Substrates and screening methods for transport proteins

Abstract

A variety of methods for assaying libraries of test compounds as ligands and/or substrates of transport proteins, including both carrier-type and receptor-type transport proteins, are provided. Both in vitro and in vivo screening methods are disclosed. Also provided are methods for screening DNA libraries to identify members that encode transport proteins. Pharmaceutical compositions including compounds identified via the screening methods are also provided.

1 . A method of screening for a substrate to a carrier-mediated transport protein expressed in a cell plasma membrane, comprising:
 (a) providing a library comprising different complexes, each complex comprising a compound and a separate reporter, the compound varying between different complexes;   (b) providing a population of cells, each cell comprising a plasma membrane, one or more of the cells expressing one or more carrier-mediated transport proteins in the plasma membrane;   (c) contacting the population of cells with a plurality of complexes from the library simultaneously; and   (d) detecting a signal from the reporter of a complex while internalized within a cell, wherein the reporter preferentially generates the signal once the reporter is internalized within the cell rather than from complexes binding to the surface of the cell, the signal thus providing an indication that a complex whose reporter generated the signal comprises a compound that is a substrate for a carrier-mediated transport protein expressed in the plasma membrane,   provided that if the reporter comprises a fluorophore, the complex comprises a compound, a fluorophore and a quencher, and the fluorophore is linked to the quencher by a linker susceptible to cleavage within the cell, whereby the quencher quenches fluorescence from the fluorophore outside the cell and is cleaved from the fluorophore within the cell after the complex is internalized within the cell, whereby the reporter preferentially generates the signal once internalized within the cell.   
   
   
       2 . (canceled) 
   
   
       3 . The method of  claim 1 , wherein the reporter contains a cleavable site and the reporter is cleaved at the cleavable site after the complex is internalized within the cell. 
   
   
       4 . The method of  claim 1 , wherein reporter comprises an agent that causes a morphological change upon internalization within a cell, and if a compound complexed with the reporter is a substrate for the carrier-mediated transport protein, the complex is transported by the carrier-mediated transport protein into a cell expressing the carrier-mediated transport protein, whereby the agent triggers a detectable morphological change in the cell. 
   
   
       5 . The method of  claim 3 , wherein the agent inhibits cytoskeleton formation. 
   
   
       6 . The method of  claim 1 , wherein the reporter comprises a fluorophore and a quencher moiety, and if a compound complexed with the reporter is a substrate for the carrier-mediated transport protein, the complex is transported by the carrier-mediated transport protein into a cell expressing the carrier-mediated transport protein, whereby the quencher moiety becomes separated from the fluorophore such that a fluorescent signal is emitted by the fluorophore within the cell, and the detection step comprises detecting the fluorescent signal. 
   
   
       7 . The method of  claim 6 , wherein the reporter further comprises a cleavable linker that links the fluorophore and quencher moiety, the cleavable linker being cleaved after entry of the reporter into the cell. 
   
   
       8 . The method of  claim 6 , wherein the fluorophore and quencher moiety are joined via a linker that contains a site cleavable by an enzyme, and the population of cells express the enzyme. 
   
   
       9 . The method of  claim 8 , wherein the enzyme is an endogenous enzyme. 
   
   
       10 . The method of  claim 8 , wherein the enzyme is expressed from an exogenous sequence harbored by the population of cells. 
   
   
       11 . The method of  claim 8 , wherein the enzyme is a hydrolase. 
   
   
       12 . The method of  claim 1 , wherein the reporter comprises a detection moiety disposed to interact with an intracellular agent, the cells have the intracellular agent, and if a compound complexed with the reporter is a substrate for the carrier-mediated transport protein, the complex is transported by the carrier-mediated transport protein into a cell expressing the carrier-mediated transport protein, whereby the detection moiety interacts with the intracellular agent to generate a detectable signal, and the detection step comprises detecting the detectable signal. 
   
   
       13 . The method of  claim 12 , wherein the detection moiety is a nucleic-acid binding dye and the intracellular agent is a nucleic acid. 
   
   
       14 . The method of  claim 1 , wherein the reporter comprises a substrate for an enzyme, and if a compound complexed with the reporter is a substrate for the carrier-mediated transport protein, the complex is transported by the carrier-mediated transport protein into a cell expressing the carrier-mediated transport protein and the enzyme, whereby the enzyme metabolizes the enzyme substrate to form a detectable product, and the detecting step comprises detecting the detectable product. 
   
