Note: Descriptions are shown in the official language in which they were submitted.
CA 02328252 2000-10-12
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RECEPTOR LIGANDS
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional
Application No. 60/030,391, filed November 5, 1996.
FIELD OF THE INVENTION
This invention relates to multimeric receptor
ligands, methods for making and identifying them and
their use as agonist or antagonists of multimeric
biological receptors.
BACKGROUND OF THE INVENTION
Many receptors of the single transmembrane class
appear to respond to ligand binding by some form of
aggregation. Aggregation can be in the form of
homodimerization or homotrimerization in the case of
identical receptor subunits or in the form of
heterodimerization or heterotrimerization in the case of
different receptor subunits. It has become clear in
several systems that receptor aggregation is part of the
signal for the target cell to respond biologically. See
review by Young, P.R. entitled "Protein hormones and their
receptors" in Curr. Opin. Biotech. 3, 408-421 (1992).
Monoclonal antibodies have been discovered which
have agonist activity to the dimeric epidermal growth
factor (EGF), tissue necrosis factor (TNF) and growth
hormone (GRH) receptors. See Schreiber, A.B. et al., J.
Biol. Chem. 258, 846-853 (1983), Defize, L.H.K. et al.,
EMBO J. 5, 1187-1992 (1986), Englemann, H. et al., J.
Biol. Chem. 256, 14497-14504 (1990) and Fuh, G. et al.,
Science 260, 1808-1810 (1992). While not wishing to be
bound to any particular theory of receptor activation,
it is believed that in all three cases, the monoclonal
antibodies, by virtue of possessing two antigen binding
sites, were able to bridge two receptor molecules to
facilitate aggregation and thus activate them.
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Receptor-mediated biological functions are
implicated in many conditions. Indications for
compounds with agonist or antagonist activity towards
single transmembrane receptors are numerous.
Despite the success of monoclonal antibodies in
producing an agonist response in certain dimeric
receptors, they are not considered ideal candidates for
development of pharmaceutical compositions. Lack of
oral bioavailability and a limited serum half-life limit
the desirability and efficacy of monoclonal antibodies
as pharmaceutical agents. Consequently, a need exists
for non-antibody ligands which have agonist or
antagonist properties towards dimeric or trimeric
receptors.
SUMMARY OF THE INVENTI,QN
Accordingly, one aspect of the present invention is
a method for agonizing or antagonizing a multimeric
receptor comprising contacting the multimeric receptor
with a non-antibody multimeric receptor ligand.
Another aspect of the invention is a method for
identifying agonists and antagonists of multimeric
receptors. The method comprises the steps of contacting
a multimeric receptor with non-antibody multimeric
receptor ligand candidates and selecting ligand
candidates which bind to the receptor.
A third aspect of the invention is isolated non
antibody multimeric receptor agonists or antagonists.
A fourth aspect of the invention is a method for
making non-antibody multimeric receptor ligands. The
method comprises the steps of reacting a bifunctional
monomer bound to a solid support with a receptor binding
moiety and cleaving the reaction product from the solid
support wherein the two functional groups are identical
and symmetrically placed after cleavage.
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S DETAILED DESS'.RIPTION OF THE INVENTION
Aspects of the present invention are non-antibody
multimeric receptor agonists or antagonists and a method
for agonizing or antagonizing a multimeric receptor by
contacting the multimeric receptor with a non-antibody
multimeric receptor ligand. A multimeric receptor is a
receptor entity which is agonized or antagonized only
when two or more subunits of the entity are aggregated
on the same cell surface through binding to a common
ligand.
Multimeric receptors which appear to signal by
heterodimerization include granulocyte-macrophage-
colony-stimulating factor (GM-CSF) receptor, the
interleukins -3, -4, -5, -6, -12 and -13 (IL-3, 4, 5, 6,
12, and 13} receptors, oncostatin M, ciliary neurotropic
factor (CNTF) receptor, leukemia inhibitory factor (LIF)
receptor, nerve growth factor (NGF) receptor, fibroblast
growth factor (FGF) receptor, the interferons a, ~i and y
( IFN-a, ~3 and y) receptors and TGF ail, 2 receptor .
Heterotrimeric signaling receptors include interleukin-2
(IL-2) receptor and tissue necrosis factor (TNF}
receptor.
