Nicotinic acid and DP1 blockade: studies in mouse models of atherosclerosis.

The use of nicotinic acid to treat dyslipidemia is limited by induction of a "flushing" response, mediated in part by the interaction of prostaglandin D(2) (PGD(2)) with its G-protein coupled receptor, DP1 (Ptgdr). The impact of DP1 blockade (genetic or pharmacologic) was assessed in experimental murine models of atherosclerosis. In Ptgdr(-/-)ApoE(-/-) mice versus ApoE(-/-) mice, both fed a high-fat diet, aortic cholesterol content was modestly higher (1.3- to 1.5-fold, P < 0.05) in Ptgdr(-/-)ApoE(-/-) mice at 16 and 24 weeks of age, but not at 32 weeks. In multiple ApoE(-/-) mouse studies, a DP1-specific antagonist, L-655, generally had a neutral to beneficial effect on aortic lipids in the presence or absence of nicotinic acid treatment. In a separate study, a modest increase in some atherosclerotic measures was observed with L-655 treatment in Ldlr(-/-) mice fed a high-fat diet for 8 weeks; however, this effect was not sustained for 16 or 24 weeks. In the same study, treatment with nicotinic acid alone generally decreased plasma and/or aortic lipids, and addition of L-655 did not negate those beneficial effects. These studies demonstrate that inhibition of DP1, with or without nicotinic acid treatment, does not lead to consistent or sustained effects on plaque burden in mouse atherosclerotic models.

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.

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.

11beta-HSD1 inhibition ameliorates metabolic syndrome and prevents progression of atherosclerosis in mice.

The enzyme 11beta-hydroxysteroid dehydrogenase (HSD) type 1 converts inactive cortisone into active cortisol in cells, thereby raising the effective glucocorticoid (GC) tone above serum levels. We report that pharmacologic inhibition of 11beta-HSD1 has a therapeutic effect in mouse models of metabolic syndrome. Administration of a selective, potent 11beta-HSD1 inhibitor lowered body weight, insulin, fasting glucose, triglycerides, and cholesterol in diet-induced obese mice and lowered fasting glucose, insulin, glucagon, triglycerides, and free fatty acids, as well as improved glucose tolerance, in a mouse model of type 2 diabetes. Most importantly, inhibition of 11beta-HSD1 slowed plaque progression in a murine model of atherosclerosis, the key clinical sequela of metabolic syndrome. Mice with a targeted deletion of apolipoprotein E exhibited 84% less accumulation of aortic total cholesterol, as well as lower serum cholesterol and triglycerides, when treated with an 11beta-HSD1 inhibitor. These data provide the first evidence that pharmacologic inhibition of intracellular GC activation can effectively treat atherosclerosis, the key clinical consequence of metabolic syndrome, in addition to its salutary effect on multiple aspects of the metabolic syndrome itself.

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.

Discovery of pyrimidine carboxamides as potent and selective CCK1 receptor agonists.

A series of six-membered heterocycle carboxamides were synthesized and evaluated as cholecystokinin 1 receptor (CCK1R) agonists. A pyrimidine core proved to be the best heterocycle, and SAR studies resulted in the discovery of analog 5, a potent and structurally diverse CCK1R agonist.

Discovery of a piperazine urea based compound as a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist.

We report the discovery of piperazine urea based compound 1, a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist. Compound 1 shows anti-obesity efficacy without potentiating erectile activity in the rodent models.

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.

Furo[2,3-b]pyridine-based cannabinoid-1 receptor inverse agonists: synthesis and biological evaluation. Part 1.

The synthesis, SAR and binding affinities of cannabinoid-1 receptor (CB1R) inverse agonists based on furo[2,3-b]pyridine scaffolds are described. Food intake, mechanism specific efficacy, pharmacokinetic, and metabolic evaluation of several of these compounds indicate that they are effective orally active modulators of CB1R.

Dihydro-pyrano[2,3-b]pyridines and tetrahydro-1,8-naphthyridines as CB1 receptor inverse agonists: synthesis, SAR and biological evaluation.

Synthesis and structure-activity relationships of cannabinoid-1 receptor (CB1R) inverse agonists based on dihydro-pyrano[2,3-b] pyridine and tetrahydro-1,8-naphtyridine scaffolds are presented. Rat food intake and pharmacokinetic evaluation of 13g, 13i, 13k and 17a revealed these compounds to be highly efficacious orally active modulators of CB1R.

Discovery of a spiroindane based compound as a potent, selective, orally bioavailable melanocortin subtype-4 receptor agonist.

We report the design, synthesis and properties of spiroindane based compound 1, a potent, selective, orally bioavailable, non-peptide melanocortin subtype-4 receptor agonist. Compound 1 shows excellent erectogenic activity in the rodent models.

Discovery of highly potent and efficacious MC4R agonists with spiroindane N-Me-1,2,4-triazole privileged structures for the treatment of obesity.

We report an SAR study of MC4R analogs containing spiroindane heterocyclic privileged structures. Compound 26 with N-Me-1,2,4-triazole moiety possesses exceptional potency at MC4R and potent anti-obesity efficacy in a mouse model. However, the efficacy is not completely mediated through MC4R. Additional SAR studies led to the discovery of compound 32, which is more potent at MC4R. Compound 32 demonstrates MC4R mediated anti-obesity efficacy in rodent models.

