U.S. patent application number 12/080948 was filed with the patent office on 2008-11-27 for methods to identify compounds that modulate rage.
This patent application is currently assigned to TransTech Pharma, Inc.. Invention is credited to Manouchehr M. Shahbaz.
Application Number | 20080293163 12/080948 |
Document ID | / |
Family ID | 26902166 |
Filed Date | 2008-11-27 |
United States Patent
Application |
20080293163 |
Kind Code |
A1 |
Shahbaz; Manouchehr M. |
November 27, 2008 |
Methods to identify compounds that modulate rage
Abstract
Provided are methods to detect of modulators of the Receptor for
Advanced Glycated Endproducts (RAGE). The invention comprises a
method for detection of RAGE modulators comprising: adsorbing a
RAGE ligand onto a solid surface; adding a compound of interest and
a protein comprising RAGE or fragment thereof, to the preadsorbed
ligand; adding an antibody which binds to RAGE or fragment thereof
and a secondary antibody which binds to the anti-RAGE antibody;
measuring the secondary antibody bound to the anti-RAGE antibody;
and comparing the amount of RAGE bound to the ligand in the
presence of varying amounts of the compound of interest. In an
embodiment, the fragment of RAGE is sRAGE. In one aspect, the
invention use of compounds detected by the method for treatment of
AGE-related syndromes including complications associated with
diabetes, kidney failure, lupus nephritis or inflammatory lupus
nephritis, amyloidoses, Alzheimer's disease, cancer, inflammation,
and erectile dysfunction.
Inventors: |
Shahbaz; Manouchehr M.; (Oak
Ridge, NC) |
Correspondence
Address: |
KILPATRICK STOCKTON LLP - 41305;CHARLES CALKINS
1001 WEST FOURTH STREET
WINSTON-SALEM
NC
27101
US
|
Assignee: |
TransTech Pharma, Inc.
|
Family ID: |
26902166 |
Appl. No.: |
12/080948 |
Filed: |
April 7, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11005843 |
Dec 7, 2004 |
7374891 |
|
|
12080948 |
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09799152 |
Mar 5, 2001 |
6908741 |
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11005843 |
|
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60207342 |
May 30, 2000 |
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Current U.S.
Class: |
436/518 |
Current CPC
Class: |
A61P 25/28 20180101;
G01N 33/566 20130101; A61P 15/10 20180101; A61P 13/12 20180101;
A61P 3/10 20180101; A61P 29/00 20180101; A61P 43/00 20180101; G01N
2333/70503 20130101; A61P 35/00 20180101 |
Class at
Publication: |
436/518 |
International
Class: |
G01N 33/543 20060101
G01N033/543 |
Claims
1. A method for detection of RAGE ligands comprising: adsorbing a
RAGE ligand onto a solid surface; adding a compound of interest and
a protein comprising a RAGE fragment comprising amino acid sequence
SEQ ID NO: 4 to the preadsorbed ligand; adding an anti-RAGE
antibody which binds to said protein; determining the amount of
said protein bound to the ligand by measuring the amount of
anti-RAGE antibody bound; and comparing the amount of said protein
bound to the ligand in the presence of varying amounts of the
compound of interest.
2. The method of claim 1, wherein the anti-RAGE antibody comprises
a monoclonal antibody.
3. The method of claim 1, wherein the anti-RAGE antibody comprises
polyclonal antibody.
4. The method of claim 1, further comprising adding a second
antibody which recognizes the anti-RAGE antibody.
5. The method of claim 4, wherein the anti-RAGE antibody and
secondary antibody are allowed to complex prior to being added.
6. The method of claim 4, further comprising performing a
calorimetric assay for the secondary antibody.
7. The method of claim 1, wherein the solid surface comprises a
microtiter well.
8. The method of claim 1, wherein the solid surface comprises a
dip-stick.
9. The method of claim 1, wherein the ligand comprises an advanced
glycated endproduct, or fragment thereof.
10. The method of claim 1, wherein the ligand comprises
carboxymethyl-lycine-modified AGE.
11. The method of claim 1, wherein the ligand comprises
.beta.-amyloid.
12. The method of claim 1, wherein the ligand comprises
calgranulin.
13. The method of claim 1, wherein the ligand comprises S100b.
14. The method of claim 1, wherein the ligand comprises
amphoterin.
15. The method of claim 1, wherein the compound of interest
comprises a synthetic peptide.
16. The method of claim 1, wherein the compound of interest
comprises a peptidomimetic.
17. The method of claim 1, wherein the compound of interest
comprises an organic compound.
18. The method of claim 1, wherein the compound of interest
comprises an inorganic compound.
19. The method of claim 1, wherein the compound of interest
comprises a lipid.
20. The method of claim 1, wherein the compound of interest
comprises a carbohydrate.
21. The method of claim 1, wherein the compound of interest
comprises a nucleic acid.
22. A method for detection of RAGE ligands comprising: adsorbing a
RAGE ligand onto a solid surface; adding a compound of interest and
a protein comprising a RAGE fragment comprising the amino acid
sequence SEQ ID NO: 4 to the preadsorbed ligand; adding anti-RAGE
antibody which binds to said protein and a secondary antibody which
binds to the anti-RAGE antibody; measuring the secondary antibody
bound to the anti-RAGE antibody; and comparing the amount of said
protein bound to the ligand in the presence of varying amounts of
the compound of interest.
23. The method of claim 22, further comprising performing a
colorimetric assay for the secondary antibody.
24. The method of claim 22, wherein the anti-RAGE antibody and
secondary antibody are allowed to complex prior to being added to
the reaction.
25. The method of claim 22, wherein the solid surface comprises a
reaction vessel.
26. The method of claim 22, wherein the solid surface comprises a
dip-stick.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of application Ser. No.
11/005,843, filed Dec. 7, 2004, which is a divisional of
application Ser. No. 09/799,152, filed Mar. 5, 2001, now U.S. Pat.
No. 6,908,741, which claims the benefit of provisional Application
No. 60/207,342, filed May 30, 2000, the entire contents of all are
hereby incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to regulation of the Receptor
for Advanced Glycated Endproducts (RAGE). More particularly, the
present invention describes methods for the rapid, high-throughput
identification of modulators of RAGE.
BACKGROUND OF THE INVENTION
[0003] Incubation of proteins or lipids with aldose sugars results
in nonenzymatic glycation and oxidation of amino groups on proteins
to form Amadori adducts. Over time, the adducts undergo additional
rearrangements, dehydrations, and cross-linking with other proteins
to form complexes known as Advanced Glycosylation End Products
(AGEs). Factors which promote formation of AGEs included delayed
protein turnover (e.g. as in amyloidoses), accumulation of
macromolecules having high lysine content, and high blood glucose
levels (e.g. as in diabetes) (Hori et al., J. Biol. Chem. 270:
25752-761, (1995)). AGEs have been implicated in a variety of
disorders including complications associated with diabetes and
normal aging.
[0004] AGEs display specific and saturable binding to cell surface
receptors on monocytes, macrophages, endothelial cells of the
microvasculature, smooth muscle cells, mesengial cells, and
neurons. The Receptor for Advanced Glycated Endproducts (RAGE) is a
member of the immunoglobulin super family of cell surface
molecules. The extracellular (N-terminal) domain of RAGE includes
three immunoglobulin-type regions: one V (variable) type domain
followed by two C-type (constant) domains (Neeper et al., J. Biol.
Chem., 267:14998-15004 (1992); Schmidt et al., Circ. (Suppl.)
96#194 (1997)). A single transmembrane spanning domain and a short,
highly charged cytosolic tail follow the extracellular domain. The
N-terminal, extracellular domain can be isolated by proteolysis of
RAGE to generate soluble RAGE (sRAGE) comprised of the V and C
domains.
[0005] RAGE is expressed in most tissues, and in particular, is
found in cortical neurons during embryogenesis (Hori et al., J.
Biol. Chem., 270:25752-761 (1995)). Increased levels of RAGE are
also found in aging tissues (Schleicher et al., J. Clin. Invest.,
99 (3): 457-468 (1997)), and the diabetic retina, vasculature and
kidney (Schmidt et al., Nature Med., 1:1002-1004 (1995)).
Activation of RAGE in different tissues and organs leads to a
number of pathophysiological consequences. RAGE has been implicated
in a variety of conditions including: acute and chronic
inflammation (Hofmann et al., Cell 97:889-901 (1999)) the
development of diabetic late complications such as increased
vascular permeability (Wautier et al., J. Clin. Invest., 97:238-243
(1996), nephropathy (Teillet et al., J. Am. Soc. Nephrol.,
11:1488-1497 (2000), atherosclerosis (Vlassara et. al., The Finnish
Medical Society DUODECIM, Ann. Med., 28:419-426 (1996), and
retinopathy (Hammes et al., Diabetologia, 42:603-607 (1999)). RAGE
has also been implicated in Alzheimer's disease (Yan et al.,
Nature, 382:685-691 (1996)); erectile dysfunction; and in tumor
invasion and metastasis (Taguchi et al., Nature, 405:354-357
(2000)).
