Note: Descriptions are shown in the official language in which they were submitted.
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ANALOGS FOR SPECIFIC OLIGOSACCHARlDE-NEUREGULIN
INTERACTIONS AND USLS THEREOF
5 Ba~k~round
1. Field of the Invention
The present application relates to the field of growth factor-glycosaminoglycan
10 interac~ions.
. R~f~kground of the Invention
Glvcosaminoglvcan Structure
Glycos~minoglycans (GAG) are naturally-occurring carbohydrate-based molecules
implicated in regulation of a number of cellular processes, in~ rling blood coagulation,
angiogenesis, tumor growth, nerve cell development, smooth muscle cell proliferation, and
gene expression, most likely by interaction with effector molecules. GAG's are linear, non-
20 branched chains of ~eatil,g two-sugar (ti~ ch~ride) units which may be up to lS0 units in
length, and are well known and described in the art. See, for example, Jackson, et al., (1991)
Pllysiological Reviews 71: 4~31-539 and Kjell~n, et al., (1991) Ann Rev. Riochem. 60: 443-
475. GAG's are often, but not always, found covalently bound to protein cores in structures
called proteoglycans. Proteoglycan structures are abundant on cell surfaces and are
25 associated with the extracellular matrix around cells.
Glycos~minoglycans (also referred to herein and the art as "glycans") can be divided
into four main classes on the basis of the repeating ~icarch~ride unit in the backbone.
Typically, one sugar is a uronic acid, and the other is either an N-acetylgluros~minç or an N-
30 acetylgalactos~min~ The classes are exemplified by the following four GAGs: (1) heparansulfate (HS) (D-glucuronic acid/N-acetyl- or N-sulfo-D-glucosamine); (2)
chondroitin/dermatan sulfate (D-glucuronic acid or L-iduronic acid/N-acetyl-D-
galactosamine); (3) keratan sulfate (D-galactose/N-acetyl-D-glucosamine), and (4)
hyaluronic acid. All GAGs, with the exception of hyaluronic acid, contain sulfate groups
35 variously esterified to the ring hydroxyl groups of the sugars. These negatively charged
groups are believed to figure prominently in the biological p,u~e,lies attributed to
glycosaminoglycans. The nacurally-occurring forms of GAGs, particularly heparin, heparan
sulfate. chondroitin sulfate and dermatan sulfate. in fact are complex hetero-oligos~rch~rides
composed of mixtures of differentially sulfated sugar residues.
uli-S~E~ff~ULE26~
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One of the most thoroughly studied glycosaminoglycans is the widely used
anticoagulant heparin. Heparin is a highly sulfated forrn of heparan sulfate, which is found
in most cells. As a commercial product, heparin is a hetero-oligos~rch~ composition of
about 20-60 monomeric units, having an overall extended length of about 100-300 ~, having
no protein associated with it, and its anticoagulant ~lu~,,Lies can be ascribed exclusively to
the specific sulfation patterns found on the carbohydrate chains. So-called "low molecular
weight" heparin typically is a hetero-oligodisaccharide composition of about 25-30
monomeric units, having an overall extended length of about 40A. Heparin is known to
have a variety of potentially useful biological activities beyond its ability to inhibit blood
coagulation including, for example, the ability to block complement activation, smooth
muscle cell proliferation and tumor growth. However, the toxicity of heparin at the levels
required to manifest these activities in vivo has limited its clinical use. Heparan sulfate, the
predominant GAG on cell surfaces. contains fewer sulfate groups than heparin and has been
shown to contain regions of high sulfation interspersed among regions of low or no
sulfation.
Other polysulfated compounds described in the art and asserted to have clinically
useful activities analogous to those attributed to heparin include fractions or fra~m~nt~ of the
naturally-occurring GAGs, pentosan polysulfate (PPS), dextran sulfate, chondroitin sulfate,
and keratan sulfate; and suramin, a polysulfonated naphthylurea whose structural motif
likely mimics that of a GAG sequence. As for heparin, the toxicity of these compounds at
the levels required for therapeutic utility has limited their clinical use. A representative
listing of publications describing these colllpollllds and their asserted biological activities
includes: US Patent No. 5.158,940 issued October '7. 1992; US Patent No. 4.826,827.
issued May 2, 1989; international patent publication Nos. WO 90/15816 (public December
27, 1990), WO 91/13624 (public September 19, 1991), and WO 93/07864 (public April 29,
1993); Wellstein, et al., (1991) J. Natl. Cancer Inst. 83: 716-720; and Jentsch, et al., (1987)
J. Gen. Virol. 68: 2183-2192.
GAG Binding Proteins and GAG Binding Specificit~
Many important regulatory proteins bind tightly to heparin. including chemokines,
growth factors (including cytokines), enzymes and proteins involved in lipid metabolism.
This binding ~.up~"Ly was, for a long time. thought to arise only from non-specific ionic
interactions involving positively charged regions on the proteins with the negativelv charged
sulfates of heparin. However, recent results with two proteins~ Antithrombin III (AT III)
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and basic fibroblast growth factor (bFGF). demonstrate that the interactions between heparin
and AT III or bFGF can show specificity. The specific interaction involves complex binding
sites on the protein molecule and infrequently occurring sequences in the heparin GAG
chain. See. for example, EPO patent publication 0 509 517 A2, published October 21, 1992;
Turnbull. et al., (1992) J. Biol. Chem. ~67:10337-10341; Gallagher, et al., (1992)
Glycobiology _: 523-528; Habuchi, et al., (1992) J. Biochem. 285: 805-813; Yayon, et al.,
(1991) Cell 64: 841 -848: and Rapraeger, et al., (1991) Science ~: 1705- 1708.
That specific protein binding sequences might exist in the carbohydrate chain ofheparin was first suggested by the observation that some preparations were more effective
than others in inhibiting coagulation. Careful studies in 1987 revealed that there is a defined
five sugar sequence (pent~c~ch~ride) with a characteristic sulfation pattern that ~~..,s~.-L~
the specific binding site for AT III. a protease inhibitor that blocks the action of thrombin
and other enzymes which initiate blood coagulation. The Kd for the binding between AT III
and this specific GAG recognition site is about 10 nM (10-8 M), which qualifies it as a high
affinity interaction. Although weaker and less specific binding of these proteins to other
regions of heparin can occur, virtually all of the anticoagulant activity of heparin is
attributable to this five sugar sequence. This pent~c~cch~ride, generally known as the AT m
binding site, now has been synthesi7~d chemically and shown to possess the a~-up~ate
activities of the naturally occurring sequence. Binding of AT III to this site is thought to
provide the basis for heparin's anticoagulant activity by positioning and "presenting" the
enzyme inhibitor to the proteases thrombin and Factor Xa.
A second example of a somewhat specific binding site has been reported for
fibroblast growth factor. This GAG sequence, isolated from fibroblast heparan sulfate. was
found to represent the tightest binding fraction present. It is not clear, however, whether
other molecules such as other heparin binding growth factors can bind to this sequence, nor
is it clear that the affinity of this binding is as high as the binding between bFGF and
heparin. The interaction between the isolated GAG sequence and bFGF might, at present,
best be described then as selective, rather than absolutely specific.
Growth ~actors and Cvtokines
It is well recognized that the endogenous heteroligodisaccharides heparan sulfate and
heparin bind with appreciable affinity to a wide spectrum of the mitogenic proteins termed
cytokines and growth factors, although the strength of these interactions varies considerably
among the different factors. Among the growth factors and cytokines described as
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heparin/HS-binding proteins are: TGF-13. endothelial cell growth factor, IL3 and GM-CSF,
hltelrelon-~, hepatocyte growth factor, fibroblast growth factor (FGF) -1 (acidic FGF), FGF-
2 (basic FGF), FGF-3 (int-2), FGF-4 (Hst-l, K-FGF), FGF-5, FGF-6 (Hst-2) and FGF-7
(keratinocyte GF). For example, heparin will release TGF-13 from inactive complexes with
5 a2-macroglobulin and will potentiate TGF-~ action. The stability in solution of acidic and
basic FGF (aFGF and bFGF) is enhanced in the presence of HS/heparin, and the
polysaccharides potentiate the mitogenic activity of the FGFs, especially of aFGF. These
effects are presumed to be due to the formation of complexes between FGF and heparin
which prolong the biological lifetime of the proteins by protecting them from proteolysis
10 and therrnal denaturation. In tissues, aFGF and bFGF can be ~letect~l in the extracellular
matrix and basement membranes, where they are bound to HS. It has been p-uposed that the
action of heparinases or proteases that degrade heparan sulfate proteoglycans will release
FGFs from the basement membranes enabling them to act on nearby target cells. In addition
to effects on FGF stability and tissue localization, a central role has now been described for
15 HS in controlling the interaction of bFGF with cell sign~lling ,cc~iptc~.
Neuregulins
A recently described family of growth factors, the neuregulins (reviewed by Mudge,
(1993) Curr. Biol. 3:361; Peles and Yarden, (1993) Bioessays 15:815), are synthssi7toA by
neurons (Marchionni, et al., (1993) Nature ~:313) and by mesenchymal cells from several
parenchymal organs (Meyer and Birchlll.,ie., (1994) PNAS 91:1064). The neuregulins and
related factors that bind pl85erbB2 have been purified, cloned and expressed (Benveniste,
et al., PNAS, 82:3930, 1985; Kimura, et al., (1990) Nature 348:2~7; Davis and Stroobant,
(1990) J. Cell Biol. 110:1353; Wen. et al., (1992) Cell 69:559; Yarden and Ullrich, (1988)
Ann. Rev Biochem. 57:443; Dobashi, et al., (1991) Proc. Natl. Acad. Sci. 88:8582; Lupu, et
al., (1992) Proc. Natl Acad. Sci. 89:2287; Wen, et al., (1994) Mol. Cell. ~3iol. 14:1909).
Recombinant neuregulins have been shown to be mitogenic for p.,li~hel~l glia (Marchionni,
et al., (1993) Na~ure 362:313) and have been shown to influence the formation of the
fieulo~ cculzlr junction (Falls, et al., (1993) Cell 72:801; Jo, et al., (1995) Nature 373: 158;
Chu, et al., Cell 14: 329, 1995).
The neuregulin gene consists of at least thirteen exons. The neuregulin transcripts
are alternatively spliced and these encode many distinct peptide growth factors, which are
referred to as the neuregulins (Marchiûnni, et al., Nature 362:313, 1993). DNA sequence
colllpalisons revealed that neu differentiation factor (NDF) (Wen. et al., (1992) Cell 69:559)
and heregulins (Holmes, et al., (1992) $cience ~:1205), which were purified as ligands of
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the pl85erbB2 (also known as neu or HER2) receptor tyrosine kinase, also are splicing
variants of the neuregulin gene. The acetylcholine receptor inducing activity (ARIA) also is
a product of the neuregulin gene (Falls, et al., (1993) Cell72:801). A common structural
feature of the neuregulins is the presence of a single epidermal growth factor-like (EGF)
domain.