Known multimeric homodimerizing receptors include
erythropoietin (EPO) receptor, granulocyte-colony-
stimulating fctor (G-CSF) receptor, macrophage-colony-
stimulating factor (M-CSF) receptor, tissue growth
factor oc (TGFoc) receptor, epidermal growth factor (EGF}
receptor, neu receptor, growth hormone (GRH) receptor,
prolactin receptor, placental lactogen receptor, stem
cell factor receptor (c-kit), tissue necrosis factor a
and ~i (TNFOC, Vii} receptors, fas receptor, CD40 receptor
and CD27 receptor.
The non-antibody multimeric receptor Iigand of the
present invention serves as the common ligand through
which two or more multimeric receptor subunits
aggregate. The multimeric receptor ligand includes a
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spacer which is substituted with two or more receptor
binding moieties.
The spacer can be any molecule having a di- or
trisubstituted center capable of substitution with the
receptor binding moieties. Preferably, the spacer
provides for spatial separation and steric orientation
of the receptor binding moieties which is sufficient to
effectively induce aggregation while not sterically
preventing such association. Most preferably, the
spacer will provide spatial separation and steric
orientation of the binding moieties which mimic the
binding moieties of the natural ligand.
Exemplary disubstituted spacers include compounds
represented by the formula (I):
- Z - (R) n - (A) m - (R) n - Z -
(I)
wherein:
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A is independently N, 0, S, dithio; carbonyl,
O
N N or nothing;
Z is independently N, 0, S or carbonyl;
R is independently d- or 1-amino acid; alkyl of 1
to 10 carbons; cis, traps-2-butenyl; cis, traps-1,2-
cyclopropyl; cis, traps-1,2-cyclobutyl; cis, traps-1,3-
cyclobutyl; cis, traps-1,3-cyclopentyl; cis, traps-1,2-
cyclopentyl; cis, traps-1,2-cyclohexyl; cis, traps-1,3-
cyclohexyl; cis, traps-1,4-cyclohexyl; endo, exo-2,3-
norbornane; 1,5-naphthyl; 2,6-naphthyl; 1,8-anthrylene;
1,5-anthrylene; 2,6-anthrylene;
M
M-M I~ X
~M II \ ( X I I I
M=M M~M'M
> ; > > ;
X X ' ~'
X X
~ii~l
~ . X ~ X
> > ; ; > >
O
X X ..~~ M~M/
~X X
XJ X
J p
> ; > ;
0
O
~ N/
M- _M X
/N / I 1
p . O ~ X /
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i x \ ~ X .~ ~ X
\ 1 1 / \ 1 1 /
x ; ; ;
i X
x ~ \ 1 1 / ~ x w
\ 1 1 / \ 1 1 /
; ;
i X ~ i X
\ 1 1 / \ 1 1 /
; ;
X ~ H
\ 1 1 /
or '''',, H ;
where
X is independently N, O or S;
M is independently C or N;
p is 0, 1, 2, or 3; and
m is 0 or 1; and
n is 0, 1, 2 or 3.
Preferred compounds of formula (I) are those where
R is
H
X
,,. \ 1 1 /
or
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Exemplary trisubstituted spacers include compounds
represented by the formula (II):
Z
(R)n
, - Z - (R) n - Q - (R) n - Z -
(II)
wherein:
Q is C; N; B; 1,3,5-phenyl; 1,3,5-cyclohexyl;
O O
1 3 5-triazin l
Y , . ;
O
~N
N O
N
O
or
O J
J-N O
O~N\J
where J is independently H or alkyl of 1 to 10
carbons; and
Z is independently N, O, S or carbonyl;
R is independently d- or 1-amino acid; alkyl of 1
to 10 carbons; cis, traps-2-butenyl; cis, traps-1,2-
cyclopropyl; cis, traps-1,2-cyclobutyl; cis, traps-1,3-
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cyclobutyl; cis, trans-1,3-cyclopentyl; cis, trans-1,2-
cyclopentyl; cis, trans-1,2-cyclohexyl; cis, trans-1,3-
cyclohexyl; cis, trans-1,4-cyclohexyl; endo, exo-2,3-
norbornane; 1,5-naphthyl; 2,6-naphthyl; 1,8-anthrylene;
1,5-anthrylene; 2,6-anthrylene;
M \ X
M-M I~ M
~M II I X I ~ I
M=M . . M~M'M
> > > ,
X X
X X
"~iil
. X . X .
> >
O
M~M~
X X
X X ,,.
