The targets fulfill the criteria that the
modulation of their amounts, function, expression and the like,
will have a therapeutic effect in the context of a particular
disease, and that they are not essential for the host or their
amount can be decreased or increased without damage to the host.
Alternatively, the targets may be present selectively or at higher
levels in cells involved in a particular disease (for example a
mutated or non mutated protein in cancer cells) such that the
modulation of the amounts of the target will affect primarily those
cells and not the entire host. Examples include, without
limitation: Amyloid Precursor Protein (APP), the protein that is a
precursor for the toxic A.beta. aggregates found in Alzheimer's
disease; mutated APP responsible for familial Alzheimer's disease;
BACE-1, the .beta.-site APP cleavage enzyme, which is one of the
two enzymes leading to the formation of toxic A.beta. aggregates;
Tau, another key protein involved in Alzheimer's disease pathology
and other diseases termed tauopathies; mutated tau responsible for
inherited forms of frontotemporal dementia; hyperphosphorylated
tau, the pathogenic form of the tau protein; .alpha.-synuclein,
which, when overexpressed and/or aggregated, causes Parkinson's
Disease and other diseases known collectively as synucleinopathies;
mutated SOD1 responsible for familial amyotrophic lateral sclerosis
(ALS); mutated huntingtin responsible for the inherited
neurological disorder Huntington's disease; viral receptors or
co-receptors such as the HIV co-receptor CCR5, which has been shown
to be non-functional in humans naturally protected against HIV
infection; oncogenes and anti-oncogenes; tumor cell markers that
are linked to tumor invasiveness and metastatic potential; insulin
receptor, the downregulation of which causes Type II diabetes;
cytokine or chemokine receptors; enzymes of the ubiquitin pathway;
enzyme markers and the like.
[0071] In some embodiments, desired target molecules are nucleic
acids, candidate target sequences are first used to search several
databases which catalog, for example, SNPs, sequences which
regulate expression or function of an encoded product and the like.
The targeted databases include NCBI's dbSNP, the UK's HGBASE SNP
database, the SNP Consortium database, and the Japanese Millenium
Project's SNP database.
[0072] In some embodiments, following dbSNP searches, gene loci
databases (e.g., Locus Link) are searched. LocusLink provides a
single query interface to curated sequence and descriptive
information about genetic loci. It presents information on official
nomenclature, aliases, sequence accessions, phenotypes, EC numbers,
MIM numbers, UniGene clusters, homology, map locations, protein
domains, and related web sites. The information output from
LocusLink includes a LocusLink accession number (LocusID), an NCBI
genomic contig number (NT#), a reference mRNA number (NM#), splice
site variants of the reference mRNA (XM#), a reference protein
number (NP#), an OMIM accession number, and a Unigene accession
number (HS#).
[0073] In other embodiments, disease association databases can be
searched to identify candidate target molecules. Following the
LocusLink search, the information returned is used to search
disease association databases. In some embodiments, the HUGO
Mutation Database Initiative, which contains a collection of links
to SNP/mutation databases for specific diseases or genes, is
searched.
[0074] In some embodiments, the OMIM database is searched. OMIM
(Online Mendelian Inheritance in Man) is a catalog of human genes
and genetic disorders developed for the World Wide Web by NCBI, the
National Center for Biotechnology Information. The database
contains textual information and references. Output from OMIM
includes a modified accession number where multiple SNPs are
associated with a genetic disorder. The number is annotated to
designate the presence of multiple SNPs associated with the genetic
disorder.
[0075] In some embodiments, following dbSNP searches, software
(e.g., including but not limited to, UniGene) is used to partition
search results into gene-oriented clusters (e.g. gene oriented
cluster analysis). UniGene is a system for automatically
partitioning GenBank sequences into a non-redundant set of
gene-oriented clusters. Each UniGene cluster contains sequences
that represent a unique gene, as well as related information such
as the tissue types in which the gene has been expressed and map
location. In addition to sequences of well-characterized genes,
hundreds of thousands novel expressed sequence tag (EST) sequences
are included in UniGene. Currently, sequences from human, rat,
mouse, zebrafish and cow have been processed.
[0076] In some embodiments, target sequences are used to search
genome databases (e.g., including but not limited to the Golden
Path Database at University of California at Santa Cruz (UCSC) and
GenBank). The GoldenPath database is searched via BLAST using the
sequence in FASTA format or using the RS# obtained from dbSNP.
GenBank is searched via BLAST using the masked sequence in FASTA
format. In some embodiments, GoldenPath and GenBank searches are
performed concurrently with TSC and dbSNP searches. In some
embodiments, the searches result in the identification of the
corresponding gene. Output from GenBank includes a GenBank
accession number. Output from both databases includes contig
accession numbers. Thus, there are many ways one of skill in the
art can identify a potential target, in addition to a user's
desired target molecule.
[0077] Other target molecules may be selected, for example, in
steroid hormone based therapies. In such cases, for example,
sulfation, encompasses involvement in estrogen level regulation in
mammary tumors, as well as androgen levels in prostate tumors. The
availability of robust HTS assays for steroid sulfation may provide
an important addition to the arsenal of molecular tools available
to pharma groups focused on steroid signal transduction.
[0078] The modulation of neurosteroids is being investigated as a
novel pharmacological approach to controlling neural excitatory
balance (Malayev, A., et al., Br J Pharmacol, 2002, 135:901-9;
Maurice, T., et al., Brain Res Brain Res Rev, 2001, 37:116-32;
Park-Chung, M., et al., Brain Res, 1999, 830:72-87). The methods
encompassed by the present invention may suitably accelerate these
efforts by allowing facile screening of endogenous and synthetic
neurosteroids for sulfoconjugation, offering insight into the
fundamental biology as well as providing a tool for lead molecule
identification and optimization. The need for better molecular
tools is accentuated by the fact that there is already a sizeable
over the counter market for DHEA as an "anti-aging" dietary
supplement purported to alleviate age related senility and memory
loss (Salek, F. S., et al., J Clin Pharmacol, 2002, 42).
[0079] In another example, the methods embodied in the present
invention may suitably identify drug targets with respect to
cholesterol sulfate in the regulation of cholesterol efflux,
platelet aggregation and skin development in treatments for
cardiovascular disease and perhaps some forms of skin cancer. In
this instance, a sulfotransferase could become the drug target, and
molecules that selectively inhibit this isoform may need to be
identified.
[0080] In another example, a target molecule may be one involved in
drug metabolism. Drug metabolism problems such as production of
toxic metabolites and unfavorable pharmacokinetics cause almost
half of all drug candidate failures during clinical trials. All of
the major pharmaceutical companies have recognized the need to
consider pharmacokinetic and pharmacogenomic consequences early in
the drug discovery process resulting in an immediate need for high
throughput in vitro methods for assessing drug metabolism. Aside
from P450-dependent oxidation, glucuronidation is perhaps the most
important route of hepatic drug metabolism. A broad spectrum of
drugs are eliminated or activated by glucuronidation including
non-steroidal anti-inflammatories, opioids, antihistamines,
antipsychotics and antidepressants (Meech, R. and Mackenzie, P. I.,
Clin Exp Pharmacol Physiol, 1997, 24:907-15; Radominska-Pandya, A.,
et al., Drug Metab Rev, 1999, 31:817-99). Despite their importance,
the broad and overlapping substrate specificity of the hepatic
UDP-glucuronosyltransferases (UGTs) that catalyze glucuronidation
remains poorly understood because of a lack of flexible in vitro
assay methods.
[0081] In another example, the target molecule can be a protein
kinase or a substrate thereof. There are more than 400 distinct
kinases encoded in the human genome; elucidating their role in
disease and identifying selective inhibitors is a major pharma
initiative. Kinase malfunction has been linked to all of the most
important therapeutic areas, including cancer, cardiovascular
diseases, inflammation, neurodegenerative diseases, and metabolic
disorders. Moreover, clinical validation of kinases as drug targets
has recently been shown in the cases of Herceptin and Gleevec,
which inhibit aberrant tyrosine kinases that contribute to breast
cancer and leukemia, respectively. Embodiments of the methods will
accelerate efforts to define kinase substrate specificity and to
identify novel inhibitors by providing a universal catalytic assay
that can be used with any kinase and any acceptor substrate.