The sites of neuregulin gene expression have been characterized by use of nucleic
acid probes to analyze RNA samples by a variety of methods, such as Northern blotting,
RNase protection, or in situ hybridization. Transcripts have been detected in the nervous
system and in a variety of other tissues (Holmes, et al., (1992) Science 256:1205; Wen, et
al., (1992) Cell 69:559; Meyer and Birchmeier, (1994) PNAS 91:1064). Sites of gene
expression have been locali7ecl in the brain and spinal chord and in other tissues. (Orr-
Urteger, et al., (1993) PNAS 90:1867; Falls, et al., (1993) Cell72:801; Marchionni, et al.,
(1993) Nature~:313; Meyer and Birchmeier. (1994) PNAS 91:1064; Chen, et al., (1994)
J. Comp. Neurol. 349:389; Corfas, et al., (1995) Neuron 14:103). Specifically in the retinal
r.~ helium. expression of neuregulin L,~ns~ has been detected at embryonic day 18
in rat (Meyer and Birchmeier, (1994) PNAS 91: 1064).
Although a large nurrber of neuregulins may be produced by alternative splicing,they can be broadly sorted into the putative membrane-bound and the soluble isoforrns. The
former contains a putative trans-membrane domain and may be pl~,sented at the cell surface.
Membrane-anchored peptide growth factors may mefli~te cell-cell interactions through cell-
adhesion or juxtacrine mech~nicm~ (reviewed by Massagué and Pandiella, (1993) Ann. Rev.
Biochem. 62:515). Alternatively, the putative membrane-bound isoforms may be cleaved
from the cell surface and function as soluble proteins (Wen, et al., (1992) Cell 69:559; Falls,
et al., (1993) Cell 72:801~. The soluble neuregulin isoforms contain sequences
corresponding to the extracellular domains of the putative membrane-bound isoforms, but
terminate before the transmembrane domain. These neuregulin isoforms may be secreted,
and hence could affect cells at a ~iict~nce or they may be present in the cytoplasm. but could
be released upon cellular injury. In the latter case. neuregulins may function as injury
factors, as has been postulated for the ciliary neurotrophic factor (Stockli. et al., (1989)
Nature 342.920). Any one of these modes of action of the neuregulins may occur.
Cellular targets of peptide growth factors are those which bear receptors for the
factor(s) and those that are shown to respond in a bioassay either in vitro or in vi~o. Based
on studies demonstrating phosphorylation on tyrosine residues or cross-linking experiments,
neuregulins are candidate ligands for the receptor tyrosine kinases pl85erbB2 (or HER-2 in
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human), pl85erbB3 (HER-3 in human), pl85erbB4 (or HER-4 in human) or related
members of the EGF receptor (EGFR) gene family. Collectively, these receptors can be
referred to as erbB receptors. Though the precise ligand-receptor relationship of each
neuregulin protein with each member of the EGFR family is yet to be clarified, several lines
5 of evidence suggest that binding of ligands is mediated by either erbB3 and erbB4, but
signaling occurs through either erbB2. erbB4 and heterodimers of the various subunits (e.g.,
Carraway and Cantley, (1994) Cell 78:5). These receptors are known to be present on
Schwann cells and muscle cells (Jo, et al., (1995) Nature 373:158), and other neuregulin
targets. such as cell lines derived from various tumor tissues. such as breast and gastric
10 epithelia. Sites of expression of the HER-4 gene have been localized by in situ
hybridization to several regions of the brain, including: hippocampus, dentate gyrus, neo
cortex, medial habenula, reticular nucleus of the thalamus, and the amygdala (Lai and
Lemke. (1991) Neuron 6:691).
Neuregulins have been shown to have a variety of biological activities depending on
the cell type being studied. Several neuregulins, including native bovine GGFI. II and m
and recombinant human GGF2 (rhGGF2) are mitogenic for Schwann cells (Marchionni, et
al., (1993) Nature 362:313), as is heregulin Bl (Levi, et al, (1995) J Neurosci. 15:1329).
Further activities of rhGGF2 on Schwann cells include the stim~ tion of migration and the
20 induction of neu,otrophic factors, such as nerve growth factor (~ah~nth~ppa, (1994)
Neurosci. Abst #691.7). On human muscle culture, rhGGF2 has a potent trophic effect on
myotubes (Sklar. et al., U.S. Pat. Applic. ~ 08/059, 022). The differentiation response to
rhGGF2 also includes induction of acetylcholine receptors in cultured myotubes (Jo, et al.,
(1995) Nature 373:158). This activity is associated with other forms of neuregulin.
including ARIA (Falls et al.. (1993) Cell 72:801) and heregulin Bl (Chu. et al., (199~)
Neuron 14:329). as well as with rhGGF2. Further, ARIA has been shown to induce
synthesis of voltage-gated sodium channels in chick skeletal muscle (Corfas and Fischbach,
(1993) J. Neurosci. 13:2118). Glial growth factor (GGF), and more specifically rhGGF2,
can restrict neural crest stem cells to differentiate into glial cells in vitro (Shah, et al., (1994)
Cell 77:349). In sulllll,a,~y, there are examples of neuregulin proteins affecting proliferation.
survival and differentiation of target cells.
Neuregulins provide signals for _rowth and differentiation of cells by binding and
activating several members of the EGFR subfamily of receptor tyrosine kinases: erbB2.
erbB3, and erbB4 (Padhy, et al.. (1982) Cell 23:865-872: Coussens, et al. (1985) Science
~: 1132- 1139: Kraus. et al., (1993) Proc Natl Acad Sci USA 90:2900-2904; Plowman. et
al., (1993) Nature 366:473-475).
--6--
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Information relating to neuregulins (also referred to as heregulins and erbB2 ligands)
provided in ~J.S. Patent No. 5,367,060, issued November 22, 1994 and U.S. Patent No.
~,530,109, issued June 25, 1996, is hereby incorporated by reference.
Proteoglvcans are mediators of growth factorfunction.
Cell surface proteoglycans and the extracellular matrix (ECM) are important
components in the regulation of growth, motility, and differentiation (for review, see
Adams, et al., (1993) Development 117:1183-1198). Of particular relevance to growth
factor signaling are the cell surface and ECM forms of heparan sulfate proteoglycans
(HeSPGs). HeSPGs are known to bind fibroblast growth factors, interleukin 2, hepatocyte
growth factor, platelet-derived growth factor B, and many others (Adams, et al. (1993)
Development 117:1183-1198). Methods for the purification of NDE (Yarden, et al., (1991)
Biochem 30:3543-3550; Peles, et al., (1992) ~ell 69:205-216), HRG-a (Holmes, et al.,
(1992) Science~:1205-1210), and ARIA (Falls, et al., (1993) ~72:801-815) all made
use of heparin-affinity chromatography.
Cancer
Activation or altered expression of erbB receptors seems to be an i.npo.lallt step in
the development of certain cancers. A specific amino acid replacement in the
transmembrane region of p185erbB2 produced a constitutively active receptor tyrosine
kinase leading to oncogenic transformation in EtNU-treated rats. Studies of transgenic mice
expressing this oncogenic form of neu (neuT) have shown that systemic ~Aminictration of
anti-erbB2 antibodies prevented the development of breast tumors for up to 90 weeks of a,~e.
Although this specific mutation has not been observed in human cancers. amplified
expression of the several different receptors of this family has been associated with
m~lign~ncy Among these cancers are: for erbB2 (HER-2, in human) -- adenocarcinoma of
breast, stomach, and ovary; for EGFR--m~lign~nt gliomas and squamous cell carcinomas,
and; for erbB3 and erbB4 -- breast carcinomas. Hence, both animal studies and molecular
diagnosis of cancer patients have implicated deregulated or ectopic expression of erbB
receptors in oncogenic transformation of a anti-HER2 (erbB2) monoclonal antibodies in
patients with metastatic breast cancer are most gratifying. Complete or partial remission
was observed in 11.6% of treated patients following 10 weeklv doses of rhuMab HER2.
These data build a strong case for pharmaceutical intervention directed at this sign~lling
pathway.
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Continuous stimulation of the erbB receptor signalling pathway could also be
mediated by excessive production of neuregulins. For example, mitogenic activity isolated
from bilateral acoustic neuromas and Schwannomas showed chromatographic properties
5 similar to native GGF purified from bovine pituitary. More recently the neoplastic
phenotype of a mutagenized, transformed Syrian h~mct~r fibroblast cell line has been
attributed to a hyperactive autocrine loop mr~ trd by neuregulins. Hence neuregulins are
e~.esscd at a place and a time which could trigger continuous proliferation of cells. It thus
would be highly desirable to develop the~d~e.l~ic agents for the treatment of cancers which
10 involves neuregulins.
Summarv of the I~
It now has been discovered that the activity of neuregulins is modulated by the
15 interaction of neuregulins with specific, determinable oligodisaccharide structures
("glycans") pendant from proteoglycans immobilized on a cell or extracellular surface. The
specific binding between a neuregulin and the oligodisaccharides is determined by the
structure and sequence of saccharides, typically ~iic~rch~rides~ and usually including the
sulfation pattern defined within the oligotlic~rch~ride unit, all of which together define a
20 binding site having relatively high affinity and specificity for a given neuregulin
("Glyceptor"). These oligodisaccharide surface-immobilized binding sites differ from we
cell surface receptors in that they typically lack the transmembrane signaling function
associated with ligand-receptor binding and, typically, bind to a site on a neuregulin
different from that recognized by the receptor binding site. This new observation presents
25 an opportunity for modulation and control of the physiological function of neuregulins.
In one aspect, the method of the invention comprises the step of subst~nti~lly
preventing or otherwise interfering with the interaction of a neuregulin of interest with its
Glyceptor sequence. By interfering in some way with the interaction between a neuregulin
30 and its Glyceptor sequence, one effectively interferes with the ability of a neuregulin to
interact with its receptor or other protein. The step of substantially preventing or otherwise
interfering with the interaction of a neuregulin with its Glyceptor sequence may be achieved
by aAmini.stering to an animal a molecule that acts as a Glyceptor sequence analog
("Glyceptor sequence antagonist"), and which competes with the Glyceptor sequence for
35 neuregulin binding. The Glyceptor sequence antagonist may act by preventing the protein
from interacting with its Glyceptor sequence, and/or by competitively displacing a protein
from its Glyceptor sequence seat. Useful Glyceptor binding sequence antagonists
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contemplated by the invention include soluble forms of the Glyceptor binding sequence~ or
any other synthetic or natural-sourced sequences that constitute or functionally mimic the
structure of the Glyceptor sequence, and which have a specific, predel~"llilled composition.
