U.S. patent application number 10/676351 was filed with the patent office on 2005-03-31 for assay solution compositions and methods for gpcr arrays.
Invention is credited to Fang, Ye, Ferrie, Ann M., Hong, Yulong, Pai, Sadashiva K., Peng, Jinlin, Webb, Brian L..
Application Number | 20050069953 10/676351 |
Document ID | / |
Family ID | 34377368 |
Filed Date | 2005-03-31 |
United States Patent
Application |
20050069953 |
Kind Code |
A1 |
Fang, Ye ; et al. |
March 31, 2005 |
Assay solution compositions and methods for GPCR arrays
Abstract
Buffered assay solutions for performing 1) binding or 2)
functional assays on GPCR arrays, along with methods for their use
are described. The buffered assay solution has an underlying
composition having: a buffer reagent with a pH in the range of
about 6.5 to about 7.9; an inorganic salt of either a monovalent or
divalent species, at a concentration from about 1 mM to about 500
mM; and optionally a combination of: c) a blocker reagent at a
concentration of about 0.01 wt. % to about 2 wt. % of the
composition, or d) protease-inhibitor at a concentration of about
0.001 mM to about 100 mM. In an embodiment for functional assay
uses, the composition is modified to also include a GTP-analogue, a
guanosine 5'-diphosphate (GDP) salt, and/or an anti-oxidant
reagent.
Inventors: |
Fang, Ye; (Painted Post,
NY) ; Ferrie, Ann M.; (Painted Post, NY) ;
Hong, Yulong; (Painted Post, NY) ; Pai, Sadashiva
K.; (Painted Post, NY) ; Peng, Jinlin;
(Painted Post, NY) ; Webb, Brian L.; (Painted
Post, NY) |
Correspondence
Address: |
CORNING INCORPORATED
SP-TI-3-1
CORNING
NY
14831
|
Family ID: |
34377368 |
Appl. No.: |
10/676351 |
Filed: |
September 30, 2003 |
Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
G01N 2333/726 20130101;
G01N 33/52 20130101 |
Class at
Publication: |
435/007.1 |
International
Class: |
G01N 033/53 |
Claims
We claim:
1. A buffered solution for multiplexed binding assays using GPCR
arrays, the solution having a composition comprising: a) a buffer
reagent with a pH in the range of about 6.5 to about 7.9; b) an
inorganic salt of either a monovalent or divalent species, at a
concentration from about 1 mM to about 500 mM; and optionally a
combination of: c) a blocker reagent at a concentration of about
0.01 wt. % to about 2 wt. % of the composition, or d)
protease-inhibitor at a concentration of about 0.001 mM to about
100 mM, or both c) and d).
2. The buffered solution according to claim 1, wherein said pH is
in a range of about 6.8-7.8.
3. The buffered solution according to claim 1, wherein said pH is
about 7.4-7.5.
4. The buffered solution according to claim 1, wherein when said
inorganic salt is a monovalent species, said concentration of said
salt is about 10-500 mM.
5. The buffered solution according to claim 1, wherein when said
inorganic salt is a divalent species, said concentration of said
salt is about 1-50 mM.
6. The buffered solution according to claim 1, wherein said
composition further comprising: a labeled ligand and a target
compound.
7. The buffered solution according to claim 1, wherein said pH
buffer is made from a solution having commonly used pH control
reagents selected from Tris-HCl, HEPES-KOH, TES-NH.sub.4OH, MOPS,
acetate, citrate, citrate-phosphate, sodium-phosphate, maleate, or
succinate buffers.
8. The buffered solution according to claim 1, wherein said
inorganic salt may be selected from NaCl, KCl, CaCl.sub.2,
MgCl.sub.2, MgSO.sub.4, or MnCl.sub.2.
9. The buffered solution according to claim 1, wherein said blocker
reagent is a hydrophilic polymer, a biopolymer, or a water-soluble
protein
10. The buffered solution according to claim 9, wherein said
blockers characterized as a reagent that reduces background signal
and does not interfere with the binding of a target molecule with
the probe receptors within a biological membrane microspot.
11. The buffered solution according to claim 9, wherein said
hydrophilic polymer is dextran, polyvinyl alchol, poly (ethylene
glycol), poly(anetholsulfate), poly(vinyl sulfate), CM-Dextran,
dextran sulfate, beta-cyclodextrin, poly(acrylic acid), poly(sodium
4-styrene sulfonate).
12. The buffered solution according to claim 9, wherein said
biopolymer is poly-glutamate acid, or DNA.
13. The buffered solution according to claim 9, wherein said
water-soluble protein is bovine serum albumin (BSA), casein, dry
milk, or wheat germ agglutinin.
14. The buffered solution according to claim 1, wherein said
solution is protease-free.
15. The buffered solution according to claim 1, wherein said
protease inhibitor may include EDTA, EGTA, phenyl methyl sulforyl
fluoride (PMSF), bacitracin, 4-(2-aminoethyl)benzenesulfonyl
fluoride (AEBSF), 1,10-phenanthroline, E-64, antipain, aprotinin,
benzamidine HCl, bestatin, chymostatin, .epsilon.-aminocaproic
acid, N-ethylmaleimid, leupeptin, pepstatin A, phosphoramidon,
trypsin inhibitor, and any combination of these.
16. A buffered solution for functional assays according to a
GTP-analogue-binding profile approach, the solution having a
composition comprising: a) a buffer reagent with a pH in the range
of about 6.5 to about 7.9; b) a divalent inorganic salt, optionally
together with a monovalent inorganic salt, at a concentration from
about 1 mM to about 500 mM; c) guanosine 5'-diphosphate (GDP) salt
at a concentration of about 0.5 mM to about 50 mM (1-10 mM); and
optionally a combination of: d) a blocker reagent at a
concentration of about 0.01 wt. % to about 2 wt. % of the
composition, e) protease-inhibitor at a concentration of about
0.001 mM to about 100 mM, or f) an anti-oxidant reagent at a
concentration of 0.01 mM to about 100 mM.
17. The solution according to claim 16, wherein said GTP-analogue
includes fluorescein-GTP.gamma.S, Bodipy-fluorescein-GTP.gamma.S,
Bodipy-TMR-GTP.gamma.S, Cy3-GTP.gamma.S, Cy5-GTP.gamma.S, Eu-GTP,
.sup.35S-GTP.gamma.S.
18. The solution according to claim 16, wherein said GDP salt is
selected from a group consisting of: lithium-, sodium-, and
Tris-GDP salts.
19. The solution according to claim 16, wherein said anti-oxidant
reagent includes sodium ascorbate, ascorbic acid, carotenoid
lycopene, .alpha.-tocopherol, .beta.-carotene, sodium azide.
