Substituted oxazolidinones as novel NPC1L1 ligands for the inhibition of cholesterol absorption

Cholesterol absorption inhibition (CAI) represents an important treatment option for hypercholesterolemia. Herein, we report the design and evaluation of a series of substituted oxazolidinones as ligands for the Niemann Pick C1 Like 1 (NPC1L1) protein, a key mediator of cholesterol transport. Novel analogs were initially evaluated in a brush border membrane NPC1L1 binding assay; subsequently, promising compounds were evaluated in vivo for acute inhibition of cholesterol absorption. These studies identified analogs with low micromolar NPC1L1 binding affinity and acute in vivo efficacy of >50% absorption inhibition at 3mg/kg.     

Modulation of human Niemann-Pick C1-like 1 gene expression by sterol: Role of sterol regulatory element binding protein 2

Niemann-Pick C1-like 1 (NPC1L1) is an essential intestinal component of cholesterol absorption. However, little is known about the molecular regulation of intestinal NPC1L1 expression and promoter activity. We demonstrated that human NPC1L1 mRNA expression was significantly decreased by 25-hydroxycholesterol but increased in response to cellular cholesterol depletion achieved by incubation with Mevinolin (an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase) in human intestinal Caco-2 cells. We also showed that a -1741/+56 fragment of the NPC1L1 gene demonstrated high promoter activity in Caco-2 cells that was reduced by 25-hydroxycholesterol and stimulated by cholesterol depletion. Interestingly, we showed that the NPC1L1 promoter is remarkably transactivated by the overexpression of sterol regulatory element (SRE) binding protein (SREBP)-2, suggesting its involvement in the sterol-induced alteration in NPC1L1 promoter activity. Finally, we identified two putative SREs in the human NPC1L1 promoter and established their essential roles in mediating the effects of cholesterol on promoter activity. Our study demonstrated the modulation of human NPC1L1 expression and promoter activity by cholesterol in a SREBP-2-dependent mechanism.     

Membrane topology of human NPC1L1, a key protein in enterohepatic cholesterol absorption

The Niemann-Pick C1 Like 1 (NPC1L1) is a predicted polytopic membrane protein that is critical for cholesterol absorption. NPC1L1 takes up free cholesterol into cells through vesicular endocytosis. Ezetimibe, a clinically used cholesterol absorption inhibitor, blocks the endocytosis of NPC1L1 thereby inhibiting cholesterol uptake. Human NPC1L1 is a 1,332-amino acid protein with a putative sterol-sensing domain (SSD) that shows sequence homo­logy to HMG-CoA reductase (HMGCR), Niemann-Pick C1 (NPC1), and SREBP cleavage-activating protein (SCAP). Here, we use protease protection and immunofluorescence in selectively permeabilized cells to study the topology of human NPC1L1. Our data indicate that NPC1L1 contains 13 transmembrane helices. The NH₂-terminus of NPC1L1 is in the lumen while the COOH-terminus projects to the cytosol. human NPC1L1 contains seven small cytoplasmic loops—four small and three large luminal loops—one of which has been reported to bind ezetimibe. Ezetimibe-glucuronide, the major metabolite of ezetimibe in vivo, can block the internalization of NPC1L1 and cholesterol. The membrane topology of NPC1L1 is similar to that of NPC1, and the putative SSD of NPC1L1 is oriented in the same manner as those of HMGCR, NPC1, and SCAP. The defined topology of NPC1L1 provides necessary information for further dissecting the functions of the different domains of NPC1L1.     

Ezetimibe blocks the internalization of NPC1L1 and cholesterol in mouse small intestine