Spiroindane based amides as potent and selective MC4R agonists for the treatment of obesity.

We report a series of potent and selective MC4R agonists based on spiroindane amide privileged structures for potential treatments of obesity. Among the synthetic methods used, Method C allows rapid synthesis of the analogs. The series of compounds can afford high potency on MC4R as well as good rodent pharmacokinetic profiles. Compound 1r (MK-0489) demonstrates MC4R mediated reduction of food intake and body weight in mouse models. Compound 1r is efficacious in 14-day diet-induced obese (DIO) rat models.

Discovery of potent, selective, and orally bioavailable 3H-spiro[isobenzofuran-1,4'-piperidine] based melanocortin subtype-4 receptor agonists.

Design, synthesis, and SAR of a series of 3H-spiro[isobenzofuran-1,4'-piperidine] based compounds as potent, selective and orally bioavailable melanocortin subtype-4 receptor (MC4R) agonists are disclosed.

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.

From Screening to Targeted Degradation: Strategies for the Discovery and Optimization of Small Molecule Ligands for PCSK9.

Proprotein convertase substilisin-like/kexin type 9 (PCSK9) is a serine protease involved in a protein-protein interaction with the low-density lipoprotein (LDL) receptor that has both human genetic and clinical validation. Blocking this protein-protein interaction prevents LDL receptor degradation and thereby decreases LDL cholesterol levels. Our pursuit of small-molecule direct binders for this difficult to drug PPI target utilized affinity selection/mass spectrometry, which identified one confirmed hit compound. An X-ray crystal structure revealed that this compound was binding in an unprecedented allosteric pocket located between the catalytic and C-terminal domain. Optimization of this initial hit, using two distinct strategies, led to compounds with high binding affinity to PCSK9. Direct target engagement was demonstrated in the cell lysate with a cellular thermal shift assay. Finally, ligand-induced protein degradation was shown with a proteasome recruiting tag attached to the high-affinity allosteric ligand for PCSK9.

Pyridopyrimidine based cannabinoid-1 receptor inverse agonists: Synthesis and biological evaluation.

The synthesis, SAR and binding affinities are described for cannabinoid-1 receptor (CB1R) specific inverse agonists based on pyridopyrimidine and heterotricyclic scaffolds. Food intake and pharmacokinetic evaluation of several of these compounds indicate that they are effective orally active modulators of CB1R.

Characterization of a novel and selective cannabinoid CB1 receptor inverse agonist, Imidazole 24b, in rodents.

We document in vitro and in vivo effects of a novel, selective cannabinoid CB(1) receptor inverse agonist, Imidazole 24b (5-(4-chlorophenyl)-N-cyclohexyl-4-(2,4-dichlorophenyl)-1-methyl-imidazole-2-carboxamide). The in vitro binding affinity of Imidazole 24b for recombinant human and rat CB(1) receptor is 4 and 10 nM, respectively. Imidazole 24b binds to human cannabinoid CB(2) receptor with an affinity of 297 nM; in vitro, it is a receptor inverse agonist at both cannabinoid CB(1) and CB(2) receptors as it causes a further increase of forskolin-induced cAMP increase. Oral administration of Imidazole 24b blocked CP-55940-induced hypothermia, demonstrating cannabinoid CB(1) receptor antagonist efficacy in vivo. Using ex vivo autoradiography, Imidazole 24b resulted in dose-dependent increases in brain cannabinoid CB(1) receptor occupancy (RO) at 2h post-dosing in rats, indicating that approximately 50% receptor occupancy is sufficient for attenuation of receptor agonist-induced hypothermia. Imidazole 24b administered to C57Bl/6 mice and to dietary-induced obese (DIO) Sprague-Dawley rats attenuated overnight food intake with a minimal effective dose of 10 mg/kg, p.o. Administration had no effect in cannabinoid CB(1) receptor-deficient mice. DIO rats were dosed orally with vehicle, Imidazole 24b (1, 3 or 10 mg/kg), or dexfenfluramine (3 mg/kg) for 2 weeks. At 3 mg/kg, Imidazole 24b reduced cumulative food intake, leading to a non-significant decrease in weight gain. Imidazole 24b at 10 mg/kg and dexfenfluramine treatment inhibited food intake and attenuated weight gain. These findings suggest that selective cannabinoid CB(1) receptor inverse agonists such as Imidazole 24b have potential for the treatment of obesity.

2-Substituted piperazine-derived imidazole carboxamides as potent and selective CCK1R agonists for the treatment of obesity.

The discovery and structure-activity relationship of 1,2-diarylimidazole piperazine carboxamides bearing polar side chains as potent and selective cholecystokinin 1 receptor (CCK1R) agonists are described. Optimization of this series resulted in the discovery of isopropyl carboxamide 40, a CCK1R agonist with sub-nanomolar functional and binding activity as well as excellent potency in a mouse overnight food intake reduction assay.

Discovery of imidazole carboxamides as potent and selective CCK1R agonists.

High-throughput screening revealed diaryl pyrazole 3 as a selective albeit modest cholecystokinin 1 receptor (CCK1R) agonist. SAR studies led to the discovery and optimization of a novel class of 1,2-diaryl imidazole carboxamides. Compound 44, which was profiled extensively, showed good in vivo mouse gallbladder emptying (mGBE) and lean mouse overnight food intake (ONFI) reduction activities.