[0006] In addition to AGEs, other compounds can bind to, and
modulate RAGE. In normal development, RAGE interacts with
amphoterin, a polypeptide which mediates neurite outgrowth in
cultured embryonic neurons (Hori et al., 1995). RAGE has also been
shown to interact with calgranulin-like ligands and .beta.-amyloid
(Yan et al., Nature, 389: 689-695, (1997); Yan et al., Nature,
382:685-691 (1996); Yan et al., Proc. Natl. Acad. Sci.,
94:5296-5301 (1997)).
[0007] Binding of ligands such as AGEs, S100/calgranulin,
.beta.-amyloid, CML (N.sup..epsilon.-Carboxymethyl lysine), and
amphoterin to RAGE has been shown to modify expression of a variety
of genes. For example, in many cell types interaction between RAGE
and its ligands generates oxidative stress, which thereby results
in activation of the free radical sensitive transcription factor
NF-.kappa.B, and the activation of NF-.kappa.B regulated genes,
such as the cytokines IL-1.beta., TNF-.alpha., and the like. In
addition, several other regulatory pathways, such as those
involving p21ras, MAP kinases, ERK1 and ERK2, have been shown to be
activated by binding of AGEs and other ligands to RAGE. In fact,
transcription of RAGE itself is regulated at least in part by
NF-.kappa.B. Thus, an ascending, and often detrimental, spiral is
fueled by a positive feedback loop initiated by ligand binding.
Antagonizing binding of physiological ligands to RAGE, therefore,
is a logical target for down-regulation of the pathophysiological
changes brought about by excessive concentrations of AGEs and other
ligands for RAGE.
[0008] Thus, there is a need for the methods to discover compounds
which modulate binding of physiological ligands to the RAGE
receptor. The method should comprise an assay which is sensitive
and yet highly specific. Preferably, the method should utilize
RAGE, or the binding site of RAGE itself, thereby allowing the
development of modulators which vary in structure and physiological
effect. Ideally, the method should lend itself to high throughput,
automative techniques suitable for large scale screening of
multiple compounds.
SUMMARY
[0009] The present invention relates to the use of a high
throughput assay for the discovery of compound that modulate RAGE.
In one aspect, the present invention comprises a method for
detection of RAGE modulators comprising: (a) adsorbing a RAGE
ligand onto a solid surface; (b) adding a compound of interest and
a protein comprising RAGE comprising SEQ ID NO: 1 or fragment
thereof to the preadsorbed ligand; (c) adding an anti-RAGE antibody
which binds to the RAGE protein or fragment thereof; (d)
determining the amount of RAGE protein or fragment thereof bound to
the ligand by measuring the amount of anti-RAGE antibody bound to
the solid surface; and (e) comparing the amount of RAGE protein or
fragment thereof bound to the ligand in the presence of varying
amounts of the compound of interest.
[0010] In another aspect, the present invention comprises a method
for detection of RAGE modulators comprising: (a) adsorbing a RAGE
ligand onto a solid surface; (b) adding a compound of interest and
a protein comprising RAGE comprising SEQ ID NO: 1 or fragment
thereof to the preadsorbed ligand; (c) adding an anti-RAGE antibody
which binds to the RAGE protein or fragment thereof and a secondary
antibody which binds to the anti-RAGE antibody; (d) measuring the
secondary antibody bound to the anti-RAGE antibody; and (e)
comparing the amount of RAGE bound to the ligand in the presence of
varying amounts of the compound of interest.
[0011] In another aspect, the invention comprises compounds
identified by the methods of the invention.
[0012] In yet another aspect, the invention comprises a kit for
detection of RAGE modulators comprising: (a) a solid surface
comprising a RAGE ligand; (b) a separately packaged solution
comprising a protein comprising RAGE comprising SEQ ID NO: 1 or
fragment thereof; (c) a detection system comprising at least one
agent which specifically binds to the RAGE protein or fragment
thereof; (d) separately packaged reagents for the detection system;
and (e) instructions for use.
[0013] The foregoing focuses on the more important features of the
invention in order that the detailed description which follows may
be better understood and in order that the present contribution to
the art may be better appreciated. There are, of course, additional
features of the invention which will be described hereinafter and
which will form the subject matter of the claims appended hereto.
It is to be understood that the invention is not limited in its
application to the details set forth in the following description
and drawings. The invention is capable of other embodiments and of
being practiced or carried out in various ways.
[0014] From the foregoing summary, it is apparent that an object of
the present invention is to provide a rapid, high-throughput method
for the detection of RAGE modulators. These, together with other
objects of the present invention, along with the various features
of novelty which characterize the invention, are pointed out with
particularity in the claims annexed to and forming a part of this
document.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Various features, aspects and advantages of the present
invention will become more apparent with reference to the following
description, appended claims, and accompanying drawings.
[0016] FIG. 1 shows a schematic representation of an embodiment of
the method of the present invention.
[0017] FIG. 2 shows an aspect of an embodiment of the present
invention comprising: (A) SEQ ID NO: 1, the amino acid sequence for
human RAGE as reported in GenBank/EMBL database, accession number
XM004205; (B) SEQ ID NO: 2, the amino acid sequence of human sRAGE;
(C) SEQ ID NO: 3, the amino acid sequence of the V-domain of human
RAGE; and (D) SEQ ID NO: 4, the N-terminal fragment of the V-domain
of human RAGE.
[0018] FIG. 3 shows an aspect of an embodiment of the present
invention comprising optimization of S-100b concentration where
sRAGE was present at 1.0.times.10.sup.-4 mg/ml, polyclonal antibody
to sRAGE was present at 3.0.times.10.sup.-3 mg/ml, and overnight
incubations were done using S-100b at concentrations which ranged
from about 2 ng/well to 4 mg/well.
[0019] FIG. 4 shows an aspect of an embodiment of the present
invention comprising optimization of sRAGE concentration where 500
ng/well S-100b was used to coat wells, and polyclonal antibody to
sRAGE was present at 3.0.times.10.sup.-3 mg/ml, and sRAGE
concentration ranged from about 2 to 10 nM.
[0020] FIG. 5 shows an aspect of an embodiment of the present
invention comprising optimization of antibody concentration where
sRAGE was present at 1.0.times.10.sup.-4 mg/ml, incubations were
done using S-100b at 50, 500, 1,000 and 2,000 ng/well as indicated,
and polyclonal antibody concentration ranged from 5.times.10.sup.-6
to 0.054 mg/ml.
[0021] FIG. 6 shows an aspect of an embodiment of the present
invention comprising optimization of DMSO tolerance where 500
ng/well S-100b was used to coat wells, and polyclonal or monoclonal
antibody to sRAGE was present at 3.0.times.10.sup.-3 mg/ml and
1.9.times.10.sup.-4 mg/ml respectively, and the concentration of
sRAGE was 2.2.times.10.sup.-4 mg/ml.
[0022] FIG. 7 shows an aspect of an embodiment of the present
invention comprising the variation in sample OD for 88 samples as a
function of individual well position, where the average OD
comprised 0.705, the standard deviation (SD) is 0.054, and the 95%
confidence value (CV) is 7.73%.
[0023] FIG. 8 shows an aspect of an embodiment of the present
invention comprising a saturation curve for sRAGE binding to
S-100b, where 50 ng/well S-100b was used to coat the wells,
polyclonal antibody to sRAGE was present at 3.0.times.10.sup.-3
mg/ml, and sRAGE concentration ranged from about 0.5 to 9 nM.
[0024] FIG. 9 shows an aspect of an embodiment of the present
invention comprising a saturation curve for sRAGE binding to
S-100b, where 100 ng/well S-100b was used to coat the wells,
polyclonal antibody to sRAGE was present at 3.0.times.10.sup.-3
mg/ml, and sRAGE concentration ranged from about 0.3 to 10 nM.
[0025] FIG. 10 shows an aspect of an embodiment of the present
invention comprising a saturation curve for sRAGE binding to
S-100b, where 500 ng/well S-100b was used to coat the wells, and
polyclonal antibody to sRAGE was present at 3.0.times.10.sup.-3
mg/ml, and sRAGE concentration ranged from about 24 to 470 nM.
[0026] FIG. 11 shows an aspect of an embodiment of the present
invention comprising a saturation curve for sRAGE binding to
S-100b, where 100 ng/well S-100b was used to coat the wells, and
polyclonal antibody to sRAGE was present at 3.0.times.10.sup.-3
mg/ml, and sRAGE concentration ranged from about 5 to 76 nM.
[0027] FIG. 12 shows an aspect of an embodiment of the present
invention comprising inhibition of sRAGE binding by Compound 1.
[0028] FIG. 13 shows an aspect of an embodiment of the present
invention comprising the structure of Compound 1.
[0029] FIG. 14 shows an aspect of an embodiment of the present
invention comprising an inhibition of sRAGE activation of the
transcription factor NF-.kappa.B by Compound 1.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The present invention relates to the use of a high
throughput assay for the discovery of compounds that modulate RAGE.
The method measures binding of ligands to RAGE, or fragments of
RAGE, under conditions in which the physiological and structural
integrity of the receptor is maintained. Those compounds which bind
are assessed for physiological activity.
