Method of screening for protein inhibitors and activators

Abstract

Chemical agents which are inhibitors or activators of a protein whose expression affects a phenotypic characteristic of the cell, especially a cultural or morphological characteristic, can be identified by their more pronounced effect on cells producing higher, usually non-naturally occurring, levels of the protein, than on cells producing little or none of the protein. Such chemical agents are used to inhibit or activate a protein in a cell. By this method, tamoxifen is used to inhibit PKC activity in a cell.

1 - 30 . (canceled)  
   
   
       31 . A method of determining whether a substance is a specific inhibitor or specific activator of a protein whose production by a cell evokes a responsive change in a phenotypic characteristic other than the level of said protein in said cell per se, which comprises: 
 (a) providing a first cell line which produces the protein and exhibits the responsive change in a phenotypic characteristic;    (b) providing a second cell line which produces the protein at a lower level than the first cell line, or does not produces the protein at all, and which exhibits the responsive change in a phenotypic characteristic to a lesser degree or not at all;    (c) incubating the substance with the first and second cell lines; and    (d) comparing the responsive change in a phenotypic characteristic of the first cell line to the substance with the responsive change in a phenotypic characteristic of the second cell line to the substance.    
   
   
       32 . The method of  claim 31 , wherein said first cell line is obtained by introducing a gene encoding the protein into a host cell, the gene being under the control of a promoter functional in the host cell, whereby the gene is expressed.  
   
   
       33 . The method of  claim 32 , wherein the gene is introduced into the host cell by means of a first genetic vector into which the gene has been inserted, and the second cell line is obtained by introducing into a similar host cell a second genetic vector essentially identical to the first genetic vector except that it does not bear the gene insert.  
   
   
       34 . The method of  claim 32 , wherein the gene is introduced into the host cell by means of a retroviral vector.  
   
   
       35 . The method of  claim 32 , wherein the host cell line essentially does not produce the protein prior to introduction of the gene.  
   
   
       36 . The method of  claim 32 , wherein the host cell line is a rat-6 fibroblast cell line.  
   
   
       37 . A method of determining whether a substance is a specific inhibitor or specific activator of a protein, which comprises: 
 (a) providing a test cell which overproduces the protein relative to a control cell which produces the protein at a lower level or essentially does not produce the protein, wherein production of the protein in the test cell evokes a responsive change in a phenotypic characteristic, other than the level of the protein in the cell, per se, which is comparatively greater than in the control cell,    (b) treating the test cell containing the overproduced protein with the substance, and    (c) examining the treated test cell to determine whether the responsive change in a phenotypic characteristic is increased or decreased in response to the substance.    
   
   
       38 . The method of  claim 37 , wherein the examination of the responsive change in a phenotypic characteristic includes comparing the responsive change in a phenotypic characteristic of the treated test cell to the responsive change in a phenotypic characteristic of a comparable untreated test cell.  
   
   
       39 . The method of  claim 37 , wherein the examination of the responsive change in a phenotypic characteristic includes comparing the responsive change in a phenotypic characteristic of the treated test cell to the responsive change in a phenotypic characteristic of a comparably treated cell that does not overproduce the protein.  
   
   
       40 . The method of  claim 37 , wherein the examination of the responsive change in a phenotypic characteristic includes comparing the responsive change in a phenotypic characteristic of the test cell in the presence of the substance with the responsive change in a phenotypic characteristic of a second test cell in the presence of a known specific inhibitor or specific activator of the protein.  
   
   
       41 . The method of  claim 37 , wherein the test cell is selected from the group consisting of cells which have been genetically engineered by insertion of a nucleic acid sequence and cells which have mutated to increase expression of the selected protein.  
   
   
       42 . The method of  claim 37 , wherein the test cell is obtained by introducing a gene encoding the protein into a host cell, the gene being under the control of a promoter functional in the host cell, whereby the gene is expressed.  
   
   
       43 . The method of  claim 42 , wherein the gene is introduced into the host cell by means of a genetic vector into which the gene has been inserted.  
   
   
       44 . The method of  claim 42 , wherein the gene is introduced into the host cell by means of a retroviral vector.  
   
   
       45 . The method of  claim 42 , wherein the host cell essentially does not produce the protein prior to introduction of the gene.  
   
   
       46 . The method of  claim 42 , wherein the host cell is selected from the group consisting of a rat 6 fibroblast cell and a CHO cell.  
   