It has not been previously demonstrated or predicted that the interaction between the
vast majority of neuregulins and surface-immobilized GAG chains can show any degree of
specificity. The unanticipated discovery of such specificity now enables the development of
a kind of inhibitory molecule, not previously envisioned, that can s~eeirically antagonize the
action of a given neuregulin. For example, one can now more specifically antagonize the
action of neuregulins while not signifieantly affecting the action of other heparin-binding
growth faetors. Moreover, the diseovery now enables the development of analogs of
speeific gylcosalllilloglycan sequences that can act as agonists or have other utilities in vivo
including, for example, as im~ging or other tissue-targeting agents
Provided herein is an enabling description of the fundamental discovery and
resulting concept which permits the identification of such thc. ~ .J~ lly useful compounds.
Also provided are an enabling description of a process for identifying and isolating the full
range of speeific GAG binding sequences and an enabling description of a proeess for
utilizing sueh sequences to screen for therapeutic~lly useful eompounds, as well as a
deseription of the characteristics defining useful natural source-derived or synthetically
produced analogs, including those that act as antagonists to the protein-glycan interaction.
Thus, it is an object of this invention to provide means for modulating a biological
effect inr~ueed by a neuregulin-receptor or neuregulin-protein interaction by mo~ ting,
including preventing or otherwise h-t.,lrti,illg with, the interaction between a neuregulin and
the glycan sequence that binds it. Another object is to teach a method for identifying and
isolating analogs of a glycan sequence having specificity for a given neuregulin, and to teach
the use of these analogs as agonists or antagonists. Another object is to provide means for
testing various types of cancer by controlling undesired cell growth and proliferation, in
vivo. Yet another object of the invention is to provide means for therapeutic and
prophylactic manipulation of neuregulins and related biological molecule function,
including providing novel compositions. and providing a process for discovering useful
novel compositions. Such compositions have utility for altering pathologic responses by
inhibiting or enhancing the action of one or more members of the neuregulin family of
growth factors.
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These and other objects and features of the invention will be apparent from the
description, drawings and claims which follow.
The foregoing and other objects of the invention. the various features thereof, as well
5 as the invention itself, may be more fully understood from the following description, when
read together with the accompanying drawings.
Brief Des~ ,lion of the 1). dWi-~.'.
10 Figure l shows the effects of distinct glycosaminoglycans on rhGGF2-induced
phosphorylation of pl85 (putative neuregulin receptor) as d~rect~d by anti-phosphotyrosine
immunoblotting.
Figure 2A shows the effects of varying doses of heparin on rhGGF2-induced DNA
15 synthesis in cultured Schwann cells.
Figure 2B shows the effects of varying doses of heparan sulfate on rhGGF2-in~iucefl DNA
synthesis in cultured Schwann cells.
20 Figure 2C shows the effects of various glycos~rninoglycans on rhGGF2-in~l~recl DNA
synthesis in cultured Schwann cells.
Figure 3 shows the effects of 4-methyllumbelliferyl-~-D-xyloside (an inhibitor of
proteoglycan biosynthesis) on rhGGF2-inr~ e~l DNA synthesis in cultured Schwann cells.
Figure 4A is a schematic diagram of how neuregulins signal cells by interacting wilh
"Glyceptors".
Figure 4B is a schematic diagram of how "Glyceptors" se~uence antagonists interfere with
30 neuregulin signaling by preventing normal neuregulin binding to heparan sulfate
proteoglycans.
Figure 4C is a schematic diagram of how ligand antagonists can interfere with neuregulin
signaling by preventing normal neuregulin binding to heparan sulfate proteoglycans by
35 occupying neuregulin binding sites on heparan sulfate proteoglycans.
-10-
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Figure 5A shows the retardation of 125I-heparin mobility by varying concentrations of
rhGGF2 (affinity co-electrophoresis gel shown in inset).
Figure 5B shows the retardation of ~25I-heparin mobility by varying concentrations of
5 rhGGF2 (affinity co-electrophoresis gel shown in inset).
. Figure 6 shows the effects of increasing rhGGF2 concentrations on the retention of
rhGGF2/complexes in the filter binding assay.
10 Figure 7 shows the effects of three different polyanion compounds on rhGGF2/heparin
complex formation in the filter binding assay.
Figure 8A shows the effects of three polyanion compounds on rhGGF2-induced DNA
synthesis.
Figure 8B shows that the polyanion effects on rhGGF2-induced DNA synthesis are
reversible as evi~nce~i by the washout ("w.o.") conditions.
Figure 9 illustrates the chemical reaction of a primary amine, an aldehyde or Ketone, a
20 carboxylic acid and an isonitrile to form an acylaminoacid.
Figure 10 illustrates potential functional groups for the four sets of reactants; acids,
aldehydes or ketones, amines and isonitriles.
25 Figure 11 ~ se.lts the 96-well format used for the construction of a pilot combinatorial
library with various aldehydes and amines as reactants.
Figure 12 represents a generic formula for acylamino acid amides.
30 Figure 13 illustrates the effect of identified combinatorial compounds in the schwann cell
proliferation assay.
Figure 14 illustrates the structures of the four identified combinatorial compounds tested in
the schwann cell proliferation assay.
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WO 97/09051 PCTrUS96/14200
Detailed Description of the Invention
The invention provides a method for modulating the interaction between neuregulins
and their Glyceptor sequences. As used herein, the term Glyceptor sequence refers to an
5 oligodisaccharide sequence, including sulfated disaccharides. contained within a given
glycosaminoglycan immobilized on a cell or extracellular matrix surface and which binds,
with specificity, to a glycan-binding protein. Thus, as used herein, Glyceptor se~uence and
"neuregulin-specific glycosaminoglycan sequence" are used interchangeably and are
understood to be synonyms. The molecular surface structure on the neuregulin that interacts
10 specifically with a given Glyceptor sequence is referred to herein as a "glycan-binding site."
Typically, the Glyceptor sequence-effector protein interaction alone has no
transmembrane signal transducing or other direct effect. Without being limited to any given
theory, interaction of a neuregulin with its Glyceptor sequence serves to enable. or otherwise
15 enh~nce. the ability of the neuregulin to interact with its receptor or other protein and/or to
facilitate sign~lling. The ligand-receptor or other protein-protein interaction may occur on
the same cell surface to which the Glyceptor sequence is immobilized. may occur on an
~dj~-ent cell. or may occur on an extracellular matrix surface.
For example, exogenous heparin and HS inhibit both early and late Schwann cell
responses to rhGGF2. The rapid phosphorylation of erbB2 family ~ ..lbel~ is a con~ict~nt,
early event in cells responding to neuregulins (Peles, et al., (1993) BioFc~ays 1~:815-824).
In the case of cultured primary rat Schwann cells, a protein of Mr = 185 kD (pl85), a size
consistent with erbB family members. is phosphorvlated within 2 minutes of exposure to
rhGGF2 (Marchionni. et al., (1993) Nature 362:317-318). If HeSPGs play a critical role in
neuregulin binding prior to receptor tyrosine kinase activation, then exogenously applied
heparin or heparin-like molecules should serve as competitive, soluble neuregulin lcc~ ol~
and thereby inhibit p 185 phosphorylation. Schwann cells were exposed to various GAGs, or
to 15 ng/ml of rhGGF2 in the presence of these GAGs. As can be seen in Figure 1. heparin
inhibits p 185 phosphorylation in a dose-dependent manner and completely blocks
phosphorylation at 1.0 mg/ml. Though significantly less potent than heparin, HS treatment
exerts some inhibition at 1.0 mg/ml. Neither keratan sulfate, dermatan sulfate, nor
chondroitin sulfate show any inhibition of p 185 phosphorylation at the doses tested.
While pl85 phosphorylation in Schwann cells is detected within 2-3 minutes of
exposure to rhGGF2, flct~ct~hle DNA synthesis in response to the factor is assayed over a 48
hour period of exposure. Heparin and HS also inhibit this mitogenic response (Fi~ures 2A
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and B). As in the case of inhibiting pl85 phosphorylation~ heparin is a more potent inhibitor
of rhGGF2-induced DNA synthesis than HS. At the highest dose of rhGGF2 tested (32
ng/ml) heparin inhibits DNA synthesis by 90% at a concentration of 1.0 mg/ml; at a
concentration of 10 mg/ml HS inhibits DNA synthesis by 50-60 %. In marked contrast,
keratan sulfate, dermatan sulfate, and chondroitin sulfate show only modest inhibitory
activity at a concentration of 10 mg/ml (Figure 2C). Thus, heparin and the closely related
HS are the only GAGs tested that block Schwann cell responsiveness to rhGGF2. This
result is suggestive of a direct interaction between rhGGF2 and HeSPGs that can be
inhibited by exogenous heparin-like molecules.
Further, a known inhibitor of proteoglycan biosynthesis inhibits Schwann cell
lesponsiveness to rhGGF2. Rather than co.,.~ with cell surface HeSPGs in the binding
of rhGGF2, the effect of exogenous heparin and heparin-related molecules might be
interpreted as solely a result of sequestering rhGGF2 away from the cell surface through
interactions akin to heparin affinity chromatography. In order to perturb directly the
expression of Schwann cell HeSPGs, the cells were cultured in the presence of ~-xyloside;
cell permeable ~-xylosides afFect proteoglycan biosynthesis by inhibiting the ~tt~rhm~.nt of
GAG chains to proteoglycan core proteins (Robinson, et al., (1975) Biochem J 148:25-34;
G~llig~ni, et al., (1975) J Biol Chem ~2:5400-5406). In the case of Schwann cells, culture
in the presence of 0.5-1.0 mM ,B-xyloside for 1 week results in a 75% to 80% reduction in
GAG ~tt~ nt to cell associated proteoglycans, and a 10- to 12-fold increase in free GAG
chains in the culture medium (Carey, et al., (1987) J Cell Biol 1987:1013-1021). As can be
seen in Figure 3, culture of Schwann cells in medium containing 0.5-1.0 mM ~-xyloside for
48 hours followed by culture for an additional 48 hours in medium containing ~-xyloside
plus rhGGF2 results in a 60% to 90% decrease in maximum DNA synthesis relative to
control cells grown in medium containing rhGGF2 plus equivalent dilutions of the ~-
xyloside solvent, dimethylsufoxide. Thus the degree to which Schwann cell responsiveness
to rhGGF2 decreased in the presence of ~-xyloside was ~l~po~Lional to reported decreases in
cell-attached GAGs inc~ncecl by this inhibitor of proteoglycan biosynthesis (Carey, et al.,
(1987) J Cell Biol 1987:1013-1021).
Thus the activity of neuregulins is modulated by the interaction of these proteins
with specific. determinable oligodisaccharide structures ("glycans") pendant from
proteoglycans immobilized on a cell or extracellular surface. The specific binding between
35 glycan-binding proteins and the oligodisaccharides is determined by the structure and
sequence of s~crh~rides, typically ~iic~c ch~rides~ and usually including the sulfation pattern
defined within the oligo~ cch~ride unit, all of which together define a binding site having
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relatively high affinity and specificity for a given glycan-binding protein These
oligosaccharide surface-immobilized binding sites differ from true cell surface receptors in
that they typically lack the transmembrane signaling function associated with ligand-
receptor binding and, typically, bind to a site on the protein different from that recognized
5 by the receptor binding site. The above observations present an opportunity for modulation
and control of the physiological function of neuregulins.
