U.S. patent application number 11/540733 was filed with the patent office on 2007-04-05 for method for identifying modulators of g protein coupled receptor signaling.
This patent application is currently assigned to cue BIOtech, Inc.. Invention is credited to Annette Gilchrist, Heidi M. Hamm.
Application Number | 20070077597 11/540733 |
Document ID | / |
Family ID | 33298332 |
Filed Date | 2007-04-05 |
United States Patent
Application |
20070077597 |
Kind Code |
A1 |
Gilchrist; Annette ; et
al. |
April 5, 2007 |
Method for identifying modulators of G protein coupled receptor
signaling
Abstract
This invention relates to methods for identifying peptides and
other compounds which block or enhance G protein coupled receptor
mediated signaling with high affinity and specificity and/or which
stabilize a particular conformer of a G protein coupled receptor.
Assays, methods of treatment and other methods developed in
conjunction with these methods also are disclosed.
Inventors: |
Gilchrist; Annette;
(Barrington, IL) ; Hamm; Heidi M.; (Nashville,
TN) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
cue BIOtech, Inc.
Madison
WI
|
Family ID: |
33298332 |
Appl. No.: |
11/540733 |
Filed: |
October 2, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10411336 |
Apr 11, 2003 |
|
|
|
11540733 |
Oct 2, 2006 |
|
|
|
09852910 |
May 11, 2001 |
|
|
|
10411336 |
Apr 11, 2003 |
|
|
|
60275472 |
Mar 14, 2001 |
|
|
|
Current U.S.
Class: |
435/7.1 ; 506/18;
506/9 |
Current CPC
Class: |
A61P 19/04 20180101;
G01N 33/566 20130101; G01N 2500/02 20130101; A61P 9/04 20180101;
A61P 25/14 20180101; A61P 35/00 20180101; A61P 25/30 20180101; A61P
31/12 20180101; A61P 3/00 20180101; G01N 2500/04 20130101; A61P
25/28 20180101; C07K 1/047 20130101; A61P 25/16 20180101; A61P 1/00
20180101; A61P 13/02 20180101; G01N 33/6845 20130101; A61P 15/08
20180101; A61P 19/10 20180101; A61P 25/18 20180101; C07K 14/4722
20130101; A61P 9/12 20180101; A61P 43/00 20180101; A61P 13/08
20180101; A61P 9/10 20180101; A61P 25/08 20180101; A61P 25/22
20180101; A61P 33/00 20180101; A61P 25/24 20180101; A61P 9/00
20180101; A61P 31/04 20180101; G01N 33/74 20130101; G01N 2333/4719
20130101; A61P 25/34 20180101; A61P 37/08 20180101; A61P 11/06
20180101; G01N 2333/726 20130101; A61P 31/10 20180101; G16B 15/00
20190201; A61P 29/00 20180101; A61P 1/04 20180101; A61P 3/04
20180101; A61P 25/20 20180101; A61P 31/18 20180101 |
Class at
Publication: |
435/007.1 |
International
Class: |
C40B 30/06 20060101
C40B030/06; C40B 40/10 20060101 C40B040/10 |
Claims
1. A two-screen method of detecting high-affinity G protein coupled
receptor (GPCR) G protein interaction site binding compounds, which
comprises: (a) providing a peptide library the members of which are
based on the primary sequence of a native G protein G.alpha.
subunit carboxyl terminal peptide sequence that binds to said GPCR
on a G protein interaction site of said GPCR, wherein said G
protein G.alpha. subunit carboxyl terminal peptide sequence is
selected from the group consisting of SEQ ID NOs: 2, 13, 15, 17,
21, 25, 26, 27, 30, 34, 38, 40, and 45-85; (b) screening said
peptide library for in vitro high affinity binding to said G
protein interaction site of said GPCR to identify library members
that bind to said G protein interaction site with higher affinity
than that of said native G protein G.alpha. subunit carboxyl
terminal peptide sequence; (c) selecting a member of said peptide
library having binding to said GPCR of higher affinity than that of
said native G protein G.alpha. subunit carboxyl terminal peptide
sequence (d) providing a library of candidate compounds to screen
for binding to said G protein interaction site of said GPCR; (e)
screening said library of candidate compounds in vitro for binding
to said GPCR in competition with a member of said peptide library
selected in step (c) to detect candidate compounds having
high-affinity binding to said G protein interaction site of said
GPCR.
2. A two-screen method of detecting high-affinity GPCR G protein
interaction site binding compounds, which comprises: (a) providing
a peptide library the members of which are based on the primary
sequence of a native G protein G.alpha. subunit carboxyl terminal
peptide sequence that binds to said GPCR on a G protein interaction
site of said GPCR; (b) screening said peptide library for in vitro
high affinity binding to said G protein interaction site of said
GPCR to identify library members that bind to said G protein
interaction site with higher affinity than that of said native G
protein G.alpha. subunit carboxyl terminal peptide sequence; (c)
selecting a member of said peptide library having binding to said
GPCR of higher affinity than that of said native G protein G.alpha.
subunit carboxyl terminal peptide sequence; (d) providing a library
of candidate compounds to screen for binding to said G protein
interaction site of said GPCR; (e) screening said library of
candidate compounds in vitro for binding to said GPCR in
competition with a member of said peptide library selected in step
(c) to detect candidate compounds having high-affinity binding to
said G protein interaction site of said GPCR.
3. A method of claim 1, wherein said screening of step (b) is
performed by testing for binding to an intact G protein coupled
receptor.
4. A method of claim 1, wherein said screening of step (b) is
performed by testing for binding to an intracellular fragment of a
GPCR.
5. A method of claim 1, wherein said screening of step (b)
comprises a competitive binding assay.
6. A method of claim 5, wherein said competitive binding assay is
characterized by co-incubation of members of said peptide library
with said native G.alpha. subunit carboxyl terminal peptide
sequence.
7. A method of claim 1, wherein binding to said GPCR is determined
by measuring a signal generated from interaction of an activating
ligand with said GPCR.
8. A method of claim 1, wherein said library of variant peptides is
a combinatorial peptide library.
9. A method of claim 8, wherein said library of variant peptides is
a protein-peptide fusion protein library.
10. A method of claim 9, wherein said protein-peptide fusion
protein library is a maltose binding protein-peptide fusion protein
library.
11. A method of claim 1, wherein said library of variant peptides
is a peptide display library.
12. A method of claim 2, wherein said native G protein G.alpha.
subunit carboxyl terminal peptide sequence that binds to said GPCR
on a G protein interaction site of said GPCR is from about 7 to
about 70 amino acids long.
13. A method of claim 2, wherein said native G protein G.alpha.
subunit carboxyl terminal peptide sequence that binds to said GPCR
on a G protein interaction site of said GPCR is from about 7 to
about 55 amino acids long.
14. A method of claim 2, wherein said native G protein G.alpha.
subunit carboxyl terminal peptide sequence that binds to said GPCR
on a G protein interaction site of said GPCR is from about 8 to
about 50 amino acids long.
15. A method of claim 2, wherein said native G protein G.alpha.
subunit carboxyl terminal peptide sequence that binds to said GPCR
on a G protein interaction site of said GPCR is from about 9 to
about 23 amino acids long.
16. A method of claim 2, wherein said native G protein G.alpha.
subunit carboxyl terminal peptide sequence that binds to said GPCR
on a G protein interaction site of said GPCR is about 11 amino
acids long.
17. A method of claim 16, wherein said G protein G.alpha. subunit
carboxyl terminal peptide sequence is selected from the group
consisting of SEQ ID NOs: 2, 13, 15, 17, 21, 25, 26, 27, 30, 34,
38, 40, and 45-85.
Description
[0001] This application is a continuation of application Ser. No.
10/411,336, filed Apr. 11, 2003, which is a continuation-in-part of
prior co-pending application Ser. No. 09/852,910, filed May 11,
2001, which claims priority from prior co-pending provisional
application Ser. No. 60/275,472, filed Mar. 14, 2001.
BACKGROUND OF THE INVENTION
[0002] 1. Technical Field
[0003] The present invention generally pertains to the field of
modulating activity of G protein-coupled receptors (GPCR) and of
identifying and preparing G protein coupled receptor antagonist and
agonist compounds, including direct, indirect, full, partial,
inverse and allosteric agonists. The invention also encompasses
compounds that bind to GPCR to stabilize a particular conformation
of the GPCR. These compounds can serve as lead compounds for drug
discovery purposes or for studying the GPCR three dimensional
structure of specific conformations by such methods as X-ray
crystallography or NMR. The invention also relates to an approach
using high-throughput screening to identify small molecules that
can bind to GPCRs and modulate their function by affecting the way
in which they contact their cognate G protein(s). As a first step
in identifying GPCR modulators, peptide analogs are identified that
mimic or antagonize G proteins and bind with high affinity to the
particular receptor under study. These peptides then are tested for
their specificity. The most specific peptides are used in a
competitive assay to screen for small molecules or other peptides
that can, for example, (1) increase the binding of the high
affinity peptide ("super agonist") or (2) can decrease the binding
of the high affinity peptide, presumably by competing for binding
at the GPCR ("antagonists").
[0004] 2. Description of the Background Art
[0005] A great number of chemical messengers exert their effects on
cells by binding to G protein-coupled receptors (GPCR). GPCR
include a wide range of biologically active receptors such as
hormone receptors, viral receptors, growth factor receptors,
chemokine receptors, sensor receptors and neuroreceptors. These
receptors are activated by the binding of ligand to an
extracellular binding site on the GPCR and mediate their actions
through the various G proteins. The molecular interactions that
occur between the receptor and the G protein are fundamental to the
transduction of environmental signals into specific cellular
responses.
[0006] G protein-coupled receptors have seven transmembrane helices
which form, on the intracellular side of the membrane, the G
protein binding domain. Experiments have suggested that activation
of the receptor by ligand binding changes conformation of the
receptor, unmasking G protein binding sites on the intracellular
face of the receptor. The transduction of the signal from the
extracellular to intracellular environments requires the actions of
heterotrimeric G proteins. The molecular interactions that occur
between the receptor and the G protein are fundamental to the
transduction of environmental signals into specific cellular
responses. Heterotrimeric G proteins are thought to interact with
GPCR in a multi-site fashion with the major site of contact being
at the carboxyl terminus of the G.alpha. subunit. Hamm et al.,
Science 241:832-835, 1998; Osawa and Weiss, J. Biol. Chem.
270:31052-31058, 1995; Garcia et al., EMBO J. 14:4460-4469, 1995;
Sullivan et al., J. Biol. Chem. 269:21519-21525, 1994; West et al.,
J. Biol. Chem. 260:14428-14430, 1985.
[0007] In the inactive state, G proteins are heterotrimeric,
consisting of one .alpha., one .beta. and one .gamma. subunit and a
bound deoxyguanosine diphosphate (GDP). Following ligand binding,
the GPCR becomes activated. Conformational changes in the activated
receptor lead to activation of the G protein, with subsequent
decreased affinity of G.alpha. for GDP, dissociation of the GDP and
replacement with GTP. Once GTP is bound, G.alpha. assumes its
active conformation, dissociates from the receptor, and activates a
downstream effector. Hydrolysis of GTP to GDP, catalyzed by the
G-protein itself, returns the G-protein to its basal, inactive
form. Thus, the G-protein serves a dual role, as both an
intermediate that relays the signal from receptor to effector and
as a clock that controls the duration of the signal. A variety of
studies have implicated the carboxyl terminus of G protein .alpha.
subunits in mediating receptor-G protein interaction and
selectivity.
[0008] The carboxyl terminal 11 amino acids are most important to
receptor interaction and to the specificity of this interaction.
Martin et al., J. Biol. Chem. 271:361-366, 1996; Kostenis et al.,
Biochemistry 36:1487-1495, 1997. Other regions on G.alpha. also are
involved in receptor contact, however. Portions of the
G.beta..gamma. dimer also have been implicated in GPCR binding. See
Onrust et al., Science 275:381-384, 1997; Lichtarge et al., Proc.
Natl. Acad. Sci. USA 93:7507-7611, 1996; Mazzoni and Hamm, J. Biol.
Chem. 271:30034-30040, 1996; Bae et al., J. Biol. Chem.
272:32071-32077, 1997. The carboxyl terminal amino acid regions of
G.alpha. proteins (and other GPCR binding regions of the
heterotrimeric G protein) not only provide the molecular basis of
receptor-mediated activation of G proteins, but also play an
important role in determining the fidelity of receptor activation.
Conklin et al., Nature 363:274-276, 1993; Conklin et al., Mol.
Pharmacol. 50:885-890, 1996.
[0009] The involvement of the carboxyl-terminal 11 amino acids of
Gt (amino acids 340-350) in interactions with the activated GPCR
(R*) is suggested by many studies, including (a) the finding that
Pertussis toxin catalyzes the ADP-ribosylation of Cys0347, which
uncouples Gt from R*; (b) a peptide corresponding to amino acids
340-350 of Gt can uncouple R* from Gt and can itself bind to R* and
mimic the effects of Gt; (c) site-directed mutagenesis; and (d) the
demonstration in related G proteins that specificity of coupling to
particular receptors resides in their carboxyl terminus in
interacting with R*.
[0010] The G proteins play important and intricate roles in
determining the specificity and temporal characteristics of the
cellular response to the ligand-binding signal. Hamm and Gilchrist,
Curr. Opin. Cell Biol. 8:189-196, 1996. Multiple receptors can
activate a single G protein subtype, and in some cases a single
receptor can activate more than one G protein, thereby mediating
multiple intracellular signals. In other cases, however,
interaction of a receptor with a G protein is regulated in a highly
selective manner such that only a particular heterotrimer is
bound.
[0011] Recognition sites are the precise molecular regions on
receptors to which the activating molecules bind. An agonist is an
endogenous substance or a drug that can interact with a receptor
and initiate a physiological response. A drug may interact at the
same site as an endogenous agonist (i.e., hormone or
neurotransmitter) or at a different site. Agonists that bind to an
adjacent or a different site are termed allosteric agonists. As a
consequence of the binding to allosteric binding sites, the
interaction with the normal ligand may be either enhanced or
reduced. The conformational change which the allosteric modulators
induce in receptors concerns not only the binding domain for the
classical ligands, but also the domain responsible for the
interaction between the receptors and the G proteins.
[0012] The visual system is an example of one in which G protein
signaling is important. Rod cells of the retina make up 95% of the
photoreceptors and are highly sensitive to light. Rods allow vision
at night or under conditions of very dim illumination. The rod
visual protein rhodopsin resides in disk membranes in the rod outer
segment (ROS). Rhodopsin is a prototypical GPCR. Helmreich and
Hofmann, Biochim. Biophys. Acta 1286:285-322, 1996; Menon et al.,
Physiol. Rev. 81:1659, 2001; Teller et al., Biochemistry 40:7761,
2001. Rhodopsin is unique among GPCRs as it is not ligand
activated.
[0013] Night vision relates to the ability of the organism to
discriminate between slight differences in the intensity of dim
light and, when dark-adapted, to detect small changes in light.
Some persons report consistent difficulties in seeing at night,
even when their eyes are fully dark-adapted. They cannot detect
objects readily visible to others and show both confusion and slow
recovery after brief exposure to relatively bright light sources.
Maneuvering in dimly illuminated spaces and driving or flying at
night present serious problems to these individuals. In addition,
some individuals have nyctalopia, or true night blindness, which is
diagnosed on the basis of a measurement of retinal sensitivity.
[0014] No definitive data on the occurrence of nyctalopia in the
population are available, since measurements have never been made
on a representative sample of the population. Studies of select
groups (e.g., school children, service men), show that the normal
population includes a percentage of persons of low visual
sensitivity whose performance will be as poor as or poorer than
that of many individuals whose nyctalopia is associated with
disease or degenerative processes. For example, about 2 percent of
Navy men were disqualified for night duties as "night blind" on
this basis. It is also a disease of aging. As the general
population ages, incidence of night blindness increases. Night
blindness also has been observed in several diseases including: (1)
Retinitis pigmentosa (In the early stages of the disease, dark
adaptation takes place, but at a retarded rate. As disease
advances, rod function is progressively lost, and the absolute
terminal threshold is elevated. More than 100,000 Americans have
retinitis pigmentosa, and most people with retinitis pigmentosa are
blind by the age of 40. See Farrar et al., EMBO J. 21(5):857-864,
2002; (2) Glaucoma (Early impairment and progressive loss of rod
sensitivity is observed in glaucoma. Cursiefen et al., Doc.
Ophthalmol. 103(l):1-12, 2001. Glaucoma is one of the leading
causes of blindness in the U.S and one of the most common causes of
blindness in individuals over age 60, one of the fastest growing
groups in the U.S.); (3) LASIK (Recent studies indicate a
significant number of patients who undergo LASIK surgery fail a
night vision test (30-60%). Miller et al., CLAO J. 27:84-88, 2001;
Brunette et al., Ophthalmology 107:1790-1796, 2000; (4) Side
effects of drugs (Several medications can cause night blindness,
including Methyltestosterone, Quinidinesis, Paramethadion and
Trimethadione (anticonvulsants), Questran (cholesterol-lowering),
Accutane (anti-acne), Hydroxychloroquine (anti-malarial), Videx
(HIV), and Nefazodone (antidepressant)). Thus, the usefulness of a
pharmaceutical approach to night blindness is clear. As the
population ages, the number of affected individuals will
increase.
[0015] Human dietary vitamin A deficiency can cause night
blindness, and this can be reversed with vitamin A supplements.
However, the night blindness associated with visual diseases such
as retinitis pigmentosa (RP), cataracts, diabetic retinopathy, and
glaucoma is only somewhat helped with vitamin A supplements, which
do not change the course of the disease. Many of the mutations that
cause retinal degeneration and visual loss are in genes that encode
photoreceptor cascade proteins; others are in genes that encode
photoreceptor structural proteins. Pang and Lam, Hum. Mutat.
19:189, 2002. Mutations in rhodopsin, PDE.beta., or G.alpha.t have
been identified in different forms of congenital stationary night
blindness. Pepe, Prog. Retin. Eye Res. 20:733-759, 2001. Stationary
night blindness is not associated with retinal degeneration and
manifests itself in the inability to see in the dark; daytime
vision is largely unaffected. Congenital stationary night blindness
(CSNB) refers to a group of non-progressive retinal disorders that
are characterized predominantly by abnormal function of the rod
system. Clinical heterogeneity even among family members with the
same mutation raises the possibility that modifying factors, either
genetic or environmental, influence the severity of the disease.
Gottlob, Curr. Opin. Ophthalmol. 12:378-383, 2001.
[0016] In night blindness resulting from defects in rhodopsin,
G.alpha.t, or PDE.beta., rod photoreceptors respond only to light
intensities far brighter than normal, and the sensitivity of rods
to light is similar to that of normal individuals who are not dark
adapted. In fundus albipunctatus and in Oguchi disease, the rod
photoreceptors can achieve normal sensitivity to dim light but only
after 2 or more hours of dark adaptation, compared with
approximately 0.5 hours for normal individuals. Dryja, Am. J.
Ophthalmol. 130:547, 2000. In each of these forms of stationary
night blindness, the poor rod sensitivity and the time course of
dark adaptation correlate with the known or presumed physiologic
abnormalities caused by the identified gene defects. Increasing the
efficacy with which rhodopsin activates the phototransduction
cascade is a possible new pharmacological approach to night
blindness. Activated rhodopsin activates the rod visual G protein,
Gt, which activates the visual transduction cascade.
Pharmacologically increasing the effective signaling of rhodopsin
can significantly impact people's ability to see and function in
low light. The ability, therefore, to identify small molecule
compounds that enhance the ability of G protein coupled receptors
to signal would be a major benefit.
[0017] Because G proteins and their receptors influence a large
number of intracellular signals mediated by a large number of
different chemical ligands, considerable potential for modulation
of disease pathology exists. Many medically significant biological
processes are influenced by G protein signal transduction pathways
and their downstream effector molecules. See Holler et al., Cell.
Mol. Life Sci. 340:1012-1020, 1999. G protein-coupled receptors and
their ligands are the target for many pharmaceutical products and
are the focus of intense drug discovery efforts. Over the past 15
years, nearly 350 therapeutic agents targeting GPCRs have been
successfully introduced into the market. Because of the ubiquitous
nature of G protein-mediated signaling systems and their influence
on a great number of pathologic states, it is highly desirable to
find new methods of modulating these systems, including both
agonist and antagonist effects. The ability to study the
three-dimensional conformations of GPCRs in response to different
individual ligands with different effects also is highly desirable,
since these studies would aid in the search and development of
drugs with particular structures which impart particular modulating
effects on GPCRs.
[0018] Drug receptor theories are grounded in the law of mass
action and include the concepts of affinity (the probability of the
drug occupying a receptor at any given instant), intrinsic efficacy
(intrinsic activity), which expresses the complex associations
involving drug or ligand concentration, and activation states of
receptors. Drugs classified as agonists interact with receptors to
alter the proportion of activated receptors, thus modifying
cellular activity. Conventional agonists increase the proportion of
activated receptors; inverse agonists reduce it. Direct agonists
act on receptors, while indirect agonists facilitate the actions of
the endogenous agonist (the neurotransmitter itself). Allosteric
modulation of receptor activation is a new approach which
circumvents the development of tolerance.
[0019] Most currently available drugs affecting GPCRs act by
antagonizing the binding between a G protein-coupled receptor and
its extracellular ligand(s). On the other hand, receptor
subtype-selective drugs have been difficult to obtain. An
additional drawback to the classical approach of designing drugs to
interfere with ligand binding has been that conventional
antagonists are ineffective for some GPCRs such as proteinase
activated receptors (PAR) due to the unique mechanism of enzymatic
cleavage of the receptor and generation of a tethered ligand. In
other cases, intrinsic or constitutive activity of receptors leads
to pathology directly, thus rendering antagonism of ligand binding
moot. For these reasons, alternative targets for blocking the
consequences of GPCR activation and signaling are highly desirable.
Increased understanding of the structural conformation of GPCRs
under the influence of different agonists, antagonists or other
ligands also allows design of compounds with highly specific
effects on GPCRs.
[0020] One potential alternative target for inhibition by new
pharmaceuticals has been the receptor-G protein interface on the
interior of the plasma membrane. Konig et al., Proc. Natl. Acad.
Sci. USA 86:6878-6882, 1989; Acharya et al., J. Biol. Chem.
272:6519-6524, 1997; Verrall et al., J. Biol. Chem. 272:6898-6902,
1997. The carboxyl terminus of G.alpha. and other regions of the G
protein heterotrimer conform to a binding site at the cytoplasmic
face of the receptor. Sondek et al., Nature 379:311-319, 1996;
Sondek et al., Nature 379:369-374, 1996; Wall et al., Science
269:1405-1412, 1996; Mixon et al., Science 270:954-960, 1995;
Lambright et al., Nature 369:621-628, 1994; Lambright et al.,
Nature 379:311-319, 1996; Sondek et al., Nature 379:369-374, 1996;
Wall et al., Science 269:1405-1412, 1996; Mixon et al., Science
270:954-960, 1995. Peptides corresponding to these binding regions
or mimicking these regions can block receptor signaling or
stabilize the active agonist-bound conformation of the receptor.
Hamm et al., Science 241:832-835, 1988; Gilchrist et al., J. Biol.
Chem. 273:14912-14919, 1998.
[0021] For example, in the case of rhodopsin, the rod
photoreceptor, the G.alpha. C-terminal peptide, G.alpha. 340-350,
stabilizes the receptor in its active metarhodopsin II
conformation. Hamm et al., Science 241:832-835, 1988; Osawa and
Weiss, J. Biol. Chem. 270:31052-31058, 1995. Two carboxyl terminal
peptides from G.alpha.S (354-372 and 384-394), but not the
corresponding peptides from G.alpha.i.sub.2, evoke high affinity
agonist binding to .beta..sub.2-adrenergic receptors and inhibit
their ability to activate G.alpha.s and adenylyl cyclase. Rasenick
et al., J. Biol. Chem. 269:21519-21525, 1994. Thus, the carboxyl
terminus of G.alpha. is important in mediating the specificity of G
protein responses. Drug discovery approaches which take advantage
of this phenomenon, however, are not available. Jones et al.,
Expert Opin. Ther. Patents 9(12):1641, 1999.
[0022] In general, GPCRs require agonist binding for activation.
However, for some receptors basic signaling activity may occur even
in the absence of an agonist (constitutive activity). In addition,
modifications to the receptor amino acid sequence can stabilize the
active state conformation without the requirement for a ligand.
Constitutive (agonist-independent) signaling activity has been
demonstrated for both mutant and wild type (or native) form
receptors (Tiberi and Caron, J. Biol. Chem. 269:27925-27931, 1994;
Hasegawa et al., J. Biol. Chem. 271:1857-1860, 1996). A number of
GPCRs that cause disease in humans, for example, receptors for
thyroid-stimulating hormone (Vassart et al., Ann N.Y. Acad Sci.
766:23-30, 1995), have been found to exhibit agonist-independent
activity. An inverse agonist is an agent that binds to the receptor
and suppresses this activity.
[0023] Experimentally, several single amino acid mutations have
produced agonist-independent activity. .beta.2 and .alpha.2
adrenergic receptors, for example, mutated at single sites in the
third cytoplasmic loop, show constitutive activity. Ren et al., J.
Biol. Chem. 268:16483-16487, 1993; Samama et al., Mol. Pharmacol.
45:390-394, 1994. In some cases, a large deletion mutation in the
carboxyl tail or in the intracellular loops of GPCRs has led to
constitutive activity. For example, in the thyrotropin releasing
hormone receptor a truncation deletion of the carboxyl terminus or
a smaller deletion in the second extracellular loop of the thrombin
receptor renders the receptor constitutively active. Nussenzveig et
al., J. Biol. Chem. 268:2389-2392, 1993; Matus-Leibovitch et al.,
J. Biol. Chem. 270:1041-1047, 1995; Nanevicz et al., J. Biol. Chem.
270:21619-21625, 1995.
[0024] These findings have led to a modification of traditional
receptor theory. Samama et al., J. Biol. Chem. 268:4625-4636, 1993.
It now is thought that receptors can exist in at least two
conformations, an inactive conformation (R) and an activated
conformation (R*), and that an equilibrium exists between these two
states that markedly favors R over R* in the majority of receptors.
It has been proposed that in some receptors (native and mutant)
there is a shift in equilibrium in the absence of agonist that
allows a sufficient number of receptors to be in the active R*
state to initiate signaling. Therefore, in response to chemical or
physical external stimuli, GPCRs undergo a conformational change
leading to the activation of heterotrimeric G proteins which go on
to initiate intracellular signaling events.
[0025] Several studies suggest that many GPCRs exhibit properties
consistent with the existence of multiple conformational states. In
rhodopsin, the existence of multiple conformers is evident from
absorbance changes. Sakmar, Prog. Nucleic Acid Res. Mol. Biol.
59:1-34, 1998. Activation occurs by transition through intermediate
conformations with the equilibrium between these forms showing a
characteristic pH sensitivity. See Armis and Hoffman, Proc. Natl.
Acad. Sci. USA 90:7849-7853, 1993; Vogel and Siebert, Biochemistry
41:3529-3535, 2002. Pharmacological studies suggest that the
existence of distinct receptor conformers can have functional
significance. Studies of fusion proteins of beta adrenergic
receptor and G proteins suggest that partial agonists stabilize a
conformational state distinct from that stabilized by a full
agonist. Seifert et al., J. Pharmacol. Exp. Ther. 297:1218-1226,
2001.
[0026] The observation in several receptors that different agonists
acting at the same receptor can direct the relative activation of
downstream pathways, a phenomenon called "signal trafficking," also
suggests the presence of multiple populations of active receptor
conformers. Kenakin, Trends Pharmacol. Sci. 16:232-238, 1995; Berg
et al., Mol. Pharmacol. 54:94-104, 1998; Cordeaux et al., J. Biol.
Chem. 276:28667-28675, 2001; Marie et al., J. Biol. Chem.
276:41100-41111, 2001. Fluorescence studies also suggest the
presence of different receptor conformational populations when
complexed with functionally distinct agonists. Ghanouni et al., J.
Biol. Chem. 276:24433-24436, 2001. This emerging support for the
existence of distinct, functionally relevant conformers in several
GPCRs suggests that, for these receptors, the molecular activation
mechanism must provide the means for switching among multiple
conformations. A method to study these conformers by methods such
as crystallographic methods and NMR would be highly useful in the
process of discovering compounds which can modulate or stabilize
particular conformers.
[0027] Protein-protein interactions involved in regulatory
phenomena are reversible and tend to involve only a small fraction
of the protein surface. Generally, to identify peptides that block
the protein-protein interactions of interest particular peptides
are synthesized in an attempt to mimic sections of one of the
native interacting proteins or active sequences are selected from
random peptide libraries after screening. Peptides are made up of
sequences of amino acids, however unlike DNA recognition, which is
linearly coded into the sequence, peptide binding is dependent on
three-dimensional structure.
[0028] The visual pigment, rhodopsin, is the most extensively
studied member of the family of G protein receptors. Recently, the
X-ray structure of crystalline bovine rhodopsin has been determined
to a resolution of 2.8 .ANG.. This has paved the way for an
understanding of the structure-function relationships of a
prototypical GPCR at the molecular level. Since rhodopsin
constitutes greater than 90% of the disk membrane protein,
measurements made on the proteins of disk membranes predominantly
reflect the properties of rhodopsin in its native environment.
Rhodopsin consists of the apoprotein opsin and the chromophore
11-cis retinal. Opsin, consisting of 348 amino acids, has a
molecular mass of about 40 kDa and folds into seven transmembrane
helices of varying length and one short cytoplasmic helix. The
retinylidene chromophore (the aldehyde of vitamin A1) is covalently
bound to Lys-296 in helix 7 via a protonated Schiff base and keeps
the receptor in an inactive conformation.
[0029] Light absorption causes a rapid 11-cis to all-trans
isomerization of the chromophore which induces a series
conformational of changes of the opsin moiety. This reaction occurs
with high efficiency (quantum yield 0.67) and the primary
photoproduct, photorhodopsin, is formed within a very short time
(200 fs). Subsequently, photorhodopsin thermally relaxes within a
few picoseconds to a distorted all-trans configuration,
bathorhodopsin. On a nanosecond time scale, bathorhodopsin
establishes an equilibrium with a blue-shifted intermediate before
the mixture decays to form lumirhodopsin. Lumirhodopsin then is
transformed into metarhodopsin I and subsequently metarhodopsin II,
the active conformation for G protein coupling. Thus, there are two
conformational switches in rhodopsin which are controlled by the
protonation of specific amino acids of the protein: the transition
from the inactive Meta I state to the active Meta II state and, in
the absence of bound retinal, the transition from the inactive to
the active state of opsin. According to current models, the
receptor is kept in an inactive conformation by electrostatic
interactions between charged groups in the protein, which are
neutralized by the proton uptake involved in the transition to an
active state conformation.
[0030] The active receptor species Meta II decays slowly within
minutes, by hydrolysis of the Schiff base and dissociation of the
receptor into the apoprotein opsin and retinal. Researchers have
shown that opsin is in a pH-dependent conformational equilibrium
between an active and an inactive state. During the decay of Meta
II at neutral pH, most structural changes of Meta II formation are
reverted and the decay product opsin eventually adopts an active
conformation similar to that of Meta II.
[0031] Four distinct steps can be observed in the process of GPCR
activation: (1) creation of the signal by a photon or by ligand
binding; (2) transduction of the signal through the membrane; (3)
interaction with the G protein; and (4) activation of the second
messenger. Although the phases clearly differ in the kind of
processes taking place, they are not discrete and independent. For
example, allostery between ligand binding and G protein binding has
been observed for several GPCRs, as well as cation-dependent
allosteric regulation of agonist and antagonist binding.
Wessling-Resnick and Johnson, J. Biol. Chem. 262:12444-12447, 1987;
Hepler and Gilman, Trends Biol. Sci. 17:383-387, 1992; Nunnari et
al., J. Biol. Chem. 262:12387-12392, 1987; Neve, Mol. Pharmacol.,
39:570-578, 1991; Neve et al., Mol. Pharmacol. 39:733-739,
1991.
[0032] A number of cytoplasmic proteins interact exclusively with
light-activated rhodopsin (R*). Because the crystal structure
depicts the inactive form of rhodopsin as not interacting
significantly with cytoplamic proteins, this structure can provide
only indirect information about the R* state. In addition, two
regions of the cytoplasmic surface domain of inactive rhodopsin
structure (amino acid residues 236-239 and 328-333) have not been
fully resolved by crystal structure analysis. Therefore, tools
which can stabilize particular conformers would be useful for
studying structure of GPCRs such as rhodopsin.
[0033] Negative antagonism is demonstrated when a drug binds to a
receptor that exhibits constitutive activity and reduces this
activity. Negative antagonists appear to act by constraining
receptors in an inactive state. Samama et al., Mol. Pharmacol.
45:390-394, 1994. Although first described in other receptor
systems, negative antagonism has been shown to occur with GPCRs
such as opioid, .beta.2-adrenergic, serotonin type 2C, bradykinin,
and D1B dopamine receptors. Schutz and Freissmuth, J. Biol. Chem.
267:8200-8206, 1992; Costa and Herz, Proc. Natl. Acad. Sci. USA
86:7321-7325, 1989; Costa et al., Mol. Pharmacol. 41:549-560, 1992;
Samama et al., Mol. Pharmacol. 45:390-394, 1994; Pei et al., Proc.
Natl. Acad. Sci. USA 91:2699-2702, 1994; Chidiac et al., Mol.
Pharmacol. 45:490-499, 1994; Barker et al., J. Biol. Chem.
269:11687-11690, 1994; Leeb-Lundberg et al., J. Biol. Chem. 269:
25970-25973, 1994; Tiberi and Caron, J. Biol. Chem. 269:
27925-27931, 1994.
[0034] That being stated, the concept of constitutively active
receptors offer insights which explain pathophysiologic conditions.
For example, a constitutively active receptor in a disease process
such as hypertension may no longer be under the influence of the
sympathetic nervous system. In hypertension, a constitutively
active GPCR may be expressed in any number of areas including the
brain, kidneys or peripheral blood vessels. A newly recognized
class of drugs (negative antagonists or inverse agonists) which
reduce undesirable constitutive activity can act as important new
therapeutic agents. Thus, a technology for identifying negative
antagonists (or understanding and stabilizing the conformational
change in a GPCR that binding a negative antagonist compound
causes) of both native and mutated GPCRs has important predictable
as well as not yet realized pharmaceutical applications.
Furthermore, because at least some constitutively active GPCRs are
tumorigenic, the identification of negative antagonists for these
GPCRs can lead to the development of anti-tumor and/or anti-cell
proliferation drugs.
[0035] Mutagenesis studies of the carboxyl terminal region of
G.alpha.t have identified several specific amino acid residues in
this binding region crucial for G.alpha.t activation by rhodopsin.
