Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CHEMICAL SYNTHESIS AND USE OF SOLUBLE
MEMBRANE PROTEIN RECEPTOR DOMAINS
Introduction
Technical Field
The present invention relates to chemical synthesis and use of soluble
extramembranous domains of membrane protein receptors.
Background
Neurotransmitters, peptide hormones. growth factors, and other molecules are
ligands for cellular receptors that regulate si~~nal transduction in and among
cells. as
well as the extracellular matrix. Most receptors are membrane proteins having
extracellular, transmembrane and cytosolic domains. In the context of
receiving and
transduction of ligand-based extracellular signals. the general simplified
function of
these domains is as follows. The extracellular domain provides a ligand-
binding site
that receives information from outside the cell based on the presence or
absence of the
ligand. The transmembrane domain anchors the receptor protein within the
plasma
membrane and permits transduction of the information received by the
extracellular
domain to the cytosolic domain. The transmembrane domain of some receptors
also
may serve as a ligand-binding site. The cytosolic domain in turn transducer
the
signaling information received on the outside of the cell to the inside.
Ligand-based
information received from inside the cell via the cytosolic domain and/or the
transmembrane domain may also contribute to the receptor-mediated signal
transduction cascade.
There are many types of cellular receptors. Some receptors are at the center
of
signaling pathways that regulate changes in cellular events such as metabolism
or
gene expression in response to hormones and <yrowth factors, while others
affect cell
adhesion and organization of the cytoskeleton. An example of a receptor family
that
effects cell adhesion and organization of the cytoskeleton is the family of
integrin
receptors. Integrin receptors are the major receptors responsible for the
attachment of
cells to the extracellular matrix. Most inte~rin receptors identified to date
possess an
extracellular domain that interacts with the ewracellular matrix, an alpha-
helix
transmembrane domain, and a short cytoplasmic domain that lacks any intrinsic
enzymatic activity.
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Membrane protein receptors that re<~ulate changes in cellular events such as
metabolism or gene expression include enzyme-linked receptors. Enzyme-linked
receptors are directly coupled to intracellular enzymes and include guanylyl
cyclases,
tyrosine kinases, tyrosine kinase-associated tyrosine phosphatases, and
serine/threonine kinases receptors. The largest family of enzyme-linked
receptors is
the receptor protein-tyrosine kinases. This family includes receptors for
epidermal
growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor
(PDGF), insulin and many other growth factors. Most enzyme-linked receptors
have
an N-terminal extracellular ligand binding domain, a single transmembrane
alpha-
helix domain, and a cytosolic C-terminal domain having tyrosine kinase
activity.
Binding of ligand such as growth factor to the extracellular domain activates
the
cytosolic kinase domain. which in turn propagates an intracellular signal.
Binding of
ligand to most enzyme-linked receptors induces dimerization of receptor
monomer
(e.g., EGF), whereas other receptors exist as dimers (e.g., insulin, PDGF and
NGF
receptors). For instance, ligand binding to receptors having a monomeric state
crosslinks the monomers and induces dimerization.
Cytokine receptors and non-receptor protein kinases represent another family
of enzyme-linked membrane protein receptors. This family includes receptors
for
cytokines such as interleukin-2 and en~thropoietin, as well as for some
polypeptide
hormones such as growth hormone. These receptors have an N-terminal
extracellular
ligand binding domain, a single transmembrane alpha-helix domain, and a
cytosolic
C-terminal domain. They differ from protein-tyrosine kinase receptors in that
the C-
terminal domain does not by itself posses kinase activity. Instead, these
receptors
transmit ligand-binding information through intracellular protein kinases
associated
with the C-terminal cytosolic domain.
The largest family of cell surface receptors transmits signals to
intracellular
targets via the intermediary action of guanine nucleotide-binding proteins
called G-
proteins. More than a thousand such G-protein coupled receptors (GPCRs) have
been
identified to date. GPCRs are characterized by seven transmembrane domains
that
terminate in an extracellular N-terminal domain and an intracellular or
cytoplasmic C-
terminal domain. Thus, GPCRs also have been referred to as "7TM" receptors.
The
GPCRs can be classified into three major subfamilies related to rhodopsin
(type A),
calcitonin (type B), and metabolic receptors (type C).
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Another family of membrane protein receptors is the ion channel receptors.
The ion channel receptors include ligand-gated and voltage-gated ion channels.
Ligand-gated ion channels are pentamers of homolo<,ous subunits. The
acetylcholine
receptor is an example of a ligand-y~ated ion channel. Voltage-gated ion
channels are
homotetramers with subunits having six transmembrane helices. Potassium and
sodium ion channels are examples of voltage-gated ion channels. Extracellular
and/or
intracellular domains that bind ligand are likely to be important in
regulating many
ion channels.
Cellular membrane protein receptors represent extremely important drug
targets. For example, ion channels are therapeutic targets for major human
diseases
such as cardiac arrhythmias, stroke, hypertension, heart failure, asthma,
diabetes,
cystic fibrosis, epilepsy, migraine, and depression. Enzyme-linked receptors
are
implicated in multiple disorders including diabetes, cancer, and blood and
nervous
system disorders. Cytokine receptors are therapeutic targets for immune system
disorders such as AIDS and arthritis. GPCRs are recognized as the largest
groups of
receptors targeted by commercially available drugs. For instance, GPCR type B
receptors are important drug targets for mediation of metabolic disease,
nervous
system disorders, cancer and other diseases. Family members include receptors
for
glucagon, glucagon-like peptide, VIP (vasoactive intestinal polypeptide), GIP
(gastro-
intestinal peptide), GHRH (growth-hormone releasing hormone), secretin, PACAP
(pituitary adenyl cyclase activating polypeptide), PTH (parathyroid hormone),
calcitonin and CRF (corticotropin-releasing factor). In addition, the type B
GPCR
family includes different subtypes and several orphan receptors.
Drug targets typically include those related to ligand binding, since drugs
can
be employed that modulate natural ligand interaction with its receptor. As
mentioned
above, most ligand binding sites of receptors reside in the eatramembranous
portions
of receptors, such as the extracellular and cvtosolic domains. Unfortunately,
very
little is known about the structure- and quantitative structure-activity
relationship
(SAR/QSAR) for most membrane protein receptors. including their
extramembranous
domains. This lack of information has hampered development of new drugs and
the
understandin~~ of these molecules in general. Bv way of example. even though
recombinant forms of GPCRs have been made. including isolated domains.
detailed
structure/function information for the extramembranous domains of these
receptors
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remains elusive. For instance. the N-terminal extramembranous domain of type B
GPCRs appears to be highly crosslinked by disulfide bridges formed by six well-
conserved cysteine residues. Replacement of any one of the six cysteine
residues.
reduction of the receptor with beta-mercaptoethanol, and deletion of the N-
terminal
domain strongly reduces the binding affinity for the respective hormone ligand
in
several family members (Wilmen. et al.. FEBS Letters ( 1996) 398:43-47;
DeAlmeida,
et al., Moleca~lcrr Endocrinology ( 1998) 12:70-76~). Also, site-directed
mutagenesis
experiments on the VIP receptor have identified several conserved residues
(Asp67,
Trp72, Pro86, Gly I 08 and Trp I 10) besides the cysteines that appear to be
crucial for
receptor activity in some members of the family (Couvineau, et al., Biochem.
Biophys.
Res. Comm. (1990 206:246-252: and Wilmen, et al., Peptides (1997) 18:301-305).
Even though theoretical modeling. and in vitro and in vivo assays have been
utilized,
the disulfide-pairing pattern of the crucial disulfide bonds of type B GPCRs
still has
yet to be established. This can be attributed in large part to the difficulty
in producing
and purifying sufficient quantities and true homogenous preparations of
materials
needed for such studies (Willshaw. et al., Biochemical Society Transactions
(1998)
26:5288; and Chow, et al., Recept. Signal Transduct. (1997) 7:143-150).
To date, production of extracellular and cytosolic domains of membrane
protein receptors has all but been limited to recombinant expression of the
domains
joined to at least one transmembrane anchoring region (See, e.g., Hsuesh, et
al., WO
97/39131). Otherwise very little product can be made, much less isolated in
useful
amounts. Nevertheless, it is unlikely that a recombinantly produced domain of
a
receptor can be purified to an extent that it represents a true homogenous
material, or
that is free of cellular contaminants, which is a problem with all recombinant
expression systems no matter how stringent and redundant the purification
conditions
might be. Another frustration with recombinantly produced receptors is that
they
cannot be, for all practical purposes. site-specifically labeled at any
position within
the molecule, particularly cytosolic and extracellular portions of a receptor
in isolation
from the transmembrane spannin~T domain(s). Labeling, for instance. has been
restricted to conjugation of labels to only a few amino acids in the full
length receptor
following expression (See, e.g., Gether, et al., J. Biol. Chern. ( 1995)
270:28268-
28275; and Kim, et al.. Biochc~mi.styv ( 1998 ) 37( 13):4680-4686),
incorporation of
radioactive or spin labels throughout the receptor or by use of in vitro
suppression
-I
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mutagenesis in henopus oocvtes of full length receptor (see, e.g., Turcatti,
et al., J.
Bio. Chem. (1996) 271:19991-19998). Nor does recombinant expression by itself
provide access to ultra pure and large amounts of ultra homogenous and
functional
extramembranous receptor domain material. The present invention addresses
these
and other problems.
Relevant Literature
Wilken, et al. (Cum. Opin. Biotech. ( 1998) 9(4):412-426) review chemical
protein synthesis. Turcatti. et al. (J Bio. Chem. (1996) 271:19991-19998)
disclose in
vitro suppression mutagenesis in Xenoprrs oocytes to introduce fluorescence-
labeled
amino acids into the seven transmembrane neurokinin-2 receptor and its
incorporation
and activity in oocyte membranes. Gether. et al., (J. Biol. Chem. ( 1995)
270:28268-
28275) disclose fluorescent labeling of cysteine residues in the transmembrane
domain of the beta-2-andreogenic receptor. Hsuesh, et al. (WO 97/39131)
disclose
recombinant expression of a transmembrane anchor coupled through a protease
1 S cleavage site to the N-terminal portion of a G-protein coupled receptor.
Various
references disclose recombinant expression of N-terminal domains of GPCRs
(Willshaw, et al., Biochemical Society Transactions (1998) 26:S288, Wilmen, et
al.,
FEBSLetters (1996) 398:43-47; Chow, et al. (Receptor Signal Transduction
(1997)
7:143-150; and Cao, et al., Biochem Biophys Res. Commun. (1995) 212(2):673-680
disclose recombinant expression of the secretin/VIP N-terminal domain). Bozon,
et
al. (J. ~~lol. Endo. (1995) 14:227) and Bobovnikova, et al. (Endocrinology
(1995)
138:588) report on recombinant expression of leutonizing hormone (LH) and
thyroid
stimulating hormone (TSH) receptor domains. respectively. U.S. Patent Nos.
x,726.290 and x,837,486 discloses soluble analogs of integrins and assays.
