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Cancer Patent Abstract
The invention includes a cell-growth-on-bead assay for screening
a one-bead-one-compound combinatorial bead library to identify synthetic
ligands for cell attachment and growth or proliferation of epithelial
and non-epithelial cells. Cells are incubated with a compound bead
library for 24 to 72-hours, allowing them to attach and grow on
the beads. Those beads with cells growing are removed, and the ligand
on the bead is identified. Also provided are ligands specific for
cancer cells.
Cancer Patent Claims
We claim:
1. A ligand specific for human epithelial cancer cells, wherein
said ligand has the chemical structure of cDGLGDDc.
2. A ligand specific for human epithelial cancer cells, wherein
said ligand has the chemical structure of c-d-G-HoCit-G-P-Q-c.
3. A ligand specific for human non-epithelial cancer cells, wherein
said ligand has the chemical structure of cDGLGDDc.
Cancer Patent Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to screening methods for one-bead-one-compound
combinatorial libraries and includes a screening assay that uses
live cells to identify synthetic ligands that promote attachment
and growth or proliferation of epithelial and non-epithelial cells.
Also included are ligands specific for epithelial and non-epithelial
cancer cells.
2. Description of Related Art
One-bead-one-compound combinatorial bead libraries (see Lam, Kit
S. et al. "A new type of synthetic peptide library for identifying
ligand-binding activity." Nature 354 (1991): 82-84), such as
one-bead-one-compound peptide libraries, are being used to study
cell adhesion properties of cancer cells. Using random peptide bead
libraries and suspended cancer cells, peptide ligands that promote
cell attachment have been identified for lymphoma (Park, Steven,
Renil Manat, Brian Vikstrom, Nail Amro, and Kit S. Lam. "Identification
of peptide ligands for .alpha.4.beta.1 integrin receptor as potential
targeting agents for non-Hodgkin's lymphoma," abstract in Peptides:
The Wave of the Future, 2nd International Peptide Symposium in conjunction
with the 17.sup.th American Peptide Symposium, San Diego, Calif.
(Jun. 9-14, 2001)) and prostate cancer cell lines (Pennington, Michael
E., Kit S. Lam and Anne E. Cress. "The use of a combinatorial
library method to isolate human tumor cell adhesion peptides."
Molecular Diversity 2 (1996): 19-28; DeRoock, Ian B., Michael E.
Pennington, Thomas C. Sroka, Kit S. Lam, G. Tim Bowden, Elisabeth
L. Bair, and Anne E. Cress. "Synthetic Peptides Inhibit Adhesion
of Human Tumor Cells to Extracellular Matrix Proteins." Cancer
Research 61 (Apr. 15, 2001): 3308-13).
In the existing methods, live cells in suspension are incubated
for about one to four hours with a bead library, and the library
is then screened for beads with peptide ligands that promote cell
attachment. This is done by visual selection the beads are examined
under a dissecting microscope and those beads with attached cells
are removed using a micropipet. Further steps are then performed
to confirm that the removed beads are in fact capable of binding
the particular type of cells tested. Then, the peptides on those
beads are sequenced. (See Pennington et al., "Use of a combinatorial
library method," 19-28.)
In another existing method of testing live cells for peptide ligands
that affect cell growth on culture plates, a bead library is prepared
having selectively cleavable peptides such that a proportion of
the peptide on each bead is attached to the bead by a cleavable
linker. When the library is treated with a cleaving agent, enough
of the peptides are released from the beads to cause the biological
effect, and the rest of the peptides remain bound to the beads to
allow for later sequencing. Suspended cells are incubated in tissue
culture wells with a few beads and with peptides released from the
beads. The effect of the released peptides on the cells (inhibition
or stimulation of cell growth) is determined, and the corresponding
beads are removed. The sequences of the attached peptides are then
determined. (See U.S. Pat. No. 5,510,240, issued Apr. 23, 1996 to
Lam, Kit S. et al.)
The existing methods, however, are not satisfactory in certain
cases. The methods are difficult to use with epithelial cells, which
include the majority of solid cancer cell cultures, such as lung
cancer cells, that exist as adherent cultures rather than as suspended
cells. With incubation periods of only a few hours, these cells
are often only weakly attached to the beads and may easily fall
off, rendering the screening method less accurate because some beads
with attached cells are missed. Also, the existing methods may not
detect cell surface receptors that may be altered by trypsin and/or
EDTA. Trypsinization is commonly used to separate tissues or cell
cultures into a single-cell suspension for testing with a combinatorial
library. The treatment with trypsin may eliminate some, or alter
the conformation of, cell surface receptors. In addition, the existing
methods do not select for ligands that promote cell growth or proliferation,
but, rather, for ligands involved in cell attachment, particularly
short-term attachment.
