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1.
Cell Metab ; 25(6): 1348-1361.e8, 2017 Jun 06.
Article in English | MEDLINE | ID: mdl-28591637

ABSTRACT

Glucagon supports glucose homeostasis by stimulating hepatic gluconeogenesis, in part by promoting the uptake and conversion of amino acids into gluconeogenic precursors. Genetic disruption or pharmacologic inhibition of glucagon signaling results in elevated plasma amino acids and compensatory glucagon hypersecretion involving expansion of pancreatic α cell mass. Recent findings indicate that hyperaminoacidemia triggers pancreatic α cell proliferation via an mTOR-dependent pathway. We confirm and extend these findings by demonstrating that glucagon pathway blockade selectively increases expression of the sodium-coupled neutral amino acid transporter Slc38a5 in a subset of highly proliferative α cells and that Slc38a5 controls the pancreatic response to glucagon pathway blockade; most notably, mice deficient in Slc38a5 exhibit markedly decreased α cell hyperplasia to glucagon pathway blockade-induced hyperaminoacidemia. These results show that Slc38a5 is a key component of the feedback circuit between glucagon receptor signaling in the liver and amino-acid-dependent regulation of pancreatic α cell mass in mice.


Subject(s)
Amino Acid Transport Systems, Neutral/metabolism , Glucagon-Secreting Cells/metabolism , Glucagon/metabolism , Receptors, Glucagon/metabolism , Signal Transduction , Amino Acid Transport Systems, Neutral/genetics , Animals , Glucagon/genetics , Glucagon-Secreting Cells/pathology , Hyperplasia , Male , Mice , Mice, Inbred ICR , Mice, Knockout , Receptors, Glucagon/genetics
2.
Microvasc Res ; 85: 59-67, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23084965

ABSTRACT

Lysophosphatidic acid (LPA) is a class of bioactive lyso-phospholipid that mediates most of its biological effects through a family of G protein-coupled receptors of which six have been identified. The role of the LPA pathway in driving chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) has gained considerable academic and industry attention. Modulation of the pulmonary artery endothelial barrier function by the LPA1 receptor has been shown to drive pulmonary fibrosis in murine models of disease. The purpose of this study was (i) to assess the effect of LPA on the barrier function of human pulmonary arterial (HPAEC) and microvascular (HMVEC) endothelial cells and (ii) to identify the LPA receptor subtype(s) responsible for changes in human pulmonary endothelial cell permeability using LPA receptor antagonists and siRNA technology. Analysis of the LPA receptor subtype expression demonstrated predominant expression of LPA2 and LPA6 receptor subtypes in both HPAECs and HMVECs. HPAECs also exhibit low expression of LPA1, LPA3, and LPA4 receptor subtypes. Treatment of cells with increasing concentrations of LPA caused loss of barrier function in HPAECs but not HMVECs, despite both cell types exhibiting very similar LPA receptor expression profiles. The LPA-mediated loss of barrier function in HPAECs appears to be independent of the LPA1 receptor and likely to be mediated via the LPA6 receptor although we cannot exclude an additional role for the LPA2 and LPA4 receptors in mediating these effects. These results suggest cell-specific mechanisms exist in human pulmonary endothelial cells to permit regulation of barrier function downstream of LPA receptors. More importantly, our data indicate that selective LPA1 receptor antagonism may be insufficient for therapeutic use in pulmonary diseases where impaired endothelial barrier function is related to disease initiation and progression.


Subject(s)
Endothelial Cells/cytology , Lung/cytology , Lysophospholipids/metabolism , Arteries/pathology , Calcium/chemistry , DNA Primers/genetics , Dose-Response Relationship, Drug , Endothelium/cytology , Humans , Idiopathic Pulmonary Fibrosis/pathology , Microcirculation , Microscopy, Fluorescence/methods , Permeability , Polymerase Chain Reaction/methods , RNA, Small Interfering/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Lysophosphatidic Acid/metabolism , Time Factors
3.
J Med Chem ; 55(17): 7920-39, 2012 Sep 13.
Article in English | MEDLINE | ID: mdl-22894757

ABSTRACT

Lysophosphatidic acid is a class of bioactive phospholipid that mediates most of its biological effects through LPA receptors, of which six isoforms have been identified. The recent results from LPA1 knockout mice suggested that blocking LPA1 signaling could provide a potential novel approach for the treatment of idiopathic pulmonary fibrosis. Here, we report the design and synthesis of pyrazole- and triazole-derived carbamates as LPA1-selective and LPA1/3 dual antagonists. In particular, compound 2, the most selective LPA1 antagonist reported, inhibited proliferation and contraction of normal human lung fibroblasts (NHLF) following LPA stimulation. Oral dosing of compound 2 to mice resulted in a dose-dependent reduction of plasma histamine levels in a murine LPA challenge model. Furthermore, we applied our novel antagonists as chemistry probes and investigated the contribution of LPA1/2/3 in mediating the pro-fibrotic responses. Our results suggest LPA1 as the major receptor subtype mediating LPA-induced proliferation and contraction of NHLF.