Martin et al., J. Biol. Chem. 271:361-6, 1996. Substitution of
three to five carboxyl-terminal amino acids from G.alpha.q with
corresponding residues from G.alpha.i allowed receptors which
signal exclusively through G.alpha.i subunits to activate the
chimeric .alpha. subunits and stimulate the G.alpha.q effector,
phospholipase C .beta.. Conklin et al., Nature 363:274-276, 1993;
Conklin et al., Mol. Pharmacol. 50:885-890, 1996. All of these
studies suggest that G.alpha. carboxyl peptide sequences are
responsible for the specificity of the signaling responses of the
individual G proteins. There are 16 unique G.alpha. subunits
(G.alpha.i.sub.1, G.alpha.i.sub.2, G.alpha.i.sub.3,
G.alpha.O.sub.1, G.alpha.O.sub.2, G.alpha.Z, G.alpha.t, G.alpha.q,
G.alpha.11, G.alpha.14, G.alpha.s, G.alpha.12, G.alpha.13,
G.alpha.15/16, G.alpha.OLF and G.alpha.gust) thought to mediate
specific interaction with different GPCRs, several hundred of which
have been cloned. Thus, peptides corresponding to G protein regions
which bind the GPCR could be used as competitive inhibitors of
receptor-G protein interactions. Hamm et al., Science 241-832-835,
1988; Gilchrist et al., J. Biol. Chem. 273:14912-14919, 1998. Drug
discovery approaches which take advantage of this opportunity,
however, are not available. Jones et al., Expert Opin. Ther.
Patents 9(12):1641-1654, 1999.
[0036] Identification of potent lead compounds for use in modern
high throughput screening assays and computerized design of new
compounds using information about the desired three-dimensional
conformation of receptor molecules, for example, are important
aspects of the modern drug discovery process. One of the major
challenges confronting those using these types of methods is the
difficulty of identifying useful binding compounds from very large
combinatorial libraries of potential candidate molecules. When
literally hundreds of thousands of compounds are screened,
characterizing the compounds which test positive for binding, for
modulatory activity or for stabilization of a conformation
(including false positives) is an expensive and time-consuming
process. Hence, a method which can identify potent and useful lead
compounds for high throughput screening and useful binding partners
for three dimensional conformational studies and which reduce the
number of false positives in the screening process would be very
desirable.
SUMMARY OF THE INVENTION
[0037] Accordingly, the invention provides a method of identifying
a G protein coupled receptor signaling modifying peptide, which
comprises providing a peptide library based on a native G protein
coupled receptor binding peptide; screening the peptide library for
high affinity binding to the G protein coupled receptor; and
selecting a member of the peptide library having binding to the G
protein coupled receptor of higher affinity than that of the native
peptide. The screening may be performed by testing for binding to
an intact G protein coupled receptor or to at least an
intracellular fragment of a G protein coupled receptor.
[0038] The G protein coupled receptor binding peptide may be a G
protein subunit or fragment thereof which is, for example from
about 7 to about 70 amino acids long or from about 7 to about 55
amino acids long or from about 8 to about 50 amino acids long or
from about 9 to about 23 amino acids long, and most preferably
about 11 amino acids long. The G protein subunit fragment
preferably is a G.alpha. subunit or a G.alpha. subunit carboxyl
terminal peptide but alternatively may be a G.beta..gamma.
dimer.
[0039] Screening may comprise a competitive binding assay, which
preferably is characterized by co-incubation of members of the
peptide library with the G protein coupled receptor binding
peptide, for example in an enzyme-linked immunosorbant assay
wherein the peptide library members are capable of providing a
detectable signal and/or wherein binding to the G protein coupled
receptor is determined by measuring a signal generated from
interaction of an activating ligand with the G protein coupled
receptor.
[0040] The peptide library preferably is a combinatorial peptide
library or a protein-peptide fusion protein library such as, for
example a peptide display library or a maltose binding
protein-peptide fusion protein library.
[0041] In another embodiment, the invention also provides a method
of identifying a G protein coupled receptor signaling modifying
compound, which comprises providing a library of candidate
compounds to screen for binding to the G protein coupled receptor;
providing a high affinity G protein coupled receptor binding
peptide; screening the library of candidate compounds for high
affinity binding to the G protein coupled receptor in competition
with the high affinity G protein coupled receptor binding peptide;
and identifying a member of the library of candidate compounds
having binding to the G protein coupled receptor of equal or higher
affinity than that of the high affinity G protein coupled receptor
binding peptide or a member of the library of candidate compounds
binding of which results in increased binding affinity of the high
affinity G protein coupled receptor binding peptide. Screens may be
performed by testing for binding to an intact G protein coupled
receptor or to at least an intracellular fragment of a G protein
coupled receptor.
[0042] The G protein coupled receptor binding peptide may be a G
protein subunit or fragment thereof which is, for example from
about 7 to about 70 amino acids long or from about 7 to about 55
amino acids long or from about 8 to about 50 amino acids long or
from about 9 to about 23 amino acids long, and most preferably
about 11 amino acids long. The G protein subunit fragment
preferably is a G.alpha. subunit or a G.alpha. subunit carboxyl
terminal peptide but alternatively may be a G.beta..gamma.
dimer.
[0043] Screening may comprise a competitive binding assay, which
preferably is characterized by co-incubation of members of the
peptide library with the G protein coupled receptor binding
peptide, for example in an enzyme-linked immunosorbant assay
wherein the peptide library members are capable of providing a
detectable signal and/or wherein binding to the G protein coupled
receptor is determined by measuring a signal generated from
interaction of an activating ligand with the G protein coupled
receptor.
[0044] The library of candidate compounds preferably is a focused
library of candidate compounds based on the structure of the high
affinity G protein coupled receptor binding peptide. The library of
candidate compounds may be a combinatorial library of, for example
drug-like molecules or a focused small molecule library whose
members, for example may be based on the chemical structure of the
high affinity G protein coupled receptor binding peptide.
[0045] The invention also provides G protein coupled receptor
signaling modifying peptides and compounds identified according to
the methods described above, as well as methods of modifying G
protein coupled receptor signaling in a cell having a G protein
coupled receptor which comprise administering such compounds to the
cell. Also provided are methods of inhibiting G protein coupled
receptor signaling which comprise contacting a compound with the G
protein coupled receptor which interferes with binding of the G
protein coupled receptor to its cognate G proteins.
[0046] In a further embodiment, the invention provides a method for
identifying a G protein coupled receptor signaling modifying
compound, which comprises providing a peptide identified according
to at least one of the methods described above, wherein the peptide
is labeled to provide a detectable peptide signal; providing a
library of candidate G protein coupled receptor signaling modifying
compounds; contacting the peptide with the G protein coupled
receptor under conditions such that the peptide binds to the G
protein coupled receptor; removing unbound peptide from the G
protein coupled receptor; measuring the signaling activity of the
peptide-bound G protein coupled receptor and measuring the
detectable peptide signal; contacting the members of the library of
candidate G protein coupled receptor signaling modifying compounds
with the peptide-bound G protein coupled receptor; measuring the
signaling activity of the peptide bound G protein coupled receptor
and measuring the detectable peptide signal; determining whether
the G protein coupled receptor signaling activity is increased or
decreased after contact with the candidate compound and whether G
protein coupled receptor peptide binding is increased or decreased
after contact with the candidate compound; and identifying
compounds for which contact with the peptide-bound G protein
coupled receptor results in both an increase in peptide binding to
the G protein coupled receptor and an increase in G protein coupled
receptor signaling, identifying compounds for which contact with
the peptide-bound G protein coupled receptor results in both a
decrease in peptide binding to the G protein coupled receptor and a
decrease a G protein coupled receptor signaling and identifying
compounds for which contact with the peptide-bound G-protein
coupled receptor results in increased binding affinity of the
peptide identified according to a method described above. Methods
for measuring the signaling activity of the peptide-bound G protein
coupled receptor may be selected from the group consisting of
measuring inositol phosphate accumulation; measuring intracellular
Ca.sup.2+ levels; measuring adenyl cyclase activity; measuring
transendothelial electrical resistance; measuring stress fiber
formation; measuring ligand binding; measuring receptor expression;
measuring receptor desensitization; measuring kinase activity;
measuring phosphatase activity; measuring nuclear transcription
factors; measuring all migration (chemotaxis); measuring superoxide
formation; measuring nitric oxide formation; measuring cell
degranulation; measuring GIRK activity; measuring actin
polymerization; measuring vasoconstriction; measuring cell
permeability; measuring apoptosis; measuring cell differentiation;
measuring membrane association of a protein that translocates upon
GPCR activation, such as protein kinase C; measuring cytosolic
accumulation of a protein that translocates upon GPCR activation,
such as protein kinase C; and measuring nuclear association of a
protein that translocates upon GPCR activation, such as Ran.
[0047] In yet a further embodiment, the invention provides
compounds selected from the group consisting of SEQ ID NOS: 2, 4,
6, 8, 10, 12, 13, 15, 17, 21, 23, 25-27, 30, 32, 34, 36, 38, 40,
45-85, 94-111, 125-150, 160-164, 175-178 and 183-264.
[0048] In yet a further embodiment, the invention provides a method
for providing a therapeutic G protein coupled receptor signaling
modifier peptide to a mammal which comprises administering to the
mammal an expression construct which expresses a compound selected
from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 13,
15, 17, 21, 23, 25-27, 30, 32, 34, 36, 38, 40, 45-85, 94-111,
125-150, 160-164, 175-178 and 183-264. Further, the invention
provides a method for treating a disease state in which excess G
protein coupled receptor signaling is a causative factor, which
comprises administering a compound selected from the group
consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 13, 15, 17, 21, 23,
25-27, 30, 32, 34, 36, 38, 40, 45-85, 94-111, 125-150, 160-164,
175-178 and 183-264.
[0049] In yet a further embodiment, the invention provides a method
of identifying a G protein coupled receptor signaling enhancer,
which comprises providing a peptide library based on a native G
protein coupled receptor binding peptide; screening the peptide
library for high affinity binding to the G protein coupled
receptor; selecting a member of the peptide library having binding
to the G protein coupled receptor of higher affinity than that of
the native peptide; providing a library of candidate compounds to
screen for binding to the G protein coupled receptor; screening the
library of candidate compounds for high affinity binding to the G
protein coupled receptor in competition with a member of the
peptide library selected above; and identifying a member of the
library of candidate compounds having binding to the G protein
coupled receptor of equal or higher affinity than that of the
peptide selected above or identifying a member of the library of
candidate compounds binding of which results in increased binding
affinity of the peptide selected above. Screening methods for use
in this embodiment may include testing for binding to an intact G
protein coupled receptor or testing for binding to at least an
intracellular fragment of a G protein coupled receptor. The G
protein coupled receptor binding peptide may be a G protein subunit
or fragment thereof, for example a G protein subunit fragment from
about 7 to about 70 amino acids long, from about 7 to about 55
amino acids long, from about 8 to about 50 amino acids long, from
about 9 to about 23 amino acids long or most preferably about 11
amino acids long. The G protein subunit fragment preferably is a
G.alpha. subunit or a G.alpha. subunit carboxyl terminal peptide
but alternatively may be a G.beta..gamma. dimer.
[0050] Screening may comprise a competitive binding assay, which
preferably is characterized by co-incubation of members of the
peptide library with the G protein coupled receptor binding
peptide, for example in an enzyme-linked immunosorbant assay
wherein the peptide library members are capable of providing a
detectable signal and/or wherein binding to the G protein coupled
receptor is determined by measuring a signal generated from
interaction of an activating ligand with the G protein coupled
receptor.
[0051] The library of candidate compounds preferably is a focused
library of candidate compounds based on the structure of the high
affinity G protein coupled receptor binding peptide. The library of
candidate compounds may be a combinatorial library of, for example
drug-like molecules or a focused small molecule library whose
members, for example may be based on the chemical structure of the
high affinity G protein coupled receptor binding peptide.
[0052] Enzyme-linked immunosorbant assays for use in the inventive
method may comprise the steps of immobilizing the G protein coupled
receptor onto a solid support; providing a protein-peptide fusion
protein display library; incubating members of the protein-peptide
fusion protein display library with the immobilized G protein
coupled receptor in the presence of the G protein coupled receptor
binding peptide under conditions such that members of
protein-peptide fusion protein display library having a binding
affinity for the G protein coupled receptor at least as high as the
G protein coupled receptor binding peptide bind to the immobilized
G protein coupled receptor; removing unbound members of the
protein-peptide fusion protein display library; incubating the
bound protein-peptide fusion protein display library with
antibodies which specifically recognize the protein portion of the
protein-peptide fusion protein display library members under
conditions such that the antibodies specifically bind to the
protein-peptide fusion protein display library members; removing
unbound antibodies; and detecting the bound antibodies. The
protein-peptide fusion protein display library preferably is a
maltose binding protein-peptide fusion protein display library and
the antibodies preferably are anti-maltose binding protein
antibodies. Binding to the G protein coupled receptor preferably is
determined by measuring a signal generated from interaction of the
signalling enhancer with the G protein coupled receptor.
[0053] The peptide library preferably is a combinatorial peptide
library, for example a protein-peptide fusion protein library such
as a maltose binding protein-peptide fusion protein library or any
suitable peptide display library. Libraries of candidate compounds
preferably are focused libraries of candidate compounds based on
the structure of the compound selected above as having binding to
the G protein coupled receptor of higher affinity than that of the
native peptide. The library may be a peptide library or a small
molecule library.
[0054] In yet a further embodiment, the invention provides
compounds identified by a method as described above. In yet further
embodiments, the invention provides a method for treating a disease
state in which alterations in G protein coupled receptor signaling
is a causative factor and a method for treating a disease state in
which alterations in G protein coupled receptor signaling is a
causative factor both of which comprise administering these
compounds. In yet a further embodiment the invention provides a
method of determining the three-dimensional structure of a G
protein coupled receptor, which comprises contacting the G protein
coupled receptor with a compound identified by at least one of the
methods described above under conditions such that binding occurs
and a conformation of the G protein coupled receptor is stabilized;
co-crystallizing the G protein coupled receptor-compound binding
pair; subjecting the co-crystallized binding pair to X-ray
crystallography; and determining the three-dimensional structure of
the co-crystallized binding pair, wherein atomic coordinates of the
G protein coupled receptor are obtained. In yet a further
embodiment, the invention provides a method of determining the
three-dimensional structure of a G protein coupled receptor, which
comprises contacting the G protein coupled receptor with a compound
identified by at least one of the methods described above under
conditions such that binding occurs and a conformation of the G
protein coupled receptor is stabilized; subjecting the binding pair
to nuclear magnetic resonance study; and determining the
three-dimensional structure of the binding pair, wherein atomic
coordinates of the G protein coupled receptor are obtained.
[0055] In yet a further embodiment, the invention provides a method
of isolating a G protein coupled receptor binding partner, which
comprises providing a solid support comprising bound compound
identified by at least one of the methods described above;
providing a library of candidate G protein coupled receptor binding
partner compounds; contacting the library of candidate compounds
with the solid support under conditions such that binding of the
candidate compounds to the compound occurs; eluting unbound and
nonspecifically bound candidate compounds from the solid support;
and recovering bound candidate compounds from the solid
support.
[0056] In yet a further embodiment, the invention provides a method
of designing small molecules that modify activation of a G protein
coupled receptor, which comprises determining the three-dimensional
structure of a G protein coupled receptor according to at least one
of the methods described above; and designing candidate structures
by computer modeling based on the atomic coordinates, wherein the
candidate structures are predicted to bind to the G protein coupled
receptor.
[0057] In yet a further embodiment, the invention provides a
nucleic acid which comprises a DNA that encodes a peptide
identified by at least one of the methods described above, wherein
the DNA is operably linked to a heterologous transcriptional
regulatory sequence, an expression vector which comprises this
nucleic acid and a cell transfected with the expression vector. The
invention also provides an antibody that specifically recognizes a
peptide identified by any of the methods described above, such as,
for example, a monoclonal antibody, a polyclonal antibody, a
humanized antibody or a single chain antibody.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] FIGS. 1A, 1B and 1C are schematic diagrams showing the basic
two-step platform.
[0059] FIGS. 2A and 2B are schematic diagrams showing an example of
the basis for the affinity screening method used to separate and
identify GPCR binding peptides.
[0060] FIG. 3 is a schematic diagram of vector pJS142.
[0061] FIG. 4 is a schematic diagram showing an ELISA
procedure.
[0062] FIG. 5 provides results showing that the LacI-Gq fusion
protein binds thrombin receptor in a concentration-dependent
manner.
[0063] FIG. 6 shows data from binding assays performed on some of
the clones identified using peptide 8 in the screening process.
[0064] FIG. 7 provides binding data for LacI peptide fusion
proteins to PAR1 receptor. pELM6 is the MBP vector alone; pELM17 is
the MBP-native Gt340-350 peptide fusion protein.
[0065] FIG. 8 is a bar graph comparing binding of high affinity
fusion proteins to the high affinity peptide 8 fusion protein (MBP
8).
[0066] FIG. 9 presents ELISA results from panning CHO cells
overexpressing human thrombin receptor (PAR1) using purified
MBP-C-terminal fusion proteins. MBP-G11=xxxx (SEQ ID NO: 1)
LQLNLKEYNLV (SEQ ID NO: 2); PAR-13=VRPS (SEQ ID NO: 3) LQLNRNEYYLV
(SEQ ID NO: 4); PAR-23=LSRS (SEQ ID NO: 5) LQQKLKEYSLV (SEQ ID
NO:6); PAR-33=LSTN (SEQ ID NO: 7) LHLNLKEYNLV (SEQ ID NO: 8);
PAR-34=LPQM (SEQ ID NO: 9) QRLNVGEYNLV (SEQ ID NO: 10); PAR-45=SRHT
(SEQ ID NO: 11) LRLNGKELNLV (SEQ ID NO:12).
[0067] FIG. 10 presents a dose-response curve of SF9 membranes
(PAR1 receptor) assayed with lacI-Gq lysates.
[0068] FIG. 11 is a concentration response curve demonstrating
binding of native Gq peptide-maltose binding protein to PAR1
reconstituted in lipid vesicles.
[0069] FIG. 12B is a schematic diagram showing an exemplary cDNA
minigene construct. SEQ ID NOS:270 and 271 are shown in FIG.
12A.
[0070] FIG. 13 shows an agarose gel of a NcoI digest of minigene
vector. Lane 1 is a 1 kb DNA ladder; lane 2 is pcDNA 3.1; lane 3 is
pcDNA-G.alpha.i; lane 4 is pcDNA-G.alpha.iR; and lane 5 is
pcDNA-G.alpha.q.
[0071] FIG. 14 shows an agarose gel of PCR products showing
transcription of peptide minigene RNA in transfected cells. Lane 1
contains size markers, lane 2 contains PCR products from cells
transfected with pcDNA-GiR, lane 3 contains PCR products from cells
transfected with pcDNA-Gi, and lane 4 contains PCR products from
cells transfected with pcDNA3.1, the empty vector.
[0072] FIG. 15 is a bar graph showing the relative [.sup.3H]
inositol phosphate production after thrombin stimulation normalized
against the basal value.
[0073] FIG. 16 presents data showing inhibition of a GPCR mediated
increase in intracellular calcium concentration in the presence or
absence of a minigene vector encoding the identified high affinity
peptide. FIG. 16A presents fluorescence ([Ca.sup.++].sub.i level)
increase 30 seconds after thrombin addition. FIG. 16B shows the
kinetics of [Ca.sup.++] fluorescence changes after cell stimulation
with thrombin.
[0074] FIG. 17 presents data showing inhibition of a GPCR-mediated
phosphoinositol (P1) hydrolysis in the presence or absence of a
minigene vector encoding the identified high affinity peptide.
[0075] FIG. 18 is a bar graph indicating relative GPCR-mediated
increase of MAPK activity in the presence or absence of a minigene
vector encoding the identified high affinity peptide in cells
expressing GPCR-binding peptides.
[0076] FIG. 19 shows reduction of thrombin-induced transendothelial
electrical resistance in cells expressing G.alpha.q, G.alpha.i,
G.alpha.iR or empty vector.
[0077] FIGS. 20A, 20B, 20C and 20D are a series of photographs of
cells stained for F-actin, showing the inhibition of stress fiber
formation after exposure to thrombin in cells expressing pcDNA-G12
or pcDNA-G13 minigene construct.
[0078] FIGS. 21A, 21B and 21C are bar graphs showing acetylcholine
(Ach) response (pA/pF) for HEK 293 cells transiently transfected
with GIRK1/GIRK4 and the indicated minigene construct.
[0079] FIG. 22 demonstrates selective G protein mediated adenylyl
cyclase inhibition in cells expressing minigene constructs
containing G.alpha. carboxyl terminal peptide inserts.
[0080] FIG. 23 presents dose-response curves of MII stabilization
by .alpha.t340350, mutant .alpha.t340-350K341L and heterotrimeric
Gt.
[0081] FIG. 24 shows stabilization of MII by small molecule
PL.sub.--0302R3C4.
[0082] FIG. 25 presents fluorescence data showing super agonists
for rhodopsin have no effect on PAR1-stimulated Ca.sup.2+
transients.
[0083] FIGS. 26A and 26B are graphs showing light responses (as
measured by a change in current) from isolated rods of dark-adapted
salamander retinas in the presence of small molecule
PL.sub.--0302R3C4.
[0084] FIGS. 27A and 27B are graphs showing light responses (as
measured by a change in current) from isolated rods of dark-adapted
salamander retinas in the absence of a small molecule.
[0085] FIG. 28B is an MBP-8 binding curve with added compound
PL.sub.--1012R2C1, the structure of which is depicted in FIG. 28A,
showing the compound's ability to enhance MBP-8 binding to
EDTA-washed rhodopsin.
[0086] FIG. 29B is an MBP-8 binding curve with added compound
PL.sub.--0894R3C7, the structure of which is depicted in FIG. 29A,
showing the compound's ability to enhance MBP-8 binding to
EDTA-washed rhodopsin.
[0087] FIG. 30B is an MBP-8 binding curve with added compound
PL.sub.--0568R1C5, the structure of which is depicted in FIG. 30A,
showing the compound's ability to enhance MBP-8 binding to
EDTA-washed rhodopsin.
[0088] FIG. 31B is an MBP-8 binding curve with added compound
PL.sub.--0551R8C1, the structure of which is depicted in FIG. 31A,
showing the compound's ability to enhance MBP-8 binding to
EDTA-washed rhodopsin.
[0089] FIG. 32B is an MBP-8 binding curve with added compound
PL.sub.--0302R3C4, the structure of which is depicted in FIG. 32A,
showing the compound's ability to enhance MBP-8 binding to
EDTA-washed rhodopsin.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0090] The present invention involves a method of identifying
compounds which can interfere with or increase binding between a G
protein-coupled receptor (GPCR) and its cognate G protein(s) and
compounds which stabilize a particular conformation of a GPCR for
conformational study. These compounds modulate G protein-mediated
signaling and thus can be used as pharmaceuticals, as lead
compounds for identification of potential useful drugs, as
components of assays which identify drug candidates or as binding
partners in conformational studies by known methods, such as for
example X-ray crystallography or nuclear magnetic resonance.
[0091] Methods for screening and drug identification use peptides
that mimic the structure of the GPCR binding regions of G proteins
and thus are able to modulate receptor-G protein interactions
specifically or specifically bind to a given receptor with high
affinity. These high affinity peptides can be delivered into cells
in the context of an expression construct to act as blockers or
agonists of specific receptor-mediated cellular responses in vitro
and in vivo or can be administered directly to a patient. The
peptides also form the basis of a screening, identification and
selection process to provide traditional pharmaceutical compounds
or to study structure-function relationships in binding. In
particular, the invention allows one to identify high affinity
analog peptides that block or mimic compounds at the receptor-G
protein interface for a particular G protein and to use these high
affinity analogs in a high throughput screen to identify other
peptides or small molecules that likewise specifically antagonize
or agonize GPCR signaling for a G protein or class of G
proteins.
[0092] Small molecules can be used in analogous high throughput
screening processes to identify further compounds. "Small molecule"
denotes any non-peptide organic compound which binds or interferes
with binding to the interfacial region of a GPCR or is a candidate
for such action. Such molecules that bind to and stabilize a
particular conformer of a GPCR also are included in the definition
of "small molecule" as used herein. Peptides or small molecules
directed at the receptor-G protein interface can be designed using
the inventive method to inhibit or enhance biological processes
that employ signaling through a GPCR or to bind to and stabilize a
particular GPCR conformer. Such compounds which bind to, interfere
into binding to or stabilize a conformer of the GPCR-G protein
interface (including but not limited to agonists, inverse agonists,
allosteric agonists, blockers, antagonists, inhibitors, negative
antagonists, partial agonists, and enhancers, as well as compounds
which bind to and stabilize a particular conformer) are termed
"modifiers" or "modifying" compounds, and may include both peptides
and small molecules. This approach to drug design is useful in
targeting G protein-GPCR interactions for which there are no
available ligands, orphan receptors the ligands of which are not
known, mutant constitutively activated receptors,
antibody-crosslinked irreversibly activated receptors such as TSH
receptors in Graves' Disease, and proteinase activated receptors
(PAR). It works equally well, however, with any GPCR-G protein
interaction and more broadly, with receptor-protein interactions in
general.
[0093] Because the method is useful for identifying high affinity
compounds that can bind to and enhance or inhibit virtually any
GPCR, the approach is useful in identifying compounds which can
prevent, ameliorate or correct dysfunctions or diseases in which a
specific class of G proteins is relevant. Conditions and disease
states for which GPCR enhancers and inhibitors are useful include,
but are not limited to: stroke; myocardial infarction; restenosis;
atherosclerosis; hypotension; hypertension; angina pectoris; acute
heart failure; cardiomyocyte apoptosis; cancers; infections such as
bacterial, fungal, protozoan and viral infections, and particularly
infections caused by HIV-1 or HIV-2; septic shock; pain; chronic
allergic disorders; asthma; inflammatory bowel disease;
osteoporosis; rheumatoid arthritis; Graves' disease; post-operative
ileus; urinary retention; testotoxicosis; ulcers; obesity; benign
prostatic hypertrophy; and psychotic and neurological disorders
including anxiety, epilepsy, schizophrenia, manic depression,
Parkinson's disease, Alzheimer's disease, delirium, dementia, drug
addiction, anorexia, bulimia, mood disorders and sleep disorders;
smoking cessation and any other disease or condition that can be
treated by G protein coupled receptor inhibition and any other
disease or condition that can be treated by G protein coupled
receptor activation. Treatment of this diverse set of disorders is
possible because the receptors to which various G proteins bind
differ enough to allow the creation of a battery of analog peptides
which can specifically interface with different GPCR or different
classes or groups of GPCR. The relationship of G proteins and G
protein signalling to various diseases and conditions such as those
listed above is known in the art.
[0094] With the inventive screening methods, the sequences
identified in a particular screen do not bind to all receptors, but
only to the particular receptor of interest. The interaction
between a G protein and a GPCR is quite specific. For example, a
difference in one amino acid can substantially reduce or eliminate
the ability of the G.alpha.i.sub.1/2 peptide to bind the A1
adenosine G protein coupled receptor-G protein interface. Gilchrist
et al., J. Biol. Chem. 273:14912-14919, 1998. Both upstream
regulation of GTP/GDP exchange on G proteins and G protein-mediated
effector activation may be inhibited with interfacial binding
compounds. Thus, high affinity analog peptides can be designed to
specifically interfere with or stabilize a particular action of one
GPCR. Likewise agonizing or enhancing peptides also specifically
affect one GPCR. These specifically-acting peptide analogs are
useful both as pharmaceutical compounds per se, and as potent lead
compounds in modern high throughput screens for other peptides and
small molecule binders having the same specific GPCR
interaction.
[0095] The inventive methods, in one embodiment, rely as first step
on screening for small molecules that enhance or inhibit the
ability of the receptor to interact with the heterotrimeric G
protein. Using rhodopsin and transducin, the screen has found small
molecules that significantly enhance rhodopsin's ability to form
MII, the active form of rhodopsin. Such small molecules can serve
as lead compounds in drug discovery efforts directed towards
potential therapeutic agents to combat night blindness. Using the
screen to identify small molecules, and then testing the identified
compounds using in vitro and in vivo analysis will result in
discovery of potent, high affinity compounds.
[0096] This invention therefore can be used to identify small
molecules that enhance the ability of rhodopsin to signal. The
inventive methods involve, in one embodiment, screening compound
libraries to discover more molecules that increase binding of G
protein peptides to activated rhodopsin. The methods also include
testing these molecules in a number of assays to determine their
effects on rhodopsin signaling, including MII stabilization,
guanosine 5'-O-(3-[.sup.35S]thio) triphosphate (GTP.gamma.S)
binding, and 3',5' cyclic GMP phosphodiesterase (PDE)
activation.
[0097] Additionally, the methods involve testing the small
molecules for their specificity by measuring their effects using
another GPCR, for example, human thrombin receptor (PAR1), which
also has been shown to couple to Gt. Seibert et al., Vision Res.
42:517,1999. Enhancement of the sensitivity of vision in vivo can
be tested according to a method of the invention using
electroretinography (ERG) of wild type and mutant mice. Chang et
al., Vision Res. 42:517-525, 2002. The inventive methods also
optionally involve optimizing the chemical structure of enhancers
or antagonists, performing pharmacokinetic, toxicological and
metabolism studies of the discovered chemical entities, and large
animal efficacy studies, and clinical trials for a pharmacological
treatment for night blindness. Therefore, the methods of the
invention can be used, for example, to identify small molecules
that enhance the binding of the high affinity Gt peptides to light
activated rhodopsin, determine whether the small molecules enhance
rhodopsin signaling in vitro, determine if the small molecules are
specific for rhodopsin, or if they can enhance other GPCR-G protein
signaling events, and test the small molecules in a mouse model for
stationary night blindness for increased sensitivity of vision as
measured by ERG.
[0098] A first step was to identify peptides with sequences based
on the C-terminus of Gt that bind with high affinity to either
light-activated or dark-adapted rhodopsin. These peptide analogs
were then tested for their specificity to binding to rhodopsin
versus other GPCRs, as well as their ability to stabilize the MII
conformation of the receptor. The methods of the invention also
identify small molecules that bind light-activated rhodopsin and by
doing so enhance the binding of the high affinity peptide analogs.
The binding affinity (EC.sub.50) of the compound is the first
criterion of a successful drug candidate. The identified small
molecules are tested in vitro for their ability to enhance
rhodopsin signaling using assays such as MII stabilization and MII
decay, GTP.gamma.S binding, and PDE activation.
[0099] Upon activation, rhodopsin undergoes a conformational change
that allows its interaction with and activation of Gt, leading
ultimately to the stimulation of PDE. The binding of Gt to
light-activated rhodopsin induces a high affinity receptor state
that can be measured spectrophotometrically by stabilization of the
active, signaling metarhodopsin II state of the receptor. Using a
split-beam SLM Aminco DW2000 spectrophotometer, for example, one
can determine if the receptor undergoes proper conformational
changes following light activation. This assay shows the small
molecule acting on the conformation of the receptor. If rhodopsin's
active intermediate, metarhodopsin II (MII), is stabilized by the
presence of the small molecule, the activation energy of the
receptor is lowered. Using the inventive assay system, compounds
were identified that allow the receptor to enter the active, MII
conformation without hetereotrimeric G protein, which normally is
required. The "enhancers" stabilize the active (signaling)
conformation of the receptor. "Inhibitors" block the binding of
transducin to rhodopsin and thus inhibit the receptor from entering
the proper conformation even in the presence of agonist (light) and
G protein.
[0100] Metarhodopsin II decay can be used to examine the
differences in compound potencies are due to changes in MII decay.
It could be postulated that differences could be due to effects of
a compounds to non-specifically attach the retinal Schiff's base
linkage of MII. Thus, one can compare the time dependent MII decay
in the presence of the individual compounds. In the process of
receptor activation, the G.alpha.t subunit binds a GTP molecule.
GTP.gamma.S binding assays can determine the ability of the
receptors to signal, with an increase in GTP.gamma.S binding
indicating receptor-mediated release of the GDP from the .alpha.
subunit and subsequent binding of GTP. Conversion of inactive
transducin (Gt.cndot.GDP) to the active state (Gt.cndot.GDP) is
accompanied by dissociation of the G.alpha. from GB.gamma.. The
free G.alpha.t.cndot.GTP then activates cGMP phosphodiesterase
(PDE) by binding to and dissociating its two inhibitory .gamma.
subunits. As a result, the released catalyzing .alpha. and .beta.
subunits of activated PDE (PDE*) can convert cGMP to GMP.
Therefore, compounds which affect rhodopsin signaling can be tested
for their affects on PDE assays.
[0101] Generally, small molecules that display an appropriate dose
curve when used to compete off the high affinity peptide fusion
proteins, with a resulting EC.sub.50<100 .mu.M for binding to
rhodopsin are suitable for continued study and are tested for the
ability to stabilize MII. Preferably, those with an EC.sub.50<10
.mu.M for MII stabilization are analyzed further. Further analysis
may include thermal stability of rhodopsin in the presence of the
small molecules (MII decay), and GTP.gamma.S binding (an assay for
the small molecule's effects on function). The rate of
GTP.gamma.S-binding is controlled by a rate-limiting GDP release of
G.alpha. subunits. Native G.alpha.t, in the presence or absence of
G.beta..gamma.t, displays very slow intrinsic rates of GDP release.
Therefore, an increase in guanosine 5'-O-(3-[.sup.35S]thio)
triphosphate(GTP.gamma.S) binding indicates receptor mediated
release of the GDP from the .alpha. subunit and subsequent binding
of the GTP.
[0102] GTP.gamma.S binding assays may be performed as follows or
using any method known in the art. G.alpha. subunits (1 mM) alone,
or G.alpha. subunits in the presence of the small molecules to be
assayed are mixed with 2 mM G.beta..gamma.t or 2 mm G.beta..gamma.t
and urea-washed ROS membranes (500 nM rhodopsin) and incubated for
3 min at 25.degree. C. Binding reactions may be started by addition
of 5 mm [.sup.35S]GTP.gamma.S (0.1 mCi). Aliquots of 50 .mu.l are
withdrawn at several timepoints, mixed with 1 ml ice-cold 20 mm
Tris.2HCl (pH 8.0) buffer containing 130 mM NaCl and 10 mM
MgSO.sub.4 and passed through Whatman.TM. cellulose nitrate filters
(0.45 mm). The filters are washed three times with the same buffer
(3 ml, ice-cold) and counted in a liquid scintillation counter
after dissolution in 3a70B mixture. See Skiba et al., J. Biol.
Chem. 271: 413-424, 1996 for exemplary methods which may be used
with the invention. The skilled person will recognize variations
and adjustments which may be made to the assay, and such variations
are considered within the scope of this invention. The k.sub.app
values for the binding reactions may be calculated by fitting the
data to the equation, GTP.gamma.S bound (% bound)=100%
(1-e.sup.-kt). The small molecule(s) also may be tested for the
ability to affect PDE activation. Gt.alpha. binding to PDEy
relieves the inhibitory effect of the gamma subunit on the
catalytic .alpha. and .beta. subunits of PDE and allows the
hydrolytic activity of these subunits to be increased almost 300
fold.
