Discovery and SAR of para-alkylthiophenoxyacetic acids as potent and selective PPARdelta agonists.

Synthesis and SAR of para-alkylthiophenoxyacetic acids is described. Achiral compounds 30, 31 and 32 were identified as potent and selective PPARdelta agonists.

Improvement of dyslipidemia, insulin sensitivity, and energy balance by a peroxisome proliferator-activated receptor alpha agonist.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the nuclear receptor family of ligand-activated transcription factors. It plays an important role in the regulation of genes involved in lipid metabolism and transport. Compound A is a potent and orally active PPARalpha agonist that activated both human and rat PPARalpha receptors. The compound induced the expression of genes involved in fatty acid metabolism in a rodent hepatoma cell line and in the liver of db/db mouse. The ability of compound A to stimulate fatty acid beta-oxidation was demonstrated in human hepatocytes and human skeletal muscle cells, which confirmed a functional activation of PPARalpha-mediated activities. Compound A was shown to be a more potent and efficacious antidyslipidemic agent in atherogenic rat and db/db mouse models as compared with fenofibrate. The increase in high-density lipoprotein cholesterol levels by compound A was at least partially due to an increase in serum apolipoprotein A-I protein concentrations in human PPARalpha transgenic mouse. The triglyceride-lowering effect was further confirmed in a higher species, obese dog models. In addition, compound A dose-dependently ameliorated hyperglycemia and hyperinsulinemia, and improved glucose tolerance in db/db mice. In a diet-induced obesity mouse model, compound A decreased body weight mainly by increasing energy expenditure and reducing fat deposition. In conclusion, the novel and potent PPARalpha agonist improves lipid profile, insulin sensitivity, and energy balance in animal models.

Synthesis and structure-activity relationships of thiadiazole-derivatives as potent and orally active peroxisome proliferator-activated receptors alpha/delta dual agonists.

Replacement of the methyl-thiazole moiety of GW501516 (a PPARdelta selective agonist) with [1,2,4]thiadiazole gave compound 21 which unexpectedly displayed submicromolar potency as a partial agonist at PPARalpha in addition to the high potency at PPARdelta. A structure-activity relationships study of 21 resulted in the identification of 40 as a potent and selective PPARalpha/delta dual agonist. Compound 40 and its close analogs represent a new series of PPARalpha/delta dual agonists. The high potency, high selectivity, significant gene induction, excellent PK profiles, low P450 inhibition or induction, and good in vivo efficacy in four animal models support 40 being selected as a pre-clinical study candidate, and may render 40 as a valuable pharmacological tool in elucidating the complex roles of PPARalpha/delta dual agonists, and the potential usage for the treatment of metabolic syndrome.

Design and synthesis of indane-ureido-thioisobutyric acids: A novel class of PPARalpha agonists.

A series of aminoindane derivatives were synthesized and shown to be potent PPARalpha agonists. The compounds were obtained as racemates in 12 steps, and tested for PPARalpha activation and PPARalpha mediated induction of the HD gene. SAR was developed by variation to the core structure as shown within. Oral bioavailability was demonstrated in a Sprague-Dawley rat, while efficacy to reduce plasma triglycerides and plasma glucose was demonstrated in db/db mice.

Synthesis and identification of [1,2,4]thiadiazole derivatives as a new series of potent and orally active dual agonists of peroxisome proliferator-activated receptors alpha and delta.

Cardiovascular disease is the most common cause of morbidity and mortality in developed nations. To effectively target dyslipidemia to reduce the risk of cardiovascular disease, it may be beneficial to activate the peroxisome proliferator-activated receptors (PPARs) PPARalpha and PPARdelta simultaneously through a single molecule. Replacement of the methylthiazole of 5 (the PPARdelta selective agonist) with [1,2,4]thiadiazole gave compound 13, which unexpectedly displayed submicromolar potency as a partial agonist at PPARalpha in addition to the high potency at PPARdelta. Optimization of 13 led to the identification of 24 as a potent and selective PPARalpha/delta dual agonist. Compound 24 and its close analogs represent a new series of PPARalpha/delta dual agonists. The high potency, significant gene induction, excellent PK profiles, and good in vivo efficacies in three animal models may render compound 24 as a valuable pharmacological tool in elucidating the complex roles of PPARalpha/delta dual agonists and as a potential treatment of the metabolic syndrome.