20. The solution according to claim 16, wherein said anti-oxidant
reagent has a concentration in a range of about 0.001 wt. % to
about 0.5 wt. %
21. The solution according to claim 16, wherein said pH is in a
range of about 6.8-7.8.
22. The solution according to claim 18, wherein said pH is about
7.4-7.5.
23. The solution according to claim 16, wherein said pH buffer is
made from a solution having commonly used pH control reagents
selected from Tris-HCl, HEPES-KOH, TES-NH.sub.4OH, MOPS, acetate,
citrate, citrate-phosphate, sodium-phosphate, maleate, or succinate
buffers.
24. The solution according to claim 16, wherein said inorganic salt
may be selected from NaCl, KCl, CaCl.sub.2, MgCl.sub.2, MgSO.sub.4,
or MnCl.sub.2.
25. The solution according to claim 16, wherein said blocker
reagent is a hydrophilic polymer, a biopolymer, or a water-soluble
protein.
26. The solution according to claim 22, wherein said blockers
characterized as a reagent that reduces background signal and does
not interfere with the binding of a target molecule with the probe
receptors within a biological membrane microspot.
27. The solution according to claim 22, wherein said hydrophilic
polymer is dextran, polyvinyl alchol, poly (ethylene glycol),
poly(anetholsulfate), poly(vinyl sulfate), CM-Dextran, dextran
sulfate, beta-cyclodextrin, poly(acrylic acid), poly(sodium
4-styrene sulfonate).
28. The solution according to claim 22, wherein said biopolymer is
poly-glutamate acid, or DNA.
29. The solution according to claim 22, wherein said water-soluble
protein is bovine serum albumin (BSA), casein, dry milk, or wheat
germ agglutinin.
30. The solution according to claim 16, wherein said solution is
protease-free.
31. The solution according to claim 16, wherein said protease
inhibitor may include EDTA, EGTA, phenyl methyl sulforyl fluoride
(PMSF), bacitracin, 4-(2-aminoethyl)benzenesulfonyl fluoride
(AEBSF), 1,10-phenanthroline, E-64, antipain, aprotinin,
benzamidine HCl, bestatin, chymostatin, .epsilon.-aminocaproic
acid, N-ethylmaleimid, leupeptin, pepstatin A, phosphoramidon,
trypsin inhibitor, and any combination of these.
32. A method of reducing background signal due to non-specific
binding of a labeled-ligand or GTP-analogue to a substrate surface,
the method comprising: a) providing a buffered solution containing
a blocker reagent; b) applying said solution to an array of GPCRs;
c) applying a second solution containing a labeled ligand or
GTP-analogue, in either the absence or presence of a target
compound; and d) monitoring or determining the binding of said
labeled ligand to a receptor, or said GTP-analogue to a G-protein
coupled with said receptor in said array.
33. The method according to claim 30, wherein said method further
comprises a washing and dry step before data acquisition.
34. A method of reducing background signal due to non-specific
binding of a labeled-ligand or GTP-analogue to a substrate surface,
the method comprising: a) providing a solution containing a blocker
reagent and a labeled ligand or GTP-analogue, in either the absence
or presence of a target compound; b) applying said solution to a
microarray of GPCRs; and c) monitoring or determining the binding
of said labeled ligand to a receptor, or said GTP-analogue to a
G-protein coupled with said receptor in said microarray.
Description
FIELD OF INVENTION
[0001] The present invention relates to biological assays. In
particular, the invention includes compositions for buffer
solutions used with binding and functional assays for GPCR arrays.
A method is also included which utilizes negatively-charged
polymers and/or water-soluble proteins to reduce the background of
such assays and improve signal to background ratios.
BACKGROUND
[0002] G-protein-coupled receptors (GPCRs) are one of the most
successful target proteins for drug discovery research to date.
Approximately 50% of current drugs target GPCRs; about 20% of the
top 50 best selling drugs target GPCRs; more than $23.5 billion in
pharmaceutical sales annually are ascribed to medications that
address this target class. (Drews, J., "Drug Discovery: A
Historical Perspective" Science 2000, 287, 1960-1963; Ma, P., and
Zemmel, R., "Value of Novelty" Nat. Rev. Drug Discov. 2002, v. 1,
571-572.) Forming a super-family of seven trans-membrane-spanning
proteins that are expressed in virtually all kinds of tissues,
GPCRs are associated with almost every major therapeutic category
or disease class, including pain, asthma, inflammation, obesity,
cancer, as well as cardiovascular, metabolic, gastrointestinal and
central nervous system diseases.
[0003] The tremendous significance of drugs targeting GPCRs lies in
the physiological roles of GPCRs--as cell-surface receptors
responsible for transducing exogenous signals into intracellular
response(s). (Haga, T., and Berstein, G., eds., G-Protein-Coupled
Receptors, CRC Press, Boca Raton, Fla., 1999.) Signaling through
these receptors regulates a wide variety of physiological
processes, such as neurotransmission, chemotaxis, inflammation,
cell proliferation, cardiac and smooth muscle contractility, as
well as visual and chemosensory perception. In addition to the role
normal receptors play in modulating physiological processes, GPCR
mutations that result in both gain and loss of function are
associated with certain human diseases. For example, GPCR
polymorphisms have been linked with hypertension, idiopathic
cardiomyopathy (endothelin A receptor), autosomal dominant
hypocalcemia and familial hypocalciuric hypercalcemia
(calcium-sensing receptor), follicular maturation arrest and
suppression of spermatogenesis (follicle-stimulating hormone
receptor), and bronchodilator desensitization and nocturnal asthma
(.beta.2-adrenoceptors).
[0004] In the human genome there are about 400-700 GPCRs of
therapeutic relevance; of these GPCRs, ligands for about 200 have
been discovered. (Pierce, K. L. et al., "Seven-Transmembrane
Receptors." Nat. Rev. Mol. Cell Biol. 2002, v. 3, 639-650.)
Although there is very little conservation at the amino acid level
among GPCR sequences, all GPCRs share certain structural and
mechanistic features. Typically, GPCRs are formed of seven-helical
trans-membrane-spanning domains (each .about.20-30 amino acids in
length) joined by intra- and extra-cellular loops. The spatial
organization of these trans-membrane regions, the extra-cellular
N-terminus and the extracellular loops, form the binding sites for
extra-cellular ligands. The intracellular loops and
carboxyl-terminus form the sites of interaction with
signal-transducing heterotrimeric G-proteins and other regulatory
proteins, such as receptor kinases and arrestins. A wide variety of
ligand species, including biogenic amines, peptides and proteins,
lipids, nucleotides, excitatory amino acids and ions, small
chemical compounds, etc., can activate GPCRs.
