NPC1L1 (NPC3) and methods of use thereof

Abstract

The present invention provides human, rat and mouse NPC1L1 polypeptides and polynucleotides encoding the polypeptides. Also provided are methods for detecting agonists and antagonists of NPC1L1. Inhibitors of NPC1L1 can be used for inhibiting intestinal cholesterol absorption in a subject.

1 . An isolated polypeptide comprising 42 or more contiguous amino acids from an amino acid sequence selected from SEQ ID NOs: 2 and 12.  
     
     
         2 . An isolated polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 2, 4 and 12.  
     
     
         3 . An isolated polynucleotide encoding a polypeptide of  claim 1 .  
     
     
         4 . An isolated polynucleotide comprising a nucleotide sequence selected from SEQ ID NOs: 1, 3 and 11.  
     
     
         5 . A recombinant vector comprising the polynucleotide of  claim 3 .  
     
     
         6 . A host cell comprising the vector of  claim 5 .  
     
     
         7 . An isolated antibody which specifically binds to a polypeptide comprising 42 or more contiguous amino acids from an amino acid sequence selected from SEQ ID NOs: 2 and 12 or to an isolated polypeptide comprising an amino acid of SEQ ID NO: 4.  
     
     
         8 . An isolated antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 39-42.  
     
     
         9 . A method for making a polypeptide comprising culturing a host cell of  claim 6  under conditions in which the polynucleotide is expressed.  
     
     
         10 . The method of  claim 9  wherein the polypeptide is isolated from the culture.  
     
     
         11 . A method for identifying an antagonist of NPC1L1 comprising: 
 (a) contacting a host cell expressing a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 2, 4 and 12 or a functional fragment thereof on a cell surface, in the presence of a known amount of detectably labeled ezetimibe, with a sample to be tested for the presence of the antagonist; and    (b) measuring the amount of detectably labeled ezetimibe specifically bound, directly or indirectly, to the polypeptide;    wherein an NPC1L1 antagonist in the sample is identified by measuring substantially reduced direct or indirect binding of the detectably labeled ezetimibe to the polypeptide, compared to what would be measured in the absence of such an antagonist.    
     
     
         12 . A method for identifying an antagonist of NPC1L1 comprising: 
 (a) placing, in an aqueous suspension, a plurality of support particles, impregnated with a fluorescer, to which a host cell expressing a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 2, 4 and 12 or a functional fragment thereof on a cell surface are attached;    (b) adding, to the suspension, radiolabeled ezetimibe and a sample to be tested for the presence of the antagonist, wherein the radiolabel emits radiation energy capable of activating the fluorescer upon direct or indirect binding of the ezetimibe to the polypeptide to produce light energy, whereas radiolabeled ezetimibe that does not directly or indirectly bind to the polypeptide is, generally, too far removed from the support particles to enable the radioactive energy to activate the fluorescer; and    (c) measuring the light energy emitted by the fluorescer in the suspension;    wherein an NPC1L1 antagonist in the sample is identified by measuring substantially reduced light energy emission, compared to what would be measured in the absence of such an antagonist.    
     
     
         13 . The method of  claim 12  wherein the fluorescer is selected from yttrium silicate, yttrium oxide, diphenyloxazole and polyvinyltoluene.  
     
     
         14 . A method of  claim 11  wherein the ezetimibe is labeled with a radiolabel selected from  3 H and  125 I.  
     
     
         15 . A method of  claim 12  wherein the ezetimibe is labeled with a radiolabel selected from  3 H and  125 I.  
     
     
         16 . A method for identifying an antagonist of NPC1L1 comprising: 
 (a) contacting a host cell expressing a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 2, 4 and 12 or a functional fragment thereof on a cell surface with a detectably labeled sterol or 5α-stanol and with a sample to be tested for the presence of the antagonist; and    (b) measuring the amount of detectably labeled sterol or 5α-stanol in the cell; wherein an NPC1L1 antagonist in the sample is identified by measuring substantially reduced detectably labeled sterol or 5α-stanol within the host cell, compared to what would be measured in the absence of such an antagonist.    
     
     
         17 . The method of  claim 16  wherein the sterol or 5α-stanol is detectably labeled with a radiolabel selected from  3 H,  14 C and  125 I.  
     
     
         18 . The method of  claim 16  wherein the sterol is cholesterol.  
     
     
         19 . A method according to  claim 11  wherein the host cell is selected from a chinese hamster ovary (CHO) cell, a J774 cell, a macrophage cell and a Caco2 cell.  
     
     
         20 . A method according to  claim 12  wherein the host cell is selected from a chinese hamster ovary (CHO) cell, a J774 cell, a macrophage cell and a Caco2 cell.  
     
     
         21 . A method according to  claim 16  wherein the host cell is selected from a chinese hamster ovary (CHO) cell, a J774 cell, a macrophage cell and a Caco2 cell.  
     
     
         22 . A mutant transgenic mouse comprising a homozygous mutation of endogenous, chromosomal NPC1L1 wherein the mouse does not produce any functional NPC1L1 protein.  
     
