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Results: 11 to 20 of 181

Publication Record


Ligand-based virtual screen for the discovery of novel M5 inhibitor chemotypes.
Geanes AR, Cho HP, Nance KD, McGowan KM, Conn PJ, Jones CK, Meiler J, Lindsley CW
(2016) Bioorg Med Chem Lett 26: 4487-4491
MeSH Terms: Animals, CHO Cells, Cricetulus, Drug Discovery, Humans, Ligands, Muscarinic Antagonists, Quantitative Structure-Activity Relationship, Receptor, Muscarinic M5
Show Abstract · Added April 6, 2017
This Letter describes a ligand-based virtual screening campaign utilizing SAR data around the M5 NAMs, ML375 and VU6000181. Both QSAR and shape scores were employed to virtually screen a 98,000-member compound library. Neither approach alone proved productive, but a consensus score of the two models identified a novel scaffold which proved to be a modestly selective, but weak inhibitor (VU0549108) of the M5 mAChR (M5 IC50=6.2μM, M1-4 IC50s>10μM) based on an unusual 8-((1,3,5-trimethyl-1H-pyrazol-4-yl)sulfonyl)-1-oxa-4-thia-8-azaspiro[4,5]decane scaffold. [(3)H]-NMS binding studies showed that VU0549108 interacts with the orthosteric site (Ki of 2.7μM), but it is not clear if this is negative cooperativity or orthosteric binding. Interestingly, analogs synthesized around VU0549108 proved weak, and SAR was very steep. However, this campaign validated the approach and warranted further expansion to identify additional novel chemotypes.
Copyright © 2016 Elsevier Ltd. All rights reserved.
1 Communities
2 Members
0 Resources
9 MeSH Terms
Rearrangement of the Extracellular Domain/Extracellular Loop 1 Interface Is Critical for Thyrotropin Receptor Activation.
Schaarschmidt J, Nagel MB, Huth S, Jaeschke H, Moretti R, Hintze V, von Bergen M, Kalkhof S, Meiler J, Paschke R
(2016) J Biol Chem 291: 14095-108
MeSH Terms: Animals, CHO Cells, Cricetinae, Cricetulus, Glycosylation, Humans, Mass Spectrometry, Models, Molecular, Mutation, Proteolysis, Receptors, Thyrotropin, Surface Plasmon Resonance
Show Abstract · Added April 8, 2017
The thyroid stimulating hormone receptor (TSHR) is a G protein-coupled receptor (GPCR) with a characteristic large extracellular domain (ECD). TSHR activation is initiated by binding of the hormone ligand TSH to the ECD. How the extracellular binding event triggers the conformational changes in the transmembrane domain (TMD) necessary for intracellular G protein activation is poorly understood. To gain insight in this process, the knowledge on the relative positioning of ECD and TMD and the conformation of the linker region at the interface of ECD and TMD are of particular importance. To generate a structural model for the TSHR we applied an integrated structural biology approach combining computational techniques with experimental data. Chemical cross-linking followed by mass spectrometry yielded 17 unique distance restraints within the ECD of the TSHR, its ligand TSH, and the hormone-receptor complex. These structural restraints generally confirm the expected binding mode of TSH to the ECD as well as the general fold of the domains and were used to guide homology modeling of the ECD. Functional characterization of TSHR mutants confirms the previously suggested close proximity of Ser-281 and Ile-486 within the TSHR. Rigidifying this contact permanently with a disulfide bridge disrupts ligand-induced receptor activation and indicates that rearrangement of the ECD/extracellular loop 1 (ECL1) interface is a critical step in receptor activation. The experimentally verified contact of Ser-281 (ECD) and Ile-486 (TMD) was subsequently utilized in docking homology models of the ECD and the TMD to create a full-length model of a glycoprotein hormone receptor.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
1 Communities
1 Members
0 Resources
12 MeSH Terms
Transthyretin Suppresses Amyloid-β Secretion by Interfering with Processing of the Amyloid-β Protein Precursor.
Li X, Song Y, Sanders CR, Buxbaum JN
(2016) J Alzheimers Dis 52: 1263-75
MeSH Terms: Alzheimer Disease, Amyloid Precursor Protein Secretases, Amyloid beta-Peptides, Amyloid beta-Protein Precursor, Animals, Brain, CHO Cells, Cricetulus, Enzyme-Linked Immunosorbent Assay, Female, Immunoprecipitation, Male, Mice, Mice, Transgenic, Prealbumin
Show Abstract · Added November 21, 2018
In Alzheimer's disease (AD), most hippocampal and cortical neurons show increased staining with anti-transthyretin (TTR) antibodies. Genetically programmed overexpression of wild type human TTR suppressed the neuropathologic and behavioral abnormalities in APP23 AD model mice and TTR-Aβ complexes have been isolated from some human AD brains and those of APP23 transgenic mice. In the present study, in vitro NMR analysis showed interaction between the hydrophobic thyroxine binding pocket of TTR and the cytoplasmic loop of the C99 fragment released by β-secretase cleavage of AβPP, with Kd = 86±9 μM. In cultured cells expressing both proteins, the interaction reduced phosphorylation of C99 (at T668) and suppressed its cleavage by γ-secretase, significantly decreasing Aβ secretion. Coupled with its previously demonstrated capacity to inhibit Aβ aggregation (with the resultant cytotoxicity in tissue culture) and its regulation by HSF1, these findings indicate that TTR can behave as a stress responsive multimodal suppressor of AD pathogenesis.