[0005] The success of GPCRs as drug targets stems from the fact
that the binding of natural ligands to their paired GPCR(s) can be
moderated using appropriate small molecule drugs. (Ma, P., and
Zemmel, R., "Value of Novelty," Nat. Rev. Drug Discov. 2002, v. 1,
571-572.) Effective engineering of these drugs is, however,
critical as aberrant binding to such a physiologically significant
target class can lead to serious side effects. Structural data on
GPCRs is limited and rational drug design is a significant
challenge. Designing drugs that do not bind to non-targeted GPCRs
is almost impossible. Currently, selectivity studies are conducted
downstream in the drug discovery process--discarding compounds
because of adverse binding at this stage makes the drug discovery
process both expensive and time consuming. Given these
considerations, and the strong possibility that so-called "orphan"
GPCRs, recently discovered as a result of the sequencing of the
human genome, may be valuable targets (Howard, A. D., et al.,
"Orphan G-Protein-Coupled Receptors and Natural Ligand Discovery."
TiPS 2001, v. 22, 132-140), there is a strong need for technologies
that enable screening against multiple GPCRs simultaneously.
[0006] Given the importance of G-protein-coupled receptors as drug
targets, a wide range of technologies has been developed to screen
compounds against GPCRs. (e.g., Hemmila, I. A., and Hurskainen, P.,
"Novel Detection Strategies for Drug Discovery," Drug Discov. Today
2002, 7, S152-S156.) The increased pace of target identification
(Venter, J. C., et al. "The Sequences of the Human Genome," Science
2001, v. 291, 1304-1351; Hopkins, A. L., and Groom, C. R., "The
Druggable Genome," Nat. Rev. Drug Discov. 2002, v. 1, 727-730) and
the increasing size of compound libraries continues to drive the
development of novel GPCR screening technologies (Schreiber, S. L.,
"Target-Oriented and Diversity-Oriented Organic Synthesis in Drug
Discovery," Science 2000, v. 287, 1964-1968). These assays can be
classified into cell based and GPCR-membrane based assays. Despite
the interest and the overwhelming number of current and future GPCR
targets, few methods have been described for simultaneously
studying multiple GPCRs. Recently, two groups of researchers have
suggested that arrays of transiently transfected cell clusters or
GPCR transfected cells on barcoded substrates could be used for
multiplexed compound screening. (Ziauddin, J., and Sabatini, D. M.,
"Microarrays of Cells Expressing Defined cDNAs," Nature 2001, v.
411, 107-110; Beske, O. E., and Goldbard, S., "High-throughput Cell
Analysis Using Multiplexed Array Technologies," Drug Discov. Today
2002, v. 7, s131-s135.)
[0007] The value of parallel analysis afforded by DNA microarrays
(e.g., Schena, M., et al. "Quantitative Monitoring of Gene
Expression Patterns with a Complementary DNA Microarray," Science
1995, v. 270, 467-470) has inspired the development of protein
arrays (e.g., Mitchell, P., "A Perspective on Protein Microarrays,"
Nat. Biotechnol. 2002, v. 20, 225-229). Beyond the use of protein
abundance profiling as an analogue to gene expression profiling,
protein arrays offer the possibility of highly parallel
investigations of protein-small molecule and protein-protein
interactions. (MacBeath, G., and Schreiber, S. L., "Printing
Proteins as Microarrays for High-throughput Function
Determination," Science 2000, v. 289, 1760-1763; Schweitzer, B., et
al. "Immunoassays with Rolling Circle DNA Amplification: A
Versatile Platform for Ultrasensitive Antigen Detection," Proc.
Natl. Acad. Sci. USA 2000, v. 97, 10113-10119; Fang, Y., et al.
"Membrane Protein Microarrays," J. Am. Chem. Soc. 2002, v. 124,
2394-2395; and Fang, Y., et al. "G-Protein-Coupled Receptor
Microarrays for Drug Discovery," Drug Discov. Today, 2003, v. 18,
755-761.)
[0008] Although the importance of combinatorial approaches to drug
design has been realized, the biological equivalent of
combinatorial chemistry--multi-target screening using protein
microarrays--has not. For multi-target screening, GPCR microarrays
maximize the potential for effective matching of biological target
space to chemical ligand space. Although, protein microarrays are
naturally suited for testing compounds against multiple proteins
simultaneously, some of the fundamental aspects of multiplexed
bioassays using protein chips are yet to be fully demonstrated. One
such fundamental aspect is a need to produce assay conditions,
which can lead to optimal binding profiles of any given target
compounds and/or labeled ligands to the numerous receptors in the
arrays. Problems due to non-specific binding of labeled ligands to
the receptor microspots and the background surface, non-optimal
interaction of target compounds and labeled ligands with the
receptors in the arrays under the assay conditions, and the lack of
general guidelines for assay buffer design and selection have
deterred scientists from testing the feasibility of multiplexed
binding assays for compound profiling and screening.
[0009] For functional assays using GPCR microarrays, one should
also keep in mind other special considerations in order to achieve
not only optimal binding profiling of labeled ligands and target
compounds to the receptors in the arrays, but also to maximize the
assay sensitivity for monitoring the agonist-induced activation of
the receptors and sequential the activation of the G proteins
coupled with the receptors. Hence, guidance for assay buffer design
and methods to reduce the background are needed for realization of
full potentials of GPCR microarrays for compound profiling and
screening using multiplexed binding assays and functional
assays.
SUMMARY OF THE INVENTION
[0010] The present invention provides in part formulations in two
embodiments of a buffered solution which can be applied to 1)
multiplexed binding assays or 2) functional assays. Typically, in a
binding assay, a cocktail of labeled ligands is used to test for or
determine the relative, specific, or selective potency of a target
compound to bind with a receptor probe in a microarray against its
pre-selected labeled-ligand(s). Commonly, each labeled ligand in
the cocktail binds with its corresponding receptor. In comparison,
a functional assay uses a labeled GTP-analogue for examining or
determining physiological functionality of a target compound acting
on a receptor probe in a microarray. The labeled GTP-analogue is
employed as a substitute for the labeled ligands. The functional
assay involves monitoring the "down-stream action" of a probe
molecule; that is, the activation of G-protein coupled with the
receptor. The binding of an agonist to a receptor results in a
conformational change in the receptor, which induces activation of
the G-protein.
[0011] The buffered solution according to a first embodiment is
designed to optimize the binding profiles of target compounds and
labeled ligands with GPCRs in a microarray, in preferably
multiplexed binding assays. Optimization refers to an ability to
derive binding profiles that are either physiologically or
pharmacologically relevant. The solution has a composition
comprising: a) a buffer reagent with a pH in the range of about 6.5
to about 7.9; b) an inorganic salt of either a monovalent or
divalent species, at a concentration from about 1 mM to about 500
mM; and optionally a combination of: c) a blocker reagent at a
concentration of about 0.01 wt. % to about 2 wt. % of the
composition, or d) protease-inhibitor at a concentration of about
0.001 mM to about 100 mM. The solution may further comprise a
labeled ligand and/or a target compound.