[0031] In one aspect, the present invention comprises a method for
detection of RAGE modulators comprising: (a) adsorbing a RAGE
ligand onto a solid surface; (b) adding a compound of interest and
a protein comprising RAGE comprising SEQ ID NO: 1 or fragment
thereof to the preadsorbed ligand; (c) adding anti-RAGE antibody
which binds to the RAGE protein or fragment thereof; and (d)
determining the amount of RAGE protein or fragment thereof bound to
the ligand by measuring the amount of anti-RAGE antibody bound to
the solid surface; and (e) comparing the amount of RAGE protein or
fragment thereof bound to the ligand in the presence of varying
amounts of the compound of interest.
[0032] In an embodiment, the fragment of RAGE is the soluble,
extracellular portion of RAGE (sRAGE), as defined by the amino acid
sequence SEQ ID NO: 2, or a sequence substantially homologous
thereto. In another embodiment, the fragment of RAGE is the
V-domain of RAGE, as defined by the amino acid sequence SEQ ID NO:
3, or a sequence substantially homologous thereto. In yet another
embodiment, the fragment of RAGE is a fragment of the V-domain of
RAGE, as defined by the amino acid sequence SEQ ID NO: 4, or a
sequence substantially homologous thereto. Preferably, the method
comprises measuring the ability of the compound of interest to
modulate the ability of RAGE, or protein substantially homologous
thereto, to activate cellular processes. More preferably, agonists
comprise stimulation of RAGE-activated cellular processes and
antagonists comprise inhibition of RAGE-activated cellular
processes. Even more preferably, the cellular process comprises
activation of NF-.kappa.B gene transcription.
[0033] In an embodiment, the antibody to RAGE comprises a
monoclonal antibody. In an embodiment, the antibody to RAGE
comprises polyclonal antibody.
[0034] Preferably, the method further comprises addition of a
second antibody which recognizes the anti-RAGE antibody. More
preferably, the anti-RAGE antibody and the secondary antibody are
allowed to complex prior to being added. Also preferably, the
method further comprises a colorimetric assay for the secondary
antibody.
[0035] In an embodiment, the solid surface comprises a reaction
vessel. In another embodiment, the solid surface comprises a
dip-stick.
[0036] Preferably, the ligand comprises an advanced glycated
endproduct, or fragment thereof. Also preferably, the ligand
comprises carboxymethyl-lycine-modified AGE. Also preferably, the
ligand comprises .beta.-amyloid. Also preferably, the ligand
comprises calgranulin. Also preferably, the ligand comprises
S-100b. Also preferably, the ligand comprises amphoterin.
[0037] Preferably, the compound of interest comprises a peptide.
Also preferably, the compound of interest comprises a
peptidomimetic. Also preferably, the compound of interest comprises
an organic compound. Also preferably, the compound of interest
comprises an inorganic compound. Also preferably, the compound of
interest comprises a lipid. Also preferably, the compound of
interest comprises a carbohydrate. Also preferably, the compound of
interest comprises nucleic acid.
[0038] The invention relates to the use of a physiologically
relevant binding assay to discover compounds identified which
modulate RAGE. In one aspect, the invention comprises compounds
identified by a method having steps comprising: (a) adsorbing a
RAGE ligand onto a solid surface; (b) adding a compound of interest
and a protein comprising RAGE comprising SEQ ID NO: 1 or fragment
thereof to the preadsorbed ligand; (c) adding anti-RAGE antibody
which binds to RAGE protein or a fragment thereof; (d) determining
the amount of RAGE protein or a fragment thereof bound to the
ligand by measuring the amount of anti-RAGE antibody bound to the
solid surface; and (e) comparing the amount of RAGE protein or a
fragment thereof bound to the ligand in the presence of varying
amounts of the compound of interest.
[0039] In an embodiment, the fragment of RAGE is the soluble,
extracellular portion of RAGE (sRAGE)), as defined by the amino
acid sequence SEQ ID NO: 2, or a sequence substantially homologous
thereto. In another embodiment, the fragment of RAGE is the
V-domain of RAGE, as defined by the amino acid sequence SEQ ID NO:
3, or a sequence substantially homologous thereto. In yet another
embodiment, the fragment of RAGE is a fragment of the V-domain of
RAGE, as defined by the amino acid sequence SEQ ID NO: 4, or a
sequence substantially homologous thereto.
[0040] Preferably, the method of identifying the compounds further
comprises measuring the ability of the compound of interest to
modulate the ability of RAGE comprising SEQ ID NO: 1 or a protein
substantially homologous thereto, to activate cellular processes.
Preferably, the compound identified by the methods of the invention
is used to treat symptoms of diabetes and symptoms of diabetic late
complications. Also preferably, the compound identified by the
methods of the invention is used to treat amyloidoses. Also
preferably, the compound identified by the methods of the invention
is used to treat Alzheimer's disease. Also preferably, the compound
identified by the methods of the invention is used to treat cancer.
Also preferably, the compound identified by the methods of the
invention is used to treat inflammation. Also preferably, the
compound identified by the methods of the invention is used to
treat kidney failure. Also preferably, the compound identified by
the methods of the invention is used to treat systemic lupus
nephritis or inflammatory lupus nephritis. Also preferably, the
compound identified by the methods of the invention is used to
treat erectile dysfunction.
[0041] The methods of the invention provide a flexible protocol
which is adapted to a variety of reagents without reduction in
reliability or precision. Thus, a variety of developing reagents or
antibody preparations may be used. In one aspect, the present
invention comprises a method for detection of RAGE modulators
comprising: (a) adsorbing a RAGE ligand onto a solid surface; (b)
adding a compound of interest and a protein comprising RAGE
comprising SEQ ID NO: 1 or fragment thereof to the preadsorbed
ligand; (c) adding anti-RAGE antibody which binds to the RAGE
protein or a fragment thereof and a secondary antibody which binds
to the anti-RAGE antibody; (d) measuring the secondary antibody
bound to the anti-RAGE antibody; (e) comparing the amount of RAGE
or fragment thereof bound to the ligand in the presence of varying
amounts of the compound of interest.
[0042] In an embodiment, the fragment of RAGE is the soluble,
extracellular portion of RAGE (sRAGE)), as defined by the amino
acid sequence SEQ ID NO: 2, or a sequence substantially homologous
thereto. In another embodiment, the fragment of RAGE is the
V-domain of RAGE, as defined by the amino acid sequence SEQ ID NO:
3, or a sequence substantially homologous thereto. In yet another
embodiment, the fragment of RAGE is a fragment of the V-domain of
RAGE, as defined by the amino acid sequence SEQ ID NO: 4, or a
sequence substantially homologous thereto.
[0043] Preferably, the method comprises measuring the ability of
the compound of interest to modulate the ability of RAGE, or
protein substantially homologous thereto, to activate cellular
processes. More preferably, agonists comprise stimulation of
RAGE-activated cellular processes and antagonists comprise
inhibition of RAGE-activated cellular processes. Even more
preferably, the cellular process comprise activation of NF-.kappa.B
gene transcription.
[0044] Preferably, the method further comprises a calorimetric
assay for the secondary antibody. In an embodiment, the anti-RAGE
antibody and secondary antibody are allowed to complex prior to
being added to the reaction vessel comprising RAGE, thus
maintaining the RAGE binding domain in an non-antibody bound (i.e.
unperturbed) state. In an embodiment, the solid surface comprises a
reaction vessel. In an embodiment, the solid surface comprises a
dip-stick.
[0045] In yet another aspect, the invention comprises a kit for
detection of RAGE modulators comprising: (a) a solid surface
comprising a RAGE ligand; (b) a separate packaged solution
comprising a protein comprising RAGE comprising SEQ ID NO: 1 or
fragment thereof; (c) a detection system comprising at least one
agent which specifically binds to the RAGE, or fragment thereof;
(c) separately packaged calorimetric reagents for the detection
system; and (d) instructions for use. In an embodiment, the kit
comprises a separately packaged antibody which binds to RAGE, or
fragment thereof. In an embodiment, the fragment of RAGE is the
soluble, extracellular portion of RAGE (sRAGE)), as defined by the
amino acid sequence SEQ ID NO: 2, or a sequence substantially
homologous thereto. In an embodiment, the fragment of RAGE is the
V-domain of RAGE, as defined by the amino acid sequence SEQ ID NO:
3, or a sequence substantially homologous thereto. In yet another
embodiment, the fragment of RAGE is a fragment of the V-domain of
RAGE, as defined by the amino acid sequence SEQ ID NO: 4, or a
sequence substantially homologous thereto.
[0046] The present invention comprises a method for the detection
of RAGE modulators which utilizes ligand and RAGE, or a fragments
of RAGE, which have not been chemically modified. Thus, the method
of the present invention is able to measure binding of ligands and
the modification of such binding, with physiological specificity.
The method is more reliable than assay systems which use
radiolabelled binding partners, and is highly sensitive due to the
use of an enzyme coupled development system. In addition, the assay
employs non-toxic reagents which do not present a threat to
laboratory personnel or the environment.
