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Laurence J. Miller - One of the best experts on this subject based on the ideXlab platform.
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beneficial effects of β sitosterol on type 1 Cholecystokinin Receptor dysfunction induced by elevated membrane cholesterol
Clinical Nutrition, 2016Co-Authors: Aditya J. Desai, Maoqing Dong, Laurence J. MillerAbstract:Summary Background & aims The type 1 Cholecystokinin Receptor (CCK1R) mediates the actions of CCK to support nutritional homeostasis, including post-cibal satiety. However, elevated levels of membrane cholesterol, such as have been observed in metabolic syndrome, interfere with CCK stimulus-activity coupling at the CCK1R, thereby disrupting this important servomechanism. We hypothesize that reversal of the negative impact of cholesterol on this Receptor could be useful in the management of obesity. Methods We have studied the effects of β-sitosterol, a phytosterol structurally related to cholesterol, on CCK Receptor function. This included CCK binding and biological activity at wild type CCK1R and CCK2R, as well as at CCK1R in a high cholesterol environment, and at a CCK1R mutant, Y140A, which mimics the behavior of wild type Receptor in high cholesterol. Results β-sitosterol (100 μM and 10 μM) significantly improved the defective signaling of the CCK1R present in high cholesterol (p Conclusion These data suggest that β-sitosterol affects CCK1R function in high cholesterol by competing with cholesterol at a Receptor cholesterol-binding site and may shift its conformation toward normal. This phytosterol extends our understanding of the structure-activity relationships for developing a drug that can target the external surface of CCK1R. Since the concentrations of β-sitosterol shown to be effective in this study are similar to serum levels of this compound achievable during oral administration, it may be worthwhile to study possible beneficial effects of β-sitosterol in metabolic syndrome.
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Molecular mechanism of action of triazolobenzodiazepinone agonists of the type 1 Cholecystokinin Receptor. Possible cooperativity across the Receptor homodimeric complex
Journal of medicinal chemistry, 2015Co-Authors: Aditya J. Desai, Polo C H Lam, Andrew Orry, Ruben Abagyan, Arthur Christopoulos, Patrick M Sexton, Laurence J. MillerAbstract:The type 1 Cholecystokinin Receptor (CCK1R) has multiple physiologic roles relating to nutrient homeostasis, including mediation of postcibal satiety. This effect has been central in efforts to develop agonists of this Receptor as part of a program to manage and/or prevent obesity. While a number of small molecule CCK1R agonists have been developed, none have yet been approved for clinical use, based on inadequate efficacy, side effects, or the potential for toxicity. Understanding the molecular details of docking and mechanism of action of these ligands can be helpful in the rational refinement and enhancement of small molecule drug candidates. In the current work, we have defined the mechanism of binding and activity of two triazolobenzodiazepinones, CE-326597 and PF-04756956, which are reported to be full agonist ligands. To achieve this, we utilized Receptor binding with a series of allosteric and orthosteric radioligands at structurally related CCK1R and CCK2R, as well as chimeric CCK1R/CCK2R constru...
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elimination of a Cholecystokinin Receptor agonist trigger in an effort to develop positive allosteric modulators without intrinsic agonist activity
Bioorganic & Medicinal Chemistry Letters, 2015Co-Authors: Aditya J. Desai, Brad R Henke, Laurence J. MillerAbstract:Abstract Cholecystokinin (CCK) acts at the type 1 Cholecystokinin Receptor (CCK1R) to elicit satiety and is a well-established drug target for obesity. To date, small molecule agonists have been developed, but have failed to demonstrate adequate efficacy in clinical trials, and concerns about side effects and potential toxicity have limited further development of full agonists. The use of positive allosteric modulators (PAMs) without intrinsic agonist activity that are active only for a brief period of time after a meal might represent a safer alternative. Here, we propose a possible novel strategy to develop such compounds by modifying the agonist ‘trigger’ of an existing small molecule agonist. We have studied analogues of the 1,5-benzodiazepine agonist, GI181771X, in which the N1-isopropyl agonist ‘trigger’ was modified. While agonist activity was greatly reduced in these compounds, they acted as negative, rather than positive modulators. The parent drug was also found to exhibit no positive modulation of CCK action. Receptor structure–activity relationship studies demonstrated that the mode of docking these derivatives was distinct from that of the parent compound, perhaps explaining their action as negative allosteric modulators. We conclude that this outcome is likely characteristic of the parental agonist, and that this strategy may be more successfully utilized with a parental ago-PAM, possessing intrinsic positive modulatory activity.
