Method for extracting quantitative information relating to an influence on a cellular response

Abstract

A method for screening a library of compounds to detect a biologically active compound that modulates intracellular translocation of a subunit of a component of an intracellular pathway affecting intracellular processes includes: culturing one or more cells containing a nucleotide sequence coding for a hybrid polypeptide comprising a luminophore linked to the subunit of the component; introducing a compound of the library of compounds into the cell culture; screening the compound to determine whether the compound modulates the intracellular translocation of the subunit of the component; measuring light emitted from the luminophore to determine a first distribution; measuring light emitted from the luminophore to determine a second distribution; computing a variation between the first distribution and the second distribution by processing the measured light, any variation is indicative that the compound is biologically active. The method is also performed with a library of compounds.

1 . A method for screening a library of compounds to detect a biologically active compound that modulates intracellular translocation of at least a subunit of a component of an intracellular pathway affecting intracellular processes, the method comprising:
 (a) culturing one or more cells of at least one cell culture containing a nucleotide sequence coding for a hybrid polypeptide comprising a luminophore linked to said at least a subunit of the component under conditions that permit expression of the nucleotide sequence, said at least one cell culture being substantially devoid of a compound of the library of compounds to be screened for biological function or biological effect on said at least a subunit of the component in the one or more cells, wherein said at least a subunit of the component exhibits a biological activity of the component;   (b) introducing said compound of the library of compounds into said at least one cell culture;   (c) screening said compound of the library of compounds to determine whether said compound modulates the intracellular translocation of said at least a subunit of the component in the one or more cells;   (d) measuring light emitted from the luminophore in the one or more cells to determine a first distribution of said at least a subunit of the component in the one or more cells;   (e) measuring light emitted from the luminophore in the one or more cells to determine a second distribution of said at least a subunit of the component in the one or more cells in response to said compound;   (f) computing a variation between the first distribution and the second distribution by processing the measured light of the first distribution and the second distribution, said variation being indicative of the translocation of said at least a subunit of the component in said one or more cells and said translocation being indicative that said compound of the library of compounds is biologically active; and   (g) repeating at least steps (b)-(f) with a plurality of compounds of the library of compounds.   
     
     
         2 . A method according to  claim 1 , wherein the method is characterized by at least one of the following:
 at least the measuring is performed with an automated system;   at least the introducing is performed with an automated system; or   at least the processing and computing is performed with a computing system.   
     
     
         3 . A method according to  claim 1 , further comprising at least one of the following:
 fixing the one or more cells; or   selecting the one or more cells to be stable cells that are stably transformed with the nucleotide sequence coding for the hybrid polypeptide.   
     
     
         4 . A method according to  claim 1 , further comprising at least one of the following:
 processing measured light data obtained from measuring light emitted from the luminophore through an algorithm;   recording a plurality of digital images of the light emitted from the luminophore;   implementing a digital filtering method on a plurality of digital images of the light emitted from the luminophore, said filtering method being selected from the group consisting of smoothing, sharpening, edge detection, and combinations thereof, or   implementing a spatial frequency method on the plurality of digital images of the light emitted from the luminophore, said spatial frequency method being selected from Fourier filtering, image cross-correlation, image autocorrelation, object finding, object classification, color space manipulation for visualization, and combinations thereof.   
     
     
         5 . A method according to  claim 1 , further comprising individually screening the plurality of compounds of the library of compounds alone in the one or more cells to determine whether each compound of the plurality of compounds modulates the intracellular translocation of said at least a subunit of the component in the one or more cells. 
     
     
         6 . A method according to  claim 1 , further comprising screening at least one combination of compounds of the library of compounds to determine whether the combination of compounds modulates the intracellular translocation of said at least a subunit of the component in the one or more cells. 
     
     
         7 . A method according to  claim 1 , wherein the component is a protein. 
     
     
         8 . A method as in  claim 7 , wherein said at least a subunit of the component is substantially the entire protein. 
     
     
         9 . A method for screening a library of compounds to detect a biologically active compound that modulates intracellular translocation of at least a subunit of a component of an intracellular pathway affecting intracellular processes, the method comprising:
 (a) culturing one or more cells of at least one cell culture containing a nucleotide sequence coding for a hybrid polypeptide comprising a luminophore linked to said at least a subunit of the component under conditions that permit expression of the nucleotide sequence, said cell culture being substantially devoid of a compound of the library of compounds to be screened for biological function or biological effect on said at least a subunit of the component in the one or more cells, wherein said at least a subunit of the component exhibits a biological activity of the component;   (b) introducing said compound of the library of compounds into said at least one cell culture;   (c) screening said compound of the library of compounds to determine whether said compound modulates the intracellular translocation of said at least a subunit of the component in the one or more cells;   (d) measuring light emitted from the luminophore in the one or more cells at a first time point and a second time point;   (e) processing the measured light of the first time point and the second time point; and   (f) computing a variation in distribution of said at least a subunit of the component in the one or more cells from the first time point to the second time point, said variation in distribution of said at least a subunit of the component being in response to said compound and being indicative of the translocation of said at least a subunit of the component in said one or more cells and said translocation being indicative that said compound of the library of compounds is biologically active; and   (g) repeating at least steps (b)-(f) with a plurality of compounds of the library of compounds.   
     