[0082] Protein kinases are a large, diverse family with a key role
in signal transduction. Protein kinases, which catalyze the
transfer of the terminal phosphate group from ATP or GTP to serine,
threonine or tyrosine residues of acceptor proteins, are one of the
largest protein families in the human genome. In the broadest
senses, they can be divided into serine/threonine or tyrosine
kinases and soluble enzymes or transmembrane receptors. In the most
recent and comprehensive genomic analysis, 428 human kinases were
identified that comprise eight different homology groups, which
also reflect differences in substrate specificity,
structure/localization and/or mode of regulation (Hanks, S. K.,
Genome Biol, 2003, 4:111). For instance, there are 84 currently
identified members of the Tyrosine Kinase group, which includes
both transmembrane growth factor receptors such as EGFR and PDGFR
and soluble enzymes such as the Src kinases, 61 currently
identified members of the cyclic nucleotide dependent group,
ser/thr kinases which includes the lipid dependent kinases--the PKC
isoforms, and 45 currently identified members of the "STE" group,
which includes the components of the mitogenic MAP kinase signaling
pathway.
[0083] Kinases are ubiquitous regulators of intracellular signal
transduction pathways, and as such have come under intense focus by
pharmaceutical companies searching for more selective therapies for
a broad range of diseases and disorders; they are second only to
G-protein coupled receptors in terms of pharma prioritization
(Cohen, P., Nat Rev Drug Discov, 2002, 1:309-15). Intracellular
targets for phosphorylation include other kinases, transcription
factors, structural proteins such as actin and tubulin, enzymes
involved in DNA replication and transcription, and protein
translation, and metabolic enzymes (Cohen, P., Trends Biochem Sci,
2000, 25:596-601). Phosphorylation can cause changes in protein
catalytic activity, specificity, stability, localization and
association with other biomolecules. Simultaneous phosphorylation
at multiple sites on a protein, with different functional
consequences, is common and central to the integration of signaling
pathways.
[0084] Each kinase may phosphorylate one or more target proteins,
sometimes at multiple sites, as well as autophosphorylate within
one or more regulatory domains that control catalytic activity or
interaction with other biomolecules. Defining the functional
consequences of cellular phosphorylation profiles for normal and
disease states is a major proteomics initiative. However, to use
this knowledge for deciding which kinases to target for drug
discovery, their specificity for acceptor substrates must also be
delineated. Kinases recognize specific linear sequences of their
target proteins that often occur at beta bends. In general, amino
acids that flank the phosphorylated residue for three to five
residues on either side define a phosphorylation site. The
PhosphoBase database, which compiles known kinase phosphorylation
sites, contains entries for 133 human kinases, less than a third of
the total kinases. Moreover, most, if not all of these specificity
profiles are incomplete, as they only show one or two peptides that
have been identified as substrates for each kinase. Though there is
significant overlap in substrate specificity among related kinases,
there is no consensus sequence that is phosphorylated by a large
number of kinases.
[0085] The biological rationale for targeting kinases to intervene
in cancer is far too extensive to attempt an overview here.
However, one of the dominant themes is the involvement of numerous
kinases in controlling the delicate balance between the rate of
cell division (cell cycle progression), cell growth (mass), and
programmed cell death (apoptosis) that is perturbed in all cancers.
Growth factor receptor tyrosine kinases (RTKs) are
membrane-spanning proteins that transduce peptide growth factor
signals from outside the cell to intracellular pathways that lead
to activation of progrowth and anti-apoptotic genes. The majority
of the fifty-eight RTKs in humans are dominant oncogenes, meaning
that aberrant activation or overexpression causes a malignant cell
phenotype. Not surprisingly, tyrosine kinases are being
aggressively pursued as anticancer drug targets and both small
molecule and monoclonal antibody inhibitors--GLEEVEC and HERCEPTIN,
respectively--have been clinically approved. Downstream signaling
from growth factor receptors occurs through multiple pathways
involving both ser/thr and tyrosine kinases. One of the dominant
kinases is the mitogen activated protein kinase (MAPK) pathway,
which includes Raf and MEK kinases; inhibitors of all of these
kinases are currently being tested in clinical trials (Dancey, J.
and Sausville, E. A., Nat Rev Drug Discov, 2003, 2:296-313).
Soluble tyrosine kinases, especially the 11 oncogenes that comprise
the Src family, also transduce mitogenic signals initiated by RTKs
and are being targeted by pharma (Warmuth, M., et al., Curr Pharm
Des, 2003, 9:2043-59). Following mitogenic signals through RTKs
that initiate entry into the G1 phase, progression through the cell
cycle is regulated by sequential activation of phase-specific
kinases in association with cyclin proteins. The cyclin dependent
kinases represent yet another important group of kinases that
pharma is pursuing in the hopes of inhibiting malignant cell
proliferation (Elsayed, Y. A. and Sausville, E. A., Oncologist,
2001, 6:517-37).
[0086] Thus, the assays embodied herein, can be used to screen drug
libraries for inhibitors or activators of protein kinases. It will
also be useful for screening peptides or proteins as acceptor
substrates for kinases. In these applications, it will have the
significant advantages over other methods such as the universal
nature of the assay, simplified homogenous assay, no radioactivity,
and the ability to quantify enzyme turnover.
[0087] Depending on the target molecule, a test compound,
identified by the methods embodied herein, would then be one that
would be useful in the treatment of that disease or disorder for
which the target molecule plays a role or directly contributes to
the disease or disorder.
[0088] Forster Resonance Energy Transfer (FRET): FRET is a
radiationless process in which energy is transferred from an
excited donor molecule to an acceptor molecule. Radiationless
energy transfer is the quantum-mechanical process by which the
energy of the excited state of one fluorophore is transferred
without actual photon emission to a second fluorophore. The quantum
physical principles are reviewed in Jovin and Jovin, 1989, Cell
Structure and Function by Microspectrofluorometry, eds. E. Kohen
and J. G. Hirschberg, Academic Press. Briefly, a fluorophore
absorbs light energy at a characteristic wavelength. This
wavelength is also known as the excitation wavelength. In FRET, the
energy absorbed by a fluorophore is subsequently transferred by a
non-radiative process to a second fluorophore. The first
fluorophore is generally termed the donor (D) and has an excited
state of higher energy than that of the second fluorophore, termed
the acceptor (A).
[0089] Critical features of the process are that the emission
spectrum of the donor fluorophore overlap with the excitation
spectrum of the acceptor, and that the donor and acceptor be
sufficiently close. The distance between D and A must be
sufficiently small to allow the radiationless transfer of energy
between the fluorophores. Since the rate of energy transfer is
inversely proportional to the sixth power of the distance between
the donor and acceptor, the energy transfer efficiency is extremely
sensitive to distance changes. Energy transfer is said to occur
with detectable efficiency in the 1-10 nm distance range, but is
typically 4-6 nm for optimal results. The distance range over which
radiationless energy transfer is effective depends on many other
factors as well, including the fluorescence quantum efficiency of
the donor, the extinction coefficient of the acceptor, the degree
of overlap of their respective spectra, the refractive index of the
medium, and the relative orientation of the transition moments of
the two fluorophores.
[0090] Fluorescent donor and corresponding acceptor moieties are
generally chosen for (a) high efficiency Forster energy transfer;
(b) a large final Stokes shift (>100 nm); (c) shift of the
emission as far as possible into the red portion of the visible
spectrum (>600 nm); and (d) shift of the emission to a higher
wavelength than the Raman water fluorescent emission produced by
excitation at the donor excitation wavelength. For example, a donor
fluorescent moiety can be chosen that has its excitation maximum
near a laser line (for example, Helium-Cadmium 442 nm or Argon 488
nm), a high extinction coefficient, a high quantum yield, and a
good overlap of its fluorescent emission with the excitation
spectrum of the corresponding acceptor fluorescent moiety. A
corresponding acceptor fluorescent moiety can be chosen that has a
high extinction coefficient, a high quantum yield, a good overlap
of its excitation with the emission of the donor fluorescent
moiety, and emission in the red part of the visible spectrum
(>600 nm).
[0091] A skilled artisan will recognize that many fluorophore
molecules are suitable for FRET. A fluorophore is a fluorescent
component, or functional group, bound to a molecule. A fluorophore
can be a fluorescent molecule, a glowing bead, a glowing liposome,
a quantum dot ("QD"), a fluorescent or phosphorescent nanoparticle
("NP"), a fluorescent latex particle or microbead. A fluorescent
molecule can be fluorescein, carboxyfluorescein and other
fluorescein derivatives, rhodamine, and their derivatives, or any
other glowing entity capable of forming a covalent bond with the
ligand.
[0092] In one embodiment, fluorescent proteins are used as
fluorophores. A large variety of fluorophores are available and can
find use in the methods described herein, for example and without
limitation: ALEXA Fluors (Molecular Probes/Invitrogen) and DYLIGHT
Fluors (Thermo Fisher Scientific). These fluorophores have an
emission spectra that span a wide range, including ultraviolet,
near-ultraviolet, visible, near-infrared, and infrared ranges.