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development of a highly selective allosteric antagonist radioligand for the type 1 Cholecystokinin Receptor and elucidation of its molecular basis of binding
Molecular Pharmacology, 2015Co-Authors: Maoqing Dong, Ashton M Vattelana, Polo C H Lam, Andrew Orry, Ruben Abagyan, Arthur Christopoulos, Patrick M Sexton, David R Haines, Laurence J. MillerAbstract:Understanding the molecular basis of ligand binding to Receptors provides insights useful for rational drug design. This work describes development of a new antagonist radioligand of the type 1 Cholecystokinin Receptor (CCK1R), (2-fluorophenyl)-2,3-dihydro-3-[(3-isoquinolinylcarbonyl)amino]-6-methoxy-2-oxo-l-H-indole-3-propanoate (T-0632), and exploration of the molecular basis of its binding. This radioligand bound specifically with high affinity within an allosteric pocket of CCK1R. T-0632 fully inhibited binding and action of CCK at this Receptor, while exhibiting no saturable binding to the closely related type 2 Cholecystokinin Receptor (CCK2R). Chimeric CCK1R/CCK2R constructs were used to explore the molecular basis of T-0632 binding. Exchanging exonic regions revealed the functional importance of CCK1R exon 3, extending from the bottom of transmembrane segment (TM) 3 to the top of TM5, including portions of the intramembranous pocket as well as the second extracellular loop region (ECL2). However, CCK1R mutants in which each residue facing the pocket was changed to that present in CCK2R had no negative impact on T-0632 binding. Extending the chimeric approach to ECL2 established the importance of its C-terminal region, and site-directed mutagenesis of each nonconserved residue in this region revealed the importance of Ser208 at the top of TM5. A molecular model of T-0632-occupied CCK1R was consistent with these experimental determinants, also identifying Met121 in TM3 and Arg336 in TM6 as important. Although these residues are conserved in CCK2R, mutating them had a distinct impact on the two closely related Receptors, suggesting differential orientation. This establishes the molecular basis of binding of a highly selective nonpeptidyl allosteric antagonist of CCK1R, illustrating differences in docking that extend beyond determinants attributable to distinct residues lining the intramembranous pocket in the two Receptor subtypes.
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molecular basis for benzodiazepine agonist action at the type 1 Cholecystokinin Receptor
Journal of Biological Chemistry, 2013Co-Authors: Kaleeckal G. Harikumar, Erin E. Cawston, Polo C H Lam, Andrew Orry, Ruben Abagyan, Arthur Christopoulos, Patrick M Sexton, Brad R Henke, Achyut Patil, Laurence J. MillerAbstract:Abstract Understanding the molecular basis of drug action can facilitate development of more potent and selective drugs. Here, we explore the molecular basis for action of a unique small molecule ligand that is a type 1 Cholecystokinin (CCK) Receptor agonist and type 2 CCK Receptor antagonist, GI181771X. We characterize its binding utilizing structurally related radioiodinated ligands selective for CCK Receptor subtypes that utilize the same allosteric ligand-binding pocket, using wild-type Receptors and chimeric constructs exchanging the distinct residues lining this pocket. Intracellular calcium assays were performed to determine biological activity. Molecular models for docking small molecule agonists to the type 1 CCK Receptor were developed using a ligand-guided refinement approach. The optimal model was distinct from the previous antagonist model for the same Receptor and was mechanistically consistent with the current mutagenesis data. This study revealed a key role for Leu7.39 that was predicted to interact with the isopropyl group in the N1 position of the benzodiazepine that acts as a “trigger” for biological activity. The molecular model was predictive of binding of other small molecule agonists, effectively distinguishing these from 1065 approved drug decoys with an area under curve value of 99%. The model also selectively enriched for agonist compounds, with 130 agonists identified by ROC analysis when seeded in 2175 non-agonist ligands of the type 1 CCK Receptor (area under curve 78%). Benzodiazepine agonists in this series docked in consistent pose within this pocket, with a key role played by Leu7.39, whereas the role of this residue was less clear for chemically distinct agonists.