   
       15 . The method of  claim 14 , wherein the enzyme is selected from the group consisting of luciferase, alkaline phosphatase, β-galactosidase, and β-glucouronidase. 
   
   
       16 . The method of  claim 15 , wherein the reporter comprises luciferin, and wherein the population of cells express luciferase, and the luciferase metabolizes the luciferin to generate the detectable product. 
   
   
       17 . The method of  claim 16 , wherein the luciferin is derivatized to bear a polar moiety to reduce passive uptake of complex. 
   
   
       18 . The method of  claim 17 , wherein luciferin is joined to the complex via a cleavable site and an intracellular enzyme cleaves luciferin from the complex before luciferin can be metabolized by luciferase. 
   
   
       19 . The method of  claim 1 , wherein the contacting step results in at least one complex being internalized within a cell through the activity of the carrier-mediated transport protein, the reporter promotes aggregation of subunits of a multimeric enzyme expressed within the population of cells, and the enzyme catalyzes production of a product that generates a detectable signal, the detecting step comprising detecting the detectable signal. 
   
   
       20 . The method of  claim 1 , wherein the contacting step results in at least one complex being internalized in a cell through the activity of the carrier-mediated transport protein, the reporter promotes transcription from a promoter within a cell resulting in expression of an expression product that generates a detectable signal, and the detecting step comprises detecting the detectable signal. 
   
   
       21 . The method of  claim 20 , wherein the expression product is an enzyme that catalyzes the production of the detectable signal. 
   
   
       22 . The method of  claim 1 , wherein the contacting step results in at least one complex being internalized in at least one cell through the activity of the carrier-mediated transport protein, the reporter confers a selective advantage within the cell(s), and the detecting step comprises propagating the population of cells under conditions that enrich for cell(s) on which the selective advantage has been conferred. 
   
   
       23 . The method of  claim 1 , wherein the contacting step results in at least one complex being internalized in a cell through the activity of the carrier-mediated transport protein, and the method further comprises washing cells to remove unincorporated complexes before the detecting step, whereby signal from reporter internalized within the cell is preferentially detected. 
   
   
       24 . The method of  claim 1 , wherein the contacting step results in at least one complex being internalized in a cell through the activity of the carrier-mediated transport protein, the reporter is a fluorescent molecule, and the method further comprises contacting cells with a fluorescence quencher incapable of entering the cells to quench fluorescence of unincorporated complexes before the detecting step, whereby signal from reporter internalized within the cell is preferentially detected. 
   
   
       25 . The method of  claim 1 , wherein
 the population of cells comprise different cells that are located in a single reaction vessel;   contacting results in at least one complex being internalized within one of the cells; and   detecting comprises detecting the signal from the at least one complex.   
   
   
       26 . The method of  claim 25 , wherein
 the different cells comprise test cells and counterpart control cells, the test cells expressing one of the one or more carrier-mediated transport proteins while the control cells fail to express the transport protein expressed by the test cells;   the at least one complex is internalized within one of the test cells; and   detecting further comprises detecting signal, if any, from the control cells.   
   
   
       27 . The method of  claim 25 , further comprising:
 separately contacting the different cells with each of the plurality of complexes; and   determining the identity of the cell(s) to which the at least one complex is internalized within.   
   
   
       28 . The method of  claim 25 , wherein different cells have different distinguishable characteristics, and the method further comprises:
 determining the identity of the cell to which the at least one complex is internalized within from its distinguishable characteristic.   
   
   
       29 . The method of  claim 28 , wherein the distinguishable characteristics are different cellular morphologies. 
   
   
       30 . The method of  claim 28 , wherein the distinguishable characteristics are different stains on the cells. 
   
   
       31 . The method of  claim 28 , wherein the distinguishable characteristics are different markers located at the surface of the cells. 
   
   
       32 . The method of  claim 31 , wherein the different markers are different epitopes, the different epitopes being differentially stained with antibodies specific for the different epitopes, antibodies for different epitopes bearing different labels. 
   
   
       33 . The method of  claim 32 , wherein one or more of the different epitopes is expressed from an endogenous nucleic acid sequence. 
   