Representative donor fluorescent moieties that can be used with
various acceptor fluorescent moieties in FRET technology include
fluorescein, Lucifer Yellow, B-phycoerythrin,
9-acridineisothiocyanate, Lucifer Yellow VS,
4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid,
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin,
succinimdyl 1-pyrenebutyrate, and
4-acetamido-4'-isothiocyanatostilbene-2-,2'-disulfonic acid
derivatives, chelates of Lanthanide ions (e.g., Europium,
Dysprosium, Samarium or Terbium). Representative acceptor
fluorescent moieties, depending upon the donor fluorescent moiety
used, include LC-Red 640, LC-Red 705, Cy5, Cy5.5, Lissamine
rhodamine B sulfonyl chloride, tetramethyl rhodamine
isothiocyanate, rhodamine x isothiocyanate, erythrosine
isothiocyanate, fluorescein, diethylenetriamine pentaacetate,
allophycocyanin, XL665, d2. Donor and acceptor fluorescent moieties
can be obtained, for example, from Molecular Probes (Junction City,
Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).
[0093] Certain naturally occurring amino acids, such as tryptophan,
are fluorescent Amino acids may also be derivatized, e.g. by
linking a fluorescent group onto an amino acid (such as linking
AEDANS to a Cys), to create a fluorophore pair for FRET. The
AEDANS-Cys pair is commonly used to detect protein conformational
change and interactions. Some other forms fluorescence groups have
also been used to modify amino acids and to generate FRET within
the protein fragments (e.g. 2,4-dinitrophenyl-lysine with
S-(N14-methyl-7-dimethylamino-coumarin-3-yl]-carboxamidomethyl)-cysteine--
).
[0094] In another embodiment, which is especially suitable for
using in live cells, green fluorescent protein (GFP) and its
various mutants are used as the fluorophores. Red fluorescent
proteins such as DsRed (Clontech) having an excitation maximum of
558 nm and an emission maximum of 583 can also be used. Examples of
fluorescent proteins are found in the Genbank and SwissPro public
databases.
[0095] FRET between two different fluorophores can be assayed by
several methods: looking at the change in color of the
fluorescence, measuring the fluorescence lifetime of the donor,
examining the changes upon photobleaching either the donor or
acceptor or both donor/acceptor fluorphore. Regardless of the
approach, most of these assays share common features of the
instrumentation. Examples of such are the EnVision Plate Reader
(Molecular Devices), ViewLux ultraHTS Microplate Imager
(PerkinElmer), OPTIMA Microplate Readers, FLUOstar and POLARstar
(BMG Labtech). Preferred measurement is by time-resolved
fluorimetry.
[0096] FRET between two different fluorophores can be assayed by
high-content cell screening using an instrument that detects
changes in fluorescence in cells or in particular subcellular
localizations. Examples of such instrumentation are the INCell
Analyzer (GE Healthcare), ImageXpress Micro High Content Screening
System (Molecular Devices), Opera, Operetta (PerkinElmer),
Cellomics ArrayScan VTI HCS Reader (Thermo Scientific).
[0097] Instead of eliciting non-radiative energy transfer to an
acceptor fluorophore by irradiating a donor fluorophore, it is
possible to achieve a non-radiative transfer of energy from a donor
enzyme to a complementary acceptor fluorophore after substrate
oxidation. Such a process is called bioluminescence resonance
energy transfer (BRET). Examples of donor enzymes are luciferase or
aequorin, substrates can be luciferin or coelenterazine and the
acceptor fluorophore can be GFP, YFP, EGFP, GFP.sup.2 or GFP10
(Pfleger K. et al, Nature Protocols, 2006, 1, 337-345). BRET can be
detected using a luminometer or scanning spectrometer.
[0098] Embodiments of the invention are also directed to various
FRET assays, such as for example: steady-state FRET, Fluorescence
Lifetime, Time-Resolved FRET, Intramolecular FRET or Intermolecular
FRET. An example of Intramolecular FRET is one between two
chromophores labeled within a single molecule (e.g. to identify
conformational changes by a molecule). In certain embodiments,
intramolecular FRET of a molecule can be measured in the absence of
any other molecules. In certain embodiments, intramolecular FRET
can be measured in the presence of one or more interacting proteins
(e.g., any ligand-receptor interaction).
[0099] FRET as provided herein can also be detected
intermolecularly, for example, between two or more chromophores
labeled in two or more different molecules.
[0100] The Z' factor is used to assess the quality of the assay
throughout development (Zhang J H et al. J Biomol Screen. 1999;
4(2):67-73). The Z' factor integrates the assay signal dynamic
range (difference between the mean of the positive controls and the
mean of the negative controls) and the statistical variability of
the signals, and ranges from 0 (poor quality) to 1 (high quality).
The higher the Z' value, the greater is the assay robustness, with
values equal to or higher than 0.5 indicating an excellent assay.
Z'=1-[3.times.(SD.sub.C++SD.sub.C-)/(Mean.sub.C+-Mean.sub.S-)]where
SD.sub.C+=standard deviation of the positive control (maximum
signal); SD.sub.C-=standard deviation of the negative control
(minimum signal); Mean.sub.C+=mean value of the positive control;
Mean.sub.C-=mean value of the negative control.
[0101] Sample Containers: Although described above as a cuvette,
embodiments of the invention are effective in any number of
receptacle, container or vessel geometries. Thus, the assays can be
conducted in a tube, vial, dish, flow cell, cassette, cartridge,
microfluidic chip, and any other similar type of containers. In
other embodiments, the container can be composed of a plethora of
materials, in any shape and of any type. Therefore, the assay
format may also be applied to a flattened plastic or glass cassette
or cartridge in which assay components might be magnetically pulled
along a channel or path by an external magnet. Hence, several
embodiments or geometries for the assay vessel are envisioned,
including cuvettes having a translucent or open surface area
pervious to irradiation at the exciting wavelength so as to enable
a fluorescent assay. For example, the cuvette translucent surface
area, may be formed as a square, rectangular, round, oval, or flat
container, beads, vial, tube, cylinder, cassette, or cartridge. The
preferred embodiment is a multiwall microtiter plate.
[0102] In embodiments, the receptacle comprises: a cuvette,
multiwell plate, tube, flask, disk, beads, vial, cassette, flow
cell, cartridge, microfluidic chip or combinations thereof, which
permit irradiation at the wavelength of the donor fluorophore and
measurement at the wavelength of the acceptor fluorophore.
[0103] In embodiments, the methods and assays described herein are
provided in a high-throughput screening assay format. The benefits
of such formats are easily identifiable, such as for example,
screening of large patient samples for diagnostic or prognostic
purposes, screening for new drugs, research, and the like.
Candidate/Test Agents:
[0104] Candidate agents include numerous chemical classes, though
typically they are organic compounds including small organic
compounds, nucleic acids including oligonucleotides, peptides or
antibodies. Small organic compounds suitably may have e.g. a
molecular weight of more than about 40 or 50 yet less than about
2,500. Candidate agents may comprise functional chemical groups
that interact with proteins and/or DNA.
[0105] Candidate agents may be obtained from a wide variety of
sources including libraries of synthetic or natural compounds. For
example, numerous means are available for random and directed
synthesis of a wide variety of organic compounds and biomolecules,
including expression of randomized oligonucleotides. Alternatively,
libraries of natural compounds in the form of e.g. bacterial,
fungal and animal extracts are available or readily produced.
[0106] Chemical Libraries: Developments in combinatorial chemistry
allow the rapid and economical synthesis of hundreds to thousands
of discrete compounds. These compounds are typically arrayed in
moderate-sized libraries of small molecules designed for efficient
screening Combinatorial methods, can be used to generate unbiased
libraries suitable for the identification of novel compounds. In
addition, smaller, less diverse libraries can be generated that are
descended from a single parent compound with a previously
determined biological activity. In either case, the lack of
efficient screening systems to specifically target therapeutically
relevant biological molecules produced by combinational chemistry
such as inhibitors of important enzymes hampers the optimal use of
these resources.
[0107] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks," such as reagents. For example, a linear combinatorial
chemical library, such as a polypeptide library, is formed by
combining a set of chemical building blocks (amino acids) in a
large number of combinations, and potentially in every possible
way, for a given compound length (i.e., the number of amino acids
in a polypeptide compound). Millions of chemical compounds can be
synthesized through such combinatorial mixing of chemical building
blocks.
[0108] A "library" may comprise from 2 to 50,000,000 diverse member
compounds. Preferably, a library comprises at least 48 diverse
compounds, preferably 96 or more diverse compounds, more preferably
384 or more diverse compounds, more preferably, 10,000 or more
diverse compounds, preferably more than 100,000 diverse members and
most preferably more than 1,000,000 diverse member compounds. By
"diverse" it is meant that greater than 50% of the compounds in a
library have chemical structures that are not identical to any
other member of the library. Preferably, greater than 75% of the
compounds in a library have chemical structures that are not
identical to any other member of the collection, more preferably
greater than 90% and most preferably greater than about 99%.