Thus, there is a need for a screening assay that is specific and
sensitive, works well with epithelial cells, can be used to detect
cell surface receptors susceptible to trypsin, and selects for ligands
that promote not only cell attachment, but also cell growth or proliferation.
SUMMARY OF THE INVENTION
The present invention is directed to a method for screening a combinatorial
bead library for ligands that promote the attachment and growth
or proliferation of epithelial and non-epithelial cells. The method
satisfies the need for an assay that is specific and sensitive,
that can be used to detect cell surface receptors susceptible to
trypsin, and that can identify ligands that promote cell growth
and proliferation. The method comprises introducing a suspension
of live cells to a combinatorial library of small molecules, peptides,
or other types of molecules, incubating the cells with the library
for about 24 to 72 hours, identifying a solid phase support of the
library with cells growing on the support, isolating the solid phase
support, and determining the chemical structure of the compound
attached to that solid phase support.
The invention also includes ligands specific for cell attachment
and growth or proliferation of epithelial and non-epithelial cancer
cells.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram depicting the steps of the cell-growth-on-bead
assay as used with epithelial cells.
FIG. 1A shows attached epithelial cells.
FIG. 1B shows the detached epithelial cells of FIG. 1A in suspension.
FIG. 1C shows the epithelial cells being mixed with the beads of
the bead library.
FIG. 1D shows a top view of three beads, in which two beads have
a monolayer of cells growing on the bead.
FIG. 1E shows a top view of the three beads of FIG. 1D, after staining,
in which the two beads with a monolayer of cells growing on the
bead are stained, and the one bead without any cells is not stained.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention includes a method, referred to as the cell-growth-on-bead
assay, for screening a one-bead-one-compound combinatorial bead
library for ligands that promote cell attachment and growth or proliferation.
Ligands that promote cell attachment and growth or proliferation
of epithelial and non-epithelial cancer cells are also described.
Cell-Growth-on-Bead Assay
The cell-growth-on-bead assay includes the following steps. A one-bead-one-compound
combinatorial library is prepared. The library is preferably synthesized
using the "split synthesis" approach described in Lam
et al., "A new type of synthetic peptide library," 82-84.
The compounds of the library may be small molecules, peptides, or
other types of molecules. An example of a suitable library is a
peptide library containing cXXXXXXc peptides, where "c"
is D-cysteine which provides intramolecular cyclization by disulfide
bonding, and "X" is any L, D, unnatural, or modified amino
acid. A suitable solid phase support, such as beads or discs made
of polystyrene, agarose, acrylamide, glass, plastic, or paramagnetic
substances, is used. Polystyrene beads have been found satisfactory.
A standard synthetic solid phase peptide synthesis method, such
as fluorenylmethyoxycarbonyl (Fmoc) chemistry or t-butyloxycarbonyl
(Boc) chemistry, is used.
A suspension of live mammalian cells is prepared according to methods
known to those skilled in the art. The cells may be epithelial or
non-epithelial cells and may be cancerous or non-cancerous. Human
cancer cells from a cell line or derived from biopsy specimens or
body fluid of cancer patients may be used. FIG. 1 shows the method
as used with epithelial cells. FIG. 1A shows attached epithelial
cells 10. FIG. 1B shows the same cells 10 in suspension.
Suspended live cells 10 are mixed with the library in culture medium,
as shown in FIG. 1C, and distributed into culture plates. The ratio
of cells to beads is preferably about 10:1, but can range from about
1:1 to 100:1. The suspension of cells 10 and beads 12 is mixed gently
for sufficient time to assure contact of beads 12 with suspended
cells 10. The culture plates are incubated in a tissue culture incubator
at about 4.degree. C. to about 37.degree. C., preferably 37.degree.
C., for a period of about 24 to about 72 hours. The suspension of
cells 10 and beads 12 may be kept still or mixed, either continuously
or intermittently, during the incubation period.
After the incubation period, beads 12 are observed under a dissecting
microscope. The presence of an increased number of cells 10 or beads
14 covered with a monolayer of cells, as shown in FIG. 1D, evidences
cell growth or proliferation. These beads 14 (referred to as "positive"
beads) are removed from the culture plates. A tetrazolium dye that
stains live, but not dead, cells can be used to facilitate the identification
and removal of the positive beads. If a dye is used, all of the
beads are removed after the incubation period and resuspended in
fresh medium in new culture plates. The dye is added. The plates
are then incubated in a tissue culture incubator at 25.degree. C.
to 37.degree. C. for about one to four hours. Live cells 10 convert
the dye to a colored metabolite, which results in beads 14 with
attached cells appearing colored, allowing them to be easily distinguished
from beads 16 without attached cells, which appear colorless, as
shown in FIG. 1E. Other dyes that stain live cells can also be used.