[0012] In a second embodiment for functional assays according to a
GTP-analogue-binding profile approach, the buffered solution is
similar to the binding solution, but further includes a
GTP-analogue, a GDP salt, and an anti-oxidant reagent. The solution
has a composition comprising: a) a buffer reagent with a pH in the
range of about 6.5 to about 7.9; b) a divalent inorganic salt,
optionally together with a monovalent inorganic salt, at a
concentration from about 1 mM to about 500 mM; c) GDP salt at a
concentration of about 0.5 mM to about 50 mM (preferably 1-10 mM);
and optionally a combination of: d) a blocker reagent at a
concentration of about 0.01 wt. % to about 2 wt. % of the
composition, e) protease-inhibitor at a concentration of about
0.001 mM to about 100 mM, or f) an anti-oxidant reagent at a
concentration of 0.01 mM to about 100 mM.
[0013] According to another aspect of the present invention, a
method for reducing background signal due to non-specific binding
of either a labeled-ligand or GTP-analogue to a substrate surface
is described. In one embodiment the method comprises: a) providing
a buffered solution containing a blocker reagent; b) applying the
solution to an array of GPCRs; c) applying a second solution
containing a labeled ligand or GTP-analogue, in either the absence
or presence of a target compound; and d) monitoring or determining
the binding of the labeled ligand to a receptor, or the
GTP-analogue to a G-protein coupled with the receptor in the array.
The method may further involve a washing and dry step before data
acquisition. Alternatively, the method may comprise: a) providing a
solution containing a blocker reagent and a labeled ligand or
GTP-analogue, in either the absence or presence of a target
compound; b) applying the solution to a microarray of GPCRs; and c)
monitoring or determining the binding of the labeled ligand to a
receptor, or the GTP-analogue to a G-protein coupled with the
receptor in the microarray.
[0014] Additional features and advantages of the present invention
will be revealed in the following detailed description. Both the
foregoing summary and the following detailed description and
examples are merely representative of the invention, and are
intended to provide an overview for understanding the invention as
claimed.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIGS. 1A & 1B are false color fluorescent images of
arrays having two replicate microspots of neurotensin receptor
Subtype-1 (NTR1), after binding with 2 nM Bodipy-TMR-neurotensin
2-13 in a binding buffer, respectively, in the presence or absence
of 100 mM NaCl.
[0016] FIG. 1C is a graph summarizing the effect of the presence or
absence of NaCl on the total binding signal of NTR1 microarrays in
FIGS. 1A and 1B.
[0017] FIGS. 2A & 2B are false-color images in terms of Cy3/Cy5
ratio of two microarrays, each having six different GPCR-membrane
preparations. The images are taken after a buffered solution for
binding assays was reacted with the arrays.
[0018] FIGS. 2C & 2D, respectively, are graphs of the percent
of total detected signal in the presence and absence of 1 .mu.M
telenzepine.
[0019] FIGS. 3A & 3B are false-color images of an array having
in four columns: NTR1, .mu.-opioid receptor, motilin receptor
(MR1), and CHO control membranes (left to right). The images are
taken after the array is incubated with a functional assay buffer
solution containing 2 nM of .sup.35S-GTP.gamma.S, in the presence
(FIG. 3A) and absence (FIG. 3B) of 100 nM dynorphin A and 100 nM
nurotensin. Dynorphin A is an agonist to mu opioid receptor;
neurotensin is an agonist to neurotensin receptor subtype 1 (NTR1)
receptor.
[0020] FIGS. 3C & 3D, respectively, are graphs of the total
counts of receptor microspots in FIGS. 3A and 3B, indicating the
respective activated and basal counts, respectively, of total
detected signal in the presence and absence of the agonist.
[0021] FIG. 4 represents the chemical structure of Cy5-GTP.gamma.S,
as synthesized for the invention.
[0022] FIG. 5 are fluorescence images of .beta.1-adrengeric
receptor arrays on a GAPS-coated surface after binding with
Cy5-GTP.gamma.S in the absence or presence of three different
blocking reagents: (A) 0.5% BSA; (B) 0.1% poly-glutamate; (C) 0.5%
poly-anetholsulfate. The concentration of Cy5-GTP.gamma.S is 20
nM.
DETAILED DESCRIPTION OF THE INVENTION
Section I--Definitions
[0023] Before describing the present invention in detail, this
invention is not necessarily limited to specific compositions,
reagents, process steps, or equipment, as such may vary. As used in
this specification and the appended claims, the singular forms "a,"
"an," and "the" include plural referents unless the context clearly
dictates otherwise. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting. All technical
and scientific terms used herein have the usual meaning
conventionally understood by persons skilled in the art to which
this invention pertains, unless context defines otherwise.
[0024] The term "ligand" refers to a chemical molecule or
biological molecule that can bind readily to a receptor with a
specific binding affinity constant.
[0025] The term "labeled-ligand" refers to either a fluorescently
labeled or radioactive isotope-labeled or hapten-labeled
(e.g.,biotin) or gold-nano-particle labeled ligand.
[0026] The term "cocktail" refers to a medium (e.g., buffered or
aqueous solution) having a mixture either of different labeled
ligands or of different compounds. Alternatively, in some
embodiments, a mixture of both ligands and compounds may be present
together in solution.
[0027] The term "compound," "target," or "target compound" as used
herein refers to a biological molecule, biochemical or chemical
entity, molecule, or pharmaceutical drug candidate to be
detected.
[0028] The term "biological molecule" or "biomolecule" refers to
any kind of biological entity, including, such as, modified and
unmodified nucleotides, nucleosides, peptides, polypeptides,
proteins, lipids, or saccharides.
[0029] The term "cognate," "corresponding," or "paired" refers to
the reciprocal moiety of a molecule to another; in particular, a
ligand that can bind specifically to a given receptor is called a
ligand-receptor pair.
[0030] The term "biospot" or "microspot" refers to a discrete or
defined area, locus, or spot on the surface of a substrate,
containing a biological or chemical probe.
[0031] The term "GPCR" refers to a guanine nucleotide-binding
protein-coupled receptor. The GPCR can have either a natural or
modified sequence.
[0032] The term "GPCR membrane" or "GPCR membrane fragment" refers
to a biological membrane or cell membrane fragment having a GPCR
embedded within a membrane layer, or a micelle having a GPCR
reconstituted within the micelle.