     
         23 . The mouse of  claim 22  wherein the mouse exhibits a reduced serum sterol or 5α-stanol level, a reduced liver sterol or 5α-stanol level or a reduced level of intestinal absorption of sterol or 5α-stanol.  
     
     
         24 . The mouse of  claim 22  wherein the region of endogenous, chromosomal NPC1L1 deleted corresponds to nucleotides 790-998 of the nucleotide sequence set forth in SEQ ID NO: 45.  
     
     
         25 . An offspring or progeny of the mouse of  claim 22  wherein the offspring or progeny has inherited a mutated NPC1L1 allele of said mouse.  
     
     
         26 . A method for screening a sample for an intestinal sterol or 5α-stanol absorption antagonist comprising: 
 (a) feeding a sterol or 5α-stanol-containing substance to a first and second mouse comprising a functional NPC1L1 gene and to a third, mutant mouse of  claim 21;     (b) administering the sample to the first mouse but not the second mouse;    (c) measuring the amount of sterol or St-stanol absorption in the intestine of said first, second and third mouse; and    (d) comparing the levels of intestinal sterol or 5α-stanol absorption in said first, second and third mouse;    wherein the sample is determined to contain the intestinal sterol or 5α-stanol absorption antagonist when the level of intestinal sterol or 5α-stanol absorption in the first mouse and third mouse are less than the amount of intestinal sterol or 5α-stanol absorption in the second mouse.    
     
     
         27 . The method of  claim 26  wherein the sterol is cholesterol.  
     
     
         28 . The method of  claim 27  wherein the cholesterol is radiolabeled.  
     
     
         29 . The method of  claim 26  wherein the level of sterol or 5α-stanol cholesterol absorption is determined by measuring the level of serum sterol or 5α-stanol in the mice.  
     
     
         30 . A method for inhibiting NPC1L1 mediated sterol or 5α-stanol uptake, in a subject, by administering, to the subject, a substance identified by the method of  claim 11 .  
     
     
         31 . A method for inhibiting NPC1L1 mediated sterol or 5α-stanol uptake, in a subject, by administering, to the subject, a substance identified by the method of  claim 12 .  
     
     
         32 . A method for inhibiting NPC1L1 mediated sterol or 5α-stanol uptake, in a subject, by administering, to the subject, a substance identified by the method of  claim 16 .  
     
     
         33 . A method for inhibiting NPC1L1 mediated sterol or 5α-stanol uptake, in a subject, by administering, to the subject, a substance identified by the method of  claim 26 .  
     
     
         34 . A kit comprising: 
 (a) ezetimibe in a pharmaceutical dosage form; and    (b) information indicating that NPC1L1 is a target of ezetimibe.    
     
     
         35 . The kit of  claim 34  wherein the dosage form is a tablet comprising 10 mg ezetimibe.  
     
     
         36 . The kit of  claim 34  further comprising simvastatin in a pharmaceutical dosage form.  
     
     
         37 . The kit of  claim 36  wherein the simvastatin in pharmaceutical dosage form comprises 5 mg, 10 mg, 20 mg, 40 mg or 80 mg simvastatin.  
     
     
         38 . The kit of  claim 36  wherein the simvastatin in pharmaceutical dosage form and the ezetimibe in pharmaceutical dosage form are associated in a single pill or tablet.  
     
     
         39 . A method for decreasing the level of intestinal sterol or 5α-stanol absorption in a subject comprising reducing the level of expression of NPC1L1 in the subject.  
     
     
         40 . The method of  claim 39  wherein the subject is a mouse, rat or human.  
     
     
         41 . The method of  claim 39  wherein the level of expression of NPC1L1 in the subject is reduced by mutating NPC1L1 in the subject.  
     
     
         42 . The method of  claim 39  wherein the sterol is cholesterol.  
     
     
         43 . A method for identifying an antagonist of NPC1L1 comprising: 
 (a) contacting a host cell expressing a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 2, 4 and 12 or a functional fragment thereof on a cell surface, in the presence of a known amount of a detectably labeled substituted azetidinone, with a sample to be tested for the presence of the antagonist; and    (b) measuring the amount of detectably labeled substituted azetidinone specifically bound, directly or indirectly, to the polypeptide;    wherein an NPC1L1 antagonist in the sample is identified by measuring substantially reduced direct or indirect binding of the detectably labeled substituted azetidinone to the polypeptide, compared to what would be measured in the absence of such an antagonist.    
     
     
         44 . A kit comprising: 
 (a) a substituted azetidinone in a pharmaceutical dosage form; and    (b) information indicating that NPC1L1 is a target of the substituted azetidinone.    
     
     
         45 . An isolated mammalian cell which lacks a gene which encodes a functional NPC1L1 protein.  
     
     
         46 . The cell of  claim 45  isolated from a mutant mouse comprising a homozygous mutation of endogenous, chromosomal NPC1L1 wherein the mouse does not produce any functional NPC1L1 protein.  
     
     
         47 . The cell of  claim 46  wherein the mutation is of a gene encoding an amino acid sequence of SEQ ID NO: 12.  
     
     
         48 . The cell of  claim 45  isolated from the duodenum, gall bladder, liver, small intestine or stomach tissue.  
     
     
         49 . The cell of  claim 48  which is an enterocyte.