After positive beads 14 are removed from the plates, attached cells
10 are separated from the beads. This can be done with the addition
of a chaotrophic agent, such as 8 M guanidine hydrochloride, or
a protease, such as trypsin.
The chemical structure of the compound (i.e. ligand) on each isolated
positive bead 14 is then determined. If the combinatorial library
used was a peptide library, then the amino acid sequence of the
ligand is preferably determined with an automated protein sequencer,
such as the Procise 494 (Applied Biosystems, Foster City, Calif.).
Alternatively, the peptide can be released via a cleavable linker
and the amino acid sequence determined by mass spectroscopy. If
the ligand on the bead consists of a small molecule, then mass spectroscopy
or encoding strategies can be used. See Liu, Ruiwu, Jan Marik, and
Kit S. Lam. "A novel peptide-based encoding system for `one-bead-one-compound`
peptidomimetic and small molecule combinatorial libraries."
J. Am. Chem. Soc. 124 (2002) 7678-7680; Song. Aimin, Jinhua Zhang,
Carlito B. Lebrilla, and Kit S. Lam. "A novel and rapid encoding
method based on mass spectrometry for `one-bead-one-compound` small
molecule combinatorial libraries." J. Am. Chem. Soc. 125 (2003)
6180-6188.
Using the cell-growth-on-bead assay, ligands that promote cell
attachment and growth or proliferation have been identified for
epithelial and non-epithelial cancer cells, including lung cancer,
ovarian cancer, brain cancer, liver cancer, and pancreatic cancer.
Structure/activity relationship studies have resulted in the identification
of ligands for epithelial and non-epithelial cancer cells having
the general structure of cXGXGXXc, in which "c" is D-cysteine;
"X" is any L, D, unnatural, or modified amino acid; and
"G" is glycine. Small molecule ligands and peptidomimetic
ligands have also been identified.
Definitions
In addition to standard abbreviations for amino acids, the following
abbreviations for amino acids are used: HoSer is homoserine, Cit
is citruline, HoCit is homocitruline, Hyp is hydroxyproline, Aad
is 2-aminohexanedioic acid, Lys(Ac) is .epsilon.-acetyllysine, 4-Pal
is 3-(4-pyridyl)alanine, D-3-Pal is D-3-(3-pyridyl)alanine, Pra
is propargylglycine, D-Pra is D-propargylglycine, Aib is 2-aminoisobutyric
acid, Phe(4-CN) is 4-cyanophenylalanine, Tyr(3-NO.sub.2) is 3-nitrotyrosine,
Tyr(Me) is O-methyltyrosine, Phe(4-NO.sub.2) is 4-nitrophenylalanine,
Bug is tertiary butylglycine, Ach is 1-amino-1-cyclohexanecarboxylic
acid, Tyr(3,5-I) is 3,5-diiodotyrosine, Aic is 2-aminoindane-2-carboxylic
acid, Phe(3-Cl) is 3-chlorophenylalanine, Chg is cyclohexylglycine,
Bta is 3-benzothienylalanine, Bpa is 4-benzoylphenylalanine, Phe(3,4-Cl)
is 3,4-dichlorophenylalanine, Hyp(Bzl) is O-benzylphenylalanine,
Cha is cyclohexylalanine, Abu is 2-aminobutyric acid, Nva is norvaline,
Phg is phenylglycine, Ach is 1-amino-1-cyclohexanecarboxylic acid,
Nle is norleucine, Phe(4-Me) is 4-methylphenylalanine, HoPhe is
homophenylalanine, 2-Nal is 3-(2-naphtyl)alanine, 1-Nal is 3-(1-naphtyl)alanine,
Tyr(3,5-I) is 3,5-diiodotyrosine, Acdt is 4-amino-4-carboxy-1,1-dioxo-tetrahydrothiopyran,
Dpr is 2,3-diaminopropionic acid, and D,L-beta-Fal(2) is D,L-3-(furan-2-yl)-3-amino-propionic
acid.
EXAMPLE 1
A one-bead-one-peptide combinatorial library, containing random
cXXXXXXc peptides, was prepared using the "split synthesis"
method of Lam et al., "A new type of synthetic peptide library,"
82-84. The random peptide library contained 19.sup.6=4.7.times.10.sup.7
possible permutations of the formula cXXXXXXc, where "c"
is D-cysteine, and "X" is one of 19 natural L-amino acids.