[0109] The preparation of combinatorial chemical libraries is well
known to those of skill in the art. For reviews, see Thompson et
al., Synthesis and application of small molecule libraries, Chem
Rev 96:555-600, 1996; Kenan et al., Exploring molecular diversity
with combinatorial shape libraries, Trends Biochem Sci 19:57-64,
1994; Janda, Tagged versus untagged libraries: methods for the
generation and screening of combinatorial chemical libraries, Proc
Natl Acad Sci USA. 91:10779-85, 1994; Lebl et al.,
One-bead-one-structure combinatorial libraries, Biopolymers
37:177-98, 1995; Eichler et al., Peptide, peptidomimetic, and
organic synthetic combinatorial libraries, Med Res Rev. 15:481-96,
1995; Chabala, Solid-phase combinatorial chemistry and novel
tagging methods for identifying leads, Curr Opin Biotechnol.
6:632-9, 1995; Dolle, Discovery of enzyme inhibitors through
combinatorial chemistry, Mol. Divers. 2:223-36, 1997; Fauchere et
al., Peptide and nonpeptide lead discovery using robotically
synthesized soluble libraries, Can J. Physiol Pharmacol. 75:683-9,
1997; Eichler et al., Generation and utilization of synthetic
combinatorial libraries, Mol Med Today 1: 174-80, 1995; and Kay et
al., Identification of enzyme inhibitors from phage-displayed
combinatorial peptide libraries, Comb Chem High Throughput Screen
4:535-43, 2001.
[0110] Other chemistries for generating chemical diversity
libraries can also be used. Such chemistries include, but are not
limited to, peptoids (PCT Publication No. WO 91/19735); encoded
peptides (PCT Publication WO 93/20242); random bio-oligomers (PCT
Publication No. WO 92/00091); benzodiazepines (U.S. Pat. No.
5,288,514); diversomers, such as hydantoins, benzodiazepines and
dipeptides (Hobbs, et al., Proc. Nat. Acad. Sci. USA, 90:6909-6913
(1993)); vinylogous polypeptides (Hagihara, et al., J. Amer. Chem.
Soc. 114:6568 (1992)); nonpeptidal peptidomimetics with
.beta.-D-glucose scaffolding (Hirschmann, et al., J. Amer. Chem.
Soc., 114:9217-9218 (1992)); analogous organic syntheses of small
compound libraries (Chen, et al., J. Amer. Chem. Soc., 116:2661
(1994)); oligocarbamates (Cho, et al., Science, 261:1303 (1993));
and/or peptidyl phosphonates (Campbell, et al., J. Org. Chem.
59:658 (1994)); nucleic acid libraries (see, Ausubel, Berger and
Sambrook, all supra); peptide nucleic acid libraries (see, e.g.,
U.S. Pat. No. 5,539,083); antibody libraries (see, e.g., Vaughn, et
al., Nature Biotechnology, 14(3):309-314 (1996) and
PCT/US96/10287); carbohydrate libraries (see, e.g., Liang, et al.,
Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853); small
organic molecule libraries (see, e.g., benzodiazepines, Baum
C&E News, January 18, page 33 (1993); isoprenoids (U.S. Pat.
No. 5,569,588); thiazolidinones and metathiazanones (U.S. Pat. No.
5,549,974); pyrrolidines (U.S. Pat. Nos. 5,525,735 and 5,519,134);
morpholino compounds (U.S. Pat. No. 5,506,337); benzodiazepines
(U.S. Pat. No. 5,288,514); and the like.
[0111] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem.
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar,
Ltd., Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek Bio
sciences, Columbia, Md., etc.).
[0112] The screening assays of the invention suitably include and
embody, animal models, cell-based systems and non-cell based
systems. Identified genes, variants, fragments, or oligopeptides
thereof are used for identifying agents of therapeutic interest,
e.g. by screening libraries of compounds or otherwise identifying
compounds of interest by any of a variety of drug screening or
analysis techniques. The gene, allele, fragment, or oligopeptide
thereof employed in such screening may be free in solution, affixed
to a solid support, borne on a cell surface, or located
intracellularly. The measurements will be conducted as described in
detail in the examples section which follows.
[0113] In some embodiments, a method of identifying candidate
therapeutic agents comprises screening a sample containing the
specific target molecule in a high-throughput screening assay
comprising the steps of: (i) adding a first and second ligand, each
having a first and second detectable label, (ii) the first and
second ligands each binding to separate and specific sites on a
specific target molecule, wherein the screening assay optionally
omits or does not require the step of (iii) washing and detecting
an emission of light when the first and second ligands specifically
bind to the specific target molecule.
[0114] In another preferred embodiment, a method of identifying
therapeutic agents comprises contacting: (i) a target molecule with
a candidate therapeutic agent; determining whether (i) the agent
modulates a function of the peptide or interaction of the peptide
with a partner molecule; or (ii) the agent modulates expression
and/or function of the nucleic acid sequence of the target as
measured by the light emission assays embodied herein.
[0115] In another preferred embodiment, a method of identifying
candidate therapeutic agents for treatment of disease, comprises
culturing an isolated cell expressing a target molecule,
administering a candidate therapeutic agent to the cultured cell;
correlating the target molecules expression, activity and/or
function in the presence or absence of a candidate therapeutic
agent as compared to control cells, wherein a drug is identified
based on desirable therapeutic outcomes. For example, a drug which
modulates expression of the target molecule whereby expression
levels are responsible for the disease state or the target molecule
modulates the activity or amount of another molecule whether
upstream or downstream in a pathway. In other examples the assays
measure kinase activity. In other examples, the assay measure
binding partners. In other examples, the assay measures amounts of
candidate therapeutic agents which provide a desired therapeutic
outcome.
[0116] Another suitable method for diagnosis and candidate drug
discovery includes contacting a test sample with a cell expressing
a target molecule, and detecting interaction of the test agent with
the target molecule, an allele or fragment thereof, or expression
product of the target molecule an allele or fragment thereof.
[0117] In another preferred embodiment, a sample, such as, for
example, a cell or fluid from a patient is isolated and contacted
with a candidate therapeutic molecule. The genes, expression
products thereof, are monitored to identify which genes or
expression products are regulated by the drug.
High-Throughput Screening
[0118] The assays embodied herein are suitable for identifying and
quantifying specific molecules in a sample. In addition, the assays
can be used for drug screening in a high throughput screening of
compounds having suitable binding affinity to the protein of
interest (see, e.g., Geysen et al., 1984, PCT application
WO84/03564). In this method, large numbers of different small test
compounds are synthesized on a solid substrate. The test compounds
are reacted with identified genes, or fragments thereof, and
washed. Bound molecules are then detected by the methods embodied
herein. Alternatively, non-neutralizing antibodies can be used to
capture the peptide and immobilize it on a solid support.
[0119] The methods of screening of the invention comprise using
screening assays to identify, from a library of diverse molecules,
one or more compounds having a desired activity. For example,
modulating the amount of a target molecule. A "screening assay" is
a selective assay designed to identify, isolate, and/or determine
the structure of, compounds within a collection that have a
preselected activity. By "identifying" it is meant that a compound
having a desirable activity is isolated, its chemical structure is
determined (including without limitation determining the nucleotide
and amino acid sequences of nucleic acids and polypeptides,
respectively) the structure of and, additionally or alternatively,
purifying compounds having the screened activity). Biochemical and
biological assays are designed to test for activity in a broad
range of systems ranging from protein-protein interactions, enzyme
catalysis, small molecule-protein binding, to cellular functions.
Such assays include automated, semi-automated assays and HTS (high
throughput screening) assays.
[0120] In HTS methods, many discrete compounds are preferably
tested in parallel by robotic, automatic or semi-automatic methods
so that large numbers of test compounds are screened for a desired
activity simultaneously or nearly simultaneously. It is possible to
assay and screen up to about 6,000 to 20,000, and even up to about
100,000 to 1,000,000 different compounds a day using the integrated
systems of the invention.
[0121] Typically in HTS, target molecules are administered or
cultured with isolated cells with modulated receptors, including
the appropriate controls.
[0122] In one embodiment, screening comprises contacting each cell
culture with a diverse library of member compounds, some of which
are ligands of the target, under conditions where complexes between
the target and ligands can form, and identifying which members of
the libraries are present in such complexes. In another non
limiting modality, screening comprises contacting a target enzyme
with a diverse library of member compounds, some of which are
inhibitors (or activators) of the target, under conditions where a
product or a reactant of the reaction catalyzed by the enzyme
produce a detectable signal. In the latter modality, inhibitors of
target enzyme decrease the signal from a detectable product or
increase a signal from a detectable reactant (or vice-versa for
activators).
