Identification of four novel genes contributing to familial elevated plasma HDL cholesterol in humans.

While genetic determinants strongly influence HDL cholesterol (HDLc) levels, most genetic causes underlying variation in HDLc remain unknown. We aimed to identify novel rare mutations with large effects in candidate genes contributing to extreme HDLc in humans, utilizing family-based Mendelian genetics. We performed next-generation sequencing of 456 candidate HDLc-regulating genes in 200 unrelated probands with extremely low (≤10th percentile) or high (≥90th percentile) HDLc. Probands were excluded if known mutations existed in the established HDLc-regulating genes ABCA1, APOA1, LCAT, cholesteryl ester transfer protein (CETP), endothelial lipase (LIPG), and UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2). We identified 93 novel coding or splice-site variants in 72 candidate genes. Each variant was genotyped in the proband's family. Family-based association analyses were performed for variants with sufficient power to detect significance at P < 0.05 with a total of 627 family members being assessed. Mutations in the genes glucokinase regulatory protein (GCKR), RNase L (RNASEL), leukocyte immunoglobulin-like receptor 3 (LILRA3), and dynein axonemal heavy chain 10 (DNAH10) segregated with elevated HDLc levels in families, while no mutations associated with low HDLc. Taken together, we have identified mutations in four novel genes that may play a role in regulating HDLc levels in humans.

Discovery of MK-4409, a Novel Oxazole FAAH Inhibitor for the Treatment of Inflammatory and Neuropathic Pain.

We report herein the identification of MK-4409, a potent and selective fatty acid amide hydrolase (FAAH) inhibitor. Starting from a high throughput screening (HTS) hit, medicinal chemistry efforts focused on optimizing of FAAH inhibition in vitro potency, improving the pharmacokinetic (PK) profile, and increasing in vivo efficacy in rodent inflammatory and neuropathic pain assays.

Discovery of XEN445: a potent and selective endothelial lipase inhibitor raises plasma HDL-cholesterol concentration in mice.

Endothelial lipase (EL) activity has been implicated in HDL metabolism and in atherosclerotic plaque development; inhibitors are proposed to be efficacious in the treatment of dyslipidemia related cardiovascular disease. We describe here the discovery of a novel class of anthranilic acids EL inhibitors. XEN445 (compound 13) was identified as a potent and selective EL inhibitor, that showed good ADME and PK properties, and demonstrated in vivo efficacy in raising plasma HDLc concentrations in mice.

The impact of partial and complete loss-of-function mutations in endothelial lipase on high-density lipoprotein levels and functionality in humans.

BACKGROUND: Endothelial lipase is a phospholipase with activity against high-density lipoprotein. Although a small number of mutations in LIPG have been described, the role of LIPG in protection against atherosclerosis is unclear. METHODS AND RESULTS: We identified 8 loss-of-function (LOF) mutations in LIPG in individuals with high-density lipoprotein cholesterol. Functional analysis confirmed that most rare mutations abolish lipase activity in vitro, indicating complete LOF, whereas 2 more common mutations N396S and R476W reduce activity by ≈50%, indicating partial LOF and implying ≈50% and ≈75% remaining endothelial lipase function in heterozygous complete LOF and partial LOF mutation carriers, respectively. complete LOF mutation carriers had significantly higher plasma high-density lipoprotein cholesterol levels compared with partial LOF mutation carriers. Apolipoprotein B-depleted serum from complete LOF carriers showed significantly enhanced cholesterol efflux acceptor capacity, whereas only trends were observed in partial LOF carriers. Carriers of LIPG mutations exhibited trends toward reduced coronary artery disease in 4 independent cohorts (meta-analysis odds ratio, 0.7; P=0.04). CONCLUSIONS: Our data suggest that the impact of LIPG mutations is directly related to their effect on endothelial lipase function and support that antagonism of endothelial lipase function improves cardioprotection.

Discovery of MK-3168: A PET Tracer for Imaging Brain Fatty Acid Amide Hydrolase.