In this example, and in all other cases where a peptide or peptidomimetic
library is used, the D-cysteines provide intramolecular cyclization
by disulfide bonding.
TentaGel polystyrene beads, with a diameter of 80 .mu.m and with
grafted polyethylene glycol of 0.25 mmol/g, were used as a solid
phase support (Rapp Polymere, Germany). A synthetic solid phase
method using fluorenylmethyoxycarbonyl (Fmoc) chemistry was adapted
for synthesizing the peptide bead library.
The non-small-cell lung cancer cell line, A549 (American Type Culture
Collection, Manassas, Va.), was used. The cell line was maintained
in appropriate culture media as recommended by American Type Culture
Collection. Cells were grown to confluency in DMEM culture medium
supplemented with 10% fetal calf serum. Attached cells were recovered
with trypsin/EDTA, washed, and resuspended as single cells in culture
medium.
About 150,000 peptide beads were mixed with approximately one million
suspended cells in 15 ml of culture medium and distributed into
six 3-cm culture plates. The culture plates were agitated gently
at about 100 rpm for about 10 minutes. The culture plates were then
incubated in a tissue culture incubator at 37.degree. C. for about
24 hours to about 72 hours.
A dissecting microscope was used to examine the beads at about
24, 48, and 72 hours. After about 24 to 72 hours, beads with a monolayer
of cells were observed. Out of a library of about 150,000 beads,
about 20 to 30 beads typically exhibited cell growth.
At the end of the incubation period, all of the beads were removed
and resuspended in fresh medium in a new culture plate. An MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl]
tetrazolium bromide) (Sigma, St. Louis, Mo.) dye solution was added
to each culture plate to a final concentration of 0.5 mg/ml. The
plates were incubated in a tissue culture incubator at 37.degree.
C. for about two hours to allow the purple color to develop. Each
purple-colored peptide bead was isolated and removed. The attached
cells were separated from the beads with 8 M guanidine hydrochloride.
The amino acid sequence of each isolated peptide bead was determined
using an automated Procise 494 protein sequencer (Applied Biosystems,
Foster City, Calif.). Several consensus peptide sequences were determined,
one of which was cNGRGEQc. This peptide was resynthesized on beads,
which were then rescreened with the A549 cells using the assay of
the invention. Virtually all of the beads with this sequence exhibited
cell attachment and growth on their surfaces.
To test the sensitivity of the assay, blank beads and a linear
XXXXXX peptide bead library of 150,000 beads were each spiked with
10 positive peptide beads carrying the sequence cNGRGEQc. These
libraries were each screened with the A549 cells. The peptide beads
with the sequence of cNGRGEQc were isolated with a recovery rate
of 90% to 100% in two separate experiments.
To test cell type specificity of beads carrying the peptide ligand
cNGRGEQc, cell growth of two other non-small-cell lung cancer cell
lines, Calu-1 and Hi 78, was observed on 70% to 90% of the peptide
beads. On the other hand, cell growth was observed on only 10% of
the peptide beads with the non-malignant bronchoepithelial cell
line, HBE-1. Thus, the cNGRGEQc peptide is a ligand specific for
promoting cell attachment and growth of malignant cells of the lung.
Through additional secondary library screening and structure/activity
relationship studies, other ligands for epithelial cells have been
identified, including the following: c-D-G-Chg-G-A-N-c; c-N-G-Bpa-G-Q-M-c;
c-N-G-Acdt-G-D-Bpa-c; cNGTGDGc; cNGQGAGc; cNGYGSFc; c-N-G-Nle-G-Y-G-c;
cNGMGAYc; cNGQGEQc; cRGNGTDc; cNGQGPLc; cNGLGRSc; cDGMGSNc; cNGLGQYc;
and cNGYGTTc.
EXAMPLE 2
The method as described in Example 1 was performed, except that
the combinatorial peptide library was screened with a different
type of epithelial cancer cells, the ovarian cancer cell line CaOV3
(American Type Culture Collection).
As described in Example 1, after culturing for about 24 to 72 hours,
beads with a monolayer of cells were observed. These positive beads
were isolated and the amino acid sequence of each positive bead
was determined, as described in Example 1.
The following ligands were identified: cDGLGDDc and cDGWGPNc.