   
       34 . The method of  claim 32 , wherein one or more of the different epitopes is expressed from an exogenous nucleic acid sequence. 
   
   
       35 . The method of  claim 1 , wherein
 the population of cells are contained in a single reaction vessel; and   contacting comprises contacting the cells within the reaction vessel with a plurality of different complexes, different complexes comprising different compounds, whereby at least one complex is bound to or internalized within the cells; and   detecting comprises detecting signal from the at least one complex.   
   
   
       36 . The method of  claim 35  further comprising:
 (d) separately contacting the population of cells within different reaction vessels with the different complexes such that cells within the same reaction vessel receive the same complex while cells in at least some of the different reaction vessels receive different complexes; and   (e) determining the identity of the at least one complex.   
   
   
       37 . The method of  claim 35 , wherein
 the reporter varies between different complexes and different reporters are disposed to generate different signals; and   detecting comprises detecting the signal from the reporter of the at least one complex, the signal from the at least one complex providing an indication of the identity of the compound of the at least one complex.   
   
   
       38 . The method of  claim 37 , wherein different reporters comprise different labels, the different labels selected from the group consisting of a radiolabel, a mass label, a spin label, a fluorophore, a chromophore and a luminescent moiety. 
   
   
       39 . The method of  claim 37 , wherein different reporters comprise substrates for different enzymes expressed within the one or more cells. 
   
   
       40 . The method of  claim 37 , wherein the population of cells is a plurality of different cells, different cells having different distinguishable characteristics, and further comprising determining the identity of the cell to which the at least one complex is bound or internalized from its distinguishing characteristic. 
   
   
       41 . The method of  claim 1 , wherein detecting comprises detecting a signal from a reporter internalized within the one or more cells to identify at least one complex that is internalized within the one or more cells, the compound complexed to the internalized complex being a substrate potentially able to transport an agent into cells expressing a carrier-mediated transport protein, the method further comprising:
 (a) providing a modified complex, the modified complex comprising the compound identified in the detecting step (d) and an agent;   (b) contacting one or more cells with the modified complex; and   (c) determining whether the modified complex is internalized within one of the one or more cells by detecting the modified complex within the one or more cells, such detection providing an indication that the compound can serve as a substrate for transporting agents into cells expressing carrier-mediated transport proteins.   
   
   
       42 . The method of  claim 41 , wherein the agent is a pharmaceutical agent. 
   
   
       43 . The method of  claim 42 , wherein the reporter is attached to the compound of the at least one compound at an attachment site and the pharmaceutical agent replaces the reporter in the modified complex such that the pharmaceutical agent is attached to the attachment site in the modified complex. 
   
   
       44 . The method of  claim 43 , wherein the providing step comprises synthesizing the modified complex. 
   
   
       45 . The method of  claim 43 , wherein the modified complex further comprises a reporter attached at a site other than the attachment site. 
   
   
       46 . The method of  claim 1 , wherein contacting results in at least one complex being internalized in a cell, detecting comprises detecting signal from the reporter of the at least one complex and the method further comprises
 (a) determining the identity of the compound in the at least one complex detected in step (d);   (b) providing a focused library, the focused library comprising a plurality of complexes, each complex in the focused library comprising a compound that is a variant of the compound identified in step (e);   (c) contacting one or more cells with one or more of the complexes of the focused library, the one or more cells expressing a carrier-mediated transport protein; and   (d) detecting a signal from a reporter of a complex internalized within one of the one or more cells, the signal providing an indication that the compound of the internalized complex is a substrate for a carrier-mediated transport protein.   
   
   
       47 . The method of  claim 1 , wherein the population of cells has been transformed with a DNA library encoding the one or more transport proteins. 
   
   
       48 . The method of  claim 47 , further comprising:
 isolating a cell that has internalized the reporter; and   isolating a DNA molecule encoding a carrier-mediated transport protein from the isolated cell to identify the carrier-mediated transport protein that exhibits activity with the compound of the complex that is internalized within the isolated cell.   
   