Discovery of para-alkylthiophenoxyacetic acids as a novel series of potent and selective PPARdelta agonists.

A novel series of potent and selective PPARdelta agonists, para-alkylthiophenoxyacetic acids, was identified. The synthesis and structure-activity relationships are described.

Human epidermoid A431 cells express functional nicotinic acid receptor HM74a.

Nicotinic acid (niacin) has been used clinically to manage dyslipidemia for many years. The molecular target of nicotinic acid was unknown until the recent revelation of human G-coupled receptor HM74a as the high affinity receptor for nicotinic acid. In searching for a cell line expressing endogenous human HM74a receptor, we have identified that the A431 cell line, a human epidermoid cell line, expresses a high level of HM74a receptor. An HM74a-specific real time PCR probe set was designed and the mRNA levels of HM74a in A431 and 32 other cultured cell lines were measured quantitatively. When the mRNA expression of HM74a in A431 cells was compared to that in human primary preadipocytes, adipocytes and adipose tissue, we found that the level in A431 was about 10- fold higher than that in adipocytes and adipose tissue. The ratio of HM74a:HM74 mRNA was measured quantitatively and it was determined to be 3:2 in A431 cells. The function of the HM74a receptor in A431 cells was evaluated for its ability to inhibit forskolin-induced cAMP production. Pertussis toxin treatment abolished the inhibition. Our data suggest that the A431 cell line may serve as a cellular model for further investigation of niacin/HM74a-mediated signal transduction in modulating metabolism. A431 cell line may also provide a valuable cell model to study prostaglandin production upon HM74a activation to improve our understanding of niacin/HM74a-mediated skin flushing.

Identification of human dopamine receptors agonists from Chinese herbs.

AIM: To find human dopamine receptors, especially D1-like receptor specific agonists from Chinese herbs as potential antihypertension drug leads. METHODS: Two D1-like receptor cell lines carrying a beta-lactamase reporter gene, and a D2 receptor cell line coexpressing a promiscuous G protein G15 were constructed using HEK293 cells. A natural compound library made from fractionated samples of herbal extracts was used for high-throughput screening (HTS) against one of the cell lines, HEK/D5R/CRE-blax. The interested hits were evaluated for their activities against various dopamine receptors. RESULTS: Fourteen hits were identified from primary screening, of which 2 of the better hit samples, HD0522 and HD0059, were selected for further material and activity analysis, and to obtain 2 compounds that appeared as 2 single peaks in HPLC, HD0522H01 and HD0059H01. HD0059H01 could activate D1, D2, and D5 receptors, with EC(50 ) values of 2.28 microg/mL, 0.85 microg/mL, and 1.41 microg/mL, respectively. HD0522H01 could only activate D1R and D5R with EC(50 ) values of 2.95 microg/mL and 8.38 microg/mL. CONCLUSION: We established cellbased assays for 3 different human dopamine receptors and identified specific agonists HD0522H01 and HD0059H01 through HTS. The specific agonist to D1-like receptors, HD0522H01, may become a new natural product-based drug lead for antihypertension treatment.

Enhancement of arachidonic acid signaling pathway by nicotinic acid receptor HM74A.

HM74A is a G protein-coupled receptor for nicotinic acid (niacin), which has been used clinically to treat dyslipidemia for decades. The molecular mechanisms whereby niacin exerts its pleiotropic effects on lipid metabolism remain largely unknown. In addition, the most common side effect in niacin therapy is skin flushing that is caused by prostaglandin release, suggesting that the phospholipase A(2) (PLA(2))/arachidonic acid (AA) pathway is involved. Various eicosanoids have been shown to activate peroxisome-proliferator activated receptors (PPAR) that play a diverse array of roles in lipid metabolism. To further elucidate the potential roles of HM74A in mediating the therapeutic effects and/or side effects of niacin, we sought to explore the signaling events upon HM74A activation. Here we demonstrated that HM74A synergistically enhanced UTP- and bradykinin-mediated AA release in a pertussis toxin-sensitive manner in A431 cells. Activation of HM74A also led to Ca(2+)-mobilization and enhanced bradykinin-promoted Ca(2+)-mobilization through Gi protein. While HM74A increased ERK1/2 activation by the bradykinin receptor, it had no effects on UTP-promoted ERK1/2 activation.Furthermore, UTP- and bradykinin-mediated AA release was significantly decreased in the presence of both MAPK kinase inhibitor PD 098059 and PKC inhibitor GF 109203X. However, the synergistic effects of HM74A were not dramatically affected by co-treatment with both inhibitors, indicating the cross-talk occurred at the receptor level. Finally, stimulation of A431 cells transiently transfected with PPRE-luciferase with AA significantly induced luciferase activity, mimicking the effects of PPARgamma agonist rosiglitazone, suggesting that alteration of AA signaling pathway can regulate gene expression via endogenous PPARs.