U.S.
Patent Nos. x,783,402 and x,462.856 disclose cell-based GPCR-linked assays for
ligands.
Summary Of The Invention
The invention relates to chemical synthesis of extramembranous receptor
domains of membrane protein receptors. and compositions and methods that
employ
them. The extramembranous receptor domains include the soluble ligand-binding
extracellular and cytosolic domains of membrane protein receptors. The
extramembranous receptor domains of the invention are produced by ligating
under
chemoselective chemical ligation conditions first and second peptides of an
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extramembranous receptor domain of a membrane protein receptor, where the
peptides have unprotected chemoselective reactive groups capable of forming a
covalent bond therein between. The li~~ation product is exposed to a folding
buffer
having a chaotropic rea~~ent and an or~~anic solvent that approximates the
water-lipid
interface of a cell membrane. Exposure to the folding buffer is followed by
isolation
from the buffer of ligation product that binds to a ligand of the membrane
protein
receptor. The ligand-binding portion of the ligation product produced by this
method
represents folded extramembranous receptor domain.
Also provided is a composition comprising a synthetic extramembranous
receptor domain of a membrane protein receptor having a chemically synthesized
segment that includes an unnatural amino acid at a pre-selected residue
position,
where the extramembranous receptor domain is free of a membrane spanning
transmembrane domain and is capable of binding to a ligand of the membrane
protein
receptor. Compositions having a totally synthetic and ultra homogenous
extramembranous receptor domain of a membrane protein receptor free of
cellular
contaminants also are provided.
The invention further includes a method of detecting binding of a ligand to a
soluble extramembranous receptor domain of a membrane protein receptor. This
aspect of the invention involves contacting the monomer of a soluble
extramembranous receptor domain of a membrane protein receptor with a ligand
of
the membrane protein receptor, where the soluble extramembranous receptor
domain
is free of a membrane spanning transmembrane domain and includes an unnatural
amino acid at a pre-selected residue position. The contacting is followed by
assaying
the soluble extramembranous receptor domain for ligand-induced association of
domain monomers, such as dimerization.
The invention also includes a method of assaying a soluble extramembranous
receptor domain monomer for ligand-induced association of domain monomers.
This
method includes contacting a soluble extramembranous receptor domain of a
membrane protein receptor with a ligand of the membrane protein, where the
soluble
extramembranous receptor domain is free of a membrane spanning transmembrane
domain and includes an unnatural amino acid at a pre-selected residue
position. The
contacting is followed by assaying the soluble extramembranous receptor domain
for
ligand-induced association of domain monomers.
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Also provided is a method of detecting bindin~~ of a li~~and to an
extramembranous receptor domain of a membrane protein receptor. This method
involves contacting a soluble extramembranous receptor domain of a membrane
protein receptor with a ligand for the membrane protein receptor, where the
soluble
extramembranous receptor domain is free of a membrane spanning transmembrane
domain and comprises an unnatural amino acid having a detectable moiety.
Detection
of ligand binding is then performed by assaying for a change in a property of
the
detectable moiety, such as fluorescence when the detectable label is a
fluorophore.
The methods and compositions of the invention provide unprecedented access
to non-limiting amounts of ultra pure and ultra homogenous soluble
extramembranous
receptor domains of membrane protein receptors. including extracellular and
cytosolic
domains having one or more unnatural amino acids and/or unnatural reactive
functional groups at pre-selected positions. This facilitates for the first
time
production and true site-specific labeling of soluble membrane receptor
domains free
of impurities, transmembrane spanning regions, and other characteristic of
domains
made solely by recombinant synthesis. The invention also provides synthetic
access
to structure/function information previously unattainable by other approaches,
as well
as discovery of novel information about extramembranous domains of receptors
for
exploitation in drug discovery, disease treatment and diagnostics. The methods
and
compositions of the invention are particularly useful for high throughput
screening of
compounds that are ligands for the receptors corresponding to the
extramembranous
receptor domains.
Definitions
Amino Acid: Include the 20 genetically coded amino acids, rare or unusual
amino acids that are found in nature. and any of the non-naturally occurring
and
modified amino acids. Sometimes referred to as amino acid residues when in the
context of a peptide, polypeptide or protein.
Chemoselective Chemical Ligation: Chemically selective reaction involving
covalent ligation of ( 1 ) a first unprotected amino acid. peptide or
polypeptide with (2)
a second amino acid, peptide or polypeptide. Anv chemoselective reaction
chemistry
that can be applied to ligation of unprotected peptide segments.
Extramembranous Receptor Domain: A domain of a membrane protein
receptor that is external to a lipid membrane bilaver of a cell. Includes
extracellular
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and cytosolic domains of a membrane protein receptor. or soluble portions of
these
domains that are capable of bindin~~ to a ligand of the membrane protein
receptor.
such as N-terminal and C-terminal extramembranous domains. Excludes membrane
spanning transmembrane domain that anchors an intact membrane protein receptor
to
the lipid membrane bilayer of a cell. Can include one or more amino acid
residues of
a transmembrane domain, provided the residues do not span the membrane or form
an
insoluble complex incapable of binding to a ligand.
Ligand: A chemical entity that interacts with its target membrane protein
receptor of a cell.
Membrane Protein Receptor: A receptor of a cell having at least one peptide
segment capable of being embedded and anchoring the receptor in the lipid
bilayer of
a cell membrane. Examples include. by way of illustration and not limitation,
enzyme-linked receptors including receptor protein-tyrosine kinases such as
receptors
for EGF, NGF, PDGF, insulin and many other growth factors; and cytokine
receptors
and non-receptor protein kinases such as receptors for cytokines such as
interleukin-2
and erythropoietin, as well as for some polypeptide hormones such as growth
hormone; G-protein coupled receptors including those related to rhodopsin
(type A),
calcitonin (type B), and metabolic receptors (type C), more particularly
including type
B receptors such as receptors for glucagon, glucagon-like peptide, VIP. GIP,
GHRH,
secretin, PACAP, PTH and CRF; and ion channel receptors including ligand-gated
channels such as the acetylcholine receptor and voltage-gated ion channels
such as the
potassium and sodium ion channels.
Peptide: A polymer of at least two monomers, wherein the monomers are
amino acids, sometimes referred to as amino acid residues, which are joined
together
via an amide bond. May have either a completely native amide backbone or an
unnatural backbone or a mixture thereof. Can be prepared by known synthetic
methods, including solution synthesis. stepwise solid phase synthesis, segment
condensation, and convergent condensation. Can be synthesized ribosomally in
cell
or in a cell free system, or generated by proteolysis of larger polypeptide
segments.
Can be synthesized by a combination of chemical and ribosomal methods.
Polypeptide: A polymer comprising three or more monomers. wherein the
monomers are amino acids. sometimes referred to as amino acid residues. which
are
joined together via an amide bond. Also referred to as a peptide or protein.
Can
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comprise native amide bonds or anv of the known unnatural peptide backbones or
a
mixture thereof. Range in size ti-om 3 to 1000 amino acid residues, preferably
from
3-100 amino acid residues, more preferably from 10-60 amino acid residues, and
most
preferably from 20-~0 amino acid residues. Segments or all of the polypeptide
can be
prepared by known synthetic methods. including solution synthesis, stepwise
solid
phase synthesis. segment condensation, and convergent condensation. Se~ments
or
all of the polypeptide also can be prepared ribosomally in a cell or in a cell-
free
translation system, or generated by proteolysis of larger polypeptide
segments. Can
be synthesized by a combination of chemical and ribosomal methods.
Protein Domain: A contiguous stretch of amino acid residues within a protein
sequence related to a functional property of the molecule.
Soluble ivlembrane Protein Receptor Domain: A domain of a membrane
protein receptor that is soluble under aqueous physiologic conditions.
Examples
include the extracellular and intracellular domains of a receptor that reside
in an
aqueous environment external or internal to a lipid membrane bilayer of a
cell,
respectively.
Brief Description Of The Drawings
Fig. 1 is a schematic showing the extramembranous receptor domains of a G-
coupled protein receptor, in the context of the intact membrane protein
receptor.
Fig. 2 is a schematic showing the extramembranous receptor domains of a G-
coupled protein receptor incorporating various unnatural amino acids, in the
context
of the intact membrane protein receptor. "~" represents a label, "Y"
represents an
unnatural backbone, and "Z" represents a chemical handle.
Fig. 3 is a schematic showing an N-terminal extracellular receptor domain of a
Type B G-coupled protein receptor.
Fig. 4 is a schematic showing the chemical ligation design for the N-terminal
extracellular receptor domain of a Type B Glucagon-like peptide I G-coupled
protein
receptor (GLP-I R), using Segment 1 (SEQ ID NO:1 ), Segment 2 (SEQ ID N0:2)
and
Segment 3 (SEQ ID N0:3).
Fig. ~ shows folding of the chemically synthesized GLP-1 R N-terminal
domain as monitored using mass spectroscopy and hi~~h performance liquid
chromatography (HPLC).
c)
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Figs. 6A and 6B show chymotrvptic digest of the chemically synthesized
GLP-1 R N-terminal domain and mappin~~ of disulfide bonds.
Fig. 7 shows the disulfide bond map of the chemically synthesized GLP-1R N-
terminal domain (SEQ ID N0:4).
Figs. 8A and 8B show binding of rhodamine-labeled GLP ligand to the GLP-
1 R N-terminal domain and fluorescence anisotropy measurement of the binding
event.
Description Of Specific Embodiments
The invention relates to the chemical synthesis of extracellular receptor
domains of membrane protein receptors. and compositions and methods that
employ
them. The extramembranous receptor domains include the soluble ligand-binding
extracellular and intracellular domains of membrane protein receptors. The
extramembranous receptor domains of the invention are produced by first
selecting
the domain targeted for synthesis. Amino acid sequence information of the
domain is
then utilized to design peptide or polypeptide segments for chemical ligation.
Peptide
segments of a selected extramembranous receptor domain are then constructed so
as
to have unprotected chemoselective reactive groups capable of forming a
covalent
bond therein between when contacted under conditions amenable to the chosen
method of chemical ligation. The peptide segments are then covalently joined
by
chemoselective chemical ligation. The ligation product formed by chemical
ligation
of peptide segments is then exposed to a folding buffer to generate a folded
ligation
product. The folding buffer contains at least one chaotropic reagent and an
organic
solvent that mimics the water-lipid interface environment of a cell membrane.
