Larissa Wilsie

Plasma lipid profiling across species for the identification of optimal animal models of human dyslipidemia.

In an attempt to understand the applicability of various animal models to dyslipidemia in humans and to identify improved preclinical models for target discovery and validation for dyslipidemia, we measured comprehensive plasma lipid profiles in 24 models. These included five mouse strains, six other nonprimate species, and four nonhuman primate (NHP) species, and both healthy animals and animals with metabolic disorders. Dyslipidemic humans were assessed by the same measures. Plasma lipoprotein profiles, eight major plasma lipid fractions, and FA compositions within these lipid fractions were compared both qualitatively and quantitatively across the species. Given the importance of statins in decreasing plasma low-density lipoprotein cholesterol for treatment of dyslipidemia in humans, the responses of these measures to simvastatin treatment were also assessed for each species and compared with dyslipidemic humans. NHPs, followed by dog, were the models that demonstrated closest overall match to dyslipidemic humans. For the subset of the dyslipidemic population with high plasma triglyceride levels, the data also pointed to hamster and db/db mouse as representative models for practical use in target validation. Most traditional models, including rabbit, Zucker diabetic fatty rat, and the majority of mouse models, did not demonstrate overall similarity to dyslipidemic humans in this study.

Discovery of a New Role of Human Resistin in Hepatocyte Low-Density Lipoprotein Receptor Suppression Mediated in Part by Proprotein Convertase Subtilisin/Kexin Type 9

OBJECTIVES In this study, our goal was to determine if human resistin plays a role in regulating the uptake of atherogenic low-density lipoproteins in human hepatocytes. BACKGROUND Serum levels of resistin, an adipose tissue-derived adipokine, are increased in human obesity and are positively correlated with atherosclerotic cardiovascular diseases. However, the function of resistin in humans is enigmatic. METHODS Human hepatocytes (HepG2 and primary) were treated (24 h) with the following: 1) purified human resistin at various concentrations, with and without lovastatin; and 2) obese human serum with elevated resistin levels or serum from which resistin was removed via antibody-immunoprecipitation. The effect of the treatments on cellular low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) messenger ribonucleic acid and protein levels were determined by using real-time polymerase chain reaction and Western blotting, respectively. RESULTS Resistin, at physiological levels observed in human obesity, down-regulated hepatocyte LDLR expression substantially (by 40%). A key mechanism by which human resistin inhibited LDLR levels was by increased cellular expression of the recently identified protease, PCSK9, which enhances intracellular LDLR lysosomal degradation. The quantitatively important role of human resistin in LDLR expression was demonstrated by antibody-immunoprecipitation removal of resistin in human serum, which decreased serum stimulation of hepatocyte LDLRs markedly (by 80%). Furthermore, resistin diminished statin-mediated up-regulation of the LDLR by 60%, implicating resistin in the relative ineffectiveness of statins in selective target populations. CONCLUSIONS These results reveal for the first time that resistin is a highly attractive therapeutic target in ameliorating elevated serum low-density lipoprotein and, thereby, atherosclerotic cardiovascular diseases in obese humans.

Discovery of novel tricyclic full agonists for the G-protein-coupled niacin receptor 109A with minimized flushing in rats.

Tricyclic analogues were rationally designed as the high affinity niacin receptor G-protein-coupled receptor 109A (GPR109A) agonists by overlapping three lead structures. Various tricyclic anthranilide and cycloalkene carboxylic acid full agonists were discovered with excellent in vitro activity. Compound 2g displayed a good therapeutic index regarding free fatty acids (FFA) reduction and vasodilation effects in rats, with very weak cytochrome P450 2C8 (CYP2C8) and cytochrome P450 2C9 (CYP2C9) inhibition, and a good mouse pharmacokinetics (PK) profile.

Discovery of a Biaryl Cyclohexene Carboxylic Acid (MK-6892): A Potent and Selective High Affinity Niacin Receptor Full Agonist with Reduced Flushing Profiles in Animals as a Preclinical Candidate

Biaryl cyclohexene carboxylic acids were discovered as full and potent niacin receptor (GPR109A) agonists. Compound 1e (MK-6892) displayed excellent receptor activity, good PK across species, remarkably clean off-target profiles, good ancillary pharmacology, and superior therapeutic window over niacin regarding the FFA reduction versus vasodilation in rats and dogs.