Jill P Smith - One of the best experts on this subject based on the ideXlab platform.
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abstract 5326 Cholecystokinin Receptor antagonist decreases fibrosis in pancreatic cancer microenvironment to improve uptake and efficacy of chemotherapy
Cancer Research, 2020Co-Authors: Zoe X Malchiodi, Narayan Shivapurkar, Hong Cao, Sunil Bansal, Benjamin A Weinberg, Amrita K Cheema, Jill P SmithAbstract:Background: Although pancreatic cancer cells respond to chemotherapeutic agents in cell culture, many of these drugs are less effective in vivo. This decreased response to chemotherapy in animal models and human subjects is in part attributed to the dense stroma of the pancreatic tumor microenvironment (TME) that impedes penetration of drugs. Pancreatic stellate cells and tissue fibroblasts have Cholecystokinin (CCK) Receptors and when these Receptors are activated, they promote collagen deposition and increase fibrosis in the TME. We have previously shown that CCK Receptor antagonists can decrease fibrosis and change the T-cell infiltrates of the TME in pancreatic tumors. We hypothesized that an oral CCK Receptor antagonist, proglumide, would improve the efficacy of gemcitabine by decreasing fibrosis in the TME and improving uptake of chemotherapy. Methods: 500,000 mT3 murine pancreatic cancer cells were injected subcutaneously into the right flank of 40 C57/BL6 female mice. One week after the mice had measurable tumors (baseline), mice were divided into 4 groups (N=10 mice each) with equal mean tumor volumes. Treatment groups included: PBS control (100µl IP twice weekly); proglumide in drinking water (0.1 mg/ml); gemcitabine (100mg/kg, 100µl IP twice weekly); and combination of gemcitabine and proglumide. Tumors were measured weekly and the volumes were calculated. Mice were euthanized per protocol when the tumor diameter reached 20mm; tumors were removed, then flash frozen for gemcitabine measurement via mass spectrometry. Results: Tumor volumes demonstrated a rapid growth rate in the PBS control mice with a slope of 184.2±2 (mm3/week), whereas growth rates for proglumide (75±2) and gemcitabine monotherapy (72±14) were 2.6-fold slower, and also 3.4-fold slower for mice treated with both gemcitabine and proglumide (55.6±12; p Conclusion: CCK Receptor blockade with proglumide decreases fibrosis in pancreatic cancer TME allowing greater tumor uptake of gemcitabine and improving efficacy by slowing tumor growth and improving survival. Strategies to improve first-line therapies for patients with pancreatic cancer may improve overall survival of this disease. Citation Format: Zoe X. Malchiodi, Hong Cao, Martha Gay, Sunil Bansal, Benjamin A. Weinberg, Amrita Cheema, Narayan Shivapurkar, Jill P. Smith. Cholecystokinin Receptor antagonist decreases fibrosis in pancreatic cancer microenvironment to improve uptake and efficacy of chemotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5326.
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Cholecystokinin Receptor antagonist therapy decreases inflammation and fibrosis in chronic pancreatitis
Digestive Diseases and Sciences, 2020Co-Authors: Sandeep Nadella, Robin D Tucker, Hong Cao, Victor Ciofoaia, Bhaskar Kallakury, Jill P SmithAbstract:Background and Aims Chronic pancreatitis is associated with recurrent inflammation, pain, fibrosis, and loss of exocrine and endocrine pancreatic function and risk of cancer. We hypothesized that activation of the CCK Receptor contributes to pancreatitis and blockade of this pathway would improve chronic pancreatitis.