We report herein the discovery of a fatty acid amide hydrolase (FAAH) positron emission tomography (PET) tracer. Starting from a pyrazole lead, medicinal chemistry efforts directed toward reducing lipophilicity led to the synthesis of a series of imidazole analogues. Compound 6 was chosen for further profiling due to its appropriate physical chemical properties and excellent FAAH inhibition potency across species. [(11)C]-6 (MK-3168) exhibited good brain uptake and FAAH-specific signal in rhesus monkeys and is a suitable PET tracer for imaging FAAH in the brain.

The design and synthesis of potent, selective benzodiazepine sulfonamide bombesin receptor subtype 3 (BRS-3) agonists with an increased barrier of atropisomerization.

Bombesin receptor subtype 3 (BRS-3) is an orphan G-protein coupled receptor expressed primarily in the hypothalamus which plays a role in the onset of both diabetes and obesity. We report herein our progress made towards identifying a potent, selective bombesin receptor subtype-3 (BRS-3) agonist related to the previously described MK-7725(1) Chobanian et al. (2012) that would prevent atropisomerization through the increase of steric bulk at the C-2 position. This would thereby make clinical development of this class of compounds more cost effective by inhibiting racemization which can occur over long periods of time at room/elevated temperature.

Discovery of MK-7725, A Potent, Selective Bombesin Receptor Subtype-3 Agonist for the Treatment of Obesity.

Extensive structure-activity relationship studies of a series derived from atropisomer 1, a previously described chiral benzodiazepine sulfonamide series, led to a potent, brain penetrant and selective compound with excellent preclinical pharmacokinetic across species. We also describe the utilization of a high throughput mouse pharmacodynamic assay which allowed for expedient assessment of pharmacokinetic and brain distribution.

Pyridinesulfonylureas and pyridinesulfonamides as selective bombesin receptor subtype-3 (BRS-3) agonists.

Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein coupled receptor belonging to the subfamily of bombesin-like receptors. BRS-3 is implicated in the development of obesity and diabetes. We report here small-molecule agonists that are based on a 4-(alkylamino)pyridine-3-sulfonamide core. We describe the discovery of 2a, which has mid-nanomolar potency, selectivity for human BRS-3 versus the other bombesin-like receptors, and good bioavailability.

Discovery of MK-5046, a Potent, Selective Bombesin Receptor Subtype-3 Agonist for the Treatment of Obesity.

We report the development and characterization of compound 22 (MK-5046), a potent, selective small molecule agonist of BRS-3 (bombesin receptor subtype-3). In pharmacological testing using diet-induced obese mice, compound 22 caused mechanism-based, dose-dependent reductions in food intake and body weight.

Discovery of benzodiazepine sulfonamide-based bombesin receptor subtype 3 agonists and their unusual chirality.

We report herein the discovery of benzodiazepine sulfonamide-based bombesin receptor subtype 3 (BRS-3) agonists and their unusual chirality. Starting from a high-throughput screening lead, we prepared a series of BRS-3 agonists with improved potency and pharmacokinetic properties, of which compound 8a caused mechanism-based, dose-dependent food intake reduction and body weight loss after oral dosing in diet-induced obese mice. This effort also led to the discovery of a novel family of chiral molecules originated from the conformationally constrained seven-membered diazepine ring.

Antiobesity effect of MK-5046, a novel bombesin receptor subtype-3 agonist.

