Identification of Compounds Modifying A Cellular Response

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.

1 . A method of identifying a compound modifying at least one cellular response, wherein each cellular response is linked to different reporter systems generating detectable outputs, said method comprising the steps of:
 (a) Providing multiple solid supports capable of supporting adherence and growth of cells, wherein each solid support is linked to multiple copies of a member of a library of test compounds via cleavable linkers and wherein at least two solid supports comprise different library members; and   (b) Attaching cells comprising said reporter system(s) onto said solid support; and   (c) Releasing a proportion of said library member from the solid support; and   (d) Screening said solid supports for solid supports comprising cells meeting at least one predetermined selection criterion, wherein said selection criterion is linked directly or indirectly to said detectable output; and   (e) Selecting solid supports comprising cells meeting said at least one selection criterion; and   (f) Identifying said library member, thereby identifying a compound modifying said at least one cellular response.   
     
     
         2 . The method according to  claim 1 , wherein said adherence is mediated through a cell adhesion compound coupled to said solid supports, wherein said cell adhesion compound enables said solid support to support growth of cells. 
     
     
         3 . The method according to  claim 1 , wherein the solid supports are resin beads. 
     
     
         4 . (canceled) 
     
     
         5 . The method according to  claim 3 , wherein the resin bead comprises or consists of polyethylene glycol 
     
     
         6 . (canceled) 
     
     
         7 . The method according to  claim 2 , wherein said cell adhesion compound is a peptide with an overall positive netcharge. 
     
     
         8 . (canceled) 
     
     
         9 . The method according to  claim 2 , wherein said cell adhesion compound is selected from the group consisting of SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9, SEQ ID 10, SEQ ID 11, SEQ ID 12, SEQ ID 13, SEQ ID 14, SEQ ID 15, SEQ ID 16, SEQ ID 17, SEQ ID 18, SEQ ID 19, SEQ ID 20, SEQ ID 21, SEQ ID 22, SEQ ID 23, SEQ ID 24, SEQ ID 25, SEQ ID 26, SEQ ID 27, SEQ ID 28, SEQ ID 29, SEQ ID 30, SEQ ID 31, SEQ ID 32, SEQ ID 33, SEQ ID 34, SEQ ID 35, SEQ ID 46, SEQ ID 47, SEQ ID 48, SEQ ID 49, SEQ ID 50, SEQ ID 51, SEQ ID 52, SEQ ID 53, SEQ ID 54, SEQ ID 55, SEQ ID 56, SEQ ID 57, SEQ ED 58, SEQ ID 59, SEQ ID 60, SEQ ID 61, SEQ ID 62, SEQ ID 63, SEQ ED 64, SEQ ID 65, SEQ ID 66, SEQ ID 67, SEQ ID 68, SEQ ID 69 and SEQ ED 70. 
     
     
         10 . The method according to  claim 2 , wherein the adhesion compound is linked to the resin bead via a cleavable linker. 
     
     
         11 . The method according to  claim 1 , wherein the cellular response is a change in interaction between two or more cellular molecules. 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . The method according to  claim 11 , wherein the cellular response is a change in interaction between proteins involved in regulation of apoptosis. 
     
     
         15 . The method according to  claim 1 , wherein said cellular response is a modulation of a signal transduction pathway. 
     
     
         16 . The method according to  claim 1 , wherein said cellular response is a modulation of signal transduction pathway, wherein said modulation is selected from the group consisting of
 Upregulation or downregulation of the level of a member of the pathway;   Relocalisation of a member of the pathway;   Complex formation between members of the pathway or between members of the pathway with other cellular compounds;   Enhanced or reduced transcription from genes regulated by the pathway;   Modification by phosphorylation of a member of the pathway;   Activation or inhibition of an enzyme of the pathway;   Degradation of a cellular compounds due to upregulation or downregulation of the pathway;   Altered secretion of a compound;   Change in ion-flux;   Morphological changes; and   Change in viability   
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . The method according to  claim 1 , wherein the cellular response is apoptosis. 
     
     
         28 . The method according to  claim 1 , wherein the reporter system is a system endogenous to said cells. 
     
     
         29 . (canceled) 
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . The method according to  claim 1 , wherein the reporter system comprises a bioluminescent moiety. 
     
     
         35 . (canceled) 
     
     
         36 . (canceled) 
     
     
         37 . (canceled) 
     
     
         38 . The method according to  claim 1 , wherein the reporter system is a proximity ligation reporter system. 
     
     
         39 . (canceled) 
     
     
         40 . The method according to  claim 1 , wherein one predetermined selection criterium is a quantitative level of said bioluminescence above or below a specific threshold. 
     
     
         41 . (canceled) 
     
     
         42 . (canceled) 
     
     
         43 . (canceled) 
     
     
         44 . The method according to  claim 1 , wherein said cells are selected from the group consisting of mammalian cells. 
     
     
         45 . (canceled) 
     
     
         46 . (canceled) 
     
     
         47 . The method according to  claim 3 , wherein at least 100 resin beads comprising different library members are provided. 
     