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Cholecystokinin Receptor Antagonist Therapy Decreases Inflammation and Fibrosis in Chronic Pancreatitis
Digestive Diseases and Sciences, 2019Co-Authors: Sandeep Nadella, Robin D Tucker, Hong Cao, Victor Ciofoaia, Bhaskar Kallakury, Jill P SmithAbstract:Background and Aims Chronic pancreatitis is associated with recurrent inflammation, pain, fibrosis, and loss of exocrine and endocrine pancreatic function and risk of cancer. We hypothesized that activation of the CCK Receptor contributes to pancreatitis and blockade of this pathway would improve chronic pancreatitis. Methods Two murine models were used to determine whether CCK Receptor blockade with proglumide could prevent and reverse histologic and biochemical features of chronic pancreatitis: the 6-week repetitive chronic cerulein injection model and the modified 75% choline-deficient ethionine (CDE) diet. In the CDE-fed model, half the mice received water supplemented with proglumide, for 18 weeks. After chronic pancreatitis was established in the cerulein model, half the mice were treated with proglumide and half with water. Histology was scored in a blinded fashion for inflammation, fibrosis and acinar ductal metaplasia (ADM) and serum lipase levels were measured. RNA was extracted and examined for differentially expressed fibrosis genes. Results Proglumide therapy decreased pancreatic weight in the CDE diet study and the cerulein-induced chronic pancreatitis model. Fibrosis, inflammation, and ADM scores were significantly reduced in both models. Lipase values improved with proglumide but not in controls in both models. Proglumide decreased pancreas mRNA expression of amylase, collagen-4, and TGFβR2 gene expression by 44, 38, and 25%, respectively, compared to control mice. Conclusion New strategies are needed to decreased inflammation and reduce fibrosis in chronic pancreatitis. CCK Receptor antagonist therapy may improve chronic pancreatitis by reversing fibrosis and inflammation. The decrease in ADM may reduce the risk of the development of pancreatic cancer.
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Cholecystokinin Receptor targeted polyplex nanoparticle inhibits growth and metastasis of pancreatic cancer
Cellular and molecular gastroenterology and hepatology, 2018Co-Authors: Julian Burks, Sandeep Nadella, Abdullah Mahmud, Charoen Mankongpaisarnrung, Juan Wang, Jongin Hahm, Robin D Tucker, Narayan Shivapurkar, Stephan T Stern, Jill P SmithAbstract:Background & Aims Pancreatic ductal adenocarcinoma (PDAC) remains the most aggressive malignancy with the lowest 5-year survival rate of all cancers in part owing to the lack of tumor-specific therapy and the rapid metastatic nature of this cancer. The gastrointestinal peptide gastrin is a trophic peptide that stimulates growth of PDAC in an autocrine fashion by interaction with the Cholecystokinin Receptor that is overexpressed in this malignancy. Methods We developed a therapeutic novel polyplex nanoparticle (NP) that selectively targets the Cholecystokinin Receptor on PDAC. The NP was characterized in vitro and stability testing was performed in human blood. The effects of the target-specific NP loaded with gastrin small interfering RNA (siRNA) was compared with an untargeted NP and with an NP loaded with a scrambled siRNA in vitro and in 2 orthotopic models of PDAC. A polymerase chain reaction metastasis array examined differentially expressed genes from control tumors compared with tumors of mice treated with the targeted polyplex NP. Results The polyplex NP forms a micelle that safely delivers specific gastrin siRNA to the tumor without off-target toxicity. Consistent with these findings, cellular uptake was confirmed only with the targeted fluorescently labeled NP by confocal microscopy in vitro and by IVIS fluorescent based imaging in mice bearing orthotopic pancreatic cancers but not found with untargeted NPs. Tumor uptake and release of the gastrin siRNA NP was verified by decreased cellular gastrin gene expression by quantitative reverse-transcription polymerase chain reaction and peptide expression by immunohistochemistry. Growth of PDAC was inhibited in a dose-related fashion in cell culture and in vivo. The targeted NP therapy completely blocked tumor metastasis and altered tumor-specific genes. Conclusions Our polyplex nanoparticle platform establishes both a strong foundation for the development of Receptor-targeted therapeutics and a unique approach for the delivery of siRNA in vivo, thus warranting further exploration of this approach in other types of cancers.