Exposure to the folding buffer is followed by isolation from the buffer of
ligation
product that binds to a ligand of the membrane protein receptor. such as a
natural
ligand of the receptor. The ligand-binding portion of the ligation product
produced by
this method represents the folded extramembranous receptor domain.
Selection of an extramembranous receptor domain can be accomplished by
identifying and retrieving amino acid sequence information for the receptor or
domain
to be synthesized, such as ti-om a private or public database. Examples of
public
accessible databases useful for this purpose include. for example, GenBank
(Benson,
et al.. ;Vcrcleic Acids Res. (1998) 26(1 ):1-7): USA National Center for
Biotechnology
Information. National Library of Medicine (National Institutes of Health.
Bethesda,
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WO 00/53624 PCT/US00/06297
MD. USA), TIGR Database (The Institute for Genomic Research. Rockville, MD,
USA). Protein Data Bank (Brookhaven \ational Laboratory, USA). and the ExPASy
and Swiss-Protein database (Swiss Institute of Bioinformatics. Geneva.
Switzerland).
Alternatively, the amino acid sequence information can be obtained de novo
using
standard biochemical and/or molecular biology techniques. such as cloning and
sequencing following= protocols well known in the art. When one or more target
extramembranous domains of a chosen receptor sequence have not been defined,
then
various rules of thumb, screening and/or modeling techniques known in the art
can be
employed to characterize putative domains. Examples include sequence homology
comparisons, and use of algorithms and protein modeling tools known in the art
that
are suitable for this purpose. For instance. mutagenesis. thermodynamic,
computational, modeling and/or any technique that reveals functional and/or
structural
information regarding a target polypeptide of interest can be used for this
process.
These techniques include immunological and chromatographic analyses,
fluorescence
resonance energy transfer (FRET), circular dichroism (CD), nuclear magnetic
resonance (NRM), electron and x-ray crystallography, electron microscopy,
Raman
laser spectroscopy and the like, which are commonly exploited for designing
and
characterizing membrane polypeptide systems. ( See, e.g., Newman, R.,
ILlethods Mol.
Biol. (1996) 56:365-387; Muller, et al.. J. Struct. Biol. (1997) 119(2):149-
157;
Fleming, et al., J. Mol. Biol. (1997) 272: 266-27: Haltia, et al.,
Biochemistry (1994)
33(32): 9731-9740.5; Swords, et al., Biochem. J. (1993) 289(1): 21~-219;
Wallin, et
al., Protein Sci. (1997) 6(4):808-81~; Goormaghtigh. et al.. Subcell Biochem.
(1994)
23:405-450). Muller, et al., Biophys. .l. (1996) 70(4):1796-1802; Sami, et
al., Biochim
BiophysActa. (1992) 1105(1):148-154. Wang, et al.,J. Mol. Biol. (1994)
237(1):1-4;
Watts, et al., Mol. Membr. Biol. (1995) 12(3):233-246; Bloom, M.. Biophys. J.
(1995)
69(5):1631-1632; and Gutierrez-Merino, et al.. Biochem Soc. Traps. (1994)
22(3):784-788).
Since most receptors typically are identified by their characteristic
transmembrane spanning domains. these re~.:ions can be used as a reference to
identify
non-transmembrane segments that are likely to exist external to the cell
membrane. In
particular, structural and functional information can be obtained using
standard
techniques including homology comparisons to other membrane protein receptors
having similar amino acid sequences and domains. preferably other receptors
for
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which at least some structural and functional information is knowm. For an
exemplary
list of membrane protein receptors of known three-dimensional structure, see
Preusch,
et al., Nuture Struct. Biol. (1998) x:12-14.
Modeling programs also may be employed to identify putative transmembrane
segments, and thus putative extramembranous domains. For example. the programs
"TmPred" and TopPredII" can be used to make predictions of membrane-spanning
regions and their orientation, which is based on the statistical analysis of a
database of
transmembrane proteins present in the SwissProt database. (von Heijne, J.
.Viol. Biol.
(1992) 225:487-494; Hoppe-Seyler, Biol. Chem. (1993) 347:166; and Claros, et
al.,
Compact. Appl. Biosci. (1994) 10(6):68-686). Other programs can be used and
include: "DAS" (Cserzo, et al., Prot. Eng. ( 1997) 10(6):673-676); "PHDhtm"
(Rost,
et al., Protein Science (1995) 4:21-533); and "SOSUI" (Mitaku Laboratory,
Department of Biotechnology, Tokyo University of Agriculture and Technology).
A target receptor also may be modeled in three dimensions to identify putative
extramembranous receptor domains therein. First, a sequence alignment between
the
polypeptide to be modeled and a polypeptide of known structure is established.
Second, a backbone structure is generated based on this alignment. This is
normally
the backbone of the most homologous structure, but a hybrid backbone also may
be
used. Third. sidechains are then placed in the model. Various techniques like
Monte
Carlo procedures, tree searching algorithms etc., can be used to model rotomer
sidechains having multiple possible conformations. If the polypeptide to be
modeled
has insertions or deletions with respect to the known structure, loops are re-
modeled,
or modeled ab irritio. Database searches for loops with similar anchoring
points in the
structure are often used to build these loops, but energy based ab initio
modeling
techniques also can be employed. Energy minimizations, sometimes combined with
molecular dynamics, are then normally used for optimization of the final
structure.
The quality of the model is then assessed, including visual inspection. to
verify that
the structural aspects of the model are not contradicting what is known about
the
functional aspects of the molecule.
The three-dimensional models are preferably generated using a computer
program that is suitable for modeling membrane polypeptides. (Vriend,
"Molecular
Modeling of GPCRs" in 7TM (1990 vol. ~). Examples of computer programs
suitable for this purpose include: "WhatIl" (Vriend, ,l. ~i-Inl. (~f'Clpll. (
1990) 8:~2-56;
1?
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WO 00/53624 PCT/US00/06297
available from EMBL, Meverhofstrasse 1, 691 17 Heidelberg , Germany) and
"Swiss-
Model" (Peitsch, et al.. ( 1996) "Molecular modeling of G-protein coupled
receptors"
in G Protein-coupled Receptors. New opportunities for commercial development.
6:6.29-6.37, N Mulford and LM Savage Eds.. IBC Biomedical Library Series;
Peitsch, et al.. Receptors and Channels (1996) 4:161-164; Peitsch, et al..
"Large-scale
comparative protein modeling" in Proteome research: new frontiers in
functional
genomics," pp. 177-186, Wilkins iVIR. Williams KL, Appel RO, Hochstrasser DF,
Eds., Springer, 1997).
Amino acid sequence information of the selected extramembranous domain is
then utilized to design peptide or polypeptide segments for chemical ligation,
where at
least one peptide employed for ligation is chemically synthesized, i.e., via
ribosomal
free synthesis. In developing the ligation strategy, peptide segments are
designed to
provide unprotected reactive groups that selectively react to yield a covalent
bond at
the ligation site, also referred to as a chemoselective ligation site. Thus
the peptides
are designed in accordance with a selected ligation chemistry, or more than
one
individual ligation chemistry, provided the segments targeted for ligation in
any given
step of synthesis provide compatible chemoselective ligation component
pairings that
form the desired covalent bond upon chemoselective chemical ligation, which
avoids
unwanted side reactions.
In particular, peptide or polypeptide segments and their chemoselective
reactive groups are designed based on the ligation chemistry selected for
covalently
stitching the segments together in their desired orientation. Any number of
ligation
chemistries may be employed in accordance with the methods of the invention.
These
chemistries include, but are not limited to. native chemical ligation (Dawson,
et al.,
Science (1994) 266:776-779; Kent, et al., WO 96/34878), extended general
chemical
ligation (Kent. et al., WO 98/28434), oxime-forming chemical ligation (Rose,
et al., J.
Amer. Chem. Soc. (1994) 116:30-33), thioester forming ligation (Schnolzer, et
al.,
Science (1992) 256:221-225), thioether forming ligation (Englebretsen, et al.,
Tet.
Letts. (1995) 36(48):8871-8874). hydrazone forming ligation (Gaertner, et al.,
Bioconj. Chem. ( 1994) S(4):333-338), thiazolidine forming ligation and
oxazolidine
forming ligation (Zhang, et al.. Proc. :Vatl. .-Iccrd. Sci. (1998) 95(16):9184-
9189; Tam,
et al.. WO 95/00846).
13
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
By way of example, for native chemical ligation. the ligation component
segments for ligation comprise a compatible native chemical ligation component
pairing in which one of the components provides a cysteine having an
unprotected
amino group and the other component provides an amino acid having an
unprotected
a,-thioester group. These groups are capable of chemically reacting to yield a
native
peptide bond at the ligation site. For oxime-forming chemical ligation, the
peptide
segments comprise a compatible oxime-forming chemical ligation component
pairing
in which one of the components provides an unprotected amino acid having an
aldehyde or ketone moiety and the other component provides an unprotected
amino
acid having an amino-oxy moiety. These groups are capable of chemically
reacting to
yield a ligation product having an oxime moiety at the ligation site. For
thioester-
forming chemical ligation, the ligation segments comprise a compatible
thioester-
forming chemical ligation component pairing in which one of the components
provides an unprotected amino acid having a haloacetyl moiety and the other
component provides an unprotected amino acid having an a-thiocarboxylate
moiety.
These groups are capable of chemically reacting to yield a ligation product
having a
thioester moiety at the ligation site. For thioether-forming chemical
ligation, the
ligation components comprise a compatible thioether-forming chemical ligation
component pairing in which one of the components provides an unprotected amino
acid having a haloacetyl moiety and the other component provides an
unprotected
amino acid having an alkyl thiol moiety. These groups are capable of
chemically
reacting to yield a ligation product having a thioether moiety at the ligation
site. For
hydrazone-forming chemical ligation, the ligation components comprise a
compatible
hydrazone-forming chemical ligation component pairing in which one of the
components provides an unprotected amino acid havinU an aldehyde or ketone
moiety
and the other component provides an unprotected amino acid having an hydrazine
moiety. These groups are capable of chemically reacting to yield a ligation
product
having a hydrazone moiety at the ligation site. For thiazolidine-forming
chemical
ligation. the ligation components comprise a compatible thiazolidine-forming
chemical ligation component pairing in which one of the components provides an
unprotected amino acid having a 1-amino. 2-thiol moiety and the other
component
provides an unprotected amino acid havin~~ an aldehyde or a ketone moiety.