In one aspect, the invention provides a method for mori~ ng the biological effect
in~ltlced by a neuregulin by interfering with, or otherwise preventing interaction of, a given
10 growth factor with its Glyceptor sequence which may be on the same cell as the growth
factor receptor, a neighboring cell. or the extracellular matrix. Thus, the method of the
invention comprises the step of subst~nti~lly preventing or otherwise in~e~re-illg with the
interaction of a neuregulin with its Glyceptor sequence. By interfering in some way with the
interaction between the neuregulin and its Glyceptor sequence. one effectively interferes
15 with the ability of the protein to interact with its receptor or other protein. The step of
~ub~l~nli~lly preventing or otherwise interfering with the interaction of the neuregulin with
its Glyceptor sequence may be achieved by ~rimini~ct~ring to an animal a molecule that acts
as a Glyceptor sequence analog ("Glyceptor sequence antagonist"), and which competes
with the Glyceptor sequence for neuregulin binding. The Glyceptor sequence antagonist
20 may act by preventing the neuregulin from interacting with its Glyceptor sequence, andlor
colllpetilively displace a neuregulin from its Glyceptor sequence. Useful Glyceptor
sequence antagonists coul~l..plated by the invention include soluble forms of the Glyceptor
binding sequence, or any other synthetic or natural-sourced sequences that constitute or
functionally mimic the structure of the Glyceptor sequence, and which have a specific,
25 predetermined co.--~,osi~ion.
The method is anticipated to be particularly useful in inhibiting undesired cellproliferation. such as can occur in a hyperproliferative disease, including cancers. As
neuregulins have been shown to stitn~ re migration of cells in culture, the method can be
30 useful in inhibiting m~-t~c~cis of tumors.
Alternatively, a molecule may be ~rlministered that is a glycan-binding protein
analog ("ligand antagonist" or "decoy ligand") which competes with the neuregulin for
Glyceptor sequence binding and. when bound, prevents or subs~antially inhibits the protein
35 from binding to the Glyceptor sequence. and/or competitively displaces protein from its
Glyceptor sequence. Such neuregulin antagonists include antibodies recognizing one or
more epitopes on the Glyceptor sequence and capable of blocking or otherwise interfering
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with the ligand binding site on the Glyceptor sequence. In one embodiment, useful
neuregulin antagonists include modified, soluble forms of a neuregulin that can still bind the
Glyceptor sequence with specificity but which, as modified, can not interact with the other
protein or receptor necessary for effecting the biological activity in vivo. Ligand
5 antagonists. including the modified glycan-binding effector protein, have an additional
utility as in vivo targeting agents. For example, an imaging or cytotoxic agent can be
complexed with the modified glycan-binding protein using standard means, such as by
covalent ~t~hmPnt, and be targeted to the site of action of the ligand thereby. Methods for
creating target-specific complexes are well-known and are well described in, for example,
10 the cancer the,~,lLic art. Still other useful ligand antagonists include synthetic organics
efining a molecule capable of mimicking the glycan binding site on the ligand.
In still another aspect, the invention contemplates a chimeric synthetic molecule
comprising at least two Glyceptor sequences covalently linked and having a conformation
15 sufficient to allow concurrent binding of each said protein-specific glycosaminoglycan
sequence to a specific glycan-binding protein. Preferably, each protein-specificglycosaminoglycan sequence binds to a different protein-specific glycosaminoglycan
binding site. The two binding sites may be tethered by means of a linker capable of acting
as a spacer as well as a crosclinking means. Preferably, the linker also allows free rotation
20 of the two sites independent of one another. The chimeric molecule is anticipated to have
particular utility as an agonist functioning, for example, to evoke receptor dimerization
and/or to help present a glycan-binding effector protein to a receptor, by binding both a
soluble effector protein and a surface bound protein.
In one embodiment, the Glyceptor sequence analogs of the invention useful as
antagonists and agonists have a binding affinity for neuregulins defined by a dissociation
con:,~al)t in the range of 10-6M to 10-12M, preferably having a dissociation constant of less
than S x 10-6M, more preferably less than 10-7M, or even 10-8M. As will be appreciated by
those skilled in the art, the higher the binding affinity of the analog, the lower the
concentration needed to induce a therapeutic effect in vivo, and the less likely the molecule
is therefore, to induce a toxic response.
In another embodiment, the overall length of the Glyceptor sequence analog
preferably does not exceed about 40A, and preferably is less than 40A, on the order of about
15-20A. Where the isolated Glyceptor sequence or an analog to be used comprises an
oligo~ rch~ride sequence, the molecule preferably has fewer than 20 monomer units,
preferably fewer than 16 monomer units, most preferably between 4-lS units. inclusive.
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Smaller oligosaccharide sequences may reduce specificity and larger sequences may
enh~nce toxicity. Preferred oligosaccharide antagonists also have an overall length of less
than 40 A. In all cases, the oligodisaccharide analogs contemplated have a specific,
predetermined composition, which serves to distinguish the compositions of the invention
5 from the endogenous soluble heterogenous oligosaccharide mixtures that may be found in
the body
Non-oligodi~rch~ride molecules useful as Glyceptor sequence analogs include
antibodies or other peptides capable of interacting specifically with the Glyceptor sequence
10 binding site on neuregulins. Still another useful class of molecules includes synthetic
organic molecules whose chemical structure functionally mimics that of a Glyceptor
sequence in binding specifically with a glycan-binding protein. These synthetic constructs
may or may not include carbohydrate and amino acid sequences. For example, suramin. a
polysulfonated naphthylurea, interferes with Glyceptor sequence-effector molecule
15 interactions. presumably by colllpe~ g with the Glyceptor sequence for the ligand binding
site. Here, the naphthylurea likely provides a scaffolding or backbone structure with an
a~p,op,iate distribution of sulfonates disposed about the heterocyclic rings to functionally
mimic the sulfated oligodisaccharide sequence that defines the protein-specific
glycosaminoglycan sequence. Thus, other synthetic organics can be generated having
20 unique backbone structures, and on which are disposed constituents of ~lo~fiate charge
and size. However derived, the Glyceptor sequence analog preferably has an ~lo~
distribution of functional substituents capable of interacting specifically with the glycan
binding site. For synthetic organics, the a~plo~liate substituents may be provided by
pendant carboxylates, phosphates, sulfonates, hydroxylates, amino groups, alkyl and
25 aromatic moieties.
In another embodiment, the synthetic organic Glyceptor sequence antagonist is
derived from the class of molecules whose structure is based on features of the glycan
binding site on the neuregulins with which the glycan analog interacts, the characteristics of
30 the class being defined by the generic struc'ture as described in detail herein below. As will
be appreciated by those having ordinary skill in the art of chemical synthesis. a
combinatorial library can be constructed containing multiple candidate sequences created
based on the generic structure. and the candidates tested in the screening assay presented
herein ~o identify useful analogs, including antagonists, having a~p,up,iate affinity for a
35 ligand. Similarly, a combinatorial library "kit" can be constructed containing isolated.
captured c~n~ te molecules defined by the generic structure, a neuregulin. and a means for
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s~ nhlg the candidates for their ability to bind said glycan binding protein with an affinity
above a preselected threshold level.
Thus, in still another aspect, the invention provides a method for identifying specific
oligodisaccharide sequences and functional analogs thereof which interact specifically with
neuregulins. As described herein, selected oligodisaccharide sequences having a defined
pattern of charged groups and a desired binding specificity and affinity for a given ligand
may be identified and used to create serum-soluble Glyceptor sequence analogs, useful per
se, or as screening reagents or templates for the rational design of polypeptide or organic-
based Glyceptor sequence analogs. In still another aspect, the invention provides a high flux
screening assay for identifying c~n~ t~- analog molecules.
The invention essentially consists of co~ oullds that are selected for their ability to
mimic a specific Glyceptor sequence-neuregulin interaction, and methods for their selection
and use. In one embodiment the compounds specifically inhibit the interaction of a
neuregulin with its cognate binding sequence in a glycosaminoglycan (GAG) chain, and
have particular utility for inhibiting that interaction in vivo. Figure 4 illustrates one
m~ch~nicm of the compounds provided by the invention, as it pertains to neuregulin-
Glyceptor sequence interactions, and where the Glyceptor sequence is a cell surface-bound
sequence. The invention also is anticipated to be useful for neuregulin-Glyceptor sequence
interactions where the Glycep~or sequence is bound to an extracellular matrix surface.
Figure 4A shows cells 210 having both Glyceptor sequences 218 and transmembrane
I~.cep~ol~ 216 on their surfaces. Neuregulin molecules 214 are aided in binding to receptors
216 by Glyceptor sequences 218. analogous to a Glyceptor sequence "hand" guiding a
neuregulin "key" into a receptor "lock" to activate cell proliferation through atransmembrane signal. In Figure 4B, Glyceptor sequence antagonists 225 are reacting in a
solution with neuregulin 214 and competitively stripping off neuregulin bound to Glyceptor
sequences 218 to modulate (here ~liminich) the activity of the neuregulin 214 on the cells
210. Figure 4C shows ligand antagonists 230 similarly modulating neuregulin activity. In
the case of neuregulins, use of the antagonists of the invention can interfere with, for
example, undesired cell growth.
Useful Glyceptor sequence analog compounds of the invention may include
antibodies or other related molecules capable of interacting and interfering with neuregulin-
Glyceptor sequence interaction by binding the "Glyceptor." Antibodies can be made by
standard means well known and described in the art (see, for example, Immunology. Roitt,
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et al., eds. Harper and Row, New York, 1989) using an isolated oligo~lic~ch~ride Glyceplor
sequence of interest as the antigen.
Other useful Glyceptor sequence analogs include soluble forms of GAG
5 oligodisaccharides or may be derived from the endogenous cognate GAG binding
sequences. Altematively, the analog may be a mimetic compound of the cognate GAGsequence. including a non-carbohydrate mimetic compound. Briefly, the Glyceptor
sequence analog compositions obtained through this enablement may be sulfated
glycos~minoglycan oligorlic~rch~rides of a predetermined, defined sequence, including that
10 of a Glyceptor sequence found in nature, or they may be synthetic mimetics thereof. A
process is provided herein for the selection of such suitable co~ unds.