XJ X
J
> ;
0
0
~ N~
M- _M x
~N ~ I . I
X / > ;
X ~ ~ X ~ ~ X
l I ~ ~ I 1
x
x
x ~ I I -- X
I t i ~ ~ ~ 1 I
- ; ' ; - ;
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X
X ~ ~ X
\ ' ' / \ ' ~ /
; ;
H
or H ;
where
X is independently N, O or S;
M is independently C or N;
p is 0, 1, 2, or 3; and
m is 0 or 1; and
n is 0, 1, 2 or 3.
Preferred compounds of formula (II) are those where
R is
H
X
,,. \ ~ ~ /
H or . Also preferred are
the compounds of formula (II) where Q is N; 1,3,5-phenyl
O
~N
N O
N
O
and
One skilled in the art could determine the required
spatial separation and steric orientation of the
receptor binding moieties through X-ray crystallographic
data for receptor entities with and without bound
natural ligand. For example, the X-ray crystal
structures of HGH {a 4-helix bundle protein) complexed
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with its homodimeric receptor, HGH binding protein
(HGHBP), have been published by Fuh et a1. in Science
256, 1677-1680 (1992). The two HGHBP molecules of the
receptor bind HGH with considerable C2 symmetry.
Analysis of the superimposition of the crystal structure
of HGH~(HGHBP)2 with an identical crystal structure,
where the identical crystal structure has been rotated
through a C2 axis to maximize the overlap of the binding
proteins, provides vectors for points of attachment for
the receptor binding moieties. Design of spacer
molecules which contain these vectors could be aided by
conducting three-dimensional compound database searches
using programs such as CAVEAT (Regents of the University
of California) or SYBYL 3-D (Tripos Associates Inc.)
This process could yield both C2 symmetric and non-C2
symmetric spacers.
The spacers derived from examination of HGH may be
useful in designing ligands for other hematopoietic
receptors, since it is known that dimeric receptor
ligands share common structural features which lead to
aggregation of the receptor subunits. It is expected
that this method could also be generalized to other
receptor-ligand complexes when their crystal structures
become available. ..
The receptor binding moieties which attach to these
spacers can be either peptides or small molecules from a
natural or synthetic source. The peptide sequences
could be chosen from but not limited to linear and
cyclic sequences known to be important for binding of
hematopoietic proteins to their receptors. Particularly
interesting are those sequences found in helices one and
four of the four a-helix bundle class of protein
ligands, since these helices are important for binding
and seem optimally situated for dimerization. Also, the
helices, unlike the loop regions of these receptors, are
very similar in orientation throughout this class of
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proteins. The identity of the possible small molecules
could be chosen from but not limited to agonists and
antagonists derived from database screens and peptide
mimetics.
The receptor binding moieties of the non-antibody
multimeric receptor ligands of the present invention can
be identical, yielding homomultimeric compounds, or they
can be different, yielding heteromultimeric compounds.
In general, non-antibody multimeric receptor ligands
could be synthesized by reacting a bifunctional monomer
bound to a solid support with a receptor binding moiety
to form a reaction product followed by cleaving the
reaction product from the solid support, wherein the two
functional groups are identical and symmetrically placed
after cleavage.
Those of ordinary skill in this art would recognize
that any single peptide or small organic molecule could
be coupled to the spacer substitution centers to provide
homomultimeric receptor ligand candidates.
Heteromultimeric compounds can be produced through
combinatorial chemistry methods in which a library of
compounds is synthesized. Combinatorial synthetic
methods known to those skilled in the art can produce
library members simultaneously as a mixture or
individually. Diverse sets of biopolymers such as
peptides containing naturally occurring and non-
naturally occurring a- and ~i-amino acids,
oligonucleotides and oligosaccharides as well as small
organic molecules can be produced.
Linear peptide and oligonucleotide libraries can be
produced by synthesis on a solid support, such as
synthesis beads, followed by cleavage from their
supports. Solution synthetic methods could also be
employed.
Small organic molecule library members are built up
on a core structure template. The core structure is
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derivatized through a series of synthetic steps to
produce a library containing a discrete number of
independently variable substituents, functional groups
or structural elements. Reaction conditions are
selected such that each derivatized core structure is
different from the others. Methods for derivatizing
core structures are disclosed in U.K. Patent Application
No. 9325621.2, which is incorporated herein by
reference.