Subject(s)
Drug Discovery , Lung/drug effects , Receptors, Lysophosphatidic Acid/antagonists & inhibitors , Administration, Oral , Animals , Fibroblasts/drug effects , Humans , Lung/cytology , Magnetic Resonance Spectroscopy , Mice , Pyrazoles/chemistry , Pyrazoles/pharmacology , Spectrometry, Mass, Electrospray Ionization , Structure-Activity Relationship
4.
Anal Cell Pathol (Amst) ; 34(5): 225-34, 2011.
Article in English | MEDLINE | ID: mdl-21988886

ABSTRACT

Despite significant differences in genetic profiles, cancer cells share common phenotypic properties, including membrane-associated changes that facilitate invasion and metastasis. The Corning Epic optical biosensor was used to monitor dynamic mass rearrangements within and proximal to the cell membrane in tumor cell lines derived from cancers of the colon, bone, cervix, lung and breast. Data was collected in real time and required no exogenously added signaling moiety (signal-free technology). Cell lines displayed unique profiles over the time-courses: the time-courses all displayed initial signal increases to maximal values, but the rate of increase to those maxima and the value of those maxima were distinct for each cell line. The rate of decline following the maxima also differed among cell lines. There were correlations between the signal maxima and the observed metastatic behavior of the cells in xenograft experiments; for most cell types the cells that were more highly metastatic in mice had lower time-course maxima values, however the reverse was seen in breast cancer cells. The unique profiles of these cell lines and the correlation of at least one profile characteristic with metastatic behavior demonstrate the potential utility of biophysical tumor cell profiling in the study of cancer biology.


Subject(s)
Biosensing Techniques , Cell Membrane/pathology , Neoplasms/pathology , Animals , CHO Cells , Cricetinae , Cricetulus , HT29 Cells , HeLa Cells , Humans , Mice , Neoplasm Invasiveness , Neoplasm Metastasis , Phenotype , Reproducibility of Results , Time Factors , Transplantation, Heterologous
5.
Bioorg Med Chem Lett ; 21(7): 1933-6, 2011 Apr 01.
Article in English | MEDLINE | ID: mdl-21388809

ABSTRACT

The highly potent but modestly selective N-(2-amino-4-methoxy-benzothiazol-7-yl)-N-ethyl-acetamide derivative 2 was selected as the starting point for the design of novel selective A(2B) antagonists, due to its excellent potency, and good drug-like properties. A series of compounds containing nonaromatic amides or ureas of five- or six-membered rings, and also bearing an m-trifluoromethyl-phenyl group (shown to impart superior potency) was prepared and evaluated for their selectivity against the A(2A) and A(1) receptors. This work resulted in the identification of compound 30, with excellent potency and high selectivity against both A(2A) and A(1) receptors.


Subject(s)
Adenosine A2 Receptor Antagonists/chemistry , Adenosine A2 Receptor Antagonists/pharmacology , Benzothiazoles/pharmacology , Drug Discovery , Benzothiazoles/chemistry , Structure-Activity Relationship
6.
Bioorg Med Chem Lett ; 20(14): 4140-6, 2010 Jul 15.
Article in English | MEDLINE | ID: mdl-20541935

ABSTRACT

7-N-Acetamide-4-methoxy-2-aminobenzothiazole 4-fluorobenzamide (compound 1) was chosen as a drug-like and non-xanthine based starting point for the discovery of A(2B) receptor antagonists because of its slight selectivity against A(1) and A(2A) receptors and modest A(2B) potency. SAR exploration of compound 1 described herein included modifications to the 7-N-acetamide group, substitution of the 4-methoxy group by halogens as well as replacement of the p-flouro-benzamide side chain. This work culminated in the identification of compound 37 with excellent A(2B) potency, modest selectivity versus A(2A) and A(1) receptors, and good rodent PK properties.


Subject(s)
Adenosine A2 Receptor Antagonists/pharmacology , Benzothiazoles/pharmacology , Receptor, Adenosine A2B/metabolism , Xanthine/chemistry , Adenosine A2 Receptor Antagonists/chemistry , Benzothiazoles/chemistry , Structure-Activity Relationship
7.
J Med Chem ; 53(9): 3502-16, 2010 May 13.
Article in English | MEDLINE | ID: mdl-20380377

ABSTRACT

The inhibition of LTB(4) binding to and activation of G-protein-coupled receptors BLT1 and BLT2 is the premise of a treatment for several inflammatory diseases. In a lead optimization effort starting with the leukotriene B(4) (LTB(4)) receptor antagonist (2), members of a series of 3,5-diarylphenyl ethers were found to be highly potent inhibitors of LTB(4) binding to BLT1 and BLT2 receptors, with varying levels of selectivity depending on the substitution. In addition, compounds 33 and 38 from this series have good in vitro ADME properties, good oral bioavailability, and efficacy after oral delivery in guinea pig LTB(4) and nonhuman primate allergen challenge models. Further profiling in a rat non-GLP toxicity experiment provided the rationale for differentiation and selection of one compound (33) for clinical development.


