Anthony V. Azzara

Acylation of Acylglycerols by Acyl Coenzyme A:Diacylglycerol Acyltransferase 1 (DGAT1) FUNCTIONAL IMPORTANCE OF DGAT1 IN THE INTESTINAL FAT ABSORPTION

Acyl coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) is one of the four intestinal membrane bound acyltransferases implicated in dietary fat absorption. Recently, it was found that, in addition to acylating diacylglycerol (DAG), DGAT1 also possesses robust enzymatic activity for acylating monoacylglycerol (MAG) (Yen, C. L., Monetti, M., Burri, B. J., and Farese, R. V., Jr. (2005) J. Lipid Res. 46, 1502–1511). In the current paper, we have conducted a detailed characterization of this reaction in test tube, intact cell culture, and animal models. Enzymatically, we found that triacylglycerol (TAG) synthesis from MAG by DGAT1 does not behave according to classic Michaelis-Menten kinetics. At low concentrations of 2-MAG (<50 μm), the major acylation product by DGAT1 was TAG; however, increased concentrations of 2-MAG (50–200 μm) resulted in decreased TAG formation. This unique product/substrate relationship is similar to MGAT3 but distinct from DGAT2 and MGAT2. We have also found that XP620 is an inhibitor that selectively inhibits the acylation of MAG by DGAT1 (IC50 of human DGAT1: 16.6 ± 4.0 nm (MAG as substrate) and 1499 ± 318 nm (DAG as substrate); IC50 values of human DGAT2, MGAT2, and MGAT3 are >30,000 nm). Using this pharmacological tool, we have shown that ∼76 and ∼89% of the in vitro TAG synthesis initiated from MAG is mediated by DGAT1 in Caco-2 cell and rat intestinal mucosal membranes, respectively. When applied to intact cultured cells, XP620 substantially decreased but did not abolish apoB secretion in differentiated Caco-2 cells. It also decreased TAG and DAG syntheses in primary enterocytes. Last, when delivered orally to rats, XP620 decreased absorption of orally administered lipids by ∼50%. Based on these data, we conclude that the acylation of acylglycerols by DGAT1 is important for dietary fat absorption in the intestine.

Testing the diet-breadth trade-off hypothesis: differential regulation of novel plant secondary compounds by a specialist and a generalist herbivore

Specialist herbivores are predicted to have evolved biotransformation pathways that can process large doses of secondary compounds from the plant species on which they specialize. It is hypothesized that this physiological specialization results in a trade-off such that specialists may be limited in ability to ingest novel plant secondary compounds (PSCs). In contrast, the generalist foraging strategy requires that herbivores alternate consumption of plant species and PSC types to reduce the possibility of over-ingestion of any particular PSC. The ability to behaviorally regulate is a key component of this strategy. These ideas underpin the prediction that in the face of novel PSCs, generalists should be better able to maintain body mass and avoid toxic consequences compared to specialists. We explored these predictions by comparing the feeding behavior of two herbivorous rodents: a juniper specialist, Neotoma stephensi, and a generalist, Neotoma albigula, fed diets with increasing concentrations of phenolic resin extracted from the creosote bush (Larrea tridentata), which produces a suite of PSCs novel to both species. The specialist lost more mass than the generalist during the 15-day trial. In addition, although the specialist and generalist both regulated phenolic resin intake by reducing meal size while on the highest resin concentration (4%), the generalist began to regulate intake on the 2% diet. The ability of the generalist to regulate intake at a lower PSC concentration may be the source of the generalist’s performance advantage over the specialist. These data provide evidence for the hypothesis that the specialist’s foraging strategy may result in behavioral as well as physiological trade-offs in the ability to consume novel PSCs.

Identification of a nonbasic melanin hormone receptor 1 antagonist as an antiobesity clinical candidate.

