5'-Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. Despite three decades of investigation, the biological roles of AMPK and its potential as a drug target remain incompletely understood, largely because of a lack of optimized pharmacological tools. We developed MK-8722, a potent, direct, allosteric activator of all 12 mammalian AMPK complexes. In rodents and rhesus monkeys, MK-8722-mediated AMPK activation in skeletal muscle induced robust, durable, insulin-independent glucose uptake and glycogen synthesis, with resultant improvements in glycemia and no evidence of hypoglycemia. These effects translated across species, including diabetic rhesus monkeys, but manifested with concomitant cardiac hypertrophy and increased cardiac glycogen without apparent functional sequelae.

Three closely related fungal metabolites, The mammalian isoprenoid pathway not only produces sterols but also produces dolichol, ubiquinone, the farnesyl group of heme A, the farnesyl and geranylgeranyl groups of prenylated proteins, and the isopentenyl side chain of isopentenyl adenine. The pathways for the synthesis of these other isoprenoids diverge from the synthesis ofcholesterol either at or before the farnesyl diphosphate (FPP) branch point. Thus, squalene synthase, which catalyzes the reductive dimerization of 2 mol of FPP to 1 mol of squalene (2, 3), is the first committed step in sterol synthesis. A specific inhibitor of squalene synthase should serve to inhibit cholesterol synthesis and not adversely affect the synthesis of other isoprenoids. FPP, the substrate for squalene synthase, is water soluble and may be readily metabolized (4). Thus, squalene synthase offers a potential target for the safe and specific inhibition of cholesterol synthesis.In this report we describe the isolation, structure, physical characterization, and biological properties of three structurally similar fungal metabolites that are potent inhibitors of squalene synthase. These metabolites, zaragozic acid A (5-7), zaragozic acid B (8, 9), and zaragozic acid C (10-12), had been reported previously only in the patent literature; however, during the review process of this manuscript, three manuscripts (13-15) appeared on the squalestatins: squalestatin I is identical with zaragozic acid A, squalestatin II is des-4'-acetylzaragozic acid A, and squalestatin III is des-6-acylzaragozic acid A. This class of squalene synthase inhibitors has potential utility as cholesterol-lowering agents. MATERIALS AND METHODSZaragozic Acid A, Cultures, and Media. An unidentified sterile fungal culture, ATCC 20986, isolated from a water sample taken from the Jalon river in Zaragoza, Spain (hence the name zaragozic acids), was used to produce zaragozic acid A. The culture was maintained at 25°C on medium B agar slants composed of 4 g of yeast extract, 10 g of malt extract, 4 g of dextrose, and 20 g of agar per liter at pH 7.0.Zaragozic acid A was produced in a two-tiered fermentation process consisting of mycelial growth and development in medium A of ref. 1 and product formation in medium C. Medium C contained 5 g of malt extract, 1 g of peptone, 15 g of dextrose, 1 g of KH2PO4, and 0.5 g of MgSO4 7H20 per liter. Fermentations consisted of mycelial growth in medium A for 72 hr at 250C with agitation, followed by inoculation (5-10%) of medium C. Maximum product was obtained from 14-day agitated fermentations at 250C.Isolation of Zaragozic Acid A. To isolate zaragozic acid A, 23 liters of harvested broth was filtered through Celite, and the mycelial cake was extracted twice with 7 liters of 50%o aqueous methanol. The filtrate was combined with the extracts, diluted with water to a final composition of 25% methanol, and adsorbed on a 1.5-liter column of Mitsubishi HP-20 resin. After a column wash with 6 liters of4:6 (vol/vol) methanol/water, crud...

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Structural modifications requiring novel synthetic chemistry were made to the morpholine acetal human neurokinin-1 (hNK-1) receptor antagonist 4, and this resulted in the discovery of 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4-(3-ox o-1 ,2,4-triazol-5-yl)methyl morpholine (17). This modified compound is a potent, long-acting hNK-1 receptor antagonist as evidenced by its ability to displace [125I]Substance P from hNK-1 receptors stably expressed in CHO cells (IC50 = 0.09 +/- 0.06 nM) and by the measurement of the rates of association (k1 = 2.8 +/- 1.1 x 10(8) M-1 min-1) and dissociation (k-1 = 0.0054 +/- 0.003 min-1) of 17 from hNK-1 expressed in Sf9 membranes which yields Kd = 19 +/- 12 pM and a t1/2 for receptor occupancy equal to 154 +/- 75 min. Inflammation in the guinea pig induced by a resiniferatoxin challenge (with NK-1 receptor activation mediating the subsequent increase in vascular permeability) is inhibited in a dose-dependent manner by the oral preadmininstration of 17 (IC50 (1 h) = 0.008 mg/kg; IC90 (24 h) = 1.8 mg/kg), indicating that this compound has good oral bioavailbility and peripheral duration of action. Central hNK-1 receptor stimulation is also inhibited by the systemic preadministration of 17 as shown by its ability to block an NK-1 agonist-induced foot tapping response in gerbils (IC50 (4 h) = 0.04 +/- 0.006 mg/kg; IC50 (24 h) = 0.33 +/- 0.017 mg/kg) and by its antiemetic actions in the ferret against cisplatin challenge. The activity of 17 at extended time points in these preclinical animal models sets it apart from earlier morpholine antagonists (such as 4), and the piperidine antagonists 2 and 3 and could prove to be an advantage in the treatment of chronic disorders related to the actions of Substance P. In part on the basis of these data, 17 has been identified as a potential clinical candidate for the treatment of peripheral pain, migraine, chemotherapy-induced emesis, and various psychiatric disorders.

