Cryo-EM structures of NPC1L1 reveal mechanisms of cholesterol transport and ezetimibe inhibition

Huang, Ching-Shin; Yu, Xianjun; Fordstrom, Preston; Choi, Kaylee; Chung, Ben C.; Roh, Sook Young; Chiu, Wah; Zhou, Mingyue; Min, Xiaoshan; Wang, Zhong Lin

doi:10.1126/sciadv.abb1989

Cited by 59 publications

(79 citation statements)

References 67 publications

(93 reference statements)

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“…A bibliographic search of the main ezetimibe pharmacophores [ 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ] was carried out, and it was found that the following requirements are advisable to obtain an analogue with reasonable activity:…”

Section: Resultsmentioning

confidence: 99%

Synthesis and Modeling of Ezetimibe Analogues

et al. 2021

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Ezetimibe is a well-known drug that lowers blood cholesterol levels by reducing its absorption in the small intestine when joining to Niemann-Pick C1-like protein (NPC1L1). A ligand-based study on ezetimibe analogues is reported, together with one-hit synthesis, highlighted in the study. A convenient asymmetric synthesis of (2S,3S)-N-α-(R)-methylbenzyl-3-methoxycarbonylethyl-4-methoxyphenyl β-lactam is described starting from Baylis–Hillman adducts. The route involves a domino process: allylic acetate rearrangement, stereoselective Ireland–Claisen rearrangement and asymmetric Michael addition, which provides a δ-amino acid derivative with full stereochemical control. A subsequent inversion of ester and acid functionality paves the way to the lactam core after monodebenzylation and lactam formation. It also shows interesting results when it comes to a pharmacophore study based on ezetimibe as the main ligand in lowering blood cholesterol levels, revealing which substituents on the azetidine-2-one ring are more similar to the ezetimibe skeleton and will more likely bind to NPC1L1 than ezetimibe.

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Section: Resultsmentioning

confidence: 99%

Synthesis and Modeling of Ezetimibe Analogues

et al. 2021

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“…Regarding the NPC1L1-mediated uptake of cholesterol, it has been reported that N-terminal domain of NPC1L1 binds cholesterol and promotes the formation of cholesterol-enriched microdomain in the plasma membrane to facilitate cholesterol transport [24,25]. In addition, recent cryo-electron microscopy structural analyses indicated that NPC1L1 forms a cholesterol-delivering tunnel from the N-terminal domain to the plasma membrane and that ezetimibe binds to the middle of the tunnel, resulting in the inhibition of cholesterol uptake [26]. As sphingolipids are colocalized with cholesterol in the plasma membrane [27], it is possible that NPC1L1 promotes the incorporation of sphingolipids, as well as cholesterol, into the plasma membrane to facilitate their transport.…”

Section: Discussionmentioning

confidence: 99%

NPC1L1 Facilitates Sphingomyelin Absorption and Regulates Diet-Induced Production of VLDL/LDL-associated S1P

et al. 2020

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Niemann-Pick C1-Like 1 (NPC1L1) is a cholesterol importer and target of ezetimibe, a cholesterol absorption inhibitor used clinically for dyslipidemia. Recent studies demonstrated that NPC1L1 regulates the intestinal absorption of several fat-soluble nutrients, in addition to cholesterol. The study was conducted to reveal new physiological roles of NPC1L1 by identifying novel dietary substrate(s). Very low-density lipoprotein and low-density lipoprotein (VLDL/LDL) are increased in Western diet (WD)-fed mice in an NPC1L1-dependent manner, so we comprehensively analyzed the NPC1L1-dependent VLDL/LDL components. Apolipoprotein M (apoM), a binding protein of sphingosine-1-phosphate (S1P: a lipid mediator), and S1P were NPC1L1-dependently increased in VLDL/LDL by WD feeding. S1P is metabolized from sphingomyelin (SM) and SM is abundant in WD, so we focused on intestinal SM absorption. In vivo studies with Npc1l1 knockout mice and in vitro studies with NPC1L1-overexpressing cells revealed that SM is a physiological substrate of NPC1L1. These results suggest a scenario in which dietary SM is absorbed by NPC1L1 in the intestine, followed by SM conversion to S1P and, after several steps, S1P is exported into the blood as the apoM-bound form in VLDL/LDL. Our findings provide insight into the functions of NPC1L1 for a better understanding of sphingolipids and S1P homeostasis.

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“…Densities that correspond to cholesterol or its derivatives have been observed at different positions along the central tunnel (34)(35)(36)(37).…”

Section: Co-folded Loop1 and Loopmentioning

confidence: 99%

“…We submitted the poly-Ala model of the luminal domains L1/L7 to the Dali server to search for homologous structures (33). All the top ranking entries are of SSDcontaining proteins, including NPC1 (Niemann Pick disease, type C), NPC1L1 (NPC1like), NCR1 (NPC1-related), Ptch 1 (patched 1), and Disp (Dispatched) (34)(35)(36)(37)(38). This result was unexpected because our previous sequence blast failed to reveal sufficient similarities between the luminal/extracellular domains of Scap and the other SSDcontaining proteins (Fig.…”

Section: Co-folded Loop1 and Loopmentioning

confidence: 99%

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Structural basis for sterol sensing by Scap and Insig

Yan

Cao

Song

et al. 2021

Preprint

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The sterol regulatory element-binding protein (SREBP) pathway senses the cellular cholesterol level through sterol regulated association between Scap and Insig. Despite the recent structural determination of the transmembrane domains of human Scap and Insig-2 bound to 25-hydroxycholesterol (25HC), the structure and regulatory mechanism of the luminal domains of Scap by cholesterol remains elusive. Here, combining cryo-EM analysis and artificial intelligence-facilitated structural prediction, we report the structure of the human Scap/Insig-2 complex in the presence of digitonin instead of 25HC. Despite the lack of sequence similarity, the structure of the luminal domain Loop 1 and a co-folded segment in Loop 7 of Scap resembles that of the luminal/extracellular domain in NPC1 and related proteins. Comparison of the sterol-loaded structures of these proteins provides clues of the regulation of Loop 1/7 interaction by cholesterol. We also show that the structure of Scap(D428A), which suppresses SREBP activation under sterol depletion, is identical to WT when complexed with Insig-2, although the gain of function may also involve a later step in protein trafficking.

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Cryo-EM structures of NPC1L1 reveal mechanisms of cholesterol transport and ezetimibe inhibition

Cited by 59 publications

References 67 publications

Synthesis and Modeling of Ezetimibe Analogues

Synthesis and Modeling of Ezetimibe Analogues

NPC1L1 Facilitates Sphingomyelin Absorption and Regulates Diet-Induced Production of VLDL/LDL-associated S1P

Structural basis for sterol sensing by Scap and Insig

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