The multiple transmembrane protein Niemann-Pick C1 like1 (NPC1L1) is essential for intestinal cholesterol absorption. Ezetimibe binds to NPC1L1 and is a clinically used cholesterol absorption inhibitor. Recent studies in cultured cells have shown that NPC1L1 mediates cholesterol uptake through vesicular endocytosis that can be blocked by ezetimibe. However, how NPC1L1 and ezetimibe work in the small intestine is unknown. In this study, we found that NPC1L1 distributed in enterocytes of villi and transit-amplifying cells of crypts. Acyl-CoA cholesterol acyltransferase 2 (ACAT2), another important protein for cholesterol absorption by providing cholesteryl esters to chylomicrons, was mainly presented in the apical cytoplasm of enterocytes. NPC1L1 and ACAT2 were highly expressed in jejunum and ileum. ACAT1 presented in the Paneth cells of crypts and mesenchymal cells of villi. In the absence of cholesterol, NPC1L1 was localized on the brush border of enterocytes. Dietary cholesterol induced the internalization of NPC1L1 to the subapical layer beneath the brush border and became partially colocalized with the endosome marker Rab11. Ezetimibe blocked the internalization of NPC1L1 and cholesterol and caused their retention in the plasma membrane. This study demonstrates that NPC1L1 mediates cholesterol entering enterocytes through vesicular endocytosis and that ezetimibe blocks this step in vivo.     

The structure of the NPC1L1 N-terminal domain in a closed conformation

Background

NPC1L1 is the molecular target of the cholesterol lowering drug Ezetimibe and mediates the intestinal absorption of cholesterol. Inhibition or deletion of NPC1L1 reduces intestinal cholesterol absorption, resulting in reduction of plasma cholesterol levels.

Principal Findings

Here we present the 2.8 Å crystal structure of the N-terminal domain (NTD) of NPC1L1 in the absence of cholesterol. The structure, combined with biochemical data, reveals the mechanism of cholesterol selectivity of NPC1L1. Comparison to the cholesterol free and bound structures of NPC1(NTD) reveals that NPC1L1(NTD) is in a closed conformation and the sterol binding pocket is occluded from solvent.

Conclusion

The structure of NPC1L1(NTD) reveals a degree of flexibility surrounding the entrance to the sterol binding pocket, suggesting a gating mechanism that relies on multiple movements around the entrance to the sterol binding pocket.     

Human NPC1L1 and NPC1 can functionally substitute for the ncr genes to promote reproductive development in C. elegans

The NPC1 and NPC1L1 are related genes whose general role is in cholesterol trafficking. However, reduction of activity of these genes results in very different phenotypes. Niemann–Pick C disease type 1 is a neurodegenerative disease with no current treatment, where cholesterol accumulates in lysosomes. The disease arises due to autosomal recessive mutations in the NPC1 gene. The NPC1L1 gene has recently been identified as the target for the drug ezetimibe (Zetia), a cholesterol absorption inhibitor, and has been shown to be an intestinal cholesterol transporter. We demonstrate that human NPC1L1, as well as human NPC1, can functionally substitute for the Caenorhabditis elegans genes ncr-1 and/or ncr-2. These genes are known to play a role in the process of dauer formation, a process which can be modulated by cholesterol in sensitized genetic backgrounds. Our results demonstrate that these human proteins retain some functional conservation, though their biological roles are vastly different.     

Bean peptides have higher in silico binding affinities than ezetimibe for the N-terminal domain of cholesterol receptor Niemann-Pick C1 Like-1

Niemann-Pick C1 like-1 (NPC1L1) mediates cholesterol absorption at the apical membrane of enterocytes through a yet unknown mechanism. Bean, pea, and lentil proteins are naturally hydrolyzed during digestion to produce peptides. The potential for pulse peptides to have high binding affinities for NPC1L1 has not been determined. In this study , in silico binding affinities and interactions were determined between the N-terminal domain of NPC1L1 and 14 pulse peptides (5≥ amino acids) derived through pepsin-pancreatin digestion. Peptides were docked in triplicate to the N-terminal domain using docking program AutoDock Vina, and results were compared to those of ezetimibe, a prescribed NPC1L1 inhibitor. Three black bean peptides (−7.2 to −7.0 kcal/mol) and the cowpea bean dipeptide Lys-Asp (−7.0 kcal/mol) had higher binding affinities than ezetimibe (−6.6 kcal/mol) for the N-terminal domain of NPC1L1. Lentil and pea peptides studied did not have high binding affinities. The common bean peptide Tyr-Ala-Ala-Ala-Thr (−7.2 kcal/mol), which can be produced from black or navy bean proteins, had the highest binding affinity. Ezetimibe and peptides with high binding affinities for the N-terminal domain are expected to interact at different locations of the N-terminal domain. All high affinity black bean peptides are expected to have van der Waals interactions with SER130, PHE136, and LEU236 and a conventional hydrogen bond with GLU238 of NPC1L1. Due to their high affinity for the N-terminal domain of NPC1L1, black and cowpea bean peptides produced in the digestive track have the potential to disrupt interactions between NPC1L1 and membrane proteins that lead to cholesterol absorption.     