[0047] Thus, in one aspect, the invention comprises the use of
ELISA type assay as a method for the detection of RAGE modulators.
In an embodiment, and referring now to FIG. 1, a RAGE ligand, such
as S-100b, is allowed to adsorb to the wells of a microtiter plate.
The wells are then washed using buffered saline to remove the
unbound ligand, and sites which comprise non-specific protein
binding sites are blocked using blocking buffer containing bovine
serum albumin (BSA) or another protein unrelated to RAGE binding.
The blocking buffer is then removed, as for example by aspiration,
and the wells washed several times to remove traces of blocking
buffer. A solution comprising the compound of interest (i.e. a
putative RAGE modulator) and RAGE comprising SEQ ID NO: 1, or
fragment thereof, is added to each well. For example, in an
embodiment, the RAGE fragment used is sRAGE (Taguchi et al.,
Nature, 405:354-360 (1996)). The mixture is incubated under
conditions which allow physiological binding of the RAGE protein
(e.g. sRAGE) to the immobilized ligand in each well.
[0048] Preferably, during the binding reaction of sRAGE to the
immobilized ligand, a complex comprising: (a) anti-RAGE antibody
(anti-RAGE); (b) biotinylated goat anti-mouse IgG (B-anti-IgG); and
(c) streptavidin labeled alkaline phosphatase (S-AP), is allowed to
form in a separate reaction vessel. After a suitable time for
binding of sRAGE to the immobilized ligand, the reaction wells are
washed to remove traces of unbound sRAGE and the compound of
interest, and the anti-RAGE: B-anti-IgG: S-AP complex added to each
well. The addition of the complex allows for immobilization of the
alkaline phosphatase enzyme via interaction of the complex with
sRAGE immobilized in the well (FIG. 1). By adding the immune
complex separately, the sRAGE is allowed to bind to the ligand and
compound of interest (i.e. putative modulator) without interference
due to antibody binding. The amount of alkaline phosphatase, and
thus the amount of immobilized sRAGE, can then be detected using a
colorimetric assay for conversion of para-nitrophenyl phosphate
(pNPP) to para-nitrophenol (pNP).
[0049] As used herein, RAGE encompasses a peptide which has the
full amino acid sequence of RAGE as shown in FIG. 2A (SEQ ID NO: 1)
or a portion of that amino acid sequence (Neeper et al., (1992)).
The binding domain of RAGE comprises that region of the protein
which is able to bind ligands with physiological specificity. A
fragment of RAGE is at least 5 amino acids in length, but more
preferably greater than 30 amino acids in length, but is
substantially less than the full amino acid sequence. Thus, in
another embodiment, the fragment of RAGE comprises sRAGE (SEQ ID
NO: 2; FIG. 2B), wherein sRAGE is the RAGE protein free from the
cell membrane (Park et al., Nature Med., 4:1025-1031 (1998)). In
another embodiment, the RAGE or fragment thereof comprises the V
domain (SEQ ID NO: 3; FIG. 2C) (Neeper et al., (1992); Schmidt et
al., (1997)), or a fragment thereof (SEQ ID NO: 4, FIG. 2D). In yet
another embodiment, the RAGE or fragment thereof is a synthetic
peptide.
[0050] The terms "substantially homologous" when referring to
polypeptides refer to at least two amino acid sequences which when
optimally aligned, are at least 75% homologous, preferably at least
about 85% homologous, more preferably at least about 90%
homologous, and still more preferably 95% homologous. Optimal
alignment of sequences for aligning a comparison may be conducted
using the algorithms standard in the art (e.g. Smith and Waterman,
Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Biol.
48:443 (1970); Pearson and Lipman, Proc. Natl. Acad. Sci. (USA),
85:2444 (1988)) or by computerized versions of these algorithms
(Wisconsin Genetics Software Package Release 7.0, Genetics Computer
Group, 575 Science Drive, Madison, Wis.).
[0051] Preferably, the concentration of ligand used for coating the
solid surface (i.e. reaction wells) allows any antagonism of
binding of the immobilized ligand to sRAGE to be detected. For
example, referring now to FIG. 3, where sRAGE comprises 0.1
.mu.g/ml, and anti-sRAGE antibody comprises 3.0 .mu.g/ml, almost
maximum signal is seen with 500 ng S-100b, and ligand
concentrations between 10 to 500 ng/well comprise a linear increase
in sRAGE binding.
[0052] Also preferably, and referring now to FIG. 4, the assay is
designed to allow selection of an amount of sRAGE which comprises a
linear region of binding of sRAGE and the immobilized ligand. For
example, if too low of a concentration of sRAGE is employed, the
detection of binding will be difficult due to low signal. If excess
sRAGE is used, however, the ability of compound of interest to
compete with the immobilized ligand will be lessened due to the
excess sRAGE binding to both the ligand and the compound being
tested.
[0053] Also preferably, the assay is designed to allow optimization
of the anti-RAGE antibody. Thus, in an embodiment, and referring
now to FIG. 5, the amount of antibody used can be titered to allow
for maximal signal, but not so high as to comprise high levels of
background binding. More preferably, the amount of antibody
allowing for maximum signal comprises a similar range of antibody
regardless of the concentration of immobilized ligand.
[0054] Also preferably, the assay is optimized to be independent of
solvents required for dissolving the compounds being tested. In an
embodiment, and referring to FIG. 6, for dimethyl sulfoxide (DMSO)
concentrations of 2%, which is the concentration of DMSO generally
employed for dissolution of organic compounds of interest in the
assay, the assay provides maximal signal. Even at concentrations as
high as 10% DMSO, the assay provides almost maximal signal.
[0055] Thus, the assay allows for optimization of each component,
such that variation of assay results due to environmental factors
and experimental variation is minimized. Preferably, once optimized
for a specific ligand, sRAGE concentration, and anti-sRAGE antibody
concentration, the assay will provide a reliable assessment of
sample binding affinity regardless of the specific competing
compound (i.e. putative modulator) which is being tested. Also
preferably, the assay comprises a high throughput assay that is
reproducible and precise. Referring now to FIG. 7, in an embodiment
the assay comprises a variance of less than 10%.
[0056] Without being bound to any particular theory, a premise of
the assay is that binding of sRAGE to the ligand immobilized in the
assay well will reflect the nature of the binding of RAGE to the
ligand in vivo. Thus, in an embodiment, the immobilized ligand will
bind RAGE comprising SEQ ID NO: 1, or fragment thereof, in a
specific and saturable manner. For example, and referring now to
FIGS. 8-11, sRAGE at concentrations ranging from about 0.3 to 480
nM binds in a saturable manner to S-100b ligand immobilized in a
microtiter well. In addition, an analysis of the data by means of
Scatchard transformation shows both high and low affinity sites
with dissociation constants (Kd) similar to the Kd for RAGE
reported previously (Hofmann et al., Cell, 97:889-901 (1999); Hori
et al., J. Biol. Chem., 270:25752-25761 (1995)).
[0057] Preferably, the assay of the present invention is able to
detect compounds that antagonize both low and high affinity binding
sites. Referring now to FIGS. 8 and 9, using sRAGE at
concentrations ranging from 0.3 to 10 nM, a high affinity binding
site comprising a Kd of about 2-6 nM is detected. Also, referring
now to FIGS. 10 and 11, using sRAGE at concentrations ranging from
24 to 480 nM, and 5 to 80 nM respectively, a lower affinity binding
site comprising a Kd of about 60 nM is detected.
[0058] In an embodiment, the anti-RAGE antibody is polyclonal.
Polyclonal antibodies are a heterogeneous population of antibody
molecules derived from the sera of animals immunized with the
antigen of interest. Adjuvants such as Freund's (complete and
incomplete), peptides, oil emulsions, lysolecithin, polyols,
polyanions and the like may be used to increase the immune
response. Thus, in an embodiment, polyclonal antibody to sRAGE is
prepared by injection of sRAGE supplemented with an adjuvant into
rabbits using methods known in the art (e.g. Schmidt et al., J.
Biol. Chem., 267:14987-14997 (1992)).
[0059] Monoclonal antibodies are homogeneous populations of
antibodies to a particular antigen, and are generally obtained by
any technique which provides for production of antibody by
continuous cell lines in culture (see e.g. U.S. Pat. No.
4,873,313). For example, in an embodiment sRAGE protein is used for
production of monoclonal antibodies using methods known in the art
(Zymed Laboratories, San Francisco, Calif.).
