Quantitative characterization of the mechanism of action and impact of a 'proteolysis-permitting' anti-PCSK9 antibody.

A recent report described a novel mechanism of action for an anti-proprotein convertase subtilisin-kexin type 9 (PCSK9) monoclonal antibody (LY3015014, or LY), wherein the antibody has improved potency and duration of action due to the PCSK9 epitope for LY binding. Unlike other antibodies, proteolysis of PCSK9 can occur when LY is bound to PCSK9. We hypothesized that this allowance of PCSK9 cleavage potentially improves LY efficiency through two pathways, namely lack of accumulation of intact PCSK9 and reduced clearance of LY.

Proteolytic cleavage of antigen extends the durability of an anti-PCSK9 monoclonal antibody.

Lilly PCSK9 antibody LY3015014 (LY) is a monoclonal antibody (mAb) that neutralizes proprotein convertase subtilisin-kexin type 9 (PCSK9). LY decreases LDL cholesterol in monkeys and, unlike other PCSK9 mAbs, does not cause an accumulation of intact PCSK9 in serum. Comparing the epitope of LY with other clinically tested PCSK9 mAbs, it was noted that the LY epitope excludes the furin cleavage site in PCSK9, whereas other mAbs span this site.

Isolation and characterization of the circulating truncated form of PCSK9.

Proprotein convertase subtilisin-kexin type 9 (PCSK9) is a secreted protein which regulates serum LDL cholesterol. It circulates in human and rodent serum in an intact form and a major truncated form. Previous in vitro studies involving the expression of human PCSK9 genetic variants and in vivo studies of furin knockout mice suggest that the truncated form is a furin cleavage product.

Varespladib (A-002), a secretory phospholipase A2 inhibitor, reduces atherosclerosis and aneurysm formation in ApoE-/- mice.

The family of secretory phospholipase A2 (sPLA2) enzymes has been associated with inflammatory diseases and tissue injury including atherosclerosis. A-001 is a novel inhibitor of sPLA2 enzymes discovered by structure-based drug design, and A-002 is the orally bioavailable prodrug currently in clinical development. A-001 inhibited human and mouse sPLA2 group IIA, V, and X enzymes with IC50 values in the low nM range.

Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain.

A 15-ketosterol is a liver X receptor ligand that suppresses sterol-responsive element binding protein-2 activity.

Hypercholesterolemia is a major risk factor for coronary artery disease. Oxysterols are known to inhibit cholesterol biosynthesis and have been explored as potential antihypercholesterolemic agents. The ability of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (15-ketosterol) to lower non-HDL cholesterol has been demonstrated in rodent and primate models, but the mechanisms of action remain poorly understood.

Niacin mediates lipolysis in adipose tissue through its G-protein coupled receptor HM74A.

A G-protein coupled receptor to niacin (nicotinic acid) was identified recently but the physiological/pharmacological role of the receptor remains poorly defined. We present our studies to demonstrate that HM74A, but not HM74, binds niacin at high affinities and effectively mediates Gi signaling events in human embryonic kidney HEK293 cells as well as in 3T3L1 adipocytes expressing HM74A. Furthermore, HM74A, but not HM74, expressed in differentiated 3T3L1 adipocytes effectively mediated inhibition of lipolysis by niacin.

Liver X receptors (LXRs) regulate apolipoprotein AIV-implications of the antiatherosclerotic effect of LXR agonists.

Liver X receptors (LXRs) regulate target genes that are critical in lipoprotein metabolism and atherosclerosis. Apolipoprotein AIV (ApoAIV) is an apolipoprotein that is associated with chylomicrons and high-density lipoproteins. Plasma ApoAIV level in humans is inversely correlated with coronary artery events and overexpression of ApoAIV in mice results in significant reduction in atherosclerosis.

Liver X receptors as potential therapeutic targets for multiple diseases.

Liver X receptor alpha (LXRalpha) and liver X receptor beta(LXRbeta are oxysterol receptors that regulate multiple target genes involved in cholesterol homeostasis. Recent studies also suggest that the pair of receptors may also be involved in glucose metabolism, inflammation and Alzheimer's disease by regulating critical molecules involved in these pathophysiological processes. Although the prototypic LXR agonists induce liver triglyceride accumulation by regulating the hepatic lipogenesis pathway, it is hoped that a subtype-specific agonist or selective modulators would provide the desired cardioprotection and other benefits without the undesirable concomitant induction of lipogenesis.

Coadministration of a liver X receptor agonist and a peroxisome proliferator activator receptor-alpha agonist in Mice: effects of nuclear receptor interplay on high-density lipoprotein and triglyceride metabolism in vivo.

