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.

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.

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.

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.

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.

Tetrahydro anthranilic acid as a surrogate for anthranilic acid: application to the discovery of potent niacin receptor agonists.

The design, synthesis, and biological activity of a series of cycloalkene acid-based niacin receptor agonists are described. This led to the discovery that tetrahydro anthranilic acid is an excellent surrogate for anthranilic acid. Several compounds were identified that were potent against the niacin receptor, had enhanced cytochrome P450 selectivity against subtypes CYP2C8 and CYP2C9, and improved oral exposure in mice.

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.

Discovery of biaryl anthranilides as full agonists for the high affinity niacin receptor.

Biaryl anthranilides are reported as potent and selective full agonists for the high affinity niacin receptor GPR109A. The SAR presented outlines approaches to reduce serum shift and both CYPCYP2C8 and CYP2C9 liabilities, while improving PK and maintaining excellent receptor activity. Compound 2i exhibited good in vivo antilipolytic efficacy while providing a significantly improved therapeutic index over vasodilation (flushing) with respect to niacin in the mouse model.

A simple in vitro model to study the stability of acylglucuronides.

INTRODUCTION: Compounds containing the carboxylic functional group (e.g. non-steroidal anti-inflammatory drugs) can be metabolized to form acylglucuronides. Acylglucuronides are intrinsically reactive metabolites capable of undergoing hydrolysis, intra-molecular rearrangement, and formation of covalent adducts with proteins, which may generate potential toxicity. The purpose of this study is to develop an in vitro screening model to assess degradation kinetics of acylglucuronides. METHOD: Zomepirac, ibuprofen, gemfibrozil, and compounds A, B, C, and D were incubated in the presence of rat microsomal protein and uridine 5'-diphosphoglucuronic acid (UDPGA), followed by addition of human plasma to evaluate degradation kinetics of the acylglucuronides. As a comparison, authentic acylglucuronide standards of zomepirac, ibuprofen, gemfibrozil, and compounds A, B, C, and D were chemically synthesized and were evaluated for degradation kinetics. RESULTS: The results demonstrate that degradation half-life values of acylglucuronides of zomepirac, ibuprofen, gemfibrozil, and compounds A, B, C, and D determined by the in vitro formation/degradation model were in the same rank-order with those of the authentic acylglucuronide standards. DISCUSSION: For the seven compounds tested, the model placed the stability of the acylglucuronides formed in vitro in a rank-order consistent with authentic acylglucuronide standards. The method allows for a rapid assessment of the stability of acylglucuronides.

Pharmacological activation and genetic manipulation of cystathionine beta-synthase alter circulating levels of homocysteine and hydrogen sulfide in mice.

Hydrogen sulfide (H(2)S) is a recently discovered gasotransmitter found in mammalian tissues and blood. Treatment with H(2)S donor molecules has shown promising results in preclinical models of inflammatory and cardiovascular diseases. Augmentation of H(2)S levels thus holds promise as a novel therapeutic approach for treatment of disease in man. Cystathionine ß-synthase (CBS) has been shown to catalyze H(2)S production in vitro. CBS enzyme activity is allosterically regulated by the endogenous activator S-adenosyl methionine. This mode of regulation suggests the possibility for designing a small molecule activator of CBS to enhance H(2)S production. This hypothesis, however, has not been directly tested in vivo. We show here that CBS contributes significantly to endogenous H(2)S production in mice: adenovirus mediated over expression of CBS in the liver significantly increased circulating levels of H(2)S, whereas CBS deficiency resulted in reduced levels. We demonstrate that CBS enzyme from endogenous sources can be activated by S-adenosyl methionine to a greater extent compared to recombinant enzyme, suggesting greater potential for activation than previously anticipated. Importantly, we show that circulating H(2)S levels are increased by pharmacological activation of CBS in vivo; i.e. in the presence of the endogenous activator. Together, our data demonstrate that CBS activity partially regulates endogenous H(2)S in mice, and suggest that pharmacological activation of CBS is a promising approach for enhancing endogenous production of H(2)S for the treatment of cardiovascular and other diseases.

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.

Label-free high-throughput screening via mass spectrometry: a single cystathionine quantitative method for multiple applications.

Label-free mass spectrometric (MS) technologies are particularly useful for enzyme assay design for drug discovery screens. MS permits the selective detection of enzyme substrates or products in a wide range of biological matrices without need for derivatization, labeling, or capture technologies. As part of a cardiovascular drug discovery effort aimed at finding modulators of cystathionine beta-synthase (CBS), we used the RapidFire((R)) label-free high-throughput MS (HTMS) technology to develop a high-throughput screening (HTS) assay for CBS activity. The in vitro assay used HTMS to quantify the unlabeled product of the CBS reaction, cystathionine. Cystathionine HTMS analyses were carried out with a throughput of 7 s per sample and quantitation over a linear range of 80-10,000 nM. A compound library of 25,559 samples (or 80 384-well plates) was screened as singlets using the HTMS assay in a period of 8 days. With a hit rate of 0.32%, the actives showed a 90% confirmation rate. The in vitro assay was applied to secondary screens in more complex matrices with no additional analytical development. Our results show that the HTMS method was useful for screening samples containing serum, for cell-based assays, and for liver explants. The novel extension of the in vitro analytical method, without modification, to secondary assays resulted in a significant and advantageous economy of development time for the drug discovery project.

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.

Biomarker discovery in rat plasma for estrogen receptor-alpha action.

To support in vivo screening efforts for estrogen receptor (ER) subtype selective therapeutic agents, we initiated work to discover surrogate markers (biomarkers) in blood plasma that would change in response to ER subtype-specific action. We used a proteomic approach employing strong anion exchange chromatography (SAX), PAGE, and MS to identify potential plasma markers for selective ER-alpha action. The methodology was used to compare blood from vehicle-treated rats to blood from rats treated with either 17beta-estradiol (an ER-alpha/ER-beta agonist) or compound 1 (17alpha-ethynyl-[3,2-c]pyrazolo-19-nor-4-androstene-17beta-ol, an ER-alpha-selective agonist). Blood samples were first fractionated by SAX to separate fractions containing dominant common plasma proteins from fractions enriched for less-abundant plasma proteins. 1-D PAGE analysis of fractions depleted of dominant plasma proteins revealed treatment-specific changes in protein profiles. Protein bands that changed reproducibly in response to ER-alpha action were excised from the gel, separated by capillary LC, and identified by microspray ESI-MS. Using this method, the plasma levels of two proteins, transthyretin and apolipoprotein E, were shown to decrease in response to ER-alpha agonism. The method lacked the sensitivity to identify the known, 1000-fold less-abundant, estrogenic marker prolactin (PRL). However, using a commercial RIA and immunoblots, we showed that PRL levels increase significantly in response to treatment with the ER-alpha selective agonist, compound 1.