WO2005045430A1 - Criblage de banques combinatoires de billes pour ligands de cellules cancereuses - Google Patents

Criblage de banques combinatoires de billes pour ligands de cellules cancereuses Download PDF

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WO2005045430A1
WO2005045430A1 PCT/US2003/032031 US0332031W WO2005045430A1 WO 2005045430 A1 WO2005045430 A1 WO 2005045430A1 US 0332031 W US0332031 W US 0332031W WO 2005045430 A1 WO2005045430 A1 WO 2005045430A1
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cells
acid
phenylalanine
library
group
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PCT/US2003/032031
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Kit S. Lam
Derick H. Lau
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The Regents Of The University Of California
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Publication of WO2005045430A1 publication Critical patent/WO2005045430A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • G01N33/5017—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity for testing neoplastic activity

Definitions

  • 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.
  • 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.
  • a cleaving agent 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 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 April 23, 1996 to Lam, Kit S. et al.)
  • the existing methods 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.
  • 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.
  • 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.
  • 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
  • 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
  • Fig. 1 is a diagram depicting the steps of the cell-growth-on-bead assay as used
  • Fig. 1A shows attached epithelial cells.
  • Fig. IB 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. ID shows a top view of three beads, in which two beads have a monolayer of cells growing on the bead.
  • Fig. IE shows a top view of the three beads of Fig. ID, 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.
  • 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 cXXXXXc 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.
  • Fig. 1 shows the method as used with epithelial cells.
  • Fig. 1A shows attached epithelial cells 10.
  • Fig. IB shows the same cells 10 in suspension.
  • Suspended live cells 10 are mixed with the library in culture medium, as shown in Fig. IC, 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
  • 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. ID, 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.
  • 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° C to 37° 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. IE. 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.
  • a chaotrophic agent such as 8 M guanidine hydrochloride
  • 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, CA). Alternatively, the peptide can be released via a cleavable linker and the amino acid sequence determined by mass spectroscopy.
  • 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.
  • Example 1 A one-bead-one-peptide combinatorial library, containing random cXXXXXc peptides, was prepared using the "split synthesis" method of Lam et ah, "A new type of synthetic peptide library," 82-84.
  • the D-cysteines provide intramolecular cyclization by disulfide bonding.
  • TentaGel polystyrene beads with a diameter of 80 ⁇ m and with grafted polyethylene glycol of 0.25 rnmol/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
  • 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° 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 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
  • the cNGRGEQc peptide is a ligand specific for promoting
  • 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
  • Example 1 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
  • X 4 , X 6 and X 7 are N, S, Q, T, HoSer, Cit,
  • 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
  • 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 2 )-G-Pra-G-c; c-d-G-(4-Pal)-G-Tyr(3-NO 2 )-Cha-c; and c-d-G-Cha-G-I-T-c.
  • 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.
  • 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 2 )-G-I-Pra-c; c-d-G-Tyr(3-NO 2 )-G-F-L-c; c-d-G-Chg-G-Hyp-N-c; and c-D-G-Cha-G-Hyp-N-c.
  • 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 2 )-G-Phg-M-c; and c-d-G-Tyr(3-NO 2 )-G-Nle-H-c.
  • 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-Cl)-G-T-Y-c; c-d-G-Tyr(3-NO 2 )-G-Aic-Q-c; c-Tyr(3-NO 2 )-G-F-G-(Pal-3D)-HoSer-c; c-d-G-HoCit-G-T-Nva-c; c-d-G-Nle-G-I-G-c; and c-Nle-G-Nle-G-Tyr(3-NO 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
  • Example 1 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
  • the small molecule library was screened with the liver cancer cell line HEPG2
  • Example 7 The method as described in Example 1 was performed, except that a different combinatorial library was used.
  • "X" was Pro, Ala, Gly, Leu, He, Asp, Asn, Glu, Gin, Trp, His, Phe, Tyr, Val, Ser, Thr or Met
  • Zj was Y, Nle, E, T, Phe(NHR , or Phe(NHR r ); and
  • Z 2 was Nle, H, D, Q, Phe(NHR 2 ), or Phe(NHR 2 >).
  • Race was benzoic acid, 5 -bromo valeric acid, 3-pyridinepropionic acid, 3-thiophenecarboxylic acid, 4-(dimethylamino)phenylacetic acid, 4-bromobenzoic acid, phenoxyacetic acid, (l-aphtoxy)acetic acid, 5-hydantoinacetic acid, phenylpropionic acid, cyclopropanecarboxylic acid, 4-methyvaleric acid, 2-phenoxybutyric acid, 3-(dimethylamino)benzoic acid, 3-thio ⁇ henecarboxylic 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-(
  • 3-thiophenecarboxylic acid 4-(dimethylamino)phenylacetic acid, 4-bromobenzoic acid, phenoxyacetic acid, (l-naphtoxy)acetic acid, 5-hydantoinacetic acid, phenylpropionic acid, cyclopropanecarboxylic acid, 4-methyvaleric acid, 2-phenoxybutyric acid, 3-(dimethylamino)benzoic acid, 3-thiophenecarbox.ylic 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 2 >" was p
  • the peptidomimetic library was synthesized on 4g of TentaGel S NH 2 resin (0.26mmol/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 DIG (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 (2xl0min). D-Cys was first coupled to the resin in the first cycle. In the second cycle, 17 natural amino acids (X) were then added.
  • the Phe(4-NO ) resin was treated with 2M SnCl 2 in DMF (24hrs) to transform the nitrogroup to an amino group.
  • the resin was then divided into 32 portions. 26 acids (R l5 20eq) and 6 acyl or sulfonyl chlorides (Rr, 20eq) were coupled to the side chain of the aminophenylalanine.
  • DIC (20eq) plus DIE A (10eq) was used as coupling reagents for the former, and DIEA (lOeq) was used as coupling reagent for the latter, hi 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, Gin, or Phe(4-NO 2 ). After the nitro group of Phe(4-NO 2 ) was reduced to amino group, 27 acids (R 2 ) and 6 chlorides (R 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, 3hrs).
  • the library was then thoroughly washed and the disulfide bridge was formed by air oxidation for 48hrs 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 ⁇ Z ⁇ c were identified, where "X”, "Zi", and "Z 2 " were as set forth above. The structure often of the ligands is shown in Table 3.
  • 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 2 GZ 1 Xc were identified, where "X”, “Zi", and "Z 2 " were as set forth in Example 7. Six of these ligands are shown in Table 4.