Kaleeckal G. Harikumar - One of the best experts on this subject based on the ideXlab platform.
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molecular basis for benzodiazepine agonist action at the type 1 Cholecystokinin Receptor
Journal of Biological Chemistry, 2013Co-Authors: Kaleeckal G. Harikumar, Erin E. Cawston, Polo C H Lam, Andrew Orry, Ruben Abagyan, Arthur Christopoulos, Patrick M Sexton, Brad R Henke, Achyut Patil, Laurence J. MillerAbstract:Abstract Understanding the molecular basis of drug action can facilitate development of more potent and selective drugs. Here, we explore the molecular basis for action of a unique small molecule ligand that is a type 1 Cholecystokinin (CCK) Receptor agonist and type 2 CCK Receptor antagonist, GI181771X. We characterize its binding utilizing structurally related radioiodinated ligands selective for CCK Receptor subtypes that utilize the same allosteric ligand-binding pocket, using wild-type Receptors and chimeric constructs exchanging the distinct residues lining this pocket. Intracellular calcium assays were performed to determine biological activity. Molecular models for docking small molecule agonists to the type 1 CCK Receptor were developed using a ligand-guided refinement approach. The optimal model was distinct from the previous antagonist model for the same Receptor and was mechanistically consistent with the current mutagenesis data. This study revealed a key role for Leu7.39 that was predicted to interact with the isopropyl group in the N1 position of the benzodiazepine that acts as a “trigger” for biological activity. The molecular model was predictive of binding of other small molecule agonists, effectively distinguishing these from 1065 approved drug decoys with an area under curve value of 99%. The model also selectively enriched for agonist compounds, with 130 agonists identified by ROC analysis when seeded in 2175 non-agonist ligands of the type 1 CCK Receptor (area under curve 78%). Benzodiazepine agonists in this series docked in consistent pose within this pocket, with a key role played by Leu7.39, whereas the role of this residue was less clear for chemically distinct agonists.
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Differential sensitivity of types 1 and 2 Cholecystokinin Receptors to membrane cholesterol
Journal of lipid research, 2011Co-Authors: Ross M. Potter, Kaleeckal G. Harikumar, Laurence J. MillerAbstract:Recent studies indicate that membrane cholesterol can associate with G protein-coupled Receptors (GPCRs) and affect their function. Previously, we reported that manipulation of membrane cholesterol affects ligand binding and signal transduction of the type 1 Cholecystokinin Receptor (CCK1R), a Class A GPCR. We now demonstrate that the closely related type 2 Cholecystokinin Receptor (CCK2R) does not share this cholesterol sensitivity. The sequences of both Receptors reveal almost identical cholesterol interaction motifs in analogous locations in transmembrane segments two, three, four, and five. The disparity in cholesterol sensitivity between these Receptors, despite their close structural relationship, provides a unique opportunity to define the possible structural basis of cholesterol sensitivity of CCK1R. To evaluate the relative contributions of different regions of CCK1R to cholesterol sensitivity, we performed ligand binding studies and biological activity assays of wild-type and CCK2R/CCK1R chimeric Receptor-bearing Chinese hamster ovary cells after manipulation of membrane cholesterol. We also extended these studies to site-directed mutations within the cholesterol interaction motifs. The results contribute to a better understanding of the structural requirements for cholesterol sensitivity in CCK1R and provides insight into the function of other cholesterol-sensitive Class A GPCRs.