A means for designing useful synthetic Glyceptor sequence analogs is to compare
sequences of a number of glycan-binding growth factors and collll,alc regions of homology
15 and non-homology. This information, together with an investigation of the known three-
dimensional structure of several such proteins permits one to identify a physical "map" of
the glycan binding site on the effector protein. (See, for example, Baldwin, et al., (1991)
PNAS 88: 502. and Clore, et al. (1991) J. Mol. Biol. 27: 611.) Characteristically, the glycan
binding site is an extended band that stretches across the surface of the protein rather than,
20 for example, defining a pocket, as may occur in enzyme-substrate interactions. Moreover,
the glycan binding site typically is defined by a particular distribution of positively charged
residues that interact favorably with the anionic charge on the GAG, and also may include
other residues that can prevent or limit interaction with particular GAGs, either by steric or
ionic interference.
The svnthetic organic analog molecules useful in the invention are synthetic
molecules that mimic the action of naturally-occurring GAG binding sequences, whether the
synthetic molecule is naturally derived, synthetically produced, substantially
oligo-iic~ch~ride in nature, or subst~nti~lly free of carbohydrate. Such compounds may
30 contain sulfate esters or negatively charged groups at precise locations in their structures that
interact with the basic side chains that characterize the glycan binding site. In this case the
analog specifically mimics the binding structure of the natural-sourced sequence.
Altematively, the analog may comprise functional groups that interact with other. different
side chains in the glycan binding site, sufficient to allow specific binding interaction of the
35 analog with the glycan binding site. but by means of different contacts. In either case the
analog can be said to functionally mimic the protein binding structure of the native
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Glyceptor sequence. Of course, a molecule that is a specific structural mimetic also will be
a functional mimetic.
In one embodiment. candidate compounds obtained from nature may be screened as
described herein. Alternatively, candidate compounds can be formulated utilizing an
approach that includes consideration of size and charge distribution of the Glyceptor
sequence and glycan binding site with which it interacts. Interaction may be achieved by
contacts analogous to those made by the endogenous glycan sequence, or by different
contacts that produce a functionally equivalent specific interaction at the site. As will be
appreciated by those having ordinary skill in the art, analogs having higher or lower binding
affinities than that of the endogenous sequence can be obtained by this method.
Preferably, and as described herein, a combinatorial "library" containing a group of
designed candidates is created, each molecule having a different composition, and the group
screened for molecules that bind the ligand of interest above a threshold affinity. A
threshold affinity of a c~n~ t~ Glyceptor sequence analog for a preselect~-l glycan-binding
protein readily may be determined by means of a standard competition assay, and/or by gel
shift assay, as described and exemplified in Examples 3, 7 and 8, below. Preferably,
candidates will exhibit binding affinities .~;pl~ sented by low dissociation constants, e.g.,
having Kd values in the range of 10-7M to l0-l2M, preferably less than 10-8 or even less
than lO-9M.
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Screening Protocol
The screening procedure for identifying lead compounds selective for binding to and
inhibitin~ neuregulins is as follows:
I. Combinatorial svnthesis reaction products--are the contents of individual wells including
, solvent. starting materials, intermediates and final products. This represents the initial
endpoint of the synthetic chemistry and the beginning of the biochemical screening steps.
0 ~. Primarv binding hits--are combinatorial svnthesis reaction products that block a
specified level of the binding of heparin to rhGGF2 when screened in a 96 well format.
3. Confirmed binding hits--are primary binding hits that have reproducible blocking of
heparin binding to rhGGF2 (to a specified level) when analyzed in individual assays.
15 Complete binding inhibition curves are performed to determine the-approximate ICSO values
for all co,./i/"lcd binding hits.
4. Selected binding cann7i~77ates--are confirmed binding hits whose ICSO values are
compalaLi~/ely low and whose final products are present in sufficient yield in the synthcsis
20 reaction products to warrant resynthesis, product purification and extensive analytical
chemistry and binding studies. Some of the selected binding candidates will advance to the
biological screening assays as pure co~ uunds.
5. Preliminarv ~ioactive car.~di~tes--are selected binding candidates-that are non-toxic and
25 block responses of cultured Schwann cells to rhGGF2 in in vitro assays.
6. Confirmed bioactive candidates--are preliminary bioactive candidates that are tested on
human tumor cell lines and found to be non-toxic and active in growth arrest assays.
30 7. Lead compounds--are confirmed bioactive candidates that are least 10-fold selective for
binding to and inhibiting rhGGF2 compared to other heparin-binding growth factors.
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A) Preliminary Screening
The solution phase binding assay described above is formatted into a hi~h
throughput screening system eo select compounds that antagonize the binding of heparin to
5 rhGGF'. Briefly, a 20nM solution of rhGGF2 is incubated for 60 minutes at roomtemperature with the test compound(s) and a trace amount of high affinity GAG fr~m~nt,
which is tyr~min~t~d at the reducing end and io-1in~te-1 The screening assays can be carried
out in 96 well Hybridot Manifold (BRL) that allows for rapid filtration through a
nitrocellulose sheet. The sheet contains 96 radioactive "spots", which are subsequently
10 counted in a Wallac microbeta microtiter plate scintillation counter. This approach has
proven to be very efficient, and can be used routinely to screen up to 12 microtiter plates
(960 wells)/week. To reduce the potential for artifacts, any binding effects of the starting
components can be assayed. In all cases, the individual components of the isonitrile
chemistry, the acid, amine and aldehyde. cannot be due to unreacted starting materials.
Libraries are analyzed first in a single point survey. Primary binding hits fall into
two groups: l) wells that blGcked > 50% of binding and 2) wells that blocked 20-50% of
binding. All of the primary binding hits are rtsc.~,~,ned and approximate IC50 values are
calculated on the confil---cd binding hits. These compounds also are tested for color or
20 ~hPmic~l quenching, and any compound that interferes is elimin~teA In order to maximize
the possibility of finding an adequate number of selected binding candidates from the
biochemical s~ ,nillg of these libraries, a "cut-off" of 40 uM can be set.
In order to ascertain quickly if the library construction rationale is correct. the first
25 few confirmed binding hits can be analyzed more extensively. Early confirmed binding hits
are resvnthesized and purified; and chemical structure is characterized by HRES-MS, to
show a single. calculated molecular ion peak, as well as characteristic fragmentations,
demonstrating that these confirmed binding hits are indeed the predicted acylaminoacid
amides.
Once the prioritized confirmed binding hits are identified (IC50<10 uM, etc.), the
compounds can be synthesi7e~i independently on a larger scale. For each synthesis, first the
crude reaction is again checked by HPLC analysis, and is re~c~es~ed in the bindin_ assay. If
these analyses provide the expected data (i.e., retention time and binding inhibition similar
35 to the initial screens), then the scaled up product is subjected to preparative-HPLC. All
major peaks are collected and assayed. Fractions that show activity in the bindin~ assay are
then taken to dryness, and the solid material is analyzed by high-resolution mass
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spectroscopy (HRES-MS), NMR or chemical analysis. Binding analyses of the pure
compounds to rhGGF2 can be repeated several times to obtain IC50 values. Data from the
analysis of each of these selected binding candidates can then be co~ a.ed in order to
prioritize those compounds that will advance to the next stage. Next, selected binding
candidates are screened in cell culture assays to validate their potential as antagonists of
neuregulin-erbB signaling.
Cultured primary rat Schwann cells provide the first bioassay system for testing the
bioactivity of rhGGF2 antagonists. The purification, cloning and expression of glial growth
factors, including rhGGF2~ is based on the proliferative response of Schwann cells to
neuregulins (Marchionni, et al., ( l993) Nature 362: 3 l 2-3 l 8), and this response provides a
highly reliable in vitro assay system to monitor the activity of rhGGF2.
Compounds (selected binding candidates) can be tested first for any overt toxicity on
cultured cell lines (e.g., NIH-3T3) or primary rat Schwann cells. Non-toxic compounds can
be tested for the inhibition of both early (neuregulin l~,c~>Lor phosphorylation) and late
(DNA synthesis) Schwann cell responses to rhGGF2. and subsequently for reversibility of
action. Using a high throughput assay for DNA synthesis, the first bioassay for selo~t~-l
binding can~i<lntes will be inhibition of rhGGF2-stimulated Schwann cell DNA synthesis.
IC50 values can be determined and compared to the binding data.
Once inhibition of rhGGF2-stimulated DNA synthesis has been established for a
specific compound, then pl85 neuregulin receptor phosphorylation can be assayed as
follows: Schwann cells are pl~pal~d as described above for the DNA synthesis assay and
plated in 24-well plates at a concentration of 250.000 cells/0.5ml in DME~/5. Test samples
(rhGGF2 and various concentrations of selected binding ca~ ~tes are premixed for 60
min. to equilibrate binding) are added to the cells for 2.5-3 minllt~c at 37~C. The media is
aspirated and 50 ul of SDS reducing sample buffer (preheated to 65~C), is added to each
well. After scraping and tliLula~ g the wells, the contents are transferred to microcentrifuge
tubes, boiled for l0 minlltes, and subjected to polyacrylamide gel electrophoresis. Proteins
are electrotransferred to nitrocellulose membranes. The membranes are pre-soaked in
transfer buffer containing 200 uM sodium orthovanadate (Sigma) and the transfer is
performed using the same buffer. Membranes are probed with the recombinant anti-phosphotyrosine antibody, RC20H (Transduction Laboratories), and immunoreactive bands
visualized using ECL chemiluminescence reagents (Amersham) per manufacturer's
instructions. Compounds that block both the receptor phosphorylation and the DNA
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synthesis activities of rhGGF2 will be analyzed further for reversibility of action in washout
exLe,i",~l1ts, as described in Sudhalter. et al. (Sudhalter, et al., (1996) Glia 17:28-38).
The results from these initial bioassays will identify compounds with bioactivity in
5 the well-characterized Schwann cell system. At the conclusion of this stage of the screen,
potential antagonists of neuregulin-erbB signaling should satisfy several key criteria,
, including: I) reversibly block both the early and the late responses to rhGGF2; and 2)
display a rank order of potency in vilro that matches the biochemical analyses. Compounds
that pass successfully through this first stage of cell-based screening are designated as
primarv bioactive can~i~lates In order to determine if primarv bioactive can~li~7tes have
activity on cells that are more relevant to cancer cells, a second tier has been added to the
cell-based screening stage, which involves the use of human tumor cell lines.
A number of human nlmor cells lines are known to respond to neuregulins. Some
15 lines also express one or more neuregulin isoforms. For exar,nple, the breast adenocarcinoma
lines MDA-MB231 and MDA-MB~53 were utilized in the erbB2 receptor phosphorylation
bioassays that were used to rnonitor the purification of NDF and heregulin (Wen, et al.,
(1992) ~ell ~ ~59-572; Holmes, et al., (1992) Science ~:1205-1210). Glial tumor lines
U87MG (astrocytoma) and U13876 (glioblastoma) express neuregulin and erbB ,,ceptor
20 transcripts detected by northern blotting. These lines can be obtained from the A",c.ican
Type Culture Collection and will use them to further evaluate primary bioactive ca)~i~ntes
Since erbB receptor activation and proliferation may be stimul~tt-d via pdla,lille or autocrine
neuregulin signaling in these lines, the lines provide opportunities for two types of
bioassays. First, in a manner similar to Schwann cells. we will determine if test compounds
25 can block the responses to exogenous rhGGF2. Second. it can be determined if the
compounds can arrest the growth of cell lines grown in low-to-moderate concentrations of
fetal calf serum (FCS) without added rhGGF2. The concentration of FCS to be used can be
determined empirically for each line.