Identification of MCHR1 antagonists with a preclinical safety profile to support clinical evaluation as antiobesity agents has been a challenge. Our finding that a basic moiety is not required for MCHR1 antagonists to achieve high affinity allowed us to explore structures less prone to off-target activities such as hERG inhibition. We report the SAR evolution of hydroxylated thienopyrimidinone ethers culminating in the identification of 27 (BMS-819881), which entered obesity clinical trials as the phosphate ester prodrug 35 (BMS-830216).

Reductions in log P improved protein binding and clearance predictions enabling the prospective design of cannabinoid receptor (CB1) antagonists with desired pharmacokinetic properties.

Several strategies have been employed to reduce the long in vivo half-life of our lead CB1 antagonist, triazolopyridazinone 3, to differentiate the pharmacokinetic profile versus the lead clinical compounds. An in vitro and in vivo clearance data set revealed a lack of correlation; however, when compounds with <5% free fraction were excluded, a more predictable correlation was observed. Compounds with log P between 3 and 4 were likely to have significant free fraction, so we designed compounds in this range to give more predictable clearance values. This strategy produced compounds with desirable in vivo half-lives, ultimately leading to the discovery of compound 46. The progression of compound 46 was halted due to the contemporaneous marketing and clinical withdrawal of other centrally acting CB1 antagonists; however, the design strategy successfully delivered a potent CB1 antagonist with the desired pharmacokinetic properties and a clean off-target profile.

Roles for central leptin receptors in the control of meal size

The adiposity hormone leptin has been implicated in the regulation of behavioral and metabolic controls of body weight. Leptin receptors are found in multiple peripheral and central tissues, particularly within hypothalamic and brainstem neuronal populations. Central leptinergic signaling acts as an indirect control to modulate the feeding inhibitory potency of the direct controls of meal size. Mouse models of neuronal leptin loss and gain of function have helped to identify and characterize how central leptin contributes to the central control of food intake.

Non-basic azolotriazinone MCHR1 antagonists for the treatment of obesity: An empirical brain-exposures-driven candidate selection for in vivo efficacy studies.

Non-basic azolotriazinones were explored using an empirical free brain exposures-driven approach to identify potent MCHR1 antagonists for evaluation in in vivo efficacy studies. An optimized lead from this series, 1j (rMCHR1 Ki=1.8 nM), demonstrated a 6.9% reduction in weight gain relative to vehicle in a rat model at 30 mg/kg after 4 days of once-daily oral treatment as a glycine prodrug. Despite a promising efficacy profile, an assessment of the biliary toxicity risk of this compound rendered this compound non-progressible.

Dihydropyrrolopyrazol-6-one MCHR1 antagonists for the treatment of obesity: Insights on in vivo efficacy from a novel FLIPR assay setup

Our investigation of the structure-activity and structure-liability relationships for dihydropyrrolopyrazol-6-one MCHR1 antagonists revealed that off-rate characteristics, inferred from potencies in a FLIPR assay following a 2 h incubation, can impact in vivo efficacy. The in vitro and exposure profiles of dihydropyrrolopyrazol-6-ones 1b and 1e were comparable to that of the thienopyrimidinone counterparts 41 and 43 except for a much faster MCHR1 apparent off-rate. The greatly diminished dihydropyrrolopyrazol-6-one anti-obesity response may be the consequence of this rapid off-rate.

An Old Dog Learns a New Trick: Regulation of Peripheral Glucose Homeostasis by the Serotonin (5-HT)2C Receptor

The American Diabetes Association has determined that diabetes costs the United States more than

Utilization of an Active Site Mutant Receptor for the Identification of Potent and Selective Atypical 5-HT2C Receptor Agonists