   
       49 . The method of  claim 1 , wherein the carrier-mediated transport protein is selected from the group of an amino acid transporter, a dipeptide transporter, an oligopeptide transporter, a simple sugar transporter, a bile acid transporter, a vitamin transporter, a phosphate transporter, a monocarboxylic acid transporter, an organic anion transporter, an organic cation transporter, fatty acid transporter, a nucleoside transporter, and a ABC transporter. 
   
   
       50 . The method of  claim 49 , wherein the transport protein is selected from the group consisting of PEPT1, sodium-dependent glucose transport protein (SGLT1), liver bile acid transporter (NTCP) and ileal bile acid transporter (ASBT). 
   
   
       51 . The method of  claim 1 , wherein the one or more carrier-mediated transport proteins are endogenous proteins. 
   
   
       52 . The method of  claim 1 , wherein the one or more carrier-mediated transport proteins are expressed from an exogenous sequence harbored by the population of cells. 
   
   
       53 . The method of  claim 1 , wherein the cells of the population of cells is selected from the group consisting of Chinese hamster ovary (CHO) cells, VERO cells, HeLA cells, COS-7 cells, MDCK cells, HEK cells, CaCo-2 cells, HCT-8 cells, T84 cells and HT29 cells. 
   
   
       54 . The method of  claim 53 , wherein the cells are treated to yield membrane preparations or vesicles and the complexes are contacted with the membrane preparations or vesicles. 
   
   
       55 . The method of  claim 1 , wherein the compound is directly joined to the reporter via a chemical bond. 
   
   
       56 . The method of  claim 1 , wherein the complex further comprises a linker joining the compound and the reporter. 
   
   
       57 . The method of  claim 1 , wherein the linker contains a cleavage site. 
   
   
       58 . The method of  claim 1 , wherein the linker is a stable linker lacking a cleavage site. 
   
   
       59 . The method of  claim 1 , wherein the reporter is selected from the group consisting of a fluorophore, a chromophore, a radioisotope, a magnetic particle, a mass label and a spin label. 
   
   
       60 . The method of  claim 59 , wherein the reporter is a fluorophore. 
   
   
       61 . The method of  claim 1 , wherein the detection step is per formed by brightfield, phase contrast or fluorescence microscopy. 
   
   
       62 . The method of  claim 1 , wherein the detection step is performed using a confocal microscope. 
   
   
       63 . The method of  claim 62 , wherein the confocal microscope has multiple wavelength detection capability. 
   
   
       64 . The method of  claim 1 , wherein the different compounds are compounds from a combinatorial library. 
   
   
       65 . The method of  claim 1 , wherein the different compounds are variants of a known substrate for a carrier-mediated transport protein. 
   
   
       66 . The method of  claim 1 , wherein the different compounds are small molecules. 
   
   
       67 . The method of  claim 1 , wherein the plurality of different compounds are peptides. 
   
   
       68 . The method of  claim 1 , wherein the population of cells of step (b) comprise a test population and the method further comprises
 (a) providing a population of control cells the same as the population of test cells of step (b), except that the control cells fail to express the one or more carrier-mediated transport proteins; and   (b) repeating steps (c) and (d) with the control cells, wherein a statistically significant difference in the signal from the reporter in the population of test cells relative to the signal from the control cells provides a further indication that the complex whose reporter generated the signal comprises a compound that is a ligand for a carrier-mediated transport protein.   
   
   
       69 - 74 . (canceled) 
   
   
       75 . A method of screening for a carrier-mediated transport protein and/or a ligand thereto, comprising:
 (a) providing a library comprising different complexes, each complex comprising a compound and a separate reporter, the compound varying between different complexes;   (b) providing a plurality of different cells comprising a plasma membrane that are located within a single reaction vessel, each cell expressing a carrier-mediated transport protein in the plasma membrane and different cells having different distinguishable characteristics;   (c) contacting the plurality of different cells with a plurality of complexes from the library simultaneously, whereby at least one complex is bound to or internalized within one of the cells;   (d) detecting a signal from the reporter of the at least one complex bound to or internalized within the cell in step (c); and   (e) determining the identity of the cell in step (c) from its distinguishable characteristic.   
   
   
       76 - 137 . (canceled) 
   
   
       138 . The method of screening of  claim 1 , wherein the carrier-mediated transport protein expressed on of the cell surface is a solute carrier transporter. 
   
