A small molecule inhibitor of PCSK9 that antagonizes LDL receptor binding via interaction with a cryptic PCSK9 binding groove

Proprotein convertase (PC) subtilisin kexin type 9 (PCSK9) inhibits the clearance of low density lipoprotein (LDL) cholesterol from plasma by directly interacting with the LDL receptor (LDLR). As the interaction promotes elevated plasma LDL cholesterol levels and a predisposition to cardiovascular disease (CVD), it has attracted much interest as a therapeutic target. While anti-PCSK9 monoclonal antibodies have been successful in the treatment of hypercholesteremia by decreasing CVD risk, their high cost and a requirement for injection have prohibited widespread use. The advent of an orally bioavailable small molecule inhibitor of the PCSK9-LDLR interaction is an attractive alternative, however efforts have been tempered as the binding interface is unfavourable for binding by small organic molecules. Despite its challenging nature, we report herein the discovery of compound 3f as a small molecule inhibitor of PCSK9. The kinase inhibitor nilotinib emerged from a computational screen that was applied to identify compounds that may bind to a cryptic groove within PCSK9 and proximal to the LDLR-binding interface. A subsequent in vitro PCSK9-LDLR binding assay established that nilotinib was a bona fide but modest inhibitor of the interaction (IC₅₀ = 9.8 µM). Through multiple rounds of medicinal chemistry, 3f emerged as a lead-like molecule by demonstrating disruption of the PCSK9-LDLR interaction at nanomolar levels in vitro (IC₅₀ = 537 nM) with no inhibitory activity (IC₅₀ > 10 µM) against a small panel of kinases. Compound 3f restored LDL uptake by liver cells at sub-micromolar levels and demonstrated excellent bioavailability when delivered subcutaneously in mice. Most significantly, compound 3f lowered total cholesterol levels in the plasma of wild-type mice, thereby providing proof-of-concept that the notion of a small molecule inhibitor against PCSK9 is therapeutically viable.     

Characterization of liver disease and lipid metabolism in the Niemann-Pick C1 mouse

Niemann-Pick type C1 (NPC1) disease is an autosomal-recessive cholesterol-storage disorder characterized by liver dysfunction, hepatosplenomegaly, and progressive neurodegeneration. The NPC1 gene is expressed in every tissue of the body, with liver expressing the highest amounts of NPC1 mRNA and protein. A number of studies have now indicated that the NPC1 protein regulates the transport of cholesterol from late endosomes/lysosomes to other cellular compartments involved in maintaining intracellular cholesterol homeostasis. The present study characterizes liver disease and lipid metabolism in NPC1 mice at 35 days of age before the development of weight loss and neurological symptoms. At this age, homozygous affected (NPC1(-/-)) mice were characterized with mild hepatomegaly, an elevation of liver enzymes, and an accumulation of liver cholesterol approximately four times that measured in normal (NPC1(+/+)) mice. In contrast, heterozygous (NPC1(+/-)) mice were without hepatomegaly and an elevation of liver enzymes, but the livers had a significant accumulation of triacylglycerol. With respect to apolipoprotein and lipoprotein metabolism, the results indicated only minor alterations in NPC1(-/-) mouse serum. Finally, compared to NPC1(+/+) mouse livers, the amount and processing of SREBP-1 and -2 proteins were significantly increased in NPC1(-/-) mouse livers, suggesting a relative deficiency of cholesterol at the metabolically active pool of cholesterol located at the endoplasmic reticulum. The results from this study further support the hypothesis that an accumulation of lipoprotein-derived cholesterol within late endosomes/lysosomes, in addition to altered intracellular cholesterol homeostasis, has a key role in the biochemical and cellular pathophysiology associated with NPC1 liver disease.     