[0123] The methods disclosed herein can be used for screening a
plurality of test compounds. In certain embodiments, the plurality
of test compounds comprises between 1 and 200,000 test compounds,
between 1 and 100,000 test compounds, between 1 and 1,000 test
compounds, between 1 and 100 test compounds, or between 1 and 10
test compounds. In certain embodiments, the test compounds are
provided by compound libraries, whether commercially available or
not, using combinatorial chemistry techniques. In certain
embodiments, the compound libraries are immobilized on a solid
support.
[0124] As discussed above, the target can be present in any
substrate as the assay parameters can be manipulated or optimized
for each type of substrate. For example, if the target is at the
surface of or in a cell, or secreted by a cell, the following
parameters would be determined: the optimal cell line, cell
density, culture medium, serum concentration, final reagents
volumes, compound incubation times (for example 12, 24, 36 or 48
hours). If the target is in a cell-free solution, the optimal
composition of the solution can be determined as well as the range
of concentrations of the positive control standard. Other
parameters that can be determined are ligand concentrations,
temperature of incubation and incubation times of the ligands (for
example 1 to 4 hours). The set-up of the reading instrument, for
example a time-resolved fluorimeter, is optimized for the
measurement window and time delay, excitation parameters (e.g.
number of flashes delivered), gain adjustment, and reader head
positioning with respect to the receptacle. The proper
pharmacological control, if available, needs to be determined.
[0125] High throughput screening can be used to measure the effects
of drugs on complex molecular events such as signal transduction
pathways, as well as cell functions including, but not limited to,
cell function, apoptosis, cell division, cell adhesion, locomotion,
exocytosis, and cell-cell communication. Multicolor fluorescence
permits multiple targets and cell processes to be assayed in a
single screen. Cross-correlation of cellular responses will yield a
wealth of information required for target validation and lead
optimization.
[0126] In another aspect, the present invention provides a method
for analyzing cells comprising providing an array of locations
which contain multiple cells wherein the cells contain one or more
fluorescent reporter molecules; scanning multiple cells in each of
the locations containing cells to obtain fluorescent signals from
the fluorescent reporter molecule in the cells; converting the
fluorescent signals into digital data; and utilizing the digital
data to determine the distribution, environment or activity of the
fluorescent reporter molecule within the cells.
[0127] Microarrays: Identification of a nucleic acid sequence
capable of binding to a target molecule can be achieved by
immobilizing a library of nucleic acids onto the substrate surface
so that each unique nucleic acid is located at a defined position
to form an array. In general, the immobilized library of nucleic
acids are exposed to a biomolecule or candidate agent under
conditions which favored binding of the biomolecule to the nucleic
acids. The nucleic acid array would then be analyzed by the methods
embodied herein to determine which nucleic acid sequences bound to
the biomolecule. Preferably the biomolecules would carry a
pre-determined label for use in detection of the location of the
bound nucleic acids.
[0128] An assay using an immobilized array of nucleic acid
sequences may be used for determining the sequence of an unknown
nucleic acid; single nucleotide polymorphism (SNP) analysis;
analysis of gene expression patterns from a particular species,
tissue, cell type, etc.; gene identification; etc.
[0129] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences, may be used as
targets in a microarray. The microarray can be used to monitor the
identity and/or expression level of large numbers of genes and gene
transcripts simultaneously to identify genes with which target
genes or its product interacts and/or to assess the efficacy of
candidate therapeutic agents in regulating expression products of
genes that mediate, for example, neurological disorders. This
information may be used to determine gene function, and to develop
and monitor the activities of therapeutic agents.
[0130] Microarrays may be prepared, used, and analyzed using
methods known in the art (see, e.g., Brennan et al., 1995, U.S.
Pat. No. 5,474,796; Schena et al., 1996, Proc. Natl. Acad. Sci.
U.S.A. 93: 10614-10619; Baldeschweiler et al., 1995, PCT
application WO95/251116; Shalon, et al., 1995, PCT application
WO95/35505; Heller et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:
2150-2155; and Heller et al., 1997, U.S. Pat. No. 5,605,662). In
other embodiments, a microarray comprises peptides, or other
desired molecules which can be assayed to identify a candidate
agent.
Utilities
[0131] In preferred embodiments, the assay provides a method of
quantifying specific proteins in a biological sample, for example,
a body fluid or a cell including molecules which are
intra-cellular, extra-cellular or cell surface molecules.
[0132] In certain embodiments, the assay provides a method of
diagnosing a disease or disorder comprising screening a biological
sample from a patient in order to identifying and/or quantify a
marker or molecule diagnostic of the particular disease or
disorder. For example, a genetic marker, protein marker and the
like.
[0133] In certain preferred embodiments, the screening is conducted
using high-. throughput screening allowing for simultaneous
diagnosing of many subjects at the same time.
[0134] In another preferred embodiment, a method of identifying
subjects at risk of developing a disease or disorder comprising
screening a biological sample from a patient and identifying and/or
quantifying a marker or molecule diagnostic of the particular
disease or disorder.
[0135] In another preferred embodiment a method for screening
candidate compounds for the treatment or prevention of a disease or
disorder comprises contacting a sample with a candidate therapeutic
agent and measuring the effects the compound has on a target. For
example if it is a cellular product such as a receptor, the
compound may regulate the receptor levels and the compound can then
be further studied for any possible therapeutic effects (increase
or decrease of the parameter being monitored e.g. expression,
oxidation level, metabolic markers, viability or apoptic markers).
An abnormal expression state of the target may be caused by
pathology such as a metabolic or infectious disease, degenerative
disease, cancer, genetic defects and/or a toxin.
Kits and Methods
[0136] The present invention further provides systems and kits
(e.g., commercial therapeutic, diagnostic, or research products,
reaction mixtures, etc.) that contain one or more or all components
sufficient, necessary, or useful to practice any of the methods
described herein. These systems and kits may include buffers,
detection/imaging components, positive/negative control reagents,
instructions, software, hardware, packaging, or other desired
components. The kits are useful for quantifying a specific protein
in a biological sample, as well as identifying that specific
protein.
[0137] The kits provide useful tools for screening test compounds
capable of modulating the effects of a compound on a target
molecule. The kits can be packaged in any suitable manner to aid
research, clinical, and testing labs, typically with the various
parts, in a suitable container along with instructions for use.
[0138] Provided herein are kits for identifying and quantifying a
specific molecule in a sample. In certain embodiments, the kits
comprise (a) a target molecule labeled with a first chromophore;
and (b) a test agent labeled with a second chromophore. In certain
embodiments, the kits comprise (a) a test agent labeled with a
first chromophore; and (b) a second test agent labeled with a
second chromophore. In certain embodiments, the kits may further
comprise lipids and/or solvents. In certain embodiments, the kits
may further comprise buffers and reagents needed for the procedure,
and instructions for carrying out the assay. In certain
embodiments, the kits may further comprise, where necessary, agents
for reducing the background interference in a test, positive and
negative control reagents, apparatus for conducting a test, and the
like.
[0139] In certain embodiments of the methods and kits provided
herein, solid phase supports are used for purifying proteins,
labeling samples or carrying out the solid phase assays. Examples
of solid phases suitable for carrying out the methods disclosed
herein include beads, particles, colloids, single surfaces, tubes,
multiwell plates, microtiter plates, slides, membranes, gels and
electrodes. When the solid phase is a particulate material (e.g.,
beads), it is, in one embodiment, distributed in the wells of
multi-well plates to allow for parallel processing of the solid
phase supports.
[0140] Methods and kits disclosed herein may be carried out in
numerous formats known in the art. In certain embodiments, the
methods provided herein are carried out using solid-phase assay
formats. In certain embodiments, the methods provided herein are
carried out in a well of a plate with a plurality of wells, such as
a multi-well plate or a multi-domain multi-well plate. The use of
multi-well assay plates allows for the parallel processing and
analysis of multiple samples distributed in multiple wells of a
plate. Multi-well assay plates (also known as microplates or
microtiter plates) can take a variety of forms, sizes and shapes
(e.g., round- or flat-bottom multi-well plates). Exemplary
multi-well plate formats that can be used in the methods provided
herein include those found on 96-well plates (12.times.8 array of
wells), 384-well plates (24.times.16 array of wells), 1536-well
plate (48.times.32 array of well), 3456-well plates and 9600-well
plates. Other formats that may be used in the methods provided
herein include, but are not limited to, single or multi-well plates
comprising a plurality of domains, cuvettes, microarrays etc. In
certain embodiments, the plates are black-wall, black-bottom
plates. In certain embodiments, the plates are black-wall,
white-bottom plates. In certain embodiments, the plates have black
walls and clear bottoms in order to allow bottom reading of the
fluorescence signals. In certain embodiments, the plates are chosen
with minimal and uniform intrinsic fluorescence intensity within
the range utilized in the method to avoid interference with the
FRET signals.