[0060] Again without being bound to any particular theory, a
premise of the assay is that one of the components in the detection
system labeled. It is contemplated that the detection system may
use reagents which are can be seen visually, and which are thereby
defined as "colorimetric. For example, in an embodiment, a complex
of anti-RAGE antibody: biotinylated goat anti-human IgG:
streptavidin labeled alkaline phosphatase is used to detect sRAGE
bound to the ligand. The alkaline phosphatase enzyme catalyses the
conversion of light yellow para-nitrophenyl phosphate (pNPP) to the
pink product para-nitrophenol. It is, however, within the scope of
the present invention to use variations of the detection system
which are known to those in the art. For example, in a embodiment,
the secondary antibody which binds to anti-RAGE antibody is labeled
with streptavidin and then detected with biotinylated alkaline
phosphatase. Other labeling moieties include fluorescein,
digoxigenin, 2,2'-Azino-di-[3-ethylbenzthiazoline sulfonate (6)]
diammonium salt, and the like. In another embodiment,
streptavidin-labeled horseradish peroxidase is employed as the
detection system. Alternatively, in another embodiment, a third
antibody comprising biotinylated anti-goat IgG, can be used to
detect a non-biotinylated anti-mouse IgG. In yet another
embodiment, the anti-RAGE antibody itself may be biotinylated, and
detected using streptavidin-labeled alkaline phosphatase.
[0061] The assay may be carried out with the ligand immobilized to
the surface of a reaction vessel or other solid surface. For
example, in an embodiment, the ligand is immobilized in a
microtiter well. In another embodiment, the ligand is immobilized
on a dip-stick. In this format, the assay may be performed by
transferring the dipstick comprising an immobilized ligand first to
solution comprising sRAGE and a test compound (or biological
sample), and then to a reagent for detecting sRAGE binding to the
dipstick (e.g. an anti-sRAGE antibody:biotinylated IgG:
streptavidin-alkaline phosphatase complex). A reduction in signal
on the dipstick as compared to a known control would indicate the
sample comprises a compound which can bind to RAGE. In an
embodiment, the assay can detect increased RAGE ligands in a
biological sample. Preferably, such ligands include AGEs,
amphoterin, (Hori et al., 1995), and .beta.-amyloid (Yan et al.,
Nature 389: 689-695, (1997); Yan et al., Nature 382:685-691 (1996);
Yan et al., Proc. Natl. Acad. Sci., 94:5296-5301 (1997)). In
another embodiment, the ligand is immobilized on beads, a nylon
membrane, or any other solid support.
[0062] The methods of the invention provide binding assays to
identify compounds that interact with RAGE under physiological
binding conditions. In this respect, physiological binding
conditions comprise those conditions which result in binding
affinities similar to those seen in vivo. Thus, in another aspect,
the present invention comprises the compounds identified by the
methods of the invention. Compounds identified by the methods of
the invention may comprise several different chemical types. For
example, in an embodiment the compound is a peptide. In another
embodiment, the compound is a peptidomimetic. In another
embodiment, the compound is an organic molecule. In another
embodiment, the compound is an inorganic molecule. In some cases,
the compound of interest may be derivatized to increase
half-life.
[0063] The compounds of the present invention may be a
peptidomimetic which is at least partly unnatural. Preferably the
compound of interest may be modified to increase stability,
efficacy, potency and bioavailability. The compound may be
synthetically prepared. The compound may include L-, D-, or
unnatural amino acids, alpha-disubstituted amino acids, N-alkyl
amino acids, or lactic acid. In an embodiment, the compound
comprises a peptidomimetic having a peptide backbone or amino acid
replaced with a suitable mimetic.
[0064] For example, as shown in FIG. 12, Compound 1, the structure
of which is shown in FIG. 13, inhibits sRAGE binding to S-100b with
IC.sub.50=1.8.+-.0.2 .mu.M, wherein IC.sub.50 is defined as the
concentration of the agent (i.e. Compound 1) which comprises 50%
inhibition of sRAGE binding.
[0065] In an embodiment, compounds identified by the binding assay
are further tested as having the ability to modulate a biological
activity of RAGE comprising SEQ ID NO: 1, or fragment thereof. For
example, compounds can be tested for their ability to modulate RAGE
induced increases in gene expression. Thus, in an embodiment and
referring now to FIG. 14, compounds identified by the binding assay
are used to modulate RAGE activation of NF-.kappa.B mediated
transcription of a reporter gene. Preferably, compounds which
modulate ligand binding to RAGE comprising SEQ ID NO: 1, or
fragment thereof, will be identified as modulating the effects of
RAGE on other cellular processes.
[0066] In an embodiment, the invention comprises treatment of human
disease by compounds identified by the methods of the invention.
Preferably, compounds identified by the methods of the invention
are used to treat diabetes. It has been shown that nonenzymatic
glycoxidation of macromolecules ultimately resulting in the
formation of advanced glycation endproducts (AGEs) is enhanced at
sites of inflammation, in renal failure, in the presence of
hyperglycemia and other conditions associated with systemic or
local oxidant stress (Dyer et al., J. Clin. Invest., 91:2463-2469
(1993); Reddy et al., Biochem., 34:10872-10878 (1995); Dyer et al.,
J. Biol. Chem., 266:11654-11660 (1991); Degenhardt et al., Cell
Mol. Biol., 44:1139-1145 (1998)). Accumulation of AGEs in the
vasculature can occur focally, as in the joint amyloid composed of
AGE-.beta..sub.2-microglobulin found in patients with
dialysis-related amyloidosis (Miyata et al, J. Clin. Invest.,
92:1243-1252 (1993); Miyata et al., J. Clin. Invest., 98:1088-1094
(1996)), or generally, as exemplified by the vasculature and
tissues of patients with diabetes (Schmidt et al., Nature Med., 1:
1002-1004 (1995)). The progressive accumulation of AGEs over time
in patients with diabetes suggests that endogenous clearance
mechanisms are not able to function effectively at sites of AGE
deposition. Such accumulated AGEs have the capacity to alter
cellular properties by a number of mechanisms. Although RAGE is
expressed at low levels in normal tissues and vasculature, in an
environment where the receptor's ligands accumulate, it has been
shown that RAGE becomes upregulated (Li et al., J. Biol. Chem.,
272:16498-16506 (1997); Li et al., J. Biol. Chem., 273:30870-30878
(1998); Tanaka et al., J. Biol. Chem., 275:25781-25790 (2000)).
RAGE expression is increased in endothelium, smooth muscle cells
and infiltrating mononuclear phagocytes in diabetic vasculature.
Also, studies in cell culture have demonstrated that AGE-RAGE
interaction caused changes in cellular properties important in
vascular homeostasis.
[0067] Also preferably, compounds identified by the methods of the
invention are used to treat atherosclerosis. Thus, it has been
shown that ischemic heart disease is particularly high in patients
with diabetes (Robertson, et al., Lab Invest., 18:538-551 (1968);
Kannel et al, J. Am. Med. Assoc., 241:2035-2038 (1979); Kannel et
al., Diab. Care, 2:120-126 (1979)). In addition, studies have shown
that atherosclerosis in patients with diabetes is more accelerated
and extensive than in patients not suffering from diabetes (see
e.g. Waller et al., Am. J. Med., 69:498-506 (1980); Crall et al,
Am. J. Med. 64:221-230 (1978); Hamby et al., Chest, 2:251-257
(1976); and Pyorala et al., Diab. Metab. Rev., 3:463-524 (1987)).
Although the reasons for accelerated atherosclerosis in the setting
of diabetes are many, it has been shown that reduction of AGEs can
reduce plaque formation.
[0068] Also preferably, compounds identified by the methods of the
invention are used to treat amyloidoses and Alzheimer's disease. It
has been shown that RAGE binds .beta.-sheet fibrillar material
regardless of the composition of the subunits (amyloid-.beta.
peptide, amylin, serum amyloid A, prion-derived peptide) (Yan et
al., Nature, 382:685-691 (1996); Yan et al., Nat. Med., 6:643-651
(2000)). In addition, deposition of amyloid has been shown to
result in enhanced expression of RAGE. For example, in the brains
of patients with Alzheimer's disease (AD), RAGE expression
increases in neurons and glia (Yan, et al., Nature 382:685-691
(1996)). The consequences of .beta.-amyloid peptide (A.beta.)
interaction with RAGE appear to be quite different on neurons
versus microglia. Whereas microglia become activated as a
consequence of A.beta.-RAGE interaction, as reflected by increased
motility and expression of cytokines, early RAGE-mediated neuronal
activation is superceded by cytotoxicity at later times. Further
evidence of a role for RAGE in cellular interactions of A.beta. is
shown by the inhibition of A.beta.-induced cerebral
vasoconstriction and transfer of the peptide across the blood-brain
barrier to brain parenchyma when the receptor was blocked (Kumar et
al., Neurosci. Program, p 141-#275.19 (2000)). Inhibition of
RAGE-amyloid interaction has been shown to decrease expression of
cellular RAGE and cell stress markers (as well as NF-.kappa.B
activation), and diminish amyloid deposition (Yan et al., Nat.
Med., 6:643-651 (2000)) suggesting a role for RAGE-amyloid
interaction in both perturbation of cellular properties in an
environment enriched for amyloid (even at early stages) as well as
in amyloid accumulation.