Non-antibody heteromultimeric receptor ligand
candidates are provided by substitution of library
members onto the spacer. The spacer is coupled to a
solid support such as a synthesis bead and the
combinatorial library members are built out from the
substitution centers present on the spacer. After
library synthesis is complete, the resulting ligand
candidates are cleaved from the support by techniques
well known to those skilled in the art.
Another aspect of the present invention is a method
for identifying agonists and antagonists of multimeric
receptors and the multimeric receptor ligands identified
thereby. In the method, a multimeric receptor is
contacted with non-antibody multimeric receptor ligand
candidates. Ligand candidates which bind to the
multimeric receptor are selected by receptor binding
assays well known to those skilled in the art.
In general, a target receptor in isolated,
immobilized or cell-bound form is contacted with a
plurality of receptor ligand candidates and those
candidates which bind to and interact with the receptor
are selected. Binding or interaction can be measured
directly by using radioactively labeled ligand
candidates or by measuring any second messenger effect
resulting from the interaction or binding of the ligand
candidate. Alternatively, the ligand candidates can be
subjected to competitive binding assays in which a known
receptor ligand, labeled preferably with an analytically
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detectable reagent, most prefereably radioactivity, is
included with the ligand candidates and a candidate's
ability to inhibit the binding of the labeled ligand is
measured.
Positive multimeric receptor ligand binding
candidates are screened for biological function by any
one of the receptor function assays well known to those
skilled in the art. It is expected that a positive
ligand binding candidate will exhibit agonist or
antagonist activity in receptor function assays.
Any agonist or antagonist compounds identified can
be isolated by affinity chromatography. Other isolation
techniques for multimeric small organic molecules
include labeling the receptor binding moieties as they
are being synthesized with coding agents such as
oligonucleotides and peptides or tagging the moieties
with structurally related molecules that can be analyzed
by electron capture capillary gas chromatography.
A non-limiting specific competitive binding assay
example follows.
COMPETITIVE BINDING ASSAY EXAMPLE A
Tissue containing the appropriate target receptor
is homogenized, filtered through cheesecloth and
centrifuged at 1500 x g for 10 minutes. Alternatively,
cell membrane preparations from cells transfected or
transformed with the target receptor gene may be
employed. The supernatant is decanted and the pellet is
resuspended in an appropriate incubation buffer, e.g.,
75 mM Tris~HCl, pH 7.4 containing 12.5 mM MgCl2 and 1.5
mM EDTA. Membranes equivalent to 100 ug protein are
incubated with 50 pmol radiolabeled receptor ligand and
an appropriate amount of the ligand binding candidate in
a total volume of 500 ~,1 for 1 hr at 37°C. The binding
reaction is terminated by dilution with the addition of
5 ml of cold incubation buffer and the bound tracer is
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separated from free by filtration on Whatman GF/C filter
paper. The filter paper is washed several times with
cold incubation buffer and then counted to determine the
amount of bound ligand. The presence of a competing
ligand is evidenced by a reduction in binding of the
radiolabeled receptor ligand relative to a control
lacking the addition of ligand binding candidate.
The present invention will now be described with
reference to the following specific, non-limiting
Examples 1 and 2. The diacids produced by the methods
of Examples 1 and 2 and other diacids within the scope
of the invention can serve as a spacer by attachment
through an amide or ester bond. They can also be
reduced to the corresponding alcohols using a reducing
agent such as borane, LiAlH4 or diisobutylaluminum
hydride; this alcohol can be converted to a leaving
group using mesyl chloride, triphenylphosphine and CC14
or tosyl chloride. This leaving group can be used to
attach the linker to the binding moieties through an
ether, amine, sulfide or hydrocarbon linkage. The
diamines produced can be attached to the binding
moieties through an amide, urea, carbamate or amine
linkage. These diacids and diamines can be elaborated
further to create other linkers or attached to a resin
used in creating combinatorial libraries.
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axAMPLE 1
Svnthesis of the Disubstituted Spacer 4.6-
Dicarboxviminodibenzvl and 4.6-Diaminoiminodibeazvl
The synthetic steps are outlined in Scheme 1 below.
Scheme 1
a'b ~ ~ ~ c, d
> ~ ~ N
N ~ N
2 COpMe O O COpMe
3
eJ
_ ~-
_N Y W N /
NHp NHZ C02H COpH
5 4
a) n-butyllithium, C02; b) CH2N2, ethyl ether; c)
C1COCOC1, ethyl ether; d) CS2, A1C13; e) H202, OH-; f)
diphenylphosphorylazide, triethylamine, t-butanol; g)
trifl:uoroacetic acid
The monoester 2 is available in two steps from
iminodibenzyl 1 (available from Aldrich Chemical Co.,
Milwaukee, WI.). The iminodibenzyl is first dilithiated
with two equivalents of an alkyl lithium, such as n-
butyllithium, to form the dianion which is subsequently
treated with carbon dioxide to form the carboxylic acid.