LDL、oxLDL对THP-1巨噬细胞PCSK9、LDLR表达的影响研究

为研究低密度脂蛋白(LDL)、氧化修饰的低密度脂蛋白(oxLDL)对THP-1源性巨噬细胞中PCSK9、 LDLR表达的影响及两者之间的关系.分别用0mg/L、10mg/L、20mg/L、30mg/L的LDL和0mg/L、10mg/L、20mg/L、30mg/L的oxLDL处理THP-1巨噬细胞,油红O染色检测细胞荷脂情况,免疫荧光检测THP-1巨噬细胞上PCSK9蛋白表达及分布情况,RT-PCR、 Western blot检测THP-1巨噬细胞上PCSK9、 LDLR mRNA、蛋白质的表达.结果发现,不同浓度LDL处理THP-1巨噬细胞后,随着LDL浓度的增大,细胞内脂滴数目略有增多.免疫荧光染色发现,PCSK9在THP-1巨噬细胞上的表达随LDL浓度的增加而增多,胞浆内定位于某一特定细胞器中;RT-PCR、 Western blot检测发现,LDL可以呈浓度依赖性下调THP-1巨噬细胞中LDLR的表达和上调PCSK9的表达.不同浓度oxLDL处理THP-1巨噬细胞后,随oxLDL浓度的增大,脂滴颗粒明显增加;oxLDL处理对THP-1巨噬细胞上PCSK9、 LDLR mRNA、蛋白质的表达影响均不明显.研究结果表明:THP-1巨噬细胞上,同时有PCSK9和LDLR的表达,且PCSK9定位于胞浆中某一特定细胞器;oxLDL对THP-1巨噬细胞LDLR和PCSK9表达没有影响;LDL能够降低THP-1巨噬细胞表面LDLR的表达,同时上调PCSK9表达,初步说明在THP-1巨噬细胞中,两者有一定的相关性.     

前蛋白转换酶枯草溶菌素9抑制剂降低脂蛋白(a)作用的机制

脂蛋白(a)(lipoprotein(a),Lp(a))在结构上与低密度脂蛋白相似,是动脉粥样硬化性心血管疾病发病的独立危险因素和潜在的治疗靶点。前蛋白转换酶枯草溶菌素9(proprotein convertase subtilisin/kexin type 9,PCSK9)抑制剂可有效降低Lp(a)的循环水平并减少心血管事件风险。本文综合近年来的相关研究,阐述PCSK9抑制剂减少Lp(a)合成和促进其降解的相关机制,分析该领域所面临的挑战和未来的发展方向。     

PCSK9 deficiency unmasks a sex- and tissue-specific subcellular distribution of the LDL and VLDL receptors in mice

Proprotein convertase subtilisin kexin type 9 (PCSK9), the last member of the family of Proprotein Convertases related to Subtilisin and Kexin, regulates LDL-cholesterol by promoting the endosomal/lysosomal degradation of the LDL receptor (LDLR). Herein, we show that the LDLR cell surface levels dramatically increase in the liver and pancreatic islets of PCSK9 KO male but not female mice. In contrast, in KO female mice, the LDLR is more abundant at the cell surface enterocytes, as is the VLDL receptor (VLDLR) at the cell surface of adipocytes. Ovariectomy of KO female mice led to a typical KO male pattern, whereas 17β-estradiol (E2) treatment restored the female pattern without concomitant changes in LDLR adaptor protein 1 (also known as ARH), disabled-2, or inducible degrader of the LDLR expression levels. We also show that this E2-mediated regulation, which is observed only in the absence of PCSK9, is abolished upon feeding the mice a high-cholesterol diet. The latter dramatically represses PCSK9 expression and leads to high surface levels of the LDLR in the hepatocytes of all sexes and genotypes. In conclusion, the absence of PCSK9 results in a sex- and tissue-specific subcellular distribution of the LDLR and VLDLR, which is determined by E2 levels.     

Ethanol-induced hepatic steatosis is modulated by glycogen level in the liver

Alcoholic liver disease (ALD) is a major health problem worldwide and hepatic steatosis is an early response to alcohol consumption. Fat and glycogen are two major forms of energy storage in the liver; however, whether glycogen metabolism in the liver impacts alcohol-induced steatosis has been elusive. In this study, we used a mouse model with overexpression of PPP1R3G in the liver to dissect the potential role of glycogen on alcohol-induced fatty liver formation. PPP1R3G is a regulatory subunit of protein phosphatase 1 and stimulates glycogenesis in the liver. Chronic and binge ethanol (EtOH) feeding reduced glycogen level in the mouse liver and such inhibitory effect of EtOH was reversed by PPP1R3G overexpression. In addition, PPP1R3G overexpression abrogated EtOH-induced elevation of serum levels of alanine aminotransferase and aspartate aminotransferase, increase in liver triglyceride concentration, and lipid deposition in the liver. EtOH-stimulated sterol regulatory element-binding protein (SREBP)-1c, a master regulator of lipogenesis, was also reduced by PPP1R3G overexpression in vivo. In AML-12 mouse hepatocytes, PPP1R3G overexpression could relieve EtOH-induced lipid accumulation and SREBP-1c stimulation. In conclusion, our data indicate that glycogen metabolism is closely linked to EtOH-induced liver injury and fatty liver formation.     