[0141] The methods provided herein, when carried out in
standardized plate formats can take advantage of readily available
equipment for storing and moving these plates as well as readily
available equipment for rapidly dispensing liquids in and out of
the plates (e.g., robotic dispenser, multi-well and multi-channel
pipettes, plate washers and the like).
Administration of Compositions
[0142] The agents identified by the methods embodied herein can be
formulated and compositions of the present invention may be
administered in conjunction with one or more additional active
ingredients, pharmaceutical compositions, or other compounds. The
therapeutic agents of the present invention may be administered to
an animal, preferably a mammal, most preferably a human.
[0143] The pharmaceutical formulations may be for administration by
oral (solid or liquid), parenteral (intramuscular, intraperitoneal,
intravenous (IV) or subcutaneous injection), transdermal (either
passively or using ionophoresis or electroporation), transmucosal
and systemic (nasal, vaginal, rectal, or sublingual), or inhalation
routes of administration, or using bioerodible inserts and can be
formulated in dosage forms appropriate for each route of
administration.
[0144] The agents may be formulated in pharmaceutically acceptable
carriers or diluents such as physiological saline or a buffered
salt solution. Suitable carriers and diluents can be selected on
the basis of mode and route of administration and standard
pharmaceutical practice. A description of exemplary
pharmaceutically acceptable carriers and diluents, as well as
pharmaceutical formulations, can be found in Remington's
Pharmaceutical Sciences, a standard text in this field, and in
USP/NF. Other substances may be added to the compositions to
stabilize and/or preserve the compositions.
[0145] The compositions of the invention may be administered to
animals by any conventional technique. The compositions may be
administered directly to a target site by, for example, surgical
delivery to an internal or external target site, or by catheter to
a site accessible by a blood vessel. Other methods of delivery,
e.g., liposomal delivery or diffusion from a device impregnated
with the composition, are known in the art. The compositions may be
administered in a single bolus, multiple injections, or by
continuous infusion (e.g., intravenously). For parenteral
administration, the compositions are preferably formulated in a
sterilized pyrogen-free form.
[0146] The compounds identified by this invention may also be
administered orally to the patient, in a manner such that the
concentration of drug is sufficient to inhibit bone resorption or
to achieve any other therapeutic indication as disclosed herein.
Typically, a pharmaceutical composition containing the compound is
administered at an oral dose of between about 0.1 to about 50 mg/kg
in a manner consistent with the condition of the patient.
Preferably the oral dose would be about 0.5 to about 20 mg/kg.
[0147] An intravenous infusion of the compound in 5% dextrose in
water or normal saline, or a similar formulation with suitable
excipients, is most effective, although an intramuscular bolus
injection is also useful. Typically, the parenteral dose will be
about 0.01 to about 100 mg/kg; preferably between 0.1 and 20 mg/kg,
in a manner to maintain the concentration of drug in the plasma at
a concentration effective to inhibit a cysteine protease. The
compounds may be administered one to four times daily at a level to
achieve a total daily dose of about 0.4 to about 400 mg/kg/day. The
precise amount of an inventive compound which is therapeutically
effective, and the route by which such compound is best
administered, is readily determined by one of ordinary skill in the
art by comparing the blood level of the agent to the concentration
required to have a therapeutic effect. Prodrugs of compounds of the
present invention may be prepared by any suitable method. For those
compounds in which the prodrug moiety is a ketone functionality,
specifically ketals and/or hemiacetals, the conversion may be
effected in accordance with conventional methods.
[0148] No unacceptable toxicological effects are expected when
compounds, derivatives, salts, compositions etc., of the present
invention are administered in accordance with the present
invention. The compounds of this invention, which may have good
bioavailability, may be tested in one of several biological assays
to determine the concentration of a compound which is required to
have a given pharmacological effect.
[0149] In another preferred embodiment, there is provided a
pharmaceutical or veterinary composition comprising one or more
identified compounds and a pharmaceutically or veterinarily
acceptable carrier. Other active materials may also be present, as
may be considered appropriate or advisable for the disease or
condition being treated or prevented.
[0150] The carrier, or, if more than one be present, each of the
carriers, must be acceptable in the sense of being compatible with
the other ingredients of the formulation and not deleterious to the
recipient.
[0151] The compounds identified by the methods herein would be
suitable for use in a variety of drug delivery systems described
above. Additionally, in order to enhance the in vivo serum
half-life of the administered compound, the compounds may be
encapsulated, introduced into the lumen of liposomes, prepared as a
colloid, or other conventional techniques may be employed which
provide an extended serum half-life of the compounds. A variety of
methods are available for preparing liposomes, as described in,
e.g., Szoka, et al., U.S. Pat. Nos. 4,235,871, 4,501,728 and
4,837,028 each of which is incorporated herein by reference.
Furthermore, one may administer the drug in a targeted drug
delivery system, for example, in a liposome coated with a
tissue-specific antibody. The liposomes will be targeted to and
taken up selectively by the organ.
[0152] The formulations include those suitable for rectal, nasal,
topical (including buccal and sublingual), vaginal or parenteral
(including subcutaneous, intramuscular, intravenous and
intradermal) administration, but preferably the formulation is an
orally administered formulation. The formulations may conveniently
be presented in unit dosage form, e.g. tablets and sustained
release capsules, and may be prepared by any methods well known in
the art of pharmacy.
[0153] Such methods include the step of bringing into association
the above defined active agent with the carrier. In general, the
formulations are prepared by uniformly and intimately bringing into
association the active agent with liquid carriers or finely divided
solid carriers or both, and then if necessary shaping the
product.
[0154] The compound identified using these methods can be
formulated according to known methods to prepare pharmaceutically
useful compositions, whereby the compound is combined in admixture
with a pharmaceutically acceptable carrier vehicle. Therapeutic
formulations are prepared for storage by mixing the active
ingredient having the desired degree of purity with optional
physiologically acceptable carriers, excipients or stabilizers
(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980)), in the form of lyophilized formulations or aqueous
solutions. Acceptable carriers, excipients or stabilizers are
nontoxic to recipients at the dosages and concentrations employed,
and include buffers such as phosphate, citrate and other organic
acids; antioxidants including ascorbic acid; low molecular weight
(less than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone, amino acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as TWEEN.TM. (ICI Americas Inc., Bridgewater,
N.J.), PLURONICS.TM. (BASF Corporation, Mount Olive, N.J.) or
PEG.
[0155] The formulations to be used for in vivo administration must
be sterile and pyrogen free. This is readily accomplished by
filtration through sterile filtration membranes, prior to or
following lyophilization and reconstitution.
[0156] Dosages and desired drug concentrations of pharmaceutical
compositions of the present invention may vary depending on the
particular use envisioned. The determination of the appropriate
dosage or route of administration is well within the skill of an
ordinary physician. Animal experiments provide reliable guidance
for the determination of effective doses for human therapy.
Interspecies scaling of effective doses can be performed following
the principles laid down by Mordenti, J. and Chappell, W. "The use
of interspecies scaling in toxicokinetics" In Toxicokinetics and
New Drug Development, Yacobi et al., Eds., Pergamon Press, New York
1989, pp. 42-96.
[0157] Formulations for oral administration in the present
invention may be presented as: discrete units such as capsules,
cachets or tablets each containing a predetermined amount of the
active agent; as a powder or granules; as a solution or a
suspension of the active agent in an aqueous liquid or a
non-aqueous liquid; or as an oil-in-water liquid emulsion or a
water in oil liquid emulsion; or as a bolus etc.
[0158] For compositions for oral administration (e.g. tablets and
capsules), the term "acceptable carrier" includes vehicles such as
common excipients e.g. binding agents, for example syrup, acacia,
gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone),
methylcellulose, ethylcellulose, sodium carboxymethylcellulose,
hydroxypropylmethylcellulose, sucrose and starch; fillers and
carriers, for example corn starch, gelatin, lactose, sucrose,
microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate,
sodium chloride and alginic acid; and lubricants such as magnesium
stearate, sodium stearate and other metallic stearates, glycerol
stearate stearic acid, silicone fluid, talc waxes, oils and
colloidal silica. Flavoring agents such as peppermint, oil of
wintergreen, cherry flavoring and the like can also be used. It may
be desirable to add a coloring agent to make the dosage form
readily identifiable. Tablets may also be coated by methods well
known in the art.