[0033] The term "GPCR microspot" refers to a microspot containing a
deposit of G-protein coupled receptors (GPCRs). The corresponding
microspots are referred to as "probe microspots," and these
microspots are arranged in a spatially addressable manner to form a
microarray.
[0034] The term "GTP-analogue" refers to a GTP molecule that is
modified with a label moiety, either for example fluorescent dye,
radioactive isotope, or Eu-chelates.
[0035] The term "probe" or "receptor probe" refers to a receptor
molecule (e.g., GPCR), which according to the nomenclature
recommended by B. Phimister (Nature Genetics 1999, 21 supplement,
pp. 1-60.), is immobilized to a substrate surface. Preferably,
probes are arranged in a spatially addressable manner to form an
array of microspots. When the array is exposed to a sample of
interest, molecules in the sample selectively and specifically
binds to their binding partners (i.e., probes). The binding of a
"target" to the probes occurs to an extent determined by the
concentration of that "target" molecule and its affinity for a
particular probe.
[0036] The term "substrate" or "substrate surface" as used herein
refers to a solid or semi-solid, or porous material (e.g., micro-
or nano-scale pores), which can form a stable support. The
substrate surface can be selected from a variety of materials.
[0037] The term "functionalization" as used herein relates to
modification of a solid substrate to provide a plurality of
functional groups on the substrate surface. The phrase
"functionalized surface" as used herein refers to a substrate
surface that has been modified to have a plurality of functional
groups present thereon. The surface may have an amine-presenting
functionality (e.g., .gamma.-amino-propylsilane (GAPS) coating), or
may be coated with amine presenting polymers such as chitosan and
poly(ethyleneimine).
Section II--Description
[0038] Previously, we demonstrated that one may fabricate GPCR
microarrays using conventional robotic pin printing technologies
and cell membrane preparations containing GPCRs from a cell line
over-expressing the receptor. (U.S. patent application Ser. Nos.
09/974,415 (U.S. Patent Publication No. 2002/0019015 A1), and
09/854,786, (U.S. Patent Publication No. 2002/0094544 A1) the
contents of which are incorporated herein by reference). Also, we
have described certain methods for fabricating biological membrane
microarrays on substrates presenting certain surface chemistries.
(U.S. patent application Ser. No. 10/300954, incorporated herein by
reference.) These kinds of arrays can be prepared under ambient
conditions, stored at about 4.degree. C., and still retain their
functionality for an extended period of time thereafter. In
addition, we have described the use of GPCR micrarrays for
compound-profiling and screening applications using multiplexed
binding and functional assays. (See, U.S. patent application Ser.
No. 10/639,718, the content of which is incorporated herein by
reference.)
[0039] The interaction of GPCRs with ligands, or target compounds,
is relatively complicated. This fact is due in part to the
diversity of compounds and ligands, and the number of different
functional roles (i.e., agonist, partial agonist, inverse agonist,
antagonist) that different target compounds may play when acting on
a single receptor. During the development of a superior assay
technology, several parameters should be kept in mind. For
instance, to achieve physiologically and pharmacologically relevant
binding profiles of target compounds and labeled ligands to the
receptor, a buffer composition should be optimized according to the
specific nature of receptor probe species in the array and/or
characteristics of target compounds. Also, a buffer composition
should maintain reasonable mechanical stability of receptor
microspots, in which the receptor membranes are generally
immobilized non-covalently on a substrate. Moreover, since
different types of assay format each may require the inclusion of a
different combination or varied set of reagents, a single, basic,
universal buffer composition for both binding and functional assay
is difficult to achieve.
[0040] Having these parameters in mind, the present invention
provides buffered solution compositions for two major classes of
assays that employ GPCR microarrays. These assay formats are
multiplexed binding assays and functional assays. For binding
assays, the present buffered solution has a composition that not
only maximizes the efficiency and specificity of binding reaction
of labeled ligands or target compounds with the receptors in the
arrays, but also can result in physiologically and
pharmacologically relevant binding profiles with improved signal to
background ratio. The buffered solution and associated methods of
use can reduce the overall background signal that is mainly due to
non-specific binding of labeled ligands to an array substrate
surface.
[0041] One the other hand, for functional assays which are based on
monitoring the activation of G protein coupled with the receptor,
the present buffered solution not only provides optimal binding of
target compounds to the receptors in the array, but also maximizes
an increased percentage of GTP-analogue binding to heterotrimeric
G-proteins coupled with receptors in the presence of an agonist
compound. The increased binding of GTP-analogue is due to the
stimulation or promotion of GTP analogue to the G proteins by
agonist-induced receptor activation and sequential G-protein
activation. At the same time, the present invention and method of
use could give rise to reduced background signal and improved assay
sensitivity.
[0042] Furthermore, to achieve superior assay performance using
GPCR arrays and to reduce non-specific binding of labeled ligands
or GTP-analogues to a background surface, we propose a method which
incorporates a blocker reagent in the assay buffer solution
composition.
[0043] A. Binding Assay Solution
[0044] As touched upon in the foregoing, the interaction of GPCRs
with ligands and target compounds is rather complicated;
interactions depend mostly on the specific nature of receptors, the
ligands and target compounds, as well as the buffer
compositions.
[0045] Although GPCRs are generally widely distributed in numerous
tissues, some GPCRs are preferably and highly distributed in
certain types of tissues. For example, some receptors, including
the muscarinic acetylcholine receptor, dopamine 2 receptor,
histamine 2 receptor, serotonin 4 receptor, and prostaglandin
receptor, are prominently distributed in the gastrointestinal
system. Some other receptors, including serotonin 1A/1D and 2A/2C
receptor, neurotensin 1 and 2 receptors, opioid receptors (mu,
delta, kappa, ORL-1), and dopamine 2/3 receptors, are prominently
distributed in the central nervous system (Stadel, J. M., et al.
TIPS 1997, v. 18, 430-437). The nature and compositions of
bio-fluids could differ significantly from one tissue system to
another. Therefore, it follows that the physiological binding
conditions for nature ligands to their paired receptors could also
differ significantly.