Thus these e~c~i"-~ ts with human tumor lines should help to demonstrate the
applicability of the primary screening system beyond neural cell growth and further validate
the overall ~loach.
The instant application also provides novel process for selecting useful analogs of
the glycan-binding protein Glyceptor sequence interaction from a collection of randomly
obtained or rationally designed candidate compounds. In one aspect. co~,lpoullds which are
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W O 97/09051 PCTrUS96/14200
selected by the process described herein will have the useful property of specifically
displacing neuregulins. from their functionally active locations.
For example. use of neuregulin antagonists of the invention can interfere with
S undesired cell growth. And thus, can be used to prevent tumor growth.
Using this type of analysis, a generic structure useful in creating candidate Glyceptor
sequence analogs can be generated, particularly useful as part of a combinatorial library of
candidates. A preferred generic structure is presented in Figure 12. The generic structure
10 defines an oligomeric structure composed of at least two monomeric units and one or more
functional groups pendant therefrom. The position and composition of the pendantfunctional groups in the generic structure are designed to provide a "library" of useful
substituents which can inuract with the side chains de~lning a glycan binding site, whether
by making the same contacts as that of the naturally occurring Glyceptor sequence, or by
15 making differen~ eonta~;L~.
The Glyceptor sequence antagonist or nc~ ulin antagonist for use as a therapeutic
agent p,'tpd.~d as described herein may be provided to an individual by any suitable means,
preferably directly or systemically, e.g., parenterally, preferably in combination with a
20 pharrn~e~ltic~lly acceptable carrier. As used herein, "a physiologically acceptable carrier"
includes any and all solvents, dispersion media, antibacterial and antifungal agents that are
non-toxic to human, and the like. Particularly contemplated are pharmaceutically acceptable
salts. which may be base salts, alkali metal salts, and alkaline metal salts. The use of such
media and agents as pharrn~eutic~lly active substances are well known in the art.
Where the th~.a~ Lic agent is to be provided directly (e.g., locally, as by injection.
to a desired tissue site), or parenterally, such as by intravenous, ~ub.~ ;.neolls, intr~rnu~clll~r?
intraorbial, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal,
intracisternal, inL,dpe,iLoneal, buccal, rectal, vaginal, intranasal or by aerosol administration,
30 for example, the therapeutic agent preferably comprises part of an aqueous solution. The
solution is physiologically acceptable so that in addition to delivery of the desired
thc-d~ Lic agent to the patient. the solution does not otherwise adversely affect the patient's
electrolvte and volume balance. The aqueous medium for the theldpeuLic agent thus may
comprise normal physiologic saline (O.9~o NaCI, 0.1 SM), pH 7-7.4.
Useful solutions for oral or parenteral ~ nini~tration may be prepared by any of the
methods well known in the pharmaceutical art, described. for example, in Remington's
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Pharm~reutical Sciences, (Gemlaro, A., ed., Mack Pub., 1990). Formulations may include,
for example, useful excipients to control the release of the thelapeulic agent in vivo. The
thcldpGuLic agents also provided herein may be ~rlminictered alone or in combination with
other molecules known to have a beneficial effect in, for example, modulating the
5 infl~mm~tory response. and/or which may enhance targeting of the agent to a desired tissue
or cell surface. Other useful cofactors may include symptom-alleviating cofactors. including
, antiseptics. antibiotics, antiviral and antifungal agents and analgesics and anesthetics.
The compounds provided herein can be formulated into pharm~relltiç~l compositions
10 by admixture with pharmaceutically acceptable nontoxic excipients and carriers. As noted
above, such compositions may be ~ ,d for p~GI~tt~dl ~rlmini~tration, particularly in the
form of liquid solutions or suspensions; for direct ~Aminictration, in the form of powders,
nasal drops or aerosols.
Compounds of the invention can be employed, either alone or in combination with
one or more other the.~eutic agents as rli~cucsec~ above, as a pharm~re~ltir~l composition in
mixture with conventional excipient, i.e., pharm~reutically acceptable organic or inorganic
carrier substances suitable for parenteral, enteral or intranasal application which do not
deleteriously react with the active compounds and are not deleterious to the recipient
20 thereof. Suitable pharmaceutically acceptable carriers include but are not limited to water,
salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose,
magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid
monolgycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose,
polyvinylpyrrolidone, etc. The pha~ re~llir~ paldLions can be sterilized and if desired
25 mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents.
emulsifiers. salts for influencing osmotic pressure. buffers, colorings, flavorings and/or
aromatic substances and the like which do not deleteriously react with the active
compounds.
The compositions can be formlll~tccl for parenteral or oral ~flminictration to humans
or other m~mm~lc in therapeutically effective amounts, e.g., amounts which provide
a~lop,iate concentrations of the agent to target tissue for a time sufficient to inhibit the
desired ligand - Glyceptor sequence interaction of interest. as described above.
.
~s will be appreciated by those skilled in the art, the concentration of the compound
described in a therapeutic composition will vary depending upon a number of factors,
including the dosage of the drug to be ~lminictered~ the chemical characteristics (e.g.,
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hydrophobicity) of the compounds employed. and the route of ~lminictration. The preferred
dosage of drug to be ~rimini.ctered also is likely to depend on such variables as the type and
extent of tissue loss or defect, the overall health status of the particular patient, the relative
biologieal efficacy of the compound selected, the formulation of the compound, the presence
5 and types of excipients in the formulation, and route of ~-lminictration. In general terms, the
compounds of this invention may be provided in an aqueous physiological buffer solution
containing about O.OOl to 01% w/v compound for parenteral ~iminictration. Typical dose
ranges are from about lO ng/kg to about I g/kg of body weight per day; a ~Icfell~,d dose
range is from about O.l ug/kg to lOO mg/kg of body weight. It will be appreciated by those
10 having ordinary skill in the art that analogs having higher binding affinities typically will
require lower total ~1minictration eoncentrations than those having eo.ll~a,dtively lower
binding affinities.
ARBREVIATIONS
Throughout the present specification the following abbreviations and terms are used:
Glyceptor sequence - protein-specific glycosaminoglycan sequence; HS - heparan sulfate;
HSPG - HS proteoglycan; GF - growth factor; dp - degree of polymerization (e.g. for a
20 ~lica~ch~ride~ dp=2, etc.); GleA - glueuronie aeid; IdoA - iduronic acid; IdoA (2s) - iduronic
aeid 2-sulfate; GleNAe - N-aeetyl glucosamine; GlcNS03 - N-sulfated glucosamine;GlcNS03 (6s) - N-sulfated glucosamine 6-sulfate; GlcA (2s) - glucuronic acid 2-sulfate;
PBS, phosphate-buffered saline.
25 Growth factors known to bind heparin
- Basic fibroblast growth factor (bFGF)
- Acidic fibroblast growth factor (aFGF)
- Heparin binding epithelial growth factor (HB-EGF)
- Recombinant human Glial Growth Factor 2 (rhGGF2)
Provided below are descriptions of experiments disclosing how to identify usefuloligo-licaeçh~ride sequences having specificity for neuregulins (Examples l, 2, 5 and 6). and
how to test the effeetiveness of identified Glyeeptor sequences and their analogs for their
effeetiveness in blocking neuregulin-Glyceptor binding (Example 3) and on cultured cells to
35 evaluate activity in vitro (Examples 4 and 7).
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Example 1 Heparin and ~S bind rhGGF2 with high affinity - d:emons~ration by
a~inity co-electrophoresis.
To measure the binding affinity of peptide growth factors for various GAGs,
horizontal agarose gel electrophoresis of [125I]-GAG chains was performed as described by
Witt and Lander (Witt, et al., (1994) Curr Biol 4:394-400).
Heparin from porcine intestinal mucosa (Sigma) was derivatized with tyramine andradiolabeled with l2sI (San Antonio. et al., (1993) l~iochem ~2:4746-4755). This material
10 was ~les~ltt-d on a PD-10 column and subjected to gel filtration on a Sephadex G-50 column.
Samples of approximate molecular weight 4000 were used in this study. HS 16mer (HS16)
was purified from porcine intestinal mucosa. HS 16 was labeled with 125I and subiected to
gel filtration on a Sephadex G-50 column (Lee, et al., (1991) Proc ~atl Acad Sci USA
88:2768-2772).
Varying concentrations of rhGGF2 were incorporated into individual lanes in a 1%agarose gel, and [125I]-heparin or [l25I]-HS (-3000 cpm per sample) were electrophoresed
through the gel in a buffer system of 0.1 M sodium acetate, 50 mM MOPSO, 0.5% CHAPS,
pH 7Ø Tmm~ t~-ly following ele~LI~plloresis, gels were dried with forced warm air and
20 the distribution of radioactivity vi~n~li7çd using a phosphorimager (Fuji BAS 1000).
Electrophoresis of the negatively charged oligos~rcll~rides through the protein-cont~ining
wells results in an alteration of mobility of the GAG chains that reflects the equilibrium
binding of the protein to the GAG. Retardation coefficients (R) can be calculated for each
concentration of protein. The appa.ent dissociation constants are equated to the protein
25 concentration at which the GAG is half maximally retarded using the forrnula R =
R,/(l+Kd/[protein]) (Lee, et al., (1991) Proc Natl Acad Sci USA 88:2768-2772).
This technique allowed the deterrnination of apparent dissociation constants by
observing the retardation in electrophoretic mobility of ['25I]-heparin (Figure SA. inset) and
30 ['2sI]-HS (Figure SB, inset) by a range of rhGGF2 concentrations embedded within agarose
gels. The dissociation constants were calculated for heparin (Figure 5A) and HS (Figure
SB~ by determining the concentration of rhGGF2 that produced a half-maximal mobility
shift of the labeled GAGs (Lee, et al. (1991) Proc Natl Acad Sci USA 88:2768-2772). The
values observed were Kd = 9.7 nM for heparin, and Kd = 22 nM for HS.