[0103] Activation of Gt.alpha. by rhodopsin can be monitored in the
presence or absence of the small molecules using fluorescence
spectroscopy at 20.degree. C. as described by Cerione, Methods
Enzymol. 237:409-423, 1994. This assay measures the
G.alpha.:GTP.gamma.S (complex between a subunit of transducin and
GTP.gamma.S) formation rate catalyzed by wild-type rhodopsin upon
illumination. The excitation wavelength is 295 nm (2 nm bandwidth),
and fluorescence emission is monitored at 340 nm (12 nm bandwidth).
Briefly, rhodopsin (40 nM) is added to a solution of Gt (250 nM) in
a reaction mixture containing 10 mM Tris (pH 7.2), 2 mM MgCl.sub.2,
100 mM NaCl, 1 mM DTT, and 0.01% n-dodecyl .beta.-maltoside. The
solution is stirred for 300 sec to equilibrate. GTP.gamma.S (5
.mu.M) is added to the reaction mixture to a final concentration of
5 .mu.M, and the increase in fluorescence is followed for and
additional 2000 sec. To calculate the activation rates, the slopes
of the initial fluorescence increase after GTP.gamma.S addition
were determined through the data points covering the first 60 sec.
The values in the presence of the small molecules may be normalized
to the value obtained for wild-type rhodopsin with no compounds
taken as 1.00. Those molecules which appear to be acting directly
on rhodopsin in these assays, or variations on these assays readily
apparent to the person of skill in the art are taken to the next
level of testing. The small molecules also are assayed for the
ability to modulate rhodopsin-transducin signaling specifically
without affecting processes mediated by other GPCRs.
[0104] Preferred small molecule "enhancers" and "inhibitors" are
uniquely specific, not only for the receptor, but for the
receptor-G protein interaction. As there are over 1000 GPCRs, and
no simple way to determine the effect of compounds on each and
every one of them individually, a few select and representative
GPCR signaling systems may be tested. Functional coupling of the
human thrombin receptor (PAR1) with Gt has been demonstrated.
Seibert, Eur. J. Biochem. 266(3):911-916, 1999. Testing for effects
on PAR1 may include determining if the small molecule(s) have an
effect on thrombin-mediated signal transduction events such as
adenylyl cyclase activity, calcium influx, and inositol phosphate
accumulation. Other tests for functional coupling to PAR1 or other
GPCRs are known in the art and may be used as well.
[0105] Adenylyl cyclase activity may be measured in a final volume
of 50 .mu.l with [.alpha.-.sup.32P]ATP (1 mM; 120-400 cpm/pmol) as
the substrate and [2,8-.sup.3H]cAMP (2 mM; 200,000 cpm/pmol) to
monitor recovery in an assay mixture containing 5 mM MgCl.sub.2, 1
mM EDTA, 1 mM 2-mercaptoethanol, 100 .mu.M papaverine, 1 pg/ml
bovine serum albumin, and an ATP-regenerating system consisting of
20 mM creatine phosphate and 120 units creatine phosphokinase/ml in
25 mM Tris-HCl buffer, pH 7.5. The concentration of ATP and cAMP
may be determined spectrophotometrically at 259 nm, using .epsilon.
values of 15.4 and 14.6 mM.sup.1cm.sup.-1, respectively. The assay
may be initiated by addition of protein and after a 10 min
incubation at 37.degree. C. the reaction is stopped with 2 volumes
stop solution (2% SDS/45 mM ATP/13 mM cAMP). The samples then may
be heated (e.g. to 100.degree. C. for 3 minutes) and the formed
cyclic [.alpha.-.sup.32P]AMP recovered. See Gilchrist et al., J.
Biol. Chem . 276:25672-25679, 2001, the disclosures of which are
hereby incorporated by reference. Finally, the compounds passing
the previous steps may be tested in an animal model of night
blindness as described by Chang et al., Vision Res. 42:517-525,
2002, the disclosures of which are hereby incorporated by
reference.
[0106] To assess the effects of small molecules on photoresponses
in an in vivo system, electroretinography of mice exposed to the
small molecule(s) may be used to measure the amplitude of both the
a- and b-waves. Plots of the amplitude against the logarithm of
relative light intensities indicate if the compounds are affecting
only rod signaling. The sensitivity for eliciting a threshold b
wave within normal limits also may be measured. Mutant animals also
may be tested to observe not only the effects of enhancers on wild
type rhodopsin signaling, but also the effects on animals with
night blindness.
[0107] The small molecules to be tested may be dissolved in sterile
PBS and administered as eye drops. Experiments may be repeated
using IV or IM injections if initial results are negative. The most
promising candidates undergo the steps needed to take them from an
identified compound to a lead compound. This approach identifies a
pharmacological treatment for night blindness which circumvents the
need for more invasive procedures such as gene therapy, laser
ablation and retinal replacement.
[0108] Mapping the sites of interaction between proteins involves
identifying parts of the interface between two proteins using
synthetic peptides corresponding to interfacial regions. The
peptides are identified because they act as competitive inhibitors
of the interaction. NMR studies of peptide structures in their
bound conformation using trNOESY, combined with analysis of
activity of substituted peptide analogs to define the minimal
structural requirements for interaction were used to understand the
structural basis of rhodopsin-transducin interaction as well as G
protein-effector interaction. Peptides corresponding to the
C-terminus of Gt can be used to stabilize rhodopsin in its active
conformation (MII) or in an inactive conformation. The
3-dimensional structures of heterotrimeric G proteins reveals that
the last 7 amino acids of G protein .alpha. subunits are
unstructured, indicating that this region of the .alpha. subunit is
critical for binding to the cytoplasmic surface of an activated
receptor with induced fit. This interaction is quite specific since
a difference in a single amino acid can affect the affinity by 1000
fold.
[0109] High throughput screening is a recent technology that has
been developed primarily within the pharmaceutical industry. It has
emerged in response to the profusion of new biological targets and
the need of the pharmaceutical industry to generate novel drugs
rapidly in a changed commercial environment. Its development has
been aided by the invention of new instrumentation, by new assay
procedures, and by the availability of databases that allow huge
numbers of data points to be managed effectively. High throughput
screening combined with combinatorial chemistry, rational design,
and automation of laboratory procedures has led to a significantly
accelerated drug discovery process compared to the traditional
one-compound-at-a-time approach. Screens may be performed manually,
however robotic screening of the compound libraries is preferred as
a time- and labor-saving device.
[0110] One critical aspect of the drug discovery process is the
identification of potent lead compounds. A purely random selection
of compounds for testing is unlikely to yield many active compounds
against a given receptor. Typically, pharmaceutical companies
screen 100,000 or more compounds per screen to identify
approximately 100 potential lead compounds. On average, only one or
two of these compounds actually produce lead compound series.
Therefore, companies have been assaying larger and larger data sets
in the search for useful compounds. Compound accessibility then
becomes an issue: historical compound collections are limited in
size and availability. In contrast, large combinatorial chemistry
libraries can be synthesized on demand, but at significant
technical difficulty and cost. As the library sizes expand, the
difficulty becomes selecting the desired compounds from these very
large combinatorial libraries. When literally hundreds of thousands
of compounds are screened, it makes characterizing the candidate
lead compounds an expensive and time-consuming process,
particularly when many of the "hits" turn out to be false
positives.
[0111] The multi-step approach to the drug discovery process
described here provides a solution to many of these problems. One
embodiment of this invention takes advantage of the properties of G
protein .alpha. subunit carboxyl termini to identify peptides which
act as high affinity, competitive inhibitors or agonists of G
protein/GPCR interactions. The method, however, can be used with
any specific protein-protein, protein-small molecule,
protein-nucleic acid interaction or the like. In addition, peptides
based on any region of a G.alpha. subunit or any region of a
G.beta..gamma. dimer which is involved in GPCR binding may be used
in the same way. Many such GPCR binding regions are known in the
art. The identification of high affinity competitors forms a first
step in a screening and selection method which overcomes many of
the disadvantages of high throughput screening by providing
specific, high affinity lead compounds against which to test
potentially useful pharmaceuticals. Because peptides selected by
this method have affinity for their binding partner up to 1,000
times higher or more than the native protein, this step is one key
to successfully screening and identifying useful pharmaceutical
compounds.
[0112] A subsequent step of the preferred process involves high
throughput screening of candidate peptide or small molecule
pharmaceutical compounds against the high affinity lead peptides
identified in the first step. Because the lead peptide compounds
are potent and specific binders to the desired receptor, screening
assays test for compounds that can decrease the binding of the
peptide ("blockers") or that increase the binding of the peptide
("agonists"). The assay system allows one to measure both binding
and function simultaneously as the peptides all serve to mimic a
required step, that of specific receptor-G protein binding. By
using this site, the system facilitates identification of those
candidate compounds which bind not only with useful affinities (nM
to .mu.M range) but by the very virtue of their selection process
will affect function by either increasing or decreasing the G
protein binding. The high throughput screening step of the drug
discovery process is thereby greatly simplified, because the number
of false positive compounds, and compounds which are identified as
binders but which bind only with low affinity, is reduced or
virtually eliminated. Only those compounds with a high chance of
success will be identified by the screen, therefore useful
compounds can be identified directly and there are many fewer
compounds which need to be characterized and further studied to
confirm that the compounds are specific, potent pharmaceutical
compounds. In addition, the method identifies a compound through
binding directly to the precise site of interest, so that the
mechanism of binding and the mechanism of action of the newly
identified pharmaceutical compound does not have to be discovered
and confirmed later by a separate process.
[0113] The identified high affinity peptides also may be used
according to the inventions to identify GPCR inverse agonists. High
affinity peptides identified in a first step of the inventive
method bind the receptor and stabilize it in an active or R*
conformation. Screens which are used to identify potent agonists
seek out compounds which can compete with this binding and also
stabilize the GPCR in its R* state. Inverse agonists, on the other
hand, stabilize the GPCR in an inactive or R state. Therefore,
screens designed to detect dissociation of the high affinity
peptide or a decrease in its affinity for the GPCR are used to
identify inverse agonists.
[0114] Although this description provides examples relative to the
interaction between a G protein coupled receptor and its cognate
G.alpha. protein, the methodology can be used to identify peptide
inhibitors of most protein-protein interactions, specifically
including any interaction between a GPCR and any region of a
G.alpha. or G.alpha..gamma. G protein subunit. The high affinity
peptides selected by this method may be used in high throughput
screening to identify small molecules that can be used as
modulators of a variety of specific biological process.
[0115] To produce very high affinity peptide GPCR blockers, the
tertiary structure of a wild-type G.alpha. carboxyl terminal
peptide or any other GPCR binding peptide in its receptor-bound
conformation may be studied, for example, using trNOESY (NMR).
Dratz et al., Nature 363:276-280, 1993. Structural data derived
from these types of studies of G protein regions are combined with
analysis of activity of substituted peptide analogs to define the
minimal structural requirements for interaction of peptides or any
ligand with GPCR. The following experimental systems are examples
of systems which can be used to define receptor-G protein
interactions: (i) rhodopsin-transducin (G.alpha.t) in retinal rod
cells, (ii) .beta.-adrenergic receptor-G.alpha.s in C6 glioma
cells, (iii) adenosine A1 receptor-G.alpha.1 in Chinese hamster
ovary cells, (iv) GABA.sub.B receptors-G.alpha.1 in rat hippocampal
CA1 pyramidal neurons, (v) muscarinic M2 receptor-G.alpha.1 in
human embryonic kidney cells, and the like. Any GPCR or group of
GPCR which is convenient or desired can be used to define the
interaction requirements, and skilled workers are aware of many
methods to understand structure-activity relationships in receptor
binding of this kind. Any of these methods are contemplated for use
in these methods and may substitute for the particular methods of
the exemplified embodiment.
[0116] The plasmid display method provides an efficient means of
identifying specific and potent peptides that can serve as
competitive inhibitors of protein-protein interactions. Using the
information gleaned from structure-activity studies, a library of
variant peptides encoding sequences related to a GPCR-binding
region, for example the G.alpha. subunit carboxyl terminus, for
each of the classes of the G.alpha. subtypes or G.beta..gamma. can
be prepared. Exemplary native sequences upon which libraries may be
based include those listed in Table III, below. Libraries
advantageously contain peptides with computer-generated random
substitutions within the sequence, and allow one to test a large
number of peptide sequences at one time. Preferably, peptide
sequences in each library are constructed such that approximately
50% of the amino acid residues are identical to the native GPCR
binding region and the remaining amino acid residues are randomly
selected from any amino acid. The peptides may range in size from
about 7 to about 55 amino acid residues or from about 8 to about 50
amino acids long or from about 7 to about 70 amino acid residues or
longer, preferably from about 9 to about 23 amino acid residues or
about 9 to about 15 amino acid residues. Undecamer peptides are
most preferred. Libraries may be constructed in which about 10% to
about 90% of the amino acid residues unchanged from the native
sequence; however, about 30% to about 70% unchanged is preferred
and about 50% is most preferred.
[0117] Alternatively, a synthetic peptide library can be based on
any protein known to interact with a GPCR, using randomly created
overlapping regions of the protein. The peptides may be about 7-70
amino acids long or about 8-50 amino acids long or preferably about
9 to about 23 or about 9 to about 15 amino acids long and most
preferably about 11 amino acids long. Oligonucleotides encoding the
peptides advantageously may be cloned to the 3' end of the LacI
gene, with a linker sequence at the N-terminus of the peptide. The
linker sequence is not mandatory for successful screening, but is
generally preferred. Restriction enzyme sites may be placed at
either end of the peptide coding sequence for cloning purposes. See
Table I below for a schematic representation of a peptide library
and an example of one peptide. Additional peptides which also can
be used are shown in Tables II and III, below. The oligonucleotides
encoding the actual peptide sequences are synthesized with 70% of
the correct base and 10% each of the remaining bases, leading to a
biased peptide library with an approximately 50% chance of having
the correct amino acid at any specific position along the peptide
sequence. Different ratios of bases may be used to achieve the
desired mutagenesis rate at any particular position in the
sequence. TABLE-US-00001 TABLE I Example for Construction of a
Synthetic Peptide Library. (SEQ ID NO:13) Q R M H L R Q Y E L L
gaggtggt nnknnknnknnk attcgtgaaaacttaaaagattgtggtcgtttc taa
ctaagtaaagc A B C D E (SEQ ID NO:14) n = any nucleotide base; k =
guanidine or thymidine; A = restriction enzyme site; B = linker
sequence; C = oligonucleotide encoding peptide sequence; D = stop
codon; E = restriction enzyme site.
[0118] TABLE-US-00002 TABLE II G.alpha. Subunit Peptides and
Corresponding DNA Constructs. G.alpha. SEQ Subunit Sequence ID NO:
Gt I K E N L K D C G L F 15 atc aag gag aac ctg aaa gac tgc ggc ctc
ttc 16 Gi1/2 I K N N L K D C G L F 17 ata aaa aat aat cta aaa gat
tgt ggt ctc ttc 18 GRi1/2 N G I K C L F N D K L 19 aac ggc atc aag
tgc ctc ttc aac gac aag ctg 20 Gi3 I K N N L K E C G L Y 21 att aaa
aac aac tta aag gaa tgt gga ctt tat 22 Go2 I A K N L R G C G L Y 23
atc gcc aaa aac ctg cgg ggc tgt gga ctc tac 24 Go1 I A N N L R G C
G L Y 25 att gcc aac aac ctc cgg ggc tgc ggc ttg tac 26 Gz I Q N N
L K Y I G L C 27 ata cag aac aat ctc aag tac att ggc ctt tgc 28 G11
L Q L N L K E Y N L V 2 ctg cag ctg aac ctc aag gag tac aac ctg gtc
29 Gq L Q L N L K E Y N A V 30 ctc cag ttg aac ctg aag gag tac aat
gca gtc 31 Golf Q R N H L K Q Y E L L 32 cag cgg atg cac ctc aag
cag tat gag ctc ttg 33 G14 L Q L N L R E F N L V 34 cta cag cta aac
cta agg gaa ttc aac ctt gtc 35 G15/16 L A R Y L D E I N L L 36 ctc
gcc cgc tac ctg gac gag atc aac ctg ctg 37 G12 L Q E N L K D I M L
Q 38 ctg cag gag aac ctg aag gac atc atg ctg cag 39 G13 L N D N L K
Q L M L Q 40 ctg cat gac aac ctc aag cag ctt atg cta cag 41 Gs Q R
N H L R Q Y E L L 13 cag cgc atg cac ctt cgt cag tac gag ctg ctc 42
5'- gatccgccgccaccatggga- -tgaa-3' (SEQ ID NOS:43, 44)
[0119] TABLE-US-00003 TABLE III Exemplary Native C Protein
Sequences for Library/Minigene Construction.* SEQ SEQ ID ID Name
Sequence NO: Name Sequence NO: hGt IKENLKDCGLF 15 CryptoGbal
LQNALRDSGIL 62 hGi1/2 IKNNLKDCGLF 17 GA3_UST LTNALKDSGIL 63 G05_DRO
IKNNLKQIGLF 45 GA1_KLU IQONLKKSGIL 64 GAF_DRO LSENVSSMGLF 46
GA3_UST LTNALKDSGIL 63 Gi-DRO IKNNLKQIGLF 45 GA1_DIC NLTLGEAGMIL 64
hGi3 IKNNLKECGLY 21 GA2_KLU LENSLKDSGVL 65 hGO-1 IANNLRGCGLY 25
GA2_UST ILTNNLRDIVL 66 hGO-2 IAKNLRGCGLY 47 Mgs-XL QRMHLPQYELL 67
GAK_CAV IKNNLKECGLY 21 hGs QRMHLRQYELL 13 GO_XEN IAYNLRGCGLY 48
hGolf QRMHLKGYELL 68 GA3_CAEEL IQANLQGCGLY 49 GA1_COPCO LQLHLRECGLL
69 GA2_CAEEL IQSNLHKSGLY 50 GA1-SOL RRRNLFEAGLL 70 GA1_CAEEL
LSTKLKGCGLY 51 GA2_SB RRRNLLEAGLL 71 GAK_XEN IKSNLMECGLY 52 GA1_SB
RRRNPLEAGLL 72 GA1_CAN VQQNLKKSGIM 53 GA1_UST IQVNLRDCGLL 73 hGZ
IQNNLKYIGLC 27 GA4_UST RENLKLTGLVG 74 hG15 LARYLDEINLL 36 GA1_ORYSA
DESMRRSREGT 75 GA2_SCHPO LQHSLKEAGMF 54 GQ1_DROME MQNALKEFNLG 76
hG12 LQENLKDIMLQ 38 GA2_DIC TQCVMKAGLYS 77 hG13 LHDNLKQLMLQ 40
GS-SCH LQHSLKEAGMF 54 GAL_DRO LQRNLNALMLQ 55 GA-SAC ENTLKDSGVLQ 56
GA2_YST ENTLKDSGVLQ 56 GA1-CE IISASLKMVGV 78 hG14 LQLNLREFNLV 34
GA2-CE NENLRSAGLHE 79 hG11 LQLNLKEYNLV 2 GA3-CE RLIRYANNIPV 80 hGQ
LQLNLKEYNAV 30 GA4-CE LSTKLKGGGLY 51 GQ_DROME LQSNLKEYNLV 57 GA5-CE
IAKNLKSMGLC 81 G11_XEN LQHNLKEYNLV 58 GA6-CE IGRNLRGTGME 82
Gq_SPOSC IQENLRLCGLI 59 GA7-CE IQHTMQKVGIQ 83 GA1_YST IQQNLKKIGII
60 GA8-CE IQKNLQKAGMM 84 GA1_NEUCR IIQRNLKQLIL 61 GAS-DIC
LKNIFNTIINY 85 *For production of minigene constructs each
nucleotide sequence should be constructed to encode the amino acids
MG at the N-terminus of the peptide by using
5'-gatccgccgccaccatggga-(SEQ ID NO:43) and -tgaa-3' (SEQ ID
NO:44).
[0120] The peptides advantageously are synthesized in a display
system for convenience and efficiency of performing the binding
reactions. For example, plasmid or phage display systems, as are
known in the art, may be employed. While peptide display systems
are preferred, any method which allows efficient contact of the
peptides with a GPCR and determination of binding may be used.
[0121] A peptide display ("peptides on plasmids") library is a
convenient system for use with this invention which exploits the
high affinity bond between LacI and lacO. The "peptides on
plasmids" display is preferred for use with this invention for two
major reasons. The technique is easily set up in the laboratory. In
addition, the fusion of the peptide at the carboxyl terminus of the
presenting protein mimics the normal presentation for carboxyl
terminal peptides during the screen. If amino terminal or interior
peptides are being tested, the peptide may be cloned at the
appropriate position to mimic native presentation.
[0122] The "peptides on plasmids" method for testing carboxyl
terminal peptides generally works as follows. Persons of skill in
the art will be able to modify these methods as needed to
accommodate different conditions using this general description and
the examples below as a guide. A library of peptides is created by
degenerate PCR based on the native GPCR-binding peptide of interest
and fused to the carboxyl terminus of LacI. The peptide library is
expressed via a plasmid vector carrying the fusion gene. The
plasmid also contains the Lac operon (LacO), and when E. coli
transcribes and translates the Lacl fusion protein, it binds back
as a tetramer to the encoding plasmid through its lacO DNA binding
sequence, displaying the inserted sequences of interest on the
plasmid. Following transcription and translation, variant peptides
encoding different sequences related to the native peptide sequence
therefore are displayed as carboxyl terminal extensions of the lacl
gene. Thus, a stable LacI-peptide-plasmid complex is formed which
can be screened for binding to receptor. Methods described in Gates
et al., J. Mol. Biol. 255:373-386, 1996, the disclosures of which
are hereby incorporated by reference, are suitable. See Examples 7
and 9 for exemplary methods.
[0123] The E. coli strain used to display the peptides was ARI814,
which has the following genotype: .DELTA.(srl-recA) endA1 nupG
lon-11 sulA1 hsdR17 .DELTA. (ompT-fepC)266 .DELTA.clpA319::kan
.DELTA.lacI lacZU118. The strain contains the hsdR17 allele that
prevents restriction of unmodified DNA introduced by transformation
or transduction. The ompT-fepC deletion removes the gene encoding
the OmpT protease, which digests peptides between paired basic
residues. The lon-11 and clpA mutations also limit proteolysis by
ATP-dependent, cytoplasmic proteases. The deletion of the lacI gene
prevents expression of the wild-type lac repressor, which would
compete with the fusion constructs for binding to the lacO sites on
the plasmid. The lacZ mutation prevents waste of the cell's
metabolic resources to make .beta.-galactosidase in the absence of
the repressor. The endA1 mutation eliminates a nuclease that has
deleterious effects on affinity purification and the recA deletion
prevents multimerization of plasmids through RecA-catalyzed
homologous recombination. This strain was selected also for its
robust growth properties and high yields of immunocompetent cells.
Transformation efficiencies of 2.times.10.sup.10 colonies per mg
DNA typically were achieved. Although this strain of E. coli is
preferred, those of skill in the art are aware of many alternatives
which are convenient for use with the methods described. Therefore,
any suitable and convenient bacterial strain known in the art is
contemplated for use with this invention.
[0124] The LacI-peptide fusion protein library may be released from
the bacteria by gentle enzymatic digestion of the cell wall using
lysozyme. After pelleting the cell debris, the lysate then can be
added directly to immobilized receptor for affinity purification or
used without purification. The display library of these peptides is
screened to identify those peptides which bind with high affinity
to a particular GPCR. In this way, it is possible to screen for and
identify high affinity peptides which bind GPCR and can interfere
with or enhance activation of the pre-selected specific G protein.
The library can be screened against any desired GPCR. Since the
combinatorial library contains peptides based on a particular
G.alpha. or G.beta..gamma. subunit, any GPCR which binds to or
mediates signaling through that subunit or class of subunits can be
used. Multiple libraries, based on the carboxyl terminal sequences
or other regions of different G protein subunits may be constructed
for screening the same or different GPCR.
[0125] To screen the plasmid display library, a G protein coupled
receptor of interest advantageously may be immobilized on
microtiter plates for screening by ELISA. A plasmid preparation
(bacterial lysate) then may be added to the wells. This screening
procedure, involving allowing the peptides displayed on the library
plasmids to bind receptor, is sometimes referred to as "panning."
Sequences that bind the receptor stick to the well so that
non-binding sequences can be removed by a washing step. The
adherent plasmids then can be expanded and used to transform E.
coli. The "panning" process generally is repeated 2 to 8 times. In
general, however, 3 to 4 sequential screens are sufficient and
preferred. In the later rounds of panning, parent peptide (wild
type sequence) preferably is co-incubated with the plasmid
preparation to bind receptors and serve as a competitive inhibitor.
In this way, only high affinity sequences on the display library
are captured by the immobilized receptor. The same competitive
inhibition advantageously may be performed using a high affinity
peptide or small molecule which has already been identified, rather
than the native peptide. See FIG. 1 for a schematic diagram
generally describing the "panning" procedure and Example 7 for a
specific embodiment. The selection process in this embodiment
preferably is carried out in low salt buffers because high salt
concentrations destabilize the Lacl-lacO complex, and could lead to
peptides becoming associated with the incorrect plasmid. For the
same reason, the panning buffers preferably contain lactose, which
causes the Lacl to bind more tightly to lacO.
[0126] The selection process of this invention allows the
identification of peptide sequences with higher and higher affinity
binding with each round of panning. For example, diversity in an
unpanned library may look like the sequences given in Table IV,
below, i.e. highly randomized. After successive rounds of
selection, the selected adherent peptides would look more like
those given in Table V, below. TABLE-US-00004 TABLE IV Diversity in
Unpanned Gq Library. SEQ. ID NO. Native LQLNLKEYNLV 2 clone #1
LLLQLVEHTLV 86 clone #2 HRLNLLEYCLV 87 clone #3 EQWNMNTFHMI 88
clone #4 SQVKLQKGHLV 89 clone #5 LRLLL*EYNLG 90 clone #6
RRLKVNEYKLL 91 clone #7 LQLRLREHNLV 92 clone #8 HVLNSKEYNQV 93
[0127] TABLE-US-00005 TABLE V Selection in Panned G.alpha.ll
Library. SEQ ID NO. Native LQLNLKEYNLV 2 Round 1 1 MKLNVSESNLV 94 2
LQTNQKEYDMD 95 3 LQLNPREDKLW 96 4 RHLDLNACNMG 97 5 LR*NDIEALLV 98 6
LVQDRQESILV 99 Round 2 1 LQLKHKENNLM 100 2 LQVNLEEYHLV 101 3
LQFNLNDCNLV 102 4 MKLKLKEDNLV 103 5 HQLDLLEYNLG 104 6 LRLDFSEKQLV
105 Round 3 1 LQKNLKEYNMV 106 2 LQYNLMEDYLN 107 3 LQMYLRGYNLV 108 4
LPLNPKEYSLV 109 5 MNLTLKECNLV 110 6 LQQSLIEYNLL 111
[0128] LacI is normally a tetramer and the minimum functional DNA
binding species is a dimer. Thus, the peptides are displayed
multivalently on the fusion protein, leading to binding to the
immobilized receptor in a cooperative fashion. This cooperative
binding permits the detection of binding events of quite low
intrinsic affinity. The sensitivity of the assay is an advantage in
that initial hits of low affinity can be identified, but the
disadvantage is that the signal in the ELISA does not necessarily
correlate with the intrinsic affinity of the bound peptides.
[0129] One preferred ELISA, where signal strength is better
correlated with affinity, involves fusing the sequences of interest
from a population of clones in frame with a gene encoding a
protein, for example E. coli maltose binding protein (MBP). Once
the sequences have been transferred into the monomeric fusion
protein, they can be overexpressed in E. coli and used as either
crude lysates or purified fusion proteins for assay by an ELISA
which detects the protein bound to receptor or any convenient
assay. Controls having the vector alone which expresses TGGG linker
only fused to MBP, or having Gt:340-350K341R peptide fused to MBP
may be used, if desired. Frozen cell stocks preferably are kept in
25% glycerol at -80.degree. C. The high affinity G.alpha. peptides
fused to MBP preferably are analyzed by ELISA, where the resulting
signal correlates to the peptide's affinity for light-activated
rhodopsin. The MBP-peptide fusions are expressed and purified over
an amylose affinity column and used to measure the relative
affinities of peptides of interest. Those samples with an
absorbance of at least two standard deviations above background may
be considered to contain high affinity binding peptides. Any
desired cut-off point may be used, however, depending on the assay
parameters and the needs of the operator.
[0130] A suitable ELISA may be performed as follows, however those
of skill in the art will be able to modify the techniques for the
conditions in their assays. Serial dilutions of MBP-peptide fusion
proteins are added to 96-well plates with immobilized
light-activated rhodopsin previously blocked with 0.1% BSA. After 1
hour at 37.degree. C., the wells are washed with phosphate buffered
saline (PBS)/0.l% Tween 20, and probed with an anti-MBP antibody,
followed by a goat-anti-rabbit antibody conjugated to horseradish
peroxidase. Color development of the assay is allowed to proceed
for 20 minutes, after which the A.sub.450 is measured on a
microtiter plate reader. See Gilchrist et al., Methods Enzymol.
315:388-404, 2000, the disclosures of which are hereby incorporated
by reference.
[0131] The purified fusion proteins can be further tested by
measuring their ability to compete for the site of binding on the
receptor using native peptide, a LacI-peptide fusion protein, or
heterotrimeric G protein. Use of competitive ELISA allows one to
calculate IC.sub.50 values for the binding of individual fusion
protein to the immobilized receptor.
[0132] Peptide fusion proteins can be analyzed in a competitive
ELISA format using a fusion protein co-incubation to prevent the
binding of lower affinity peptide fusion proteins to the GPCR. Any
convenient protein which does not interfere with peptide binding
may be used, including for example, glutathione-S-transferase,
green fluorescent protein or ubiquitin, however a maltose binding
protein fusion protein such as MB-G.alpha..sub.t340-35OK341R is
preferred. Competitive ELISA indicates which peptide sequences have
the highest affinity for light activated rhodopsin. Several
different assay formats are suitable. For example, synthetic
Gt:340-350K341R peptide may be used to compete with the MBP fusion
proteins containing the G.alpha. high affinity peptides for
binding. In addition, MBP fusion proteins containing the G.alpha.
high affinity peptides may be used to compete with
LacI-Gt:340-350K341R peptide fusion protein for binding to
light-activated rhodopsin. Recombinant heterotrimeric Gt also may
be tested against the high affinity peptides. The relative affinity
of the variant peptides may be assessed using an ELISA format where
a constant concentration of MBP-G.alpha. peptide fusion proteins is
competed by serial dilutions of native peptide, LacI-G.alpha.t
peptide fusion protein or recombinant heterotrimeric Gt. The wells
advantageously may be probed with an anti-MBP antibody to measure
the amount of MBP-G.alpha. peptide fusion protein remaining bound.
The dose-response curves may be analyzed by non-linear regression
to calculate an EC.sub.50.
[0133] Cloning the library into pJS142 creates a BspEI restriction
site near the beginning of the random coding region of the library.
Conveniently, digestion with BspEI and ScaI allows the purification
of a 900 base pair DNA fragment that may be subcloned into pELM3, a
vector that directs the MBP fusion protein to the cytoplasm, a
reducing environment. Alternatively, the fragment can be cloned
into pELM15, a vector which directs the MBP fusion protein to the
periplasm, an oxidizing environment. pELM3 and pELM15 are simple
modifications of the pMALc2 and pMALp2 vectors, respectively,
available commercially (New England Biolabs). Digestion of pELM3
with AgeI and ScaI allows efficient cloning of the BspEI-ScaI
fragment from the pJS142 library. Any suitable method may be used
which is convenient to achieve the desired result. Modifications of
these methods are well known by those of skill in the art of
molecular biology and are contemplated for use here.
[0134] Proof that the high affinity peptides competitively bind to
GPCR and interfere with or enhance its recognition of G protein can
be obtained using a competitive binding assay in the presence of a
heterotrimeric G protein. For example, if rhodopsin is the GPCR
used in the screen, heterotrimeric G protein, transducin (Gt) may
be used. Gt binds rhodopsin with multiple epitopes and is
membrane-bound via myristoylation of the .alpha. subunit and
farnesylation of the .gamma. subunit carboxyl terminus. Poor
binding by carboxyl terminal native peptide LacI constructs and/or
heterotrimeric Gt indicates high affinity binding of the
MBP-peptide fusion proteins. An analogous strategy of panning,
peptide synthesis and binding studies may be employed for
determining high affinity peptides that bind any GPCR, for example
the thrombin receptors (PAR1, PAR3, PAR4), dopamine receptors (D1,
D2, D3, D4, D5), vasopressin receptors (V1a, V1b, V2) and histamine
receptors (H1, H2, H3), using carboxyl terminal peptide libraries
for any G.alpha. subunit, for example G.alpha.i, G.alpha.s and
G.alpha.q. Once peptide analogs with higher binding affinities have
been elucidated, they can be exploited to inhibit GPCR-G protein
interaction.
[0135] The peptides selected by this method, characterized by high
affinity, specific blockade of or enhancement of a desired
GPCR-mediated signaling event, may be used as therapeutic agents
such as traditional pharmaceuticals or gene therapies to treat
disorders which would benefit by modifying GPCR activity or used to
screen additional libraries of compounds able to compete with the
high affinity peptide analogs or to modulate (i.e., increase or
decrease) the binding affinity of the high affinity peptide analogs
or the high affinity peptide analog-fusion proteins.
[0136] Any method known in the art for selecting and synthesizing
small molecule libraries for screening is contemplated for use in
this invention. Small molecules to be screened are advantageously
collected in the form of a combinatorial library. For example,
libraries of drug-like small molecules, such as .beta.-turn mimetic
libraries and the like, may be purchased from for example ChemDiv,
Pharmacopia or Combichem or synthesized and are described in Tietze
and Lieb, Curr. Opin. Chem. Biol. 2:363-371, 1998; Carrell et al.,
Chem Biol. 2:171-183, 1995; U.S. Pat. Nos. 5,880,972, 6,087,186 and
6,184,223, the disclosures of which are hereby incorporated by
reference.
[0137] Any of these libraries known in the art are suitable for
screening, as are random libraries or individual compounds. In
general, hydrophilic compounds are preferred because they are more
easily soluble, more easily synthesized, and more easily
compounded. Compounds having an average molecular weight of about
500 often are most useful, however, compounds outside this range,
or even far outside this range also may be used. Generally,
compounds having c logP scores of about 5.0 are preferred, however
the methods are useful with all types of compounds. Simple filters
like Lipinski's "rule of five" have predictive value and may be
used to improve the quality of leads discovered by this inventive
strategy by using only those small molecules which are
bioavailable. See Lipinski et al., Adv. Drug Delivery Rev. 23:3-25,
1997.