Bombesin receptor subtype-3 (BRS-3) is an orphan G protein-coupled receptor implicated in the regulation of energy homeostasis. Here, we report the biologic effects of a highly optimized BRS-3 agonist, (2S)-1,1,1-trifluoro-2-[4-(1H-pyrazol-1-yl)phenyl]-3-(4-{[1-(trifluoromethyl)cyclopropyl]methyl}-1H-imidazol-2-yl)propan-2-ol (MK-5046). Single oral doses of MK-5046 inhibited 2-h and overnight food intake and increased fasting metabolic rate in wild-type but not Brs3 knockout mice. Upon dosing for 14 days, MK-5046 at 25 mg · kg(-1) · day(-1) reduced body weight of diet-induced obese mouse by 9% compared with vehicle-dosed controls. In mice, 50% brain receptor occupancy was achieved at a plasma concentration of 0.34 ± 0.23 µM. With chronic dosing, effects on metabolic rate, rather than food intake, seem to be the predominant mechanism for weight reduction by MK-5046. The compound also effectively reduced body weight in rats and caused modest increases in body temperature, heart rate, and blood pressure. These latter effects on temperature, heart rate, and blood pressure were transient in nature and desensitized with continued dosing. MK-5046 is the first BRS-3 agonist with properties suitable for use in larger mammals. In dogs, MK-5046 treatment produced statistically significant and persistent weight loss, which was initially accompanied by increases in body temperature and heart rate that abated with continued dosing. Our results demonstrate antiobesity efficacy for MK-5046 in rodents and dogs and further support BRS-3 agonism as a new approach to the treatment of obesity.

Body temperature as a mouse pharmacodynamic response to bombesin receptor subtype-3 agonists and other potential obesity treatments.

Treatment of rodents with a bombesin receptor subtype-3 (BRS-3) agonist reduces food intake and increases fasting metabolic rate, causing weight loss with continued treatment. In small mammals, core body temperature (T(b)) is regulated in part by nutritional status, with a reduced T(b) during fasting. We report that fed Brs3 knockout mice have a lower T(b), which is discordant with their nutritional status. Treatment of wild-type mice with a BRS-3 agonist increased T(b), more so when the baseline T(b) was reduced such as by fasting or during the inactive phase of the light cycle. With repeated BRS-3 agonist dosing, the T(b) increase attenuated despite continued weight loss efficacy. The increase in T(b) was not prevented by inhibitors of prostaglandin E (PGE) production but was partially reduced by a ß-adrenergic blocker. These results demonstrate that BRS-3 has a role in body temperature regulation, presumably secondary to its effect on energy metabolism, including effects on sympathetic tone. By making use of this phenomenon, the reversal of the fasting T(b) reduction was developed into a sensitive single-dose pharmacodynamic assay for BRS-3 agonism and other antiobesity compounds acting by various mechanisms, including sibutramine, cannabinoid-1, and melanin-concentrating hormone-1 receptor blockers, and melanocortin, ß3-adrenergic, and cholecystokinin-1 receptor agonists. These drugs increased both the fasted T(b) and the fasted, resting metabolic rates. The T(b) assay is a robust, information-rich assay that is simpler and has a greater throughput than measuring metabolic rate and is a practical, effective tool for drug discovery.

Synthesis and cannabinoid-1 receptor binding affinity of conformationally constrained analogs of taranabant.

The design, synthesis, and binding activity of ring constrained analogs of the acyclic cannabinoid-1 receptor (CB1R) inverse agonist taranabant 1 are described. The initial inspiration for these taranabant derivatives was its conformation 1a, determined by (1)H NMR, X-ray, and molecular modeling. The constrained analogs were all much less potent than their acyclic parent structure. The results obtained are discussed in the context of a predicted binding of 1 to a homology model of CB1R.

Synthesis of 7-benzyl-5-(piperidin-1-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[3,4-c][2,7]naphthyridin-1-ylamine and its analogs as bombesin receptor subtype-3 agonists.

The original structure of a high-throughput screening hit obtained from an external vendor was revised based on multiple NMR studies. The active compound was re-synthesized via a novel route and its structure and biological activity as a BRS-3 agonist were unambiguously confirmed. Multi-gram quantities of the hit were prepared for pharmacokinetic and efficacy studies. The synthetic strategy allowed for the preparation of multiple analogs for SAR exploration.

Synthesis and SAR of heterocyclic carboxylic acid isosteres based on 2-biarylethylimidazole as bombesin receptor subtype-3 (BRS-3) agonists for the treatment of obesity.