0 Communities
1 Members
0 Resources
MeSH Terms
Identification of a Novel Transcript and Regulatory Mechanism for Microsomal Triglyceride Transfer Protein.
Suzuki T, Brown JJ, Swift LL
(2016) PLoS One 11: e0147252
MeSH Terms: Alternative Splicing, Animals, CHO Cells, Carrier Proteins, Cricetulus, Electrophoresis, Polyacrylamide Gel, Female, HEK293 Cells, Humans, Mice, Protein Isoforms, RNA, Messenger, Rabbits, Reverse Transcriptase Polymerase Chain Reaction
Show Abstract · Added February 22, 2016
Microsomal triglyceride transfer protein (MTP) is essential for the assembly of triglyceride-rich apolipoprotein B-containing lipoproteins. Previous studies in our laboratory identified a novel splice variant of MTP in mice that we named MTP-B. MTP-B has a unique first exon (1B) located 2.7 kB upstream of the first exon (1A) for canonical MTP (MTP-A). The two mature isoforms, though nearly identical in sequence and function, have different tissue expression patterns. In this study we report the identification of a second MTP splice variant (MTP-C), which contains both exons 1B and 1A. MTP-C is expressed in all the tissues we tested. In cells transfected with MTP-C, protein expression was less than 15% of that found when the cells were transfected with MTP-A or MTP-B. In silico analysis of the 5'-UTR of MTP-C revealed seven ATGs upstream of the start site for MTP-A, which is the only viable start site in frame with the main coding sequence. One of those ATGs was located in the 5'-UTR for MTP-A. We generated reporter constructs in which the 5'-UTRs of MTP-A or MTP-C were inserted between an SV40 promoter and the coding sequence of the luciferase gene and transfected these constructs into HEK 293 cells. Luciferase activity was significantly reduced by the MTP-C 5'-UTR, but not by the MTP-A 5'-UTR. We conclude that alternative splicing plays a key role in regulating MTP expression by introducing unique 5'-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP levels and activity.
0 Communities
1 Members
0 Resources
14 MeSH Terms
M1 and M3 muscarinic receptors may play a role in the neurotoxicity of anhydroecgonine methyl ester, a cocaine pyrolysis product.
Garcia RC, Dati LM, Torres LH, da Silva MA, Udo MS, Abdalla FM, da Costa JL, Gorjão R, Afeche SC, Yonamine M, Niswender CM, Conn PJ, Camarini R, Sandoval MR, Marcourakis T
(2015) Sci Rep 5: 17555
MeSH Terms: Animals, Apoptosis, CHO Cells, Cocaine, Cricetinae, Cricetulus, DNA Fragmentation, Female, Hippocampus, Neurotoxicity Syndromes, Neurotoxins, Rats, Receptor, Muscarinic M1, Receptor, Muscarinic M3, Time Factors
Show Abstract · Added February 18, 2016
The smoke of crack cocaine contains cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME). AEME possesses greater neurotoxic potential than cocaine and an additive effect when they are combined. Since atropine prevented AEME-induced neurotoxicity, it has been suggested that its toxic effects may involve the muscarinic cholinergic receptors (mAChRs). Our aim is to understand the interaction between AEME and mAChRs and how it can lead to neuronal death. Using a rat primary hippocampal cell culture, AEME was shown to cause a concentration-dependent increase on both total [(3)H]inositol phosphate and intracellular calcium, and to induce DNA fragmentation after 24 hours of exposure, in line with the activation of caspase-3 previously shown. Additionally, we assessed AEME activity at rat mAChR subtypes 1-5 heterologously expressed in Chinese Hamster Ovary cells. l-[N-methyl-(3)H]scopolamine competition binding showed a preference of AEME for the M2 subtype; calcium mobilization tests revealed partial agonist effects at M1 and M3 and antagonist activity at the remaining subtypes. The selective M1 and M3 antagonists and the phospholipase C inhibitor, were able to prevent AEME-induced neurotoxicity, suggesting that the toxicity is due to the partial agonist effect at M1 and M3 mAChRs, leading to DNA fragmentation and neuronal death by apoptosis.