[0138] Combinatorial chemistry small molecule "libraries" can be
screened against drug targets. The idea is that diversity of
chemical structures increases the chances of finding the needle in
the 10.sup.200 possible small organic molecule haystack. These
collections provide an excellent source of novel, readily available
leads. For example, ChemDiv uses more than 800 individual chemical
cores, a unique Building Block Library, and proprietary chemistry
in designing its Diversity Collections (small molecule libraries)
to assemble 80,000-100,000 compounds a year. CombiLab lead library
sets of 200-400 compounds also can be produced as a follow-up. In
addition, ChemDiv's compounds are designed to ensure their
similarity to drugs adjusted according to proprietary algorithms of
"drug-likeness definitions" (group similarity and advanced neural
net approaches), and a variety of intelligent instruments for
ADME&T (Absorption, Distribution, Metabolism, Excretion and
Toxicity) properties prediction, such as partition coefficient,
solubility, dissociation coefficients, and acute toxicity.
[0139] Thus, focused synthesis of new small molecule libraries can
provide a variety of compounds structurally related to the initial
lead compound which may be screened to choose optimal structures.
Preferably, a library of compounds is selected that are predicted
to be "drug-like" based on properties such as pKa, log P, size,
hydrogen bonding and polarity. The inventive multi-step approach
which yields high affinity peptides in the first step, and small
molecules in a subsequent step reduces the number of artificial
hits by eliminating the lower affinity small molecules that would
be selected and have to be assayed in a normal high throughput
screening method. In addition, it focuses the search for molecules
that can modulate the binding of a peptide the mimics the G protein
rather than screening for binding to any site on the receptor.
Other advantages of this technology are that it is simple to
implement, amenable to many different classes of receptors, and
capable of rapidly screening very large libraries of compounds.
[0140] Screening of the peptides or small molecules may be
performed conveniently using receptors from any source. Generally,
it is convenient to purify receptor from cells and reconstitute the
receptor in lipid vesicles or to use membranes isolated from insect
or mammalian cells that express or overexpress the receptor. PAR1
and rhodopsin are convenient receptors, however any suitable
receptor is contemplated for use with this invention. The receptors
used for screening may be purified from a natural source or
purified from cells which overexpress the receptor and
reconstituted in lipid vesicles. Membranes containing the receptor
may be prepared from cells which natively express the receptor, for
example Sf9 cells which express PAR1, or from cells which have been
genetically engineered to express the receptor, for example
mammalian or insect cells overexpressing PAR1. Peptides identified
from screening a receptor (PAR1) expressed by three different
methods are shown in Tables XI, XII, and XIII. The results indicate
the methods give similar results showing a high degree of
conservation, (N348; L349) being identified for all three methods
of receptor expression. Initially, it is advantageous to determine
the binding affinity of the peptide fusion protein or high affinity
peptide against which the peptides or small molecules are screened.
This allows the amount of receptor and peptide MBP peptide fusion
protein or small molecule in the assay to be optimized.
[0141] Generally, it is convenient to test the libraries using a
one well-one compound approach to identify compounds which compete
with the peptide fusion protein or high affinity peptide for
binding to the receptor. A single compound per well can be used, at
about 1 .mu.M each or at any convenient concentration depending on
the affinity of the receptor for the compounds and the peptide
against which they are being tested. Compounds may be pooled for
testing, however this approach requires deconvalution. Compounds
may be pooled in groups of about 2 to about 100 compounds per well,
or more, or about 10 to about 50 compounds per well at about 10 nM
each or at any convenient concentration depending on the affinity
of the receptor for the compounds being tested. Several different
concentrations may be used if desired. Peptides desirably are
screened using a pooled approach because of the layer members of
peptides which are screened in the first instance. Peptides may be
screened individually as well, but preferably are screened in pools
of about 10.sup.4-10.sup.12 peptides per well or about
10.sup.8-10.sup.10 peptide per well or most preferably about
10.sup.9 peptides per well.
[0142] ELISA, or any other convenient assay, such as fluorescence
assays or radioimmunoassay may be used to determine (1) if one or
more peptides in each well reduce the amount of binding by the high
affinity peptide fusion protein or high affinity peptide, or (2) if
one or more peptides in each well bind to the receptor. Compounds
may be tested at a series of concentrations as well, and this
generally is preferred if the affinity of the peptide or peptide
fusion protein is not known. In an ELISA, wells in which the
OD.sub.450 is half or less than half than that of control wells (no
tested compounds) generally are considered "positive" and may be
further studied. Any suitable cut-off point may be used, however,
depending on the assay components and the goals of the assay.
[0143] Screening against the high affinity peptide analogs can be
performed using the desired GPCR immobilized onto microtiter wells,
biochips, or any convenient assay surface. Binding assays performed
in solution also are suitable. One, several, or thousands of
candidate small molecule pharmaceutical compounds can be screened
for binding to the receptor in the presence or absence of a high
affinity peptide analog. The assays preferably are performed in the
presence of a high affinity binding peptide to ensure that only
those candidate compounds which can successfully compete for
binding against the high-affinity binding peptide will be captured
by the receptor. Alternatively, organic compounds or small
molecules which have been identified by screening as competitively
binding in the presence of a high affinity peptide analog also may
be used as lead compounds in screening for further small molecule
candidate compounds with even higher affinity. In either screening
process, binding may be detected by any convenient method, for
example by ELISA, fluorescence assays or radioimmunoassays.
[0144] By using a two-step protocol to identify compounds which
block G protein signaling, high throughput screening of compounds
and characterization of the selected compounds is significantly
reduced in both time and cost, because only potent and strongly
binding compounds are selected. The first step of identification of
high affinity peptides which strongly compete with G proteins for
their site of binding on G protein-coupled receptors insures this
because the high affinity peptides are designed and tested for the
particular desired binding specificity, ability to modify function
within a cellular system and ability to modify functions in
vivo.
[0145] Preferably, only the most strongly binding and effective
peptide analogs or small molecules are used in the second or
subsequent screening step. This two- or multi-step protocol reduces
the number of false positives and identification of compounds which
bind only weakly by eliminating the lower affinity small molecules
that would be detected and then further studied if a conventional
high throughput screening method were used. This method, therefore,
is simple to implement, inexpensive, composed of only a few
components, amenable to many different classes of receptors, and
capable of rapidly screening large libraries of compounds. This
method enables efficient identification of new classes of small
organic peptidomimetic molecules that function as inhibitors or
enhancers of receptor action, for example, thrombin receptor
modifiers, dopamine receptor modifiers, histamine receptor
modifiers, or vasopressin receptor modifiers. These identified
compounds can target a single GPCR, a class of GPCR, or block or
enhance a single G protein pathway activated by GPCR.
[0146] Thorough evaluation of the selected compounds (either
peptides or small molecules) for use as therapeutic agents may
proceed according to any known method. Properties of the compounds,
such as pK.sub.a, log P, size, hydrogen bonding and polarity are
useful information. They may be readily measured or calculated, for
example from 2D connection tables, if not already known prior to
identification by the inventive method as a useful compound.
Association/dissociation rate constants may be determined by
appropriate binding experiments. Parameters such as absorption and
toxicity also may be measured, as well as in vivo confirmation of
biological activity. The screen may be optimized for small
molecules according to methods known in the art. Additionally, it
is preferable to use a software system for presentation of data
that allows fast analysis of positives. See Example 36 and FIG.
2.
[0147] Pharmaceutical preparations are prepared by formulating the
peptides or small molecules identified by the inventive screen
according to methods well known in the art, with any suitable
pharmaceutical excipient or combination of pharmaceutical
excipients. Preparations may be made for administration by any
route, such as intravenous, intramuscular, subcutaneous, oral,
rectal, vaginal, transdermal, transmucosal, sublingual and the
like, however, parenteral routes generally are preferred for
peptide preparations. Any suitable vehicle may be used, for example
saline or lactated Ringer's, for intravenous administration.
[0148] Dosages for treatment of GPCR-related diseases or conditions
will depend on many factors such as the nature of the disorder, the
GPCR involved, the route of administration, factors relating to the
general physical condition and health of the patient and the
judgment of the treating physician. Persons of skill in the art are
well aware of these factors and consider manipulation of dosage to
obtain an optimum result to be routine. Generally, dosages for
intravenous administration may vary between about 0.01 mg/kg and
1000 mg/kg, however, this range can be expanded depending on the
patient's needs. Such an expanded range is considered within the
scope of this invention.
[0149] Alternatively, peptides according to this invention may be
provided to cells, in vivo or ex vivo, by delivery of an expression
construct. Gene therapy can be performed in vivo as a direct
introduction of the genetic material. The in vivo gene transfer
would introduce the oligonucleotides encoding the peptides to cells
at the site they are found in the body, for example to skin cells
on an arm, or to lung epithelial cells following inhalation of the
gene transfer vector. Alternatively, ex vivo gene transfer, the
transfer of genes into viable cells that have been temporarily
removed from the patient and are then returned following treatment
(e.g. bone marrow cells) could also be employed.
[0150] Gene transfer vectors can be engineered to enter specific
tissues or cells. Transductional targeting allows the gene transfer
vectors to interact with specific cell surface receptors.
Transductional targeting also can take advantage of the rate of
cellular division by using gene transfer vectors that target
rapidly dividing cells such as tumor cells. Transcriptional
targeting recruits distinct cellular promoter and enhancer elements
to influence transcription of the therapeutic gene. Transfection
efficiencies are also enhanced by engineering vectors with
monoclonal antibodies, carbohydrate ligands, and protein ligands
that help deliver genes to specific cells.
[0151] The gene transfer vectors used to produce the high affinity
peptides inside cells could be viral vectors (e.g. Retrovirus,
Adenovirus, Adeno-Associated Virus, Herpes Simplex Virus, or
Vaccinia Virus). As an alternative, non-viral vectors also may be
used, these include such methods as injection of naked DNA, or
introduction of either DNA or peptides by attachment to positively
charged lipids, or cationic liposomes, electroporation or ballistic
DNA injection (limited to ex-vivo applications), as well as
introduction of branched peptides.
[0152] Tet-inducible retroviral vectors for the native C-terminal
sequences that co-expresses GFP driven by an internal ribosomal
entry site (IRES) from encephalomyocarditis virus (p-Tet-Ti-GFP)
may be used. These vectors can be modified so that they encode the
high affinity peptide sequences. In addition, the high affinity
peptide can be driven by a sequence allowing for spatial or
temporal expression. For in vitro studies, viral supernatants may
be collected from a pantropic producer line such as GP-293
(Clontech) in serum-free media. Viral supernatants may be
concentrated by ultracentrifugation at 4.degree. C. for 2 hr at
22,000 rpm, and the pellets resuspended in 1/100 the original
volume in serum-free media with a titer of at least 10.sup.8 IU
(infectious units)/ml and stored at -80.degree. C.
[0153] Murine leukemia virus (MLV) derived retroviral vectors are
commonly used vehicles for stable delivery of therapeutic genes
into endothelial cells. For the retrovirus studies in vivo, high
affinity peptides advantageously are subcloned into a
replication-defective murine Moloney retrovirus vector which is
Tet-inducible and co-expresses GFP driven by an internal ribosomal
entry site (IRES) from encephalomyocarditis virus (pTet-GFP). These
constructs may then be transiently transfected into a producer line
to generate cell-free titers of 10.sup.6-10.sup.10 IU/ml. If
needed, a pantropic retroviral expression system which utilizes
VSV-G, an envelope glycoprotein from the vesicular stomatitis virus
(GP-293; Clontech), may be utilized to overcome low transfection
efficiencies. By using this innovative cell-based gene transfer
method one can obtain stable, long-term, and localized gene
expression of the high affinity C-terminal peptides.
[0154] To conclusively demonstrate that the compounds identified by
this method can modulate G protein signaling events implicated in
disease syndromes in vivo, antagonism or enhancement of selective G
protein signal transduction events may be confirmed. One method of
testing the ability of compounds to compete with native G protein
binding involves expressing peptides that block the receptor-G
protein interface in cells bearing the receptor. Plasmid constructs
that encode GPCR-binding region peptides, such as carboxyl terminal
peptide sequences from the various G.alpha. subunits (see Table VI)
can be used to express them in cells in vivo, ex vivo or in vitro,
so that the metabolic effects of selective GPCR blockade can be
studied qualitatively and quantitatively. Such studies provide
proof that the binding which the compounds possess is useful in
vivo to modulate selective G protein signals.
[0155] Expression of the peptides is conveniently achieved using
the minigene approach by methods such as those described in
Examples 23 and 24, however any suitable method may be used.
Minigene vectors allow the high affinity peptides to be evaluated
in cellular systems prior to high throughput screening. Any desired
peptide sequence may be expressed using these methods. Those of
skill in the art are well aware of alternative methods for
construction, transfection and expression of protein and peptide
constructs comprising the high affinity peptide analogs, and such
methods are contemplated for use with them.
[0156] Human embryonic kidney cells advantageously are cultured in
DMEM (Gibco) with 10% fetal bovine serum (Gibco), and Pen/Strep
(5000 U/ml; 5000 pg/ml/ Gibco) in an atmosphere of 95% air/5%
CO.sub.2 at 37.degree. C. The cells may be plated at 60-70% density
the day before transfection and transiently transfected for 1.5
hours with DNA (3 .mu.g) for pcDNA 3.1 vector with the insert
(pcDNA3.1-high affinity peptide) or vector alone using an Effectene
kit from Qiagen. After transfection, cells are washed once before
adding complete HMEC media. When required, selection for cells
carrying the minigenes may be performed by adding Neomycin to the
media 48 hrs after transfection. To monitor efficiency of
transfection cells are transfected with the GFP plasmid
(Clonetech). When necessary, transfectants may be selected using
300 .mu.g/mL geneticin (G418). The expression of the vectors in HEK
transfectants can be confirmed using reverse transcription (RT)PCR
and Northern blot analysis for mRNA expression, and expression of
the peptides can be characterized by HPLC as described previously.
See Gilchrist et al., Methods Enzymol. 344:58-69, 2002, the
disclosures of which are hereby incorporated by reference.
TABLE-US-00006 TABLE VI Exemplary Sequences of C-terminal Minigene
Peptides. Peptide Name Sequence SEQ ID NO: G.alpha.i MGIKNNLKDCGLF
112 G.alpha.iR MGNGIKCLFNDKL 113 G.alpha.q MGLQLNLKEYNAV 114
G.alpha.q** MGLQLNLKEYNTL 115 G.alpha.12 MGLQENLKDIMLQ 116
G.alpha.13 MGLHDNLKQLMLQ 117
[0157] As discussed above, many receptors interact with and
activate multiple G proteins. Using the minigene strategy to
introduce the high affinity-binding carboxyl terminal peptides into
cells, it is possible to inhibit specific G protein-coupled
receptor interactions with individual G proteins, thus
demonstrating the feasibility of specific G protein blockade in
vivo with compounds identified by the inventive method. For those
receptors which activate multiple G proteins each of which
activates a distinct set of signaling pathways mediating a specific
set of responses (for example, the thrombin receptor), one pathway
can be inhibited without substantially affecting the others.
[0158] To selectively antagonize G protein signal transduction
events in vivo by expressing peptides that block the receptor-G
protein interface, minigene plasmid vectors were designed to
express the C-terminal peptide sequence of the various G.alpha.
subunits following their transfection into mammalian cells. A
control minigene vector also was created, encoding the carboxyl
terminus of G.alpha.i.sub.1/2 in random order (G.alpha.iR, see
Table VI). One important element necessary for the minigene
approach to block intracellular signaling pathways effectively in
vivo is expression of adequate amounts of the desired peptides.
Therefore, expression of the minigene should be confirmed by a
convenient method of detecting mRNA, protein or both. Any
convenient method known in the art can be used.
[0159] To determine the cellular efficacy of the minigene approach
for expressing GPCR binding peptides, and to show the specific
inhibition of one G protein pathway in response to a given receptor
activation signal without affecting others, compounds
advantageously may be assayed in a system designed to exhibit a
measurable cellular signaling endpoint. One example of such a
system is the thrombin receptor, PAR1, in endothelial cells. This
receptor activates multiple G proteins. Several signaling
endpoints, including transcription analysis of induced PAR1 gene
expression; biochemical analysis of effector molecules including
[Ca.sup.2+], MAP kinase ("MAPK") activity, adenylyl cyclase
activity, and inositol phosphate accumulation; as well as
functional assays such as cell proliferation and endothelial
permeability are available to measure specific activation or
modulation of activation of different G proteins by ligand binding
at this receptor. Signaling activity may be measured by any
convenient method, including: measuring inositol phosphate
accumulation; measuring intracellular calcium concentration levels;
measuring transendothelial electrical resistance; measuring stress
fiber formation; measuring ligand binding (agonist, antagonist,
inverse agonist, etc.); measuring receptor expression; measuring
receptor desensitization; measuring kinase activity; measuring
phosphatase activity; measuring nuclear transcription factors;
measuring cell migration (chemotaxis); measuring superoxide
formation; measuring nitric oxide formation; measuring cell
degranulation; measuring GIRK activity; measuring actin
polymerization; measuring vasoconstriction; measuring cell
permeability; measuring apoptosis; measuring cell differentiation;
measuring membrane association of a protein that translocates upon
GPCR activation, such as protein kinase C; measuring cytosolic
accumulation of a protein that translocates upon GPCR activation,
such as protein kinase C; measuring cytosolic accumulation of a
protein that translocates upon GPCR activation, such as src; and
measuring nuclear association of a protein that translocates upon
GPCR activation, such as Ran. The functional effects of G.alpha.
C-terminal minigenes in the mechanism of thrombin-induced cell
retraction, as measured by the change in transendothelial
electrical resistance (TEER) also can be used to measure G protein
inhibition.
[0160] For example, thrombin-mediated PAR1 gene induction was
inhibited in human microvascular endothelial cells (HMEC)
expressing the G.alpha.i minigene construct. Expression of the
G.alpha.q minigene construct, however, affected thrombin-mediated
inositol phosphate accumulation. Expression of G.alpha.q also
specifically decreased both thrombin-induced intracellular
[Ca.sup.++].sub.i rise and thrombin-induced MAPK activity.
[0161] Thrombin activation of the G.alpha.i mechanism in HMEC
decreases cAMP levels increased in response to isoproterenol (which
acts through G.alpha.s). Assay for cAMP level increases in response
to isoproterenol alone may be compared to increases after thrombin
pre-incubation in cells expressing G.alpha.i to show that
expression of the GPCR binding peptide blocks G.alpha.i
signaling.
[0162] Recent work by Gohla et al., J. Biol. Chem. 274:17901-17907,
1999, elegantly demonstrated that thrombin receptors induce stress
fiber accumulation via G.alpha.12 in an EGF receptor-independent
manner. Stress fiber formation appears to be Rho dependent. Both
G12 and G13 have been implicated in the Rho signaling pathway.
Therefore, expression of G.alpha.12 and G.alpha.13 GPCR-binding
peptides in HMEC were used to determine whether these peptides
could block the appearance of stress fibers in response to
thrombin.
[0163] The extracellular signal-regulated kinase (ERK) subfamily of
mitogen-activated protein kinases (MAPKs) regulates numerous cell
signaling events involved in proliferation and differentiation.
This forms the basis of another assay which can determine whether
GPCR binding peptides can affect a specific G protein mediated
pathway. Transfection of HMEC cells with minigenes encoding GPCR
binding peptides along with HA-MAPK followed by immunoprecipitation
of the HA-MAPK permits measurement of the effects only on cells
expressing GPCR binding peptides.
[0164] Many studies have shown that the M.sub.2 muscarinic receptor
(mAChR) couples exclusively to the Gi/GO family. See Dell'Acqua et
al., J. Biol. Chem. 268:5676-5685, 1993; Lai et al., J. Pharm. Exp.
Ther. 258:938-944, 1991; Offermanns et al., Mol. Pharm. 45:890-898,
1994; Thomas et al., J. Pharm. Exp. Ther. 271:1042-1050, 1994. The
M.sub.2 mAChR can efficiently couple to mutant G.alpha.q** in which
the last five amino acids are substituted with the corresponding
residues from G.alpha.i or G.alpha.O, suggesting that this receptor
contains domains that are specifically recognized by the carboxyl
terminus of G.alpha.i/O subunits. See Liu et al., Proc. Natl. Acad.
Sci. USA 92:11642-11646, 1995.
[0165] To test inhibition of G protein-coupled receptor-mediated
cellular responses by carboxyl terminal G.alpha. peptides expressed
using minigene constructs, prototypical directly G.beta..gamma.
activated channels (GIRK channels) regulated by a pertussis
toxin-sensitive M.sub.2 mAChR was chosen as the model. In this
model, the importance of the G.alpha. carboxyl terminus and the
downstream effector system have been well established. See
Krapivinsky et al., J. Biol. Chem. 270:29059-29062, 1995;
Krapivinsky et al., J. Biol. Chem. 273:16946-16952, 1998; Sowell et
al., Proc. Natl. Acad. Sci. USA 94:7921-7926, 1997. Inhibition of
M.sub.2 mAChR activation of inwardly rectifying potassium currents
can be tested to demonstrate inhibition of a downstream functional
response following agonist stimulation of GPCR on cells transiently
transfected with a G.alpha. carboxyl terminal peptide minigene or
treated with a pharmaceutical compound identified by screening
against high affinity G.alpha. peptides.
[0166] GIRK channels modulate electrical activity in many excitable
cells. See Breitwiese et al., J. Membr. Biol. 152:1-11, 1996; Jan
et al., Curr. Opin. Cell Biol. 9:155-160, 1997; Wickman et al.,
Curr. Opin. Neurobiol. 5:278-285, 1995. Because the channel opens
as a consequence of a direct interaction with G.beta..gamma., whole
cell patch clamp recording of I.sub.KACh can be used to demonstrate
inhibition of a downstream functional response following agonist
stimulation of GPCR on cells transiently transfected with a
G.alpha. carboxyl terminal peptide minigene or treated with a
pharmaceutical compound identified by screening against high
affinity G.alpha. peptides. Superfusion of cells expressing
GIRK1/GIRK4 with their ligand, acetylcholine (ACh), activates
inwardly rectifying potassium currents.
[0167] Using well-established receptor models accepted to be
indicative of in vivo cellular results, this type of data can show
that the individual G proteins activated via a given GPCR have
specific roles in mediating cellular events and can be modulated in
a specific fashion by ligands mimicking GPCR binding regions of
individual G.alpha. subunits. In particular, for receptors such as
the thrombin receptor, which activate multiple G proteins, each of
which activates a distinct set of signaling pathways mediating a
specific set of responses, it is possible using the inventive
methods to block one pathway while leaving all the others
functional. The high affinity peptide analogs identified in vitro
by consecutive affinity purification and competitive binding are
capable of specifically inhibiting the downstream consequences of G
protein signaling.
[0168] The assays described above clearly establish the ability of
compounds identified by in vitro competitive binding studies to
modulate a particular GPCR-G protein interaction selectively, even
when the GPCR regulates multiple G proteins within the cell.
Moreover, the peptides compete very effectively with the native
sequence. In addition, the minigene approach described above and
exemplified in the examples below allows a systematic test of the
roles of other G proteins such as G.alpha.12 and G.alpha.13, which
may be involved in the mechanism of increase of endothelial
permeability, and clearly demonstrates the viability of this
approach to select and identify G.alpha. subunit modulating
compounds. The peptides therefore are suitable for use in treatment
of any disorder or syndrome characterized by G protein signaling
excess.
[0169] In another aspect, the invention relates to methods to
identify the G proteins with which a specific orphan receptor is
coupled, using the materials provided by the invention. For
example, the described methods can be used to test any GPCR with a
battery of G.alpha. subunit peptides to determine which species of
G protein(s) mediates the effects of the receptor. The methods
described in Examples 12-14 are suitable. Those of skill in the art
are capable of designing other assays, or variations and
modifications using these assays as guides.
[0170] Rhodopsin can be measured spectrophotometrically in many of
its conformational states. The high affinity, biologically active
rhodopsin state can be easily differentiated from its precursor,
MI, by the "extra" MII assay. See Example 35. This assay relies on
the observation that under conditions of high pH and low
temperature, MII is stabilized in the presence of Gt and can be
spectrophotometrically measured. The ability of the C-terminal
peptide to stabilize Meta II in the same manner as the
heterotrimeric Gt, provides the tools to investigate the structural
basis of the interaction of G proteins with the agonist binding
sites of activated receptors.
[0171] The screening platform according to one embodiment of the
invention can identify small molecules that increase the binding of
the high affinity peptides which mimic G protein or stabilize the
active conformation of the GPCR. These small molecules have an
appropriate dose curve, and have an EC.sub.50 in the low .mu.M
range. Samples of urea-washed rod outer segments typically have
little or no (<5%) stabilization of MII unless G protein is
added, however, the small molecules identified by the invention in
one example screen stabilize the active (signaling) conformation of
rhodopsin (MII). Addition of the small molecule alone results in
70% stabilization. The EC.sub.50 for stabilization of MII also
appears to be in the low .mu.M range. Further, these same small
molecules were added to human embryonic kidney (HEK) cells with
measurement of their calcium response to a second GPCR do not
enhance signaling of an unrelated GPCR and do not appear to cause
an acute toxic response.
[0172] Drug discovery has evolved from an essentially random
screening of products, into a process that includes the rational
and combinatorial design of large numbers of synthetic molecules as
potential bioactive agents, such as agonists, antagonists and
inverse agonists, as well as the structural characterization of
their biological targets, which may be polypeptides, proteins, or
nucleic acids. Several approaches to facilitating the understanding
of the structure of the therapeutic targets have been developed.
These include sequencing of proteins and nucleic acids (Findlay et
al., Protein Sequencing: A Practical Approach, IRL Press, Oxford,
1989; Adams et al., In Automated DNA sequencing and Analysis,
Academic Press, San Diego, 1994), elucidation of secondary and
tertiary structures via NMR (Jefson, Ann. Rep. Med. Chem. 23:275,
1998; Erikson and Fesik, Ann. Rep. Med. Chem. 27:271-289, 1992),
X-ray crystallography (Erikson and Fesik, Ann. Rep. Med. Chem.
27:271-289, 1992) and computer algorithms for predicting protein
folding (Copeland, Methods of Protein Analysis: A Practical Guide
to Laboratory Protocols, Chapman and Hall, New York, 1994;
Creighton, Protein Folding, W.H. Freeman and Co., 1992).
Experiments such as ELISA (Kemeny and Challacombe, ELISA and other
Solid Phase Immunoassays: Theoretical and Practical Aspects; Wiley,
New York, 1988) and radioligand binding assays (Berson and Yalow,
Clin. Chim. Acta, 22:51-60, 1968; Chard, An Introduction to
Radioimmunoassay and Related Techniques, Elseveier press,
Amsterdam/New York, 1982), surface-plasmon resonance (Karlsson et
al., Anal. Biochem. 300:132-138, 2002), and scintillation proximity
assays (Kariv et al., J. Biomol. Screen. 4:27-32, 1999) also can be
used to understand the nature of the receptor-G protein
interaction.
[0173] Peptides that block the protein-protein interactions of
interest do so by binding to the surface of one of the interacting
proteins and mimicking the interactions of the complete protein
with the receptor. One can study the conformation of the active
receptor-bound peptides when they are exchanging with the bound
form, using transferred-NOESY NMR methods or X-ray diffraction if
the peptides are more tightly bound. The bound peptide
conformations can provide useful templates for the design of
non-peptide small molecule drug leads which block the
protein-protein interactions of interest. The binding sites of the
peptides on the receptors also can be investigated using
photochemical crosslinking, by substitution of peptide residues
with photoactivatable amino acid analogs, crosslinking of the
peptide to the receptor binding sites, cleavage of the receptor
into peptide fragments and mass spectrometry analysis of the
location of the binding sites. Combining structural data from a
variety of experiments allows the development of models of the
interacting protein surfaces using computer graphics and guides the
design of novel non-peptide molecules to modulate the
interactions.
[0174] In X-ray diffraction crystallography, a crystalline form of
the molecule under study is exposed to a beam of X-rays and the
intensity of detracted radiation is measured at a variety of angles
from the angle of incidence. The beam of X-rays is diffracted into
a plurality of diffraction "reflections," with each reflection
representing a reciprocal lattice vector. From the diffraction
intensities of the reflections, the magnitudes of a series of
numbers, known as "structure factors," are determined. The
structure factors in general are complex numbers, having a
magnitude and a phase in the complex plane, and are defined by the
electron distribution within the unit cell of the crystal.
[0175] Crystals can be formed of receptor or portions of receptor
bound to peptides that stabilize a particular conformation of
interest. The methods of this invention, which identify peptides
using combinatorial techniques that scan the complete set of
possible amino acid sequences to find those that bind specifically
to a particular receptor with high affinity, can identify peptides
that bind to particular conformations of a GPCR. These peptides can
be bound (and co-crystallized) with the receptor for structural
determination studies by NMR or crystallography. Co-crystallization
in this manner may be performed according to any method known in
the art, for example the methods of Kimple et al., Nature
416:878-881, 2002, the disclosures of which are hereby incorporated
by reference.
[0176] Therefore, in another embodiment, assays for identifying
peptides that bind to a particular conformer of a GPCR are
performed according to the methods described above for selection of
the high affinity peptide analogs that bind activated rhodopsin.
Once the high affinity peptides have been identified, they can be
used in peptidomimetic studies. Compounds that mimic the
conformation and desirable features of a particular peptide, e.g.,
an oligopeptide, but that avoid undesirable features, e.g.,
flexibility (loss of conformation) and metabolic degradation, are
known as "peptidomimetics." Peptidomimetics that have physical
conformations that mimic the three dimensional structure of the
high affinity peptide analogs, that have surface active groups that
allow binding to the receptor, or that have physical conformations
that mimic the three dimensional structure of the high affinity
peptide analogs can be used to make pharmaceutical compositions.
Drugs with the ability to mimic the function of the high affinity
peptide analogs that bind to the designated receptors can be
identified using rational drug design according to this invention.
The compounds preferably include the surface active functional
groups of the high affinity peptide analogs, or substantially
similar groups, in the same or substantially similar orientation,
so that the compounds possess the same or similar biological
activity. The surface-active functional groups in the high affinity
peptide analogs possess a certain orientation when the receptor is
present, in part due to their secondary or tertiary structure.
Rational drug design involves both the identification and chemical
modification of suitable compounds that mimic the function of the
parent molecules.
[0177] The physical conformation of the peptidomimetics are
determined, in part, by their primary, secondary and tertiary
structure. The primary structure of a peptide is defined by the
number and precise sequence of amino acids in the high affinity
peptide analogs. The secondary structure is defined by the extent
to which the polypeptide chains possess any helical or other stable
structure. The tertiary structure is defined by the tendency for
the polypeptides to undergo extensive coiling or folding to produce
a complex, somewhat rigid three-dimensional structure.
[0178] Computer modeling technology allows scientists to visualize
the three-dimensional atomic structure of a selected molecule and
derive information that allows the rational design of new compounds
that will mimic the molecule or which will interact with the
molecule. The three-dimensional structure can be determined based
on data from X-ray crystallographic analyses and/or NMR imaging of
the selected molecule, or from ab initio techniques based solely or
in part on the primary structure, as described, for example, in
U.S. Pat. No. 5,612,895. The computer graphics systems enable one
to predict how a new compound will link to the target molecule and
allow experimental manipulation of the structures of the compound
and target molecule to perfect binding specificity.
[0179] Many databases and computer software programs are available
for use in drug design. For example, see Ghoshal et al., Pol. J.
Pharmacol. 48(4):359-377, 1996; Wendoloski et al., Pharmacol. Ther.
60(2):169-183, 1993; and Huang et al., J. Comput. Aided Mol. Des.
11:21-78, 1997. Databases including constrained, metabolically
stable non-peptide moeties may be used to search for and to suggest
suitable analogs of the high affinity peptides identified in the
screen. Searches can be performed using a three dimensional
database for non-peptide (organic) structures (e.g., non-peptide
analogs, and/or dipeptide analogs) having three dimensional
similarity to the known structure of the active regions of these
molecules. See, for example, Allen, Acta Crystallogr. B.
58:380-388, 2002.
[0180] Alternatively, three dimensional structures generated by
other means such as molecular mechanics can be consulted. In
addition, search algorithms for three dimensional database
comparisons are available in the literature. Rufino et al., J.
Comput. Aided Mol. Des. 8:5-27, 1994. Commercial software for such
searches is also available from vendors such as Accelrys Inc. (9685
Scranton Road, San Diego, Calif. 92121-3752). The searching is done
in a systematic fashion by simulating or synthesizing analogs
having a substitute moiety at every residue level. Preferably, care
is taken that replacement of portions of the backbone does not
disturb the tertiary structure and that the side chain
substitutions are compatible to retain the high affinity
peptide/receptor interactions.
[0181] Using information regarding the bond angles and spatial
geometry of the critical amino acids, one can use computer programs
as described herein to develop peptidomimetics. Thermal protein
unfolding, or thermal "shift" assays have been used to determine
whether a given ligand binds to a target receptor protein. In a
physical thermal shift assay, a change in a biophysical parameter
of a protein is monitored as a function of increasing temperature.
For example, in calorimetric studies, the physical parameter
measured is the change in heat capacity as a protein undergoes
temperature-induced unfolding transitions. Differential scanning
calorimetry may be used to measure the affinity of a ligand for a G
protein coupled receptor. Grauschopf et al., Biochemistry
39:8878:87, 2000; Brandts et al., Biochemistry 29:6927-40, 1990.
Thus, using methods common to those skilled in the arts, the high
affinity peptides may be assayed for their ability to modulate
thermal shift of the receptor.
[0182] Because of the difficulty in obtaining high-resolution
crystallographic structures from GPCRs, a variety of biophysical
methods have been applied to characterize the interactions between
the G protein, the receptor and the ligand. These include
fluorescence resonance energy transfer (FRET) experiments performed
with fluorescence-labeled peptide analogs (Bettio et al.,
Biopolymers 60:420-37, 2001), bioluminescence resonance energy
transfer (BRET) experiments (Ayoub et al., J. Biol. Chem.
277:21522-8, 2002), photoaffinity labeling (Turek et al., J. Biol.
Chem 277:16791-16797, 2002), fluorescence spectroscopy (Ghanouni et
al. J. Biol. Chem. 276:24433-24436, 2001), site-direct spin
labeling (Hubbell et al., Nat. Struct. Biol. 7:735-739, 2000),
Fourier transform infrared difference spectroscopy (Vogel et al.,
Biochemistry 35:11149-11159,1996), and intrinsic tryptophan
fluorescence spectroscopy. Farrens et al., J. Biol. Chem.
270:5073-5076, 1995.
[0183] The following non-limiting examples are provided to
illustrate certain aspects of this invention.
EXAMPLES
Example 1
Construction of a Peptide Library
[0184] Construction of a biased peptide library has been described
previously. Martin et al., J. Biol. Chem. 271:361-366, 1996; Schatz
et al., Meth. Enzymol. 267:171-191, 1996. The vector used for
library construction was pJS142 (see FIG. 3). This vector had a
linker sequence between the LacI and the biased undecamer peptide
coding sequence, as well as restriction sites for cloning the
library oligonucleotide. The oligonucleotide synthesized to encode
the mutagenesis library was synthesized with 70% of the correct
base and 10% of each of the other bases at each position. This
mutagenesis rate leads to a biased library such that there is
approximately a 50% chance that any of the 11 codons will be the
appropriate (native) amino acid and approximately a 50% chance that
it will be another amino acid. In addition, a linker of four random
NNK (where N denotes A, C, G or T and K denotes G or T) codons were
synthesized at the 5' end of the sequence to make a total of 15
randomized codons. Using this method, a library with greater than
10.sup.9 independent clones per microgram of vector used in the
ligation was constructed based on the carboxyl terminal sequence of
G.alpha.t (IKENLKDCGLF; SEQ ID NO:15). The nucleic acid used for
creating this library was:
5'-GAGGTGGTNNKNNKNNKNNKattcaaggagaacctgaaggactgcggcctcttcTAACT
AAGTAAAGC-3', wherein N=A/C/G/T and K=G/T; SEQ ID NO:118).