[0069] Also preferably, compounds identified by the methods of the
invention are used to treat cancer. For example, amphoterin is a
high mobility group I nonhistone chromosomal DNA binding protein
(Rauvala et al., J. Biol. Chem., 262:16625-16635 (1987); Parkikinen
et al., J. Biol. Chem. 268:19726-19738 (1993)) which has been shown
to interact with RAGE. It has been shown that amphoterin promotes
neurite outgrowth, as well as serving as a surface for assembly of
protease complexes in the fibrinolytic system (also known to
contribute to cell mobility). In addition, a local tumor growth
inhibitory effect of blocking RAGE has been observed in a primary
tumor model (C6 glioma), the Lewis lung metastasis model (Taguchi
et al., Nature 405:354-360 (2000)), and spontaneously arising
papillomas in mice expressing the v-Ha-ras transgene (Leder et al.,
Proc. Natl. Acad. Sci., 87:9178-9182 (1990)).
[0070] Also preferably, compounds identified by the methods of the
invention are used to treat inflammation. Also preferably, the
compounds identified by the methods of the invention are used to
treat kidney failure. Also preferably, the compounds identified by
the methods of the invention are used to treat systemic lupus
nephritis or inflammatory lupus nephritis. For example, the
S100/calgranulins have been shown to comprise a family of closely
related calcium-binding polypeptides characterized by two EF-hand
regions linked by a connecting peptide (Schafer et al., TIBS,
21:134-140 (1996); Zimmer et al, Brain Res. Bull., 37:417-429
(1995); Rammes et al., J. Biol. Chem., 272:9496-9502 (1997);
Lugering et al., Eur. J. Clin. Invest., 25:659-664 (1995)).
Although they lack signal peptides, it has long been known that
S100/calgranulins gain access to the extracellular space,
especially at sites of chronic immune/inflammatory responses, as in
cystic fibrosis and rheumatoid arthritis. RAGE is a receptor for
many members of the S100/calgranulin family, mediating their
proinflammatory effects on cells such as lymphocytes and
mononuclear phagocytes. Also, studies on delayed-type
hypersensitivity response, colitis in IL-10 null mice,
collagen-induced arthritis, and experimental autoimmune
encephalitis models suggest that RAGE-ligand interaction
(presumably with S-100/calgranulins) has a proximal role in the
inflammatory cascade.
[0071] Also preferably, compounds identified by the methods of the
invention are used to treat erectile dysfunction. Relaxation of the
smooth muscle cells in the cavemosal arterioles and sinuses results
in increased blood flow into the penis, raising corpus cavemosum
pressure to culminate in penile erection. Nitric oxide is
considered the principle stimulator of cavemosal smooth muscle
relaxation (Chitaley et al, Nature Medicine, January; 7(1):119-122
(2001)). RAGE activation produces oxidants (Yan et al., J. Biol.
Chem., 269:9889-9897, 1994) via an NADH oxidase-like enzyme,
therefore suppressing the circulation of nitric oxide. Potentially
by inhibiting the activation of RAGE signaling pathways by
decreasing the intracellular production of AGEs, generation of
oxidants will be attenuated. Rage blockers may promote and
facilitate penile erection by blocking the access of ligands to
RAGE. The calcium-sensitizing Rho-kinase pathway may play a
synergistic role in cavemosal vasoconstriction to maintain penile
flaccidity. The antagonism of Rho-kinase results in increased
corpus cavemosum pressure, initiating the erectile response
independently of nitric oxide (Wingard et al., 2001). One of the
signaling mechanisms activated by RAGE involves the Rho-kinase
family such as cdc42 and rac (Huttunen et al., J Biol. Chem.,
274:19919-24 (1999)). Thus, inhibiting activation of Rho-kinases
via suppression of RAGE signaling pathways will enhance and
stimulate penile erection independently of nitric oxide.
[0072] In one aspect, the present invention also provides a method
for inhibiting the interaction of an AGE with RAGE in a subject
which comprises administering to the subject a therapeutically
effective amount of a compound identified by the methods of the
invention. A therapeutically effective amount is an amount which is
capable of preventing interaction of AGE/RAGE in a subject.
Accordingly, the amount will vary with the subject being treated.
Administration of the compound may be hourly, daily, weekly,
monthly, yearly or a single event. Preferably, the effective amount
of the compound comprises from about 1 ng/kg body weight to about
100 mg/kg body weight. More preferably, the effective amount of the
compound comprises from about 1 .mu.g/kg body weight to about 50
mg/kg body weight. Even more preferably, the effective amount of
the compound comprises from about 10 .mu.g/kg body weight to about
10 mg/kg body weight. The actual effective amount will be
established by dose/response assays using methods standard in the
art (Johnson et al., Diabetes. 42: 1179, (1993)). Thus, as is known
to those in the art, the effective amount will depend on
bioavailability, bioactivity, and biodegradability of the
compound.
[0073] In an embodiment, the subject is an animal. In an
embodiment, the subject is a human. In an embodiment, the subject
is suffering from an AGE-related disease such as diabetes,
amyloidoses, renal failure, aging, or inflammation. In another
embodiment, the subject comprises an individual with Alzheimer's
disease. In an alternative embodiment, the subject comprises an
individual with cancer. In yet another embodiment, the subject
comprises an individual with systemic lupus erythmatosis, or
inflammatory lupus nephritis.
[0074] In an embodiment, administration of the compound comprises
intralesional, intraperitoneal, intramuscular, or intravenous
injection. In an embodiment, administration of the compound
comprises infusion or liposome-mediated delivery. In an embodiment,
administration of the compound comprises topical application to the
skin, nasal cavity, oral membranes or ocular tissue.
[0075] The pharmaceutically acceptable carriers of the invention
comprise any of the standard pharmaceutically accepted carriers
known in the art. In an embodiment, the carrier comprises a
diluent. In an embodiment, the carrier comprises a liposome, a
microcapsule, a polymer encapsulated cell, or a virus. For example,
in one embodiment, the pharmaceutical carrier may be a liquid and
the pharmaceutical composition in the form of a solution. In
another embodiment, the pharmaceutically acceptable carrier is a
solid and the composition is in the form of a powder or tablet. In
a further embodiment, the pharmaceutical carrier is a gel or
ointment and the composition is in the form of a suppository,
cream, or liquid. Thus, the term pharmaceutically acceptable
carrier encompasses, but is not limited to, any of the standard
pharmaceutically accepted carriers, such as phosphate buffered
saline solution, water, emulsions such as oil/water emulsions or
triglyceride emulsion, various types of wetting agents, tablets,
coated tablets and capsules.
[0076] For example, tablets or capsules may utilize
pharmaceutically acceptable binding agents (e.g.
polyvinylpyrrolidone, hydroxypropyl methylcellulose, starch);
fillers (e.g. lactose, microcrystalline cellulose, calcium hydrogen
phosphate); lubricants (e.g. magnesium stearate, silica or talc).
Liquid preparations for oral administration may comprise syrups or
suspensions prepared by conventional means with pharmaceutically
acceptable additives such as suspending agents (e.g. hydrogenated
fats, sorbitol syrup), emulsifying agents (e.g. lecithin), and
preservatives. Preparations may contain buffer, salts, and
flavoring agents as appropriate. Suitable examples of liquid
carriers include water, alcohols, and oils containing additives as
described above.
[0077] When administered, compounds are often rapidly cleared from
the circulation. Thus, in an embodiment, compounds are modified by
the covalent attachment of water-soluble polymers such as
polyethylene glycol (PEG), copolymers of PEG and polypropylene
glycol, polyvinylpyrrolidone or polyproline, carboxymethyl
cellulose, dextran, polyvinyl alcohol, and the like. Such
modifications also may increase the compound's solubility in
aqueous solution, and reduce immunogenicity of the compound.
Polymers such as PEG may be covalently attached to one or more
reactive amino residues, sulfhydryl residues or carboxyl residues.
Numerous activated forms of PEG have been described, including
active esters of carboxylic acid or carbonate derivatives,
particularly those in which the leaving groups are
N-hydroxsuccinimide, p-nitrophenol, imidazole or
1-hydroxy-2-nitrobenzene-3 sulfone for reaction with amino groups,
maleimido or haloacetyl derivatives for reaction with sulfhydryl
groups, and amino hydrazine or hydrazide derivatives for reaction
with carbohydrate groups.
EXAMPLES
[0078] Features and advantages of the inventive concept covered by
the present invention are further illustrated in the examples which
follow.
Example 1
ELISA Assay Protocol
[0079] Generally, the protocol for detection of RAGE modulators is
as follows. RAGE ligand (e.g. S-100b, .beta.-amyloid, CML) is
diluted to 5 .mu.g/ml in buffer A (fixing buffer) (100 mM
Na.sub.2CO.sub.3/NaHCO.sub.3, pH 9.8) and 100 .mu.l added to
microtiter plate wells and allowed to incubate overnight at
4.degree. C. to allow the ligand to become fixed to the surface of
the wells. Wells are then washed 3 times with 400 .mu.l/well buffer
C (wash buffer) (20 mM Imidazole, 150 mM NaCl, pH 7.2), with a 5
second soak in buffer C between each wash. Buffer B (blocking
buffer) (50 mM Imidazole pH 7.2, 5% BSA, 5 mM CaCl.sub.2, 5 mM
MgCl.sub.2) is then added to the wells and allowed to incubate for
2 hours at 37.degree. C. to block nonspecific protein binding
sites. The blocking buffer is then aspirated from the wells, and
the plate washed 3 times (400 .mu.l/well) with buffer C, with a 5
second soak in buffer C between each wash. The compound of
interest, 25 .mu.l dissolved in 2% DMSO FAC (Final Assay
Concentration) and sRAGE (75 .mu.l) (2.2.times.10.sup.-4 mg/ml FAC)
are then added to each well, and incubated 1 hour at 37.degree. C.