EXAMPLE 3
The method as described in Example 1 was performed, except that
a different combinatorial library was used and different types of
epithelial cancer cells were used. A one-bead-one-compound combinatorial
peptide library was prepared according to the method described in
Example 1, with the following formula: cX.sub.2GX.sub.4GX.sub.6X.sub.7c,
where "c" is D-cysteine; "G" is glycine; X.sub.2
is D,d,N, n, S,Q,q,T, HoSer, Cit, E, e, HoCit, Hyp, Aad, Lys(Ac),
A, 4-Pal, D-3-Pal, Pra, D-Pra, Y, Aib, M, Phe(4-CN), Tyr(3-NO.sub.2),
Tyr(Me), Phe(4-NO.sub.2), Bug, Ach, Tyr(3,5-I), Aic, Phe(3-Cl),
Chg, Bta, Bpa, Phe(3,4-Cl), Hyp(Bzl), or Cha; and X.sub.4, X.sub.6
and X.sub.7 are N, S, Q, T, HoSer, Cit, HoCit, Hyp, H, A, Pal, D-3-Pal,
Pra, R, Y, Aib, Abu, P, M, V, Nva, Tyr(3-NO.sub.2), W, Phg, Phe(4-NO.sub.2),
Bug, I, Ach, L, Nle, Phe(4-Me), Aic, Phe(3-Cl), HoPhe, Chg, Bta,
Bpa, 2-Nal, 1-Nal, Phe(3,4-Cl), Hyp(Bzl), or Cha.
The peptide library was screened with one of the following epithelial
ovarian cancer cell lines: SKOV-3 and ES-2 (American Type Culture
Collection).
As described in Example 1, after culturing for about 24 to 72 hours,
beads with a monolayer of cells were observed. These positive beads
were isolated and the amino acid sequence of each positive bead
was determined, as described in Example 1.
The following ligands for cells of the SKOV-3 cell line were identified:
cdGIGPQc; c-d-G-Phg-G-P-F-c; c-d-G-Cit-G-Hyp(Bzl)-M-c; c-d-G-Phe(4-Me)-G-T-Pra-c;
cdGLGFTc; c-d-G-Nva-G-Phe (4-CN)-F-c; c-d-G-Tyr(3NO.sub.2)-G-Pra-G-c;
c-d-G-(4-Pal)-G-Tyr(3-NO.sub.2)-Cha-c; and c-d-G-Cha-G-1-T-c.
The following ligands for cells of the ES-2 cell line were identified:
c-d-G-V-G-Hyp-HoSer-c; c-d-G-Phe(4Me)-G-P-Cha-c; c-d-G-Phe(3-Cl)-G-Q-F-c;
cdGLGYYc; c-d-G-L-G-HoSer-T-c; c-d-G-Tyr(Me)-G-T-M-c; and c-d-G-Cha-G-HoCit-S-c.
EXAMPLE 4
The method as described in Example 1 was performed, except that
a different combinatorial library was used and different types of
epithelial cancer cells were used. The peptide library of Example
3 was used, except that the concentration of peptides on the surface
of each bead was 20% of that used in Example 3, resulting in an
increase in screening stringency.
The peptide library was screened with one of the following epithelial
ovarian cancer cell lines: CaOV3, SKOV-3, ES-2 and OVCAR-3 (American
Type Culture Collection).
As described in Example 1, after culturing for about 24 to 72 hours,
beads with a monolayer of cells were observed. These positive beads
were isolated and the amino acid sequence of each positive bead
was determined, as described in Example 1.
The following ligands for cells of the CaOV3 cell line were identified:
cdGMGSAc; c-d-G-M-G-S-Cha-c; c-d-G-M-G-HoSer-Nle-c; c-d-G-Tyr(3-NO.sub.2)-G-i-Pra-c;
c-d-G-Tyr(3-NO.sub.2)-G-F-L-c; c-d-G-Chg-G-Hyp-N-c; and c-D-G-Cha-G-Hyp-N-c.
The following ligands for cells of the SKOV-3 cell line were identified:
c-d-G-A-G-Bta-L-c; c-d-G-L-G-S-Bpa-c; c-d-G-Nle-G-Phe(3-Cl)-S-c;
c-d-G-Tyr(3-NO.sub.2)-G-Phg-M-c; and c-d-G-Tyr(3-NO.sub.2)-G-Nle-H-c.
The following ligands for cells of the ES-2 cell line were identified:
c-d-G-Aib-G-P-S-c; c-d-G-Cha-G-Bta-Q-c; c-d-G-Bta-G-Hyp-Y-c; c-d-G-Phe(4-Me)-G-Aib-S-c;
c-d-G-Aib-G-Aib-N-c; cdGLGWGc; c-d-G-Cha-G-HoCit-Q-c; and c-d-G-HoCit-G-P-Q-c.