These
groups are capable of chemically reacting to yield a ligation product having a
1~
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
thiazolidine moiety at the ligation site. For oxazolidine-forming chemical
ligation,
the ligation components comprise a compatible oxazolidine-forming chemical
ligation
component pairing in which one of the components provides an unprotected amino
acid having a 1-amino. 2-hydroxyl moiety and the other component provides an
unprotected amino acid presenting an aldehyde or a ketone moiety. These groups
are
capable of chemically reacting to yield a ligation product having an
oxazolidine
moiety at the ligation site.
Other design considerations include uniqueness of the ligation site,
solubility
of the ligation components, and specificity and completeness of the ligation
reaction.
In particular, ligation component pairings are preferably designed to maximize
selectivity of the ligation reaction. This includes design of linker or
capping
sequences that may be employed to generate chemoselective reactive groups. or
used
to assist in the solubility of the ligation components. The ligation
components and
complementary pairings thereof also can be selected by modeling them in two or
three-dimensions to simulate the ligated product and/or the pre-ligation
reaction
components. Modeling programs as described above can be used for this purpose.
When designing a smaller extramembranous receptor domain. all peptides can
be synthesized chemically and employed for total chemical synthesis and
ligation.
This includes, for example, domains ranging in size up to about 200 to 250
amino
acids. These totally synthetic domains also may be ligated together to form
even
larger totally synthetic domains. Since chemical synthesis is utilized, the
chemically
synthesized peptides or polypeptides can be linear. cyclic or branched, and
often
composed of. but not limited to, the 20 genetically encoded L-amino acids.
Chemical
synthetic approaches also permit incorporation of novel or unusual chemical
moieties
including D-amino acids, other unnatural amino acids. oxime. hydrazone. ether,
thiazolidine, oxazolidine, ester or alkyl backbone bonds in place of the
normal amide
bond, N- or C-alkyl substituents, side chain modifications. and constraints
such as
disulfide bridges and side chain amide or ester linka~~es. See, for example,
Wilken, et
al., (Curr. Opin. Biotech. ( 1998) 9(4):412-426). which reviews various
chemistries for
chemical synthesis of peptides and polypeptides.
For example, native chemical ligation and synthesis of polypeptides having a
native peptide backbone structure is disclosed in Kent. et al., WO 96/34878.
See also
Dawson, et al. (Science (1994) 266:77-779) and Tam. et al. (Proc. .~~'cnl.
,~ccid. .Sci.
1~
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
USA (1990 92:1248-12489). Unnatural peptide backbones also can be made by
known methods (See, e.g., Schnolzer, et al., Science (1992) 26:221-225; Rose,
et al..
J. Am Chem. Soc. ( 1994) I I 6:30-3=1: Liu, et al., Proc. Natl. Acad Sci. USA
( 1994)
91:6584-6588: Englebretsen, et al., Tct. Lefts. ( 1990 36(48):8871-8874;
Gaertner, et
al., Bioconj. Chem. (1994) J(4):3J3-33g: Zhang, et al., I'roc. Ncrtl. Acaa!
Sci. (1998)
95(16):9184-9189; and Tam. et al., WO 9/00846). Extended general chemical
ligation and synthesis also may be employed as disclosed in Kent, et al., WO
98/28434.
Additionally, rapid methods of synthesizing assembled polypeptides via
chemical ligation of three or more unprotected peptide segments using a solid
support,
where none of the reactive functionalities on the peptide segments need to be
temporarily masked by a protecting group, and with improved yields and
facilitated
handling of intermediate products is described in Canne, et al., WO 98/6807.
Briefly, this method involves solid phase sequential chemical ligation of
peptide
segments in an N-terminus to C-terminus direction, with the first solid phase-
bound
unprotected peptide segment bearing a C-terminal a.-thioester that reacts with
another
unprotected peptide segment containing an N-terminal cysteine and a C-terminal
thioacid. The techniques also permits solid-phase native chemical ligation in
the C- to
N-terminus direction. Large polypeptides can also be synthesized by chemical
ligation of peptide segments in aqueous solution on a solid support without
need for
protecting groups on the peptide segments. A variety of peptide synthesizers
are
commercially available for batchwise and continuous flow operations as well as
for
the synthesis of multiple peptides within the same run and are readily
automated.
For larger extramembranous receptor domains, it may be desirable to
chemically synthesize one or more smaller peptide or polypeptide segments that
incorporate an unnatural or modified amino acid having a selected reactive
group (R1)
at a chosen termini, and utilize one or more recombinantly produced
polypeptide
segments that include a terminal amino acid having a selected reactive group
(R2),
where R1 and R2 represent compatible chemoselective reactive groups. An
example
is utilization of native chemical ligation in which the recombinant segment is
provided with a terminal cysteine residue (R?) that is capable of
chemoselective
ligation to a thioester provided by the selected reactive group (R 1 ) of the
chemically
synthesized peptide segment.
16
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
Some commonly used host cell systems for cloning, expression and recovery
of membrane protein receptor polypeptides include E. coli. Xenopus oocytes.
baculovirus, vaccinia. and yeast. as well as many higher eukaryotes including
transgenic cells in culture and in whole animals and plants. (See, e.g., G.W.
Gould;
"Membrane Protein Expression Systems: A User's Guide," Portland Press, 1994,
Rocky S. Tuan, ed.; and "Recombinant Gene Expression Protocols," Humana Press,
1996). For example. yeast expression systems are well known and can be used to
express and recover a target membrane protein receptor polypeptide of interest
following standard protocols. (See, e.g., Nekrasova, et al, Eur. J. Biochem.
(1996)
238:28-37; Gene Expression Technology Methods in Enzymology 185 ( 1991 );
Molecular Biology and Genetic Engineering of Yeasts CRC Press, Inc. (1992);
Herescovics, et al., FASEB (1993) 7:40-~~0; Larriba, G. Yeast (1993) 9:441-
463;
Buckholz, R.G., Curr. Opinion Biotech. (1993) 4:38-X42; Asenjo, et al., "An
Expert
System for Selection and Synthesis of Protein Purification Processes Frontiers
in
Bioprocessing II" pp. 358-379, American Chemical Society, (1992); Mackett, M,
"Expression of Membrane Proteins in Yeast Membrane Protein Expression Systems:
A Users Guide" pp. 177-218, Portland Press ( 1995)).
Cleavage sites also may be suitably positioned into the segment utilized for
ligation, so that cleavage yields the desired terminal group for ligation.
Some
commonly encountered protease cleavage sites are: Thrombin
(KeyValProArg/GIySer); Factor Xa Protease (IleGluGlyArg); Enterokinase
(AspAspAspAspLys); rTEV (GIuAsnLeuTyrPheGln/Gly), which is a recombinant
endopeptidase from the Tobacco Etch Virus; and 3C Human rhino virus Protease
(LeuGluValLeuPhe Gln/GlyPro) (Pharmacia Biotech).
Various chemical cleavage sites are also known and include, but are not
limited to, the intein protein-splicing elements (Dalgaard, et al., Nucleic
Acids Res.
(1997) 2(6):4626-4638) and cyanogen bromide cleavage sites. Inteins can be
constructed which fail to splice, but instead cleave the peptide bond at
either splice
junction (Xu. et al., E~t~IBO J. ( 1996) 1 ~( 19):~ 146-51 ~3; and Chong, et
al., J. Biol.
Chem. (1996) 271:2219-22168). For example, the intein sequence derived from
the
Sacchcrromyces cerevisiac~ VMAI gene can be modified such that it undergoes a
self
cleavage reaction at its N-terminus at low temperatures in the presence of
thiols such
17
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
as I .4-dithiothreitol (DTT). 2-mercaptoethanol or cysteine (Chong, et al..
Gene ( 1997)
192:271-281).
Cyanogen bromide (CnBr) cleaves at internal methionine (Met) residues of a
polypeptide sequence. Cleavage with CnBr yields two or more fragments, with
the
fragments containing C-terminal residues internal to the original polypeptide
sequence having an activated alpha-carboxyl functionality, e.g. cyanogen
bromide
cleavage at an internal Met residue to give a fragment with a C-terminal
homoserine
lactone. For some polypeptides, the fragments will re-associate under folding
conditions to yield a folded polypeptide-like structure that promotes reaction
between
the segments to give a reasonable yield (often 40-60%) of the full-length
polypeptide
chain (now containing homoserine residues where there were Met residues
subjected
to cyanogen bromide cleavage) (Woods. et al.. J. Biol. Chem., (1996),
271:32008-
32015).
In general, a cleavage site for generating a ligation site amenable to a
desired
chemical ligation chemistry usually is selected to be unique, i.e., it occurs
only once
in the target polypeptide. However, when more than one cleavage site is
present in a
target polypeptide that is recognized and cleaved by the same cleavage
reagent, if
desired one or more of such sites can be permanently or temporarily blocked
from
access to the cleavage reagent and/or removed during synthesis. Cleavage sites
can
be removed during synthesis of a given peptide segment by replacing, inserting
or
deleting one or more residues of the cleavage reagent recognition sequence,
and/or
Incorporating one or more unnatural amino acids that achieve the same result
(See
Figs. 1 and 2). A cleavage site also may be blocked by agents that bind to the
peptide, including ligands that bind the peptide and remove accessibility to
all or part
of the cleavage site. However a cleavage site is blocked or removed, one of
ordinary
skill in the art will recognize that the method is selected such that upon
cleavage the
peptide or polypeptide is capable of chemoselective chemical ligation to a
target
ligation component of interest.
A ligation component also can be selected to contain moieties that facilitate
and/or ease purification and/or detection. For example. purification handles
or tags
that bind to an affinity matrix can be used for this purpose (See Fi'~s. 1 and
2). Many
such moieties are known and can be introduced via post-synthesis chemical
modification and/or during synthesis. (See, e.'~.. Protein Purification
Protocols.
18
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
( 1996), Doonan. ed., Humana Press Inc.: Schriemer, et al.. Anul Chem. ( 1998)
70(8):169-1575; Evangelists, et al.. J. C'hromato,~Tr. B. Biomed Sci. r~ppl.
(1997)
699( 1-2):3 83-401; Kaufmann. J. C'hromutogr. 13. Biomed. Scl. ~lppl. ( 1997)
699( 1-
2):347-369; Nilsson, et al, Protein Expr. Pnrif. ( 1997) 1 1 ( 1 ):1-16;
Lanfermeijer, et
al., Protein Expr. Purif. (1998) 12( 1 ): 29-37). For example, one or more
unnatural
amino acids having a chemical moiety that imparts a particular property that
can be
exploited for purification can be incorporated during synthesis. Purification
sequences also can be incorporated by recombinant DNA techniques. In some
instances, it may be desirable to include a chemical or protease cleavage site
to
remove the tag, depending on the tag and the intended end use. An unnatural
amino
acid or chemically modified amino acid also may be employed to ease detection,
such
as incorporation of a chromophore. hapten or biotinylated moiety detectable by
fluorescence spectroscopy, immunoassays, and/or MALDI mass spectrometry.