     
         48 . (canceled) 
     
     
         49 . The method according to  claim 1 , wherein the library is selected from the group consisting of peptides, glycopeptides, lipopeptides, nucleic acids (DNA or RNA), oligosaccharides; chemically modified peptides, oligomers of amino acids, glycopeptides, and small organic molecules. 
     
     
         50 . (canceled) 
     
     
         51 . (canceled) 
     
     
         52 . (canceled) 
     
     
         53 . The method according to  claim 1 , wherein the library comprises polymers of in the range of 3 to 6 amino acids selected from the group of amino acids mentioned in table 3, table 4, table 5 and table 6. 
     
     
         54 . The method according to  claim 1 , wherein compounds modifying at least two cellular responses are identified, wherein step c) involves screening said resin beads for beads comprising cells meeting at least two predetermined selection criterion, wherein each selection criterion is related to a different detectable output. 
     
     
         55 . The method according to  claim 1 , wherein at least one cleavable linker is selected from the group consisting of acid-labile, base-labile, fluoride-labile and photo-labile linkers. 
     
     
         56 . (canceled) 
     
     
         57 . (canceled) 
     
     
         58 . The method according to  claim 1 , wherein step c) comprises releasing in the range of 5 to 95% of the copies of the library member. 
     
     
         59 . A library useful in the method according to  claim 1 , comprising multiple solid supports linked to multiple copies of a member of a library of test compounds via cleavable linkers and wherein at least two solid supports comprise different library members; and furthermore wherein the solid supports are coupled to a cell adhesion compound, wherein said cell adhesion compound is selected from either:
 i) the group consisting of peptides of: SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9, SEQ ID 10, SEQ ID 11, SEQ ID 12, SEQ ID 13, SEQ ID 14, SEQ ID 15, SEQ ID 16, SEQ ID 17, SEQ ID 18, SEQ ID 19, SEQ ID 20, SEQ ID 21, SEQ ID 22, SEQ ID 23, SEQ ID 26, SEQ ID 27, SEQ ID 28, SEQ ID 29, SEQ ID 30, SEQ ID 31, SEQ ID 32, SEQ ID 33, SEQ ID 34, SEQ ID 35, SEQ ID 46, SEQ ID 47, SEQ ID 48, SEQ ID 49, SEQ ID 50, SEQ ID 51, SEQ ID 52, SEQ ID 53, SEQ ID 54, SEQ ID 55, SEQ ID 56, SEQ ID 57, SEQ ID 58, SEQ ID 59, SEQ ID 60, SEQ ID 61, SEQ ID 62, SEQ ID 63, SEQ ID 64, SEQ ID 65, SEQ ID 66, SEQ ID 67, SEQ ID 68, SEQ ID 69 and SEQ ID 70 or:   ii) a peptide comprising at least one D-form amino acid, said peptide being selected from the group consisting of:   SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9, SEQ ID 10, SEQ ID 11, SEQ ID 12, SEQ ID 13, SEQ ID 14, SEQ ID 15, SEQ ID 16, SEQ ID 17, SEQ ID 18, SEQ ID 19, SEQ ID 20, SEQ ID 21, SEQ ID 22, SEQ ID 23, SEQ ID 24, SEQ ID 25, SEQ ID 26, SEQ ID 27, SEQ ID 28, SEQ ID 29, SEQ ID 30, SEQ ID 31, SEQ ID 32, SEQ ID 33, SEQ ID 34, SEQ ID 35, SEQ ID 46, SEQ ID 47, SEQ ID 48, SEQ ID 49, SEQ ID 50, SEQ ID 51, SEQ ID 52, SEQ ID 53, SEQ ID 54, SEQ ID 55, SEQ ID 56, SEQ ID 57, SEQ ID 58, SEQ ID 59, SEQ ID 60, SEQ ID 61, SEQ ID 62, SEQ ID 63, SEQ ID 64, SEQ ID 65, SEQ ID 66, SEQ ID 67, SEQ ID 68, SEQ ID 69 and SEQ ID 70.   
     
     
         60 . A method of manufacturing a compound modifying at least one cellular response, wherein said method comprises the steps of:
 a) Identifying said compound by the method according to  claim 1     b) Preparing said compound by chemical synthesis   c) Thereby manufacturing said compound   
     
     
         61 . A method of modulating the interaction between two or more cellular molecules comprising the steps of
 a) Identifying a compound by the method according to  claim 11     b) Incubating said compound together with cells expressing said two cellular molecules   c) Thereby modulating the interaction between the two cellular molecules   
     
     
         62 . Compound identified by the method according to  claim 1 . 
     
     
         63 . The compound according to  claim 62 , wherein the compound is an oligomer of in the range of 3 to 6 amino acids selected from the group of amino acids mentioned in table 3, table 4, table 5 and table 6. 
     
     
         64 . A library comprising at least two oligomers of in the range of 3 to 6 amino acids selected from the group of amino acids mentioned in table 3, table 4, table 5 and table 6.