Meanwhile, polyclonal antibody or monoclonal antibody for sRAGE
(e.g. 3.0.times.10.sup.-3 mg/ml and 1.9.times.10.sup.-4 mg/ml FAC,
respectively), biotinylated goat F (ab').sub.2 anti-mouse IgG (e.g.
8.0.times.10.sup.-4 mg/ml FAC) (Biosource International, Camarillo,
Calif. (TAGO)), and alkaline phosphatase labeled streptavidin
(3.0.times.10.sup.-3 mg/ml FAC) (ZYMED, San Francisco, Calif.) are
added to 5 ml of buffer D (complex buffer) (50 mM Imidazole, pH
7.2; 0.2% BSA, 5 mM CaCl.sub.2, 5 mM MgCl.sub.2) in a 15 ml conical
tube and allowed to incubate 30 minutes at room temperature.
[0080] The solution containing sRAGE and the compound of interest
is then aspirated from each well, and after 3 washes with wash
buffer, with a 5 second soak between each wash, the
anti-sRAGE:IgG:streptavidin-alkaline phosphatase complex is added
to each well (100 .mu.l complex per well). After 1 hour at room
temperature, the solution in each well is aspirated, and the wells
washed 3 times, with 5 second soaks between each wash.
[0081] The alkaline phosphatase substrate, para-nitrophenyl
phosphate (pNPP) (1 mg/ml in 1 M diethanolamine, pH 9.8), is added
and the color allowed to develop for 1 hr in the dark. After the
addition of 10 .mu.l stop solution per well (0.5 N NaOH; 50%
methanol), the OD.sub.405 is measured.
Example 2
Optimization of Assay Conditions
[0082] Experiments detailing the approach used for optimization of
assay conditions are shown in FIGS. 3-6. For example, an experiment
showing optimization of the assay for varying ligand concentrations
is shown in FIG. 3. Thus, using sRAGE at a concentration of
1.0.times.10.sup.-4 mg/ml FAC and polyclonal or monoclonal antibody
(3.0.times.10.sup.-3 mg/ml and 1.9.times.10.sup.-4 mg/ml FAC
respectively), it was found that maximal signal (1.06 OD) was
achieved when S-100b at 500 ng/well was used for coating, and that
ligand concentrations between 10 to 500 ng/well comprised a linear
increase in sRAGE binding.
[0083] Experiments showing optimization of the assay at varying
sRAGE concentrations are shown in FIG. 4. Using 500 ng/well S-100b
to coat the wells, anti-sRAGE polyclonal or monoclonal antibody
(3.0.times.10.sup.-3 mg/ml and 1.9.times.10.sup.-4 mg/ml FAC
respectively), and sRAGE ranging from 2 to 10 nM FAC, maximum
signal is seen at about 10 nM sRAGE (FIG. 4).
[0084] Experiments showing optimization of the assay for varying
antibody concentration are shown in FIG. 5. Wells were coated using
S-100b at either 50, 500, 1,000 or 2,000 ng/well overnight as
indicated. sRAGE at 6 nM was added and after 1 hour at 37.degree.
C., polyclonal antibody to sRAGE comprising final concentrations
ranging from 5.0.times.10.sup.-2 to 5.5.times.10.sup.-5 mg/ml was
added. It can be seen that regardless of the concentration of
ligand used to coat the wells, maximal signal was obtained at
5.4.times.10.sup.-3 mg/ml antibody. Using 500 ng/well S-100b to
coat the wells, and sRAGE at 1.0.times.10.sup.-4 mg/ml, maximal
signal was found at 1.4.times.10.sup.-2 mg/ml antibody.
[0085] An experiment showing optimization of the assay for varying
DMSO concentrations is shown in FIG. 6. Thus, using sRAGE at a
concentration of 6 nM FAC and polyclonal or monoclonal antibody
(3.0.times.10.sup.-3 mg/ml and 1.9.times.10.sup.-4 mg/ml FAC,
respectively), it was found that maximal signal was achieved using
DMSO concentrations between 0% to 2% FAC.
Example 3
Accuracy and Precision of the Assay
[0086] In the experiment shown in FIG. 10, wells were coated by
incubation with 500 ng/well S-100b overnight, and after washing,
sRAGE diluted in buffer D added to the final concentrations shown.
Polyclonal anti-sRAGE antibody (3.0.times.10.sup.-3 mg/ml FAC) was
then added, and after 1 hr at 37.degree. C., the plates washed and
developed as described in Example 1. It was found that over four
separate determinations, the goodness of fit comprised an R.sup.2
of 0.9383, with Kd value of 60 and 95% confidence interval of 41.2
to 78.6.
[0087] An assessment of signal variability is shown in FIG. 7. In
this experiment, 88 wells were coated by incubation with 500
ng/well S-100b overnight, and after washing, sRAGE diluted in
buffer D added to all the wells with a final concentration of
2.2.times.10.sup.-4 mg/ml. Polyclonal anti-sRAGE antibody
(3.0.times.10.sup.-3 mg/ml FAC) was then added, and after 1 hr at
37.degree. C., the plates washed and developed as described in
Example 1. An average OD.sub.405 of 0.705.+-.0.054 and percent
coefficient variance of 7.7 was observed. Data indicates that the
signal variability from well to well is lower than 10%.
Example 4
Detection of Low and High Affinity Binding of Ligands to sRAGE
[0088] The utility of the assay for detecting compounds which
antagonize both low and high affinity RAGE binding domains is shown
in FIGS. 8-11. In these experiments, wells were coated with varying
amounts of S-100b (50-500 mg/well overnight) and sRAGE added to
each well. The amount of sRAGE bound to the immobilized ligand was
detected using anti-sRAGE polyclonal or monoclonal antibody
(3.0.times.10.sup.-3 mg/ml and 1.9.times.10.sup.-4 mg/ml FAC,
respectively) and the development system as described in Example 1.
Plots were linearized by means of Scatchard analysis and values for
Kd and Bmax determined. Statistical analysis of experimental
precision was determined by utilizing GraghPad Prisam version 3
software (GraphPad Software, Inc. San Diego, Calif.).
[0089] Both low and high affinity binding of sRAGE to S-100b are
detected using the assay method of the invention. In the
experiments shown in FIGS. 8 and 9, plates were incubated overnight
with S-100b at a concentration of 50 and 100 ng/well, respectively.
After washing, sRAGE comprising a final concentration ranging from
about 0.3 to 10 nM was added to each well, and after 1 hour at
37.degree. C., bound sRAGE was measured by the addition of
polyclonal antibody complex. For both experiments, a high affinity
binding component comprising a Kd of about 6 nM (FIG. 8) to 2 nM
(FIG. 9) is detected.
[0090] In the experiments shown in FIGS. 10 and 11, plates were
incubated overnight with S-100b at a concentration of 500 and 100
ng/well, respectively. After washing, sRAGE comprising a final
concentration ranging from about 23 to 470, or 5 to 76 nM,
respectively, was added to each well, and after 1 hour at
37.degree. C., bound sRAGE measured by addition of the sRAGE
polyclonal antibody complex (anti-sRAGE:IgG:streptavidin-alkaline
phosphatase) and detection reagents. For both experiments, a low
affinity binding site, having a Kd of about 60 nM, was
detected.
Example 5
Inhibition of sRAGE Binding by Modulators
[0091] Interaction of AGE's with RAGE generates intracellular
oxidative stress (Yan, et al., J. Biol. Chem., 269:9889-9897
(1994)) resulting in the activation of the free radical-sensitive
transcription factor NF-.kappa.B and the activation of NF-.kappa.B
regulated genes (Yan, et al., J. Biol. Chem., 269:9889-9897 (1994);
Wautier, et al., Proc. Natl. Acad. Sci., 91:7742-7746 (1994)).
Since RAGE transcription is also regulated by interaction at two
different NF-.kappa.B binding sites in the RAGE promoter sequence
(Li, et al., J. Biol. Chem., 272:16498-16506 (1997)) an ascending
detrimental spiral is fueled by this positive feedback loop. A
secondary cell based functional assay was used to evaluate small
molecules which modulate binding of sRAGE to an immobilized ligand.
The pNF-.kappa.B-Luc gene was introduced into a C6 glioma cell line
expressing high levels of RAGE (see e.g. Huttunen et al., J Biol.