   
       47 . The method of  claim 31  or  claim 37 , wherein the responsive change in a phenotypic characteristic is a graded cellular response.  
   
   
       48 . The method of  claim 31  or  claim 37 , wherein the responsive change in a phenotypic characteristic is one observable with the naked eye.  
   
   
       49 . The method of  claim 31  or  claim 37 , wherein the responsive change in a phenotypic characteristic is selected from a change in a cultural or morphological characteristic of the cell, a change in the ability of the cell line to grow in an anchorage-independent fashion, a change in the ability of the cell line to grow on soft agar, a change in foci formation in cell culture, a change the ability of the cells to take up a selected stain, a change in the differentiation state of the cell, and a change in an antigenic characteristic of the cell.  
   
   
       50 . The method of  claim 31  or  claim 37 , wherein the responsive change in a phenotypic characteristic includes a change in the phosphorylation of an intracellular substrate of the protein.  
   
   
       51 . The method of  claim 31  or  claim 37 , wherein the responsive change in a phenotypic characteristic is detected on a subcellular fraction of the cell.  
   
   
       52 . The method of  claim 31  or  claim 37 , wherein the protein is an enzyme.  
   
   
       53 . The method of  claim 52 , wherein the responsive change in a phenotypic characteristic is measurable upon treatment of the test cell with an activator or inhibitor of the enzyme.  
   
   
       54 . The method of  claim 52 , wherein the responsive change in a phenotypic characteristic is detectable prior to treatment of the test cell with an activator or inhibitor of the enzyme.  
   
   
       55 . The method of  claim 52 , wherein increased activity of the enzyme is correlated with increased tumorigenesis.  
   
   
       56 . The method of  claim 52 , wherein the enzyme is a protein kinase.  
   
   
       57 . The method of  claim 52 , wherein the enzyme is a tyrosine kinase.  
   
   
       58 . The method of  claim 52 , wherein the enzyme is a protein kinase C enzyme or a fragment, domain, or subunit of a receptor which has protein kinase C activity.  
   
   
       59 . The method of  claim 52 , wherein the enzyme is selected from the group consisting of ornithine decarboxylase, insulin receptor, and serotonin receptor.  
   
   
       60 . The method of  claim 31  or  claim 37 , wherein the protein is the expression product of an oncogene.  
   
   
       61 . The method of  claim 31  or  claim 37 , wherein the substance is a suspected specific inhibitor or a specific activator of the biological activity of the protein.  
   
   
       62 . A test kit for determining whether a substance is a specific inhibitor or specific activator of a protein whose production evokes a responsive change in a phenotypic characteristic other than level of said protein in said cell per se, which comprises: 
 (a) a first cell line which produces the protein and exhibits the responsive change in a phenotypic characteristic; and    (b) a second cell line which produces the protein at a lower level than the first cell line, or does not produce the protein, and which exhibits the responsive change in a phenotypic characteristic to a lesser degree or not at all.    
   
   
       63 . The test kit of  claim 62 , wherein the cell lines are animal cell lines.  
   
   
       64 . The test kit of  claim 62 , wherein the cell lines are mammalian cell lines.  
   
   
       65 . The test kit of  claim 62 , which further comprises a known specific inhibitor or specific activator of the protein.  
   
   
       66 . The test kit of  claim 62 , wherein the level of production of the protein in the first cell line is at least five times the level of production of the protein in the second line.  
   
   
       67 . The test kit of  claim 62 , wherein the responsive change in a phenotypic characteristic is selected from the group consisting of growth rate, saturation density, plating efficiency in soft agar, colony size in soft agar, and combinations thereof.  
   
   
       68 . A test cell which comprises a target protein, wherein the presence of the target protein evokes a responsive change in a phenotypic characteristic of the cell other than the level of the target protein in the test cell per se, wherein the responsive change in a phenotypic characteristic is comparatively greater in the test cell than in a control cell, and wherein the responsive change in a phenotypic characteristic has been found to be responsive to treatment with a test substance that is capable of specifically inhibiting or specifically activating a biochemical function of the target protein by a method comprising the steps of: 
 (a) providing a test cell in which the target protein is present at a higher level relative to a control cell in which the protein is present at a lower level or is essentially not present;    (b) treating the test cell of step (a) with a test substance; and    (c) examining the treated test cell of step (b) to determine whether the change in a responsive characteristic that is responsive to specific inhibition or specific activation of the target protein of step (a) has been altered by treatment with the substance of step (b).