Use of modified tethers in screening compound libraries

• US 5,958,703 A
• Filed: 12/03/1996
• Issued: 09/28/1999
• Est. Priority Date: 12/03/1996
• Status: Expired due to Fees

• Current Assignee: Smithkline Beecham Corporation (Smithkline Beecham PLC)
• Original Assignee: Glaxo Group Limited (Glaxosmithkline Holdings Ltd.)
• Inventors: Mattheakis, Larry, Dower, William J., Schatz, Peter J., Heinkel, Gregory L.
• Primary Examiner(s): Achutamurthy, Ponnathapura
• Assistant Examiner(s): Ricigliano, Joseph W
• Application Number:
  US08/758,307
• Time in Patent Office: 1,029 Days
• Field of Search: 435/5, 435/6, 435/7.1, 435/7.2, 435/7.21, 435/7.5, 435/21, 435/23, 435/24, 436/501
• US Class Current: 435/7.1
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Abstract

The invention provides methods for screening libraries of complexes for compounds having a desired property, especially, the capacity to agonize, bind to, or antagonize a cellular receptor. The complexes in such libraries comprise a compound under test, a tag recording at least one step in synthesis of the compound, and a tether susceptible to modification by a reporter molecule. Modification of the tether is used to signify that a complex contains a compound having a desired property. The tag can be decoded to reveal at least one step in the synthesis of such a compound.

Claims

1. A method of screening compounds for a desired property, the method comprising:

providing a library of complexes, each complex comprising a compound under test, a tag recording at least one step in the synthesis of the compound, and a tether susceptible to modification by a reporter molecule produced in screening the test compounds, wherein at least one, but not all, of the complexes have a tether that has been modified by the reporter molecule, the modification indicating that the at least one complex bears a compound having the desired property;

separating the at least one complex having a modified tether from the library by virtue of the modified tether; and

decoding the tag of the at least one complex having the modified tether to identify at least one step in the synthesis of a compound having the desired property.

2. The method of claim 1, wherein the desired property is capacity to transduce a signal through a cellular receptor and the test compounds are screened by:

contacting the complexes with cells having a receptor and a DNA segment encoding a reporter molecule;

whereby the tether of the compound of the at least one complex is modified by the compound transducing a signal through the receptor of a cell causing expression of the reporter molecule from the DNA segment, which reporter molecule is released from the cell and modifies the tether of the at least one complex.

3. The method of claim 2, wherein each complex further comprises a support bearing the compound under test, the tag and the tether.

4. The method of claim 3, wherein the support is a bead.

5. The method of claim 3, wherein the support is at least two linked beads.

6. The method of claim 3, wherein the contacting step further comprises freeing the test compounds from the supports such that the at least one compound is free of its support in transducing the signal through the receptor of the cell.

7. The method of claim 6, wherein the test compounds are linked to the supports by a photocleavable linker and the test compounds are freed from the supports by exposure to radiation.

8. The method of claim 6, wherein first and second aliquots of each compound are linked to the supports by first and second linkers, whereby the first and second aliquots of compound can be separately freed from the supports on cleaving the first and second linkers.

9. The method of claim 6, wherein the freeing step is incomplete leaving the supports bearing residual amounts of the compounds.

10. The method of claim 3, wherein the tags are nucleic acids or inert hydrocarbons.

11. The method of claim 8, wherein the test compounds are selected from the group consisting of polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines, oligocarbamates, thiazolidinones and imidizolidinones.

12. The method of claim 3, wherein the reporter molecule is operably linked to a signal sequence that directs the secretion of the reporter molecule from the cell.

13. The method of claim 12, wherein:

the reporter molecule is operably linked to a phospholipid anchoring sequence,

the reporter molecule is secreted from the cell, where it binds to phospholipid on the cell surface through the phospholipid anchoring sequence; and

the method further comprises treating the cells with a phospholipase to release the reporter molecule.

14. The method of claim 3, wherein the reporter molecule is released by lysis of the cells.

15. The method of claim 3, wherein the receptor is a cell surface receptor.

16. The method of claim 15, wherein the receptor is heterologous to the cells.

17. The method of claim 16, wherein the receptor is a G-protein coupled receptor, a cytokine receptor or a growth factor receptor.

18. The method of claim 3, wherein the reporter molecule is expressed from a fos promoter or a segment thereof effective to promote transcription, which promoter or segment is activated by the signal transduced through the receptor.

19. The method of claim 3, wherein the supports are contacted with the cells in a gel matrix.

20. The method of claim 6, wherein in the contacting step, the supports are contacted with the cells in wells of a plate, whereby after release from the cell, the reporter molecule is confined within the well containing the cell and can only modify the tethers of complexes within that well.

21. The method of claim 20, wherein first and second aliquots of the test compounds are linked to the supports by first and second linkers, and the method comprises at least two cycles of screening,

the first cycle comprising:

contacting the supports with the cells such that at least some wells each contain a plurality of supports,

freeing the first aliquots of test compounds from the supports, whereby in a well containing the at least one complex, the compound of the at least one complex transduces the signal through the receptor of a cell causing expression of the reporter molecule, which reporter molecule is released from the cell and modifies the tether of the at least one complex as well as the tethers of other compounds in the same well, and

the second cycle comprising

isolating the supports having modified tethers and converting the modified tethers to unmodified tethers,

contacting the isolated supports having unmodified tethers with the cells in wells of a fresh plate whereby at least some wells each contain a single support,

freeing the second aliquots of the test compounds from the isolated supports having unmodified tethers, whereby the compound of the at least one complex transduces the signal through the receptor of a cell causing expression of the reporter molecule, which reporter molecule is released from the cell and modifies the tether of the at least one complex.