   
       139 . The method of  claim 138 , wherein the solute carrier transporter is selected from the group consisting of: an amino acid transporter, a dipeptide transporter, an oligopeptide transporter, a simple sugar transporter, a bile acid transporter, a vitamin transporter, a phosphate transporter, a monocarboxylic acid transporter, an organic anion transporter, an organic cation transporter, fatty acid transporter, and a nucleoside transporter. 
   
   
       140 - 142 . (canceled) 
   
   
       143 . A method of screening for a substrate to a carrier-mediated transport protein, comprising:
 (a) providing a library comprising different complexes, each complex comprising a compound and a separate reporter, the compound varying between different complexes;   (b) providing a population of cells, one or more of which expresses one or more carrier-mediated transport proteins;   (c) contacting the population of cells with a plurality of complexes from the library; and   (d) detecting a signal from the reporter of a complex while internalized within a cell, wherein the reporter preferentially generates the signal once the reporter is internalized within the cell rather than from complexes binding to the surface of the cell, the signal thus providing an indication that a complex whose reporter generated the signal comprises a compound that is a substrate for a carrier-mediated transport protein;   provided that if the reporter comprises a fluorophore, the fluorophore is linked to a quencher by a linker susceptible to cleavage within the cell, whereby the quencher quenches fluorescence from the fluorophore outside the cell and is cleaved from the fluorophore within the cell after the complex is internalized within the cell, whereby the reporter preferentially generates the signal once internalized within the cell;   wherein the reporter comprises a substrate for an enzyme, and if a compound complexed with the reporter is a substrate for the carrier-mediated transport protein, the complex is transported by the carrier-mediated transport protein into a cell expressing the carrier-mediated transport protein and the enzyme, whereby the enzyme metabolizes the enzyme substrate to form a detectable product, and the detecting step comprises detecting the detectable product; and   wherein the enzyme is selected from the group consisting of luciferase, alkaline phosphatase, β-galactosidase, and β-glucouronidase.   
   
   
       144 . The method of  claim 143 , wherein the reporter comprises luciferin, and wherein the population of cells express luciferase, and the luciferase metabolizes the luciferin to generate the detectable product. 
   
   
       145 . A method of screening for a substrate to a carrier-mediated transport protein, comprising:
 (a) providing a library comprising different complexes, each complex comprising a compound and a separate reporter, the compound varying between different complexes;   (b) providing a population of cells, one or more of which expresses one or more carrier-mediated transport proteins;   (c) contacting the population of cells with a plurality of complexes from the library; and   (d) detecting a signal from the reporter of a complex while internalized within a cell, wherein the reporter preferentially generates the signal once the reporter is internalized within the cell rather than from complexes binding to the surface of the cell, the signal thus providing an indication that a complex whose reporter generated the signal comprises a compound that is a substrate for a carrier-mediated transport protein;   provided that if the reporter comprises a fluorophore, the fluorophore is linked to a quencher by a linker susceptible to cleavage within the cell, whereby the quencher quenches fluorescence from the fluorophore outside the cell and is cleaved from the fluorophore within the cell after the complex is internalized within the cell, whereby the reporter preferentially generates the signal once internalized within the cell;   wherein the population of cells comprise different cells that are located in a single reaction vessel; contacting results in at least one complex being internalized within one of the cells; and detecting comprises detecting the signal from the at least one complex; and   wherein different cells have different distinguishable characteristics, and the method further comprises:   determining the identity of the cell to which the at least one complex is internalized within from its distinguishable characteristic; and   wherein the distinguishable characteristics are different stains on the cells.   
   
   
       146 . A method of screening for a substrate to a carrier-mediated transport protein, comprising:
 (a) providing a library comprising different complexes, each complex comprising a compound and a separate reporter, the compound varying between different complexes;   (b) providing a population of cells, one or more of which expresses one or more carrier-mediated transport proteins;   (c) contacting the population of cells with a plurality of complexes from the library; and   (d) detecting a signal from the reporter of a complex while internalized within a cell, wherein the reporter preferentially generates the signal once the reporter is internalized within the cell rather than from complexes binding to the surface of the cell, the signal thus providing an indication that a complex whose reporter generated the signal comprises a compound that is a substrate for a carrier-mediated transport protein;   provided that if the reporter comprises a fluorophore, the fluorophore is linked to a quencher by a linker susceptible to cleavage within the cell, whereby the quencher quenches fluorescence from the fluorophore outside the cell and is cleaved from the fluorophore within the cell after the complex is internalized within the cell, whereby the reporter preferentially generates the signal once internalized within the cell;   wherein the population of cells comprise different cells that are located in a single reaction vessel; contacting results in at least one complex being internalized within one of the cells; and detecting comprises detecting the signal from the at least one complex; and   wherein different cells have different distinguishable characteristics, and the method further comprises:   determining the identity of the cell to which the at least one complex is internalized within from its distinguishable characteristic; and   wherein the distinguishable characteristics are different markers located at the surface of the cells.   
   
