The target of ezetimibe is Niemann–PickC1-Like 1 (NPC1L1)Margarita Garcia-Calvoa,b,c, JeanMarie Lisnocka,c, Herbert G. Bulla,c, Brian E. Hawesc,d, Duane A. Burnettc,e,Matthew P. Braunc,f, James H. Cronac,d, Harry R. Davis, Jr.c,d, Dennis C. Deanc,f, Patricia A. Detmersc,g,Michael P. Grazianoc,d, Meredith Hughesa,c, D. Euan MacIntyrec,h, Anthony Ogawac,i, Kim A. O’Neillc,d,Sai Prasad N. Iyerc,d, Diane E. Shevellc,g, Marsha M. Smithc,j, Yui S. Tangc,f, Amanda M. Makarewiczc,i,Feroze Ujjainwallac,i, Scott W. Altmannc,d, Kevin T. Chapmanc,k, and Nancy A. Thornberrya,cDepartments ofaMetabolic Disorders,fDrug Metabolism Labeled Compound Synthesis,gImmunology,hPharmacology,iMedicinal Chemistry, andkTargetValidation, Merck Research Laboratories, Rahway, NJ 07065; and Departments ofdCardiovascular兾Metabolic Diseases,eChemistry, andjDiscoveryTechnologies, Schering-Plough Research Institute, Kenilworth, NJ 07033Edited by Joseph L. Goldstein, University of Texas Southwestern Medical Center, Dallas, TX, and approved April 20, 2005 (received for reviewJanuary 12, 2005)Ezetimibe is a potent inhibitor of cholesterol absorption that hasbeen approved for the treatment of hypercholesterolemia, but itsmolecular target has been elusive. Using a genetic approach, werecently identified Niemann–Pick C1-Like 1 (NPC1L1) as a criticalmediator of cholesterol absorption and an essential component ofthe ezetimibe-sensitive pathway. To determine whether NPC1L1 isthe direct molecular target of ezetimibe, we have developed abinding assay and shown that labeled ezetimibe glucuronide bindsspecifically to a single site in brush border membranes and tohuman embryonic kidney 293 cells expressing NPC1L1. Moreover,the binding affinities of ezetimibe and several key analogs torecombinant NPC1L1 are virtually identical to those observed fornative enterocyte membranes. KDvalues of ezetimibe glucuronidefor mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540,40, and 220 nM, respectively. Last, ezetimibe no longer binds tomembranes from NPC1L1 knockout mice. These results unequivo-cally establish NPC1L1 as the direct target of ezetimibe and shouldfacilitate efforts to identify the molecular mechanism of choles-terol transport.cholesterol 兩 intestinal brush border membranesBlood cholesterol levels are regulated by several processes,including de novo synthesis, cholesterol absorption, andbiliary clearance and excretion. Absorption of dietary and biliarycholesterol oc curs in the proximal jejunum of the small intestine(1). Ezetimibe is a potent cholesterol and phy tosterol uptakeinhibitor (2, 3) and is used for the treatment of hypercholester-olemia. Ezetimibe ef fectively lowers circulating plasma choles-terol in humans by 15–20% (4–6), and c oadministration ofezetimibe with 3-hydroxy-3-methylglutaryl CoA (HMG-CoA)reduct ase inhibitors (statins), inhibitors of cholesterol synthesis,results in additive effects on cholesterol reduction (7–11).For the past decade, there have been intense efforts todeter mine the molecular target of ezetimibe. Uptake and sortingof cholesterol and phytosterols by intestinal enterocytes is ac omplex process (for a synopsis, see ref. 12). This phenomenonis believed to involve a variety of mediators, including theATP-binding cassette (ABC) transporters ABCA1, ABCG5,and G8, and the scavenger receptor B1 (SRB1), but knockoutshave ruled out each of these proteins as promoting net sterolupt ake f rom the intestine (13–16). Other multidrug-resistancetransporters in the ABC and RND (resistance–nodulation–division) superfamilies have also been proposed to be gatekeep-ers of intracellular sterol and lipid homeostasis in mammals, buttheir specific molecular functions remain uncertain (for a review,see ref. 17).Recently, we demonstrated that Niemann–Pick C1-Like 1(NPC1L1) (18) is essential in the ezetimibe responsive pathwayof cholesterol absorption (19). This protein was identified as apotential candidate gene by a search of expressed sequence tagdat abases by using the following criteria: presence of a sterol-sensing domain (SSD), a plasma membrane secretion signal, andenriched expression in intestinal enterocytes. Mice deficient inNPC1L1 had ⬇70% reduction in sterol absorption, with theresidual being insensitive to ezetimibe (19). These findingsc onvincingly demonstrated that NPC1L1 is central to cholesterolupt ake in enterocytes and is in a pathway sensitive to ezetimibe,but did not establish the molecular basis.To determine whether NPC1L1 is the direct molecular targetof ezetimibe, we have est ablished a radioligand binding assay forezetimibe using enterocyte brush border membranes (BBMs)f rom several species. Binding af finities were determined forezetimibe and several key analogs to native membranes, mem-branes from cells expressing recombinant NPC1L1, and entero-c yte BBMs from NPC1L1-deficient mice. Together, the resultsdefin itively establish NPC1L1 as the direct target of ezetimibe invivo.MethodsMaterials. The [3H]ezetimibe glucuronide (EZE-gluc) [1-([2,6-3H]-4-fluorophenyl)-(3R)-[3-(4-fluorophenyl)-(3S)-hydroxypropyl]-(4S)-([3,5-3H]-4-hydroxyphenyl)-2-azetidinone; 34.5 Ci兾mmol; 1Ci ⫽ 37 GBq and its analogs were prepared at Merck ResearchLaboratories. Digitonin was purchased from Wako Pure Chemical(Osaka).Preparation of BBMs. Membrane s were prepared from Rhesusmacaque (Macaca mulatta), rat (male Sprague–Dawley), andmouse (male C57BL兾6N) intestines by using a Mg2⫹precipitationmethod as described with some modifications (20–22). The prox-imal intestines from freshly killed animals were cut into ⬇10-cmsegments, washed with ice-cold saline buffer (buffer A: 26 mMNaHCO3兾0.96 mM NaH2PO4兾5 mM Hepes兾5.5 mM glucose兾117mM NaCl兾5.4 mM KCl, pH 7.40), placed on cold glass plates, andopened longitudinally, and the mucosa was scraped with glassmicroscope slips. The mucosa could be used fresh or frozen withidentical results. To prepare the membranes, the mucosal scrapingsThis paper was submitted directly (Track II) to the PNAS office.Abbreviations: NPC1L1, Niemann–Pick C1-Like 1; HEK, human embryonic kidney; SSD,sterol-sensing domain; EZE-glue, ezetimibe glucuronide; BBM, brush border membranes.bTo whom correspondence should be addressed at: Department of Metabolic Disorders,Merck Research Laboratories, Mail Stop RY50G241, P.O. Box 2000, Rahway, NJ 07065.E-mail: margarita㛭[email protected]., J.L., H.G.B., B.E.H., D.A.B., M.P.B., J.H.C.,