Improved efficacy for ezetimibe and rosuvastatin by attenuating the induction of PCSK9

Reducing circulating LDL-cholesterol (LDL-c) reduces the risk of cardiovascular disease in people with hypercholesterolemia. Current approaches to reduce circulating LDL-c include statins, which inhibit cholesterol synthesis, and ezetimibe, which blocks cholesterol absorption. Both elevate serum PCSK9 protein levels in patients, which could attenuate their efficacy by reducing the amount of cholesterol cleared from circulation. To determine whether PCSK9 inhibition could enhance LDL-c lowering of both statins and ezetimibe, we utilized small interfering RNAs (siRNAs) to knock down Pcsk9, together with ezetimibe, rosuvastatin, and an ezetimibe/rosuvastatin combination in a mouse model with a human-like lipid profile. We found that ezetimibe, rosuvastatin, and ezetimibe/rosuvastatin combined lower serum cholesterol but induce the expression of Pcsk9 as well as the Srebp-2 hepatic cholesterol biosynthesis pathway. Pcsk9 knockdown in combination with either treatment led to greater reductions in serum non-HDL with a near-uniform reduction of all LDL-c subfractions. In addition to reducing serum cholesterol, the combined rosuvastatin/ezetimibe/Pcsk9 siRNA treatment exhibited a significant reduction in serum APOB protein and triglyceride levels. Taken together, these data provide evidence that PCSK9 inhibitors, in combination with current therapies, have the potential to achieve greater reductions in both serum cholesterol and triglycerides.     

A high-fat and cholesterol diet causes fatty liver in guinea pigs. The role of iron and oxidative damage

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease. Iron, cholesterol, and oxidative damage are frequently suggested to be related to the progression of NAFLD, but the precise relationship between them remains unclear. Guinea pigs fed on a high cholesterol and fat diet (without oxidized lipids) generated a disease model of NAFLD with hallmark observations in liver histology and increased liver damage markers. Hepatic cholesterol and iron levels were found to be significantly elevated and directly correlated. Plasma hepcidin and transferrin levels were decreased. Plasma iron concentrations were found to be elevated, likely due to an increased intestinal iron absorption caused by the decrease in plasma hepcidin. However, hepatic transferrin receptor-2 levels were unchanged. No significant increase in hepatic lipid peroxidation was detected using F₂-isoprostanes as a reliable biomarker, nor was there a rise in protein carbonyls, a general index of oxidative protein damage. Some increases in cholesterol oxidation products were observed, but largely negated after normalizing for the elevated hepatic cholesterol content. Indeed, increased hemosiderin deposition and unchanged ferritin levels in liver suggested that the excess iron mainly existed as hemosiderin, which is redox-inactive.     

Hdac3, Setdb1, and Kap1 mark H3K9me3/H3K14ac bivalent regions in young and aged liver

Post‐translational modifications of histone tails play a crucial role in gene regulation. Here, we performed chromatin profiling by quantitative targeted mass spectrometry to assess all possible modifications of the core histones. We identified a bivalent combination, a dually marked H3K9me3/H3K14ac modification in the liver, that is significantly decreased in old hepatocytes. Subsequent sequential ChIP‐Seq identified dually marked single nucleosome regions, with reduced number of sites and decreased signal in old livers, confirming mass spectrometry results. We detected H3K9me3 and H3K14ac bulk ChIP‐Seq signal in reChIP nucleosome regions, suggesting a correlation between H3K9me3/H3K14ac bulk bivalent genomic regions and dually marked single nucleosomes. Histone H3K9 deacetylase Hdac3, as well as H3K9 methyltransferase Setdb1, found in complex Kap1, occupied both bulk and single nucleosome bivalent regions in both young and old livers, correlating to presence of H3K9me3. Expression of genes associated with bivalent regions in young liver, including those regulating cholesterol secretion and triglyceride synthesis, is upregulated in old liver once the bivalency is lost. Hence, H3K9me3/H3K14ac dually marked regions define a poised inactive state that is resolved with loss of one or both of the chromatin marks, which subsequently leads to change in gene expression.     