Thus, one or more of the peptides or polypeptides utilized for ligation may be
( 1 ) totally synthetic, i.e., produced in toto by ribosomal free chemical
synthesis; (2)
semi-synthetic, i.e., produced at least partially using ribosomal synthesis
such as via
recombinant DNA techniques; or (3) natural, i.e., produced in toto by
ribosomal
synthesis. The extramembranous receptor domains of the invention can thus be
totally synthetic or semi-synthetic. and may include one or more unnatural
amino
acids incorporated at pre-selected residue positions, as illustrated in Figs.
1 and 2.
Once the extramembranous receptor domain is designed and the ligation
components prepared, the segments are ligated under the appropriate chemical
ligation conditions corresponding to the chosen ligation chemistry(s) imparted
by the
design. As can be appreciated, reaction conditions for a given ligation
chemistry are
selected to maintain the desired interaction of the ligation components. For
example,
pH and temperature, and solubilizing reagents can be varied to optimize
ligation.
Addition or exclusion of reagents that solubilize the ligation components to
different
extents may further be used to control the specificity and rate of the desired
ligation
reaction. Reaction conditions are readily determined by assaying for the
desired
chemoselective reaction product compared to one or more internal and/or
external
controls.
Homogeneity and the structural identity of the desired, ligation products can
be
confirmed by any number of means includin~~ immunoassays. fluorescence
19
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
spectroscopy, gel electrophoresis. HPLC using either reverse phase or ion
exchange
columns. amino acid analysis, mass spectrometry, crystallography. NMR and the
like.
Positions of amino acid modifications. insertions and/or deletions. if
present, can be
identified by sequencing with either chemical methods (Edman chemistry) or
tandem
mass spectrometry.
For folding, the ligation product is dissolved in an aqueous solution
containing
a solubilizing amount of a chaotropic reagent at a pH compatible with the
ligation
product in question. Chaotropic reagents suitable for this purpose include,
for
example, urea and guanidinium chloride. The concentration of the chaotropic
reagent
and pH of the solution can be adjusted for optimal solubilization of the
ligation
product, but within a range that does not damage the protein. When cysteines
are
present in the ligation product. a disulfide reducing agent such as
dithiothreitol may
be used. The solution containing the dissolved ligation product is then
diluted by
admixing with folding buffer. The folding buffer includes a buffering reagent,
a
chaotropic reagent and an organic solvent that are combined in amounts that
mimic
the water-lipid interface of a cell membrane. Buffering reagents are well
known and
include salts, such as Tris and Mops. The organic solvent utilized in the
folding
buffer is chosen to have a chemical moiety that hydrogen bonds with water, and
another chemical group providing an aliphatic moiety. Preferred organic
solvents are
water soluble. Examples of water soluble organic solvents include monohydroxy
alcohols such as methyl, ethyl, n-propyl, isopropyl, tert-butyl, and allyl
alcohols. Diol
and triol alcohols such as ethylene glycol, propylene glycol, trimethyl glycol
and
glycerol also may be utilized. Preferred water soluble organic solvent for use
in the
methods of the invention are methanol and glycerol, with the most preferred
being
methanol. It will be appreciated that organic solvents and other folding
buffer
components that denature proteins are avoided. or are present in non-
denaturing
amounts. It also will be appreciated that one or more chaotropes, organic
solvents and
the like can be employed for folding. Other additives may be included such as
detergents, lipids and the like. This includes chaperone proteins. Moreover,
the
folding buffer may be utilized for folding of the ligation product in a
perfusion device,
for example, as with the oxidative refolding chromatography approach described
in
Altamirano. et al. (Nature l3iotechnolo~ry ( I 999) 17:187-191 ), that employs
an
immobilized chaperone system. The li~~ation buffer also may include additives
such
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
as reduced and/or oxidized glutathione. for example, when disulfide bond
forming
cysteines are present. It also will be appreciated that the actual components
and ratios
thereof employed in the folding buffer of the method of the invention can be
determined for a given ligation product. and adjusted as necessary. The
ligation
product is exposed to the folding buffer for a period of time sufficient to
permit
folding, which can be monitored by any number of standard techniques described
above and known in the art. A preferred monitoring method is liquid
chromatography, e.;~. HPLC, which also permits isolation of folding products
concurrent with monitoring. Folding products are separated by any number of
non-
denaturing chromatographic techniques, and then assayed for binding to a
ligand of
the membrane protein from which the extramembranous receptor domain was
derived. Assays known in the art and/or those described herein can be used for
this
purpose. Folding product that binds to the ligand is then categorized as a
folded
extramembranous receptor domain. The folded ligation product can then be
utilized
immediately or stored, in unfolded or folded form for later use.
In another embodiment of the invention, compositions are provided that are
produced according to the method of the invention. One composition of the
invention
includes a synthetic extramembranous receptor domain of a membrane protein
receptor having a chemically synthesized segment that includes an unnatural
amino
acid at a pre-selected residue position. where the extramembranous receptor
domain is
free of a membrane spanning transmembrane domain and is capable of binding to
a
ligand of the membrane protein receptor. Compositions of the invention also
include
a totally synthetic and ultra homogenous extramembranous receptor domain of a
membrane protein receptor free of cellular contaminants. The extramembranous
receptor domain of the compositions of the invention may comprise one or more
synthetic segments that include genetically encoded L-amino acids, linear,
cyclic or
branched amino acids. D-amino acids. other unnatural amino acids, as well as
oxime,
hydrazone, ether, thiazolidine, oxazolidine, ester or alkyl backbone bonds in
place of
the normal amide bond. N- or C-alkyl substituents, side chain modifications,
and
constraints such as disulfide bridges and side chain amide or ester linkages.
Preferred unnatural amino acids are those having a detectable label. In this
embodiment, chemical synthesis is utilized to incorporate at least one
detectable label
in a pre-ligation component. In this wav the resulting ligation product can be
21
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
designed to contain one or more detectable labels at pre-specified positions
of choice.
Isotopic labels detectable by NMR are of particular interest. Also of
particular
interest is the incorporation of one or more unnatural amino acids comprising
a
detectable label at one or more specific sites in a target ligation component
of interest.
By unnatural amino acid is intended any of the non-genetically encoded L-amino
acids and D-amino acids that are modified to contain a detectable label. such
as
photoactive groups, as well as chromophores including fluorophores and other
dyes,
or a hapten such as biotin. Unnatural amino acids comprising a chromophore and
chemical synthesis techniques used to incorporate them into a peptide or
polypeptide
sequence are well known, and can be used for this purpose. For example, it may
be
convenient to conjugate a fluorophore to the N-terminus of a resin-bound
peptide
before removal of other protecting groups and release of the labeled peptide
from the
resin. Fluorescein, eosin, Oregon Green, Rhodamine Green, Rhodol Green,
tetramethylrhodamine, Rhodamine Red. Texas Red, coumarin and NBD fluorophores,
the dabcyl chromophore and biotin are all reasonably stable to hydrogen
fluoride
(HF), as well as to most other acids, and thus suitable for incorporation via
solid
phase synthesis. (Peled, et al., Biochemistry (1994) 33:7211; Ben-Efraim, et
al.,
Biochemistry (1994) 33:6966). Other than the coumarins, these fluorophores
also are
stable to reagents used for deprotection of peptides synthesized using FMOC
chemistry (Strahilevitz, et al., Biochemistry ( 1994) 33:10951 ). The t-BOC
and a-
FMOC derivatives of s-dabcyl-L-lysine also can be used to incorporate the
dabcyl
chromophore at selected sites in a polypeptide sequence. The dabcyl
chromophore
has broad visible absorption and can used as a quenching group. The dabcyl
group
also can be incorporated at the N-terminus by using dabcyl succinimidyl ester
(Maggiora, et al., J. Med. Chern. ( 1992) 35:3727). EDANS is a common
fluorophore
for pairing with the dabcyl quencher in FRET experiments. This fluorophore is
conveniently introduced during automated synthesis of peptides by using 5-((2-
(t-
BOC)-y-glutamylaminoethyl) amino) naphthalene-1-sulfonic acid (Maggiora, et
al.,
szrprcr). An a-(t-BOC)-E-dansvl-L-lysine can be used for incorporation of the
dansyl
fluorophore into polypeptides during chemical synthesis (Gauthier. et al..
Arch
Biochem Biophys (1993) 306:304). As with EDANS fluorescence of this
fluorophore
overlaps the absorption of dabcyl. Site-specific biotinylation of peptides can
be
achieved using the t-BOC-protected derivative of biocytin (Geahlen. et al.,
Anal.
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
Biochem. ( 1992) 202:68), or other well known biotinylation derivatives such
as NHS-
biotin and the like. Racemic benzophenone phenylalanine analog also can be
incorporated into peptides following its t-BOC or FMOC protection (Jung, et
al., Intl.
J. Peptide Prot. Rcs. (1995) 4:106). Resolution of the diastereomers can be
accomplished during HPLC purification of the products: the unprotected
benzophenone also can be resolved by standard techniques in the art. Keto-
bearing
amino acids for oxime coupling, aza/hvdroxy tn~ptophan, biotyl-lysine and D-
amino
acids are among other examples of unnatural amino acids that can be utilized.
It will
be recognized that other protected amino acids for automated peptide synthesis
can be
prepared by custom synthesis following standard techniques in the art.
It will be appreciated that other detectable labels can be incorporated into a
ligation component post-chemical ligation, although less preferred. This can
be done
by chemical modification using a reactive substance that forms a covalent
linkage
once having bound to a reactive group of the target molecule. For example, a
peptide
or polypeptide ligation component can include several reactive groups, or
groups
modified for reactivity, such as thiol, aldehyde, amino groups, suitable for
coupling
the detectable label by chemical modification (Lundblad, et al., in "Chemical
Reagents for Protein Modification", CRC Press. Boca Raton, FL, (1984)). Site-
directed mutagenesis and/or chemical synthesis also can be used to introduce
and/or
delete such groups from a desired position. Any number of detectable labels
including biotinylation probes of a biotin-avidin or streptavidin system,
antibodies,
antibody fragments, carbohydrate binding domains, chromophores including
fluorophores and other dyes, lectin, nucleic acid hybridization probes, drugs,
toxins
and the like, can be coupled in this manner. For instance, a low molecular
weight
hapten, such a fluorophore, digoxigenin. dinitrophenyl (DNP) or biotin, can be
chemically attached to the membrane polypeptide or ligation label component by
employing haptenylation and biotinylation reagents. The haptenylated
polypeptide
then can be directly detected using fluorescence spectroscopy, mass
spectrometry and
the like, or indirectly using a labeled reagent that selectively binds to the
hapten as a
secondary detection reagent. Commonly used secondary detection reagents
include
antibodies, antibody fragments, avidins and streptavidins labeled with a
fluorescent
dye or other detectable marker.