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Fluorescence Polarization Screening for Allosteric Small Molecule Ligands of the Cholecystokinin Receptor
Assay and drug development technologies, 2011Co-Authors: Kaleeckal G. Harikumar, Erin E. Cawston, Laurence J. MillerAbstract:ABSTRACT The success in screening for drug candidates is highly dependent on the power of the strategy implemented. In this work, we report and characterize a novel fluorescent benzodiazepine antagonist of the type 1 Cholecystokinin Receptor (3-(3-(7-fluoro-1-(2-isopropyl(4-methoxyphenyl)amino)-2-oxoethyl)-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo[b][1,4]-diazepin-3-yl)ureido)benzoic acid) that can be used as a Receptor ligand in a fluorescence polarization assay, which is ideally suited for the identification of small molecule allosteric modulators of this physiologically important Receptor. By binding directly to the small molecule-docking region within the helical bundle of this Receptor, this indicator can be displaced by many small molecule candidate drugs, even those that might not affect the binding of an orthosteric Cholecystokinin-like peptide ligand. The biological, pharmacological, and fluorescence properties of this reagent are described, and proof-of-concept is provided in a fluorescence...
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Examination of the Effects of Cholesterol on Cholecystokinin Receptor Function using Model Cell Lines
Biophysical Journal, 2011Co-Authors: Valerie Echeveste, Kaleeckal G. Harikumar, Osman Najam, Ross M. Potter, Laurence J. MillerAbstract:The Family A G protein-coupled type 1 Cholecystokinin Receptor (CCK1R) has been shown to be particularly sensitive to membrane cholesterol, with elevated levels induced by cholesterol-loaded methyl-β-cyclodextrin interfering with Receptor-G protein coupling and hormone-induced signaling (JBC, 2005). This is important, because CCK is a physiologic satiety factor, and a defect in CCK-induced activation of this Receptor can result in obesity with associated hyperlipidemia. These problems can then worsen the CCK signaling defect and contribute to a cycle of further clinical problems. Of note, the structurally closely-related CCK2R is not sensitive to membrane cholesterol when manipulated in the same manner (unpublished data). Here, we have attempted to study the function of these Receptors in 25RA cells derived by T.Y. Chang from CHO cells (JBC 1980). These cells have elevated membrane cholesterol secondary to a gain-of-function defect in the SCAP gene (sterol regulatory element binding protein (SREBP) cleavage-activating protein). We established and characterized 25RA cell lines stably expressing CCK1R and CCK2R. Increased membrane cholesterol was visualized with a quantitative filipin-binding morphologic assay. In this cholesterol-rich environment, CCK radioligand binding and CCK-stimulated intracellular calcium assays were performed, with results compared to those in parental CHO cells. Like the CHO cells with acute physical enhancement of cholesterol, these genetically modified cells demonstrated defective CCK-induced signaling, despite normal CCK radioligand binding. We also analyzed CCK Receptor trafficking in the environment of 25RA cells. We found that both CCK1R and CCK2R internalize in response to agonist occupation and to recycle to the plasma membrane normally despite the increase in membrane cholesterol.. (Supported by NIH grants DK32878 and DK78385, and a Kinney Career Development Award).
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Monitoring the State of Cholecystokinin Receptor Oligomerization after Ligand Binding Using Decay of Time-Resolved Fluorescence Anisotropy
Annals of the New York Academy of Sciences, 2008Co-Authors: Kaleeckal G. Harikumar, Laurence J. MillerAbstract:Oligomeric complexes of G protein–coupled Receptors (GPCRs) are now commonly recognized and can provide a mechanism for regulation of signaling systems. Receptor oligomerization has been most extensively studied using coimmunoprecipitation and bioluminescence or fluorescence resonance energy-transfer techniques. Here, we have utilized decay of time-resolved fluorescence anisotropy of yellow fluorescent protein-labeled Cholecystokinin Receptor constructs to examine the state of oligomerization of this Receptor in living cells. The rotational correlation times established that the Cholecystokinin Receptor is constitutively present in an oligomeric state that is dissociated in response to agonist occupation. In contrast, antagonist occupation failed to modify this signal, leaving the oligomeric structure intact. This dynamic technique complements the other biochemical and steady-state fluorescence techniques to establish the presence of oligomeric Receptor complexes in living cells.
Daniel Fourmy - One of the best experts on this subject based on the ideXlab platform.
