Discovery of a brain-sparing GIRK1/4 inhibitor for pharmacological cardioversion of atrial fibrillation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia, and a significant risk factor for ischemic stroke and heart failure. Marketed anti-arrhythmic drugs can restore sinus rhythm, but with limited efficacy and significant toxicities, including potential to induce ventricular arrhythmia. Atrial-selective ion channel drugs are expected to restore and maintain sinus rhythm without risk of ventricular arrhythmia. One such atrial-selective channel target is GIRK1/4 (G-protein regulated inwardly rectifying potassium channel 1/4). Here we describe 14b, a potent GIRK1/4 inhibitor developed to cardiovert AF to sinus rhythm while minimizing central nervous system exposure - an issue with preceding GIRK1/4 clinical candidates.

Lack of authentic atrial fibrillation in commonly used murine atrial fibrillation models.

The mouse is a useful preclinical species for evaluating disease etiology due to the availability of a wide variety of genetically modified strains and the ability to perform disease-modifying manipulations. In order to establish an atrial filtration (AF) model in our laboratory, we profiled several commonly used murine AF models. We initially evaluated a pharmacological model of acute carbachol (CCh) treatment plus atrial burst pacing in C57BL/6 mice. In an effort to observe micro-reentrant circuits indicative of authentic AF, we employed optical mapping imaging in isolated mouse hearts. While CCh reduced atrial refractoriness and increased atrial tachyarrhythmia vulnerability, the left atrial (LA) excitation patterns were rather regular without reentrant circuits or wavelets. Therefore, the atrial tachyarrhythmia resembled high frequency atrial flutter, not typical AF per se. We next examined both a chronic angiotensin II (Ang II) infusion model and the surgical model of transverse aortic constriction (TAC), which have both been reported to induce atrial and ventricular structural changes that serve as a substrates for micro-reentrant AF. Although we observed some extent of atrial remodeling such as fibrosis or enlarged LA diameter, burst pacing-induced atrial tachyarrhythmia vulnerability did not differ from control mice in either model. This again suggested that an AF-like pathophysiology is difficult to demonstrate in the mouse. To continue searching for a valid murine AF model, we studied mice with a cardiac-specific deficiency (KO) in liver kinase B1 (Cardiac-LKB1), which has been reported to exhibit spontaneous AF. Indeed, the electrocardiograms (ECG) of conscious Cardiac-LKB1 KO mice exhibited no P waves and had irregular RR intervals, which are characteristics of AF. Histological evaluation of Cardiac-LKB1 KO mice revealed dilated and fibrotic atria, again consistent with AF. However, atrial electrograms and optical mapping revealed that electrical activity was limited to the sino-atrial node area with no electrical conduction into the atrial myocardium beyond. Thus, Cardiac-LKB1 KO mice have severe atrial myopathy or atrial standstill, but not AF. In summary, the atrial tachyarrhythmias we observed in the four murine models were distinct from typical human AF, which often exhibits micro- or macro-reentrant atrial circuits. Our results suggest that the four murine AF models we examined may not reflect human AF well, and raise a cautionary note for use of those murine models to study AF.

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.

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.

The design and implementation of a generic lipopeptide scanning platform to enable the identification of 'locally acting' agonists for the apelin receptor.

This Letter describes methodology to enable the identification of tool or therapeutic lipopeptides which modulate the function of membrane bound proteins. The choice of lipopeptides as a chemotype is the amalgamation of multiple medicinal chemistry considerations including duration of action, low systemic exposure and access to intracellular components. The 'lipopeptide shuffle' has been applied here to the APJ receptor and has rapidly resulted in the discovery of a 33 nM APJ agonist hit from an initial 369 member lipopeptide synthetic array.

Niacin lipid efficacy is independent of both the niacin receptor GPR109A and free fatty acid suppression.