23
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
Depending on the reactive group. chemical modification can be reversible or
irreversible. A common reactive group targeted in peptides and polypeptides
are thiol
groups, which can be chemically modified by haloacetyl and maleimide labeling
reagents that lead to irreversible modifications and thus produce more stable
products.
S For instance, reactions of sulfhydryl groups with a-haloketones. amides, and
acids in
the physiological pH range (pH 6.S-8.0) are well known and allow for the
specific
modification of cysteines in peptides and polypeptides (Hermason, et al., in
"Bioconjugate Techniques", Academic Press, San Diego. CA. pp. 98-100. (1996)).
Covalent linkage of a detectable label also can be triggered by a change in
conditions,
for example, in photoaffinity labeling as a result of illumination by light of
an
appropriate wavelength. For photoaffinity labeling, the label, which is often
fluorescent or radioactive, contains a group that becomes chemically reactive
when
illuminated (usually with ultraviolet light) and forms a covalent linkage with
an
appropriate group on the molecule to be labeled. An important class of
photoreactive
1 S groups suitable for this purpose is the aryl azides, which form short-
lived but highly
reactive nitrenes when illuminated. Flash photolysis of photoactivatable or
"caged"
amino acids also can be used for labeling peptides that are biologically
inactive until
they are photolyzed with UV light. Different caging reagents can be used to
modify
the amino acids, such derivatives of o-nitrobenzylic compounds, and detected
following standard techniques in the art. (Kao, et al., "Optical Microscopy:
Emerging
Methods and Applications," B. Herman, J.J. Lemasters, eds., pp. 27-8S (1993)).
The
nitrobenzyl group can be synthetically incorporated into the biologically
active
molecule via an ether, thioether, ester (including phosphate ester). amine or
similar
linkage to a heteroatom (usually O, S or N). Caged fluorophores can be used
for
2S photoactivation of fluorescence (PAF) experiments, which are analogous to
fluorescence recovery after photobleaching (FRAP). Those caged on the s-amino
group of lysine. the phenol of tyrosine, the y-carboxylic acid of glutamic
acid or the
thiol of cysteine can be used for the specific incorporation of caged amino
acids in the
sequence. Alanine. glycine, leucine. isoleucine. methionine, phenylalanine,
tryptophan and valine that are caged on the a-amine also can be used to
prepare
peptides that are caged on the N-terminus or caged intermediates that can be
selectively photolyzed to yield the active amino acid either in a polymer or
in
solution. (Patchornik, et al., J. ~nr. Che»~. Soc. (1970) 92:6333). Spin
labeling
2~
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
techniques of introducing a groupin<, with an unpaired electron to act as an
electron
spin resonance (ESR) reporter species may also be used. such as a nitroxide
compound (-N-O) in which the nitrogen forms part of a sterically hindered ring
(Oh,
et al., supra).
Selection of a detectable label system generally depends on the assay and its
intended use. In particular, the chemical ligation methods and compositions of
the
invention can be employed in a screening or detection assay of the invention.
These
include diagnostic assays, screening new compounds for drug development, and
other
structural and functional assays that employ binding of a ligand to a
extramembranous
receptor domain produced by the method of the invention. The ligands may be
derived from naturally occurring ligands or derived from synthetic sources.
such as
combinatorial libraries. Screening and detection methods of particular
interest
involve detection of ligand binding by fluorescence spectroscopy.
In one embodiment, a soluble extramembranous receptor domain of a
membrane protein receptor produced by the method of the invention is utilized
to
detect binding of a ligand thereto. This aspect of the invention involves
contacting
monomers of a soluble extramembranous receptor domain of a membrane protein
receptor with a ligand of the membrane protein receptor. The soluble
extramembranous receptor domain used in this method is free of a membrane
spanning transmembrane domain and includes an unnatural amino acid at a pre-
selected residue position, such as an unnatural amino acid comprising a
detectable
label. The contacting is followed by assaying the soluble extramembranous
receptor
domain for ligand-induced association of domain monomers. For example,
association of domain monomers. such as dimerization, can be detected by
monitoring
a change in the property of the detectable label.
In another embodiment, a method of assaying a soluble extramembranous
receptor domain monomer for ligand-induced association of domain monomers is
provided. This method includes contacting a soluble extramembranous receptor
domain of a membrane protein receptor with a ligand of the membrane protein
receptor. In this method, as in the above method. the soluble extramembranous
receptor domain is free of a membrane spanning transmembrane domain and
includes
an unnatural amino acid at a pre-selected residue position. The contacting is
followed
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
by assaying the soluble extramembranous receptor domain for ligand-induced
association of domain monomers.
In yet another embodiment. a method of detecting binding of a ligand to an
extramembranous receptor domain of a membrane protein receptor is provided.
This
method involves contacting a soluble extramembranous receptor domain of a
membrane protein receptor with a ligand for the membrane protein receptor,
where
the soluble extramembranous receptor domain is free of a membrane spanning
transmembrane domain and comprises an unnatural amino acid having a detectable
moiety. Detection of ligand binding is then performed by assaying for a change
in a
property of the detectable moiety, such as fluorescence when the detectable
label is a
fluorophore.
Of particular interest are methods and compositions employing a totally
synthetic N-terminal extramembranous domain of a GPCR, such as a type B GPCR
exemplified in the Examples. For instance, very little is known about the
structure of
type B GPCRs and few homogenous and truly high-throughput assays exist. The
following gives a list of possible applications of synthetic N-terminal
receptor domain
in drug-discovery.
N-terminal receptor domains with FRET probes liQand displacement assays
In this assay format the N-terminal receptor domain and its ligand are labeled
with a fluorescent donor and acceptor, respectively. Binding of the ligand to
receptor
will result in energy transfer between the ligand and the receptor. Small
molecules
that disrupt this interaction can be identified due to their interference with
the energy
transfer. Time-resolved luminescent probes, such as the lanthanide chelator
complexes are ideal for this purpose, since they allow to reject background
signals
2~ due to light-scattering and are compatible with current homogenous high-
throughput
screening equipment. Depending on the binding stoichiometry, other FRET assay
formats can be envisioned. An intriguing possibility is that the binding of
the
hormone to the receptor results in receptor dimerization (or oligomerization).
This
means that one can envision receptor agonists that act by inducing
dimerization
(oligomerization) of the receptor.
Dimerization (oli~omerization) assay
For dimerization (oligomerization) assays employing the N-terminal domains
of GPCRs, in this assay format. one labels a portion of the receptor domains
with a
26
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WO 00/53624 PCT/US00/06297
fluorescent acceptor and the other half with a t7uorescent donor. In the
absence of a
ligand inducing dimerization (oligomerization). no FRET is observed. However,
the
presence of such a ligand is indicated by a rise in a FRET signal. Time-
resolved
luminescent probes, such as lanthanide chelator complexes are ideal for this
purpose,
since they allow to reject background signals due to light-scattering and are
compatible with current homogenous high-throughput screening equipment. Donor-
Donor dimerized (oligomerized) pairs will not contribute to acceptor emission.
Acceptor-Acceptor pairs can be gated away using an appropriate time delay.
This
leaves an unambiguous contribution from the Donor-Acceptor pair only.
Receptor domain labeled with isotopic NMR probes
Structure/Activity Relationship by Nuclear Magnetic Resonance (SAR by
NMR) is a novel approach to drug screening that requires isotopic labeling of
the drug
target protein to identify interactions of small molecule drug precursors with
a drug
target. This approach detects changes in the chemical shift of an amino acid
located
in the binding site of a drug target that is induced by binding of a potential
agonist or
antagonist. Current SAR by NMR approaches require 2-dimensional NMR
techniques on homogeneously isotopically labeled proteins. placing a severe
constraint on the throughput of molecules amenable for screening. Chemical
synthesis of proteins uniquely allows for the site-specific incorporation of
isotopic
labels into large quantities of protein. potentially requiring only 1-
dimensional NMR
techniques for SAR by NMR. This will provide significant time-savings per
sample,
propelling SAR by NMR into the realm of true high-throughput screening.
N-terminal receptor binding domains for phase display screening
Phage display is a very sensitive technique that allows for the amplification
and identification of peptides that bind to an immobilized drug target.
Chemical
synthesis techniques are uniquely suited to y~enerate large quantities of ion
channels
with site-specific attachment sites, e.g. via biotin labeling. Attachment of
such a
labeled domain to a solid support can then be used to select for phages that
display a
peptide exhibiting binding affinity to the N-terminal receptor domain. This
will allow
for the rapid identification of peptides that bind to a specific N-terminal
receptor
domain and that can be used as lead compounds for drug discovery. This
approach
could also be used to identify ligands for orphan receptors.
N-terminal receptor domains on a support matrix
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As described for phage display attachment. an N-terminal receptor domain
having a chemical handle such as a biotin label. can be used to attach the
protein to a
support matrix such as chip or a polymer. Such a device could be used to
identify
small peptides binding to an N-terminal receptor domain. In this approach, one
synthesizes a library of small peptides. A solution of these peptides is
incubated with
the matrix. Unbound peptides are then washed off. Peptides binding to the
receptor
domain can then be easily analyzed by MALDI analysis. The same approach could
also be used to identify ligands for orphan receptors.
Structural information for structure-based drug design
In addition, chemically synthesized receptor domains could be used to gain
structural information that is crucial for structure based drug design. Such
information includes NMR and crystallographic data on the free and ligand-
bound
state as well as structural data of the receptor domain complexed with a novel
agonist
or antagonist. Crystallographic studies will be aided by synthesis of N-
terminal
receptor domains with heavy isotope labels (such as selenomethionine and
iodotyrosine).
Therapeutic Uses
The receptor domains of the present invention also find use as therapeutic
agents, including use as agonists or antagonists of the corresponding
naturally
occurring receptor ligands, and as vaccines.
As an example, the GPCR type B receptor domains of the present invention
can be utilized in the mediation of metabolic disease, nervous system
disorders,
cancer and other disease indications associated with the GPCR type B
receptors.