Fomiroid A,a Novel Compound from the Mushroom Fomitopsis nigra,Inhibits NPC1L1-Mediated Cholesterol Uptake via a Mode of Action Distinct from That of Ezetimibe

Hypercholesterolemia is one of the key risk factors for coronary heart disease, a major cause of death in developed countries. Suppression of NPC1L1-mediated dietary and biliary cholesterol absorption is predicted to be one of the most effective ways to reduce the risk of hypercholesterolemia. In a screen for natural products that inhibit ezetimibe glucuronide binding to NPC1L1, we found a novel compound, fomiroid A, in extracts of the mushroom Fomitopsis nigra. Fomiroid A is a lanosterone derivative with molecular formula C₃₀H₄₈O₃. Fomiroid A inhibited ezetimibe glucuronide binding to NPC1L1, and dose-dependently prevented NPC1L1-mediated cholesterol uptake and formation of esterified cholesterol in NPC1L1-expressing Caco2 cells. Fomiroid A exhibited a pharmacological chaperone activity that corrected trafficking defects of the L1072T/L1168I mutant of NPC1L1. Because ezetimibe does not have such an activity, the binding site and mode of action of fomiroid A are likely to be distinct from those of ezetimibe.     

The N-terminal domain of NPC1L1 protein binds cholesterol and plays essential roles in cholesterol uptake

Niemann-Pick C1-like 1 (NPC1L1) is a multitransmembrane protein playing a crucial role in dietary and biliary cholesterol absorption. Cholesterol promotes the formation and endocytosis of NPC1L1-flotillin-cholesterol membrane microdomains, which is an early step in cholesterol uptake. How cholesterol is sensed in this step is unknown. Here, we find that the N-terminal domain (NTD) of NPC1L1 binds cholesterol. Mutation of residue Leu-216 in NPC1L1-NTD eliminates cholesterol binding, decreases the formation of NPC1L1-flotillin-cholesterol membrane microdomains, and prevents NPC1L1-mediated cholesterol uptake in culture cells and mice livers. NPC1L1-NTD specifically binds cholesterol but not plant sterols, which may account for the selective cholesterol absorption in intestine. Furthermore, 25- or 27-hydroxycholesterol competes with cholesterol to bind NPC1L1-NTD and inhibits the cholesterol induced endocytosis of NPC1L1. Together, these results demonstrate that plasma membrane-localized NPC1L1 binds exogenous cholesterol via its NTD, and facilitates the formation of NPC1L1-flotillin-cholesterol membrane microdomains that are then internalized into cells through the clathrin-AP2 pathway. Our study uncovers the mechanism of cholesterol sensing by NPC1L1 and proposes a mechanism for selective cholesterol absorption.     

Ezetimibe blocks internalization of the NPC1L1/cholesterol complex

Niemann-Pick C1-like 1 (NPC1L1) is a target for ezetimibe, a drug that blocks intestinal cholesterol absorption. A new study by Ge et al. (2008) in this issue of Cell Metabolism shows that non-lipoprotein-bound cholesterol induces endocytosis of NPC1L1 and that ezetimibe blocks the internalization of the NPC1L1/cholesterol complex. The in vivo significance of these findings is discussed.     