Example 2
Sequences for the Creation of G.alpha. Subunit Peptide
Libraries
[0185] Libraries were created using the methods of Example 1 and
the sequences listed below in Table VII. TABLE-US-00007 TABLE VII
C-Terminal G.alpha. Subunit Peptide Library Constructs. G.alpha.
SEQ Sub- ID unit RE Linker Peptide Coding Region Stop RE NO: Gs
5-GAGGTGGT NNKNNKNNKNNK attcgtgaaaacttaaaagattgtggtcgtttc TAA
CTAAGTAAAGC-3' 14 G11 5-GAGGTGGT NNKNNKNNKNNK
ctgcagctgaacctgaaggagtacaatctggtc TAA CTAAGTAAAGC-3' 119 G12
5-GAGGTGGT NNKNNKNNKNNK ctgcaggagaacctgaaggacatcatgctgcag TAA
CTAAGTAAAGC-3' 120 G13 5-GAGGTGGT NNKNNKNNKNNK
ctgcatgacaacctcaagcagcttatgctacag TAA CTAAGTAAAGC-3' 121 G15
5-GAGGTGGT NNKNNKNNKNNK ctcgcccggtacctggacgagattaatctgctg TAA
CTAAGTAAAGC-3' 122 Gz 5-GAGGTGGT NNKNNKNNKNNK
atacagaacaatctcaagtacattggcctttgc TAA CTAAGTAAAGC-3' 123
Example 3
Isolation of Membranes from Insect Cells Expressing Thrombin
Receptor
[0186] Sf9 cells (2.times.10.sup.8 cells) were cultured with 200 ml
of Grace's insect cell culture medium (Life Technologies, Inc.,
Grand Island, N.Y.) containing 0.1% Pluronic F-68 (Life
Technologies, Inc., Grand Island, N.Y.)), 10% fetal calf serum, and
20 .mu.g/ml gentamicin in a 1-liter spinner flask at 27.degree. C.
for 25 hours. Sf9 cells were infected with the ThR/pBluebac
recombinant virus at a multiplicity of infection of 3-5, and
cultured at 27.degree. C. for 4 days. The cells were harvested,
washed with phosphate buffered saline, and then resuspended in 10
mM Tris-HCl, pH 7.4. Cells were then homogenized with a hand-held
homogenizer set at low speed for 20 seconds. The broken cells then
were sedimented at 17,000.times.g for 15 minutes. The supernatant
was discarded, and the pellet resuspended in a buffer consisting of
50 mM Tris-HCl, pH 7.4 and 10% glycerol. Concentration of receptor
in the membrane preparation ranged from 1-10,000 pmole/mg. For
screening, a final concentration of 200 .mu.g/ml was used. The
thrombin receptors were tested for their ability to bind to the
native Gq-C terminal peptide using a MBP-Gq fusion protein.
Example 4
Isolation of Membranes from Mammalian Cells Overexpressing Thrombin
Receptor
[0187] PAR1 receptor cDNA (2.1 kb insert) was obtained by
polymerase chain reaction and cloned into the mammalian expression
vector pBJ5. The resulting plasmid was transfected into Chinese
hamster ovary cells by the calcium phosphate coprecipitation
method. The PAR1-transfected cells were grown with Dulbecco's
modified Eagle's medium containing 10% fetal calf serum, 100
units/mL penicillin and 100 .mu.g/mL streptomycin. The cells were
detached using PBS with 5 mM EDTA and washed twice in PBS. The
pellet was either used immediately for membrane preparation or
stored frozen at -20.degree. C. Pellets were homogenized in 20 mM
Tris-HCl, pH 7.5, with 5 mM EDTA and 0.5 mM PMSF, using a Dounce
homogenizer (10 strokes) and sonicated for 10 seconds. Nuclear
debris and intact cells were removed by centrifugation at 3000 rpm
for 10 minutes. The supernatant was sedimented at 12,000.times.g
for 30 minutes and the resulting pellet suspended in 25 mM
Tris-HCl, pH 7.5, 25 mM MgCl.sub.2, 10% sucrose, 0.5 mM PMSF, 50
.mu.g/mL antipain, 1 .mu.g/mL aprotinin, 40 .mu.g/mL bestatin, 100
.mu.g/mL chymostatin, 0.5 .mu.g/mL leupeptin and 0.7 .mu.g/mL
pepstatin. The membranes were aliquoted and frozen at -80.degree.
C.
Example 5
Preparation of Rod Outer Segments
[0188] Bovine rod outer segments (rhodopsin-containing membranes)
were prepared from fresh retinas under dim red light as described
by Arsharky et al., J. Biol. Chem. 269:19882-19887, 1994. The
retinas were placed in a beaker for dissection filled with 200 mL
of 30% (w/v) sucrose in isolation buffer (90 mM KCl, 30 mM NaCl, 2
mM MgCl.sub.2, 0.1 mM EDTA, 1 mM DTT, 50 .mu.M phenylmethylsulfonyl
fluoride, 10 mM MOPS, pH 7.5) on ice with constant moderate
stirring of the solution during dissection. Following dissection,
the retina solution was left in the dark for one hour on ice. The
retina-sucrose solution was distributed into eight 50 mL tubes and
sedimented at 3000.times.g for four minutes at 4.degree. C. The
supernatant was decanted into eight fresh centrifuge tubes and
placed on ice. The tubes were filled to 1.5 cm below top with
isolation buffer, then sedimented at 17,000.times.g for 20 minutes
("spin 1").
[0189] The pellets were resuspended in a small volume of 30%
sucrose and consolidated from eight tubes into four tubes. The
tubes were filled to 1.5 cm below top with 30% sucrose, sedimented
at 5000.times.g for four minutes at 4.degree. C., and the
supernatant decanted into four clear tubes. These tubes were filled
to 1.5 cm below top with isolation buffer and sedimented at
17,000.times.g for 20 minutes at 4.degree. C. ("spin 2").
[0190] A stepwise sucrose gradient was prepared in six gradient
tubes using the solutions in Table VIII, below, with a sequence
from top to bottom of #2, #3, #4. TABLE-US-00008 TABLE VIII Sucrose
Gradient Solutions. Solution #2 (0.84 M) #3 (1.0 M) #4 (1.14 M) 42%
Sucrose 51.30 g 61.05 g 69.75 g 1.0 M MOPS 750 .mu.L 750 .mu.L 750
.mu.L 2.0 M KCl 2250 .mu.L 2250 .mu.L 2250 .mu.L 3.0 M NaCl 750
.mu.L 750 .mu.L 750 .mu.L 2.0 M MgCl.sub.2 75 .mu.L 75 .mu.L 75
.mu.L Total Weight 83.25 g 84.75 g 86.25 g
[0191] The pellets from "spin 1" and "spin 2" were resuspended in
isolation buffer using 1 mL 26% sucrose buffer per tube. After
making a slurry, each tube was homogenized with a 1 mL pipette and
the tubes consolidated. The pellet solution was carefully laid onto
the sucrose gradients and was not allowed to invade the gradient
layers. The gradient tubes were subjected to 24,000.times.g for 30
minutes at 4.degree. C. in a swinging bucket rotor, after which the
orange layer containing the membranes was collected carefully, to
avoid disturbing the pellet or the dark solution near the pellet.
The membranes were distributed into six 50 mL tubes and placed on
ice. The tubes then were filled to 1.5 cm below top with isolation
buffer and sedimented at 17,000.times.g for 20 minutes at 4.degree.
C. The supernatant was discarded and the pellets resuspended in 1
mL isolation buffer containing 5 .mu.g/mL pepstatin and 10 .mu.g/mL
E-64. This suspension was stored in a foil-wrapped 15 mL conical
tube at -80.degree. C. until needed, then thawed, homogenized in
EDTA buffer (10 mM Tris, pH 7.5, 1 mM EDTA 1 mM DTT) and sedimented
at 30,000.times.g for 30 minutes. The supernatants were discarded
and the pellets resuspended and sedimented again as described
above. The pellets then were resuspended in urea buffer (10 mM
Tris, pH 7.5, 1 mM EDTA, 1 mM DTT, 7 M urea), homogenized and
sedimented at 45,000 kg for 40 minutes. These pellets were
resuspended and homogenized in Buffer A (200 mM NaCl, 10 mM MOPS,
pH 7.5, 2 mM MgCl.sub.2, 1 mM DTT, 100 .mu.M PMSF), then sedimented
at 30,000.times.g for 30 minutes. The pellets each were resuspended
and homogenized by pipetting in 1 mL buffer A and stored at
-80.degree. C. in 100 .mu.L aliquots in foil-covered tubes for use
in assays. For screening, the receptor was added to wells at 10
.mu.g/ml. Binding assays were performed as in Example 18.
Example 6
Purification of PAR1 Thrombin Receptor from Insect Cells and
Reconstitution of Receptors into Lipid Vesicles
[0192] Sf9 cells (2.times.10.sup.8 cells) were cultured in Grace's
insect cell culture medium (Life Technologies, Inc., Grand Island,
N.Y.) containing 0.1% Pluronic F-68 (Life Technologies), 10% fetal
calf serum and 20 .mu.g/mL gentamicin in a 1 L spinner flask at
27.degree. C. for 25 hours. The cells were infected with
ThR/pBluebac (recombinant virus) at a multiplicity of infection of
3-5 and cultured at 27.degree. C. for four days. The cells were
harvested, washed with phosphate buffered saline containing 2.7 mM
EDTA and stored at -70.degree. C. until used. The cells were
resuspended in lysis buffer (2.5 mM Tris-HCl, pH 7.2, 7.5 mM NaCl,
10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin,
10 mg/mL aprotinin, 50 mM NaF) and washed. All subsequent steps
were done on ice with cold buffers and centrifuge rotors at or
below 4.degree. C. The cells were homogenized for one minute at
maximum speed and sedimented for 45 minutes at 30,000.times.g. The
pellet was resuspended in lysis buffer and the homogenation/washing
step repeated three times. The resulting pellet was resuspended in
30 mL solubilization buffer (20 mM Tris-HCl, pH 7.4, 15 mM EGTA, 1
mM phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin, 10 mg/mL
aprotinin, 50 mM NaF, 0.1% (w/v) digitonin, 0.1% (w/v) Na
deoxychoate) and then homogenized for one minute. The suspension
was stirred for 90 minutes at 4.degree. C. and then sedimented for
60 minutes at 30,000.times.g. The supernatant was loaded onto an
anti-PAR1 monoclonal antibody column equilibrated with
solubilization buffer containing 0.2% digitonin. After application
of the supernatant, the column was washed with 10 column volumes of
10 mM Tris-HCl buffer, pH 7.4, containing 0.2% (w/v) Na dodecyl
maltoside. The receptor was eluted using 10 mM triethylamine, pH
11.8. The eluted fractions were neutralized immediately using 1 M
HEPES, pH 6.4. The pooled fractions were dialyzed against 50 mM
HEPES buffer, pH 7.4, containing 50% (v/v) glycerol, 0.1 M NaCl and
0.2% (w/v) Na dodecyl maltoside. Aliquots were stored at
-80.degree. C.
[0193] For preparation of lipid vesicles, 200 .mu.L
phosphatidylserine (50 mg/mL in CHCl.sub.3; Matreya) was dried in a
rotary evaporator for 30 minutes or using a stream of dry N.sub.2.
After addition of 200 .mu.L buffer A (50 mM HEPES, 100 mM NaCl,
0.2% (w/v) Na dodecylmaltoside), the tube was sealed under an
N.sub.2 atmosphere and sonicated in a bath sonicator for 30
minutes. Reconstitution of receptors into lipid vesicles was
performed the same day, using purified receptor prepared as in
Example 5. Purified receptor stocks (200 .mu.g/mL) were thawed on
ice and 50 .mu.L was incubated for 20 minutes at 4.degree. C. with
the appropriate agonist peptide (100 nM final concentration). In
the case of thrombin receptor, the agonist is thrombin receptor
agonist peptide (CalbioChem). After addition of 80 .mu.L sonicated
lipids and 50 .mu.L buffer A, the samples were mixed using a vortex
machine and placed on ice for 10 minutes. The samples then were
loaded onto a 1 mL Extracti-gel.TM. column which had been washed
with 0.2% BSA and pre-equilibrated with 5 mL Buffer A without Na
dodecylmaltoside. The reconstituted vesicles were eluted from the
column with 2.5 mL HEK buffer.
[0194] Samples (100-200 .mu.L) were collected for purity analysis
by SDS-PAGE. The concentration for each batch generally was about
10-1000 .mu.g/ml. For use, receptor was placed in microtiter plates
at about 1-100 .mu.g/ml. The purified, reconstituted thrombin
receptors were tested for their ability to bind to the native Gq-C
terminal peptide using a MBP-Gq fusion protein. As a control, empty
vesicles also were tested for their ability to bind to the native
Gq-C terminal peptide using a MBP-Gq fusion protein.
Example 7
Identification of GPCR-Binding High Affinity Peptide Analogs
(Panning)
[0195] Electrocompetent cells were produced as follows. A single
colony of ARI814 bacteria was grown overnight at 37.degree. C. in 5
ml sterile SOP (20 g/L Bacto-tryptone; 10 g/L Bacto-yeast extract;
5 g/L NaCl; 2.5 g/L anhydrous K.sub.2HPO.sub.4; 1 g/L
Mg.sub.2SO.sub.4.7H.sub.2O). One milliliter of this overnight
growth was added to 500 ml SOP and the bacteria were allowed to
grow until the OD.sub.600 read 0.6-0.8. All further washing steps
were done in the cold. The cells were placed in an ice-water bath
for at least 15 minutes, then subjected to centrifugation at
4000.times.g for 15 minutes at 4.degree. C. followed by
resuspension in 500 ml 10% glycerol. After sitting on ice for 30
minutes, the cells were washed twice more in 500 ml and 20 ml 10%
glycerol with sedimentation as above, and finally sedimented at
5000.times.g for 10 minutes at 4.degree. C. and resuspended in 1 mL
10% glycerol. Cells were quick-frozen using dry ice and isopropanol
in 100 .mu.L aliquots for later use.
[0196] To transfect, aliquots (40 .mu.L) of thawed ARI814 cells
were placed into each of three chilled microcentrifuge tubes. A
peptide display library based on the undecamer carboxyl terminal
peptide of G.alpha.t (SEQ ID NO:15) was prepared according to
Example 1. Two microliters of library plasmid were added to the
tubes and mixed. For the first round of "panning," 200 .mu.l of the
plasmid library was added. For subsequent rounds, three sets of
transfections were performed (adherent plasmids from wells
containing receptor (+); adherent plasmids from wells containing no
receptor (-); and the PRE sample which was not incubated). See
below.
[0197] In each round of panning, less library was used (round 2:100
.mu.l; round 3:50 .mu.l; round 4:10 .mu.l). After the panning was
completed, the DNA for the LacI fusion protein was eluted. This DNA
(50 .mu.l) was used to transfect E. Coli cells by electroporation,
using cold, sterile 0.1 cm electrode gap cuvettes. The cuvettes
were pulsed one time using a BioRad E. coli Pulsar set to 1.8 kV,
25 .mu.F capacity, time constant 4-5 mseconds, with the Pulser
Controller unit at 200 m.OMEGA.. Immediately, 1 mL of SOC was added
and the mixture transferred to a labeled 17.times.100 mm
polystyrene tube. The tube was shaken for one hour at 37.degree. C.
Aliquots were taken from each set to plate 100 .mu.L undiluted to
10.sup.-6 dilution samples on LB-Amp plates. Counts of the PRE
plates indicated library diversity, while comparison of the (+) and
(-) plates indicated whether specific clones were being enriched by
the panning procedure.
[0198] The remaining .about.900 .mu.L in the + receptor tube was
added to a 1 L flask containing 200 mL LB-AMP media, prewarmed to
37.degree. C., and grown at 37.degree. C., shaking until
OD.sub.600=0.5. The tube of cells then were placed in an ice water
bath for at least 10 minutes, and kept chilled at or below
4.degree. C. during the subsequent washing steps. The cells were
sedimented at 5000.times.g for six minutes, resuspended in 100 mL
WTEK buffer, sedimented at 5000.times.g for six minutes,
resuspended in 50 mL TEK buffer, resedimented at 5000.times.g for
six minutes and resuspended in 4 mL HEK buffer. The cells were
divided into the cryovials and stored at -70.degree. C. One tube
was used for the next round of panning and the other saved as a
backup.
[0199] The panning process is illustrated in FIG. 1. For screening
of the library by "panning," rhodopsin receptors prepared according
to Example 5 were immobilized directly on Immulon 4 (Dynatech)
microtiter wells (0.1-1 .mu.g of protein per well) in cold 35 mM
HEPES, pH 7.5, containing 0.1 mM EDTA, 50 mM KCl and 1 mM
dithiothreitol (HEK/DTT). After shaking for one hour at 4.degree.
C., unbound membrane fragments were washed away with HEK/DTT. The
wells were blocked with 100 .mu.l 2% BSA in HEKL (35 mM HEPES; 0.1
mM EDTA; 50 mM KCl; 0.2 M .alpha.-lactose; pH 7.5, with 1 mM DTT).
For rounds 1 and 2, BSA was used for blocking; in later rounds 1%
nonfat dry milk was used. For the first round of panning, about 24
wells of a 96-well plate were used. In subsequent rounds, 8 wells
with receptor and 8 wells without receptor were prepared.
[0200] The Gt library was thawed (2 mL aliquot) and mixed with 6 mL
lysis buffer on ice. Lysis buffer contains 4.25 mL HE (25 mM HEPES:
0.1 mM EDTA; pH 7.5); 1 mL 50% glycerol; 750 .mu.L 10 mg/mL
protease-free BSA in HE; 10 .mu.L 0.5 M DTT; and 6.25 .mu.L 0.2 M
PMSF. Freshly prepared lysozyme solution (150 .mu.L 10 mg/mL
lysozyme in cold HE) was added and the tube was gently inverted
several times and incubated on ice for no more than two minutes.
The extent of lysis is evidenced by an increase in viscosity that
can be observed by noting the slow migration of bubbles to the top
of the tube after mixing. Lysis was terminated by mixing in 2 mL
20% lactose and 250 .mu.L 2M KCl. The tube was centrifuged
immediately at 13,000.times.g for 15 minutes at 4.degree. C. and
the supernatant transferred to a new tube. A small aliquot of 0.1%
(the PRE sample) was saved in a separate, labeled tube. The blocked
rhodopsin receptor-coated plate was rinsed four times with HEKL/1%
BSA and exposed to room light for less than five minutes on ice to
activate the rhodopsin for light-activated rhodopsin (Table IX), or
left in the dark for dark-adapted (inactive) rhodopsin (Table X).
Immediately thereafter, the crude bacterial lysate from the peptide
library (200 .mu.L) was added to each well and allowed to shake
gently for one hour at 4.degree. C. For round 2, this same
procedure was followed. In round 3, the amount of lysate used was
reduced to 100 .mu.L. In subsequent rounds, the lysate was diluted
1:10 in HEKL/BSA. In all rounds, 5-10 .mu.L 200 .mu.M native
peptide was added to the wells to chase off peptides that were
bound with lower affinity.
[0201] After incubation with the bacterial lysate, the wells were
washed four times into cold HEKL/1% BSA. Sonicated salmon sperm DNA
(200 .mu.L 0.1 mg/mL in HEKL/1% BSA was added to each well and
shaken gently for 30 minutes at 4.degree. C. The plates were washed
four times with cold HEKL and twice with cold HEK, then eluted by
adding 50 .mu.L/well 1 mM IPTG/0.2 M KCl in HE with vigorous
shaking at room temperature for 30 minutes. The eluants from each
group of wells (+ or - receptor) were combined in one or more
microcentrifuge tubes as necessary. The volume of the PRE sample
which had been saved previously was brought up to match the volume
of the eluant samples and precipitated in parallel with them. To
precipitate, 1/10 volume of 5M NaCl was mixed with each of the
samples, then 1 .mu.L 20 mg/mL glycogen was mixed with the samples.
An equal volume of RT isopropanol was then added and mixed
thoroughly. The samples were subjected to centrifugation at
13,000.times.g for 15 minutes and the supernatant aspirated. The
pellet was washed with 500 .mu.L cold 80% ethanol and again
subjected to centrifugation at 13,000.times.g for 10 minutes. The
pellets of plasmid DNA were resuspended in sterile,
double-distilled water, 200 .mu.L for the PRE sample and 4 .mu.L
for the + or - receptor samples and stored at -20.degree. C.
[0202] Both light-activated rhodopsin and dark-adapted rhodopsin
were used to screen the library in this manner. See Tables IX and
X, below. Six of the sequences obtained using light-activated
rhodopsin were 100-1000 times more potent than the native sequence
at binding rhodopsin and are listed in Table IX. When the G.alpha.t
library was used to pan light-activated rhodopsin, residues L344,
C347 and G348 were invariant. Also, in each of the highest affinity
sequences, the basic residue at position 341 (R341) was changed to
a neutral residue. When the G.alpha.t library was used to pan
dark-adapted odopsin, the L344, C347 and G348 residues were no
longer variant (L344 present in 62.5% of sequences, C347 present in
25% of sequences, G348 present in 75% of sequences) and the residue
at position 341 was usually unchanged. Thus, the conformation of
the receptor in its inactive, dark-adapted state allows it to bind
to a different set of peptide analogs than the light-activated
receptor. In addition, it appears that in the light-activated
receptor, it is the last seven amino acids of the peptide which are
most important (344-350) while the first six amino acids (340-345)
are more important for dark-adapted rhodopsin binding.
TABLE-US-00009 TABLE IX Light-Activated Rhodopsin High Affinity
Sequences. Clone No. SEQ ID NO: Sequence Library Sequence 124
IRENLKDCGLF 8 125 LLENLRDCGMF 9 126 IQGVLKDCGLL 10 127 ICENLKECGLF
18 128 MLENLKDCGLF 23 129 VLEDLKSCGLF 24 130 MLKNLKDCGMF 3 131
LLDNIKDCGLF 4 132 ILTKLTDCGLF 6 133 LRESLKQCGLF 11 134 IHASLRDCGLF
13 135 IRGSLKDCGLF 14 136 IFLNLKDCGLF 15/28 137 IRENLEDCGLF 16 138
IIDNLKDCGLF 17 139 MRESLKDCGLF 19 140 IRETLKDCGLL 26 141
ILADVIDCGLF 27 142 MCESLKECGLF
[0203] TABLE-US-00010 TABLE X Dark-Adapted Rhodopsin High Affinity
Sequences. Clone No. SEQ ID NO: Sequence Library Sequence 124
IRENLKDCGLF 2 143 IREKWKDLALF 3 144 VRDNLKNCFLF 7 145 IGEQIEDCGPF
17 146 IRNNLKRYGMF 21 147 IRENLKDLGLV 26 148 IRENFKYLGLW 33/37 149
SLEILKDWGLF 41 150 IRGTLKGWGLF
Example 8
Screens of PAR1 with a Gq Peptide Library
[0204] The methods of Example 7 were used to screen different
sources of PAR1 receptor using the Gq library. Purified PAR1,
reconstituted in lipid vesicles (Example 6), membranes prepared
from Sf9 insect cells expressing PAR1 (Example 3) and membranes
prepared from mammalian cells overpressing PAR1 were used. The
results of the screens are presented in Tables XI, XII and XIII,
respectively. The peptide used as the competitor for all three
screens was LQLNLKEYNLV (SEQ ID NO:2). The 4-residue linker
sequences are random and are optionally present at the amino
terminus of the binding peptide. These results show that the
identified high affinity peptides are similar for all three sources
of screened PAR1. When a Gq-biased library is used to pan PAR1, the
positions that appear to be critical for receptor recognition, and
thus are invariant, are N348, L349 and V350. TABLE-US-00011 TABLE
XI Reconstituted Purified Recombinant PAR1 Receptor; Screening
Results. SEQ ID Clone Linker SEQ ID NO: LQLNLKEYNLV NO:2 R2-16 *SWV
151 LQFNLNDCNLV 102 R2-17 FVNC 152 LQRNKKQYNLG 160 R2-18 EVRR 153
MKLKLKEDNLV 103 R2-20 *RVQ 154 HQLDLLEYNLG 104 R2-21 RLTR 155
LQLRYKCYNLV 161 R3-37 SR*K 156 LQQSLIEYNLL 111 R3-38 MTHS 157
VHVKLKEYNLV 162 R3-44 SGPQ 158 LQLNVKEYNLV 163 R3-46 ML*N 159
LRIYLKGYNLV 164
[0205] TABLE-US-00012 TABLE XII PAR1 Receptor Sf9 Insect Cell
Membranes; Screening Results. SEQ ID Clone Linker SEQ ID NO:
LQLNLKEYNLV NO:2 S1-13 S*IR 165 MKLNVSESNLV 94 S1-18 RWIV 166
LQLNLKVYNLV 175 S1-23 G*GH 167 LELNLKVYNLF 176 S2-26 RSEV 168
LQLKHKENNLM 100 S2-30 CEPG 169 LHLNMAEVSLV 177 S2-36 HQMA 170
LQVNLEEYHLV 101 S3-6 VPSP 171 LQKNLKEYNMV 106 S3-8 QMPN 172
LQMYLRGYNLV 108 S3-10 MWPS 173 LKRYLKESNLV 178 S3-15 C*VE 174
MNLTLKECNLV 110
[0206] TABLE-US-00013 TABLE XIII Mammalian (CHO) Cells
Overexpressing PAR1; Screening Results. SEQ ID Clone Linker SEQ ID
NO: LQLNLKEYNLV NO:2 C4-5 PRQL 179 LQLKRGEYILV 183 C4-19 VRPS 3
LQLNRNEYYLV 4 C5-10 SRHT 11 LRLNGKELNLV 12 C5-12 FFWV 180
CSLKLKAYNLV 184 C4-16 QRDT 181 LQMNHNEYNLV 185 C7-3 NERN 182
PQLNLNAYNLV 186 C7-10 LPQM 9 QRLNVGEYNLV 10 C7-13 LSTN 7
LHLNLKEYNLV 8 C7-14 LSRS 5 LQQKLKEYSLV 6
Example 9
Identification of GPCR-Binding High Affinity Peptide Analogs
(Panning)
[0207] The methods of Example 7 were repeated using recombinant
reconstituted .beta..sub.2 adrenergic receptor panned with the Gs
Library. Results of the panning screens and ELISA binding affinity
of the selected peptides are shown in Table XIV, below.
TABLE-US-00014 TABLE XIV .beta.2-Adrenergic Receptor screened with
Gs library. SEQ ID NO Competitor QRMHLRQYELL 13 ELISA AG1
QGMQLRRFKLR 187 .435 AG20 RWLHWQYRGRG 188 .431 AG19 PRPRLLRFKIP 189
.361 AG2 QGEHLRQLQLQ 190 .330 AG4 QRLRLGPDELF 191 .291 BAR1
QRIHRRPFKFF 192 .218 AG3 QRMPLRLFEFL 193 .217 BAR2 QRVHLRQDELL 194
.197 AG11 DRMHLWRFGLL 195 .192 AG9 QRMPLRQYELL 196 .190 BAR3
QWMDLRQHELL 197 .185 AG18 QRMNLGPCGLL 198 .155 BAR20 NCMKFRSCGLF
199 .079 AG13 QRLHLRGYEFL 200 .054 BAR11 HRRHIGPFALL 201 .048 BAR8
ERLHRRLFQLH 202 .047 BAR40 PCIQLGQYESF 203 .028 BAR31 QRLRLRKYRLF
204 .026
Example 10
Identification of GPCR-Binding High Affinity Peptide Analogs
(Panning)
[0208] The methods of Example 7 repeated using rhodopsin screening
with a Gt library. Results of the panning screens and ELISA binding
affinity of the selected peptides are shown in Table XV, below. To
identify the rank order of binding, the lysates were analyzed using
ELISA methods in which the secondary antibody was conjugated to
HRP. Following addition of the substrate, the microplate was read
using a spectrophotometer. The binding is the OD.sub.450 for wells
with receptor--OD.sub.450 for wells in which no receptor (control
wells with empty lipid vesicles). TABLE-US-00015 TABLE XV Rhodopsin
screened with Gt library. SEQ ID NO: Competitor IRENLKDCGLF 124
ELISA L33 IVEILEDCGLF 205 1.007 L4 MLDNLKACGLF 206 .908 L3
ILENLKDCGLF 207 .839 L14 LRENLKDCGLL 208 .833 L38 LLDILKDCGLF 209
.823 L15 VRDILKDCGLF 210 .621 L34 ILESLNECGLF 211 .603 L17
ILQNLKDCGLE 212 .600 L7 MLDNLKDCGLF 213 .525 L10 IHDRLKDCGLF 214
.506 L20 IRGSLKDCGLF 135 .423 L6 ICENLKDCGLF 215 .342 L8
IVKNLEDCGLF 216 .257 L13 ISKNLRDCGLL 217 .187 L10 IRDNLKDCGLF 218
.162
Example 11
Additional Peptide Analogs
[0209] Chinese hamster ovary-expressed PAR1 was screened against
the Gt, G12 and G13 libraries, using the competitor peptide
indicated in Table XVI below. Additional peptide analogs were
identified using the Gt, G12 or G13 library as indicated and
IRENLKDCGLF (SEQ ID NO:124), LQENLKDIMLQ (SEQ ID NO:38) or
LQDNLKQLMLQ (SEQ ID NO:233), respectively as competitor with
screening for high affinity binding to PAR1 receptor obtained from
Chinese hamster ovary cells as described in Example 1, indicated in
Table XVII, below. TABLE-US-00016 TABLE XVI Peptides Identified
with CHO EXPRESSED PAR1. Gt library G12 library G13 library
(IRENLKDCGLF; (LQENLKDIMLQ; (LQDNLKQLMLQ; SEQ ID NO:124) SEQ ID
NO:38) SEQ ID NO:233) IREFLTDCGLF 219 LQENLKEMMLQ 225 LQDNLRHLMLQ
234 IRLDLKDVSLF 220 LEENLKYRMLD 226 LQDKINHLMLQ 235 ICERLNDCGLC 221
LQEDLKGMTLQ 227 LQANRKLGMLQ 236 PRDNTKVRGLF 222 LQETMKDQSLQ 228
LIVKVKQLIWQ 237 FWGNLQDSGLF 223 PQVNLKSIMRQ 229 MRAKLNNLMLE 238
RRGNGKDCRHF 224 WQHKLSEVMLQ 230 LQDNLRHLIQ 239 LKEHLMERMLQ 231
LQDNRNQLLF 240 LLGMLEPLMEQ 232
[0210] TABLE-US-00017 TABLE XVII PAR1 Binding Peptides Screened
using a G11 Library (LQLNLKEYNLV; SEQ ID NO: 2) SF9 CHO EXPRESSED
SEQ ID NO: Recomb/Reconst SEQ ID NO: EXPRESSED SEQ ID NO:
LQLNVKEYNLV 163 LQLNVKEYNLV 163 LQLNLKVYNLV 175 LQLNRKNYNLV 241
LQLRVKEYKRG 244 LQLKHKENNLM 100 LQLRYKCYNLV 161 LQLRYKCYNLV 161
LQKNLKEYNMV 106 LQLDLKESNMV 242 LQIYLKGYNLV 245 LQVNLEEYHLV 101
LQLNLKKYNRV 243 LQFNLNDCNLV 102 LFLNLKEYSLV 257 LQLRVKEYKRG 244
LQRNKKQYNLG 160 LELNLKVYNLV 258 LQRNKKQYNLG 160 LQRNKNQYNLG 254
LPLNPKEYSLV 109 LQIYLKGYNLV 245 LQQSLIEYNLL 111 LPLNLIDFSLM 259
LQFNLNDCNLV 102 LRLDFSEKQLV 105 LPRNLKEYDLG 260 LQYNLKESFVV 246
LYLDLKEYCLF 255 LRLNDIEALLV 261 LQQSLIEYNLL 111 HQLDLLEYNLG 104
LVLNRIEYNLL 262 LQRDHVEYKLF 247 VQVKLKEYNLV 251 LHLNMAEVSLV 177
LVIKPKEFNLV 248 MKLKLKEDNLV 103 MNLTLKECNLV 110 IQLNLKNYNIV 249
SAKELDQYNLG 256 MKLNVSESNLV 94 HQLDLLEYNLG 104 VHVKLKEYNLV 162
LKRYLKESNLV 178 MQLNLKEYNLV 250 LKRKLKESNMG 263 VQVKLKEYNLV 251
LKRKVKEYNLG 264 QLLNQYVYNLV 252 LELNLKVYNLF 176 MKLKLKEDNLV 103
LQMYLRGYNLV 108 WRLSLKVYNLV 253 LQLKRGEYILV 183 LQLNRNEYYLV 4
LRLNGKELNLV 12 CSLKLKAYNLV 184 LQMNHNEYNLV 185 PQLNLNAYNLV 186
QRLNVGEYNLV 10 LHLNLKEYNLV 8 LQQKLKEYSLV 6
Example 12
Preparation of LacI Lysates
[0211] In the last round of panning, several clones were selected
from the (+) receptor plates and grown up overnight in LB-Amp
media. Three hundred microliters of the overnight culture was
diluted in 3 mL LB-Amp media for "ELISA lysate culture." Another 30
.mu.L was added to an equal volume of 50% glycerol was stored in
labeled microcentrifuge tubes at -70.degree. C. The remaining 4.5
mL was used to make DNA using a standard miniprep protocol (Qiagen
Spinprep.TM. kits) and sequenced using a 19 base pair reverse
primer which is homologous to the vector at a site 56 basepairs
downstream from the TAA stop codon that terminates the random
region of the library (GAAAATCTTCTCTCATCCG; SEQ ID NO:265). The DNA
was stored at -20.degree. C. The ELISA lysate culture was allowed
to shake for one hour at 37.degree. C. Expression was induced by
adding 33 .mu.L 20% arabinose (0.2% final concentration) with
shaking at 37.degree. C. for 2-3 hours. The culture then was
subjected to sedimentation at 4000.times.g for five minutes, the
pellet resuspended in 3 mL cold WTEK buffer, resedimented at
4000.times.g for five minutes and the pellet resuspended in 1 mL
cold TEK buffer. After transfer to 1.5 mL microcentrifuge tubes,
the pellet was sedimented at 13,000.times.g for two minutes and the
supernatant aspirated. The cell pellet was resuspended in 1 mL
lysis buffer (42 mL HE, 5 mL 50% glycerol, 3 mL 10 mg/mL BSA in HE,
750 .mu.L 10 mg/mL lysozyme in HE and 62.5 .mu.L 0.2 M PMSF) and
incubated on ice for one hour. One hundred ten microliters 2M KCl
was added to the lysis mixture and inverted to mix, then sedimented
at 13,000.times.g for 15 minutes at 4.degree. C. The clear crude
lysate (about 0.9 mL supernatant) was transferred to a new tube and
stored at -70.degree. C.