Human cardiomyocytes express high level of Na+/glucose cotransporter 1 (SGLT1).

We have quantitatively measured gene expression for the sodium-dependent glucose cotransporters 1 and 2 (SGLT1 and SGLT2) in 23 human tissues using the method of real time PCR. As predicted, our results revealed that the expression of SGLT1 was very high in the small intestine (1.2E + 6 molecules/microg total RNA) relative to that in the kidney (3E + 4 molecules/microg total RNA). Surprisingly, we observed that the expression of SGLT1 in human heart was unexpectedly high (3.4E + 5 molecules/microg total RNA), approximately 10-fold higher than that observed in kidney tissue. DNA sequencing confirmed that the PCR amplified fragment was indeed the human SGLT1 gene. Moreover, in situ hybridization studies using a digoxigenin (DIG)-labeled antisense cRNA probe corresponding to human SGLT1 cDNA confirm that human cardiomyocytes express SGLT1 mRNA. In contrast, the expression of SGLT2 in human tissues appears to be ubiquitous, with levels ranging from 6.7E + 4 molecules/microg total RNA (in skeletal muscle) to 3.2E + 6 molecules/microg total RNA (in kidney), levels 10-100-fold higher than the expression of SGLT1 in the same tissues. Our finding that human cardiomyocytes express high levels of SGLT1 RNA suggests that SGLT1 may have a functional role in cardiac glucose transport. Since several SGLT inhibitors are currently in development as potential anti-diabetic agents, it may be important to assess the functional consequences of inhibition of SGLT1 in the heart.

Characterization of adenosine A1 receptors in human proximal tubule epithelial (HK-2) cells.

BACKGROUND: Adenosine A1 receptors (A1ARs) modulate various aspects of renal functions, such as hormone release, hemodynamics and tubular absorption. Here we set up to demonstrate the expression of A1ARs in an immortalized cell line (HK-2) derived from normal adult human proximal tubule. We also examined the mechanism whereby A1ARs signal in HK-2 cells and their potential role in renal physiology such as sodium-dependent phosphate transport. METHODS: Ligand binding assay of A1ARs was performed using plasma membranes of HK-2 cells and a selective high-affinity A1AR radioligand [3H]DPCPX. HK-2 cells in 96-well plates were treated with various agents (forskolin, adenosine receptor agonists, and antagonists) to activate or inhibit adenylate cyclase. Intracellular cyclic AMP accumulation was measured using cAMP flashplates. mRNA levels of adenosine receptors in HK-2 cells was determined by real-time PCR technique. Sodium-dependent phosphate transport across cell membrane was measured after 15-minute incubation of phosphorus-33 in transport buffer with HK-2 cells at room temperature. RESULTS: In HK-2 cells, A1ARs were expressed at a density of 211 +/- 74 fmol/mg membrane proteins. [3H]DPCPX bound to A1ARs on HK-2 cell membranes with Kd of 8.3 +/- 2.2 nM. Activation of A1ARs inhibited isoproterenol-stimulated adenylate cyclase activity through pertussis toxin-sensitive Gi protein in HK-2 cells. Coexpression of adenosine A2a receptors at a seemingly lower level than A1ARs was revealed by synergistically activating adenylate cyclase with forskolin. Real-time RT-PCR further demonstrated the expression of both A1AR and A2aAR in HK-2 cells. Sodium-dependent phosphate transport was augmented by activation of A1ARs in HK-2 cells. CONCLUSION: A1ARs are expressed in human proximal tubule epithelial (HK-2) cells and modulate sodium-dependent phosphate transport.