The following ligands for cells of the OVCAR-3 cell line were identified:
c-d-G-Phe(3-C l)-G-T-Y-c; c-d-G-Tyr(3-NO.sub.2)-G-Aic-Q-c; c-Tyr(3-NO.sub.2)-G-F-G-(Pal-3D)-HoSer-c;
c-d-G-HoCit-G-T-Nva-c; c-d-G-Nle-G-1-G-c; and c-Nle-G-Nle-G-Tyr(3-NO.sub.2)-L-c.
EXAMPLE 5
The method as described in Example 1 was performed, except that
a different combinatorial library was used and non-epithelial cancer
cells were used. A one-bead-one-compound combinatorial peptide library
was prepared as described in Example 3.
The peptide library was screened with the non-epithelial glioblastoma
brain cancer cell line A-172 (American Type Culture Collection).
As described in Example 1, after culturing for about 24 to 72 hours,
beads with a monolayer of cells were observed. These positive beads
were isolated and the amino acid sequence of each positive bead
was determined, as described in Example 1.
The following ligands were identified: cDGLGDDc; cDGWGPNc; c-N-G-Nle-G-(4-Pal)-M-c;
c-e-G-y-G-Hyp-W-c; c-d-G-(4-Pal)-G-Phe (4-Me)-T-c; c-e-G-N-G-S-(1-Nal)-c;
c-D-G-L-G-P-HoPhe-c; and c-e-G-L-G-Nle-M-c.
EXAMPLE 6
The method as described in Example 1 was performed, except that
a different combinatorial library was used and a different cell
type was used. A one-bead-one-compound combinatorial library consisting
of small molecules was prepared according to the method described
in Liu et al., "A novel peptide-based encoding system,"
7678-7680. The library consisted of molecules of the general structure
##STR00001## where "Aa.sub.1" was one of 96 amino acids
including 20 L-natural amino acids and 19 D-isomers, 15 beta-amino
acids, and 42 other amino acids; "R.sub.1COOH" was one
of 33 carboxylic acids, acyl chlorides, or sulfonyl chlorides; and
"R.sub.2COOH" was one of 50 carboxylic acids; as described
in Liu et al., "A novel peptide-based encoding system,"
7678-7680.
The small molecule library was screened with the liver cancer cell
line HEPG2 (American Type Culture Collection).
As described in Example 1, after culturing for about 24 to about
72 hours, beads with a monolayer of cells were observed. These positive
beads were isolated as described in Example 1. The chemical structure
of the small molecule on each positive bead was determined according
to the method described in Liu et al., "A novel peptide-based
encoding system," 7678-7680.
Ligands having the general structure
##STR00002## were identified, where "Aa.sub.1" was D-cys,
Cys, Trp, Nva, Aic, Dpr, Ile, Nle, or D, L-beta-Fal(2); "R.sub.1COOH"
was 3-pyridine propionic acid, 4-bromophenyl acetic acid, 2-pyrazine
carboxylic acid, 2-thiophene carboxylic acid, phenoxy acetic acid,
benzoic acid, or cyclopropane carboxylic acid; and "R.sub.2COOH"
was indole-2-carboxylic acid, 4-phenoxybenzoic acid, 2-butynoic
acid, 2-pyrazine carboxylic acid, 4-hydroxyl phenyl acetic acid,
or 3-thiophene carboxylic acid. The structure of twelve of the ligands
is shown in Tables 1 and 2.