Indeed, there is an emerging sense that soluble forms of receptor domains
represent an
important new class of protein therapeutics for the treatment of human
diseases (See,
e.g., Heaney, et al., J. Leukocyte Biology (1998) 64(2):135-46). Some specific
examples include recombinantly expressed soluble proteins containing a soluble
IL-6
(Interleukin 6) receptor domain has been shown to act as agonists of IL-6 and
normal
IL-6 receptor activity (Mackiewicz, et al., FEBS ( 1992) 30:257). Accordingly,
it is
envisioned that synthetic IL-6 receptor domains produced according to the
methods of
the invention can be utilized as agonists on IL-6 receptor signaling. As
another
example, a proinflammatory cytokine. tumor necrosis factor alpha (TNFa) is
involved
in mediation of acute and chronic inflammation. and recombinant antibody-like
28
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WO 00/53624 PCTNS00/06297
proteins comprising a TNFa receptor binding domain have found use as ligand
traps
for TNFa (Edwards CK 3rd., Annals of the Rheumatic Diseases, 1999 Nov. ~8
Suppl
1:I73-81; Solorzano, et al., J. Appl. Phvs. ( 1998) 84(4):1119-1130). Thus,
synthetic
TNFa receptor domains produced according to the methods of the invention can
be
used as antagonists to treat chronic inflammatory disease associated with the
TNFa
inflammation pathway. Furthermore. the synthetic receptor domains of the
present
invention can be utilized in a clinical application to generate neutralizing
antibodies
for blocking a membrane receptor protein involved in disease or for use as a
vaccine.
For instance, inhibition of the IL-2 receptor via a neutralizing antibody
produces an
effect having immunotherapeutic value (Rosenberg, S.A., Immunology Today
(1988)
9:58-59). Also, the extracellular domain of the minor, virus-coded M2 protein
is
nearly invariant in all influenza A strains. Administration of a fusion
proteins of the
M2 domain to the hepatitis B virus core (HBc) protein provided 90-100%
protection
to mice against otherwise deadly viral infection (Neirynck, et al., Nature
~l~ledicine
(1999) 5(10):1157-1163). One may thus utilize the methods and synthetic
receptor
domains of the invention as a broadband influenza vaccine constructs made up
of the
extracellular domain of the influenza A M2 protein as well as the homologous
protein
domains for influenza B and C joined in a multivalent fashion through a
linker, or in a
template assisted synthetic protein (TASP) construct design.
Given the absolute precision and power of chemical synthesis in constructing
ultra pure and homogenous receptor domains compounds according to the present
invention, these compounds thus find use in clinical applications that cannot
be
addressed using recombinant DNA techniques alone.
The following Examples are intended to illustrate various aspects of the
invention and are not intended to limit the scope of the invention.
Examples
Example 1
Peptide Synthesis
The following peptide segments (See Fig. 3) for chemical synthesis of the
GLP-1 receptor N-terminal domain (GLP-1R NTD) were synthesized using a custom-
modified Applied Biosystems 430A peptide synthesizer following established
protocols (Schnolzer, et al., Int. J. Peptide Protein Res. (1992) 40:180-193).
29
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
Segment 1 (SEQ ID NO:1 ):
AGPRPQGATVSLWETVQKWREYRRQCQRSLTEDPPPATDLF
Segment 2 (SEQ ID N0:2):
CNRTFDEYACWPDGEPGSFVNVSCPWYLPWASSVPQGHVYRF
Segment 3 (SEQ ID N0:3):
CTAEGLWLQKDNSSLPWRDLSECEESKRGERSSPEEQLLFL
A putative signaling domain consisting of 20 amino acid residues (Goke, et
al., FEBS Letters ( 1996) 398:43-47) was conveniently excluded in the
synthesis
design. Peptide segments were purified by preparative gradient reversed phase
HPLC
on a Rainin dual-pump high-pressure mixing system with 214 nm UV detection
using
a Vydac C-4 preparative or semi-preparative column (10 mm particle size, 2.2
cm x
25 cm, and 1 cm x 25 cm, respectively) and analytical reversed phase HPLC was
performed on a Vydac C-18 analytical column (~ mm particle size, 0.46 cm x 15
cm),
using a Hewlett Packard Model 1100 quaternary pump high-pressure mixing system
with 214 nm and 280 nm UV detection. Electrospray mass spectra (ESMS) of the
peptide products were obtained using a PE-Sciex API-1 quadrupole ion-spray
mass
spectrometer. Peptide masses were calculated from all the observed protonation
states
and peptide mass spectra were reconstructed using the MACSPEC software (PE-
Sciex, Thornhill, ON, Canada). Theoretical masses were calculated using the
MACPROMASS software (Terri Lee, City of Hope). GLP-1 R NTD segments 1 (SEQ
ID NO:1) and 2 (SEQ ID N0:2) (amino acid residues 21-61 and 62-103,
respectively)
were synthesized on a thioester generating resin by the in situ neutralization
protocol
for Boc (tert-butoxycarbonyl) chemistry stepwise SPPS (Schnolzer, et al.,
supra),
using established side-chain protection strategies. The N-terminal cysteine of
segment 2 was protected with an ACM (acetamidomethyl) group to prevent
cyclization. GLP-1 R NTD segment 3 (SEQ ID N0:3) (amino acid residues 104-144)
was synthesized analogously on a -OCH~-PAM resin (Schnolzer, et al., supra).
The
peptides were deprotected and simultaneously cleaved from the resin support
using
HF/p-cresol according to standard Boc-chemistrv~ procedures (Schnolzer. et
al.,
szzpra). All three GLP-1 R NTD segments were purified by preparative reversed-
phase HPLC with a linear gradient of 25-4~% Buffer B (100% acetonitrile
containing
0.1 % TFA) versus 0.1 % aqueous TFA in 4~ minutes. Fractions containing pure
peptide were identified using ESMS. pooled and lyophilized for subsequent
li~ation.
3O
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WO 00/53624 PCTNS00/06297
The purified peptides were characterized by electrospray MS: Segment 1
thioester
peptide (SEQ ID NO:1) obs. MW: 4962 + 1 D, calc. MW : 4,961.3 D (average
isotope
composition); Segment 2 thioester peptide with 2 protecting groups ( 1
His(DNP) and
1 Cys(ACM) groups) (SEQ ID NO:?): obs. MW 5.294 + 1 kD , calc. MW 5,294.4 D
(average isotope composition); Se<~ment 3 carboxylate peptide (SEQ ID N0:3)
obs.
MW: 4769 + 1 D, calc. MW : 4,749.3 D (average isotope composition).
Example 2
Chemical Protein Synthesis
Equimolar amounts of the purified unprotected GLP-1R NTD peptide (amino
acid residues 104-144, Segment 3. SEQ ID N0:3) was added to a solution of the
purified unprotected thioester peptide GLP-1 R NTD (amino acid residues 62-
103)alpha-COSR (Segment 2, SEQ ID N0:2) (2 mM) in O.1M sodium phosphate/6M
guanidinium chloride, pH 7.5 and 1 % thiophenol. The ligation mixture was
stirred
overnight at room temperature and the reaction was monitored by reversed-phase
HPLC and ESMS. The reaction mixture was subsequently treated with an equal
volume of a solution of 40% beta-mercaptoethanol in 6M guanidinium chloride,
100
mM phosphate, pH 7.5) for 20 minutes to remove any residual His(DNP)
protecting
groups. Reactants and products were separated by preparative reversed-phase
HPLC
with a linear gradient of 25-45% Buffer B versus 0.1% aqueous TFA in 45
minutes.
Fractions containing GLP-IR NTD (amino acid residues 62-144, Segments 2 and 3,
SEQ ID NOS:2 and 3) were identified by ESMS (obs. MW 9,690 + 1 kD , calc. MW
9690.7 D (average isotope composition)), pooled and lyophilized. Subsequently,
the
purified GLP-1R NTD (62-144) was dissolved in 0.5 M acetic acid containing 2M
urea and a 1.5 molar excess (relative to the total cysteine concentration) of
Hg(acetate)2. After 30 minutes, the solution was made 20% in beta-
mercaptoethanol
to scavenge mercury ions. Subsequently, the solution was desalted by
preparative
reversed-phase HPLC with a step <,4radient of I 0-45% Buffer B versus 0. I %
aqueous
TFA and the resulting lyophilized GLP-1 R NTD (amino acid residues 62-144,
Segments 2 and 3, SEQ ID NOS: 2 and 3).
Equimolar amounts of the purified unprotected GLP-1 R NTD (amino acid
residues 62-144, Segments 2 and 3. SEQ ID NOS: 2 and 3) was added to a
solution of
the purified unprotected thioester peptide GLP-I R NTD(21-61 )alpha-COSR
(Segment 1, SEQ ID NO:1) (2 mM) in 0.1 M sodium phosphate/6M guanidinium
31
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
chloride. pH 7.~ and 1% thiophenol. Ligation and workup proceeded as described
above to generate the following ligation product:
Synthetic GLP-1R N-terminal domain (amino acid residues 21-144, SEQ ID
N0:4):
AGPRPQGATVSLWETVQKWREYRRQCQRSLTEDPPPATDLF
CNRTFDEYACWPDGEPGSFVNVSCPWYLPWASSVPQGHVYR
FCTAEGLWLQKDNSSLPWRDLSECEESKRGERSSPEEQLLFL
Reactants and products were separated by preparative reversed-phase HPLC
with a linear gradient of 25-45% Buffer B versus 0.1 % aqueous TFA in 45
minutes.
Fractions containing full-length GLP-1 R NTD (amino acid residues 21-144, SEQ
ID
N0:4) were identified by ESMS (obs. MW 14,37 + 1 kD , calc. MW 14,375.0 D
(average isotope composition)), pooled and lyophilized.
Example 3
Protein Folding
The purified full-length GLP-1R NTD (amino acid residues 21-144, SEQ ID
N0:4) was dissolved at 4 mg/ml in freshly degassed 6M guanidinium/HCI, pH 4 (
100
mM sodium acetate) under an argon atmosphere. A 1 molar equivalent of DTT
(dithiothreitol) was added and the solution was stirred for 30 min.
Subsequently, the
solution was diluted to a peptide concentration of 0.2 mg/ml with freshly
degassed
2M guanidinium/HCI, pH 8.6 (200 mM Tris) containing 20% methanol, and an 8
molar equivalent of reduced glutathione and a 1 molar equivalent of oxidized
glutathione (equivalents to cysteine concentration in the peptide) was added
(Wetlaufer, et al., Biochemistry (1970) 9(25):~Ol ~). The solution was stirred
under
argon overnight. The progress of folding was monitored by analytical reversed-
phase
HPLC with a linear gradient of 25-45% Buffer B versus 0.1% aqueous TFA for 30
minutes until no change in the shape of the HPLC-trace was detected and most
of the
protein peaks had collapsed under one main peak, suggesting homogenous
folding.