[0159] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active agent in a
free flowing form such as a powder or granules, optionally mixed
with a binder, lubricant, inert diluent, preservative,
surface-active or dispersing agent. Molded tablets may be made by
molding in a suitable machine a mixture of the powdered compound
moistened with an inert liquid diluent. The tablets may be
optionally be coated or scored and may be formulated so as to
provide slow or controlled release of the active agent.
[0160] Other formulations suitable for oral administration include
lozenges comprising the active agent in a flavored base, usually
sucrose and acacia or tragacanth; pastilles comprising the active
agent in an inert base such as gelatin and glycerin, or sucrose and
acacia; and mouthwashes comprising the active agent in a suitable
liquid carrier.
[0161] Parenteral formulations will generally be sterile.
[0162] Dose: An effective dose of a composition of the presently
disclosed subject matter is administered to a subject in need
thereof. A "therapeutically effective amount" or a "therapeutic
amount" is an amount of a therapeutic composition sufficient to
produce a measurable response (e.g., a biologically or clinically
relevant response in a subject being treated). The response can be
measured in many ways, as discussed above, e.g. cytokine profiles,
cell types, cell surface molecules, etc. Actual dosage levels of
active ingredients in the compositions of the presently disclosed
subject matter can be varied so as to administer an amount of the
active compound(s) that is effective to achieve the desired
therapeutic response for a particular subject. The selected dosage
level will depend upon the activity of the therapeutic composition,
the route of administration, combination with other drugs or
treatments, the severity of the condition being treated, and the
condition and prior medical history of the subject being treated.
However, it is within the skill of the art to start doses of the
compound at levels lower than required to achieve the desired
therapeutic effect and to gradually increase the dosage until the
desired effect is achieved. The potency of a composition can vary,
and therefore a "treatment effective amount" can vary. However,
using the assay methods described herein, one skilled in the art
can readily assess the potency and efficacy of a candidate compound
of the presently disclosed subject matter and adjust the
therapeutic regimen accordingly.
[0163] All documents mentioned herein are incorporated herein by
reference. All publications and patent documents cited in this
application are incorporated by reference for all purposes to the
same extent as if each individual publication or patent document
were so individually denoted. By their citation of various
references in this document, Applicants do not admit any particular
reference is "prior art" to their invention.
EXAMPLES
[0164] While various embodiments of the present invention have been
described above, it should be understood that they have been
presented by way of example only, and not limitation. The following
non-limiting examples are illustrative of the invention.
Example 1
Identification and Quantification of Prion Protein
(PrP)-PrP-FEHTA
[0165] Materials and Methods
[0166] Best Ligand Pair Analysis. A panel of ligands coupled to a
fluorescent chromophore were tested for their capacity to bind the
target at the cell surface or in solution. The ligands yielding a
robust target-specific signal were selected. The absence of
interference for binding to the target when the ligands were added
simultaneously was then verified upon systematic pairing (one
labeled "binding" ligand is mixed with one non-labeled
"interfering" ligand). Finally, a best pair analysis was conducted
in the following manner: the signal generated by any given ligand
pair combination (one ligand being labeled with a donor chromophore
and the other with an acceptor chromophore, and vice-versa) was
measured for increasing concentrations of the target. The labeled
ligand pair generating the strongest signal was retained.
[0167] Quantification of Recombinant PrP (rPrP) in Solution:
Recombinant PrP is added to the wells of a microtiter plate in
phosphate buffer saline (PBS). PrP is immediately detected using
PrP specific antibodies SAF32 (aa53-93) and D18 (aa133-157) labeled
with the donor and acceptor fluorophores, respectively. PBS alone
is used as a control for signal background. For data analysis,
ratios (R) of the 665 nm (acceptor emission) to the 620 nm (donor
emission) measurements are calculated.
[0168] Determination of PrP at the Cell Surface: Cells are added to
the wells of a microtiter plate. Compounds of a screening library
are added, or the solvent control (usually DMSO) and the cells are
incubated for 24 hours. Then, the amount of PrP present at the cell
surface is detected on living cells. To detect cell surface PrP,
antibodies are used as ligands. The best antibody pair is SAF32
directed against aa53-93 (Cayman Chemical) and D18 directed against
aa133-157 (Williamson R. A., J. Virol, 1998, 72 (11), 9413-18)
labeled with the donor and acceptor fluorophores, respectively. In
this particular example, HTRF.RTM. was used, with Terbium cryptate
as the donor fluorophore and d2 as the acceptor fluorophore.
Antibodies were labeled by Cisbio. A PrP.sup.0/0 cell line (KO)
derived from primary hippocampal neurons of PrP gene-deficient mice
serves as negative control for PrP expression. Blanks consist in
culture medium in the absence of cells. For data analysis, ratios
(R) of the 665 nm (acceptor emission) to the 620 nm (donor
emission) measurements are calculated, to correct for non-specific
absorption of 620 nm light by the assay mix. The value for the
specific signal of the sample or positive control is given by Delta
F %=[(R.sub.C+-R.sub.C-)/R.sub.C-].times.100 where R.sub.C+ and
R.sub.C- are the 665/620 ratios of the positive and negative
control. This ratiometric measurement allows to correct for
fluorescence interference induced by the assay matrix or screening
compounds.
[0169] Cell Line: LD9 cells, a fibroblastic cell line (Mahal S. et
al. Proc Natl Acad Sci USA. 2007; 104(52):20908-13). This cell line
presents reduced shedding of PrP into the medium and therefore less
background signal for PrP when compared to neuroblastoma cells.
[0170] Pharmacological control: Brefeldine A (BFA), a compound that
prevents trafficking of proteins from the ER to the Golgi
(Nebenfuhr A. et al. Plant physiology. 2002; 130: 1102-8).
[0171] Other Assay Conditions: Optimal incubation times are 24
hours for the compound, 3 hours for the antibody. Optimal antibody
concentrations (dubbed 1.times.) are 0.33 .mu.g/ml (D18-d2) and
0.036 .mu.g/ml (SAF32-Tb).
[0172] Results and Discussion
[0173] With the assay in the 384-well format, the US Drug
Collection was screened, a 1280-compound library comprising mainly
FDA-approved drugs. The library was screened at a 20 .mu.M
concentration. Thirty-eight compounds reduced PrP expression by
more than 50%, which was chosen as the screening threshold. The
library was then counter-screened using a cell viability assay to
reveal the toxic compounds. Nine out of the 38 candidate hits
exhibited less than 10% toxicity and were considered hits (FIG. 3).
Therefore the hit rate was 0.7%.
[0174] To confirm the hits, cell surface PrP levels were measured
by an independent method. Cells were exposed to the compounds,
washed with PBS, labeled at +4.degree. C. with monoclonal antibody
D18 for one hour, then fixed with 4% PFA prior to the addition of
the Alexa-488 labeled secondary antibody to reveal the PrP
antibody. Reduction of cell surface PrP can then be visualized
under the epifluorescence microscope and quantified either by flow
cytometry or with a high content subcellular analysis system such
as the IN Cell Analyzer 1000 or 2000 (GE Lifesciences). Six of nine
hits were confirmed, demonstrating that the primary assay yields
hits that reproducibly reduce PrP levels at the cell surface.
Moreover, as the final target of prions in the living organism is
the brain, it is important to show the activity of compounds on a
type of cells close to neuronal cells. All six compounds reduced
PrP levels on neuroblastoma cells (N2a). The activity of Tacrolimus
is shown in FIGS. 4A-4D as an example. Tacrolimus reduced PrP by
70% at 20 .mu.M in the primary assay (on LD9 cells), and by 75 and
73% at 30 in the secondary assay (on N2a cells) by flow cytometry
and IN Cell analyzer quantification, respectively.
[0175] Other active compounds were Astemizole, Lasalocid sodium,
Monensin, Emetine and cetrimonium. Quantification of PrP reduction
by Astemizole and Lasalocid sodium is shown in FIGS. 5A and 5B.
These compounds were subjected to further selection by
counter-screening and prioritization as described below.
[0176] Counter-screening: Toxic compounds generate an artifactual
decrease in the intensity of cell surface PrP signal and are
excluded. The high-throughput luminescent cell viability assay,
CELLTITER-GLO.RTM. (Promega) can be used to this purpose as shown
in FIG. 3. Addition of the CELLTITER-GLO.RTM. reagent results in
cell lysis and generation of a luminescent signal at 610 nm
proportional to the amount of ATP, reflecting the number of living
cells in culture.
[0177] Compound Prioritization: To select the best compounds from
the hits generated during the primary screening, the following
strategy can be used: (1). Selection of the compounds exerting the
highest PrP reducing effect. (2). Selection of the compounds
exerting the PrP reducing effect at the lowest concentration and
harboring the least toxicity on neuronal cells. To this end,
EC.sub.50 and TC.sub.50 have to be determined on N2a cells using,
for example, the assays described above (IF and Cell Titer Glo).
