P-glycoproteins and uses therefor

Abstract

The invention pertains to cynomologous monkey P-glycoproteins and related P-glycoproteins which include cynomologous-specific amino acids, as well as nucleic acids which encode those polypeptides. The present invention also includes fragments and biologically functional variants of the cynomologous monkey P-glycoprotein. The invention further relates to methods of using such cynomologous monkey P-glycoprotein nucleic acids and polypeptides, especially in methods for determining bioavailability of drugs and for screening for inhibitors of cynomologous PGP. Also included are cynomologous PGP inhibitors which inhibit cynomologous PGP activity by inhibiting the expression or function of cynomologous PGP.

What is claimed is:  
     
         1 . An isolated nucleic acid molecule selected from the group consisting of 
 (a) nucleic acid molecules that code for the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4,    (b) allelic variants of (a), and    (c) complements of (a) or (b).    
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein the isolated nucleic acid molecule codes for SEQ ID NO:2.  
     
     
         3 . The isolated nucleic acid molecule of  claim 1 , wherein the isolated nucleic acid molecule codes for SEQ ID NO:4.  
     
     
         4 . The isolated nucleic acid molecule of  claim 1 , wherein the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4.  
     
     
         5 . An isolated P-glycoprotein polypeptide or fragment thereof which comprises at least one amino acid of a cynomologous P-glycoprotein selected from the group consisting of amino acids 12, 24, 30, 74, 78, 86, 89, 90, 91, 92, 95, 97, 99, 102, 103, 104, 185, 324, 363, 518, 635, 650, 656, 659, 677, 730, 738, 742, 745, 761, 765, 835, 851, 921, 967, 1003, 1027, 1038, 1048, 1103, 1128, 1168 and 1277 of SEQ ID NO:2 and amino acids 93, 94 and 95 of SEQ ID NO:4, wherein the P-glycoprotein is identical to a human P-glycoprotein except for the at least one amino acid of a cynomologous P-glycoprotein  
     
     
         6 . The isolated P-glycoprotein polypeptide or fragment thereof of  claim 5 , wherein the human P-glycoprotein is selected from the group of SEQ ID NO:5 and SEQ ID NO:6.  
     
     
         7 . An isolated P-glycoprotein polypeptide or fragment thereof which comprises at least one amino acid of a cynomologous P-glycoprotein selected from the group consisting of amino acids  3, 6, 8, 10, 13, 17, 19, 20, 21, 26, 30, 36, 38, 48, 52, 56, 64, 74, 78, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 98, 100, 101, 102, 103, 104, 105, 106, 110, 113, 145, 190, 197, 210, 231, 319, 324, 327, 345, 363, 395, 451, 455, 456, 468, 473, 494, 518, 530, 631, 641, 642, 648, 650, 655, 656, 664, 665, 672, 673, 674, 675, 683, 687, 689, 691, 692, 694, 701, 705, 715, 729, 730, 734, 742, 743, 745, 754, 757, 765, 835, 912, 918, 921, 940, 941, 944, 966, 967, 968, 970, 972, 981, 1008, 1015, 1023, 1024, 1048, 1093, 1096, 1103, 1128, 1142, 1146, 1147, 1156, 1160, 1163, 1166, 1250 and 1271 of SEQ ID NO: 2 and amino acids 93 and 94 of SEQ ID NO:4, wherein the P-glycoprotein is identical to a dog P-glycoprotein except for the at least one amino acid of a cynomologous P-glycoprotein  
     
     
         8 . The isolated P-glycoprotein polypeptide or fragment thereof of  claim 7 , wherein the dog P-glycoprotein is selected from the group of SEQ ID NO:7 and SEQ ID NO:8.  
     
     
         9 . The isolated P-glycoprotein polypeptide or fragment thereof of  claim 5  or  7 , wherein the amino acid sequence of the polypeptide or fragment thereof is an amino acid sequence selected from the group consisting of SEQ ID NO:2, fragments of SEQ ID NO:2, SEQ ID NO:4 and fragments of SEQ ID NO:4.  
     
     
         10 . An isolated nucleic acid molecule which encodes the isolated P-glycoprotein polypeptide or fragment thereof of any of claims  5 - 9 .  
     
     
         11 . An expression vector comprising the isolated nucleic acid molecule of  claim 1  operably linked to a promoter.  
     
     
         12 . An expression vector comprising the isolated nucleic acid molecule of  claim 10  operably linked to a promoter.  
     
     
         13 . A host cell transformed or transfected with the expression vector of  claim 11 .  
     
     
         14 . A host cell transformed or transfected with the expression vector of  claim 12 .  
     
     
         15 . An agent which selectively binds the isolated polypeptide of  claim 5 .  
     