Liver X receptors (LXRs) are master transcription factors regulating cholesterol and fatty acid metabolism. Treatment of C57B6 mice with a specific synthetic LXR agonist, N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide (T0901317), resulted in elevated high-density lipoprotein (HDL) cholesterol as well as plasma and liver triglycerides. Peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists are known to induce peroxisomal fatty acid beta-oxidation and also mediate HDL cholesterol metabolism.

A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes.

Apolipoprotein E (apoE) is an important protein involved in lipoprotein clearance and cholesterol redistribution. ApoE is abundantly expressed in astrocytes in the brain and is closely linked to the pathogenesis of Alzheimer's disease (AD). We report here that small molecule ligands that activate either liver X receptors (LXR) or retinoid X receptor (RXR) lead to a dramatic increase in apoE mRNA and protein expression as well as secretion of apoE in a human astrocytoma cell line (CCF-STTG1 cells).

Enlargement of high density lipoprotein in mice via liver X receptor activation requires apolipoprotein E and is abolished by cholesteryl ester transfer protein expression.

The factors involved in the generation of larger high density lipoprotein (HDL) particles, HDL1 and HDLc, are still not well understood. Administration of a specific synthetic liver X receptor (LXR) agonist, T0901317, in mice resulted in an increase of not only HDL cholesterol but also HDL particle size (Cao, G., Beyer, T.

A natural product ligand of the oxysterol receptor, liver X receptor.

Natural products have been identified as ligands for a number of members of the nuclear hormone receptor (NHR) superfamily. Often these natural products are used as dietary supplements to treat myriad ailments ranging from perimenopausal hot flashes to hypercholesterolemia and reduced cognitive function. Examples of some natural product ligands for NHRs include genestein (estrogen receptors NR3A1 and NR3A2), guggulsterone (farnesoid X receptor NR1H4), and St.

A chemical switch regulates fibrate specificity for peroxisome proliferator-activated receptor alpha (PPARalpha ) versus liver X receptor.

Fenofibrate is clinically successful in treating hypertriglyceridemia and mixed hyperlipidemia presumably through peroxisome proliferator-activated receptor alpha (PPARalpha)-dependent induction of genes that control fatty acid beta-oxidation. Lipid homeostasis and cholesterol metabolism also are regulated by the nuclear oxysterol receptors, liver X receptors alpha and beta (LXRalpha and LXRbeta). Here we show that fenofibrate ester, but not fenofibric acid, functions as an LXR antagonist by directly binding to LXRs.

Antidiabetic action of a liver x receptor agonist mediated by inhibition of hepatic gluconeogenesis.

The oxysterol receptors LXR (liver X receptor)-alpha and LXRbeta are nuclear receptors that play a key role in regulation of cholesterol and fatty acid metabolism. We found that LXRs also play a significant role in glucose metabolism. Treatment of diabetic rodents with the LXR agonist, T0901317, resulted in dramatic reduction of plasma glucose.

Liver X receptor and retinoic X receptor mediated ABCA1 regulation and cholesterol efflux in macrophage cells-messenger RNA measured by branched DNA technology.

ABCA1 is an ATP binding cassette transporter that plays an essential role in cholesterol and phospholipid efflux and HDL biogenesis. ABCA1 expression in macrophage cells is subject to regulation by cAMP, cholesterol loading, and ligands of the nuclear receptors liver X receptor (LXR) and retinoid X receptor (RXR). We report here the development of a rapid and high volume branched DNA (bDNA) method to measure ABCA1 mRNA.

Genetic disorders associated with ATP binding cassette cholesterol transporters.

Coronary artery disease is the most prevalent form of mortality and morbidity in Western countries. Studies in the last several decades have identified high LDL cholesterol and low HDL cholesterol as major risk factors leading to the disease. Human genetic studies have provided significant insight into the regulation of lipoprotein metabolism.

Phospholipid transfer protein is regulated by liver X receptors in vivo.

Liver X receptors (LXR) belong to the nuclear receptor superfamily that can regulate important lipid metabolic pathways. The plasma phospholipid transfer protein (PLTP) is known to mediate transfer of phospholipids from triglyceride-rich lipoproteins to high density lipoprotein (HDL) and plays a critical role in HDL metabolism. We report here that a specific LXR agonist, T0901317, elevated HDL cholesterol and phospholipid in C57/BL6 mice and generated enlarged HDL particles that were enriched in cholesterol, ApoAI, ApoE, and phospholipid.

Estrogen receptor-mediated repression of human hepatic lipase gene transcription.

Estrogen replacement therapy in women decreases hepatic lipase (HL) activity, which may account for the associated increase in HDL cholesterol. To investigate whether estrogen decreases HL transcription, transient cotransfection assays with HL promoter and estrogen receptor-alpha (ERalpha) expression constructs were performed in HepG2 cells. 17beta-estradiol (E(2)) decreased transcription driven by the -1557/+41 human HL promoter by up to 50% at 10(-7) M.