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 . 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.   
     
     
         14 . A mutant transgenic mouse comprising a homozygous mutation of endogenous, chromosomal NPC1L1 wherein the mouse does not produce any functional NPC1L1 protein. 
     
     
         15 . An offspring or progeny of the mouse of  claim 14  wherein the offspring or progeny has inherited a mutated NPC1L1 allele of said mouse. 
     
     
         16 . 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 5α-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. 
     
     
         17 . 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 . 
     
     
         18 . 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 . 
     
     
         19 . 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 13 . 
     
     
         20 . 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 . 
     
     
         21 . A kit comprising:
 (a) ezetimibe in a pharmaceutical dosage form; and   (b) information indicating that NPC1L1 is a target of ezetimibe.   
     
     
         22 . 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. 
     
     
         23 . 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.   
     
     
         24 . 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.   
     
     
         25 . An isolated mammalian cell which lacks a gene which encodes a functional NPC1L1 protein. 
     
     
         26 . A method for inhibiting intestinal cholesterol absorption, in a mammalian subject, comprising administering an isolated oligonucleotide consisting of between 30 and 100 nucleotides, which is RNA or DNA, and is capable of hybridizing to the sense strand of a polynucleotide that encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 4 or 12; to the subject.