NPC1L1 (NPC3) and methods of identifying ligands thereof
View Patent ↗The present invention provides human, rat and mouse NPCIL1 polypeptides and polynucleotides encoding the polypeptides. Methods for detecting ligands which bind to NPC1L1 and block intestinal cholesterol absorption are provided. Also included is a method of identifying ligands which bind to NPCILI using membranes derived from brush border membrane preparations. Compounds that bind to NPCILI can be used for inhibiting intestinal cholesterol absorption in a subject.
1. A method for identifying a ligand of NPC1L1 comprising:
(a) contacting human NPC1L1 with a candidate compound and a detectably labeled substituted 2-azetidinone glucuronide compound selected from the group consisting of compound 1, compound 8
and a compound of Formula II a
wherein,
R 9 is selected from the group consisting of —C≡C—CH 2 —NR 10 R 11 wherein R 11 is selected from the group consisting of —H, —C 1-3 alkyl, —C(O)—C 1-3 alkyl, —C(O)—NR 10 R 10 , —SO 2 —C 1-3 alkyl and —SO 2 -phenyl; —C≡C(O)NR 10 —SO 2 —C 1-3 alkyl; —C≡C—C(O)NR 10 —SO 2 —phenyl; —(CH 2 ) 3 —NR 10 —SO 2 -phenyl;
R 10 is independently selected at each occurrence from —H and —C 1-3 alkyl; and
R 12 is selected from
(b) measuring the amount of detectably labeled substituted 2-azetidinone glucuronide compound that is bound to NPC1L1,
wherein binding of said candidate compound to human NPC1L1 decreases binding of said detectably labeled substituted 2-azetidinone glucuronide to human NPC1L1, and indicates that the candidate compound is a ligand of human NPC1L1.
2. The method of claim 1 , wherein the substituted 2-azetidinone-glucuronide comprises a detectable label from the group consisting of 3 H, 35 S and 125 I.
3. The method of claim 2 , wherein the substituted 2-azetidinone-glucuronide is a compound of Formula IIa.
4. The method of claim 3 , wherein the substituted 2-azetidinone-glucuronide is a compound of Formula IIa, wherein R 9 is selected from the group consisting of —C≡C—CH 2 —NR 10 R 11 , —C≡C—C(O)NR 10 R 11 , —(CH 2 ) 3 —NR 10 —SO 2 —C 1-3 alkyl and —(CH 2 ) 3 —NR 10 —SO 2 -phenyl, and R 11 is selected from —SO 2 —C 1-3 alkyl and —SO 2 -phenyl.
5. The method of claim 4 , wherein the substituted 2-azetidinone-glucuronide of Formula IIa is labeled with 35 S.
6. The method of claim 5 wherein R 9 is —C≡C—CH 2 —NR 10 R 11 .
7. The method of claim 1 wherein the detectably labeled substituted 2-azetidinone glucuronide is 35 S-labeled compound 2
8. The method of claim 1 wherein R 12 is
9. The method of claim 1 wherein the detectably labeled substituted 2-azetidinone glucuronide is selected from the group consisting of compound 1, and compound 8.
10. The method of claim 9 wherein the detectably labeled substituted 2-azetidinone glucuronide comprises a detectable label selected from the group consisting of 3 H and 125 I.