Example 13
PAR1 Receptor-Specific Binding of LacI-Peptide Fusion Proteins
[0212] The binding properties of the peptide encoded by individual
clones were assayed as follows. Purified PAR1 receptor prepared
from Sf9 insect cells (1-10,000 pg/mL in 50 mM Tris-HCl, pH 7.4,
10% glycerol) was reconstituted in lipid vesicles according to
Example 6. A serial dilution of the membranes containing receptor
ranging from 0.2 to 20,000 .mu.g/mL (+/- receptor) was added to
wells on a microtiter plate and shaken gently for one hour at
4.degree. C. After washing, a 1:1 to 1:10,000 serial dilution of a
LacI-Gq lysate prepared from the LacI-Gq clone according to the
methods described in Example 12 was added to the wells, the plate
was shaken gently for one hour at 4.degree. C., and washed.
Anti-LacI antibodies (Stratagene) were added (1:1000) and the plate
shaken gently for one hour at 4.degree. C. After washing,
HRP-conjugated goat anti-rabbit antibodies (Kierkegaard and Perry
Laboratories) were added (1:2500) and the plate shaken gently for
one hour at 4.degree. C. The plate was washed, color was developed
using horseradish peroxidase, and then read in an ELISA reader at
OD.sub.450. The general methodology for the ELISA is illustrated in
FIG. 4. The results, see FIG. 5, show that the LacI-Gq fusion
protein binds thrombin receptor in a concentration-dependent
manner. The ability of the LacI-Gq fusion protein to bind the empty
vesicles was significantly less than vesicles reconstituted with
thrombin receptor.
Example 14
Screening in the Presence of a High Affinity Peptide
[0213] To identify peptides having even higher affinity to
light-activated rhodopsin than those identified by the panning
procedure described in Example 7, a high affinity peptide was
included in the library incubations in rounds three and four.
Peptide 8 (LLENLRDCGMF; SEQ ID NO:125) had been identified in the
first screening as a peptide exhibiting binding to light-activated
rhodopsin 1000-fold higher than the native sequence. Screening of
the G.alpha.t library was performed as in Example 7, except that 10
.mu.L 100 .mu.M (100 nM final concentration) peptide 8 was included
in the wells in rounds three and four. This screen revealed several
clones that both bind rhodopsin with very high affinity and
stabilize it in its active form, metarhodopsin II. See Table XVIII,
below. Comparing Tables IX and XVIII, it is clear using peptide 8
in the screen resulted in a change at position 341 to a neutral
residue. Residues L344, C347 and G348 remained stable whether
peptide 8 was included in the screen or not. Use of peptide 8
resulted in a higher incidence of isoleucine at position 340 (17%
with native peptide versus 71% with peptide 8) and a lower
incidence of glutamine at position 342 (67% with native peptide
versus 29% with peptide 8) This type of information not only
contributes to the discovery of highly potent analog peptides for
use as drugs or drug screening compounds, but also furthers the
understanding of the structural framework which underlies the sites
of contact between G.alpha. and receptor.
[0214] Binding assays performed on some of the clones identified in
this way are shown in FIG. 6. All peptides identified using peptide
8 in the screening process bound with equal or greater affinity to
light-activated rhodopsin as did peptide 8. Compare the first bar
(HAP=peptide 8) with the remaining bars. TABLE-US-00018 TABLE XVIII
Exemplary Light-Activated Rhodopsin High Affinity Sequences
Identified in Screens with Addition of Peptide 8. Clone No. SEQ ID
NO: Sequence Library Sequence 124 IRENLKDCGLF Peptide 8 125
LLENLRDCGMF 3 266 ILENLKDCGLL 7 213 MLDNLKDCGLF 8 216 IVKNLEDCGLF
10 218 IRDNLKDCGLF 13 217 ISKNLRDCGLL 17 212 ILQNLKDCGLF 19 206
MLDNLKACGLF
Example 15
Subcloning into MBP Vectors and Preparation of MBP Crude
Lysates
[0215] pELM3 was digested at room temperature with AgeI (New
England Biolabs) and the cut vector was separated from uncut vector
on a 0.7% agarose gel. DNA was purified (Qiagen Extract-a-gel kit)
and digested with ScaI (New England Biolabs). The 5.6 kb MBP vector
fragment was separated on a 1% agarose gel and purified as above.
During the final affinity purification round of the peptide
library, a 20 mL portion of the 200 mL amplification culture was
set aside before harvesting the cells. This 20 mL portion was
allowed to grow to saturation, usually overnight, and DNA was
prepared from the cells (Qiagen midi-prep kit). The pJS142 plasmid
DNA was digested with BspEI and ScaI. The 0.9 kb peptide-encoding
fragment was separated from the 3.1 and 1.7 kb vector fragments on
a 1% agarose gel and purified.
[0216] Different ratios of the 5.6 kb MBP vector fragment and the
peptide-encoding 0.9 kb fragment (1:2, 1:1, 2.5:1, 5:1, 10:1) were
ligated in ligase buffer containing 0.4 mM ATP at 14.degree. C.
overnight with T4 DNA ligase. The ligation was terminated by
increasing the temperature to 65.degree. C. for ten minutes. To
lower the background, the ligation mixture was digested with XbaI
before isopropanol precipitation using 1 .mu.L glycogen as a
carrier. After one wash with 80% ethanol, the pellet was
resuspended in 20 .mu.L double-distilled water. ARI814 cells were
transformed as described in Example 7 using 1 .mu.L of the
precipitated XbaI digested ligation mix. After allowing the cells
to shake for one hour at 37.degree. C. in 1 mL SOC, 100 .mu.L of
the suspension was spread on LB-Amp Plates. Crude lysates were
prepared as described for LacI lysates in Example 12.
Example 16
MBP-Peptide Fusion Protein Purification
[0217] An overnight culture (1 mL) of a single MBP-peptide fusion
protein clone was inoculated into 200 mL LB-AMP media. The culture
was shaken at 37.degree. C. until OD.sub.600=0.5. Protein
expression was induced by addition of 150 .mu.L 1 M IPTG (final
concentration 0.3 mM), with continued shaking at 37.degree. C. for
two hours. The culture then was sedimented at 5000.times.g for 20
minutes and resuspended in 5 mM column buffer (10 mM Tris, pH 7.4;
200 mM NaCl; 1 mM EDTA; 1 mM DTT) and 16.25 .mu.L 0.2 M PMSF was
added. The resuspended cell pellet was then stored at -70.degree.
C. The stored pellet was thawed in cold water and placed in an ice
bath. The pellet was sonicated in short pulses of less than 15
seconds with a Fisher Scientific 55 Sonic Dismembrator (40%
constant time, output 5, repeating five times with a total one
minute duration). The sonicated pellet was subjected to
centrifugation at 9000.times.g for 30 minutes, after which the
supernatant was saved and diluted to 100 mL using column buffer.
Usually, the protein concentration was approximately 2.5 mg/mL. A
column was prepared by pouring 7.5 ml amylose resin in a BioRad
disposable column and washing with eight volumes of column buffer.
The diluted crude extract was loaded by gravity flow at about 1
mL/min and the column was washed again with eight volumes of column
buffer. The fusion protein was eluted with 10 mL 10 mM maltose in
column buffer and concentrated using Amicon centriplus 30.TM.
columns, then aliquoted and stored at -70.degree. C.
Example 17
Method for Screening Library Crude Lysates by ELISA
[0218] Microtiter wells were coated with 0.1-1.0 .mu.g/well
rhodopsin receptor in a final volume of 100 .mu.L HEK containing 1
mM DTT with shaking at 4.degree. C. for one hour. The wells then
were blocked with bovine serum albumin (BSA) by adding 100 .mu.L 2%
BSA in HEK with 1 mM DTT to the wells and continuing shaking at
4.degree. C. for at least 30 minutes, then washed four times with
HEK containing 1 mM DTT. Crude lysates were diluted 1:50 in HEK
containing 1 mM DTT and added to the coated wells (100 .mu.L per
well). The plates were shaken at 4.degree. C. for one hour, washed
four times with PBS/0.05% Tween.TM.20 1 mM maltose and then probed
with 100 .mu.L 1:1000 rabbit anti-MBP antibodies (New England
BioLabs) in PBS containing 0.05% Tween.TM. 20 and 1 mM maltose,
with shaking for 30 minutes at 4.degree. C. After another wash, the
wells were probed with 100 .mu.L 1:7500 goat anti-rabbit secondary
antibodies conjugated to horseradish peroxidase in PBS containing
1% BSA and 1 mM maltose with shaking for 30 minutes at 4.degree. C.
The plate was washed four times with PBS containing 0.05% Tween.TM.
20 and 1 mM maltose. Horseradish peroxidase substrate (Bio-Fx; 100
.mu.L) was added and the color developed for 20-30 minutes. The
reaction was stopped by addition of 100 .mu.L 2N sulfuric acid and
the plate read at OD.sub.450. If the color reaction occurred too
quickly (less than 10 minutes) or if the background in negative
control wells was too high (greater than 0.2) the assay was
repeated using 1:100 or 1:200 dilutions of the crude lysates.
Example 18
Binding Assay of High Affinity Rhodopsin Binding Peptide Fusion
Proteins
[0219] The entire population of peptide-coding sequences identified
in round 4 of panning (see Example 7) was transferred from pJS142
to pELM3 (New England Biolabs). This plasmid is a pMal-c2
derivative with a modified polylinker, inducible by isopropyl
.beta.-thiogalacto-pyranoside and containing the E. coli malE gene
with a deleted leader sequence and leads to cytoplasmic expression
of MBP fusion proteins. The MBP-carboxyl terminal peptide analog
fusion proteins were expressed in E. coli.
[0220] For the assay, in the dark, 1 .mu.g/well of ROS membranes
(rhodopsin) as described in Example 5 was directly immobilized on
microtiter wells in cold HEK/DTT for one hour at 4.degree. C. The
wells were rinsed, blocked with 1% BSA in HEK/DTT for one hour at
4.degree. C. and rinsed again. Bound rhodopsin was activated by
exposure to light for 5 minutes on ice before addition of the MBP
fusion proteins (crude bacterial lysates were diluted 1:50 in HEK
with 1 .mu.M dithiothreitol; purified proteins were used at 0.2-120
nM). The MBP-G.alpha.t340-350K341R (pELM17) fusion protein and MBP
with linker sequence only (pELM6) were present in control wells at
50 nM final concentration. After 30 minutes, wells were washed and
rabbit anti-MBP antibody (New England Biolabs) was added. The
anti-MBP antibody was used at a 1:1000 dilution for crude lysates
and a 1:3000 dilution for purified proteins. After 30 minutes,
wells were rewashed and goat anti-rabbit antibody conjugated to
horseradish peroxidase (1:7500 dilution for crude lysates; 1:10,000
dilution for purified proteins; Kierkagaard & Perry
Laboratories) was added. After 30 minutes, the plate was washed
four times with PBS containing 0.05% Tween.TM.20. Horseradish
peroxidase substrate (100 .mu.l) was added and color was allowed to
develop for about 20 minutes. The reaction was stopped by addition
of 100 .mu.l 2N sulfuric acid. The results are presented in FIG. 7.
Values indicate absorbance at OD.sub.450. The positive control for
the assay was pELM 17, which encodes the MBP fusion protein
G.alpha..sub.t340-350K341R. pELM6, which expresses MBP protein
fused to a linker sequence only, served as the negative control.
"No lysate" control wells were included to reflect any intrinsic,
non-specific binding within the assay. See FIG. 7.
[0221] The IC.sub.50 values of the high affinity MBP fusion
proteins ranged from 3.8 to 42 nM, up to 3 orders of magnitude more
potent than the 6 .mu.M IC.sub.50 of MBP-G.alpha.t340-350K341R. In
all the highest affinity sequences, position 341, which is a
positively charged residue in the native sequence, was changed to a
neutral residue. Leu344, Cys347, and Gly348 were found to be
invariant and hydrophobic residues were always located at positions
340, 349, and 350, indicating the critical nature of these
residues.
Example 19
Binding of High Affinity Peptide Fusion Proteins to Rhodopsin Can
Be Competitively Inhibited by Heterotrimeric Gt
[0222] When light-activated rhodopsin was screened for peptides
based on the C-terminus of Gt, a large number of high-affinity
sequences were obtained. Binding of MBP fusion proteins containing
the high affinity peptide from the library (sequences from clones
8, 9, 10, 18, 23, 24, as well as pELM17 which encodes the wild-type
peptide sequence, and pELM6 which contains no peptide; MBP-8,
MBP-9, MBP-10, MBP-18, MBP-23, MBP-24, MBP-pELM17) were assessed
for their ability to bind rhodopsin (0.5 .mu.g rhodopsin/well) in
the presence or absence of heterotrimeric Gt.
[0223] Lysate (50 .mu.l) from each clone was added and incubated in
the light. After 45 minutes, 1 .mu.M heterotrimeric Gt was added
and the solution incubated for 30 minutes. Anti-MBP antibody was
added, followed by goat anti-rabbit alkaline phosphatase conjugated
antibody and substrate. The color was allowed to develop.
Absorbance data are presented in FIG. 8.
[0224] Most peptide sequences obtained were highly homologous to
the native G.alpha.t C-terminal sequence. Several of these
sequences are of very high affinity (>1000-fold higher than the
parent peptide) and are potent and specific antagonists of
receptor-mediated G protein activation. The high-affinity peptide
fusion proteins were tested for binding to light-activated
rhodopsin and for their ability to stabilize the MII conformation
(Table XXI).
[0225] The screen used MBP-8 because this peptide bound to
rhodopsin with high affinity and stabilized MII. MBP-18 and MBP-24,
which both showed even higher binding affinities than did MBP-8 to
rhodopsin, were not used because the affinity was so high that the
small molecules might not have been able to competitively inhibit
their binding. Of course, the screen may be repeated using another
peptide as is convenient, for example peptides that are of higher
affinity to find even more potent small molecules. TABLE-US-00019
TABLE XIX Absorbance at OD.sub.450 in a Panning ELISA and EC50
values for MII binding and MII Stabilization for Selected MBP-High
Affinity Peptide Fusion Proteins. SEQ MII MII ID ELISA binding
stabilization NO: OD.sub.450 EC.sub.50 EC.sub.50 Gt IKENLKDCGLF 15
.01 6000 nM >100 .mu.M 9 LQQVLKDCGLL 267 .35 10 nM 1.05 .mu.M 10
ICENLKDCGLF 215 .36 42 nM 5.40 .mu.M 8 LLENLRDCGMI 268 .54 7.8 nM
0.94 .mu.M 18 MLENLKDCGLF 128 .58 3.8 nM 1.24 .mu.M 24 MLKNLKDCGMF
130 .61 6.6 nM 0.49 .mu.M 23 VLEDLKSCGLF 129 .66 20 nM 3.50
.mu.M
[0226] Heterotrimeric Gt competitively inhibited high affinity
peptide fusion protein binding to light-activated rhodosin. See
FIG. 8. The heterotrimeric Gt contains multiple determinants of
rhodopsin binding and is membrane bound via myristoylation of the
.alpha. subunit and farnesylation of the .gamma. subunit carboxyl
terminus. Thus, the selected peptide sequences from the
combinatorial library bind to the receptor with very high
affinity.
Example 20
Binding of MBP Clones to PAR1
[0227] To identify high affinity peptides that bind PAR1, membranes
prepared from mammalian cells (Chinese hamster ovary)
overexpressing PAR1 were panned with the G11 peptide library. ELISA
binding affinity results of selected clones are shown in FIG. 9 for
their binding to membranes prepared from SF9 cells expressing
either PAR1 or the Gq-coupled muscarinic M1 receptor. To quantitate
the binding, purified MBP clones were analyzed using ELISA methods
in which the secondary antibody was conjugated to HRP. The binding
for the control MBP-Gq fusion protein is shown. See FIG. 9. The
data are the average of two separate experiments done in duplicate.
MBP clones PAR-13 and PAR-34 both show both high affinity binding
for PAR1 as well as specificity. MBP clones PAR-23 and PAR-33
appear to be both of low affinity and low specificity. See Table
XIII for the sequences.
Example 21
Binding Specificity of LacI-Peptide Fusion Proteins
[0228] PAR1 binding clones of LacI-peptide fusion protein selected
from the G11 Library were diluted 1:100 in HEK/DTT and tested for
dose-responsive binding to Sf9 insect cell membranes from cells
expressing no receptor, the M1 receptor (which couples to Gi) or
PAR1 receptor, prepared according to Example 3. Increasing amounts
of membrane as indicated in FIG. 10 were coated in microtiter
wells, incubated and rinsed. LacI-peptide fusion protein lysates
were added, incubated and rinsed, and the receptor-bound
LacI-peptide fusion protein was measured as described above using a
LacI antibody. Results for a single, representative clone are
presented in FIG. 10, and demonstrate the specificity of the
selected peptides for PAR1.
Example 22
Binding of Native G.alpha.q-Maltose Binding Protein-Peptide Fusion
Protein to PAR1
[0229] Microtiter wells were coated with purified, reconstituted
PAR1 in the presence of 100 nmoles thrombin receptor activating
peptide, as described above in Example 6. Purified maltose binding
protein-G.alpha.q (MBP-G.alpha.q) was added at the concentrations
indicated in FIG. 11 and incubated one hour on a shaker at
4.degree. C. The wells were rinsed and then probed with a rabbit
anti-maltose binding protein antibody, followed by alkaline
phosphatase conjugated secondary antibodies, as described above.
Substrate was added and the color was allowed to develop about 20
minutes. Absorbance at 405 nm was measured and dose-response curves
were calculated using GraphPad Prism (version 2.0). See results in
FIG. 11. The calculated IC.sub.50 of G.alpha.q binding to activated
PAR1 was 214 nM.
Example 23
Design of Oligonucleotides for G.alpha. Peptide Minigene
Constructs
[0230] cDNA encoding the last 11 amino acids of G.alpha. subunits
was synthesized (Great American Gene Company) with newly engineered
5'- and 3'-ends. The 5'-end contained a BamHI restriction enzyme
site followed by the human ribosome-binding consensus sequence
(5'-GCCGCCACC-3'; SEQ ID NO:269), a methionine codon (ATG) for
translation initiation, and a glycine codon (GGA) to protect the
ribosome binding site during translation and the nascent peptide
against proteolytic degradation. A HindIII restriction enzyme site
was synthesized at the 3' end immediately following the
translational stop codon (TGA). Thus, the full-length 56 bp
oligonucleotide for the Gi.alpha..sub.1/2 carboxyl terminal
sequence was
5'-gatccgccgccaccatgggaatcaagaacaacctgaaggactgcggcctcttctgaa-3'
(SEQ ID NO:270) and the complimentary strand was
5'-agctttcagaagaggccgcagtccttcaggttgttcttgattcccatggtggcggcg-3'
(SEQ ID NO:271). See FIG. 12. As a control, oligonucleotides
encoding the G.alpha.i.sub.1/2 carboxyl terminus in random order
(G.alpha.iR) with newly engineered 5'- and 3'-ends also were
synthesized.
[0231] The DNA was diluted in sterile ddH.sub.2O to form a stock
concentration at 100 .mu.M. Complimentary DNA was annealed in
1.times. NEBuffer 3 (50 mM Tris-HCl, 10 mM MgCl.sub.2, 100 mM NaCl,
1 mM DTT; New England Biolabs) at 85.degree. C. for 10 minutes then
allowed to cool slowly to room temperature. The DNA then was
subjected to 4% agarose gel electrophoresis and the annealed band
was excised. DNA was purified from the band using a kit, according
to the manufacture's protocol (GeneClean II Kit, Bio101). After
digestion with each restriction enzyme, the pcDNA 3.1(-) plasmid
vector was subjected to 0.8% agarose gel electrophoresis, the
appropriate band cut out, and the DNA purified as above (GeneClean
II Kit, Bio101). The annealed/cleaned cDNA was ligated for 1 hour
at room temperature into the cut/cleaned pcDNA 3.1 .mu.lasmid
vector (Invitrogen) previously cut with BamHI and HindIII.
[0232] For the ligation reaction, several different ratios of
insert to vector cDNA (ranging from 25 .mu.M:25 pM to 250 pM:25 pM
annealed cDNA) were plated. Following the ligation reaction, the
samples were heated to 65.degree. C. for 5 min to deactivate the T4
DNA ligase. The ligation mixture (1 .mu.l) was electroporated into
50 .mu.l competent cells as described in Example 7 and the cells
immediately placed into 1 ml of SOC (Gibco). After 1 hour shaking
at 37.degree. C., 100 .mu.l of the electroporated cells containing
the minigene plasmid DNA was spread on LB/Amp plates and incubated
at 37.degree. C. for 12-16 hours. To verify that insert was
present, colonies were grown overnight in LB/Amp and their plasmid
DNA purified (Qiagen SpinKit). The plasmid DNA was digested with
Ncol (New England Biolabs, Inc.) for 1 hour at 37.degree. C. and
subjected to 1.5% (3:1) agarose gel electrophoresis. Vector alone
produced 3 bands. When the 56 bp annealed oligonucleotide insert is
present, there is a new NcoI site resulting in a shift in the band
pattern such that the digest pattern goes from three bands (3345
bp, 1352 bp, 735 bp) to four bands (3345 bp, 1011 bp, 735 bp, 380
bp). See FIG. 13. DNA with the correct electrophoresis pattern was
sequenced to confirm the appropriate sequence. This method may be
used to insert any high affinity peptide to create a minigene
constant.
Example 24
Expression of Peptides from Minigene Constructs
[0233] Expression of the GPCR binding peptides was achieved using
constructs which included minigene inserts corresponding to the
carboxyl terminal sequences of various G protein .alpha. subunits
(G.alpha.i, G.alpha.o, G.alpha.s, G.alpha.q, G.alpha.11,
G.alpha.12, G.alpha.13, G.alpha.14), as well as a control minigene
containing the G.alpha.i sequence in random order (G.alpha.iR). The
minigene insert DNAs were made by synthesizing short complimentary
oligonucleotides corresponding to the peptide sequences from the
carboxyl terminus of each G.alpha. with BamHI and HindIII
restriction sites at the 5' and 3' ends, respectively.
Complementary oligonucleotides were annealed and ligated into the
mammalian expression vector pcDNBA3.1 according to the methods of
Gilchrist et al., J. Biol. Chem. 274:6610-6616, 1999, the
disclosures of which are hereby incorporated by reference.
[0234] Human embryonic kidney (HEK) 293 cells were transfected
using a standard calcium phosphate procedure according to the
methods of Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, New York, vol. 1-3 (1989), the
disclosures of which are hereby incorporated by reference. To
confirm the transcription of minigene constructs in transfected
cells, total RNA was isolated from the cells 48 hours post
transfection with pcDNA-G.alpha.i or pcDNA-G.alpha.iR using methods
known in the art. Reverse transcriptase PCR was used to make cDNA
and PCR analysis was performed using the cDNA as template with
primers specific for the relevant G.alpha. carboxyl terminal
peptide insert (forward: 5'-ATCCGCCGCCACCATGGGA (SEQ ID NO:272);
reverse: 5'-GCGAAAGGAGCGGGGCGCTA (SEQ ID NO:273)). These primers
for the G.alpha. minigenes amplify a 434 bp fragment only if the
inserted peptide-encoding oligonucleotides are present; no band is
observed in cells transfected with the empty pcDNA3.1 vector. The
PCR products were separated on 1.5% agarose gels. The presence of a
single 434 bp band indicated that G.alpha. carboxyl terminus
peptide minigene RNA had been transcribed. See FIG. 14. Control
experiments were done using a T7 forward primer with the vector
reverse primer to verify the presence of the pcDNA3.1 vector, and
G3DPH primers (Clonetech) to approximate the amount of total
RNA.
[0235] To verify that the peptide was being produced in the
transfected cells, the cells were lysed and homogenized 48 hours
post transfection according to known methods. Cytosolic extracts
were analyzed by gradient reversed phase HPLC as follows: 100 .mu.L
of cytosolic fraction extract was loaded onto a C4 column (Vydac)
equilibrated with 0.1% TFA in ddH.sub.2O. The peptide was eluted
using 0.1% TFA in an acetonitrile gradient which increased from
0-60% over 45 minutes. Peaks were collected, lyophilized, and
analyzed using ion mass spray analysis (University of
Illinois-Urbana Champagne). Mass spectrometry analysis for peak 1
from G.alpha..sub.1/2 peptide vector (pcDNA-G.alpha.i) transfected
cells, and from cells transfected with pcDNA-G.alpha.iR indicated
that a 1450 Dalton peptide (the expected molecular weight for both
13 amino acid peptide sequences) was present in each cytosolic
extract. The minigene-encoded peptides were the major peptides
found in the cytosol, strongly indicating that the vectors produced
the appropriate peptide sequences in large amounts.
Example 25
Interfacial G Protein Peptide Inhibition of Thrombin-Mediated
Inositol Phosphate Accumulation
[0236] HMEC were seeded onto 6-well plates 24 hours before
transfection at 1.times.10.sup.5 well. Cells were transiently
transfected with pcDNA3.1, pcDNA-G.alpha.i, pcDNA-G.alpha.iR, or
pcDNA-Gq as described in Example 24. After 24 hours, cells were
incubated in 2 mL culture medium containing 4 .mu.Ci/mL
[.sup.3H]-myoinositol to obtain steady-state labeling of cellular
inositol lipids. Transiently transfected cells were assayed for
inositol phosphate (IP) accumulation 48 hours after transfection.
Two hours prior to stimulation with .alpha.-thrombin, cells were
washed, and medium replaced with medium containing 5 mM LiCl. Cells
were stimulated with 10 nM .alpha.-thrombin for 10 minutes.
Inositol phosphate (IP) formation was stopped by aspiration of the
medium and addition of ice-cold methanol (final concentration
5%).
[0237] Perchloric acid-lysed cells were centrifuged at 2500 rpm,
4.degree. C. for 5 min. The supernatant containing IP was eluted
through a Poly-Prep chromatography column (Bio-Rad) containing 1.6
ml anion exchange resin (DOWEX AG1-X8, formate form, 200-400 mesh).
The perchloric acid-precipitated pellets (containing
phosphatidylinositols and lipids) were resuspended in 1 ml
chloroform-methanol-10 M HCl (200:100:1, v/v/v). These suspensions
were mixed with 350 .mu.L HCl and 350 .mu.L chloroform and
sedimented for 5 min at 2500 rpm to separate the phases. The lower,
hydrophobic phase was recovered and dried in counting vials to
determine the amount of radioactivity in total
phosphatidylinositols. The relative amount of [.sup.3H]-IP
generated was calculated as follows: ([.sup.3H]-IP
(cpm)/[.sup.3H]-IP (cpm)+[.sup.3H]-inositol (cpm)). Each value was
normalized using the basal value (no thrombin stimulation) obtained
in pcDNA transfected cells. See FIG. 15. The results presented are
the normalized mean.+-.SEM of at least 3 independent experiments
performed in triplicate. The ** symbol indicates p<0.005.
Results indicate that addition of thrombin increased IP production
in control cells (pcDNA, pcDNA-GiR). Cells transfected with
PcDNA-Gq had no thrombin-mediated IP production increase, while
cells transfected with pcDNA-Gi had a normal response. This
indicates that transfection of the Gq C-terminal minigene vector
into HMEC with subsequent expression of the Gq C-terminal peptide
can inhibit thrombin-mediated increases in IP.
Example 26
Interfacial G Protein Peptide Inhibition of Thrombin-Induced P1
Hydrolysis and Intracellular Ca.sup.++ Rise
[0238] To determine whether expression of the G.alpha.q C-terminal
minigene vector could affect intracellular [Ca.sup.++].sub.i
levels, HMEC were transfected with empty vector (pcDNA) or with
pcDNA-G.alpha.i, pcDNA-G.alpha.q, or pcDNA-G.alpha.iR minigene DNA
(1 .mu.g), which encode high affinity peptides identified for their
ability to bind the receptors. Transfected cells were seeded at a
low confluency on coverslips in a 24-well plate 48 hours post
transfection. The cells were allowed to adhere for two hours. The
medium was aspirated and each coverslip was incubated with 10 .mu.M
Oregon Green 488 BAPTA-1 acetoxymethyl ester (a calcium-sensitive
dye) and 0.1% (v/v) Pluronic F-127 and allowed to incubate for
20-30 minutes at 37.degree. C., then rinsed twice with wash buffer.
Basal conditions were established before addition of thrombin
(.about.70 nM) in Ca.sup.++ buffer. Recordings were made every 10
seconds and continued for 170 seconds after stimulation with
thrombin. Images were quantitated using NIH Image. Data from at
least 70 individually recorded cells were used to calculate the
changes in fluorescence (y-axis). See FIG. 16A, which presents
fluorescence in ([Ca.sup.++].sub.i level) increase 30 seconds after
thrombin addition. Each bar in FIG. 16A represents the mean
((F.sub.S-F.sub.B/F.sub.B-1).+-.SEM of over 70 individually
recorded cells. The ** symbol indicates p<0.005. FIG. 16B shows
the kinetics of [Ca.sup.++].sub.i; fluorescence changes after cell
stimulation with thrombin. Data presented are the mean
((F.sub.S-F.sub.B/F.sub.B-1).+-.SEM at each recording point for
cells transfected with CDNA for the empty vector (pcDNA) or the Gq
C-terminal minigene vector (pcDNA-G.alpha.q). The arrow indicates
the time thrombin was added. Each time point represents over 100
individually recorded cells.
[0239] As shown in FIG. 16, following cell activation by addition
of thrombin there was a transient increase in intracellular
[Ca.sup.++].sub.i levels. Thirty seconds after the addition of
thrombin, cells transfected with pcDNA-G.alpha.q had a calcium
response that was 44% decreased as compared to cells transfected
with pcDNA (FIG. 16A). pcDNA-G.alpha.q transfected cells had a 45%
decrease compared to those transfected with pcDNA when all time
points measured after thrombin stimulation are averaged (FIG. 16B).
This decrease appears to be specific as cells transfected with
pcDNA-G.alpha.i or pcDNA-G.alpha.iR did not have any effect on
thrombin stimulated [Ca.sup.++].sub.i levels. Thus, cells
expressing the G.alpha.q C-terminal peptide appear to be inhibited
in their ability to stimulate [Ca.sup.++].sub.i levels following
activation with thrombin, indicating a specific block of this
downstream mediator by expression of G.alpha.q.
[0240] pcDNA, pcDNA-GiR, pcDNA-Gi, pcDNA-Gq, or pcDNA-Gs minigene
constructs were transfected into HMEC and used to assay inositol
phosphate (IP) accumulation. After 24 hours, cells were reseeded
onto 24-well plates and labeled with [.sup.3H]-myoinositol (2
.mu.Ci/ml). After 48 hours, cells were rinsed, and incubated with
or without thrombin (10 nM) for 10 minutes. Total IP accumulation
was assayed as described above using Dowex.TM. columns to separate
[.sup.3H]IP. The relative amount of [.sup.3H]IP generated was
calculated as follows: ([.sup.3H]IP (cpm)/[.sup.3H]IP
(cpm)+[.sup.3H]inositol (cpm)). Each value was normalized by the
basal value (no thrombin stimulation) obtained in pcDNA transfected
cells. See FIG. 17. The results presented are the normalized
mean.+-.SEM of at least three independent experiments performed in
triplicate. The ** symbol indicates p<0.005.
Example 27
Prevention of Thrombin-Induced MAPK Activity by GPCR-binding
C-terminal Peptides
[0241] Hemagglutanin (HA)-MAPK (1.times.10.sup.5/mL was
co-transfected into HMEC with the pcDNA, pcDNA-G.alpha.i,
pcDNA-G.alpha.q or pcDNA-G.alpha.iR minigene constructs using the
methods described in Example 24. After 30 hours, cells were
serum-starved for 18 hours and then treated with 10 nM thrombin for
20 minutes. Cells were then lysed with RIPA buffer (50 mM Tris, pH
7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.5% deoxycholate,
0.1% SDS, 10% glycerol, 10 .mu.g/mL aprotinin and 10 .mu.g/mL
leupeptin) and HA-MAPK protein immunoprecipitated using 12CA5
antibody (Roche Molecular Biochemicals; Indianapolis, Ind.) (one
hour, 4.degree. C.) and Protein A sepharose beads (three hours,
4.degree. C.). Immune complexes were washed three times in RIPA
buffer. Kinase activity in the immunoprecipitates was measured
using maltose binding protein (MBP) substrate and a kinase assay
kit (Upstate Biotechnology, Inc., Lake Placid, N.Y.). MAPK activity
(nmol/min/mg) was obtained for each, and the relative increase of
MAPK activity (thrombin-mediated fold increase) was calculated as
follows: (stimulated activity (nmol/min/mg)-basal activity
(nmol/min/mg))/basal activity (nmol/min/mg). Results are presented
as the mean.+-.SEM of at least three independent experiments in
FIG. 18. A * symbol indicates p<0.05.
[0242] Addition of 10 nmol thrombin resulted in a 3.66 fold
increase in HA-MAPK activity in cells transfected with the pcDNA
control vector. Similarly, cells transfected with pcDNA-GiR had an
essentially equivalent increase in thrombin mediated MAPK activity
with (4.46 fold increase). However, endothelial cells transfected
with a minigene construct encoding the G.alpha.i, G.alpha.q,
G.alpha.12 or G.alpha.13 GPCR binding peptides showed a significant
decrease in thrombin-mediated HA-MAPK activity (59%, 57%, 50% and
77%, respectively) compared to cells transfected with pcDNA.
Example 28
Reduction of Thrombin-Induced Transendothelial Electrical
Resistance
[0243] Transendothelial electrical resistance (TEER) was measured
by passing an alternating current (50 .mu.A; 2 pulses every minute)
across monolayers of HMEC expressing G.alpha.q, G.alpha.i,
G.alpha.iR or no minigene construct. Basal TEER did not change
significantly with minigene transfection. Upon addition of 10 nM
thrombin, however, there was a decrease in the TEER of cells
expressing the G.alpha.q minigene compared to non-transfected cells
in the presence of 10 nM thrombin. See FIG. 19 (representative of
multiple experiments). The decrease in transendothelial electrical
resistance in response to thrombin was significantly reduced in
endothelial cells transfected with the minigene for the carboxyl
terminus of G.alpha.q, while there was no effect in cells
transfected with G.alpha.i, G.alpha.iR, or empty vector. These
results suggested that G.alpha.q is partially responsible for the
effects of thrombin on endothelial cell shape changes.