170 billion per year in associated health care costs (1). Even more discouraging is the fact that the percentage of individuals diagnosed with type II diabetes continues to climb both in the U.S. and worldwide. Although numerous medications are prescribed to treat hyperglycemia, finding effective treatments for diabetes and the resulting microvascularrelated complications remains a challenge. Thus discovery of new modalities by which to treat this condition is of the utmost importance. Diabetes begins with a loss of insulin sensitivity in white adipose tissue and skeletal muscle. Initially, the body compensates for the decrease in insulin-mediated signaling by enlarging the beta cells (found in the Islets of Langerhans in the pancreas), resulting in increased insulin release to maintain euglycemia (2). Eventually, however, the beta cells begin to fail, leading to a loss of insulin production. The lack of insulin results in elevated plasma glucose concentrations, which if sustained, leads to damage of the microvasculature of various internal organs (e.g., kidneys and eyes) and the peripheral aspects of the limbs, including the feet. Current therapies for diabetes include agents that enhance insulin sensitivity, such as metformin and the thiazolindinediones; elevators of insulin release, such as the sulfonylureas and metiglinide; and inhibitors of carbohydrate absorption, such as acarbose (3). More recently characterized agents mimic the role (and enhance the activity) of the incretin hormone glucagon-like peptide-1 (GLP-1) (Box 1). Such mimetics include the dipeptylpeptidase inhibitor sitigliptin, which prolongs the half-life of endogenous GLP-1, and the non-hydrolyzable GLP-1 analog exendin-4. GLP-1 is released in a glucose-dependent manner from the L cells of the gut, enters the circulation, evokes GLP-1 receptor–mediated release of insulin from beta cells, inhibits glucagon release, and reduces gastric motility (4). Serotonin (5-HT)-mediated neural networks––and the 5-HT 2C receptor (5-HT 2C R) in particular––have long been implicated in the control of food intake and energy homeostasis. Evidence for this comes from both pharmacological and genetic experiments. Fenfluramine, a stimulator of serotonin release and an inhibitor of serotonin reuptake, was marketed as a weight loss agent from 1973 until 1997 (5), when it and its more potent enantiomer dexfenfluramine were removed from the market because of their cardiac side effects, which were likely due to activity at the 5-HT 2B R (6). These weight loss effects are 5-HT 2C R–mediated, as is demonstrated by the reduction in efficacy of m-chlorophenylpiperzine (mCPP) and fenfluramine when administered to 5-HT 2C knockout (KO) mice (7, 8). Thus, highly selective 5-HT 2C receptor agonists have been extensively studied over the last decade as a drug development target for obesity and more recently as a possible treatment for schizophrenia. Two compounds, lorcaserin and vabicaserin, developed for the treatment of obesity and schizophrenia, respectively, have entered late-stage trials and might reach the marketplace in the next two to five years. New research by Zhou et al. implies that these and other 5-HT 2C R agonists may have utility in the treatment of type II diabetes (9). If true, these findings would indicate that a distinctly neuronal mechanism may be an effective treatment option for regulating plasma glucose concentrations. Zhou et al. identify 5-HT 2C R agonists as possibly useful agents for type II diabetes and demonstrate that the action of these compounds is dependent upon the melanocortin-4 receptor (MC4R) system (Figure 1). The hypothesis that 5-HT 2C R agonists may improve glucose tolerance was based on the finding that the

Characterization of CD28 null T cells in idiopathic pulmonary fibrosis

Agonism of the 5-HT2C receptor represents one of the most well-studied and clinically proven mechanisms for pharmacological weight reduction. Selectivity over the closely related 5-HT2A and 5-HT2B receptors is critical as their activation has been shown to lead to undesirable side effects and major safety concerns. In this communication, we report the development of a new screening paradigm that utilizes an active site mutant D134A (D3.32) 5-HT2C receptor to identify atypical agonist structures. We additionally report the discovery and optimization of a novel class of nonbasic heterocyclic amide agonists of 5-HT2C. SAR investigations around the screening hits provided a diverse set of potent agonists at 5-HT2C with high selectivity over the related 5-HT2A and 5-HT2B receptor subtypes. Further optimization through replacement of the amide with a variety of five- and six-membered heterocycles led to the identification of 6-(1-ethyl-3-(quinolin-8-yl)-1H-pyrazol-5-yl)pyridazin-3-amine (69). Oral administration of 69 to rats reduced food intake in an ad libitum feeding model, which could be completely reversed by a selective 5-HT2C antagonist.