0 Communities
1 Members
0 Resources
15 MeSH Terms
The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin.
Rumah KR, Ma Y, Linden JR, Oo ML, Anrather J, Schaeren-Wiemers N, Alonso MA, Fischetti VA, McClain MS, Vartanian T
(2015) PLoS Pathog 11: e1004896
MeSH Terms: Animals, Bacterial Toxins, Binding Sites, CHO Cells, Cell Death, Clostridium perfringens, Cricetulus, Humans, Injections, Intravenous, Ligands, Mice, Inbred C57BL, Mice, Knockout, Mutagenesis, Insertional, Myelin and Lymphocyte-Associated Proteolipid Proteins, Protein Interaction Domains and Motifs, Protein Precursors, Rats, Recombinant Fusion Proteins, Recombinant Proteins, Tissue Distribution, Toxicokinetics
Show Abstract · Added September 28, 2015
Clostridium perfringens ε-toxin (ETX) is a potent pore-forming toxin responsible for a central nervous system (CNS) disease in ruminant animals with characteristics of blood-brain barrier (BBB) dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS), a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL). While native Chinese Hamster Ovary (CHO) cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/-) mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity.
0 Communities
1 Members
0 Resources
21 MeSH Terms
Heparan Sulfate Proteoglycans Are Important for Islet Amyloid Formation and Islet Amyloid Polypeptide-induced Apoptosis.
Oskarsson ME, Singh K, Wang J, Vlodavsky I, Li JP, Westermark GT
(2015) J Biol Chem 290: 15121-32
MeSH Terms: Amyloid, Animals, Apoptosis, Base Sequence, CHO Cells, Cells, Cultured, Cricetinae, Cricetulus, DNA Primers, Glucuronidase, Heparan Sulfate Proteoglycans, Humans, Islet Amyloid Polypeptide, Islets of Langerhans, Mice, Mice, Inbred C57BL, Mice, Transgenic, Real-Time Polymerase Chain Reaction
Show Abstract · Added June 6, 2017
Deposition of β cell toxic islet amyloid is a cardinal finding in type 2 diabetes. In addition to the main amyloid component islet amyloid polypeptide (IAPP), heparan sulfate proteoglycan is constantly present in the amyloid deposit. Heparan sulfate (HS) side chains bind to IAPP, inducing conformational changes of the IAPP structure and an acceleration of fibril formation. We generated a double-transgenic mouse strain (hpa-hIAPP) that overexpresses human heparanase and human IAPP but is deficient of endogenous mouse IAPP. Culture of hpa-hIAPP islets in 20 mm glucose resulted in less amyloid formation compared with the amyloid load developed in cultured islets isolated from littermates expressing human IAPP only. A similar reduction of amyloid was achieved when human islets were cultured in the presence of heparin fragments. Furthermore, we used CHO cells and the mutant CHO pgsD-677 cell line (deficient in HS synthesis) to explore the effect of cellular HS on IAPP-induced cytotoxicity. Seeding of IAPP aggregation on CHO cells resulted in caspase-3 activation and apoptosis that could be prevented by inhibition of caspase-8. No IAPP-induced apoptosis was seen in HS-deficient CHO pgsD-677 cells. These results suggest that β cell death caused by extracellular IAPP requires membrane-bound HS. The interaction between HS and IAPP or the subsequent effects represent a possible therapeutic target whose blockage can lead to a prolonged survival of β cells.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
1 Communities
0 Members
0 Resources
18 MeSH Terms
Toxicity studies of coumarin 6-encapsulated polystyrene nanospheres conjugated with peanut agglutinin and poly(N-vinylacetamide) as a colonoscopic imaging agent in rats.
Sakuma S, Kumagai H, Shimosato M, Kitamura T, Mohri K, Ikejima T, Hiwatari K, Koike S, Tobita E, McClure R, Gore JC, Pham W
(2015) Nanomedicine 11: 1227-36
MeSH Terms: Acetamides, Animals, Body Weight, CHO Cells, Caco-2 Cells, Colon, Colonoscopy, Colorectal Neoplasms, Coumarins, Cricetulus, Drinking, Eating, Fluorescent Dyes, Humans, Male, Nanospheres, Peanut Agglutinin, Polystyrenes, Polyvinyls, Rats, Rectum, Thiazoles
Show Abstract · Added February 15, 2016
UNLABELLED - We are investigating an imaging agent that detects early-stage primary colorectal cancer on the mucosal surface in real time under colonoscopic observation. The imaging agent, which is named the nanobeacon, is fluorescent nanospheres conjugated with peanut agglutinin and poly(N-vinylacetamide). Its potential use as an imaging tool for colorectal cancer has been thoroughly validated in numerous studies. Here, toxicities of the nanobeacon were assessed in rats. The nanobeacon was prepared according to the synthetic manner which is being established as the Good Manufacturing Practice-guided production. The rat study was performed in accordance with Good Laboratory Practice regulations. No nanobeacon treatment-related toxicity was observed. The no observable adverse effect levels (NOAEL) of the nanobeacon in 7-day consecutive oral administration and single intrarectal administration were estimated to be more than 1000mg/kg/day and 50mg/kg/day, respectively. We concluded that the nanobeacon could be developed as a safe diagnostic agent for colonoscopy applications.