[0046] On the other hand, although GPCRs share some characteristic
motifs, GPCRs differs in sequences and structure. For example, in
the trans-membrane (TM) II an Asp residue is conserved in most
GPCRs (e.g., neurotensin receptor subtype 1; NTR1), but not certain
other GPCRs (e.g., neurotensin receptor subtype 2; NTR2). Natural
agonists bind to receptors with the TM II Asp residue more
sensitively to sodium ions in an assay buffer than those do to
receptors absent the TM II Asp residue. (Martin, S. et al.,
"Pivotal Role of an Aspartate Residue in Sodium Sensitivity and
Coupling to G-Proteins of Neurotensin Receptors," Molecular
Pharmacology, 1999, 55, 210-215). The presence of sodium ions, at a
concentration as low as about 20 mM, inhibits the binding of
radio-labeled neurotensin to NTR1 by at least 50% (IC.sub.50 of 18
mM). In contrast, one observes a very weak effect of sodium ions on
the binding of radio-labeled neurotensin to mouse NTR2 (IC.sub.50
of 225 mM). Other receptors also show similar allosteric modulation
of ligand-receptor binding by monovalent cations include
.alpha..sub.2A and .alpha..sub.2B adrenergic receptors, .mu. opioid
receptor, D.sub.2 dopamine receptor, SST2 somatostatin receptor,
.beta..sub.2 adrenergic receptor, and adenosine receptors (Ceresa,
B. P., & Limbird, L. E., "Mutation of an Aspartate Residue
Highly Conserved Among G-Protein-Coupled Receptors Results in
Non-Reciprocal Disruption of .alpha..sub.2-Adrenergic
Receptor-G-Protein Interactions," J. Biol. Chem., 1994, 269,
29577-29564). Monovalent cations
(Na.sup.+.gtoreq.Li.sup.+>>K.sup.+) have reciprocal effects
on agonist versus antagonists interactions at the receptor.
[0047] The effect of NaCl on the binding of natural agonists to
receptors in a microarray also has been observed as presented in
FIG. 1. False color images in FIGS. 1A and 1B show the binding of
labeled-agonist to NTR1 receptors. The graph of FIG. 1C signifies
that NaCl strongly influences the binding of BT-NT to NTR1.
[0048] Ligands for GPCRs are very diverse, including biogenic
amines, peptides and proteins, lipids, nucleotides, excitatory
amino acids and ions, small chemical compounds, etc. (Morris, A.
J., and Malbon, C. C., "Physiological Regulation of
G-Protein-linked Signaling," Physiol. Rev., 1999, 79, 1373-1430.)
Generally, a particular GPCR could couple to one or more trimeric G
proteins in a particular cell line. The binding affinities of
agonists to a GPCR depend on the coupling state of the receptor
with its G proteins. Compounds that bind with a receptor might have
different functionalities, such as agonism, antagonism,
super-agonism or inverse agonism. The binding sites involved might
be different for different compounds binding to the same
receptor.
[0049] In sum, buffer compositions for binding assays could not
only affect the interaction of receptors with ligands or target
compounds, but also the functionality of the membrane proteins. For
example, some GPCR-ligand interactions depend strongly on the
presence of particular divalent cations such as Mg.sup.2 or
Mn.sup.2+. In addition, the buffer composition also may have a
negative impact on the physical stability and packing of
receptor-containing lipid membranes that are immobilized on the
substrate surfaces non-covalently, therefore decreasing array
performance and assay robustness.
[0050] In solving the issues associated with achieving
physiologically and pharmacologically relevant binding profiles for
target compounds and labeled ligands to the receptor, an ideal
buffer composition should meet at least following criteria: (1) the
binding profiling (affinity, specificity, selectivity) of labeled
ligands and target compounds to their paired receptors should be
physiologically and pharmacologically relevant; (2) the microarrays
should be stable through the bioassays under assay buffer
conditions; (3) the background signals should be minimal.
[0051] According to the present invention, a buffered solution has
a composition comprising: a) a buffer reagent with a pH in the
range of about 6.5 to about 7.9; b) an inorganic salt of either a
monovalent or divalent species, at a concentration from about 1 mM
to about 500 mM; and optionally a combination of: c) a blocker
reagent at a concentration of about 0.01 wt. % to about 2 wt. % of
the composition, or d) protease-inhibitor at a concentration of
about 0.001 mM to about 100 mM.
[0052] Preferably, the pH value is in a range of about 6.8-7.8, and
more preferably about 7.4-7.5. The pH buffer is made from a
solution having commonly used pH control reagents selected from
Tris-HCl, HEPES-KOH, TES-NH.sub.4OH, MOPS, acetate, citrate,
citrate-phosphate, sodium-phosphate, maleate, or succinate buffers.
Preferably, the reagents are Tris-HCl, HEPES-KOH, MOPS, or
sodium-phosphate. The pH buffer reagent is preferably at a
concentration of about 1-200 mM, or more preferably about 10-50
mM.
[0053] The inorganic salt may be either a monovalent or a divalent
species, or a combination of the two, each at a concentration of
about 1-100 mM, preferably about 1-50 mM. The inorganic salt may be
selected from NaCl, KCl, CaCl.sub.2, MgCl.sub.2, MgSO.sub.4, or
MnCl.sub.2.
[0054] The composition may further include a labeled ligand and/or
a target compound.
[0055] Generally, the blocker reagent is characterized as a reagent
that reduces background signal and does not interfere with the
binding of a target molecule with the probe receptors within a
biological membrane microspot. The blocker reagent can be either a
hydrophilic polymer, a biopolymer, or a water-soluble protein. A
hydrophilic polymer can be: dextran, polyvinyl alchol, poly
(ethylene glycol), poly(anetholsulfate), poly(vinyl sulfate),
CM-Dextran, dextran sulfate, beta-cyclodextrin, poly(acrylic acid),
poly(sodium 4-styrene sulfonate). A biopolymer can be a
poly-glutamate acid, or DNA, and a water-soluble protein can be
albumin (e.g., bovine serum albumin (BSA)), casein, dry milk, or
wheat germ agglutinin. The preferred blocker reagent is a water
soluble protein.
[0056] The buffered assay solution preferably is protease-free.
Protease inhibitors, however, can be included. Examples of protease
inhibitors may include ethylene diamine-tetra-acetic acid (EDTA),
ethylene bis-(oxyethylenenitrilo)-tetra-acetic acid (EGTA), phenyl
methyl sulforyl fluoride (PMSF), bacitracin,
4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), aprotinin,
1,10-phenanthroline, E-64, antipain, aprotinin, benzamidine HCl,
bestatin, chymostatin, .epsilon.-aminocaproic acid,
N-ethylmaleimid, leupeptin, pepstatin A, phosphoramidon, trypsin
inhibitor, and any combination of these. Preferably, EDTA or EGTA
is always included in the composition of the buffered assay
solution to reduce damage from free-radicals and protease-induced
degradation of receptors.
[0057] A single buffered binding solution composition, according to
the invention--including for example, 50 mM Tris-HCl, pH 7.4, 10 mM
MgCl.sub.2, 1 mM EDTA, 0.1 wt. % BSA--is used for multiplexed
competitive binding assay. The results are presented in FIG. 2.