Reactive oxygen species‐induced changes in glucose and lipid metabolism contribute to the accumulation of cholesterol in the liver during aging

Aging is a major risk factor for many chronic diseases due to increased vulnerability to external stress and susceptibility to disease. Aging is associated with metabolic liver disease such as nonalcoholic fatty liver. In this study, we investigated changes in lipid metabolism during aging in mice and the mechanisms involved. Lipid accumulation was increased in liver tissues of aged mice, particularly cholesterol. Increased uptake of both cholesterol and glucose was observed in hepatocytes of aged mice as compared with younger mice. The mRNA expression of GLUT2, GK, SREBP2, HMGCR, and HMGCS, genes for cholesterol synthesis, was gradually increased in liver tissues during aging. Reactive oxygen species (ROS) increase with aging and are closely related to various aging‐related diseases. When we treated HepG2 cells and primary hepatocytes with the ROS inducer, H₂O₂, lipid accumulation increased significantly compared to the case for untreated HepG2 cells. H₂O₂ treatment significantly increased glucose uptake and acetyl‐CoA production, which results in glycolysis and lipid synthesis. Treatment with H₂O₂ significantly increased the expression of mRNA for genes related to cholesterol synthesis and uptake. These results suggest that ROS play an important role in altering cholesterol metabolism and consequently contribute to the accumulation of cholesterol in the liver during the aging process.     

Liver osteopontin is required to prevent the progression of age‐related nonalcoholic fatty liver disease

Osteopontin (OPN), a senescence‐associated secretory phenotype factor, is increased in patients with nonalcoholic fatty liver disease (NAFLD). Cellular senescence has been associated with age‐dependent hepatosteatosis. Thus, we investigated the role of OPN in the age‐related hepatosteatosis. For this, human serum samples, animal models of aging, and cell lines in which senescence was induced were used. Metabolic fluxes, lipid, and protein concentration were determined. Among individuals with a normal liver, we observed a positive correlation between serum OPN levels and increasing age. This correlation with age, however, was absent in patients with NAFLD. In wild‐type (WT) mice, serum and liver OPN were increased at 10 months old (m) along with liver p53 levels and remained elevated at 20m. Markers of liver senescence increased in association with synthesis and concentration of triglycerides (TG) in 10m OPN‐deficient (KO) hepatocytes when compared to WT hepatocytes. These changes in senescence and lipid metabolism in 10m OPN‐KO mice liver were associated with the decrease of 78 kDa glucose‐regulated protein (GRP78), induction of ER stress, and the increase in fatty acid synthase and CD36 levels. OPN deficiency in senescent cells also diminished GRP78, the accumulation of intracellular TG, and the increase in CD36 levels. In 20m mice, OPN loss led to increased liver fibrosis. Finally, we showed that OPN expression in vitro and in vivo was regulated by p53. In conclusion, OPN deficiency leads to earlier cellular senescence, ER stress, and TG accumulation during aging. The p53‐OPN axis is required to inhibit the onset of age‐related hepatosteatosis.     

Inhibition of TREM-1 attenuates inflammation and lipid accumulation in diet-induced nonalcoholic fatty liver disease

In the liver tissues of obese diabetic or nondiabetic patients, triggering receptor expressed on myeloid cells-1 (TREM-1) is usually found to be upregulated, thus leading to upregulation of various inflammatory cytokines and lipid accumulation. On the other hand, nonalcoholic fatty liver disease (NAFLD), characterized by excess lipid accumulation, and inflammatory injury in liver, is becoming an epidemic disease, globally. In the present study, we aimed to investigate the biological role and the underlying mechanisms of TREM-1 in NAFLD. upregulation of TREM-1 occurred in high-fat diet (HFD)-induced mice NAFLD model and oleic acid-treated HepG2 and primary mouse hepatocytes cell model at messenger RNA and protein levels. Functional studies established that overexpression of TREM-1 displayed hyperlipidemia, and increased in inflammatory indicators and lipid accumulation-related genes, which was ameliorated by knockdown of TREM-1. Our results also showed that obvious lipid accumulation and inflammatory injury occurred in the liver tissue of HFD-fed mice, while treatment with lentiviral vector short hairpin TREM showed marked improvement in tissue morphology and architecture and less lipid accumulation, thus deciphering the mechanism through which knockdown of TREM-1 ameliorated the inflammatory response and lipid accumulation of NAFLD mice through inactivation of the nuclear factor-κB (NF-κB) and PI3K/AKT signal pathways, respectively. In conclusion, TREM-1/NF-κB and TREM-1/PI3K/AKT axis could be an important mechanism in ameliorating the inflammatory response and lipid accumulation, respectively, thus shedding light on the development of novel therapeutics to the treatment of NAFLD.