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FYs~mrl~ 2 Heparin binds rhGGF2 with high affinity - demonstration by solut~on
phase binding and trapping of heparin/rhGGF2 complexes on nitroce~ os~
Another method for quantifying the binding interactions between proteins and GAGs
5 is to allow such binding to occur in solution, and then trap the resultant protein-GAG
complexes on nitrocellulose filters by suction filtration. Filter binding was used as an assay
for compounds that block rhGGF2 binding to a radiolabeled heparin fragment. Equilibrium
binding was achieved in PBS with the rhGGF2 present at varying concentrations, and the
concentration of the [l25I]-heparin fragment m~int~ined below the Kd. Once equilibrium was
reached (1.5 h), rhGGF2-GAG complexes were ca~u.. ,d by suction filtration onto a 0.45
~lM nitrocellulose filter. The filter was then dried at 42~C for 30 ~ ules and evaluated using
a matrix array scintillation detector (Wallac 1205 Betaplate). Only protein-GAG complexes
are retained on the filter, and thus the amount of heparin retained on the filter was plotted.
As can be seen in Figure 6. ['25I]-heparin retention on the filter is dependent on the
15 concentration of added rhGGF2. If the starting concentration of GAG chain is below the Kd
for rhGGF2 binding, then the apparent Kd will be the rhGGF2 concentration at which half
the input GAG chains are retained on the filter. In this case, that concentration was 5.5 nM,
a value co...~able with the Kd determined in Example 1.
FY~ , le 3 Neparin-rhGGF2 binding is i~hihite~l by synthetic antagonists, but not by
other highly s~f~fe~ GAGs.
The filter binding assay is also a useful tool for comparing quantitatively the ability
of various compounds to competitively inhibit the binding of rhGGF2 to labeled heparin:
Filter binding was used as an assay for compounds that block rhGGF2 binding to aradiolabeled heparin fragment. Equilibrium binding was achieved in PBS with the rhGGF2
concentration m~int~ined at 40 nM (just above the Kd) for each reaction, and theconc~..t,dlion of the [~25I]-heparin fragment ,l~A;l~Aille~i below the Kd. Test compounds were
30 added to the assay solution at 10 ,.LM and were serially diluted 2-fold for a total of 9
concentrations per compound. Once equilibrium was reached (1.5 h), rhGGF2-GAG
complexes were captured by suction filtration onto a 0.45 ~M nitrocellulose filter and
analyzed as described in Example 2; the amount of heparin retained on the filter in the
absence of competitor was plotted as 100%. Disruption of protein [125I]-heparin complexes
35 by col~lyetilor results in decreased radioactivity retained on the filter; the point at which 50~c
of the counts are retained was calculated to be the IC50 for the drug.
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Figure 7 shows the dose-dependent inhibition of rhGGF2 binding to heparin by
suramin and two related polyanion compounds, GL12 and NF066. The cc,~ c;LiLion curves
were used to calculate the IC50 of each of these compounds (Table 1).
C;rowth Factor Tested
rhGGF2 kE~E
GL12 0.9 mg/ml 3.8 mg/ml
Suramin 2.2 mg/ml 21.6 mgfml
NF066 2.0 mg/ml 25.0 mg/ml
15 Table 1. Comp~rison of ICSo values of polyanion com~ lc on rhGGF2 and
bFGF binding to heparin. Kd of binding to heparin is 5.5 nM for rhGGF2, and 5.0 nM for
bFGF. ICso values were determined using the filter binding assay. IC50 values were
determined by calculating the dose nececs~ry to achieve half-maximal inhibition in Figure
SB.
Note that the rank order of inhibitory activity of these compounds, GL12 >> suramin >
NF066. is the same for both rhGGF2 and for bFGF. The filter binding assay also
demonstrates that the same GAGs do not compete with rhGGF2-heparin binding at the doses
tested. This lack of Colllp~,liLion by a variety of highly sulfated GAGs demonstrates the
25 specificity of the interaction between rhGGF2 and heparin-type sulfated polymers (Table 2).
Glycosalllilloglycan IC~.. to displace rhGGF2 from heparin
Heparin .06 mg/ml
Chondroitin sulfate 30.0 mglml
Dermatan sulfate > 12.5 mg/ml
Keratan sulfate > 25.0 mg/ml
Table 2. Comr~rison of IC50 values of GAGs on rhGGF2 bi~ to heparin. Kd
of binding to heparin is 5.5 n~ for rhGGF2. IC50 values were determined using the filter
binding assay, and were determined by calculating the dose necessary to achieve half-
m~xim~l inhibition as in Table l.
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E,xample 4 Synthetic antagonists of heparin-GGF b~nding inhibit Schwann cell
responsiveness to rhGGF2.
The data concerning suramin and its analogs (Example 3) present an additional
5 pharmacological means of directly perturbing rhGGF2 interactions with Schwann cell
surface HeSPGs:
Sciatic nerve Schwann cells from 3 day old rats were purified by the methods of
Brockes (Brockes (1987) Meth Fn7~ymol 147:217-225). Cells were plated on tissue culture
10 plastic precoated with poly-D-lysine (PDL, Collaborative Research) in low glucose
Dulbecco's Modified Eagel's Medium (DMEM, Fisher/Me~ t~ch) supplem~ted with 10%
heat-inactivated fetal bovine serum (Hyclone). This medium is referred to as DMEM/10.
After 24 hours the medium was replaced with fresh DMEM/I0 containing 10 mM cytosine
arabinoside (Sigma). Two to 3 days later, the medium was removed and replaced with
15 DMEM/I0 supplemented with partially purified bovine GGF (th'e carboxymethylcellulose
fraction of bovine pituitary extract, GGF-CM (Goodearl, et al., (1993) J Biol Chem
~:18095-18102)) and 5 mM forskolin (Calbiochem). When the cells reached confluence,
they were trypsinized and treated with anti-Thy I (Tl lD7e, Serotec), and rabbit complement
(Gibco) to remove cont~min~ting fibroblasts. Cells were then plated in DMEM/I0
20 supplemented with GGF-CM and forskolin. Upon further expansion, cell stocks were used
for assay purposes ~t~ d below or were frozen for future use.
The DNA synthesis assay was performed according to the method of Brockes
(Brockes, (1987) Meth F.n7ymol 147:217-225) with slight modifications. Schwann cells
25 were prepared for assay by growing to confluence on PDL coated plastic in the presence of
GGF-CM and 5 mM forskolin. The cells were then withdrawn from growth factor and
forskolin for 3 days, trypsinized, and plated in 96-well plates at a concentration of 10,000
cells/100 ml in DMEM supplemented with 5% serum (DMEM/5). The next day test samples
were added to the wells and 24 hours later either ['~5I]-UdR~ or [methyl-3H]thymidine was
30 added. The cells were harvested approximately 18 hours later using a Tomtec Harvestor and
the samples counted using a Wallac 1450 MicroBeta liquid scintillation counter.
Figure 8A demonstrates that not only do GL12, suramin, and NF066 inhibit
rhGGF2-induced DNA synthesis in a dose-dependent manner, but the rank order of their
35 potency matches that of their potency in inhibiting rhGGF2 binding to heparin in the cell-
free assay (Figure 7 and Table 1, abo~e). When cultured in the presence of 60 ng/ml
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rhGGF2. half maximal inhibition of Schwann cell DNA synthesis by GLl2 is ~ 5 mM,~Uldlllill is ~20 mM, and NF066 is ~35 mM.
The effects of suramin and its analogs are reversible. and not due to non-specific
5 cytotoxicity. This was demonstrated by "washout" experiments where Schwann cells were
maintained in normal medium. lO0 mM suramin, or lO0 mM GLl2 for 24 hours. After
washing with fresh control medium, cultures were transferred to one of four experimental
media with labeled nucleoside (control m~-liurn, 60 ng/ml rhGGF2, lO0 mM test co-ll~oulld,
or rhGGF2 plus test compound). Total DNA synthesis was assayed 48 hours later as in the
10 previous experiments. As can be seen in Figure 8B, cells m~int~ined in test compounds for
the first 24 hours and then transferred to control medium (washout conditions) displayed
levels of DNA synthesis idencical to those of cells m~int:~ined in control medium or test
cu"l~ound through the entire culture period. No sign of overt cytotoxicity was observed by
light microscopy in any of the above conditions. Cells transferred from control medium to
15 medium containing rhGGF2 showed expected increases in DNA synthesis. Schwann cells
transferred from test compound medium to compound-free medium cont~ining rhGGF2
displayed DNA synthesis levels comparable to rhGGF2-treated cells that had not been
pretreated with test coll,~oullds. Thus the inhibition of rhGGF2-indureA DNA synthesis by
suramin and its analogs appears to occur through the direct inhibition of rhGGF2 binding to
20 cell surface HeSPGs.
FYS~ 5 Combinatorial c*~ ~ library construchon.
Combinatorial library construction exploited the significant advantage of isonitriles in that
25 they can be used to carry out numerous single step, high yield solution phase reactions
(Gokel. G., et al.. in Isonitrile Chemistry. (Ed. Ugi, I.), Academic Press, New York, 40). A
combinatorial isonitrile (Ugi) chemistry technology was developed which produced organic
colll~ou,,ds in a 96-well format. By not relying on solid phase chemistry, it was feasible to
syntheci7e many compounds in parallel with one (or a small number) of compounds per
30 well. This avoided artifacts which can arise from assaying complex mixtures of weakly
active compounds (non specific binders) and simplifies structural identification of "hits".
We investigated the reaction of a primary amine, an aldehyde or ketone, a carboxylic acid
and an isonitrile to form an acylaminoacid amide as shown in Figure 9.
This type of compound was synthesized by mixing equimolar amounts of the
four reactants in a polar solvent. For the libraries described below R2 is not H and thus
each reaction produces two isomeric compounds. For simple alkyl substituted starting
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materials. yields in excess of 90% have been reported (Lee. et al., (1991) PN~S USA
88:2768-2772). In addition, there are thousands of commercially available amines,
aldehydes and carboxylic acids which enable sets of compounds to be synthesized with
virtually any type of functionality.
Validation of Starting Materials Prior to using the isonitrile chemistry, the
starting materials were determined to have had the solubility and reactivity plo~elLies
that are consistent with high yield product formation. Typically, each potential reactant
was substituted for the corresponding amine, aldehyde or carboxylic acid from the
10 "model reaction" described above. Results from reverse phase HPLC analyses showed
that this chemistry is extremely robust in that excellent yields of product are obtained
from aromatic as well as Z~ h~tic amines, ketones as well as aldehydes and a range of
sterically hindered carboxylic acids. From this type of ex~fi-~-cnt, a set of 20 amines,
16 aldehydes/ketones and 24 carboxylic acids were qualified which consistently
15 produced high yields. In addition. a series of isonitriles were evaluated and n-butyl,
cyclohexyl, benzyJ and methyl acetate all gave excellent results. The functional groups
.ep.esented in these four sets of re~rt~ntc are shown in Figure 10 (the arrow on each
structure in~ir~teS the point of ~t~ch..~ t to the amino acid scaffold).