Example 29
Inhibition of Thrombin-Mediated Stress Fiber Formation
[0244] HMEC cells were transfected with pcDNA, pcDNA-G.alpha.12 or
pcDNA-G.alpha.13 minigene constructs 1 .mu.g each/100 mm dish. As a
marker for transfected cells, the pGreenLantern-1 plasmid,
containing the gene for green fluorescent protein (GFP) was
co-transfected together with minigene constructs. After 48 hours,
cells were serum starved for 18 hours and treated with 10 nM
thrombin for 20 minutes. After exposure to thrombin, the cells were
fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton
X-100 and stained for F-actin with 1 mM rhodamine-phalloidin for 30
minutes. Cells were extensively washed, mounted using
Vectashield.TM. antifade mounting medium (Vector Laboratories,
Inc.). Cells were observed with an inverted microscope (Diaphot
200, Nikon, Inc.) equipped for both differential interference
contrast microscopy and epifluorescence observation using a 60x
oil-immersion objective. Fluorescence and DIC images were recorded
for each cell field with a cooled, integrating CCD array camera
(Imagepoint, Photometrix, Ltd.) connected to the microscope. See
FIG. 20 for fluorescence images showing inhibition of
thrombin-mediated stress fiber formation by G.alpha.12 and
G.alpha.13 peptides.
[0245] Serum-starved cells transfected with pcDNA exhibited a thin
cortical F-actin rim at their margins, and contained few stress
fibers (FIG. 20, panel A). Those present were inconspicious and in
apparently random orientation. For HMEC transfected with pcDNA
after a 20-minute exposure to thrombin actin had reorganized into
prominent stress fibers, typically arranged in a parallel pattern
along the longitudinal axis of the cell (FIG. 20, panel B). A very
different pattern was observed for cells transfected with
pcDNA-G.alpha.12 (FIG. 20, panel C) or pcDNA-G.alpha.13 (FIG. 20,
panel D) minigenes after exposure to thrombin. In both
pcDNA-G.alpha.12 and pcDNA-G.alpha.13 transfected cells, thrombin
stimulation did not result in the appearance of stress fibers. In
cells transfected with pcDNA-G.alpha.13, the peripheral actin rim
appears thicker and more linear, providing a clear outline of
cell-cell junctions. Thus, in agreement with earlier reports,
thrombin induced rapid stress fiber formation in endothelial cells.
Transfection of either pcDNA-G.alpha.12 or pcDNA-G.alpha.13
minigenes resulted in cells that no longer showed thrombin-induced
stress fiber formation. Given that stress fiber formation is
dependent on the small GTPase Rho, these results concur with other
findings that G.alpha.12 and G.alpha.13 are intimately linked to
Rho signaling and demonstrates the ability of GPCR binding peptides
to specifically block this G protein pathway when expressed
intracellularly.
Example 30
Inhibition of G Protein Activity by GPCR Binding Peptides in Single
Intact Cells
[0246] Human embryonic kidney (HEK) 293 cells, which stably express
the M.sub.2 mACR (.about.400 fmol receptor/mg protein), were grown
in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco),
streptomycin/penicillin (100 U each; Gibco) and G418 (500 mg/L;
Gibco). Cells were grown under 10% CO.sub.2 at 37.degree. C. In all
transfections for electrophysiological studies, the CD8 reporter
gene system was used to visualize transfected cells using
Dynabeads.TM. coated with anti-CD8-antibodies (Dynal). The
following amounts of cDNA were used to transfect the cells:
pC1-GIRK1 (rat)--1 .mu.g; nH3-CD* (human)--1 .mu.g; pcDNA3.1,
pcDNA-G.alpha.i, pcDNA-G.alpha.iR, pcDNA-G.alpha.q, or
pcDNA-G.alpha.s--4 .mu.g. Thus, typically the total amount of cDNA
used for transfecting one 10 cm disk was 7 .mu.g. The cDNAs for
GIRK1 and GIRK4 were gifts from F. Lesage and M. Lazdunski (Nice,
France). A standard calcium phosphate procedure was used for
transient transfection of HEK cells according to the methods of
Schenborn et al., Meth. Mol. Biol. 130:135-145, 2000. All assays
were performed 48-72 hours post transfection.
[0247] Whole cell currents were recorded from stably M.sub.2
mAChR-expressing HEK 293 cells that had been transiently
transfected with cDNA for GIRK1, GIRK4 and either pcDNA-G.alpha.i,
pcDNA-G.alpha.s, or pcDNA-G.alpha.q. For the measurement of
inwardly rectifying K.sup.+ currents, whole cell currents were
recorded using an extracellular solution contained 120 mM NaCl; 20
mM KCl; 2 mM CaCl.sub.2; 1 mM MgCl.sub.2; and 10 mM HEPES-NaOH, pH
7.4. The solution for filling the patch pipettes was composed of
100 mM potassium glutamate; 40 mM KCl; 5 mM MgATP; 10 mM HEPES-KOH,
pH 7.4; 5 mM NaCl; 2 mM EGTA; 1 mM MgCl.sub.2; and 0.01 mM GTP.
Membrane currents were recorded under voltage clamp, using
conventional whole cell patch techniques. See Bunemann et al., J.
Physiol. 489:701-777, 1995 and Bunemann et al., J. Physiol.
482:81-89, 1995, the disclosures of which are hereby incorporated
by reference. To minimize variations due to different transfections
or culture conditions, control experiments (transfection with
pcDNA-G.alpha.iR) were done in parallel. Patch-pipettes were
fabricated from borosilicate glass capillaries, (GF-150-10, Warner
Instrument Corp.) using a horizontal puller (P-95 Fleming &
Poulsen). The DC resistance of the filled pipettes ranged from 3-6
M.OMEGA..
[0248] Membrane currents were recorded using a patch-clamp
amplifier (Axopatch 200, Axon Instruments). Signals are
analog-filtered using a lowpass Bessel filter (1-3 kHz corner
frequency). Data were digitally stored using an IBM compatible PC
equipped with a hardware/software package (ISO2 by MFK,
Frankfurt/Main, Germany) for voltage control, data acquisition and
data evaluation. To measure K.sup.+ currents in the inward
direction, the potassium equilibrium potential was set to about -50
mV and the holding potential was -90 mV. Agonist-induced currents
were evoked by application of acetylcholine (ACh; 1 .mu.M) using a
solenoid operated superfusion device which allowed for solution
exchange within 300 mseconds. Linear voltage ramps (from -120 mV to
+60 mV within 500 mseconds) were applied every 10 seconds. By
subtracting non-agonist dependent currents, the current voltage
properties of the agonist induced currents could be resolved. To
exclude experiments in which currents were recorded from cells that
may not have expressed the functional channel, only those cells
that exhibited a basal non-agonist dependent Ba.sup.++ (200 .mu.M)
sensitive inwardly rectifying current were used for analysis. For
analysis of the data, the maximal current density (peak amplitude)
of ACh-induced inwardly rectifying K.sup.+ currents was measured at
-80 mV and compared.
[0249] Superfusion of HEK 293 cells transiently transfected with
GIRK1/GIRK4 and either pcDNA-Gi or pcDNA-GiR DNA with 1 .mu.M ACh
revealed that cells transfected with pcDNA-G.alpha.i DNA have a
dramatically impaired response to the M.sub.2 mAChR agonist. See
FIG. 21, which summarizes data showing the maximum amplitude of ACh
evoked currents for the different transfection conditions (cells
transfected with GIRK1/GIRK4 and pcDNA-Gi or cells transfected with
GIRK1/GIRK4 and pcDNA-GiR). The pcDNA-Gi minigene vector results in
high intracellular expression of the G.alpha.i peptide, leading to
diminished ability of the receptor to signal the heterotrimenic
G.alpha.i.
[0250] The maximum current evoked by ACh was 3.7+/-1.5 pA/pF (n=14)
in cells transfected with pcDNA-Gi, compared to 24.1+/-8.8 pA/pF
(n=11) in cells transfected with pcDNA-GiR. This indicates that the
G.alpha.i minigene construct completely blocked the agonist
mediated M.sub.2 mAChR GIRK1/GIRK4 response while the control
minigene construct (pcDNA-GiR) had no effect. Compare FIG. 21A to
FIGS. 21B and 21C. Cells transfected with minigene constructs
encoding G.alpha. carboxyl termini for G.alpha.q or G.alpha.s
pcDNA-G.alpha.q or pCDNA-G.alpha.s were not significantly different
than those of cells transfected with the control vectors. These
findings confirm the specificity of the inhibition of M.sub.2
mAChR-activated G protein-coupled inwardly rectifying K.sup.+
current responses by expression of the G.alpha.i minigene.
Example 31
Selective G Protein Signaling Inhibition in Human Microvascular
Endothelial Cells
[0251] Different measures of G-protein signaling final actions were
assayed in human microvascular endothelial cells (HMEC) which
natively express the thrombin receptor, PAR1. The cells were seeded
onto 6-well plates at 1.times.10.sup.5 cells/well and transiently
transfected after 24 hours with minigene constructs containing
G.alpha. carboxyl terminal peptides (pcDNA, pcDNA-G.alpha.i, or
pcDNA-G.alpha.iR; 1 .mu.g per well) using Effectene (Qiagen)
according to the manufacturer's protocol. After 24 hours, the cells
were labeled with 3 .mu.Ci/ml [.sup.3H]-adenine for 30 minutes at
37.degree. C. After another 24 hours, the cells were washed with
serum-free medium containing 1 mM isobutyl-methyl xanthine. To
stimulate cAMP accumulation, cells were treated with 1 .mu.M
isoproterenol for 30 minutes at 37.degree. C. To see the inhibitory
effects of thrombin on cAMP accumulation, cells were pretreated
with thrombin (50 nM) for 15 minutes prior to addition of
isoproterenol. The reactions were terminated by aspiration of media
followed by addition of ice-cold 5% trichloroacetic acid.
[0252] Results are provided in FIG. 22 as (cAMP/cAMP+ATP) X 1000.
Three separate experiments were done in duplicate. The ** symbol
indicates p<0.005. Basal cAMP levels were essentially equivalent
for all conditions tested. Endothelial cells stimulated with
isoproterenol to activate .beta.-adrenergic receptors increase
their cAMP levels through the Gs pathway. Cells transfected with
pcDNA, pcDNA-G.alpha.i, or pcDNA-G.alpha.iR showed little
difference with 82-, 64-, and 77-fold increases in
isoproterenol-mediated cAMP accumulation, respectively. When the
endothelial cells were pre-incubated with thrombin prior to
addition of isoproterenol, a decrease in cAMP levels was observed
due to thrombin activation of the Gi pathway. Endothelial cells
transfected with pcDNA and pre-incubated with thrombin showed a 39%
decrease in cAMP level over cells stimulated with only
isoproterenol. Similarly, cells transfected with pcDNA-G.alpha.iR
and pre-incubated with thrombin showed had a 43% decrease over
cells stimulated with only isoproterenol. However, cells
transfected with pcDNA-G.alpha.i and pre-incubated with thrombin
had only a 0.1% decrease in cAMP levels as compared to cells
stimulated with only isoproterenol. Thus, cells expressing the
G.alpha.i C-terminal peptide appear to be unable to inhibit adenyl
cyclase following activation with thrombin, indicating that
thrombin-mediated Gi signaling was specifically blocked by
expression of the pcDNA-G.alpha.i minigene.
Example 32
Screening Method to Identify Inverse Agonists
[0253] Urea-washed rod outer segment membrane fragments containing
rhodopsin receptor are immobilized onto microtiter wells and
blocked as described in Example 7. The receptor is light-activated.
Labeled native G.alpha.t carboxyl terminal peptide is added to each
well and allowed to shake gently for one hour at 4.degree. C. The
wells are washed to remove unbound peptide. Crude bacterial lysates
(labeled) from a G.alpha.t carboxyl terminal peptide prepared
according to the methods described in Example 7 (200 .mu.L) are
added to each well and incubated with shaking for one hour at
4.degree. C.
[0254] The wells then are washed to remove unbound label. The
supernatants or well-bound labels are quantitated by ELISA to
detect dissociation of labeled native peptide from the receptor
after incubation with library peptides compared to control wells
incubated in the absence of library peptides.
Example 33
Small Molecule Library Screening Method
[0255] Small molecule libraries are screened for inhibition of
GPCR-mediated G protein signaling as follows. PAR1 thrombin
receptor prepared from insect cells according to Example 3 are
immobilized onto microtiter wells, blocked and washed. A small
molecule library purchased from Chem Div (San Diego, Calif.) are
added simultaneously with MBP-peptide fusion protein (0.1-1000 nM)
in a 96- or 384-well plate and allowed to shake for one hour at
4.degree. C. Initial screens are performed with the small molecules
at about 5-5000 nM. The wells are washed four times in cold PBS
containing 0.05% Tween 20.TM. and 1 mM maltose. The amount of
maltose binding protein adhering to the wells is quantitated with
anti-MBP antibodies as described in Example 17, versus control
wells incubated without library compounds.
Example 34
Identification of Very High Affinity Activated Rhodopsin-Binding
Gt-Based Peptides
[0256] A combinatorial peptide library based on the C-terminal
sequence of Gt was constructed by introducing all possible
mutations at each position, but with an overall bias toward the
G.alpha.t sequence with a K341R change and panned for high-affinity
binding. See Martin et al., J. Biol. Chem. 271:361-366, 1996;
Gilchrist et al., Methods Enzymol. 315:388-404, 2000 the
disclosures of which are hereby incorporated by reference, and
Examples 7 and 17 for methods used. Specific residues within the
C-terminal sequence were highly conserved. Perhaps more interesting
is not only the selection against the native amino acid at a given
position (R341, the second residue in the peptide shown below) but
the apparent selection for a specific amino acid at that location
(leucine). See Table XX. Table XXI shows amino acid sequences
obtained from screening dark-adapted bovine rhodopsin with the same
combinatorial peptide library based on the C-terminal sequence of
Gt. As observed with Gt, specific residues within the carboxyl
terminal sequence were conserved and specific residues were
selected against. Notably, at identical positions there are extreme
differences between the selection of light-activated and
dark-adapted rhodopsin (i.e., position C347) indicating that upon
activation the receptor undergoes a conformational change unmasking
new sites which the G protein can interact. TABLE-US-00020 TABLE XX
Alignment of the Highest-Affinity Amino Acid Sequences Screened
based on the C-Terminal Sequence of Gt with Light-Activated
Rhodopsin. ##STR1##
[0257] TABLE-US-00021 TABLE XXI Alignment of Amino Acid Sequences
Screened based on the C-Terminal Sequence of Gt with Dark-Adapted
Rhodopsin. ##STR2##
[0258] In all the high affinity sequences selected for binding to
the light adapted rhodopsin, position 341, which normally is a
positively charged residue was changed to a neutral one. There is
an obvious selection for a specific amino acid change from R to L.
Peptides synthesized with this single change were assayed for high
affinity binding, and the results are shown in Table XX. There was
not a selection for a neutral amino acid at position 341 in
peptides selected for binding to dark-adapted rhodopsin. See Table
XXI. Arg is found 75% of the time. For peptides selected for
light-activated rhodopsin, Leu344, Cys347, and Gly348 were found to
be invariant, and hydrophobic residues were always located at
positions 340, 349 and 350, indicating the critical nature of these
residues. This differs considerably from the peptides selected for
binding to dark-adapted rhodopsin, which did not show any invariant
positions. Most striking is the apparent selection against the
Cys347 position. Cys347 and Gly 348 both are part of a type II'
.beta.-turn which is required for MII stabilization. This suggests
that a site on rhodopsin which is required to bind the Cys 347 of
Gt is unmasked only after the receptor has received a photon of
light and formed MII. See Gilchrist et al., Methods Enzymol.
15:388, 2000. Other works indicate that the critical nature of
Cys347 for binding light-activated rhodopsin is due to its
hydrophobicity. Aris et al., J. Biol. Chem. 276:2333, 2001.
Replacement of Cys 347 with a hydrophobic amino acid (Cys347Met,
Cys347Val and Cys347Abu)(Abu=2-aminobutyric acid) stabilizes MII to
the same extent and with similar potency as the parent peptide. The
apparent selection of Lys at 347 in the dark-adapted rhodopsin
peptides clearly indicates that binding of the G.alpha.t peptide to
dark-adapted rhodopsin is very different from light-activated
rhodopsin. These results show that the site on rhodopsin recognized
by the C-terminal tail of Gt differs depending on whether the
receptor is dark-adapted or light-activated. This implies that high
affinity peptides selected for binding to light-activated rhodopsin
only bind the activated state of the receptor and not dark-adapted
receptor.
Example 35
Assays for Determining Peptide, Peptide-Fusion Protein or Small
Molecule Affinities for Metarhodopsin II
[0259] For the "extra MII" assay, EDTA-washed rhodopsin (Example 5;
5 .mu.M) is incubated in a 50 mM HEPES buffer, pH 8.2, with 100 mM
NaCl, 1 mM MgCl.sub.2, and 1 mM DTT at 5.3.degree. C., in the
absence or presence of varying concentrations of Gt340-350 analogs
or Gt. The sample is maintained at 5.4.degree. C. using a
water-jacketed and thermostated circulator cuvette holder in an SLM
Aminco DW2000 spectrophotometer at 390 and 440 nm. A flash of light
bleaching 10% of the rhodopsin is presented and after a 1 min
incubation, a second spectrum is measured and the difference in
spectrum calculated. "Extra" MII is calculated as the difference
between the absorbance at 390 and 440. Dose response curves of MII
stabilization by .alpha.t340350 (.lamda.), mutant
.alpha.340-350K341L(.nu.), and heterotrimeric Gt (v) were analyzed
by non-linear regression using the program GraphPad PRISM and are
shown in FIG. 23.
[0260] For the MII decay assay, the absorbance spectra of
EDTA-washed ROS (10 .mu.M) is measured in an SLM Aminco DW2000
spectrophotometer in 10 mM K.sub.2PO.sub.4, pH 6.5 containing 0.1 M
KCl, 0.1 mM EDTA, 1 mM DTT, in the presence of peptide, fusion
protein expressing high affinity peptide or small molecule. The
spectra are measured in the dark, then completely bleached in room
light. The spectra of the bleached sample is measured at intervals
of 30 minutes over a 6 hour period.
Example 36
Analysis of Data From Small Molecule Library Screen
[0261] Competition ELISA assays were employed to screen a small
molecule library (a 10,000 compound library representative of
ChemDiv's Diverse Collection of drug-like molecules) for compounds
that bind activated rhodopsin and increase/decrease the binding of
MBP-8 high affinity peptide fusion protein. MBP 8 was selected
based on its mid-range affinity. The screen may be repeated using
an MBP which displays higher affinity and ability to stabilize MII
(i.e., MBP 18; Table XIX). These types of screens may be used with
libraries of any size, therefore it is possible to increase the
size of the compound library by 10 fold or greater and continue
screening for small molecule hits in a similar manner.
[0262] A software program that displays results of screening as a
colorometric readout with a unique color coding that represents the
amount of inhibition or stimulation of bound light-activated
rhodopsin-bound peptide fusion protein is advantageous and
preferred. Two representative 96-well plates in which
light-activated-rhodopsin-bound MBP-8 high-affinity peptide fusion
proteins were assayed for competitive binding by 80 different
compounds. Experiments were done in duplicate, and the results of
the two separate plates averaged.
[0263] Dose response curves of MII (FIG. 24) indicate that both
PL.sub.--0302 R3.C4 (.sigma.), and heterotrimeric Gt (.nu.)
stabilize the active form of rhodopsin. EDTA-washed rhodopsin (5
.mu.M) was incubated in a 50 mM HEPES buffer, pH 8.2, with 100 mM
NaCl, 1 mM MgCl.sub.2, and 1 mM DTT at 5.3.degree. C., and "extra"
MII was measured. For compounds that enhanced MBP-8 binding over
25% using the color coded readout, dose studies were performed to
generate EC.sub.50 curves. Table XXII below provides the calculated
EC.sub.50 for metarhodopsin II stabilization of each compound.
TABLE-US-00022 TABLE XXII EC.sub.50 values for selected small
molecules on the binding of MBP-8 to MII. Small molecule MW Binding
of MBP-8 Name (daltons) EC.sub.50 (.mu.M) PL_0568 R1.C5 291.2 0.96
PL_0551 R8.C1 328.5 0.95 PL_0894 R3.C7 424.9 10.1 PL_0302 R3.C4
290.27 11.9 PL_1012 R2.C1 433.5 5.12
Example 37
Very High Affinity Agonists for Rhodopsin Have No Effect on
PAR-1-Stimulated Ca.sup.++ Transients
[0264] Small molecules PL.sub.--0568 R1.C5, PL.sub.--0551 R8.C1,
PL-0894 R3.C7, PL.sub.--0302 R3.C4, and PL.sub.--1012 R2.C1 were
tested for their effect on the ability of an unrelated receptor
(PAR1) to activate Ca.sup.++ signaling. Human embryonic kidney
cells were cultured in a 96-well format and allowed to adhere for 2
hours. The medium was aspirated and the plate incubated at
37.degree. C. for 30 minutes in 0.5 mL loading buffer (20 mM HEPES
(pH 7.4), 130 mM NaCl, 5 mM KCl, 2 mM CaCl.sub.2, 1 mM MgSO.sub.4,
0.83 mM Na.sub.2HPO.sub.4, 0.17 mM NaH.sub.2PO.sub.4, 1 mg/ml BSA,
25 mM mannose) containing 0.1% (v/v) Pluronic F127 and 10 .mu.M
Oregon Green Bapta-1 acetoxymethyl ester. The small molecules were
added to the appropriate wells after 30 minutes and the cells
incubated at 37.degree. C. for another 30 minutes. The 96-well
plate was tested for calcium concentration using a Flexstation.TM.
system. Basal conditions were established before addition of
thrombin (.+-.70 nM). Recordings were made every 5 seconds and
continued for >100 seconds after stimulation with thrombin. See
FIG. 25.
Example 38
Modulation of MBP-8 Binding to Rhodopsin by Small Molecules
[0265] To test the effects of the small molecules in cells, light
response experiments were carried out on isolated rods from the
dark-adapted retina of a salamander. Single rods were isolated by
shredding a small piece of retina. Photoreceptors were mechanically
isolated from the dark-adapted retinas and placed in a gravity-fed
superfusion chamber on the stage of an inverted microscope.
Membrane currents were recorded with a suction electrode as
described by Baylor et al., (Baylor et al., J. Physiol. (Lond.).
288:589-611, 1979; Baylor et al. J. Physiol. (Lond.). 288:589-611,
1979) in Ringer solution containing 120 mM NaCl, 2.0 mM KCl, 2 mM
NaHCO.sub.3, 1.6 mM MgCl.sub.2, 1.0 mM CaCl.sub.2, 10 mM glucose,
and 3 mM HEPES, pH 7.6, as described by Rieke and Baylor, Biophys.
J. 71:2553-2572, 1996. Membrane current collected by the suction
electrode was amplified, low-pass filtered at 20 Hz (3 dB point;
8-pole Bessel low-pass), digitized at 100 Hz and stored on a
computer for subsequent analysis. Light responses were elicited by
10-msecond flashes of 50-500 nm light. The flash strength was
controlled with calibrated neutral density filters. The cell was
positioned in the suction electrode to collect as much dark current
as possible. Solution changes (by which addition of the small
molecule was effected) were achieved with a series of
electronically controlled pinch valves (Biochem Valves, Boonton,
N.J.) the outlets of which were connected to a common perfusion
pipe about 100 .mu.m in diameter. Solution changes with this system
were completed in 200-300 mseconds. Solutions were driven by
positive pressure through a pair of glass pipes with openings about
50 .mu.m in diameter. The pipes were mounted on a piezoelectric
translation stage (Burleigh Instruments, Fishers, N.Y.). Solution
changes at the cut end of the outer segment were completed in less
than 10 mseconds with this system.
[0266] Light stimuli were delivered from a dual beam optical bench.
Monochromatic lights were obtained by passing the light from a
tungsten-halogen bulb through interference filters with 10 nm
nominal bandwidths. Wavelength (520 nm for rods and 440 nm or 620
nm for cones) and intensity of the stimulating light were set with
calibrated narrow band interference and neutral density filters,
respectively. Salamander L, S, and ultraviolet-sensitive cones have
peak sensitivities at 600 nm, 430 nm, and 360 nm (Makino and Dodd,
J. Gen. Physiol. 108:27-34, 1996) and are readily identified by the
relative amplitudes of their responses to 620 nm, 440 nm, and 380
nm lights. After identification, S cones were stimulated with 440
nm light and L cones were stimulated with 620 nm.
Ultraviolet-sensitive cones were not studied. Light intensities
were controlled with a set of calibrated neutral density filters,
and light flashes were produced by an electronically controlled
shutter in the light path.
[0267] The results are presented in FIGS. 26 and 27, which show the
light response of an isolated rod from the dark-adapted retina of a
salamander in the presence or absence of 5 .mu.M compound
PL.sub.--0302R3C4, respectively. Panel A of each figure shows the
membrane current (response) plotted against time for the light
responses as a result of increasing light flashes. In panel B, the
peak responses have been normalized so that the current at the
highest light flash is 1.0. The circles correspond to the peak
response for each light flash. In FIG. 27, panel B, the bissecting
lines indicate that in the presence of PL.sub.--0302R3C4 a lower
intensity is required to get the same change of current. The
results indicate that compound PL.sub.--0302R3C4 increases the peak
response (as measured by a change in current) 20%-50%, depending on
the intensity of the light flash and thus the amount of rhodopsin
receptors activated. The results are representation of these
separate experiments. The results suggest taht the compound
PL.sub.--302R3C4 can serve as an allosteric agonist and increase
the signaling activity of the receptor in cells.
[0268] Small molecules also were tested for their ability to
enhance the binding of MBP-8. PELM6 (the MBP control) and MBP-8
were plated on 96 well plates that contained EDTA-washed rhodopsin.
A small molecule compound library was added, and the amount of
pELM6 or MBP-8 that remained bound was measured. Standard methods
were used.
[0269] FIGS. 28-32 show MBP-8 binding curves for the depicted small
molecules' ability to enhance binding of the high affinity peptide
fusion protein, MBP-8 to EDTA-washed rhodopsin.