FROM THE CLINICAL EDITOR - Colon cancer remains a major cause of death. Early detection can result in early treatment and thus survival. In this article, the authors tested potential systemic toxicity of coumarin 6-encapsulated polystyrene nanospheres conjugated with peanut agglutinin (PNA) and poly(N-vinylacetamide) (PNVA), which had been shown to bind specifically to colonic cancer cells and thus very promising in colonoscopic detection of cancer cells.
Copyright © 2015 Elsevier Inc. All rights reserved.
0 Communities
2 Members
0 Resources
22 MeSH Terms
Safety and angiogenic effects of systemic gene delivery of a modified erythropoietin.
de Lucas Cerrillo AM, Bond WS, Rex TS
(2015) Gene Ther 22: 365-73
MeSH Terms: Animals, CHO Cells, Cells, Cultured, Cricetinae, Cricetulus, Endothelium, Vascular, Erythropoiesis, Erythropoietin, Gene Transfer Techniques, Humans, Mice, Mice, Inbred C57BL, Mutation, Missense, Neovascularization, Physiologic, Retinal Vessels
Show Abstract · Added April 2, 2019
Erythropoietin (EPO) is critical for red blood cell production and is also an effective neuroprotective agent. However, it may contribute to pathological angiogenesis. Here we investigate the angiogenic potential of EPO and a mutant form with attenuated erythropoietic activity, EPO-R76E, on primary human retinal microvascular endothelial cells (HRMECs) and in the adult retina. Assays of death, proliferation and tube formation were performed on HRMECs exposed to EPO, EPO-R76E or media alone. Postnatal day-9 wild-type mice were injected intramuscularly with adeno-associated virus vectors expressing either enhanced green fluorescent protein or EpoR76E. At 3 months, levels of EPO-R76E in the eye were quantified, and the health of the retinal vasculature was assessed by fluorescein angiography and isolectin immunolabeling. Immunohistochemistry, histology and electroretinogram (ERG) assessments were performed as measures of retinal health. Neither EPO nor EPO-R76E induced proliferation or tube formation in HRMECs under the conditions used. EPO-R76E decreased HRMEC death in a dose-dependent manner. Long-term systemic gene delivery of EPO-R76E was safe in terms of retinal vasculature, histology and the ERG in vivo. Our results show that EPO-R76E can block HRMEC death, consistent with its role in erythropoiesis and neuroprotection. In addition, long-term gene delivery of EPO-R76E is safe in the adult retina.
0 Communities
1 Members
0 Resources
MeSH Terms
Development of a phenotypic high-content assay to identify pharmacoperone drugs for the treatment of primary hyperoxaluria type 1 by high-throughput screening.
Madoux F, Janovick JA, Smithson D, Fargue S, Danpure CJ, Scampavia L, Chen YT, Spicer TP, Conn PM
(2015) Assay Drug Dev Technol 13: 16-24
MeSH Terms: Animals, Biological Assay, CHO Cells, Cell Survival, Cricetulus, Drug Design, Drug Evaluation, Preclinical, High-Throughput Screening Assays, Humans, Hyperoxaluria, Primary, Molecular Chaperones, Phenotype, Technology, Pharmaceutical
Show Abstract · Added February 18, 2016
Primary hyperoxaluria is a severe disease for which the best current therapy is dialysis or organ transplantation. These are risky, inconvenient, and costly procedures. In some patients, pyridoxine treatment can delay the need for these surgical procedures. The underlying cause of particular forms of this disease is the misrouting of a specific enzyme, alanine:glyoxylate aminotransferase (AGT), to the mitochondria instead of the peroxisomes. Pharmacoperones are small molecules that can rescue misfolded proteins and redirect them to their correct location, thereby restoring their function and potentially curing disease. In the present study, we miniaturized a cell-based assay to identify pharmacoperone drugs present in large chemical libraries to selectively correct AGT misrouting. This assay employs AGT-170, a mutant form of AGT that predominantly resides in the mitochondria, which we monitor for its relocation to the peroxisomes through automated image acquisition and analysis. Over the course of a pilot screen of 1,280 test compounds, we achieved an average Z'-factor of 0.72±0.02, demonstrating the suitability of this assay for HTS.
0 Communities
1 Members
0 Resources
13 MeSH Terms