FIGS. 2A and 2B show false-color fluorescence images in Cy3/Cy5
ratio of two membrane microarrays of six different GPCR-membrane
preparations (from left to right: HEK cell membrane as control,
muscarinic receptor subtype 1 (M1), motilin receptor (MOTR),
neurotensin receptor subtype 1 (NTR1), opioid receptor like subtype
1 (ORL1) and delta 2 opioid receptor (OP1)). Each microarray is
incubated with a buffered solution containing a mixture or cocktail
of labeled-ligands in the presence or absence of 1000 nM of
motilin. The labeled-ligands are 2 nM Cy3B-telenezepine (from
Amersham), 4 nM BT-motilin 1-16, 4 nM Cy5-neurotensin 2-13, 2 nM
Cy5-nociceptin 1-13, and 4 nM Cy5-naltrexone. Graphs of FIGS. 2C
and 2D signify the specific and selective inhibition of the binding
of labeled ligands to M1 receptors by unlabeled telenzepine, as
evidenced in the relative total signal percentage in the presence
(C) and absence (D) of 1 .mu.M telenzepine in column 2. Telenzepine
is an antagonist for M1 receptor alone. Samples 1A and 1B, in the
graphs, show the HEK control signals in Cy3 and Cy5,
respectively.
[0058] B. Functional Assay Solution
[0059] Functional assays afford several advantages over binding
assays. For instance, functional assay use a single labeled
biological molecule, e.g., GTP-analogue, to study, screen or
profile target compounds, instead of a mixed solution of
labeled-ligands for multiplexed binding assays. Further, functional
assays can provide more information (i.e., biological functionality
beyond mere binding profiles) about the interaction of a target
compound with its receptor(s).
[0060] Upon agonist binding to a receptor, the GPCR undergoes
conformational changes that prompt GTP binding to receptor-coupled
G.sub..alpha. protein. This property gives rise to one of the most
useful measures of GPCR activation of G protein. To date, a couple
of analogues have been used for both heterogeneous- and
homogeneous-format functional assays. These analogues include
radioactive .sup.35S-GTP.gamma.S and Eu-GTP.
[0061] For functional assay applications that employ a GTP-analogue
binding format, the present invention offers a buffered assay
solution that can improve assay performance and sensitivity. The
composition of the functional assay solution is similar to that of
the binding assay solution, but not identical because a functional
assay should not only offer physiological relevant binding profiles
of target compounds, but also should maximize the activation
signals, which is manifested as a binding of the GTP-analogue with
the G-proteins coupled with receptors.
[0062] The buffered assay solution has a composition comprising: a)
a buffer reagent with a pH in the range of about 6.5 to about 7.9;
b) a divalent inorganic salt, optionally together with a monovalent
inorganic salt, at a concentration from about 1 mM to about 500 mM;
c) guanosine 5'-diphosphate (GDP) salt at a concentration of about
0.5 mM to about 50 mM (1-10 mM); and optionally a combination of:
d) a blocker reagent at a concentration of about 0.01 or 0.1 wt. %
to about 2 wt. % of the composition, e) protease-inhibitor at a
concentration of about 0.001 mM to about 100 mM, or f) an
anti-oxidant reagent at a concentration of 0.01 mM to about 100 mM.
Although most components and the pH values of the functional assay
solution are similar, the major distinctions between the binding
assay and functional assay solutions involve the inclusion of a
GTP-analogue, a GDP salt, an anti-oxidant reagent, and varied
inorganic salt species.
[0063] The GTP-analogue may include: fluorescein-GTP.gamma.S,
Bodipy-fluorescein-GTP.gamma.S, Bodipy-TMR-GTP.gamma.S,
Cy3-GTP.gamma.S, Cy5-GTP.gamma.S, Eu-GTP, .sup.35S-GTP.gamma.S.
FIG. 3 presents the structure of Cy5-GTP.gamma.S, as synthesized
according to the invention. In the synthesis, for example, a
GTP.gamma.S tetralithium salt (Calbiochem) is dissolved in about 20
mM sodium bicarbonate at a pH.about.9 to a concentration of about
15 mM. A Cy5 maleimide monoreactive dye (Amersham) is dissolved in
DMF to a concentration of about 15 mM. Both solutions are flushed
with nitrogen, and mixed together. After a certain amount of time
(e.g., 0.5 to 24 hrs), the resulting solution is loaded into a
reverse phase high-performance-liquid- -chromatography (HPLC). One
major product is collected and examined by UV spectrometry,
mass-spectrometry, and NMR, which confirmed the structure as shown
in FIG. 4.
[0064] The GDP salt may be selected from a group consisting of:
lithium-, sodium-, and Tris-GDP salts. The anti-oxidant reagent may
include sodium ascorbate, ascorbic acid, carotenoid lycopene,
.alpha.-tocopherol, .beta.-carotene, sodium azide, at a
concentration in a range of about 0.001 wt. % to about 0.5 wt.
%.
[0065] FIG. 3 illustrates agonist-prompted binding of
.sup.35S-GTP.gamma.S to GPCR microarrays using a buffered solution
according to the present invention. Microarrays are incubated with
at in a functional buffer containing 2 nM .sup.35S-GTP.gamma.S in
the absence and presence of a cocktail agonists (100 nM neurotensin
and 100 nM dynorphin A). The buffered solution comprises 75 mM
Tris-HCl, pH 7.4, 1 mM EDTA, 100 mM NaCl, 10 mM MgCl.sub.2, and 3
.mu.M GDP. Under the optimized buffer conditions, both the
activation of agonist-induced receptors (NTR1 and mu, but not CHO
control and MOTR in the same arrays) and the sequential activation
of G proteins coupled with the receptors can be observed
simultaneously, as evidenced by the increased signals due to the
binding of .sup.35S-GTP.gamma.S.
[0066] An example of the effect of blocker reagents on binding of
labeled-ligands or GTP-analogues is shown in FIG. 5. Blocker
reagents in columns A-C are: A=0.5% BSA; B=0.1% poly-glutamate;
C=0.5% poly-anetholsulfate. Data, derived from fluorescence images
of .beta.1 arrays after bioassays in Cy5 channel, suggest that: (1)
there is a negligible auto-fluorescence of membrane microspots; (2)
high fluorescence background of membrane arrays is present after
the binding of Cy5-GTP.gamma.S in the absence of a blocking
reagent--partially due to the non-specific binding of the probe to
GAPS; and (3) presence of BSA in binding solution reduces the
fluorescence background by about 50%, while the presence of the
other two blockers can greatly reduces the fluorescence background
from non-specific binding by about 70-90%, without detrimentally
effecting the total signal of fluorescence of the membrane
microspots.