The sets of re~t~ntc were chosen to have minim~l overlap in structure. Thus the
amine substituents are different and complementary to the aldehyde/ketone, or
carboxylic acid substituents. The result was that the current set of re~t~ntc contained 58
different pharmacophores. The functionality was chosen to: I ) mimic the side chains of
amino acids; 2) to contain a variety of heterocyclic ring systems as is found in many
existing protein-binding drugs; and 3) to be rich hydrogen bond forming functional
groups such as phenols. alcohols, esters, ethers, etc. The later feature is based on the
assumption that hydrogen bond forming functionality is ap~.~.p.iate for a drug which
will bind a carbohydrate binding pocket on the growth factor. One additional desired
feature for the library is anionic functional groups. Since carboxylic acids are inherently
incompatible with this chemistry, methyl and ethyl esters which can subsequently be
hydrolyzed to the corresponding carboxylic acids (see below) were chosen to be
introduced into the library.
Pilot combinatorial librarv construction. In order to confirm that the
selected reactants could be used to create libraries. a series of experiments was carried
out in which compounds were synthesized in a 96 well format. where all wells contain 3-
furoic acid and N-butyl isonitrile and the rows and columns contain a specified aldehyde
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and amine. as indicated in Figure l 1. The experimental details were as follows: A 2M
stock solution of each reactant was prepared in the a~plop-iate solvent. An equal
amount (50 ml) of the four ap~ .iate reactants was added to 80 wells of a 96 well
polypropvlene plate in which the well volume was l ml. The reactions in each column
contained a specified amine and the indicated aldehyde was added to each row. A single
carboxylic acid. 3-furoic acid and n-butyl isonitrile was added to all 80 wells. Columns
l l and 12 are reserved for controls which are added prior to screening. After 20 hours,
the reactions were diluted by addition of 200 ml of DMSO and 600 ml of methanol, a
sequence which was empirically determined to maximize product recovery in those
wells in which the product ~l~,ci~i~aLed. A~ ming a 50% yield, product concentration in
these plates is 50 mM. The products are stable and do not ~,~,cipildte when stored at
-20~ C. An aliquot was then taken from each well and analyzed by HPLC. In each
chromatogram the assignment of product was confirmed by an algorithm which enables
the retention time of the product to be predicted within 2 minutes from the incl~lllelltdl
contributions of the reactant side chains. The yields for each reaction are shown in the
coll~ oliding wells. A yield of >50% was observed in 70/80 (88%) of the reactions and
>60% in 63/80 (79%) of the reactions. In several wells (G3. H3) the yield was low due
to selective ~ ilation of the product. In other wells (E3, E6) the principle side-
product was the Schiff base formed from the amine and aldehyde in the first step of the
reaction sequence. Thus, the principal hllpulilies are starting materials or Schiff base.
Eleven additional plates were synthesized by this protocol in which the different amines,
aldehydes/ketones and carboxylic acid were evaluated. For these plates samples were
taken from ten wells per plate for analysis by HPLC. Of the 110 reactions analyzed, 96
(87%) showed product yield of >50%.
A protocol also was developed ~o introduce carboxylates by hydrolyzing the
ester-containing compounds in a 96 well plate format. Ester containing wells were
diluted 5-fold with methanol to a product concentration of approximately l O rnM. A 50
ul aliquot of the compound solution was combined with 10 ml of IM K2HPO4
(pH=13.3) to increase the pH to >1-7. The hydrolysis proceeded overnight at roomtel,lpeldture and the solution was neutralized by addition of lO ml of I M KH2PO4
(p~=2.~). These conditions were empirically determined to provide quantitative
hydrolvsis withou~ precipitation of phosphate or the ester.
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FYs-mple 6 Pilot Library Synthesis~Screening
Since all of the components and operations of our strategy for combinatorial
synthesis appeared to be in workin~ order, a combined synthesis/screening technology
5 was implemented in a moderate scale (several thousand compounds) compound screen.
The combinatorial synthesis technology was applied to the construction of two libraries
of compounds with chemical functionality biased towards GAG-like structures. In the
first library, each compound contains a carboxylic acid moiety which was introduced in
the Ugi reaction as an ester which was subsequently hydrolyzed to the corresponding
10 acid. The second library has been constructed with re~ct~ntc containing sulfonate groups
to mimic the sulfonate groups found in GAGs and the sulfonate groups found in
suramin, GL-12, and NF-066. The d~t~ilec~ composition of the two libraries is shown in
Table I below:
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Table 1
O R3 H
R IJ~N ~N_R4
~ R2 ~
Ca.l.uA~laLe Library
Plate # ~1 R2 R3 R4 # C~
81C0--8101 20 16 320
81G2-8103 20 ~ 16 ~ 320
8104, 8105, 8113 24 20 480
8106. 8107, 8114 24 20 ~
8108-8109 '' 20 16 ~ 320
8110-8111 ~ 20 16 ~ 320
8112 8 2 32 ~ 128
82~Ch ~ 11 8 ~OH 21764
S.'". -' Library
Plate # Rl R2 R3 R4# C~-~F- ~~
8301 83Q4 HO' 5;~ 10 8 4 640
8401 84Q4 ~ 10 8 4 0
inflir~trs conn~-ctior to root structure
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Following the synthesis of a set of 2544 carboxylates and 1280 sulfonate
derivatives we employed the rhGGF2 binding assay adapted to a 96 well format to
identify an initial set of hits. Briefly. a 20 nM solution of rhGGF2 was incubated for 60
minutes at room temperature with the test compound(s) and a trace amount of a high
5 affinity GAG fragment which has been tyraminated at the reducing end and iodinated.
Assays for bFGF and VEGF were run under similar conditions except that the protein
concentrations are set at 7.5 nM and IS nM, respectively. The s~ ,nillg assays were
carried out in 96 well Hybridot Manifold (BRL) which allowed rapid filtration through a
nitrocellulose sheet which then contains 96 radioactive "spots". Radioactivity on the
10 filter was qu~ntit~tP(l on a up to 10 sheets of nitrocellulose per run. This approach has
proven to be very efficient and has been routinely used to screen up to 12 microtiter
plates (960 wells)/week. Since each plate contained 80 reaction products, 16 wells were
available for positive and negative controls which included purified samples of the
relevant amines~ aldehydes. carboxvlic acids and isonitrile reactants. Other controls
15 included wells with no compound added and wells that contained excess "cold" heparin.
The two libraries were analyzed first in a single point survey. Positive hits fell
into two groups: I ) wells that block >50% of binding and 2) those that block 20-50% of
binding. Wells from the second group are l~sc,celled to determine if the blocking was
20 reproducible, and if so, they were selected along with those wells in the first group for
complete binding inhibition curves to delc""ille compound IC50 values. Compoundswith IC50 < 30 uM were selected for further analysis. These selected compounds were
tested for color or chemical quenching and some were elimin~cl A few of were
purified by HPLC and were re-analyzed. The current status of these screens are given in
25 Table 2. In Table 3, the ICS0 data is reported for the eight most advanced compounds
in the screen.
These efforts have resulted in the identification of 17 preliminary lead
compounds. 2 carboxylates and IS sulfonates, which are at various stages of analysis.
30 The composite hit rate of 0.45% is interpreted to mean that the general design of our
approach is working as planned and it bodes well for additional screening efforts.
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Table2. rhGGF2 LIBRU~RY SCEUEENnNG
T ihr~ry Initi:~l surveva First resereenb Fnrth~r sere~nir~C
2544 earboxylates 133 @ 20-509'c (5.2%) 8 seleeted
41 @>50% (1.6%) 10seleeted
Total hits 174 18 seleeted 2 seleeted for
(0.7% of 2544) product pllrifir~tiond
(0.07'Yo of 2544)
.
1280 sulfonates 90 @ 20-50% (7.0%) 7 seleeted
29 @ >50% (2.3%) 20 seleeted
Total hits 119 27 seleeted 15 seleeted for
(2.1% of 1280) product purificationd
(1.17% of 1280)
a rnnnpo~n~ between 30-40 IlM: those c~. l.. , .1~ that are 20-50% inhibitors are .CJ~ ,.. _d as
sincle pomts: those ~ l.u ~ that are >50% inhibitors are ,cJ~._d with a complete binding inhibition curve
to determine ~ ~ I .u ~ ~l lC50.
b singie point rescreens that repeat as 20-50% inhibitors are selected for complete binding inhibition curves to
de~ermine ~ ~J l - - .1 lC50; cu .l .,~ with IC50 e 3011M are selected for further analysis
c IC50 is repeated 2-3 times to determine Ic~J~udu~iL;lity; ~ are tested for color or chemical ~ n ~L ~f
signal; c~ are ~ ~ for non ~c~J~ud~._;b;lity and signal ~In~.n~ ng
d punfied .., ~ will be analYzed further ' - ' '1!, including activity ~ (IC50 values;
c~ r.... -~ ;.... of structure (mass s~ .U:~u~ and; specificity binding assays against other growth factors
Table 3. IC~;Oa S. ~ of 8 Selected ('~ 1 -
Library Compound rhGGF2 rhGGF2 bFGF bFGF
U~ Ul ified purified UUIJUl ified purified
Carboxylate 8108 B3 15 3 50 3.5
8'70nhF~ 12 20 15 >100
Sulfonate 8301F10 40 63 >100
8303B8 15 14 11 18
8303E9 25 27 34 >100
8304B9 35 38 38 >100
8401F10 40 85
8404H3 15 45
a Values are in uM
CA 0223038~ 1998-02-24
W O 97/09OSl PCT~US96/14200
Example 7 Effect of identified combinatorial compoun~s in the schwann cell
proliferahon assay
Three compounds that were identified in the binding assay were resyntheci7~cl on a
5 larger scale, then purified by HPLC and tested for inhibition of Schwann cell proliferation.
The samples were provided in 100% methanol and were diluted into media at least 100-fold
at the highest concentration used in the bioassay. These compounds were analyzed for
biological activity in the Schwann cell proliferation assay using the method described by
Sudhalter, et al.. (Sudhalter, et al., (1996) Glia 17:28-38). Figure 13 shows the results of this
10 assay.
The compounds and controls were diluted into culture media and mixed with
rhGGF2 (60 ng/ml final concentration) to achieve the final concentrations ranging from
0.195uM to 200uM. After incubating at room le.,lp~,dtu.~ for 90 minutes the mixtures were
15 used to replace the media on the cells, and then the assay was performed according to the
standard protocol. Stocks of individual compounds were in 200 mM methanol (except
ROY20.2 and ROY20.3, which were at 20 mM).
Compound ROY20.1 was 100% methanol, the solvent control. When it was present
20 at up to 0.1 % in the assay it did not interfere with the mitogenic response of Schwann cells
to rhGGF2. Compound ROY22.2 was ~ dlllin, the positive control, which shows an IC50
of roughly 50 uM in this assay. Compound ROY2''.3 appeared to be the most potent in the
batch, but also was quite toxic to the cells. Compounds ROY22.4-6 represent the HPLC-
purified compounds that were positive in the binding assay. Inhibition of rhGGF2-
25 stim~ t.ocl Schwann cell proliferation was seen in the l00 uM range.
-38-