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Abstract

L'invention concerne une méthode d'analyse par croissance cellulaire sur bille permettant le criblage d'une banque combinatoire du type comprenant un composé par bille, afin d'identifier des ligands de synthèse permettant la fixation et la multiplication ou la prolifération de cellules épithéliales ou non épithéliales. Les cellules sont incubées avec une banque de composés sur billes pendant 24 à 72 heures, de manière à permettre aux cellules de se fixer et de se multiplier sur les billes. Les billes comportant des cellules en voie de multiplication sont ensuite extraites, et le ligand dont elles sont porteuses est identifié. L'invention concerne également des ligands spécifiques des cellules cancéreuses.
PCT/US2003/032031 2003-10-08 2003-10-08 Criblage de banques combinatoires de billes pour ligands de cellules cancereuses WO2005045430A1 (fr)

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AU2003287040A AU2003287040A1 (en) 2003-10-08 2003-10-08 Screening combinatorial bead libraries for cancer ligands

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US7632814B2 (en) 2006-09-07 2009-12-15 University Of South Florida HYD1 peptides as anti-cancer agents
WO2011038142A3 (fr) * 2009-09-24 2011-08-18 The Regents Of The University Of California Peptides du type ligand spécifiques du cancer de la vessie
EP2688911A4 (fr) * 2011-03-24 2015-08-26 Opko Pharmaceuticals Llc Recherche de biomarqueurs dans un fluide biologique au moyen de banques à base de perles ou de particules, kits diagnostiques et thérapeutique
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005116656A1 (fr) * 2004-05-25 2005-12-08 2Curex Identification de composes modifiant une reponse cellulaire
US9150899B2 (en) 2004-05-25 2015-10-06 2Curex Identification of compounds modifying a cellular response
WO2007009457A2 (fr) * 2005-07-18 2007-01-25 2Curex Aps Identification de ligands de proteine modifiant une reponse cellulaire
WO2007009457A3 (fr) * 2005-07-18 2007-05-24 2Curex Aps Identification de ligands de proteine modifiant une reponse cellulaire
US7632814B2 (en) 2006-09-07 2009-12-15 University Of South Florida HYD1 peptides as anti-cancer agents
WO2011038142A3 (fr) * 2009-09-24 2011-08-18 The Regents Of The University Of California Peptides du type ligand spécifiques du cancer de la vessie
US8946379B2 (en) 2009-09-24 2015-02-03 The Regents Of The University Of California Bladder cancer specific ligand peptides
US9539340B2 (en) 2009-09-24 2017-01-10 The Regents Of The University Of California Bladder cancer specific ligand peptides
US10335365B2 (en) 2009-09-24 2019-07-02 The Regents ot the Univershy of California Bladder cancer specific ligand peptides
US10059740B2 (en) 2010-03-19 2018-08-28 H. Lee Moffitt Cancer Center And Research Institute, Inc. Integrin interaction inhibitors for the treatment of cancer
EP2688911A4 (fr) * 2011-03-24 2015-08-26 Opko Pharmaceuticals Llc Recherche de biomarqueurs dans un fluide biologique au moyen de banques à base de perles ou de particules, kits diagnostiques et thérapeutique
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