Farnesyl diphosphate, the substrate for squalene synthase, accumulates in the presence of zaragozic acid A, a squalene synthase inhibitor. A possible metabolic fate for farnesyl diphosphate is its conversion to farnesol, then to farnesoic acid, and finally to farnesol-derived dicarboxylic acids (FDDCAs) which would then be excreted in the urine. Seven dicarboxylic acids were isolated by high performance liquid chromatography (HPLC) from urine of either rats or dogs treated with zaragozic acid A or rats fed farnesol. Their structures were determined by nuclear magnetic resonance analysis. Two 12-carbon, four 10-carbon, and one 7-carbon FDDCA were identified. The profile of urinary dicarboxylic acids from rats fed farnesol was virtually identical to that produced by treating with zaragozic acid A, establishing that these dicarboxylic acids are farnesolderived. By feeding [1-14 C]farnesol and comparing the mass of the dicarboxylic acids produced with the ultraviolet absorption of the HPLC peaks, a method to quantitate the ultraviolet-absorbing FDDCAs was devised. When rats were treated with zaragozic acid A, large amounts of FDDCAs were excreted in the urine. The high level of FDDCAs that were found suggests that their synthesis is the major metabolic fate for carbon diverted from cholesterol synthesis by a squalene synthase inhibitor. A metabolic pathway is proposed to explain the production of each of these FDDCAs.Squalene synthase is an attractive target for the development of a cholesterol synthesis inhibitor that could serve as a cholesterol lowering agent. Cholesterol synthesis inhibitors, such as lovastatin (1), a 3-hydroxy-3-methylglutaryl-coenzyme A inhibitor, are effective cholesterol-lowering agents in man and/or animals. Squalene synthase catalyzes the first committed step in cholesterol synthesis, and selective inhibition of this enzyme should result in inhibition of cholesterol synthesis without affecting the synthesis of other isoprenoids such as dolichol, ubiquinone, and the prenylated proteins. A novel class of fungal metabolites, known as zaragozic acids, has been recently discovered and characterized as potent inhibitors of squalene synthase (2-8). The zaragozic acids are subnanomolar inhibitors of squalene synthase in vitro, they inhibit cholesterol synthesis from acetate or mevalonate in cell culture and in animal models, and also have been shown to lower plasma cholesterol when administered orally in certain animal species (4, 5,8). Other classes of squalene synthase inhibitors have also been discovered (for a review, see Ref. 9).An important question to be answered for these compounds is the metabolic effect of inhibition of squalene synthase. The squalene synthase reaction consists of the reductive dimerization of two molecules of farnesyl diphosphate (FPP) 1 to form a molecule of squalene (10). The primary consequence of inhibition of this reaction would be an accumulation of FPP. Thus, the metabolic fate of FPP in the presence of a squalene synthase inhibitor is of interest. Previous wor...

The regioselective dibenzylphosphorylation of 2 followed by catalytic reduction in the presence of N-methyl-D-glucamine afforded 2-(S)-(1-(R)-(3, 5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluoro)phenyl-4-(5-(2- phosphoryl-3-oxo-4H,-1,2,4-triazolo)methylmorpholine, bis(N-methyl-D-glucamine) salt, 11. Incubation of 11 in rat, dog, and human plasma and in human hepatic subcellular fractions in vitro indicated that conversion to 2 would be expected to occur in vivo most readily in humans during hepatic circulation. Conversion of 11 to 2 occurred rapidly in vivo in the rat and dog with the levels of 11 being undetectable within 5 min after 1 and 8 mg/kg doses iv in the rat and within 15 min after 0.5, 2, and 32 mg/kg doses iv in the dog. Compound 11 has a 10-fold lower affinity for the human NK-1 receptor as compared to 2, but it is functionally equivalent to 2 in preclinical models of NK-1-mediated inflammation in the guinea pig and cisplatin-induced emesis in the ferret, indicating that 11 acts as a prodrug of 2. Based in part on these data, 11 was identified as a novel, water-soluble prodrug of the clinical candidate 2 suitable for intravenous administration in humans.

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BackgroundCholesterol deposition in arterial wall drives atherosclerosis. The key goal of this study was to examine the relationship between plaque cholesterol content and patient characteristics that typically associate with disease state and lesion vulnerability. Quantitative assays for free cholesterol, cholesteryl ester, triglyceride, and protein markers in atherosclerotic plaque were established and applied to plaque samples from multiple patients and arterial beds (Carotid and peripheral arteries; 98 lesions in total).ResultsWe observed a lower cholesterol level in restenotic than primary peripheral plaque. We observed a trend toward a higher level in symptomatic than asymptomatic carotid plaque. Peripheral plaque from a group of well-managed diabetic patients displayed a weak trend of more free cholesterol deposition than plaque from non-diabetic patients. Plaque triglyceride content exhibited less difference in the same comparisons. We also measured cholesterol in multiple segments within one carotid plaque sample, and found that cholesterol content positively correlated with markers of plaque vulnerability, and negatively correlated with stability markers.ConclusionsOur results offer important biological validation of cholesterol as a key lipid marker for plaque severity. Results also suggest cholesterol is a more sensitive plaque marker than routine histological staining for neutral lipids.