SAR around non-peptidic potent bombesin receptor subtype-3 (BRS-3) agonist lead 2 is presented. Attempts to replace the carboxylic acid with heterocyclic isosteres to improve oral bioavailability and brain penetration are described.

Synthesis and SAR of derivatives based on 2-biarylethylimidazole as bombesin receptor subtype-3 (BRS-3) agonists for the treatment of obesity.

This Letter describes a series of potent and selective BRS-3 agonists containing a biarylethylimidazole pharmacophore. Extensive SAR studies were carried out with different aryl substitutions. This work led to the identification of a compound 2-{2-[4-(pyridin-2-yl)phenyl]ethyl}-5-(2,2-dimethylbutyl)-1H-imidazole 9 with excellent binding affinity (IC(50)=18 nM, hBRS-3) and functional agonist activity (EC(50)=47 nM, 99% activation). After oral administration, compound 9 had sufficient exposure in diet induced obese mice to demonstrate efficacy in lowering food intake and body weight via BRS-3 activation.

Discovery of substituted biphenyl imidazoles as potent, bioavailable bombesin receptor subtype-3 agonists.

We report SAR studies on a novel non-peptidic bombesin receptor subtype-3 (BRS-3) agonist lead series derived from high-throughput screening hit RY-337. This effort led to the discovery of compound 22e with significantly improved potency at both rodent and human BRS-3.

Regulation of energy homeostasis by bombesin receptor subtype-3: selective receptor agonists for the treatment of obesity.

Bombesin receptor subtype 3 (BRS-3) is a G protein coupled receptor whose natural ligand is unknown. We developed potent, selective agonist (Bag-1, Bag-2) and antagonist (Bantag-1) ligands to explore BRS-3 function. BRS-3-binding sites were identified in the hypothalamus, caudal brainstem, and several midbrain nuclei that harbor monoaminergic cell bodies. Antagonist administration increased food intake and body weight, whereas agonists increased metabolic rate and reduced food intake and body weight. Prolonged high levels of receptor occupancy increased weight loss, suggesting a lack of tachyphylaxis. BRS-3 agonist effectiveness was absent in Brs3(-/Y) (BRS-3 null) mice but was maintained in Npy(-/-)Agrp(-/-), Mc4r(-/-), Cnr1(-/-), and Lepr(db/db) mice. In addition, Brs3(-/Y) mice lost weight upon treatment with either a MC4R agonist or a CB1R inverse agonist. These results demonstrate that BRS-3 has a role in energy homeostasis that complements several well-known pathways and that BRS-3 agonists represent a potential approach to the treatment of obesity.

Factors influencing the interconversion of a new class of dibenzodiazepine sulfonamide atropisomers.

A novel family of atropisomers based on a conformationally constrained seven membered ring system is investigated using a combination of preparative chiral chromatography, circular dichroism, and other analytical techniques. The influence of structure on the rate of atropisomer interconversion was explored with a series of analogs showing a range of interconversion rates ranging from very fast (undetectable on the HPLC timescale) to very slow (half life of many days).

Synthesis and evaluation of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-aminopropanamide as human cannabinoid-1 receptor (CB1R) inverse agonists.

Obesity is a chronic medical condition that is affecting large population throughout the world. CB1 as a target for treatment of obesity has been under intensive studies. Taranabant was discovered and then developed by Merck as the 1st generation CB1R inverse agonist. Reported here is part of our effort on the 2nd generation of CB1R inverse agonist from the acyclic amide scaffold. We replaced the oxygen linker in taranabant with nitrogen and prepared a series of amino heterocyclic analogs through a divergent synthesis. Although in general, the amine linker gave reduced binding affinity, potent and selective CB1R inverse agonist was identified from the amino heterocycle series. Molecular modeling was applied to study the binding of the amino heterocycle series at CB1 binding site. The in vitro metabolism of representative members was studied and only trace glucuronidation was found. Thus, it suggests that the right hand side of the molecule may not be the appropriate site for glucuronidation.