This monocarboxylic acid can then be esterified by
standard techniques, such as diazomethane in ether. The
monoester 2 can be acylated at the 4-position by a two
step procedure. The iminodibenzyl 2 is first treated
with oxalyl chloride to form the amide; this
intermediate cyclizes to the a,-ketoamide upon treatment
with a Lewis acid such as A1C13, TiCl4 or FeCl3. The a-
ketoamide 3 can be converted to the diacid by treatment
with an oxidizing agent such as H202 or NaI04 and
hydrolysis with hydroxide anion (Hess, B.A. et al. J.
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Am. Chem. Soc. 92, 1672 (1969)). The diacid can also be
converted to the diamine using standard Curtius
conditions (diphenylphosphorylazide and triethylamine or
NaN3 and C1COCOC1) and the t-butyl carbamate produced
can be hydrolyzed with trifluoroacetic acid.
L$ a
Svathesisof the Disubstituted Sflacer traps-2.6
Dicarboxvdecalin and traps-2.6-Diaa~inodecalin
The synthetic steps are outlined in Scheme 2 below.
Scheme 2
o ~H
H H
.v _.v
H H
C 6 ~ 7
H QH H
H2N '
c, d, a
~~ NH Z
H H
OH
7 8
OH Me0 ZC
H = H H
f. B h i, i . ~ zC
_ '~. _ ''b
v CO H
H V. iI H
OH
7 9 10
a)i) lithium diisopropylamide, phenylselenenyl
chloride; ii) m-chloroperoxybenzoic acid,
triethylamine; b) lithium tri-sec-butylborohydride; c)
C13CCN, heat; d) H2, Pd; e) NaOH; f) CH302CC1,
pyridine; g) Tebbe reagent, heat; h) H2, Pd; i)
lithium diisopropylamide, 03; j) Ag0
The~diol 7 is synthesized in two steps from trans-
1,5-decalindione (available from Aldrich Chemical Co.,
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Milwaukee, WI). The first-step conversion to the
unsaturated ketones involves conversion to the a-phenyl
selenide using a sequential addition of a strong base,
such as lithium diisopropylamide or
bis[trimethylsilyl]amide, and phenylselenenyl chloride,
oxidation of the selenium using hydrogen peroxide or m
chloroperoxybenzoic acid followed by a quench of the
oxidant and basic elimination. The oc,~3-unsaturated
ketones are then reduced to the corresponding axial
alcohols using a bulky hydride, such as L-Selectride0
(1.0 M lithium tri-sec-butylborohydride in
tetrahydrofuran) or K-Selectride0 (1.0 M potassium tri-
sec-butylborohydride in tetrahydrofuran), which prefer
equatorial attack. Intermediate 7 is then converted to
the desired diamine by conversion to the corresponding
trichloroimidate followed by rearrangement to the
transposed allylic amide (Overman, L. J. Am. Chem. Soc.
98, 2901-2910 (1976)). The allylic amide is then
reduced to the amide by hydrogenation using Pd,
Wilkinson's catalyst (tris[triphenylphosphine)rhodium[1]
chloride) or Pt as a catalyst and the amide is
hydrolyzed using basic hydrolysis such as NaOH, KOH or
Li00H to form the diamine 8. The diol 7 can also be
converted to the diacid 10 in five steps. The carbonate
is formed using methylchloroformate and a base such as
pyridine or triethylamine. The carbonate is then
methylenated using Tebbe reagent and the enol ether
undergoes an allylic rearrangement. The ester is then
hydrogenated using Pd, Wilkinson's catalyst or Pt as a
catalyst to form 9. The ester is then enolized using a
strong base such as lithium diisopropylamide or sodium
bis[trimethylsilyl)amide and the enolate is cleaved with
ozone. The dialdehyde produced is treated with silver
oxide to form the diacid.
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The present invention may be embodied in other
specific forms without departing from the spirit or
essential attributes thereof and, accordingly, reference
should be made to the appended claims, rather than to
the foregoing specification, as indicating the scope of
the invention.
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