   
       147 . The method of  claim 146 , wherein the different markers are different epitopes, the different epitopes being differentially stained with antibodies specific for the different epitopes, antibodies for different epitopes bearing different labels. 
   
   
       148 . The method of  claim 147 , wherein one or more of the different epitopes is expressed from an endogenous nucleic acid sequence. 
   
   
       149 . The method of  claim 147 , wherein one or more of the different epitopes is expressed from an exogenous nucleic acid sequence. 
   
   
       150 . A method of screening for a substrate to a carrier-mediated transport protein, comprising:
 (a) providing a library comprising different complexes, each complex comprising a compound and a separate reporter, the compound varying between different complexes;   (b) providing a population of cells, one or more of which expresses one or more carrier-mediated transport proteins;   (c) contacting the population of cells with a plurality of complexes from the library; and   (d) detecting a signal from the reporter of a complex while internalized within a cell, wherein the reporter preferentially generates the signal once the reporter is internalized within the cell rather than from complexes binding to the surface of the cell, the signal thus providing an indication that a complex whose reporter generated the signal comprises a compound that is a substrate for a carrier-mediated transport protein;   provided that if the reporter comprises a fluorophore, the fluorophore is linked to a quencher by a linker susceptible to cleavage within the cell, whereby the quencher quenches fluorescence from the fluorophore outside the cell and is cleaved from the fluorophore within the cell after the complex is internalized within the cell, whereby the reporter preferentially generates the signal once internalized within the cell;   wherein the population of cells are contained in a single reaction vessel; and   contacting comprises contacting the cells within the reaction vessel with a plurality of different complexes, different complexes comprising different compounds, whereby at least one complex is bound to or internalized within the cells; and   detecting comprises detecting signal from the at least one complex.   
   
   
       151 . The method of  claim 150 , wherein
 the reporter varies between different complexes and different reporters are disposed to generate different signals; and   detecting comprises detecting the signal from the reporter of the at least one complex, the signal from the at least one complex providing an indication of the identity of the compound of the at least one complex.   
   
   
       152 . The method of  claim 151 , wherein the population of cells is a plurality of different cells, different cells having different distinguishable characteristics, and further comprising determining the identity of the cell to which the at least one complex is bound or internalized from its distinguishing characteristic. 
   
   
       153 . A method of screening for a substrate to a carrier-mediated transport protein, comprising:
 (a) providing a library comprising different complexes, each complex comprising a compound and a separate reporter, the compound varying between different complexes;   (b) providing a population of cells, one or more of which expresses one or more carrier-mediated transport proteins;   (c) contacting the population of cells with a plurality of complexes from the library; and   (d) detecting a signal from the reporter of a complex while internalized within a cell, wherein the reporter preferentially generates the signal once the reporter is internalized within the cell rather than from complexes binding to the surface of the cell, the signal thus providing an indication that a complex whose reporter generated the signal comprises a compound that is a substrate for a carrier-mediated transport protein;   provided that if the reporter comprises a fluorophore, the fluorophore is linked to a quencher by a linker susceptible to cleavage within the cell, whereby the quencher quenches fluorescence from the fluorophore outside the cell and is cleaved from the fluorophore within the cell after the complex is internalized within the cell, whereby the reporter preferentially generates the signal once internalized within the cell; and   wherein contacting results in at least one complex being internalized in a cell, detecting comprises detecting signal from the reporter of the at least one complex and the method further comprises   (a) determining the identity of the compound in the at least one complex detected in step (d);   (b) providing a focused library, the focused library comprising a plurality of complexes, each complex in the focused library comprising a compound that is a variant of the compound identified in step (e);   (c) contacting one or more cells with one or more of the complexes of the focused library, the one or more cells expressing a carrier-mediated transport protein; and   (d) detecting a signal from a reporter of a complex internalized within one of the one or more cells, the signal providing an indication that the compound of the internalized complex is a substrate for a carrier-mediated transport protein.   
   