     
         10 . A method according to  claim 9 , wherein the method is characterized by at least one of the following:
 at least the measuring is performed with an automated system;   at least the introducing is performed with an automated system; or   at least the processing and computing is performed with a computing system.   
     
     
         11 . A method according to  claim 9 , further comprising at least one of the following:
 fixing the one or more cells; or   selecting the one or more cells to be stable cells that are stably transformed with the nucleotide sequence coding for the hybrid polypeptide.   
     
     
         12 . A method according to  claim 9 , further comprising at least one of the following:
 processing measured light data obtained from measuring light emitted from the luminophore through an algorithm;   recording a plurality of digital images of the light emitted from the luminophore;   implementing a digital filtering method on a plurality of digital images of the light emitted from the luminophore, said filtering method being selected from the group consisting of smoothing, sharpening, edge detection, and combinations thereof, or   implementing a spatial frequency method on the plurality of digital images of the light emitted from the luminophore, said spatial frequency method being selected from Fourier filtering, image cross-correlation, image autocorrelation, object finding, object classification, color space manipulation for visualization, and combinations thereof.   
     
     
         13 . A method according to  claim 9 , further comprising individually screening a plurality of compounds of the library of compounds alone in the one or more cells to determine whether each compound of the plurality of compounds modulates the intracellular translocation of said at least a subunit of the component in the one or more cells. 
     
     
         14 . A method according to  claim 9 , further comprising screening at least one combination of compounds of the library of compounds to determine whether the combination of compounds modulates the intracellular translocation of said at least a subunit of the component in the one or more cells. 
     
     
         15 . A method according to  claim 9 , wherein the component is a protein. 
     
     
         16 . A method as in  claim 15 , wherein said at least a subunit of the component is substantially the entire protein. 
     
     
         17 . A method for screening a library of compounds to detect a biologically active compound that modulates intracellular translocation of at least a subunit of a component of an intracellular pathway affecting intracellular processes, the method comprising:
 (a) culturing one or more cells of at least one cell culture containing a nucleotide sequence coding for a hybrid polypeptide comprising a luminophore linked to said at least a subunit of the component under conditions that permit expression of the nucleotide sequence and that are substantially devoid of a compound of the library of compounds to be screened for biological function or biological effect on said at least a subunit of the component in the one or more cells, wherein said at least a subunit of the component exhibits a biological activity of the component;   (b) introducing said compound of the library of compounds into said at least one cell culture;   (c) screening said compound of the library of compounds to determine whether said compound modulates the intracellular translocation of said at least a subunit of the component in the one or more cells;   (d) measuring light emitted from the luminophore in the one or more cells to determine a first distribution of said at least a subunit of the component when said at least one cell culture is substantially devoid of said compound of the library of compounds to be screened;   (e) measuring light emitted from the luminophore in the incubated one or more cells to determine a second distribution of said at least a subunit of the component in the one or more cells in response to said compound;   (f) processing distribution data obtained from the first distribution and the second distribution through an algorithm; and   (g) computing a variation of said at least a subunit of the component from the first distribution to the second distribution, such variation being indicative of the translocation of said at least a subunit of the component in said one or more cells and said translocation being indicative that said compound of the library of compounds is biologically active; and   (h) repeating at least steps (b)-(h) with a plurality of compounds of the library of compounds.   
     
     
         18 . A method according to  claim 17 , wherein the method is characterized by at least one of the following:
 at least the measuring is performed with an automated system;   at least the introducing is performed with an automated system; or   at least the processing and computing is performed with a computing system.   
     
     
         19 . A method according to  claim 17 , further comprising at least one of the following:
 fixing the one or more cells; or   selecting the one or more cells to be stable cells that are stably transformed with the nucleotide sequence coding for the hybrid polypeptide.   
     
     
         20 . A method according to  claim 17 , further comprising at least one of the following:
 processing measured light data obtained from measuring light emitted from the luminophore through an algorithm;   recording a plurality of digital images of the light emitted from the luminophore;   implementing a digital filtering method on a plurality of digital images of the light emitted from the luminophore, said filtering method being selected from the group consisting of smoothing, sharpening, edge detection, and combinations thereof, or   implementing a spatial frequency method on the plurality of digital images of the light emitted from the luminophore, said spatial frequency method being selected from Fourier filtering, image cross-correlation, image autocorrelation, object finding, object classification, color space manipulation for visualization, and combinations thereof.   
     
     
         21 . A method according to  claim 17 , further comprising individually screening a plurality of compounds of the library of compounds alone in the one or more cells to determine whether each compound of the plurality of compounds modulates the intracellular translocation of said at least a subunit of the component in the one or more cells. 
     
     
         22 . A method according to  claim 17 , further comprising screening at least one combination of compounds of the library of compounds to determine whether the combination of compounds modulates the intracellular translocation of said at least a subunit of the component in the one or more cells. 
     
     
         23 . A method according to  claim 17 , wherein the component is a protein. 
     
     
         24 . A method according to  claim 23 , wherein said at least a subunit of the component is substantially the entire protein.