The formation of 3 disulfide bridges during folding was identified by ESMS.
The folded full-length GLP-1R NTD (amino acid residues 21-144, SEQ ID
N0:4) was purified by preparative reversed-phase HPLC with a linear gradient
of 2~-
45% Buffer B versus 0.1 % aqueous TFA in 4~ minutes. Fractions containing
folded
full-length GLP-1R NTD (amino acid residues 21-144, SEQ ID N0:4) were
identified
by ESMS (obs. MW 14,369 + 1 kD . calc. MW 14,369.0 D (average isotope
composition)), pooled and lyophilized.
32
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Example 4
Analysis of Folded Product
Fig. 7 presents the sequence of the GLP-1 R NTD (SEQ ID N0:4). Strictly
conserved residues in type-B GPCR's are bolded. as well as the ligation sites
and all
cysteines, including the ligation sites at Cys62 and Cys 104. Ligation of the
3
segments at these sites provides efficient access to multi-mg quantities of
full-length
GLP-1R NTD (21-144) peptide. Fig. 5 shows analytical reversed-phase HPLC
traces
monitoring the folding reaction of GLP-1R NTD. The top trace presents an HPLC-
trace of full-length GLP-1R NTD (21-144) peptide dissolved in 6M guanidinium
chloride, pH 4. The sharp peak at 21.1 minutes suggests high purity of the
synthetic
unfolded material. After an hour, multiple peaks in the HPLC-trace indicate a
wide
range of folding intermediates (data not shown). After overnight folding, most
of
these folding intermediates have disappeared and the corresponding HPLC-peaks
have collapsed into the main peak at 18.9 minutes. A broader peak at earlier
retention
time is due to glutathion adduct formation. The formation of 3 disulfide
bridges in the
folded protein was confirmed by the observation of a mass loss of 6 D relative
to the
unfolded protein (See insets; unfolded full-length GLP-1 R NTD (amino acid
residues
21-144, SEQ ID N0:4) obs. MW 14,375 + 1 D , calc. MW 14,175.0 D (average
isotope composition)), folded full-length GLP-1R NTD (amino acid residues 21-
144,
SEQ ID N0:4) obs. MW 14,369 + 1 D, calc. MW 14,369 D (average isotope
composition)). Folded protein was separated from unfolded protein by reversed
phase
HPLC. Re-dissolving the lyophilized, folded protein gave solutions that showed
GLP-1 binding activity.
Example 5
Proteolvtic Digest of Folded Product & Disulfide Mapping
For proteolytic digest, 50 pg folded GLP-1R NTD (21-144) was dissolved in
100 pl 125 mM Tris-HCI, pH 7.5 containing 2 M urea and 10 mM CaCh. 4.5 ~g
CLCK treated chymotrypsin (49 u/g, Worthington Biochemicals) was added. The
solution was stirred for 1 hour under argon and acidified with 100 ~l 200 mM
aqueous acetic acid. The peptide mixture was separated by analytical reversed-
phase
HPLC with a linear gradient of 5-45% Buffer B. Individual peptide fragments
were
identified by electrospray mass spectroscopy. Electrospray mass spectra (ESMS)
of
the digestion peptide products were obtained using a PE-Sciex API-1 quadrupole
ion-
spray mass spectrometer. Peptide masses were calculated from all the observed
-, -,
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
protonation states and peptide mass spectra were reconstructed using the
MACSPEC
software (PE-Sciex, Thornhill, ON, Canada).
Figs. 6A and 6B present the results of the chymotryptic digest of the digested
folded protein prior and after treatment of the digest mixture with TCEP (tris
carboxyethylphosphine) and the difference spectrum between the chromatograms.
In
the difference chromatogram, one clearly observes positive peaks for the
disulfide
bonded peptide fragments containing disulfide bridges between Cys94 and Cys126
(amino acid residues 70-80 of SEQ ID NO: 4 and amino acid residues 81-87 of
SEQ
ID NO: 4), and Cys71 and Cys85 (amino acid residues 104-110 of SEQ ID NO: 4
and
amino acid residues 121-142 of SEQ ID NO: 4). Upon reduction. these positive
peaks
disappear, and 2 negative bands appear per peak, corresponding to the reduced
segments that previously made up the disulfide bonded peptides. Combining this
result with the formation of 3 disulfide bonds upon folding, one can conclude
that a
third disulfide bridge is formed between Cys46 and Cys62. Partial tryptic
digestion of
the folded protein in SM urea for 1 hour produced a fragment that contained
Cys46
and Cys62 (amino acid residues 4~-48 of SEQ ID NO: 4 and amino acid residues
49-
64 of SEQ ID NO: 4). Reduction yielded a peptide fragment corresponding to
amino
acid residues 49-64. Longer digestion times in trypsin resulted in disulfide
scrambling. Fig. 7 shows the disulfide bond map of the totally synthetic GLP-1
R
NTD.
Example 6
Fluorescence Anisotropy Binding Assay
Fluorescence anisotropy binding assays were performed as follows. 300 pl of
a 0.7 ~M solution of GLP-I (7-36) labeled with tetramethyl rhodamine at Lys33
in
binding buffer ( 125 mM Tris, pH 7.3, I 50 mM NaCI, 1 mM EDTA) were placed
into
the thermostated (T = 25°C) sample compartment of a Fluorolog 3 L-
format
spectrofluorimeter with single excitation and emission spectrographs (ISA-Spex-
Jobin-Yvon, New Jersey). For additional rejection of stray light, a »0 nm long-
pass
filter was added to the emission beam path. A stock solution of 300 p.g of
folded
GLP-IR NTD in ~0 pl binding buffer was prepared and added in small aliquots to
the
ligand solution. To account for non-specific binding, a stock solution of SDF-
lalpha
(a highly disulfide crosslinked chemokine) was prepared and the concentration
was
adjusted to be equivalent to the total molar concentration of amino acid
residues.
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WO 00/53624 PCT/US00/06297
Concentrations were determined by absorption spectroscopy. After each
addition, the
sample was allowed to equilibrate under stirring for 10 minutes at
25°C. Longer
equilibration times did not lead to any significant changes in anisotropy.
Fluorescence anisotropy and a magic-anv~le total emission scan from 590 nm to
630
nm were taken after excitation at 520 nm. Scans were taken with 4 nm step size
and I
s dwell time. To improve the S/N ratio, the total anisotropy from 590 nm to
630 nm
was integrated for analysis.
Example 7
Binding Assay
Figs. 8A and 8B present the result of the anisotropy ligand-binding assay of
the GLP-I receptor in a semi-logarithmic representation. Clearly, beginning
saturation of binding is observed with an approximate Kd50 of 17 pM. More
interestingly, the sigmoidal shape of the ligand-binding curve in the linear
representation suggests that binding of the receptor domain by the hormone is
cooperative. Further studies to determine the exact stoichiometry of the
ligand
receptor complex, the extent of cooperativity and the binding constant are in
progress.
The above Examples illustrate that chemical synthesis of membrane protein
receptor domains can be utilized to provide facile and unprecedented access to
the
extramembranous domains of membrane protein receptors. The present invention
opens the way for detailed structure-function studies of soluble receptor
domains and
for the development of homogenous and true high-throughput drug screening
assays
and diagnostics.
All publications and patent applications mentioned in this specification are
herein incorporated by reference to the same extent as if each individual
publication
or patent application was specifically and individually indicated to be
incorporated by
reference.
The invention now being fully described, it will be apparent to one of
ordinary
skill in the art that many changes and modifications can be made thereto
without
departing from the spirit or scope of the appended claims.
CA 02372164 2001-09-10
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SEQUENCE LISTING
<110> Gryphon Sciences
<120> CHEMICAL SYNTHESIS AND USE OF SOLUBLE MEMBRANE PROTEIN
RECEPTOR DOMAINS
<130> GRFN-031/O1W0
<140> Not Yet Available
<141> 2000-03-11
<150> US 60/124,272
<151> 1999-03-11
<160> 4
<170> PatentIn Ver. 2.1
<210> 1
<211> 41
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 1
Ala Gly Pro Arg Pro Gln Gly Ala Thr Val Ser Leu Trp Glu Thr Val
1 5 10 15
Gln Lys Trp Arg Glu Tyr Arg Arg Gln Cys Gln Arg Ser Leu Thr Glu
20 25 30
Asp Pro Pro Pro Ala Thr Asp Leu Phe
35 40
<210> 2
<211> 42
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 2
Cys Asn Arg Thr Phe Asp Glu Tyr Ala Cys Trp Pro Asp Gly Glu Pro
1
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
1 5 10 15
Gly Ser Phe Val Asn Val Ser Cys Pro Trp Tyr Leu Pro Trp Ala Ser
20 25 30
Ser Val Pro Gln Gly His Val Tyr Arg Phe
35 40
<210> 3
<211> 41
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 3
Cys Thr Ala Glu Gly Leu Trp Leu Gln Lys Asp Asn Ser Ser Leu Pro
1 5 10 15
Trp Arg Asp Leu Ser Glu Cys Glu Glu Ser Lys Arg Gly Glu Arg Ser
20 25 30
Ser Pro Glu Glu Gln Leu Leu Phe Leu
35 40
<210> 4
<211> 124
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 4
Ala Gly Pro Arg Pro Gln Gly Ala Thr Val Ser Leu Trp Glu Thr Val
1 5 10 15
Gln Lys Trp Arg Glu Tyr Arg Arg Gln Cys Gln Arg Ser Leu Thr Glu
20 25 30
Asp Pro Pro Pro Ala Thr Asp Leu Phe Cys Asn Arg Thr Phe Asp Glu
35 40 45
Tyr Ala Cys Trp Pro Asp Gly Glu Pro Gly Ser Phe Val Asn Val Ser
50 55 60
2
CA 02372164 2001-09-10
WO 00/53624 PCT/US00/06297
Cys Pro Trp Tyr Leu Pro Trp Ala Ser Ser Val Pro Gln Gly His Val
65 70 75 80
Tyr Arg Phe Cys Thr Ala Glu Gly Leu Trp Leu Gln Lys Asp Asn Ser
85 90 95
Ser Leu Pro Trp Arg Asp Leu Ser Glu Cys Glu Glu Ser Lys Arg Gly
100 105 110
Glu Arg Ser Ser Pro Glu Glu Gln Leu Leu Phe Leu
115 120
3