     
         16 . The method of  claim 15 , wherein the agent does not bind a human or dog P-glycoprotein.  
     
     
         17 . The agent of  claim 15 , wherein the agent is a polypeptide.  
     
     
         18 . The agent of  claim 17 , wherein the polypeptide is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, Fab antibody fragments, F(ab) 2  antibody fragments and antibody fragments including a CDR3 region.  
     
     
         19 . An agent which selectively binds the isolated nucleic acid molecule of  claim 1  or  claim 10 .  
     
     
         20 . The agent of  claim 19 , wherein the agent is an antisense nucleic acid which selectively binds to the isolated nucleic acid molecule.  
     
     
         21 . A method for predicting the bioavailability of a compound, comprising 
 measuring the transmembrane transport of a test compound by a first P-glycoprotein,    comparing the transmembrane transport of the test compound by the first P-glycoprotein and a second P-glycoprotein to predict the bioavailability of the test compound, wherein the relative amount or rate of transport by the first P-glycoprotein and the second P-glycoprotein is predictive of bioavailability of the test compound.    
     
     
         22 . The method of  claim 21 , wherein the first P-glycoprotein is selected from the group consisting of dog P-glycoproteins and primate P-glycoproteins.  
     
     
         23 . The method of  claim 21 , wherein the first P-glycoprotein is the polypeptide of claims  5  or  7 .  
     
     
         24 . The method of  claim 21 , wherein the second P-glycoprotein is a human P-glycoprotein.  
     
     
         25 . A method for inhibiting P-glycoprotein transporter activity in a mammalian cell comprising 
 contacting the mammalian cell with an amount of the agent of  claim 19  effective to inhibit P-glycoprotein transporter activity in the mammalian cell.    
     
     
         26 . A method for increasing bioavailability of a drug in a subject comprising 
 administering to a subject in need of such treatment the agent of  claim 19  in an amount effective to increasing bioavailability of a drug.    
     
     
         27 . The method of  claim 26 , wherein the inhibitor is administered prior to administering the drug.  
     
     
         28 . The method of  claim 26 , wherein the inhibitor is administered concurrently with the drug.  
     
     
         29 . A method for increasing P-glycoprotein transporter activity in a cell comprising 
 contacting the cell with a molecule selected from the group consisting of the nucleic acid molecule of  claim 1  and the nucleic acid molecule of  claim 10 , in an amount effective to increase P-glycoprotein transporter activity in the cell.    
     
     
         30 . A method for identifying lead compounds for a pharmacological agent useful in the treatment of disease associated with P-glycoprotein transporter activity comprising 
 providing a cell or other membrane-encapsulated space comprising a P-glycoprotein as claimed in  claim 5  or  claim 7;     contacting the cell or other membrane-encapsulated space with a candidate pharmacological agent under conditions which, in the absence of the candidate pharmacological agent, cause a first amount of P-glycoprotein transporter activity;    determining a second amount of P-glycoprotein transporter activity as a measure of the effect of the pharmacological agent on the P-glycoprotein transporter activity, wherein a second amount of P-glycoprotein transporter activity which is less than the first amount indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which reduces P-glycoprotein transporter activity and wherein a second amount of P-glycoprotein transporter activity which is greater than the first amount indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which increases P-glycoprotein transporter activity.    
     
     
         31 . The method of  claim 30 , further comprising the step of loading the cell or other membrane-encapsulated space with a detectable compound, wherein the compound is detected as a measure of the P-glycoprotein transporter activity.  
     
     
         32 . A method for identifying compounds which selectively bind a P-glycoprotein comprising, 
 contacting the P-glycoprotein claimed in  claim 5  or  claim 7  with a compound,    determining the binding of the compound to the P-glycoprotein.    
     
     
         33 . The method of  claim 32  further comprising determining the effect of the compound on the P-glycoprotein transporter activity of the P-glycoprotein.  
     
     
         34 . The method of  claim 32  further comprising determining the effect of the compound on the ATPase activity of the P-glycoprotein.  
     
     
         35 . A method for determining ATPase activity of a P-glycoprotein comprising 
 contacting the host cell of  claim 12  or  14 , or a membrane fraction thereof, with a test drug, and    measuring ATPase activity of the P-glycoprotein.    
     
     
         36 . The method of  claim 35 , wherein the step of measuring ATPase activity is performed at least twice at different times.  
     
     
         37 . A method for determining transmembrane transport of a compound by a P-glycoprotein, comprising 
 contacting the host cell of  claim 12  or  14 , or a membrane fraction thereof, with a test drug, and    measuring transport of the test drug under sink conditions in at least one direction of transport selected from the group consisting of the apical to basolateral direction and the basolateral to apical direction.    
     
     
         38 . The method of  claim 37 , wherein the step of measuring transport of the test drug is performed at least twice at different times.