22. The method of claim 3, wherein the reporter molecule is protease and the tether has a site cleavable by the protease.

23. The method of claim 3, wherein the reporter molecule is BirA and the tether has a site susceptible to biotinylation by BirA.

24. The method of claim 3, wherein the cells further comprise a second segment encoding a second reporter molecule, and the reporter molecule induces expression of the second reporter molecule, whereby the second reporter molecule modifies the tether.

25. The method of claim 3, wherein the cells further comprises a second segment encoding a second reporter molecule, which is constitutively expressed, and the reporter molecule lyses the cell causing release of the second reporter molecule whereby the second reporter molecule modifies the tether.

26. The method of claim 3, wherein the cells in the contacting step further comprise a second DNA segment comprising a coding sequence for a second reporter molecule, which coding sequence is disrupted by an inactivating sequence flanked by sites recognized by the site-specific recombinase, and the reporter molecule is a site-specific recombinase whereby the the compound of the at least one complex causes expression of the site-specific recombinase, which excises the inactivating sequence from the coding sequence resulting in expression of the second reporter molecule, which is released from the cell and modifies the tether of the at least one complex from which the compound was released.

27. The method of claim 26, wherein the site-specific recombinase is cre and the sites recognized by the recombinase are loxP sites.

28. The method of claim 26, wherein the site-specific recombinase is Flp and the sites recognized by the recombinase are frt sites.

29. The method of claim 22, wherein the reporter molecule expressed from the DNA segment in the cells in the contacting step is tPA, the tether has a site cleaved by plasmin, and the method further comprises contacting the cells with plasminogen, whereby the tPA cleaves the plasminogen generating plasmin which modifies the tether.

30. The method of claim 3, wherein in the separating step, the support of the at least one complex having the modified tether is isolated from supports bearing unmodified tethers by screening with a labelled receptor having specific affinity for the modified tether.

31. The method of claim 3, wherein in the separating step, the support of the at least one complex bearing the modified tether is isolated from supports bearing unmodified tethers by screening with a labelled receptor having specific affinity for the unmodified tethers.

32. The method of claim 31, wherein the label is gold.

33. The method of claim 3, further comprising mixing the compound transducing the signal with a pharmaceutical carrier.

34. The method of claim 3, wherein at least 10⁶ compounds are provided.

35. The method of claim 3, wherein the test compounds are polymers and the tags identify each monomer component of the polymers.

36. The method of claim 3, wherein the at least one complex comprises a plurality of complexes collectively bearing a plurality of compounds which transduce the signal through the receptor of a plurality of cells, and the reporter molecule secreted by each of the plurality of cells modifies the tether of the support from which the compound transducing the signal in that cell was released.

37. The method of claim 3, wherein the cells used in the contacting step have a second DNA segment encoding a second reporter molecule, and the signal transduced through the receptor of the cell by the compound of the at least one complex causes expression of the reporter and second reporter molecules, whereby the second reporter molecule activates the reporter molecule and the activated reporter molecule modifies the tether.

38. The method of claim 3, wherein the cells used in the contacting step have a second receptor and a second DNA segment encoding a second reporter molecule, whereby

(1) a compound under test, which has the capacity to transduce a signal through the receptor and the second receptor,

transduces a signal through the receptor and the second receptor causing expression of the reporter molecule and the second reporter molecule, whereby the second reporter molecule inactivates the reporter molecule rendering it incapable of modifying the tether,

(2) the compound of the at least one complex is a compound under test, which has the capacity to transduce a signal through the receptor but not through the second receptor, and

transduces a signal through the receptor of the cell causing expression of the reporter molecule, which is released from the cell and modifies the tether of the support of the at least one complex from which the compound transducing the signal was released, thereby indicating that the compound has the capacity to transduce a signal through the receptor but not through the second receptor.

39. The method of claim 38, wherein the cells used in the contacting step comprise first and second cells, the first cells having the DNA segment encoding the reporter molecule and the second cells having the second DNA segment encoding the second reporter molecule, wherein the reporter and second reporter molecules are released from the first and second cells before the second reporter molecule inactivates the reporter molecule.

40. The method of claim 1, wherein the desired property is capacity to inhibit enzymatic modification of a substrate, and test compounds are screened by:

contacting the complexes with the enzyme and releasing the test compounds from the complexes within a matrix that retains a released compound in proximity to the complex from which it was released, whereby the enzyme modifies the tether of complexes from which compounds lacking enzyme inhibiting activity were released without modifying the tether of at least one complex from which a compound having enzyme inhibiting activity was released due to protection of the tether by the compound.

41. The method of claim 40, wherein the compounds are released from the complexes after contacting the complexes with the enzyme.

42. The method of claim 40, wherein the enzyme is a protease, phosphatase, kinase, glycosylase, or polymerase.

43. The method of claim 42, wherein the enzyme is a protease and the tether is a protein containing a site cleavable by the protease.

44. The method of claim 3, wherein the cells further comprise a second segment encoding a second reporter molecule, which is expressed from the second segment, and the reporter molecule modifies the second reporter molecule, whereby the modified second reporter molecule modifies the tether.