Chem., 274:19919-24 (1999)) by transfection using standard
techniques. The pNF-.kappa.B-Luc reporter is designed such that
upon activation of the NF-.kappa.B, the luciferase gene is
expressed. Luciferase activity is then quantified by measuring
luminescence with a luminometer. Generally, expression of the
luciferase gene is low. Upon introduction of a RAGE ligand,
however, RAGE activates NF-.kappa.B, resulting in an increase in
luciferase expression. Thus, the assay measures the ability of a
compound to modulate RAGE induced-luciferase activity. If the
modulator comprises an agonist, the compound induces activation of
NF-.kappa.B transcription, and luciferase activity is increased. If
the modulator is an antagonist, the compound inhibits the ability
of RAGE agonists to induce activation of NF-.kappa.B transcription,
and luciferase activity is increased.
[0092] In the experiment shown in FIG. 14, inhibition of
S-100b/RAGE interaction in C6 glioma cells by Compound 1 (FIG. 13)
had an IC.sub.50 of 3.3 .mu.M. Thus, the cell based assay shown in
FIG. 14 showed good correlation with the binding ELISA IC.sub.50
value for inhibition of sRAGE binding for Compound 1 of 1.8 .mu.M
(FIG. 12).
[0093] With respect to the descriptions set forth above, optimum
dimensional relationship of parts of the invention (to include
variations in specific components and manner of use) are deemed
readily apparent and obvious to those skilled in the art, and all
equivalent relationships to those illustrated in the drawings and
described in the specification are intended to be encompassed
herein. The foregoing is considered as illustrative only of the
principal of the invention. Since numerous modifications and
changes will readily occur to those skilled in the art, it is not
intended to limit the invention to the exact embodiments shown and
described, and all suitable modifications and equivalents falling
within the scope of the appended claims are deemed within the
present inventive concept.
[0094] It is to be further understood that the phraseology and
terminology employed herein are for the purpose of description and
are not to be regarded as limiting. Those skilled in the art will
appreciate that the conception on which this disclosure is based
may readily be used as a basis for designing the methods and
systems for carrying out the several purposes of the present
invention. The claims are regarded as including such equivalent
constructions so long as they do not depart from the spirit and
scope of the present invention.
Sequence CWU 1
1
41404PRTHomo sapiens 1Gly Ala Ala Gly Thr Ala Val Gly Ala Trp Val
Leu Val Leu Ser Leu1 5 10 15Trp Gly Ala Val Val Gly Ala Gln Asn Ile
Thr Ala Arg Ile Gly Glu20 25 30Pro Leu Val Leu Lys Cys Lys Gly Ala
Pro Lys Lys Pro Pro Gln Arg35 40 45Leu Glu Trp Lys Leu Asn Thr Gly
Arg Thr Glu Ala Trp Lys Val Leu50 55 60Ser Pro Gln Gly Gly Gly Pro
Trp Asp Ser Val Ala Arg Val Leu Pro65 70 75 80Asn Gly Ser Leu Phe
Leu Pro Ala Val Gly Ile Gln Asp Glu Gly Ile85 90 95Phe Arg Cys Gln
Ala Met Asn Arg Asn Gly Lys Glu Thr Lys Ser Asn100 105 110Tyr Arg
Val Arg Val Tyr Gln Ile Pro Gly Lys Pro Glu Ile Val Asp115 120
125Ser Ala Ser Glu Leu Thr Ala Gly Val Pro Asn Lys Val Gly Thr
Cys130 135 140Val Ser Glu Gly Ser Tyr Pro Ala Gly Thr Leu Ser Trp
His Leu Asp145 150 155 160Gly Lys Pro Leu Val Pro Asn Glu Lys Gly
Val Ser Val Lys Glu Gln165 170 175Thr Arg Arg His Pro Glu Thr Gly
Leu Phe Thr Leu Gln Ser Glu Leu180 185 190Met Val Thr Pro Ala Arg
Gly Gly Asp Pro Arg Pro Thr Phe Ser Cys195 200 205Ser Phe Ser Pro
Gly Leu Pro Arg His Arg Ala Leu Arg Thr Ala Pro210 215 220Ile Gln
Pro Arg Val Trp Glu Pro Val Pro Leu Glu Glu Val Gln Leu225 230 235
240Val Val Glu Pro Glu Gly Gly Ala Val Ala Pro Gly Gly Thr Val
Thr245 250 255Leu Thr Cys Glu Val Pro Ala Gln Pro Ser Pro Gln Ile
His Trp Met260 265 270Lys Asp Gly Val Pro Leu Pro Leu Pro Pro Ser
Pro Val Leu Ile Leu275 280 285Pro Glu Ile Gly Pro Gln Asp Gln Gly
Thr Tyr Ser Cys Val Ala Thr290 295 300His Ser Ser His Gly Pro Gln
Glu Ser Arg Ala Val Ser Ile Ser Ile305 310 315 320Ile Glu Pro Gly
Glu Glu Gly Pro Thr Ala Gly Ser Val Gly Gly Ser325 330 335Gly Leu
Gly Thr Leu Ala Leu Ala Leu Gly Ile Leu Gly Gly Leu Gly340 345
350Thr Ala Ala Leu Leu Ile Gly Val Ile Leu Trp Gln Arg Arg Gln
Arg355 360 365Arg Gly Glu Glu Arg Lys Ala Pro Glu Asn Gln Glu Glu
Glu Glu Glu370 375 380Arg Ala Glu Leu Asn Gln Ser Glu Glu Pro Glu
Ala Gly Glu Ser Ser385 390 395 400Thr Gly Gly Pro2339PRTHomo
sapiens 2Gly Ala Ala Gly Thr Ala Val Gly Ala Trp Val Leu Val Leu
Ser Leu1 5 10 15Trp Gly Ala Val Val Gly Ala Gln Asn Ile Thr Ala Arg
Ile Gly Glu20 25 30Pro Leu Val Leu Lys Cys Lys Gly Ala Pro Lys Lys
Pro Pro Gln Arg35 40 45Leu Glu Trp Lys Leu Asn Thr Gly Arg Thr Glu
Ala Trp Lys Val Leu50 55 60Ser Pro Gln Gly Gly Gly Pro Trp Asp Ser
Val Ala Arg Val Leu Pro65 70 75 80Asn Gly Ser Leu Phe Leu Pro Ala
Val Gly Ile Gln Asp Glu Gly Ile85 90 95Phe Arg Cys Gln Ala Met Asn
Arg Asn Gly Lys Glu Thr Lys Ser Asn100 105 110Tyr Arg Val Arg Val
Tyr Gln Ile Pro Gly Lys Pro Glu Ile Val Asp115 120 125Ser Ala Ser
Glu Leu Thr Ala Gly Val Pro Asn Lys Val Gly Thr Cys130 135 140Val
Ser Glu Gly Ser Tyr Pro Ala Gly Thr Leu Ser Trp His Leu Asp145 150
155 160Gly Lys Pro Leu Val Pro Asn Glu Lys Gly Val Ser Val Lys Glu
Gln165 170 175Thr Arg Arg His Pro Glu Thr Gly Leu Phe Thr Leu Gln
Ser Glu Leu180 185 190Met Val Thr Pro Ala Arg Gly Gly Asp Pro Arg
Pro Thr Phe Ser Cys195 200 205Ser Phe Ser Pro Gly Leu Pro Arg His
Arg Ala Leu Arg Thr Ala Pro210 215 220Ile Gln Pro Arg Val Trp Glu
Pro Val Pro Leu Glu Glu Val Gln Leu225 230 235 240Val Val Glu Pro
Glu Gly Gly Ala Val Ala Pro Gly Gly Thr Val Thr245 250 255Leu Thr
Cys Glu Val Pro Ala Gln Pro Ser Pro Gln Ile His Trp Met260 265
270Lys Asp Gly Val Pro Leu Pro Leu Pro Pro Ser Pro Val Leu Ile
Leu275 280 285Pro Glu Ile Gly Pro Gln Asp Gln Gly Thr Tyr Ser Cys
Val Ala Thr290 295 300His Ser Ser His Gly Pro Gln Glu Ser Arg Ala
Val Ser Ile Ser Ile305 310 315 320Ile Glu Pro Gly Glu Glu Gly Pro
Thr Ala Gly Ser Val Gly Gly Ser325 330 335Gly Leu Gly3112PRTHomo
sapiens 3Ala Gln Asn Ile Thr Ala Arg Ile Gly Glu Pro Leu Val Leu
Lys Cys1 5 10 15Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg Leu Glu Trp
Lys Leu Asn20 25 30Thr Gly Arg Thr Glu Ala Trp Lys Val Leu Ser Pro
Gln Gly Gly Gly35 40 45Pro Trp Asp Ser Val Ala Arg Val Leu Pro Asn
Gly Ser Leu Phe Leu50 55 60Pro Ala Val Gly Ile Gln Asp Glu Gly Ile
Phe Arg Cys Gln Ala Met65 70 75 80Asn Arg Asn Gly Lys Glu Thr Lys
Ser Asn Tyr Arg Val Arg Val Tyr85 90 95Gln Ile Pro Gly Lys Pro Glu
Ile Val Asp Ser Ala Ser Glu Leu Thr100 105 110430PRTHomo sapiens
4Ala Gln Asn Ile Thr Ala Arg Ile Gly Glu Pro Leu Val Leu Lys Cys1 5
10 15Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg Leu Glu Trp Lys20 25
30
* * * * *