Luteolin and Quercetin Affect the Cholesterol Absorption Mediated by Epithelial Cholesterol Transporter Niemann–Pick C1-Like 1 in Caco-2 Cells and Rats

Niemann–Pick C1-Like 1 (NPC1L1) mediates cholesterol absorption, and ezetimibe is a potent NPC1L1 inhibitor applicable for medication of hypercholesterolemia. Epidemiological studies demonstrated that consumption of polyphenols correlates with a decreased risk for atherosclerosis due to their antioxidant effect. This activity can hardly be attributable to the antioxidant activity only, and we hypothesized that polyphenols inhibit intestinal transport of cholesterol. We elucidated the kinetic parameters of intestinal cholesterol absorption, screened several polyphenols for their ability to specifically inhibit intestinal cholesterol absorption, and determined the inhibitory effects of selected flavonoids in vitro and in vivo. The concentration-dependent uptake of cholesterol by Caco-2 cells obeyed a monophasic saturation process. This indicates the involvement of an active-passive transport, i.e., NPC1L1. Parameters of cholesterol uptake by Caco-2 cells were as follows: J _max, K _t, and K _d were 6.89±2.96 19.03±11.58 µM, and 0.11±0.02 pmol/min/mg protein, respectively. Luteolin and quercetin inhibited cholesterol absorption by Caco-2 cells and human embryonic kidney 293T cells expressing NPC1L1. When preincubated Caco-2 cells with luteolin and quercetin before the assay, cholesterol uptake significantly decreased. The inhibitory effects of these flavonoids were maintained for up to 120 min. The level of inhibition and irreversible effects were similar to that of ezetimibe. Serum cholesterol levels significantly decreased more in rats fed both cholesterol and luteolin (or quercetin), than in those observed in the cholesterol feeding group. As quercetin induced a significant decrease in the levels of NPC1L1 mRNA in Caco-2 cells, the in vivo inhibitory effect may be due to the expression of NPC1L1. These results suggest that luteolin and quercetin reduce high blood cholesterol levels by specifically inhibiting intestinal cholesterol absorption mediated by NPC1L1.     

Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis

Niemann-Pick C1 Like 1 (NPC1L1) is a protein localized in jejunal enterocytes that is critical for intestinal cholesterol absorption. The uptake of intestinal phytosterols and cholesterol into absorptive enterocytes in the intestine is not fully defined on a molecular level, and the role of NPC1L1 in maintaining whole body cholesterol homeostasis is not known. NPC1L1 null mice had substantially reduced intestinal uptake of cholesterol and sitosterol, with dramatically reduced plasma phytosterol levels. The NPC1L1 null mice were completely resistant to diet-induced hypercholesterolemia, with plasma lipoprotein and hepatic cholesterol profiles similar to those of wild type mice treated with the cholesterol absorption inhibitor ezetimibe. Cholesterol/cholate feeding resulted in down-regulation of intestinal NPC1L1 mRNA expression in wild type mice. NPC1L1 deficiency resulted in up-regulation of intestinal hydroxymethylglutaryl-CoA synthase mRNA and an increase in intestinal cholesterol synthesis, down-regulation of ABCA1 mRNA, and no change in ABCG5 and ABCG8 mRNA expression. NPC1L1 is required for intestinal uptake of both cholesterol and phytosterols and plays a major role in cholesterol homeostasis. Thus, NPC1L1 may be a useful drug target for the treatment of hypercholesterolemia and sitosterolemia.     

Cholesterol-regulated translocation of NPC1L1 to the cell surface facilitates free cholesterol uptake

Although NPC1L1 is required for intestinal cholesterol absorption, data demonstrating mechanisms by which this protein facilitates the process are few. In this study, a hepatoma cell line stably expressing human NPC1L1 was established, and cholesterol uptake was studied. A relationship between NPC1L1 intracellular trafficking and cholesterol uptake was apparent. At steady state, NPC1L1 proteins localized predominantly to the transferrin-positive endocytic recycling compartment, where free cholesterol also accumulated as revealed by filipin staining. Interestingly, acute cholesterol depletion induced with methyl-beta-cyclodextrin stimulated relocation of NPC1L1 to the plasma membrane, preferentially to a newly formed "apical-like" subdomain. This translocation was associated with a remarkable increase in cellular cholesterol uptake, which in turn was dose-dependently inhibited by ezetimibe, a novel cholesterol absorption inhibitor that specifically binds to NPC1L1. These findings define a cholesterol-regulated endocytic recycling of NPC1L1 as a novel mechanism regulating cellular cholesterol uptake.     

