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Identification of Compounds Modifying A Cellular Response

Inactive Publication Date: 2009-09-24
2CUREX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]It is of great importance to provide new and efficient methods for identification of compounds influencing specific cellular processes. In particular, such methods wherein a very large quantity of candidate compounds may be tested for a specific effect on a cell within a relatively short period of time.
[0012]It is therefore an object of the present invention to provide very efficient procedures for testing or discovering the influence of compounds of a library on a physiological process in a cell. In particular, the methods provides means for testing very large numbers of different compounds for one or more physiological effects within a rather short time period. This may be obtained by attaching living cells to resin beads coupled to a test compound. The test compounds may be released from the resin beads and thus influence physiological processes in said cells. Said influence(s) may be detected and beads containing cells displaying the desired influence(s) may be selected. Once selected the compounds coupled to the selected beads may be identified. These methods may for example be very useful in connection with screening for new drugs, testing of substances for toxicity, identifying drug targets for known or novel drugs.

Problems solved by technology

In particular, such methods wherein a very large quantity of candidate compounds may be tested for a specific effect on a cell within a relatively short period of time.

Method used

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  • Identification of Compounds Modifying A Cellular Response
  • Identification of Compounds Modifying A Cellular Response
  • Identification of Compounds Modifying A Cellular Response

Examples

Experimental program
Comparison scheme
Effect test

example 1

General Methods for Solid Phase Peptide Synthesis (SPPS)

General for Chemical Synthesis:

[0316]All chemicals described, commercially available and used without further purification. All solvents were HPLC-grade. PEGA-resins were purchased from VersaMatrix A / S, Copenhagen. Each washing step lasted 2 min unless otherwise stated. Purifications were performed on a standard reverse phase HPLC using gradients of acetonitrile-Water with various amounts of TFA.

Coupling of HMBA Linker to PEGA-Resin:

[0317]Dry PEGA-resin was swelled in DCM and washed with DMF (3×). 3.0 eq. HMBA, 2.9 eq. TBTU and 3.0 eq. NEM were mixed in appropriate DMF and allowed to react for 10 min. The mixture was added to resin and after 2 h the resin was washed with DMF (6×), DCM (6×) and lyophilised.

General Procedure for Coupling of Amino Acid to HMBA-Linker:

[0318]Dry PEGA-resin with HMBA-linker was swelled in DCM. 3.0 eq. Fmoc-protected amino acid, 2.25 eq. Melm and 3.0 eq. MSNT were mixed in appropriate amount of DCM an...

example 2a

Screening of Adhesion Peptide Library

[0323]Approx. 100 adhesion peptide library beads were mixed with 1×10E6 cells (BHK, CHO, U2OS, Hek) in each well of a Falcon 12 well plate using 2 ml growth medium. The adhesion peptide library was prepared according to the general methods for solid phase peptide synthesis outlined above. The library consisted of heptamers of D-amino acids. The peptide library beads were PEGA beads each coupled to a potential adhesion peptide. The cells and beads were mixed gently every 15 min for 2 hrs. Supernatant with nonattached cells were removed and new growth medium added. Cells / beads were incubating for another 16 hrs. (37° C., 5% CO2). Cell adhesive beads were identified using a microscope with 10× objective and positive beads were transferred to a filter paper (to suck off medium). Peptides were identified by amino acid sequencing. Examples of useful peptides are given in table 2.

example 2b

Identification of an Adhesion Peptide with Low Absorption of Fluorescent Components from Growth Medium and High Adhesion Properties

[0324]An adhesion D-amino peptide library was synthesized (500.000 members) as described above in Example 2a and screened for low fluorescence / high adherence properties. This was done in 4 steps:

1) Selection of low fluorescent beads by Fluorescence Activated Bead Sorting (FABS).

[0325]The 500.000 member adhesion peptide library was FABSorted and 150.000 low fluorescent beads were isolated.

2) Selection of beads with good cell adhesion properties.

[0326]The 150.000 low fluorescent beads were incubated with GFP expressing U2OS cells followed by FABS sorting for high fluorescence (high cell adhesion). 536 beads were isolated.

3) Identification and isolation of beads with high Hek293 cell adherence properties.

[0327]The 536 beads were cleared for U2OS cells and incubated with GFP expressing Hek293 cells. 47 beads with high cell adhesion properties were isolated u...

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Abstract

The present invention relates to methods for identifying compounds capable of modulating a cellular response. The methods involve attaching living cells to solid supports comprising a library of test compounds. The test compounds are linked to the solid support via cleavable linkers and may thus be released from the solid supports. Solid supports comprising cells, wherein the cellular response of interest has been modulated are selected and the test compound of the solid support can then be identified. The cellular response may for example be changes in complex formation between proteins.

Description

[0001]All patent and non-patent references cited in the application are hereby incorporated by reference in their entirety.FIELD OF INVENTION[0002]The present invention relates to a method and tools for extracting information relating to an influence, for example on intracellular molecule(s), in particular an influence caused by contacting a cellular molecule with a substance, which has been released from a solid support to which a cell expressing the cellular component is attached. In particular the method related to a solid support that allow chemical synthesis of individual substances on beads of the solid support[0003]The method of the invention may be used as a very efficient procedure for testing or discovering the influence of a library of substances on a physiological process, for example in connection with screening for new drugs, testing of substances for toxicity, identifying drug targets for known or novel drugs. Other valuable uses of the method and technology of the in...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B30/00C40B40/00C07D471/04C07K1/04C07K5/00C07K5/103C07K7/00C07K7/06C07K7/08G01N33/50G01N33/543G01N33/68G01N35/00
CPCC07K1/047C07K5/1008C07K7/06C07K7/08C12Q1/025G01N33/54313G01N33/6845G01N2035/00158G01N2500/10G01N33/5023C40B30/06
Inventor THASTRUP, OLEMELDAL, MORTENHAGEL, GRITHCHR. NORRILD, JENSHENTZER, MORTEN
Owner 2CUREX
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