   
       154 . A method of screening for a substrate to a carrier-mediated transport protein, comprising:
 (a) providing a library comprising different complexes, each complex comprising a compound and a separate reporter, the compound varying between different complexes;   (b) providing a population of cells, one or more of which expresses one or more carrier-mediated transport proteins;   (c) contacting the population of cells with a plurality of complexes from the library; and   (d) detecting a signal from the reporter of a complex while internalized within a cell, wherein the reporter preferentially generates the signal once the reporter is internalized within the cell rather than from complexes binding to the surface of the cell, the signal thus providing an indication that a complex whose reporter generated the signal comprises a compound that is a substrate for a carrier-mediated transport protein;   provided that if the reporter comprises a fluorophore, the fluorophore is linked to a quencher by a linker susceptible to cleavage within the cell, whereby the quencher quenches fluorescence from the fluorophore outside the cell and is cleaved from the fluorophore within the cell after the complex is internalized within the cell, whereby the reporter preferentially generates the signal once internalized within the cell; and   wherein the population of cells has been transformed with a DNA library encoding the one or more transport proteins.   
   
   
       155 . The method of  claim 154 , further comprising:
 isolating a cell that has internalized the reporter; and   isolating a DNA molecule encoding a carrier-mediated transport protein from the isolated cell to identify the carrier-mediated transport protein that exhibits activity with the compound of the complex that is internalized within the isolated cell.   
   
   
       156 . A method of screening for a substrate to a carrier-mediated transport protein, comprising:
 (a) providing a library comprising different complexes, each complex comprising a compound and a separate reporter, the compound varying between different complexes;   (b) providing a population of cells, one or more of which expresses one or more carrier-mediated transport proteins;   (c) contacting the population of cells with a plurality of complexes from the library; and   (d) detecting a signal from the reporter of a complex while internalized within a cell, wherein the reporter preferentially generates the signal once the reporter is internalized within the cell rather than from complexes binding to the surface of the cell, the signal thus providing an indication that a complex whose reporter generated the signal comprises a compound that is a substrate for a carrier-mediated transport protein;   provided that if the reporter comprises a fluorophore, the fluorophore is linked to a quencher by a linker susceptible to cleavage within the cell, whereby the quencher quenches fluorescence from the fluorophore outside the cell and is cleaved from the fluorophore within the cell after the complex is internalized within the cell, whereby the reporter preferentially generates the signal once internalized within the cell;   wherein the carrier-mediated transport protein is selected from the group of an amino acid transporter, a dipeptide transporter, an oligopeptide transporter, a simple sugar transporter, a bile acid transporter, a vitamin transporter, a phosphate transporter, a monocarboxylic acid transporter, an organic anion transporter, an organic cation transporter, fatty acid transporter, a nucleoside transporter, and a ABC transporter; and   wherein the transport protein is selected from the group consisting of PEPT1, sodium-dependent glucose transport protein (SGLT1), liver bile acid transporter (NTCP) and ileal bile acid transporter (ASBT).   
   
   
       157 . A method of screening for a substrate to a carrier-mediated transport protein, comprising:
 (a) providing a library comprising different complexes, each complex comprising a compound and a separate reporter, the compound varying between different complexes;   (b) providing a population of cells, one or more of which expresses one or more carrier-mediated transport proteins;   (c) contacting the population of cells with a plurality of complexes from the library; and   (d) detecting a signal from the reporter of a complex while internalized within a cell, wherein the reporter preferentially generates the signal once the reporter is internalized within the cell rather than from complexes binding to the surface of the cell, the signal thus providing an indication that a complex whose reporter generated the signal comprises a compound that is a substrate for a carrier-mediated transport protein;   provided that if the reporter comprises a fluorophore, the fluorophore is linked to a quencher by a linker susceptible to cleavage within the cell, whereby the quencher quenches fluorescence from the fluorophore outside the cell and is cleaved from the fluorophore within the cell after the complex is internalized within the cell, whereby the reporter preferentially generates the signal once internalized within the cell; and   wherein the one or more carrier-mediated transport proteins are expressed from an exogenous sequence harbored by the population of cells.   
   