NPC1L1 and cholesterol transport

The polytopic transmembrane protein, Niemann-Pick C1-Like 1 (NPC1L1), is enriched in the apical membrane of small intestine absorptive enterocytes where it mediates extracellular sterol transport across the brush border membrane. It is essential for intestinal sterol absorption and is the molecular target of ezetimibe, a potent cholesterol absorption inhibitor that lowers blood cholesterol in humans. NPC1L1 is also highly expressed in human liver. The hepatic function of NPC1L1 may be to limit excessive biliary cholesterol loss. NPC1L1-dependent sterol uptake seems to be a clathrin-mediated endocytic process and is regulated by cellular cholesterol content. Recently, NPC1L1 inhibition has been shown to have beneficial effects on components of the metabolic syndrome, such as obesity, insulin resistance, and fatty liver, in addition to atherosclerosis.     

Human NPC1L1 and NPC1 can functionally substitute for the ncr genes to promote reproductive development in C. elegans

The NPC1 and NPC1L1 are related genes whose general role is in cholesterol trafficking. However, reduction of activity of these genes results in very different phenotypes. Niemann-Pick C disease type 1 is a neurodegenerative disease with no current treatment, where cholesterol accumulates in lysosomes. The disease arises due to autosomal recessive mutations in the NPC1 gene. The NPC1L1 gene has recently been identified as the target for the drug ezetimibe (Zetia), a cholesterol absorption inhibitor, and has been shown to be an intestinal cholesterol transporter. We demonstrate that human NPC1L1, as well as human NPC1, can functionally substitute for the Caenorhabditis elegans genes ncr-1 and/or ncr-2. These genes are known to play a role in the process of dauer formation, a process which can be modulated by cholesterol in sensitized genetic backgrounds. Our results demonstrate that these human proteins retain some functional conservation, though their biological roles are vastly different.     

Multiple plasma membrane receptors but not NPC1L1 mediate high-affinity, ezetimibe-sensitive cholesterol uptake into the intestinal brush border membrane

We compared cholesterol uptake into brush border membrane vesicles (BBMV) made from the small intestines of either wild-type or Niemann-Pick C1-like 1 (NPC1L1) knockout mice to elucidate the contribution of NPC1L1 to facilitated uptake; this uptake involves cholesterol transport from lipid donor particles into the BBM of enterocytes. The lack of NPC1L1 in the BBM of the knockout mice had no effect on the rate of cholesterol uptake. It follows that NPC1L1 cannot be the putative high-affinity, ezetimibe-sensitive cholesterol transporter in the brush border membrane (BBM) as has been proposed by others. The following findings substantiate this conclusion: (I) NPC1L1 is not a brush border membrane protein but very likely localized to intracellular membranes; (II) the cholesterol absorption inhibitor ezetimibe and its analogues reduce cholesterol uptake to the same extent in wild-type and NPC1L1 knockout mouse BBMV. These findings indicate that the prevailing belief that NPC1L1 facilitates intestinal cholesterol uptake into the BBM and its interaction with ezetimibe is responsible for the inhibition of this process can no longer be sustained.     

Niemann-Pick C1 like 1 gene expression is down-regulated by LXR activators in the intestine

Niemann-Pick C1 like 1 (NPC1L1) is a protein critical for intestinal cholesterol absorption. The nuclear receptors peroxisome proliferator-activated receptor alpha (PPARalpha) and liver X receptors (LXRalpha and LXRbeta) are major regulators of cholesterol homeostasis and their activation results in a reduced absorption of intestinal cholesterol. The goal of this study was to define the role of PPARalpha and LXR nuclear receptors in the regulation of NPC1L1 gene expression. We show that LXR activators down-regulate NPC1L1 mRNA levels in the human enterocyte cell line Caco-2/TC7, whereas PPARalpha ligands have no effect. Furthermore, NPC1L1 mRNA levels are decreased in vivo, in duodenum of mice treated with the LXR agonist T0901317. In conclusion, the present study identifies NPC1L1 as a novel LXR target gene further supporting a crucial role of LXR in intestinal cholesterol homeostasis.