[0067] C. Methods
[0068] Fluorescence-based detection schemes for microarrays have
certain advantages and disadvantages. An advantage is that a
fluorescence technology is highly sensitive relative to other
methods. A disadvantage is that proteins, especially biological
membrane preparations, have relatively high intrinsic
auto-fluorescence, particularly in low (.about.400-590 nm)
wavelength channels (e.g., FITC and Cy-3). An approach to overcome
this problem is to use a fluorescence dye having a longer
wavelength emission, such as Cy-5. Use of a probe labeled with a
dye of long wavelength emission would allow one to avoid the
auto-fluorescence of membranes in the microspots as well as the
surface.
[0069] The non-specific binding of labeled targets to background,
however, raises significant concerns for microarray technologies.
For example, we have observed high fluorescence background when
Bodipy-FL-GTP.gamma.S is used as a probe for GPCR functional
assays. The background is mainly due to the non-specific binding of
highly negatively charged Bodipy-FL-GTP.gamma.S to the positively
charged GAPS surface. Reagents that can significantly reduce the
background due to the non-specific binding of the probe to the
surface would be extremely helpful.
[0070] Hence, another approach to improve assay performance by
means of reducing background involves using blocker reagents.
According to the present invention, we provide a method of reducing
background signal due to non-specific binding of a labeled-ligand
or GTP-analogue to a substrate surface. The method comprises: a)
providing a buffered solution containing a blocker reagent; b)
applying said solution to an array of GPCRs; c) applying a second
solution containing a labeled ligand or GTP-analogue, in either the
absence or presence of a target compound; and d) monitoring or
determining the binding of said labeled ligand to a receptor, or
said GTP-analogue to a G-protein coupled with said receptor in said
array. The method may further comprise a washing and dry step
before data acquisition. In an alternative simplified embodiment,
one may providing a solution containing a blocker reagent and a
labeled ligand or GTP-analogue, in either the absence or presence
of a target compound; applying said solution to a microarray of
GPCRs; and monitoring or determining the binding.
[0071] D. Empirical Examples
[0072] 1. Membrane Preparations
[0073] After obtaining commercial membrane fractions containing
human neurotensin receptor subtype 1 (NTR1), .beta.1 adrenergic
receptor, opioid-like receptor subtype 1 (ORL1), motilin receptor
(MOTR) CHO cell membranes, HEK cell membranes (Perkin Elmer Life
Sciences (Boston, Mass.)), and delta-2 opioid receptor (OP1)
(Euroscreen (Gosselies, Belgium)), these membrane suspensions were
either directly used or reformulated in buffer according to the
formulation in co-assigned U.S. patent application Ser. No.
10/651,554.
[0074] 2. Synthesis and Characterization of Fluorescently Labeled
Ligands
[0075] Neurotensin (2-13), nociceptin (1-13), naltrexone and
motilin (1-16) were obtained commercially from Sigma (St. Louis,
Mo.), while solutions of the peptides in bicarbonate or phosphate
buffer were treated with solutions of N-hydroxysuccinimidyl (NHS)
derivatives of the fluorescent dyes in DMSO to synthesize
Cy5-nociceptin (1-13), Bodipy-TMR-motilin (1-16) and
Cy5-neurotensin (2-13). Cy5-naltrexone is synthesized following the
method developed by Luke et al. (Luke, M. C., Hahn, E. F., Price,
M. & Pasternak, G. W. Irreversible opioid agonists and
antagoinsts: V. hydrazone and acylhydrazone derivatives of
naltrexone. Life Sciences 1998, 43, 1249-1256). The labeled
compounds are purified by means of reverse phase HPLC (Alliance
System 2690 and Nova-Pak C.sub.18 column, Waters Inc, Milford,
Mass.), characterized by mass spectroscopy (IonSpec HiRes MALDI
FT-mass spectrometer, IonSpec, Lake Forest, Calif.), and tested for
specificity using GPCR microarrays (s) and ligand-binding assays on
filter plates. MALDI-MS (m/z) (M+H): 2201 for Cy5-NT (2-13), 994
for Cy5-naltrexone, 2480 for Bodipy-TMR-motilin (1-16), and 2020
for Cy5-nociceptin (1-13).
[0076] 3. Microarray Fabrication
[0077] Using a quill-pin printer (Cartesian Technologies, Model PS
5000) equipped with software for programmable aspiration and
dispensing, one can fabricate GPCR microarrays. For printing, 5-7
.mu.l of each GPCR suspension is added to different wells of a 384
well microplate. Replicate microspots were obtained using a single
insertion of the pin into the solution. To prevent contamination
due to carry-over between different GPCR suspensions, an automatic
wash and dry cycle was incorporated. After printing, the arrays
were incubated in a humid chamber at room temperature for one hour,
and then used for ligand binding experiments.
[0078] 4. Binding Assays
[0079] For the binding assays, each individual array was incubated
with 10 .mu.l of a solution containing labeled ligand(s) at a
particular concentration in the absence and presence of varying
amounts (0-1000 nM) of unlabeled compounds. The binding buffer used
for all experiments was Tris-HCl (50 mM, pH 7.4) containing 10 mM
MgCl.sub.2, 0.1% BSA and 1 mM EDTA.
[0080] 5. Functional Assays
[0081] For the functional assays, each individual array was
incubated with 15 .mu.l of a solution containing
.sup.35S-GTP.gamma.S at a particular concentration in the absence
and presence of 100 nM neurotensin and 100 nM dynorphin A. The
functional assay buffer used for all experiments was 75 mM
Tris-HCl, 7.4 containing 10 mM MgCl.sub.2, 100 mM NaCl, 0.1% BSA, 3
.mu.M GDP, and 1 mM EDTA.
[0082] 6. Fluorescence Measurements
[0083] After executing the binding assays, the microarrays are
washed and dried, and then examined using Genepix scanner 4000
(Axon Instruments, Union City, Calif.). Using Genepix software,
resulting data is analyzed. Each data point in the plots represents
the average of at least three replicate micospots. Using non-linear
regression analysis (Graphpad PRISM, Graphpad Software Inc., San
Diego, Calif.), one can estimate K.sub.d and IC.sub.50.
[0084] 7. Radioactivity Measurements
[0085] After executing functional assays, the microarrays are
washed and dried, and then exposed overnight to a screen specific
for .sup.35S isotope. Afterwards, the screen is imaged using
Typhoon 9400 (Amersham Biosciences). Data analysis is carried out
using Genepix software, wherein each data point in the plots
represents the average of at least three replicate micospots.
[0086] The present invention has been described both in general and
in detail by way of examples. Persons skilled in the art will
understand that the invention is not limited necessarily to the
specific embodiments disclosed. Modifications and variations may be
made without departing from the scope of the invention as defined
by the following claims or their equivalents, including equivalent
components presently known, or to be developed, which may be used
within the scope of the present invention. Hence, unless changes
otherwise depart from the scope of the invention, the changes
should be construed as being included herein.
* * * * *