TABLE-US-00001 TABLE 1 Liver Cancer Cell Ligands (HEPG2 cells)
##STR00003## Entry Aa.sub.1 Structure R.sub.1 COOH Structure R.sub.2
COOH Structure 1 cys ##STR00004## 3-Pyridinepropionic acid ##STR00005##
Indole-2-carboxylicacid ##STR00006## 2 cys ##STR00007## 4-Bromophenylacetic
acid ##STR00008## 4-Phenoxybenzoic acid ##STR00009## 3 Cys ##STR00010##
2-Pyrazinecarboxylic acid ##STR00011## 4-Phenoxybenzoic acid ##STR00012##
4 Cys ##STR00013## 2-Thiophenecarboxylic acid ##STR00014## 2-Butynoicacid
##STR00015## 5 Cys ##STR00016## Phenoxy aceticacid ##STR00017##
2-Pyrazinecarboxylicacid ##STR00018## 6 Trp ##STR00019## Phenoxy
aceticacid ##STR00020## 2-Pyrazinecarboxylicacid ##STR00021## 7
Nva ##STR00022## Phenoxy aceticacid ##STR00023## 4-Phenoxybenzoic
acid ##STR00024## 8 Aic ##STR00025## Phenoxy aceticacid ##STR00026##
4-Hydroxylphenylacetic acid ##STR00027## 9 Dpr ##STR00028## Benzoic
acid ##STR00029## 4-Hydroxylphenylacetic acid ##STR00030## 10 Ile
##STR00031## Cyclopropanecarboxylic acid ##STR00032## 3-Thiophenecarboxylicacid
##STR00033## 11 Nle ##STR00034## 4-Bromophenylacetic acid ##STR00035##
3-Thiophenecarboxylicacid ##STR00036## 12 D,L-beta-Fal(2) ##STR00037##
3-Pyridinepropionic acid ##STR00038## 3-Thiophenecarboxylicacid
##STR00039##
TABLE-US-00002 TABLE 2 Chemical Structure of Ligands for Liver
Cancer Cells (HEPG2) ##STR00040## 1 ##STR00041## 2 ##STR00042##
3 ##STR00043## 4 ##STR00044## 5 ##STR00045## 6 ##STR00046## 7 ##STR00047##
8 ##STR00048## 9 ##STR00049## 10 ##STR00050## 11 ##STR00051## 12
EXAMPLE 7
The method as described in Example 1 was performed, except that
a different combinatorial library was used. A one-bead-one-compound
combinatorial peptidomimetic or modified peptide library of the
chemical structure cNGZ.sub.2GZ.sub.1Xc was prepared. "X"
was Pro, Ala, Gly, Leu, Ile, Asp, Asn, Glu, Gln, Trp, His, Phe,
Tyr, Val, Ser, Thr or Met; "Z.sub.1" was Y, Nle, E, T,
Phe(NHR.sub.1), or Phe(NHR.sub.1,); and "Z.sub.2" was
Nle, H, D, Q, Phe(NHR.sub.2), or Phe(NHR.sub.2). "R1 "
was benzoic acid, 5-bromovaleric acid, 3-pyridinepropionic acid,
3-thiophenecarboxylic acid, 4-(dimethylamino)phenylacetic acid,
4-bromobenzoic acid, phenoxyacetic acid, (1-aphtoxy)acetic acid,
5-hydantoinacetic acid, phenylpropionic acid, cyclopropanecarboxylic
acid, 4-methyvaleric acid, 2-phenoxybutyric acid, 3-(dimethylamino)benzoic
acid, 3-thiophenecarboxylic acid, 2-pyrazinecarboxylic acid, furylacrylic
acid, 3,4-dichlorophenylacetic acid, 3-indolepropionic acid, 2,5-dimethoxyphenylacetic
acid, 3-hydroxy-2-quinoxalinecarboxylic acid, 4-hydrxyphenylacetic
acid, cyclohexanecarboxylic acid, 2-methylbutyric acid, or 4-bromophenylacetic
acid; "R.sub.1," was p-toluensulfonyl chloride, 3,4-dimethoxybenzoyl
chloride, 2-naphtalenesulfonyl chloride, 2-thiphenesulfonyl chloride,
2-thiopheneacetyl chloride, or propargylchloroformate; "R.sub.2"
was benzoic acid, 5-bromovaleric acid, 3-pyridinepropionic acid,
3-thiophenecarboxylic acid, 4-(dimethylamino)phenylacetic acid,
4-bromobenzoic acid, phenoxyacetic acid, (1-naphtoxy)acetic acid,
5-hydantoinacetic acid, phenylpropionic acid, cyclopropanecarboxylic
acid, 4-methyvaleric acid, 2-phenoxybutyric acid, 3-(dimethylamino)benzoic
acid, 3-thiophenecarboxylic acid, 2-pyrazinecarboxylic acid, furylacrylic
acid, 3,4-dichlorophenylacetic acid, 3-indolepropionic acid, 2,5-dimethoxyphenylacetic
acid, 3-hydroxy-2-quinoxalinecarboxylic acid, 4-hydrxyphenylacetic
acid, cyclohexanecarboxylic acid, 2-methylbutyric acid, 4-bromophenylacetic
acid, or 4-nitrophenylacetic acid; and "R.sub.2," was
p-toluensulfonyl chloride, 3,4-dimethoxybenzoyl chloride, 2-naphtalenesulfonyl
chloride, 2-thiphenesulfonyl chloride, 2-thiopheneacetyl chloride,
or propargylchloroformate.
The peptidomimetic library was synthesized on 4 g of TentaGel S
NH.sub.2 resin (0.26 mmol/g, Rapp Biopolymere) with standard solid
phase peptide synthesis methods, using a split-mix synthesis approach.