Nicotinic acid (niacin) induces beneficial changes in serum lipoproteins and has been associated with beneficial cardiovascular effects. Niacin reduces low-density lipoprotein, increases high-density lipoprotein, and decreases triglycerides. It is well established that activation of the seven-transmembrane G(i)-coupled receptor GPR109A on Langerhans cells results in release of prostaglandin D2, which mediates the well-known flushing side effect of niacin. Niacin activation of GPR109A on adipocytes also mediates the transient reduction of plasma free fatty acid (FFA) levels characteristic of niacin, which has been long hypothesized to be the mechanism underlying the changes in the serum lipid profile. We tested this "FFA hypothesis" and the hypothesis that niacin lipid efficacy is mediated via GPR109A by dosing mice lacking GPR109A with niacin and testing two novel, full GPR109A agonists, MK-1903 and SCH900271, in three human clinical trials. In mice, the absence of GPR109A had no effect on niacin's lipid efficacy despite complete abrogation of the anti-lipolytic effect. Both MK-1903 and SCH900271 lowered FFAs acutely in humans; however, neither had the expected effects on serum lipids. Chronic FFA suppression was not sustainable via GPR109A agonism with niacin, MK-1903, or SCH900271. We conclude that the GPR109A receptor does not mediate niacin's lipid efficacy, challenging the long-standing FFA hypothesis.

(1aR,5aR)1a,3,5,5a-Tetrahydro-1H-2,3-diaza-cyclopropa[a]pentalene-4-carboxylic acid (MK-1903): a potent GPR109a agonist that lowers free fatty acids in humans.

G-protein coupled receptor (GPCR) GPR109a is a molecular target for nicotinic acid and is expressed in adipocytes, spleen, and immune cells. Nicotinic acid has long been used for the treatment of dyslipidemia due to its capacity to positively affect serum lipids to a greater extent than other currently marketed drugs. We report a series of tricyclic pyrazole carboxylic acids that are potent and selective agonists of GPR109a. Compound R,R-19a (MK-1903) was advanced through preclinical studies, was well tolerated, and presented no apparent safety concerns. Compound R,R-19a was advanced into a phase 1 clinical trial and produced a robust decrease in plasma free fatty acids. On the basis of these results, R,R-19a was evaluated in a phase 2 study in humans. Because R,R-19a produced only a weak effect on serum lipids as compared with niacin, we conclude that the beneficial effects of niacin are most likely the result of an undefined GPR109a independent pathway.

The use of stable-isotopically labeled oleic acid to interrogate lipid assembly in vivo: assessing pharmacological effects in preclinical species.

The use of stable isotopically labeled substrates and analysis by mass spectrometry have provided substantial insight into rates of synthesis, disposition, and utilization of lipids in vivo. The information to be gained from such studies is of particular benefit to therapeutic research where the underlying causes of disease may be related to the production and utilization of lipids. When studying biology through the use of isotope tracers, care must be exercised in interpreting the data to ensure that any response observed can truly be interpreted as biological and not as an artifact of the experimental design or a dilutional effect on the isotope. We studied the effects of dosing route and tracer concentration on the mass isotopomer distribution profile as well as the action of selective inhibitors of microsomal tri-glyceride transfer protein (MTP) in mice and diacylglycerol acyltransferase 1 (DGAT1) in nonhuman primates, using a stable-isotopically labeled approach. Subjects were treated with inhibitor and subsequently given a dose of uniformly ¹³C-labeled oleic acid. Samples were analyzed using a rapid LC-MS technique, allowing the effects of the intervention on the assembly and disposition of triglycerides, cholesteryl esters, and phospholipids to be determined in a single 3 min run from just 10 µl of plasma.

The discovery of high affinity agonists of GPR109a with reduced serum shift and improved ADME properties.

Amino-anthranilic acid derivatives have been identified as a new class of low serum shifted, high affinity full agonists of the human orphan G-protein-coupled receptor GPR109a with improved ADME properties.

GPR109a agonists. Part 2: pyrazole-acids as agonists of the human orphan G-protein coupled receptor GPR109a.

5-Alkyl and aryl-pyrazole-acids have been identified as a new class of selective, small-molecule, agonists of the human orphan G-protein-coupled receptor GPR109a, a high affinity receptor for the HDL-raising drug 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.

Cholesterol efflux potential and antiinflammatory properties of high-density lipoprotein after treatment with niacin or anacetrapib.