Sequence CWU 1
1
273 1 4 PRT Mammal misc_feature (1)..(1) Xaa can be any naturally
occurring amino acid misc_feature (2)..(2) Xaa can be any naturally
occurring amino acid misc_feature (3)..(3) Xaa can be any naturally
occurring amino acid misc_feature (4)..(4) Xaa can be any naturally
occurring amino acid misc_feature (1)..(4) MBP-G11 1 Xaa Xaa Xaa
Xaa 1 2 11 PRT Homo sapiens 2 Leu Gln Leu Asn Leu Lys Glu Tyr Asn
Leu Val 1 5 10 3 4 PRT Mammal misc_feature (1)..(4) PAR-13 3 Val
Arg Pro Ser 1 4 11 PRT Artificial Sequence Gq peptide library
sequence 4 Leu Gln Leu Asn Arg Asn Glu Tyr Tyr Leu Val 1 5 10 5 4
PRT Mammal misc_feature (1)..(4) PAR-23 5 Leu Ser Arg Ser 1 6 11
PRT Artificial Sequence Gq peptide library sequence 6 Leu Gln Gln
Lys Leu Lys Glu Tyr Ser Leu Val 1 5 10 7 4 PRT Mammal misc_feature
(1)..(4) PAR-33 7 Leu Ser Thr Asn 1 8 11 PRT Artificial Sequence Gq
peptide library sequence 8 Leu His Leu Asn Leu Lys Glu Tyr Asn Leu
Val 1 5 10 9 4 PRT Mammal misc_feature (1)..(4) PAR-34 9 Leu Pro
Gln Met 1 10 11 PRT Artificial Sequence Gq peptide library sequence
10 Gln Arg Leu Asn Val Gly Glu Tyr Asn Leu Val 1 5 10 11 4 PRT
Mammal misc_feature (1)..(4) PAR-45 11 Ser Arg His Thr 1 12 11 PRT
Artificial Sequence Gq peptide library sequence 12 Leu Arg Leu Asn
Gly Lys Glu Leu Asn Leu Val 1 5 10 13 11 PRT Homo sapiens 13 Gln
Arg Met His Leu Arg Gln Tyr Glu Leu Leu 1 5 10 14 67 DNA Artificial
Sequence G alpha t library construct misc_feature (9)..(9) n = a,
c, g or t misc_feature (10)..(10) n = a, c, g or t misc_feature
(12)..(12) n = a, c, g or t misc_feature (13)..(13) n = a, c, g or
t misc_feature (15)..(15) n = a, c, g or t misc_feature (16)..(16)
n = a, c, g or t misc_feature (18)..(18) n = a, c, g or t
misc_feature (19)..(19) n = a, c, g or t 14 gaggtggtnn knnknnknnk
attcgtgaaa acttaaaaga ttgtggtcgt ttctaactaa 60 gtaaagc 67 15 11 PRT
Homo sapiens 15 Ile Lys Glu Asn Leu Lys Asp Cys Gly Leu Phe 1 5 10
16 33 DNA Homo sapiens 16 atcaaggaga acctgaaaga ctgcggcctc ttc 33
17 11 PRT Homo sapiens 17 Ile Lys Asn Asn Leu Lys Asp Cys Gly Leu
Phe 1 5 10 18 33 DNA Homo sapiens 18 ataaaaaata atctaaaaga
ttgtggtctc ttc 33 19 11 PRT Artificial Sequence G alpha i 1/2
sequence in random order 19 Asn Gly Ile Lys Cys Leu Phe Asn Asp Lys
Leu 1 5 10 20 33 DNA Artificial Sequence G alpha i 1/2 sequence in
random order 20 aacggcatca agtgcctctt caacgacaag ctg 33 21 11 PRT
Homo sapiens 21 Ile Lys Asn Asn Leu Lys Glu Cys Gly Leu Tyr 1 5 10
22 33 DNA Homo sapiens 22 attaaaaaca acttaaagga atgtggactt tat 33
23 11 PRT Homo sapiens 23 Ile Ala Lys Asn Leu Arg Gly Cys Gly Leu
Tyr 1 5 10 24 33 DNA Homo sapiens 24 atcgccaaaa acctgcgggg
ctgtggactc tac 33 25 11 PRT Homo sapiens 25 Ile Ala Asn Asn Leu Arg
Gly Cys Gly Leu Tyr 1 5 10 26 33 DNA Homo sapiens 26 attgccaaca
acctccgggg ctgcggcttg tac 33 27 11 PRT Homo sapiens 27 Ile Gln Asn
Asn Leu Lys Tyr Ile Gly Leu Cys 1 5 10 28 33 DNA Homo sapiens 28
atacagaaca atctcaagta cattggcctt tgc 33 29 33 DNA Homo sapiens 29
ctgcagctga acctcaagga gtacaacctg gtc 33 30 11 PRT Homo sapiens 30
Leu Gln Leu Asn Leu Lys Glu Tyr Asn Ala Val 1 5 10 31 33 DNA Homo
sapiens 31 ctccagttga acctgaagga gtacaatgca gtc 33 32 11 PRT Homo
sapiens 32 Gln Arg Met His Leu Lys Gln Tyr Glu Leu Leu 1 5 10 33 33
DNA Homo sapiens 33 cagcggatgc acctcaagca gtatgagctc ttg 33 34 11
PRT Homo sapiens 34 Leu Gln Leu Asn Leu Arg Glu Phe Asn Leu Val 1 5
10 35 33 DNA Homo sapiens 35 ctacagctaa acctaaggga attcaacctt gtc
33 36 11 PRT Homo sapiens 36 Leu Ala Arg Tyr Leu Asp Glu Ile Asn
Leu Leu 1 5 10 37 33 DNA Homo sapiens 37 ctcgcccgct acctggacga
gatcaacctg ctg 33 38 11 PRT Homo sapiens 38 Leu Gln Glu Asn Leu Lys
Asp Ile Met Leu Gln 1 5 10 39 33 DNA Homo sapiens 39 ctgcaggaga
acctgaagga catcatgctg cag 33 40 11 PRT Homo sapiens 40 Leu His Asp
Asn Leu Lys Gln Leu Met Leu Gln 1 5 10 41 33 DNA Homo sapiens 41
ctgcatgaca acctcaagca gcttatgcta cag 33 42 33 DNA Homo sapiens 42
cagcgcatgc accttcgtca gtacgagctg ctc 33 43 20 DNA Artificial
Sequence 5' minigene construct sequence 43 gatccgccgc caccatggga 20
44 4 DNA Artificial Sequence 3' minigene construct sequence 44 tgaa
4 45 11 PRT Drosophila melanogaster 45 Ile Lys Asn Asn Leu Lys Gln
Ile Gly Leu Phe 1 5 10 46 11 PRT Drosophila melanogaster 46 Leu Ser
Glu Asn Val Ser Ser Met Gly Leu Phe 1 5 10 47 11 PRT Homo sapiens
47 Ile Ala Lys Asn Leu Arg Gly Cys Gly Leu Tyr 1 5 10 48 11 PRT
Xenopus laevis 48 Ile Ala Tyr Asn Leu Arg Gly Cys Gly Leu Tyr 1 5
10 49 11 PRT Caenorhabditis elegans 49 Ile Gln Ala Asn Leu Gln Gly
Cys Gly Leu Tyr 1 5 10 50 11 PRT Caenorhabditis elegans 50 Ile Gln
Ser Asn Leu His Lys Ser Gly Leu Tyr 1 5 10 51 11 PRT Caenorhabditis
elegans 51 Leu Ser Thr Lys Leu Lys Gly Cys Gly Leu Tyr 1 5 10 52 11
PRT Xenopus laevis 52 Ile Lys Ser Asn Leu Met Glu Cys Gly Leu Tyr 1
5 10 53 11 PRT Canis familiaris 53 Val Gln Gln Asn Leu Lys Lys Ser
Gly Ile Met 1 5 10 54 11 PRT Schizosaccharomyces pombe 54 Leu Gln
His Ser Leu Lys Glu Ala Gly Met Phe 1 5 10 55 11 PRT Drosophila
melanogaster 55 Leu Gln Arg Asn Leu Asn Ala Leu Met Leu Gln 1 5 10
56 11 PRT Saccharomyces cerevisiae 56 Glu Asn Thr Leu Lys Asp Ser
Gly Val Leu Gln 1 5 10 57 11 PRT Drosophila melanogaster 57 Leu Gln
Ser Asn Leu Lys Glu Tyr Asn Leu Val 1 5 10 58 11 PRT Xenopus laevis
58 Leu Gln His Asn Leu Lys Glu Tyr Asn Leu Val 1 5 10 59 11 PRT
Sporothrix schenckii 59 Ile Gln Glu Asn Leu Arg Leu Cys Gly Leu Ile
1 5 10 60 11 PRT Saccharomyces cerevisiae 60 Ile Gln Gln Asn Leu
Lys Lys Ile Gly Ile Ile 1 5 10 61 11 PRT Neurospora crassa 61 Ile
Ile Gln Arg Asn Leu Lys Gln Leu Ile Leu 1 5 10 62 11 PRT
Filobasidiella neoformans 62 Leu Gln Asn Ala Leu Arg Asp Ser Gly
Ile Leu 1 5 10 63 11 PRT Ustilago maydis 63 Leu Thr Asn Ala Leu Lys
Asp Ser Gly Ile Leu 1 5 10 64 11 PRT Kluyveromyces lactis 64 Ile
Gln Gln Asn Leu Lys Lys Ser Gly Ile Leu 1 5 10 65 11 PRT
Kluyveromyces lactis 65 Leu Glu Asn Ser Leu Lys Asp Ser Gly Val Leu
1 5 10 66 11 PRT Ustilago maydis 66 Ile Leu Thr Asn Asn Leu Arg Asp
Ile Val Leu 1 5 10 67 11 PRT Mus musculus 67 Gln Arg Met His Leu
Pro Gln Tyr Glu Leu Leu 1 5 10 68 11 PRT Homo sapiens 68 Gln Arg
Met His Leu Lys Gly Tyr Glu Leu Leu 1 5 10 69 11 PRT Coprinus
congregatus 69 Leu Gln Leu His Leu Arg Glu Cys Gly Leu Leu 1 5 10
70 11 PRT Lycopersicon esculentum 70 Arg Arg Arg Asn Leu Phe Glu
Ala Gly Leu Leu 1 5 10 71 11 PRT Glycine max 71 Arg Arg Arg Asn Leu
Leu Glu Ala Gly Leu Leu 1 5 10 72 11 PRT Glycine max 72 Arg Arg Arg
Asn Pro Leu Glu Ala Gly Leu Leu 1 5 10 73 11 PRT Ustilago maydis 73
Ile Gln Val Asn Leu Arg Asp Cys Gly Leu Leu 1 5 10 74 11 PRT
Ustilago maydis 74 Arg Glu Asn Leu Lys Leu Thr Gly Leu Val Gly 1 5
10 75 11 PRT Oryza sativa 75 Asp Glu Ser Met Arg Arg Ser Arg Glu
Gly Thr 1 5 10 76 11 PRT Calliphora vicina 76 Met Gln Asn Ala Leu
Lys Glu Phe Asn Leu Gly 1 5 10 77 11 PRT Dictyostelium discoideum
77 Thr Gln Cys Val Met Lys Ala Gly Leu Tyr Ser 1 5 10 78 11 PRT
Caenorhabditis elegans 78 Ile Ile Ser Ala Ser Leu Lys Met Val Gly
Val 1 5 10 79 11 PRT Caenorhabditis elegans 79 Asn Glu Asn Leu Arg
Ser Ala Gly Leu His Glu 1 5 10 80 11 PRT Caenorhabditis elegans 80
Arg Leu Ile Arg Tyr Ala Asn Asn Ile Pro Val 1 5 10 81 11 PRT
Caenorhabditis elegans 81 Ile Ala Lys Asn Leu Lys Ser Met Gly Leu
Cys 1 5 10 82 11 PRT Caenorhabditis elegans 82 Ile Gly Arg Asn Leu
Arg Gly Thr Gly Met Glu 1 5 10 83 11 PRT Caenorhabditis elegans 83
Ile Gln His Thr Met Gln Lys Val Gly Ile Gln 1 5 10 84 11 PRT
Caenorhabditis elegans 84 Ile Gln Lys Asn Leu Gln Lys Ala Gly Met
Met 1 5 10 85 11 PRT Dictyostelium discoideum 85 Leu Lys Asn Ile
Phe Asn Thr Ile Ile Asn Tyr 1 5 10 86 11 PRT Artificial Sequence Gq
library peptide 86 Leu Leu Leu Gln Leu Val Glu His Thr Leu Val 1 5
10 87 11 PRT Artificial Sequence Gq library peptide 87 His Arg Leu
Asn Leu Leu Glu Tyr Cys Leu Val 1 5 10 88 11 PRT Artificial
Sequence Gq library peptide 88 Glu Gln Trp Asn Met Asn Thr Phe His
Met Ile 1 5 10 89 11 PRT Artificial Sequence Gq library peptide 89
Ser Gln Val Lys Leu Gln Lys Gly His Leu Val 1 5 10 90 10 PRT
Artificial Sequence Gq library sequence 90 Leu Arg Leu Leu Leu Glu
Tyr Asn Leu Gly 1 5 10 91 11 PRT Artificial Sequence Gq library
peptide 91 Arg Arg Leu Lys Val Asn Glu Tyr Lys Leu Leu 1 5 10 92 11
PRT Artificial Sequence Gq library peptide 92 Leu Gln Leu Arg Leu
Arg Glu His Asn Leu Val 1 5 10 93 11 PRT Artificial Sequence Gq
library peptide 93 His Val Leu Asn Ser Lys Glu Tyr Asn Gln Val 1 5
10 94 11 PRT Artificial Sequence G alpha 11 library peptide 94 Met
Lys Leu Asn Val Ser Glu Ser Asn Leu Val 1 5 10 95 11 PRT Artificial
Sequence G alpha 11 library peptide 95 Leu Gln Thr Asn Gln Lys Glu
Tyr Asp Met Asp 1 5 10 96 11 PRT Artificial Sequence G alpha 11
library peptide 96 Leu Gln Leu Asn Pro Arg Glu Asp Lys Leu Trp 1 5
10 97 11 PRT Artificial Sequence G alpha 11 library peptide 97 Arg
His Leu Asp Leu Asn Ala Cys Asn Met Gly 1 5 10 98 10 PRT Artificial
Sequence G alpha 11 library peptide 98 Leu Arg Asn Asp Ile Glu Ala
Leu Leu Val 1 5 10 99 11 PRT Artificial Sequence G alpha 11 library
peptide 99 Leu Val Gln Asp Arg Gln Glu Ser Ile Leu Val 1 5 10 100
11 PRT Artificial Sequence G alpha 11 library peptide 100 Leu Gln
Leu Lys His Lys Glu Asn Asn Leu Met 1 5 10 101 11 PRT Artificial
Sequence G alpha 11 library peptide 101 Leu Gln Val Asn Leu Glu Glu
Tyr His Leu Val 1 5 10 102 11 PRT Artificial Sequence G alpha 11
library peptide 102 Leu Gln Phe Asn Leu Asn Asp Cys Asn Leu Val 1 5
10 103 11 PRT Artificial Sequence G alpha 11 library peptide 103
Met Lys Leu Lys Leu Lys Glu Asp Asn Leu Val 1 5 10 104 11 PRT
Artificial Sequence G alpha 11 library peptide 104 His Gln Leu Asp
Leu Leu Glu Tyr Asn Leu Gly 1 5 10 105 11 PRT Artificial Sequence G
alpha 11 library peptide 105 Leu Arg Leu Asp Phe Ser Glu Lys Gln
Leu Val 1 5 10 106 11 PRT Artificial Sequence G alpha 11 library
peptide 106 Leu Gln Lys Asn Leu Lys Glu Tyr Asn Met Val 1 5 10 107
11 PRT Artificial Sequence G alpha 11 library peptide 107 Leu Gln
Tyr Asn Leu Met Glu Asp Tyr Leu Asn 1 5 10 108 11 PRT Artificial
Sequence G alpha 11 library peptide 108 Leu Gln Met Tyr Leu Arg Gly
Tyr Asn Leu Val 1 5 10 109 11 PRT Artificial Sequence G alpha 11
library peptide 109 Leu Pro Leu Asn Pro Lys Glu Tyr Ser Leu Val 1 5
10 110 11 PRT Artificial Sequence G alpha 11 library peptide 110
Met Asn Leu Thr Leu Lys Glu Cys Asn Leu Val 1 5 10 111 11 PRT
Artificial Sequence G alpha 11 library peptide 111 Leu Gln Gln Ser
Leu Ile Glu Tyr Asn Leu Leu 1 5 10 112 13 PRT Artificial Sequence G
alpha i minigene peptide 112 Met Gly Ile Lys Asn Asn Leu Lys Asp
Cys Gly Leu Phe 1 5 10 113 13 PRT Artificial Sequence G alpha i R
minigene peptide 113 Met Gly Asn Gly Ile Lys Cys Leu Phe Asn Asp
Lys Leu 1 5 10 114 13 PRT Artificial Sequence G alpha q minigene
peptide 114 Met Gly Leu Gln Leu Asn Leu Lys Glu Tyr Asn Ala Val 1 5
10 115 13 PRT Artificial Sequence G alpha q** minigene peptide 115
Met Gly Leu Gln Leu Asn Leu Lys Glu Tyr Asn Thr Leu 1 5 10 116 13
PRT Artificial Sequence G alpha 12 minigene peptide 116 Met Gly Leu
Gln Glu Asn Leu Lys Asp Ile Met Leu Gln 1 5 10 117 13 PRT
Artificial Sequence G alpha 13 minigene peptide 117 Met Gly Leu His
Asp Asn Leu Lys Gln Leu Met Leu Gln 1 5 10 118 68 DNA Artificial
Sequence G alpha t library construct misc_feature (9)..(9) n is a,
c, g, or t misc_feature (10)..(10) n is a, c, g, or t misc_feature
(12)..(12) n is a, c, g, or t misc_feature (13)..(13) n is a, c, g,
or t misc_feature (15)..(15) n is a, c, g, or t misc_feature
(16)..(16) n is a, c, g, or t misc_feature (18)..(18) n is a, c, g,
or t misc_feature (19)..(19) n is a, c, g, or t 118 gaggtggtnn
knnknnknnk attcaaggag aacctgaagg actgcggcct cttctaacta 60 agtaaagc
68 119 67 DNA Artificial Sequence Gs library construct misc_feature
(9)..(9) n is a, c, g, or t misc_feature (10)..(10) n is a, c, g,
or t misc_feature (12)..(12) n is a, c, g, or t misc_feature
(13)..(13) n is a, c, g, or t misc_feature (15)..(15) n is a, c, g,
or t misc_feature (16)..(16) n is a, c, g, or t misc_feature
(18)..(18) n is a, c, g, or t misc_feature (19)..(19) n is a, c, g,
or t 119 gaggtggtnn knnknnknnk ctgcagctga acctgaagga gtacaatctg
gtctaactaa 60 gtaaagc 67 120 67 DNA Artificial Sequence G12 library
construct misc_feature (9)..(9) n is a, c, g, or t misc_feature
(10)..(10) n is a, c, g, or t misc_feature (12)..(12) n is a, c, g,
or t misc_feature (13)..(13) n is a, c, g, or t misc_feature
(15)..(15) n is a, c, g, or t misc_feature (16)..(16) n is a, c, g,
or t misc_feature (18)..(18) n is a, c, g, or t misc_feature
(19)..(19) n is a, c, g, or t 120 gaggtggtnn knnknnknnk ctgcaggaga
acctgaagga catcatgctg cagtaactaa 60 gtaaagc 67 121 67 DNA
Artificial Sequence G13 library construct misc_feature (9)..(9) n
is a, c, g, or t misc_feature (10)..(10) n is a, c, g, or t
misc_feature (12)..(12) n is a, c, g, or t misc_feature (13)..(13)
n is a, c, g, or t misc_feature (15)..(15) n is a, c, g, or t
misc_feature (16)..(16) n is a, c, g, or t misc_feature (18)..(18)
n is a, c, g, or t misc_feature (19)..(19) n is a, c, g, or t 121
gaggtggtnn knnknnknnk ctgcatgaca acctcaagca gcttatgcta cagtaactaa
60 gtaaagc 67 122 67 DNA Artificial Sequence G15 library construct
misc_feature (9)..(9) n is a, c, g, or t misc_feature (10)..(10) n
is a, c, g, or t misc_feature (12)..(12) n is a, c, g, or t
misc_feature (13)..(13) n is a, c, g, or t misc_feature (15)..(15)
n is a, c, g, or t misc_feature (16)..(16) n is a, c, g, or t
misc_feature (18)..(18) n is a, c, g, or t misc_feature (19)..(19)
n is a, c, g, or t 122 gaggtggtnn knnknnknnk ctcgcccggt acctggacga
gattaatctg ctgtaactaa 60 gtaaagc 67 123 67 DNA Artificial Sequence
Gz library construct misc_feature (9)..(9) n is a, c, g, or t
misc_feature (10)..(10) n is a, c, g, or t misc_feature (12)..(12)
n is a, c, g, or t misc_feature (13)..(13) n is a, c, g, or t
misc_feature (15)..(15) n is a, c, g, or t misc_feature (16)..(16)
n is a, c, g, or t misc_feature (18)..(18) n is a, c, g, or t
misc_feature (19)..(19) n is a, c, g, or t 123 gaggtggtnn
knnknnknnk atacagaaca atctcaagta cattggcctt
tgctaactaa 60 gtaaagc 67 124 11 PRT Artificial Sequence G alpha t
library peptide 124 Ile Arg Glu Asn Leu Lys Asp Cys Gly Leu Phe 1 5
10 125 11 PRT Artificial Sequence G alpha t library peptide 125 Leu
Leu Glu Asn Leu Arg Asp Cys Gly Met Phe 1 5 10 126 11 PRT
Artificial Sequence G alpha t library peptide 126 Ile Gln Gly Val
Leu Lys Asp Cys Gly Leu Leu 1 5 10 127 11 PRT Artificial Sequence G
alpha t library peptide 127 Ile Cys Glu Asn Leu Lys Glu Cys Gly Leu
Phe 1 5 10 128 11 PRT Artificial Sequence G alpha t library peptide
128 Met Leu Glu Asn Leu Lys Asp Cys Gly Leu Phe 1 5 10 129 11 PRT
Artificial Sequence G alpha t library peptide 129 Val Leu Glu Asp
Leu Lys Ser Cys Gly Leu Phe 1 5 10 130 11 PRT Artificial Sequence G
alpha t library peptide 130 Met Leu Lys Asn Leu Lys Asp Cys Gly Met
Phe 1 5 10 131 11 PRT Artificial Sequence G alpha t library peptide
131 Leu Leu Asp Asn Ile Lys Asp Cys Gly Leu Phe 1 5 10 132 11 PRT
Artificial Sequence G alpha t library peptide 132 Ile Leu Thr Lys
Leu Thr Asp Cys Gly Leu Phe 1 5 10 133 11 PRT Artificial Sequence G
alpha t library peptide 133 Leu Arg Glu Ser Leu Lys Gln Cys Gly Leu
Phe 1 5 10 134 11 PRT Artificial Sequence G alpha t library peptide
134 Ile His Ala Ser Leu Arg Asp Cys Gly Leu Phe 1 5 10 135 11 PRT
Artificial Sequence G alpha t library peptide 135 Ile Arg Gly Ser
Leu Lys Asp Cys Gly Leu Phe 1 5 10 136 11 PRT Artificial Sequence G
alpha t library peptide 136 Ile Phe Leu Asn Leu Lys Asp Cys Gly Leu
Phe 1 5 10 137 11 PRT Artificial Sequence G alpha t library peptide
137 Ile Arg Glu Asn Leu Glu Asp Cys Gly Leu Phe 1 5 10 138 11 PRT
Artificial Sequence G alpha t library peptide 138 Ile Ile Asp Asn
Leu Lys Asp Cys Gly Leu Phe 1 5 10 139 11 PRT Artificial Sequence G
alpha t library peptide 139 Met Arg Glu Ser Leu Lys Asp Cys Gly Leu
Phe 1 5 10 140 11 PRT Artificial Sequence G alpha t library peptide
140 Ile Arg Glu Thr Leu Lys Asp Cys Gly Leu Leu 1 5 10 141 11 PRT
Artificial Sequence G alpha t library peptide 141 Ile Leu Ala Asp
Val Ile Asp Cys Gly Leu Phe 1 5 10 142 11 PRT Artificial Sequence G
alpha t library peptide 142 Met Cys Glu Ser Leu Lys Glu Cys Gly Leu
Phe 1 5 10 143 11 PRT Artificial Sequence G alpha t library peptide
143 Ile Arg Glu Lys Trp Lys Asp Leu Ala Leu Phe 1 5 10 144 11 PRT
Artificial Sequence G alpha t library sequence 144 Val Arg Asp Asn
Leu Lys Asn Cys Phe Leu Phe 1 5 10 145 11 PRT Artificial Sequence G
alpha t library sequence 145 Ile Gly Glu Gln Ile Glu Asp Cys Gly
Pro Phe 1 5 10 146 11 PRT Artificial Sequence G alpha t library
sequence 146 Ile Arg Asn Asn Leu Lys Arg Tyr Gly Met Phe 1 5 10 147
11 PRT Artificial Sequence G alpha t library sequence 147 Ile Arg
Glu Asn Leu Lys Asp Leu Gly Leu Val 1 5 10 148 11 PRT Artificial
Sequence G alpha t library sequence 148 Ile Arg Glu Asn Phe Lys Tyr
Leu Gly Leu Trp 1 5 10 149 11 PRT Artificial Sequence G alpha t
library sequence 149 Ser Leu Glu Ile Leu Lys Asp Trp Gly Leu Phe 1
5 10 150 11 PRT Artificial Sequence G alpha t library sequence 150
Ile Arg Gly Thr Leu Lys Gly Trp Gly Leu Phe 1 5 10 151 3 PRT
Artificial Sequence G alpha t library linker sequence 151 Ser Trp
Val 1 152 4 PRT Artificial Sequence G alpha t library linker
sequence 152 Phe Val Asn Cys 1 153 4 PRT Artificial Sequence G
alpha t library linker sequence 153 Glu Val Arg Arg 1 154 3 PRT
Artificial Sequence G alpha t library linker sequence 154 Arg Val
Gln 1 155 4 PRT Artificial Sequence G alpha t library linker
sequence 155 Arg Leu Thr Arg 1 156 3 PRT Artificial Sequence G
alpha t library linker sequence 156 Ser Arg Lys 1 157 4 PRT
Artificial Sequence G alpha t library linker sequence 157 Met Thr
His Ser 1 158 4 PRT Artificial Sequence G alpha t library linker
sequence 158 Ser Gly Pro Gln 1 159 3 PRT Artificial Sequence G
alpha t library linker sequence 159 Met Leu Asn 1 160 11 PRT
Artificial Sequence G alpha t library peptide 160 Leu Gln Arg Asn
Lys Lys Gln Tyr Asn Leu Gly 1 5 10 161 11 PRT Artificial Sequence G
alpha t library peptide 161 Leu Gln Leu Arg Tyr Lys Cys Tyr Asn Leu
Val 1 5 10 162 11 PRT Artificial Sequence G alpha t library
pepetide 162 Val His Val Lys Leu Lys Glu Tyr Asn Leu Val 1 5 10 163
11 PRT Artificial Sequence G alpha t library peptide 163 Leu Gln
Leu Asn Val Lys Glu Tyr Asn Leu Val 1 5 10 164 11 PRT Artificial
Sequence G alpha t library peptide 164 Leu Arg Ile Tyr Leu Lys Gly
Tyr Asn Leu Val 1 5 10 165 3 PRT Artificial Sequence G alpha 11
library linker sequence 165 Ser Ile Arg 1 166 4 PRT Artificial
Sequence G alpha 11 library linker sequence 166 Arg Trp Ile Val 1
167 3 PRT Artificial Sequence G alpha 11 library linker sequence
167 Gly Gly His 1 168 4 PRT Artificial Sequence G alpha 11 library
linker sequence 168 Arg Ser Glu Val 1 169 4 PRT Artificial Sequence
G alpha 11 library linker sequence 169 Cys Glu Pro Gly 1 170 4 PRT
Artificial Sequence G alpha 11 library linker sequence 170 His Gln
Met Ala 1 171 4 PRT Artificial Sequence G alpha 11 library linker
sequence 171 Val Pro Ser Pro 1 172 4 PRT Artificial Sequence G
alpha 11 library linker sequence 172 Gln Met Pro Asn 1 173 4 PRT
Artificial Sequence G alpha 11 library linker sequence 173 Met Trp
Pro Ser 1 174 3 PRT Artificial Sequence G alpha 11 library linker
sequence 174 Cys Val Glu 1 175 11 PRT Artificial Sequence G alpha t
library peptide 175 Leu Gln Leu Asn Leu Lys Val Tyr Asn Leu Val 1 5
10 176 11 PRT Artificial Sequence G alpha t library peptide 176 Leu
Glu Leu Asn Leu Lys Val Tyr Asn Leu Phe 1 5 10 177 11 PRT
Artificial Sequence G alpha t library peptide 177 Leu His Leu Asn
Met Ala Glu Val Ser Leu Val 1 5 10 178 11 PRT Artificial Sequence G
alpha t library peptide 178 Leu Lys Arg Tyr Leu Lys Glu Ser Asn Leu
Val 1 5 10 179 4 PRT Artificial Sequence G alpha 11 library linker
sequence 179 Pro Arg Gln Leu 1 180 4 PRT Artificial Sequence G
alpha 11 library linker sequence 180 Phe Phe Trp Val 1 181 4 PRT
Artificial Sequence G alpha 11 library linker sequence 181 Gln Arg
Asp Thr 1 182 4 PRT Artificial Sequence G alpha 11 library linker
sequence 182 Asn Phe Arg Asn 1 183 11 PRT Artificial Sequence G
alpha t library peptide 183 Leu Gln Leu Lys Arg Gly Glu Tyr Ile Leu
Val 1 5 10 184 11 PRT Artificial Sequence G alpha t library peptide
184 Cys Ser Leu Lys Leu Lys Ala Tyr Asn Leu Val 1 5 10 185 11 PRT
Artificial Sequence G alpha library peptide 185 Leu Gln Met Asn His
Asn Glu Tyr Asn Leu Val 1 5 10 186 11 PRT Artificial Sequence G
alpha t library peptide 186 Pro Gln Leu Asn Leu Asn Ala Tyr Asn Leu
Val 1 5 10 187 11 PRT Artificial Sequence Gs library peptide 187
Gln Gly Met Gln Leu Arg Arg Phe Lys Leu Arg 1 5 10 188 11 PRT
Artificial Sequence Gs library peptide 188 Arg Trp Leu His Trp Gln
Tyr Arg Gly Arg Gly 1 5 10 189 11 PRT Artificial Sequence Gs
library peptide 189 Pro Arg Pro Arg Leu Leu Arg Phe Lys Ile Pro 1 5
10 190 11 PRT Artificial Sequence Gs library peptide 190 Gln Gly
Glu His Leu Arg Gln Leu Gln Leu Gln 1 5 10 191 11 PRT Artificial
Sequence Gs library peptide 191 Gln Arg Leu Arg Leu Gly Pro Asp Glu
Leu Phe 1 5 10 192 11 PRT Artificial Sequence Gs library peptide
192 Gln Arg Ile His Arg Arg Pro Phe Lys Phe Phe 1 5 10 193 11 PRT
Artificial Sequence Gs library peptide 193 Gln Arg Met Pro Leu Arg
Leu Phe Glu Phe Leu 1 5 10 194 11 PRT Artificial Sequence Gs
library peptide 194 Gln Arg Val His Leu Arg Gln Asp Glu Leu Leu 1 5
10 195 11 PRT Artificial Sequence Gs library peptide 195 Asp Arg
Met His Leu Trp Arg Phe Gly Leu Leu 1 5 10 196 11 PRT Artificial
Sequence Gs library peptide 196 Gln Arg Met Pro Leu Arg Gln Tyr Glu
Leu Leu 1 5 10 197 11 PRT Artificial Sequence Gs library peptide
197 Gln Trp Met Asp Leu Arg Gln His Glu Leu Leu 1 5 10 198 11 PRT
Artificial Sequence Gs library peptide 198 Gln Arg Met Asn Leu Gly
Pro Cys Gly Leu Leu 1 5 10 199 11 PRT Artificial Sequence Gs
library peptide 199 Asn Cys Met Lys Phe Arg Ser Cys Gly Leu Phe 1 5
10 200 11 PRT Artificial Sequence Gs library peptide 200 Gln Arg
Leu His Leu Arg Gly Tyr Glu Phe Leu 1 5 10 201 11 PRT Artificial
Sequence Gs library peptide 201 His Arg Arg His Ile Gly Pro Phe Ala
Leu Leu 1 5 10 202 11 PRT Artificial Sequence Gs library peptide
202 Glu Arg Leu His Arg Arg Leu Phe Gln Leu His 1 5 10 203 11 PRT
Artificial Sequence Gs library peptide 203 Pro Cys Ile Gln Leu Gly
Gln Tyr Glu Ser Phe 1 5 10 204 11 PRT Artificial Sequence Gs
library peptide 204 Gln Arg Leu Arg Leu Arg Lys Tyr Arg Leu Phe 1 5
10 205 11 PRT Artificial Sequence Gt library peptide 205 Ile Val
Glu Ile Leu Glu Asp Cys Gly Leu Phe 1 5 10 206 11 PRT Artificial
Sequence Gt library peptide 206 Met Leu Asp Asn Leu Lys Ala Cys Gly
Leu Phe 1 5 10 207 11 PRT Artificial Sequence Gt library peptide
207 Ile Leu Glu Asn Leu Lys Asp Cys Gly Leu Phe 1 5 10 208 11 PRT
Artificial Sequence Gt library peptide 208 Leu Arg Glu Asn Leu Lys
Asp Cys Gly Leu Leu 1 5 10 209 11 PRT Artificial Sequence Gt
library peptide 209 Leu Leu Asp Ile Leu Lys Asp Cys Gly Leu Phe 1 5
10 210 11 PRT Artificial Sequence Gt library peptide 210 Val Arg
Asp Ile Leu Lys Asp Cys Gly Leu Phe 1 5 10 211 11 PRT Artificial
Sequence Gt library peptide 211 Ile Leu Glu Ser Leu Asn Glu Cys Gly
Leu Phe 1 5 10 212 11 PRT Artificial Sequence Gt library sequence
212 Ile Leu Gln Asn Leu Lys Asp Cys Gly Leu Phe 1 5 10 213 11 PRT
Artificial Sequence Gt library sequence 213 Met Leu Asp Asn Leu Lys
Asp Cys Gly Leu Phe 1 5 10 214 11 PRT Artificial Sequence Gt
library sequence 214 Ile His Asp Arg Leu Lys Asp Cys Gly Leu Phe 1
5 10 215 11 PRT Artificial Sequence Gt library peptide 215 Ile Cys
Glu Asn Leu Lys Asp Cys Gly Leu Phe 1 5 10 216 11 PRT Artificial
Sequence Gt library peptide 216 Ile Val Lys Asn Leu Glu Asp Cys Gly
Leu Phe 1 5 10 217 11 PRT Artificial Sequence Gt library peptide
217 Ile Ser Lys Asn Leu Arg Asp Cys Gly Leu Leu 1 5 10 218 11 PRT
Artificial Sequence Gt library peptide 218 Ile Arg Asp Asn Leu Lys
Asp Cys Gly Leu Phe 1 5 10 219 11 PRT Artificial Sequence Gt
library peptide 219 Ile Arg Glu Phe Leu Thr Asp Cys Gly Leu Phe 1 5
10 220 11 PRT Artificial Sequence Gt library peptide 220 Ile Arg
Leu Asp Leu Lys Asp Val Ser Leu Phe 1 5 10 221 11 PRT Artificial
Sequence Gt library sequence 221 Ile Cys Glu Arg Leu Asn Asp Cys
Gly Leu Cys 1 5 10 222 11 PRT Artificial Sequence Gt library
peptide 222 Pro Arg Asp Asn Thr Lys Val Arg Gly Leu Phe 1 5 10 223
11 PRT Artificial Sequence Gt library peptide 223 Phe Trp Gly Asn
Leu Gln Asp Ser Gly Leu Phe 1 5 10 224 11 PRT Artificial Sequence
Gt library peptide 224 Arg Arg Gly Asn Gly Lys Asp Cys Arg His Phe
1 5 10 225 11 PRT Artificial Sequence G12 library peptide 225 Leu
Gln Glu Asn Leu Lys Glu Met Met Leu Gln 1 5 10 226 11 PRT
Artificial Sequence G12 library peptide 226 Leu Glu Glu Asn Leu Lys
Tyr Arg Met Leu Asp 1 5 10 227 11 PRT Artificial Sequence G12
library peptide 227 Leu Gln Glu Asp Leu Lys Gly Met Thr Leu Gln 1 5
10 228 11 PRT Artificial Sequence G12 library peptide 228 Leu Gln
Glu Thr Met Lys Asp Gln Ser Leu Gln 1 5 10 229 11 PRT Artificial
Sequence G12 library peptide 229 Pro Gln Val Asn Leu Lys Ser Ile
Met Arg Gln 1 5 10 230 11 PRT Artificial Sequence G12 library
peptide 230 Trp Gln His Lys Leu Ser Glu Val Met Leu Gln 1 5 10 231
11 PRT Artificial Sequence G12 library peptide 231 Leu Lys Glu His
Leu Met Glu Arg Met Leu Gln 1 5 10 232 11 PRT Artificial Sequence
G12 library peptide 232 Leu Leu Gly Met Leu Glu Pro Leu Met Glu Gln
1 5 10 233 11 PRT Artificial Sequence G13 library peptide 233 Leu
Gln Asp Asn Leu Lys Gln Leu Met Leu Gln 1 5 10 234 11 PRT
Artificial Sequence G13 library peptide 234 Leu Gln Asp Asn Leu Arg
His Leu Met Leu Gln 1 5 10 235 11 PRT Artificial Sequence G13
library peptide 235 Leu Gln Asp Lys Ile Asn His Leu Met Leu Gln 1 5
10 236 11 PRT Artificial Sequence G13 library peptide 236 Leu Gln
Ala Asn Arg Lys Leu Gly Met Leu Gln 1 5 10 237 11 PRT Artificial
Sequence G13 library sequence 237 Leu Ile Val Lys Val Lys Gln Leu
Ile Trp Gln 1 5 10 238 11 PRT Artificial Sequence G13 library
peptide 238 Met Arg Ala Lys Leu Asn Asn Leu Met Leu Glu 1 5 10 239
10 PRT Artificial Sequence G13 library peptide 239 Leu Gln Asp Asn
Leu Arg His Leu Ile Gln 1 5 10 240 10 PRT Artificial Sequence G13
library peptide 240 Leu Gln Asp Asn Arg Asn Gln Leu Leu Phe 1 5 10
241 11 PRT Artificial Sequence G11 library peptide 241 Leu Gln Leu
Asn Arg Lys Asn Tyr Asn Leu Val 1 5 10 242 11 PRT Artificial
Sequence G11 library peptide 242 Leu Gln Leu Asp Leu Lys Glu Ser
Asn Met Val 1 5 10 243 11 PRT Artificial Sequence G11 library
peptide 243 Leu Gln Leu Asn Leu Lys Lys Tyr Asn Arg Val 1 5 10 244
11 PRT Artificial Sequence G11 library peptide 244 Leu Gln Leu Arg
Val Lys Glu Tyr Lys Arg Gly 1 5 10 245 11 PRT Artificial Sequence
G11 library peptide 245 Leu Gln Ile Tyr Leu Lys Gly Tyr Asn Leu Val
1 5 10 246 11 PRT Artificial Sequence G11 library peptide 246 Leu
Gln Tyr Asn Leu Lys Glu Ser Phe Val Val 1 5 10 247 11 PRT
Artificial Sequence G11 library peptide 247 Leu Gln Arg Asp His Val
Glu Tyr Lys Leu Phe 1 5 10 248 11 PRT Artificial Sequence G11
library peptide 248 Leu Val Ile Lys Pro Lys Glu Phe Asn Leu Val 1 5
10 249 11 PRT Artificial Sequence G11 library peptide 249 Ile Gln
Leu Asn Leu Lys Asn Tyr Asn Ile Val 1 5 10 250 11 PRT Artificial
Sequence G11 library peptide 250 Met Gln Leu Asn Leu Lys Glu Tyr
Asn Leu Val 1 5 10 251 11 PRT Artificial Sequence G11 library
peptide 251 Val Gln Val Lys Leu Lys Glu Tyr Asn Leu Val 1 5 10 252
11 PRT Artificial Sequence G11 library peptide 252 Gln Leu Leu Asn
Gln Tyr Val Tyr Asn Leu Val 1 5 10 253 11 PRT Artificial Sequence
G11 library peptide 253 Trp Arg Leu Ser Leu Lys Val Tyr Asn Leu Val
1 5 10 254 11 PRT Artificial Sequence G11 library peptide 254 Leu
Gln Arg Asn Lys Asn Gln Tyr Asn Leu Gly 1 5 10 255 11 PRT
Artificial Sequence G11 library peptide 255 Leu Tyr Leu Asp Leu Lys
Glu Tyr Cys Leu Phe 1 5 10 256 11 PRT Artificial Sequence G11
library peptide 256 Ser Ala Lys Glu Leu Asp Gln Tyr Asn Leu Gly 1 5
10 257 11 PRT Artificial Sequence G11 library peptide 257 Leu Phe
Leu Asn Leu Lys Glu Tyr Ser Leu Val 1 5 10 258 11 PRT Artificial
Sequence G11 library peptide 258 Leu Glu Leu Asn Leu Lys Val
Tyr Asn Leu Val 1 5 10 259 11 PRT Artificial Sequence G11 library
peptide 259 Leu Pro Leu Asn Leu Ile Asp Phe Ser Leu Met 1 5 10 260
11 PRT Artificial Sequence G11 library peptide 260 Leu Pro Arg Asn
Leu Lys Glu Tyr Asp Leu Gly 1 5 10 261 11 PRT Artificial Sequence
G11 library peptide 261 Leu Arg Leu Asn Asp Ile Glu Ala Leu Leu Val
1 5 10 262 11 PRT Artificial Sequence G11 library peptide 262 Leu
Val Leu Asn Arg Ile Glu Tyr Asn Leu Leu 1 5 10 263 11 PRT
Artificial Sequence G11 library peptide 263 Leu Lys Arg Lys Leu Lys
Glu Ser Asn Met Gly 1 5 10 264 11 PRT Artificial Sequence G11
library peptide 264 Leu Lys Arg Lys Val Lys Glu Tyr Asn Leu Gly 1 5
10 265 19 DNA Artificial Sequence Reverse primer 265 gaaaatcttc
tctcatccg 19 266 11 PRT Artificial Sequence Gt library peptide 266
Ile Leu Glu Asn Leu Lys Asp Cys Gly Leu Leu 1 5 10 267 11 PRT
Artificial Sequence Gt library peptide 267 Leu Gln Gln Val Leu Lys
Asp Cys Gly Leu Leu 1 5 10 268 11 PRT Artificial Sequence Gt
library peptide 268 Leu Leu Glu Asn Leu Arg Asp Cys Gly Met Ile 1 5
10 269 9 DNA Homo sapiens 269 gccgccacc 9 270 57 DNA Artificial
Sequence Gi alpha 1/2 carboxy terminal sequence oligonucleotide 270
gatccgccgc caccatggga atcaagaaca acctgaagga ctgcggcctc ttctgaa 57
271 57 DNA Artificial Sequence complementary strand to Gi alpha 1/2
oligonucleotide 271 agctttcaga agaggccgca gtccttcagg ttgttcttga
ttcccatggt ggcggcg 57 272 19 DNA Artificial Sequence forward primer
for G alpha carboxyl terminal peptide insert 272 atccgccgcc
accatggga 19 273 20 DNA Artificial Sequence reverse primer for G
alpha carboxyl terminal peptide insert 273 gcgaaaggag cggggcgcta
20
* * * * *