(3). Selection of the compounds exhibiting the highest specificity
for PrP. Although complete specificity for PrP is not required for
a compound to achieve a good therapeutic index, this can be used as
criteria for compound selection. Other markers expressed at the
surface of neurons, such as, but not restricted to, amyloid
precursor protein (APP) and CD24 (also called heat-stable antigen
or nectadrin) are detected by IF using the same methodology as that
used to detect cell surface PrP. After treatment of the cells with
the compound, the extent of reduction of other proteins is compared
with that of PrP. (4). Selection of the compounds showing the
highest capacity to cure prion-infected cells and to prevent
cellular infection by prions. Selected compounds can be tested for
their capacity to inhibit prion replication in prion-infected cells
using a Western blot or the Scrapie Cell Assay (SCA) (Kloehn P. C.
et al, Proc Natl Acad Sci USA. 2003; 100(20):11666-71). These
assays detect infected cells by virtue of their PrP.sup.Sc content.
The total PrP.sup.Sc content of the cell culture can be measured by
Western blot of cell lysates; infected cells can be recorded as
"spots" in the SCA). FIG. 6 illustrates the curing effect of RML
scrapie-infected PK1 cells by the enzyme phosphatidylinositol
phospholipase C (PIPLC), an enzyme that cleaves off GPI-anchored
proteins, hence PrP, from the cell surface, at 1 .mu.g/ml, a
concentration that removes approximately 50% of cell surface PrP.
FIGS. 7A and 7B illustrate that tacrolimus (Tac) and astemizole
(Ast), two compounds screened using the method described herein
that reduce cell surface PrP amounts as shown in FIGS. 4A-4D and
5A-5B, block infection of PK1 neuroblastoma cells by RML and 22 L
prions. PK1 cells were pretreated for 3 days with the indicated
doses of drugs and infected with RML (FIG. 7A) or 22 L (FIG. 7B)
prions using a 10.sup.-4 dilution of brain homogenate from an RML-
or 22 L-infected mouse. Treatment was continued for 12 days after
infection. Cells were analyzed by western blot for proteinase
K-resistant PrP.sup.Sc (a hallmark of prion infection) 9 and 18
days post-infection (i.e. 3 days before and 6 days after treatment
cessation). PPS (pentosan polysulfate), a drug that prevents prion
infection, was used at the dose of 10 .mu.g/ml as positive control
for treatment efficacy. CTRL: untreated cells. Both astemizole and
tracrolimus blocked prion infection, and there was no rebound of
infectivity after treatment cessation.
[0178] These methods to screen for molecules inhibiting cell
surface PrP expression have implications beyond the field of prion
diseases. Indeed, PrP has been shown to mediate A.beta.
oligomer-induced neurotoxicity (Kudo et al., Hum. Mol. Genet.
21(5):1138-1144 (2012)) and memory impairment in transgenic
Alzheimer mice (Lauren et al, Nature 2009; 457(7233):1128-32,
Gimbel et al., J. Neurosci. 30(18):6367-6374 (2010)). Therefore
compounds reducing PrP amounts may prevent A.beta.-induced
neurodegeneration in Alzheimer's disease. FIGS. 8A-8E show that
A.beta. oligomers are toxic for SK-NSH cells, but not for SH-SY5Y,
the SK-NSH-derived cell line that does not express PrP or expresses
undetectable levels thereof.
Example 2
Identification and Quantification of Tau Protein (Tau-FEHTA)
[0179] Another non-limiting example illustrative of the invention
is provided. Here the microtubule-associated protein tau (MAPT, or
tau) is being detected. Tau is a protein expressed primarily, but
not exclusively, in the central and peripheral nervous system.
Under physiological conditions, tau is subject to several
posttranslational modifications including phosphorylation. It is
believed to be a multifunctional protein involved in regulating the
stability of microtubules and modulation of signaling pathways.
Abnormally phosphorylated tau is involved in the pathogenesis of
Alzheimer's disease and other neurodegenerative diseases
collectively termed tauopathies. Hyperphosphorylated tau
dissociates from microtubules and forms aggregates referred to as
neurofibrillary tangles in Alzheimer's disease. Reduction or
deletion of endogenous tau ameliorates synaptic and cognitive
impairments in several mouse models of Alzheimer's disease
(Roberson et al., Science, 2007, 316:750-754; Ittner et al. Cell,
2010, 142:387-397; Roberson et al., J. Neurosci., 2011, 31:700-711)
and rescues neuronal loss in a mouse model of Alzheimer's disease
(Leroy et al., Am. J. Pathol., 2012, 181:1928-1940). Therefore both
reduction of overall tau as well as reduction of
hyperphosphorylated tau would be beneficial for the treatment of
Alzheimer's disease and other tauopathies.
[0180] Quantification of intracellular Tau: SH-SYSY or SK-NSH cells
were added to the wells of a microtiter plate. Compounds of a
screening library were added, or the solvent control (usually DMSO)
and the cells were incubated for 24 hours. Then, cells were lysed,
for example, by the addition of concentrated lysis buffer and the
amount of intracellular tau was detected. In this particular
example, tau was detected using human tau specific antibodies
directed against aa159-163 (0.05 .mu.g/ml) and aa16-46 (1 .mu.g/ml)
labeled with the donor and acceptor fluorophores, respectively.
Antibody incubation time was 60-90 minutes. HTRF.RTM. was used,
with Terbium cryptate as the donor fluorophore and d2 as the
acceptor fluorophore. Blanks consist in culture medium in the
absence of cells. For data analysis, ratios (R) of the 665 nm
(acceptor emission) to the 620 nm (donor emission) measurements
were calculated, to correct for non-specific absorption of 620 nm
light by the assay mix. The value for the specific signal of the
sample or positive control is given by Delta F
%=[(R.sub.C+-R.sub.C-)/R.sub.C-].times.100 where R.sub.C+ and
R.sub.C- are the 665/620 ratios of the positive and negative
control (blank). This ratiometric measurement allows to correct for
fluorescence interference induced by the assay matrix or screening
compounds.
[0181] Tau Knock-down: SH-SY5Y cells were plated at 70-80%
confluence in wells of a E-well microtiter plate. Transfection with
[SMARTpool: ON-TARGETplus MAPT siRNA] as well as [ON-TARGETplus
Non-targeting Pool] as control siRNA (5 .mu.M) was performed
according to the manufacturer's instructions using the transfection
reagent DHARMAFECT 2 (Thermo Fisher Scientific Biosciences Inc, 5
.mu.l per well). Cells were lysed at day 3 post-transfection and
cell lysates plated in 384-well microtiter plates for Tau
quantification.
[0182] Pharmacological control: Staurosporine was used as a control
for the reduction of the amounts of Tau protein produced in the
cell culture. Staurosporine was a kinase inhibitor inducing cell
apoptosis at the concentrations used in FIG. 9C.
[0183] Compound prioritization: To select the best compounds from
the hits generated during the primary screening, the following
strategy can be used: (1). Selection of the compounds exerting the
highest tau reducing effect, confirmed using an orthogonal assay.
(2). Selection of the compounds exerting the tau reducing effect at
the lowest concentration and harboring the least toxicity on
neuronal cells. To this end, EC.sub.50 and TC.sub.50 were
determined. (3). Selection of the compounds exhibiting the highest
specificity for tau compared to other intracellular proteins. After
treatment of the cells with the compound, the extent of reduction
of other proteins was compared with that of tau. (4). Selection of
compounds effective in primary neuronal cell cultures or neurons
derived from differentiation of stem cells or induced pluripotent
stem cells (iPSCs). (5). Medicinal chemistry analysis to select
scaffolds lacking undesirable substructures that could hinder
optimization efforts due to anticipated toxicity and/or potential
for poor drug metabolism and pharmacokinetic (DMPK) properties, in
particular with regards to passing the blood-brain-barrier (6).
Selection of compounds according to their mode of action.
[0184] In particular, a modification of the assay can be performed
to enable specific quantification of hyperphosphorylated tau. In
this case, the assay made use of one or two ligands specific for
the hyperphosphorylated form of the tau protein.
[0185] Although the invention has been illustrated and described
with respect to one or more implementations, equivalent alterations
and modifications will occur to others skilled in the art upon the
reading and understanding of this specification and the annexed
drawings. In addition, while a particular feature of the invention
may have been disclosed with respect to only one of several
implementations, such feature may be combined with one or more
other features of the other implementations as may be desired and
advantageous for any given or particular application.
[0186] The Abstract of the Disclosure is provided to allow the
reader to quickly ascertain the nature of the technical disclosure.
It is submitted with the understanding that it will not be used to
interpret or limit the scope or meaning of the following
claims.
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