OBJECTIVE: To examine the effects of treatments with niacin or anacetrapib (an inhibitor of cholesteryl ester transfer protein) on the ability of high-density lipoprotein (HDL) to promote net cholesterol efflux and reduce toll-like receptor-mediated inflammation in macrophages. METHODS AND RESULTS: A total of 18 patients received niacin, 2 g/d, for 4 weeks; 20 patients received anacetrapib, 300 mg/d, for 8 weeks; and 2 groups (n=4 and n=5 patients) received placebo. HDL samples were isolated by polyethylene glycol precipitation or ultracentrifugation, tested for the ability to promote cholesterol efflux in cholesterol-loaded THP-I or mouse peritoneal macrophages, or used to pretreat macrophages, followed by lipopolysaccharide exposure. HDL cholesterol levels were increased by 30% in response to niacin and by approximately 100% in response to anacetrapib. Niacin treatment increased HDL-mediated net cholesterol efflux from foam cells, primarily by increasing HDL concentration, whereas anacetrapib treatment increased cholesterol efflux by both increasing HDL concentration and causing increased efflux at matched HDL concentrations. The increased efflux potential of anacetrapib-HDL was more prominent at higher HDL cholesterol concentrations (>12 microg/mL), which was associated with an increased content of lecithin-cholesterol acyltransferase (LCAT) and apolipoprotein E and completely dependent on the expression of ATP binding cassette transporters (ABCA1 and ABCG1). Potent antiinflammatory effects of HDL were observed at low HDL concentrations (3 to 20 microg/mL) and were partly dependent on the expression of ABCA1 and ABCG1. All HDL preparations showed similar antiinflammatory effects, proportionate to the HDL cholesterol concentration. CONCLUSIONS: Niacin treatment caused a moderate increase in the ability of HDL to promote net cholesterol efflux, whereas inhibition of cholesteryl ester transfer protein via anacetrapib led to a more dramatic increase in association with enhanced particle functionality at higher HDL concentrations. All HDLs exhibited potent ability to suppress macrophage toll-like receptor 4-mediated inflammatory responses, in a process partly dependent on cholesterol efflux via ABCA1 and ABCG1.

Discovery of pyrazolyl propionyl cyclohexenamide derivatives as full agonists for the high affinity niacin receptor GPR109A.

A series of pyrazolyl propionyl cyclohexenamides were discovered as full agonists for the high affinity niacin receptor GPR109A. The structure-activity relationship (SAR) studies were aimed to improve activity on GPR109A, reduce Cytochrome P450 2C8 (CYP2C8) and Cytochrome P450 2C9 (CYP2C9) inhibition, reduce serum shift and improve pharmacokinetic (PK) profiles.

Potent tricyclic pyrazole tetrazole agonists of the nicotinic acid receptor (GPR109a).

Tricyclic pyrazole tetrazoles which are potent partial agonists of the high affinity niacin receptor, GPR109a, have been discovered and optimized. One of these compounds has proven to be effective at lowering free fatty acids in vitro and in vivo.

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.

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.

GPR109a agonists. Part 1: 5-Alkyl and 5-aryl-pyrazole-tetrazoles as agonists of the human orphan G-protein coupled receptor GPR109a.

5-Alkyl and aryl-pyrazole-tetrazoles have been identified as a new class of selective, small-molecule, agonists of the human G-protein-coupled receptor GPR109a, a high affinity receptor for the HDL-raising drug nicotinic acid.

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.

Phenolic acids suppress adipocyte lipolysis via activation of the nicotinic acid receptor GPR109A (HM74a/PUMA-G).

Phenolic acids are found in abundance throughout the plant kingdom. Consumption of wine or other rich sources of phenolic acids, such as the "Mediterranean diet," has been associated with a lower risk of cardiovascular disease. The underlying mechanism(s), however, has remained unclear. Here, we show that many phenolic acids, including those from the hydroxybenzoic and hydroxycinnamic acid classes, can bind and activate GPR109A (HM74a/PUMA-G), the receptor for the antidyslipidemic agent nicotinic acid. In keeping with this activity, treatment with a number of phenolic acids, including cinnamic acid, reduces lipolysis in cultured human adipocytes and in fat pats isolated from wild-type mice but not from mice deficient of GPR109A. Oral administration of cinnamic acid significantly reduces plasma levels of FFA in the wild type but not in mice deficient of GPR109A. Activation of GPR109A by phenolic acids may thus contribute to a cardiovascular benefit of these plant-derived products.

Role of GPR81 in lactate-mediated reduction of adipose lipolysis.

Heavy exercise or oxygen deficit often links with higher levels of arterial lactate and lower levels of plasma free fatty acids (FFA). Treatment with lactate reduces circulating levels of FFA in vivo and lipolysis in adipose tissues in vitro. However, the underlying mechanism has remained unclear. Here we employ pharmacological and genetic approaches to show that GPR81, an orphan G-protein-coupled receptor with relatively restricted expression in the adipose tissues, functions as a receptor for lactate and can mediate an anti-lipolytic effect of lactate. GPR81 may thus function as a sensor of lactate that can modulate the FFA pool under exercise or conditions of oxygen deficit.