   
       158 . A method of screening for a substrate to a carrier-mediated transport protein, comprising:
 (a) providing a library comprising different complexes, each complex comprising a compound and a separate reporter, the compound varying between different complexes;   (b) providing a population of cells, one or more of which expresses one or more carrier-mediated transport proteins;   (c) contacting the population of cells with a plurality of complexes from the library; and   (d) detecting a signal from the reporter of a complex while internalized within a cell, wherein the reporter preferentially generates the signal once the reporter is internalized within the cell rather than from complexes binding to the surface of the cell, the signal thus providing an indication that a complex whose reporter generated the signal comprises a compound that is a substrate for a carrier-mediated transport protein;   provided that if the reporter comprises a fluorophore, the fluorophore is linked to a quencher by a linker susceptible to cleavage within the cell, whereby the quencher quenches fluorescence from the fluorophore outside the cell and is cleaved from the fluorophore within the cell after the complex is internalized within the cell, whereby the reporter preferentially generates the signal once internalized within the cell; and   wherein the cells of the population of cells is selected from the group consisting of Chinese hamster ovary (CHO) cells, VERO cells, HeLA cells, COS-7 cells, MDCK cells, HEK cells, CaCo-2 cells, HCT-8 cells, T84 cells and HT29 cells.   
   
   
       159 . The method of  claim 158 , wherein the cells are treated to yield membrane preparations or vesicles and the complexes are contacted with the membrane preparations or vesicles. 
   
   
       160 . A method of screening for a substrate to a carrier-mediated transport protein, comprising:
 (a) providing a library comprising different complexes, each complex comprising a compound and a separate reporter, the compound varying between different complexes;   (b) providing a population of cells, one or more of which expresses one or more carrier-mediated transport proteins;   (c) contacting the population of cells with a plurality of complexes from the library; and   (d) detecting a signal from the reporter of a complex while internalized within a cell, wherein the reporter preferentially generates the signal once the reporter is internalized within the cell rather than from complexes binding to the surface of the cell, the signal thus providing an indication that a complex whose reporter generated the signal comprises a compound that is a substrate for a carrier-mediated transport protein;   provided that if the reporter comprises a fluorophore, the fluorophore is linked to a quencher by a linker susceptible to cleavage within the cell, whereby the quencher quenches fluorescence from the fluorophore outside the cell and is cleaved from the fluorophore within the cell after the complex is internalized within the cell, whereby the reporter preferentially generates the signal once internalized within the cell; and   wherein the linker is a stable linker lacking a cleavage site.   
   
   
       161 . A method of screening for a substrate to a carrier-mediated transport protein, comprising:
 (a) providing a library comprising different complexes, each complex comprising a compound and a separate reporter, the compound varying between different complexes;   (b) providing a population of cells, one or more of which expresses one or more carrier-mediated transport proteins;   (c) contacting the population of cells with a plurality of complexes from the library; and   (d) detecting a signal from the reporter of a complex while internalized within a cell, wherein the reporter preferentially generates the signal once the reporter is internalized within the cell rather than from complexes binding to the surface of the cell, the signal thus providing an indication that a complex whose reporter generated the signal comprises a compound that is a substrate for a carrier-mediated transport protein;   provided that if the reporter comprises a fluorophore, the fluorophore is linked to a quencher by a linker susceptible to cleavage within the cell, whereby the quencher quenches fluorescence from the fluorophore outside the cell and is cleaved from the fluorophore within the cell after the complex is internalized within the cell, whereby the reporter preferentially generates the signal once internalized within the cell; and   wherein the population of cells of step (b) comprise a test population and the method further comprises   (a) providing a population of control cells the same as the population of test cells of step (b), except that the control cells fail to express the one or more carrier-mediated transport proteins; and   (b) repeating steps (c) and (d) with the control cells, wherein a statistically significant difference in the signal from the reporter in the population of test cells relative to the signal from the control cells provides a further indication that the complex whose reporter generated the signal comprises a compound that is a ligand for a carrier-mediated transport protein.