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. Test compounds modulating a cellular response, for example via a cell surface molecule may be identified by selecting solid supports comprising cells, wherein the cellular response of interest has been modulated. The cellular response may for example be changes in signal transduction pathways modulated by a cell surface molecule.

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 covalently linked to one member of a library of test compounds and wherein at least two solid supports comprise different library members; and   (b) Attaching cells comprising said reporter system(s) onto said solid support;   
       wherein
 cells are directly attached to the solid support, and/or 
 at least 10% of the solid supports comprise cell adhesion molecules as well as said library member, and cells adhere to said cell adhesion molecules; 
 
       and
 (c) 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 
 (d) Selecting solid supports comprising cells meeting said at least one selection criterion; and 
 (e) 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 support, 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 1 , wherein the solid supports are spots or regions on a surface or a plated gel or a membrane. 
     
     
         6 . The method according to  claim 2 , wherein said cell adhesion compound is a peptide with an overall positive netcharge. 
     
     
         7 . (canceled) 
     
     
         8 . The method according to  claim 6 , 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 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. 
     
     
         9 . The method according to  claim 1 , wherein said cellular response is modulation of a signal transduction pathway mediated by a cell surface molecule. 
     
     
         10 . The method according to  claim 9 , wherein said cell surface molecule is a G-protein coupled receptor (GPCR). 
     
     
         11 . The method according to  claim 10 , wherein said GPCR is selected from the group consisting of GPCR of table 3. 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . The method according to  claim 1 , wherein the cellular response is modulation of transcriptional activity. 
     
     
         18 . (canceled) 
     
     
         19 . The method according to  claim 1 , wherein the cellular response is change in the intracellular level of a compound 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . The method according to  claim 1 , wherein the cellular response is relocalisation of a compound. 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . (canceled) 
     
     
         29 . The method according to  claim 1 , wherein the reporter system is a system endogenous to said cells. 
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . The method according to  claim 1 , wherein the reporter system comprises a nucleic acid comprising a nucleotide sequence encoding a detectable polypeptide operably linked to a response element, the activity of which is modulated by the cellular response. 
     
     
         34 . (canceled) 
     
     
         35 . The method according to  claim 9 , wherein the reporter system comprises a nucleic acid comprising a nucleotide sequence encoding a detectable polypeptide operably linked to a response element selected from the group consisting of cAMP response element (CRE) and serum response element (SRE). 
     
     
         36 . The method according to  claim 9 , wherein the reporter system comprises a nucleic acid comprising a nucleotide sequence encoding a detectable polypeptide operably linked to transcriptional response element (TRE). 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . The method according to  claim 1 , wherein said detectable polypeptide is selected from the group consisting of fluorescent proteins and enzymes. 
     
     
         40 . (canceled) 
     
     
         41 . The method according to  claim 1 , wherein the reporter system comprises a bioluminescent moiety. 
     
     
         42 . (canceled) 
     
     
         43 . (canceled) 
     
     
         44 . The method according to  claim 1 , wherein one predetermined selection criteria is a quantitative level of said bioluminescence above or below a specific threshold. 
     
     
         45 . The method according to  claim 1 , wherein the predetermined selection criteria is specific localisation of a fluorescent signal. 
     
     
         46 . The method according to  claim 1 , wherein said cells are selected from the group consisting of mammalian cells. 
     
     
         47 . (canceled) 
     
     
         48 . (canceled) 
     
     
         49 . The method according to  claim 9 , wherein the cells attached to the resin beads comprise a nucleic acid comprising a first nucleotide sequence encoding said cell surface molecule operably linked to a second nucleotide sequence not naturally associated therewith directing expression of said first sequence. 
     
     
         50 . The method according to  claim 1 , wherein at least 100 resin beads comprising different library members are provided. 
     
     
         51 . (canceled) 
     
     
         52 . 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, glycopeptides, nucleic acids (DNA or RNA) [oligosaccharides,] and small organic molecules. 
     
     
         53 . The method according to  claim 1 , wherein the library is a library of small organic molecules. 
     
     
         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 the resin bead comprises or consists of polyethylene glycol 
     
     
         56 . (canceled) 
     
     
         57 . A cell adhesion compound selected from either:
 i) the group consisting of peptides of: SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ED 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. 
 
     
     
         58 . A resin bead comprising a cell adhesion compound 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. 
     
     
         59 . The resin bead according to  claim 58 , wherein said resin bead comprises polyethylene glycol. 
     
     
         60 . (canceled) 
     
     
         61 . 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   
     
     
         62 . A method of modulating the activity of a GPCR receptor comprising the steps of
 a) Providing a compound identified by the method according to  claim 10     b) Incubating said compound together with cells expressing said GPCR   c) Thereby modulating the activity of said GPCR   
     
     
         63 . Compound identified by the method according to  claim 1   
     
     
         64 . A method of synthesising a cyclic peptide or peptide mimetic library, comprising the steps
 i) Providing a plurality of peptides or peptide mimetics covalently linked to an azide moiety and an acetylene moeity; and   ii) cyclizing said peptide or peptide mimetic through a Cu(I) catalysed reaction between said azide- and said acetylene moiety; and   iii) thereby obtaining a library of cyclic peptides or peptide mimetics.   
     
     
         65 . The method according to  claim 64 , wherein each peptide or peptide mimetic are immobilised on a solid support. 
     
     
         66 . The method according to  claim 64 , wherein the solid support is resin beads and each resin bead comprises only one library member in one or more copies. 
     
     
         67 . A library prepared by the method according to  claim 64 . 
     
     
         68 . The method according to  claim 1 , wherein the library of test compounds is a cyclic peptide or peptide mimetic library prepared by a method comprising the steps
 i) providing a plurality of peptides or peptide mimetics covalently linked to an azide moiety and an acetylene moeity; and   ii) cyclizing said peptide or peptide mimetic through a Cu(I) catalysed reaction between said azide- and said acetylene moiety; and   iii) thereby obtaining a library of cyclic peptides or peptide mimetics.   
     
     
         69 . A method of synthesising a library of heterocyclic ureas, comprising the steps of
 i) Providing a plurality of urea containing peptide aldehydes; and   ii) Subjecting said urea containing peptides to an intramolecular Pictet-Spengler reaction; and   iii) Thereby obtaining a library of heterocyclic ureas   
     
     
         70 . The method according to  claim 69 , wherein said urea containing peptide aldehydes are immobilised on a solid support. 
     
     
         71 . A library obtained by the method according to  claim 69 . 
     
     
         72 . The method according to  claim 1 , wherein the library of test compounds is a library of heterocyclic ureas prepared by a method comprising the steps of
 i) providing a plurality of urea containing peptide aldehydes; and   ii) subjecting said urea containing peptides to an intramolecular Pictet-Spengler reaction; and   iii) thereby obtaining a library of heterocyclic ureas   
     
     
         73 . The method according to  claim 1 , wherein the library of test compounds is a library of heterocyclic compounds obtained by cyclisation of a peptide aldehyde through an intramolecular Pictet-Spengler reaction.