Discovery of pyrazolopyrimidines as the first class of allosteric agonists for the high affinity nicotinic acid receptor GPR109A.

Pyrazolopyrimidines were discovered as the first class of allosteric agonists for the high affinity nicotinic acid receptor GPR109A. In addition to its intrinsic activity, compound 9n significantly enhances nicotinic acid binding to the receptor, thereby potentiating the functional efficacy of nicotinic acid.

Anthranilic acid replacements in a niacin receptor agonist.

Niacin is an effective drug for raising HDL cholesterol. However, niacin must be taken in large doses and significant side effects are often observed, including facial flushing, loss of glucose tolerance, and liver toxicity. An anthranilic acid was identified as an agonist of the niacin receptor. In order to improve efficacy and provide structural diversity, replacements for the anthranilic acid were investigated and several compounds with improved properties were identified.

Pyrazole acids as niacin receptor agonists for the treatment of dyslipidemia.

Niacin is an effective drug for raising HDL cholesterol and reducing coronary risks, but patients show low compliance with treatment due to severe facial flushing upon taking the drug. A series of bicyclic pyrazole carboxylic acids were synthesized and tested for their ability to activate the niacin receptor. One analog, 23, showed improved potency and lacked flushing at doses that effectively altered the lipid profile of rats.

Discovery and Pharmacology of a Novel Class of Diacylglycerol Acyltransferase 2 Inhibitors

DGAT2 plays a critical role in hepatic triglyceride production, and data suggests that inhibition of DGAT2 could prove to be beneficial in treating a number of disease states. This article documents the discovery and optimization of a selective small molecule inhibitor of DGAT2 as well as pharmacological proof of biology in a mouse model of triglyceride production.

Identification of DGAT2 Inhibitors Using Mass Spectrometry

Mass spectrometry offers significant advantages over other detection technologies in the areas of hit finding, hit validation, and medicinal chemistry compound optimization. The foremost obvious advantage is the ability to directly measure enzymatic product formation. In addition, the inherent sensitivity of the liquid chromatography/mass spectrometry (LC/MS) approach allows the execution of enzymatic assays at substrate concentrations typically at or below substrate Km. Another advantage of the LC/MS approach is the ability to assay impure enzyme systems that would otherwise be difficult to prosecute with traditional labeled methods. This approach was used to identify inhibitors of diacylglycerol O-acyltransferase-2 (DGAT2), a transmembrane enzyme involved in the triglyceride (TG) production pathway. The LC/MS approach was employed because of its increased assay window (compared with control membranes) of more than sevenfold compared with less than twofold with a conventional fluorogenic assay. The ability to generate thousands of dose-dependent IC50 data was made possible by the use of a staggered parallel LC/MS system with fast elution gradients. From the hit-deconvolution efforts, several structural scaffold series were identified that inhibit DGAT2 activity. Additional profiling of one chemotype in particular identified two promising reversible and selective compounds (compound 15 and compound 16) that effectively inhibit TG production in mouse primary hepatocytes.

C-terminal Loop Mutations Determine Folding and SecretionProperties of PCSK9

Human genetics and pharmacologic clinical intervention demonstrate the key role of PCSK9 in cholesterol regulation. To understand the role of the C-terminal domain of PCSK9, two human mutations in this region (S462P and A522T PCSK9) have been profiled. Confirming and extending previous observations, S462P and WT PCSK9 bind to LDLR with equivalent affinity; however, while S462P PCSK9 cleavage is unaffected, its secretion is defective, and association with the ER protein-folding sensor calreticulin, increased. In a similar manner, A522T PCSK9 also exhibits defective secretion and an enhanced association with calreticulin. To assess the in vivo lipid phenotype of the S462P and A522T PCSK9 mutations, Pcsk9-/- mice were infected with AAV8’s encoding the different variants. Although liver transcript levels for all were equivalent, circulating levels of S462P PCSK9, and to a lesser degree A522T PCSK9, were reduced relative to WT PCSK9 correlating with the in vitro phenotype. Further, the extent of reduced circulating S462P or A522T PCSK9 correlated well with increases in mouse liver LDLR and reductions of LDL/ total cholesterol. When interpreted within the context of molecular modeling, it appears that the human non-synonymous polymorphisms S462P and A522T destabilize the C-terminal domain of PCSK9 impacting folding and secretion.