The coupling of all Fmoc protected amino acids (3eq) was initiated
by DIC (3eq), HOBt (3eq) and the progress of the reaction was monitored
by the Kaiser test. The Fmoc protecting groups were removed by 20%
piperidine in DMF (2.times.10 min).
D-Cys was first coupled to the resin in the first cycle. In the
second cycle, 17 natural amino acids (X) were then added. In the
third cycle, the resins were divided up into 5 portions (v:v:v:v:v=1:1:1:1:32)
and Tyr, Nle, Glu, Thr, and Phe(4-NO.sub.2) were added to the respective
resin portion together with the coupling reagents. After coupling
was completed, the Phe(4-NO.sub.2) resin was treated with 2M SnCl.sub.2
in DMF (24 hrs) to transform the nitrogroup to an amino group. The
resin was then divided into 32 portions. 26 acids (R.sub.1, 20eq)
and 6 acyl or sulfonyl chlorides (R.sub.1, 20eq) were coupled to
the side chain of the aminophenylalanine. DIC (20eq) plus DIEA (10eq)
was used as coupling reagents for the former, and DIEA (10eq) was
used as coupling reagent for the latter. In the fourth cycle, Gly
was attached. The fifth cycle was carried out according to the method
used in the third cycle, with the following amino acids: Nle, His,
Asp, Gln, or Phe(4-NO.sub.2). After the nitro group of Phe(4-NO.sub.2)
was reduced to amino group, 27 acids (R.sub.2) and 6 chlorides (R.sub.2,)
were added. Finally, the last three amino acids were coupled and
the side chain protecting groups were removed by TFA:TIS:water:EDT
(94:1:2.5:2.5 v/v/v/v, 3 hrs). The library was then thoroughly washed
and the disulfide bridge was formed by air oxidation for 48 hrs
mediated by DMSO (10%). The library was washed and stored in 70%
ethanol.
The peptidomimetic library was screened with the non-small-cell
lung cancer cell line A549 (American Type Culture Collection).
As described in Example 1, after culturing for about 24 to 72 hours,
beads with a monolayer of cells were observed. These positive beads
were isolated and the chemical structure of the peptidomimetic compound
on each positive bead was determined using amino acid sequencing,
as described in Example 1.
Ligands having the general structure cNGZ.sub.2GZ.sub.1Xc were
identified, where "X", "Z.sub.1", and "Z.sub.2"
were as set forth above. The structure of ten of the ligands is
shown in Table 3.
TABLE-US-00003 TABLE 3 Chemical Structure of Ligands for Non-Small-Cell
Lung Cancer Cells (A549) c-N-G-Z.sub.2-G-Z.sub.1-X-c Z.sub.2 Z.sub.1
X ##STR00052## ##STR00053## ##STR00054## ##STR00055## ##STR00056##
##STR00057## ##STR00058## ##STR00059## ##STR00060## ##STR00061##
##STR00062## ##STR00063## ##STR00064## ##STR00065## ##STR00066##
##STR00067## ##STR00068## ##STR00069## ##STR00070## ##STR00071##
##STR00072## ##STR00073## ##STR00074## ##STR00075## ##STR00076##
##STR00077## ##STR00078## ##STR00079## ##STR00080## ##STR00081##
EXAMPLE 8
The method as described in Example 7 was performed, except that
the peptidomimetic library was screened with the pancreatic cancer
cell line Panc-1 (American Type Culture Collection).
As described in Example 1, after culturing for about 24 to 72 hours,
beads with a monolayer of cells were observed. These positive beads
were isolated and the chemical structure of the peptidomimetic compound
on each positive bead was determined using amino acid sequencing,
as described in Example 1.
Ligands having the general structure cNGZ.sub.2GZ.sub.1Xc were
identified, where "X", "Z.sub.1", and "Z.sub.2"
were as set forth in Example 7. Six of these ligands are shown in
Table 4.
TABLE-US-00004 TABLE 4 Chemical Structure of Ligands for Pancreatic
Cancer Cells (Panc-1) c-N-G-Z.sub.2-G-Z.sub.1-X-c Z.sub.2 Z.sub.1
X ##STR00082## ##STR00083## ##STR00084## ##STR00085## ##STR00086##
##STR00087## ##STR00088## ##STR00089## ##STR00090## ##STR00091##
##STR00092## ##STR00093## ##STR00094## ##STR00095## ##STR00096##
##STR00097## ##STR00098## ##STR00099##
The invention has been described above with reference to the preferred
embodiments. Those skilled in the art may envision other embodiments
and variations of the invention that fall within the scope of the
claims. |