Subject(s)
Drug Discovery , Leukotriene Antagonists/chemistry , Phenyl Ethers/pharmacology , Receptors, Leukotriene B4/antagonists & inhibitors , Animals , Drug Evaluation, Preclinical , Guinea Pigs , HL-60 Cells , Humans , Leukotriene Antagonists/pharmacology , Phenyl Ethers/chemistry , Primates , Protein Binding , Rats , Receptors, G-Protein-Coupled/metabolism , Receptors, Leukotriene B4/metabolism , Structure-Activity Relationship
8.
Prostaglandins Other Lipid Mediat ; 92(1-4): 33-43, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20214997

ABSTRACT

Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.


Subject(s)
Benzodioxoles/pharmacology , Phenylpropionates/pharmacology , Pneumonia/drug therapy , Primates , Receptors, Leukotriene B4/antagonists & inhibitors , Animals , Benzodioxoles/therapeutic use , Benzodioxoles/toxicity , Dogs , Drug-Related Side Effects and Adverse Reactions , Female , Guinea Pigs , HL-60 Cells , Humans , Hypersensitivity/complications , Lipopolysaccharides/pharmacology , Lung/drug effects , Male , Mice , Ozone/pharmacology , Phenylpropionates/therapeutic use , Phenylpropionates/toxicity , Pneumonia/chemically induced , Pneumonia/complications , Pneumonia/metabolism , Rats , Receptors, Leukotriene B4/metabolism , Smoking/adverse effects , Toxicity Tests
9.
Mol Cancer Ther ; 5(11): 2644-58, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17121911

ABSTRACT

The cyclin-dependent protein kinases are key regulators of cell cycle progression. Aberrant expression or altered activity of distinct cyclin-dependent kinase (CDK) complexes results in escape of cells from cell cycle control, leading to unrestricted cell proliferation. CDK inhibitors have the potential to induce cell cycle arrest and apoptosis in cancer cells, and identifying small-molecule CDK inhibitors has been a major focus in cancer research. Several CDK inhibitors are entering the clinic, the most recent being selective CDK2 and CDK4 inhibitors. We have identified a diaminopyrimidine compound, R547, which is a potent and selective ATP-competitive CDK inhibitor. In cell-free assays, R547 effectively inhibited CDK1/cyclin B, CDK2/cyclin E, and CDK4/cyclin D1 (K(i) = 1-3 nmol/L) and was inactive (K(i) > 5,000 nmol/L) against a panel of >120 unrelated kinases. In vitro, R547 effectively inhibited the proliferation of tumor cell lines independent of multidrug resistant status, histologic type, retinoblastoma protein, or p53 status, with IC(50)s

Subject(s)
Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Apoptosis , Cell Line, Tumor , Cell Proliferation/drug effects , Clinical Trials, Phase I as Topic , Cyclin-Dependent Kinases/metabolism , Female , G1 Phase/drug effects , G2 Phase/drug effects , Genes, MDR/drug effects , Humans , Mice , Mice, Nude , Phosphorylation/drug effects , Pyrimidines/pharmacokinetics , Pyrimidines/therapeutic use , Rats , Rats, Inbred F344 , Retinoblastoma/drug therapy , Retinoblastoma/metabolism , Tumor Suppressor Protein p53/metabolism
10.
Anal Chim Acta ; 578(1): 25-30, 2006 Sep 18.
Article in English | MEDLINE | ID: mdl-17723691

ABSTRACT

The Caco-2 cell monolayer model was used to classify 13 compounds as P-glycoprotein (Pgp) substrates or non-substrates. The apparent permeability coefficients (Papp) in the basal-to-apical direction (Papp(B-A)) and in the apical-to-basal direction (Papp(A-B)) were determined for each compound and a compound was designated as a Pgp substrate if Papp(B-A)/Papp(A-B), the permeability ratio, exceeded 2.0. The same compounds were chromatographed on open tubular glass columns containing membranes from cell lines that either expressed Pgp (Pgp+-OT column) or did not express Pgp (Pgp(-)-OT column). The differential retentions in min, Deltat values, of the compounds were determined using the following relationship Deltat=t((Pgp(+)-OT))-t((Pgp(-)-OT)). A statistically significant correlation was observed between the Deltat values and the permeability ratios, r2=0.7749 (p=0.0063), indicating that the differential chromatography approach could be used to quantitatively assess permeability ratios. The results also indicated that a Deltat value > or =0.5 min was a reliable measure of a permeability ratios >2 and could be used as a rapid qualitative determination of whether a test compound was a Pgp substrate. The chromatographic study took 1h to complete and a single pair of columns could be used to screen at least 150 compounds a week and 600 compounds during the 4-week lifetime of the columns.

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