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Evidence That Interspecies Polymorphism in the Human and Rat Cholecystokinin Receptor-2 Affects Structure of the Binding Site for the Endogenous Agonist Cholecystokinin
Journal of Biological Chemistry, 2005Co-Authors: Ingrid Langer, Irina G. Tikhonova, Marie-agnès Travers, Elodie Archer-lahlou, Chantal Escrieut, Bernard Maigret, Daniel FourmyAbstract:The Cholecystokinin (CCK) Receptor-2 exerts very important central and peripheral functions by binding the neuropeptides Cholecystokinin or gastrin. Because this Receptor is a potential therapeutic target, great interest has been devoted to the identification of efficient antagonists. However, interspecies genetic polymorphism that does not alter Cholecystokinin-induced signaling was shown to markedly affect activity of synthetic ligands. In this context, precise structural study of the agonist binding site on the human Cholecystokinin Receptor-2 is a prerequisite to elucidating the molecular basis for its activation and to optimizing properties of synthetic ligands. In this study, using site-directed mutagenesis and molecular modeling, we delineated the binding site for CCK on the human Cholecystokinin Receptor-2 by mutating amino acids corresponding to that of the rat homolog. By doing so, we demonstrated that, although resembling that of rat homolog, the human Cholecystokinin Receptor-2 binding site also displays important distinct structural features that were demonstrated by susceptibility to several point mutations (F120A, Y189A, H207A). Furthermore, docking of CCK in the human and rat Cholecystokinin Receptor-2, followed by dynamic simulations, allowed us to propose a plausible structural explanation of the experimentally observed difference between rat and human Cholecystokinin-2 Receptors.
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Purification of A-subtype pancreatic Cholecystokinin Receptor by immunoaffinity chromatography.
Biochimie, 1992Co-Authors: Marlène Dufresne, Sandrine Poirot, Nicole Vaysse, Juan Jiménez, Daniel FourmyAbstract:Abstract So far, no efficient affinity chromatography for CCK Receptor purification has been reported that prevented obtention of sequenceable amounts of purified Receptor. In this work, 10% of plasma membrane Receptor sites were specifically cross-linked with the photoreactive cleavable agonist 125I-ASD-[Thr28, Ahx31]-CCK-25-33, solubilized by NP-40, chromatographied on immobilized wheat germ agglutinin and further immunopurified using anti-CCK antibodies to an overall rate of 3000–3600-fold. Analysis of eluted material demonstrated a protein migrating at Mt 85 000–100 000 and the absence of 35S-labeled impurity. This single and efficient affinity chromatography should provide enough homogeneous Receptor protein for microsequence determination and leads to consider immunoaffinity chromatography on immobilized anti-ligand antibodies as a potential tool for purification of membrane Receptors.
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Biochemical characterization of a subtype pancreatic Cholecystokinin Receptor and of its agonist binding domain.
Journal of receptor research, 1992Co-Authors: Sandrine Poirot, Nicole Vaysse, Marlène Dufresne, Juan Jiménez, Daniel FourmyAbstract:AbstractThis study was undertaken in order to improve photoaffinity labelling efficiency of pancreatic Cholecystokinin Receptor by the cleavable probe 125I-ASD-(Thr28, Ahx31)-CCK-25-33 and to further characterize the denaturated Receptor and is agonist binding domain. Membrane bound pancreatic Cholecystokinin Receptor was specifically labelled by 125I-ASD-(Thr28, Ahx31)-CCK-25-33 as a component of Mr ≈ 85,000-100,000. The efficiency of the photolabelling was 3–4%. Performing photolysis on [125I-ASD-(Thr28, Ahx31)-CCK-25-33-Receptor] complexes solubilized by CHAPS did not affect specificity of the labelling reaction but enhanced its efficiency so that up to 10% of the Receptor site population could be cross-linked. Several lectins were tested for their ability to recognize and purify the Cholecystokinin Receptor denaturated by Nonidet P-40. Wheat germ agglutinin provided the best recovery and purification rate. The Receptor was fully adsorbed on immobilized wheat germ agglutinin, while only a fraction was ...
Sandeep Nadella - One of the best experts on this subject based on the ideXlab platform.
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Cholecystokinin Receptor antagonist therapy decreases inflammation and fibrosis in chronic pancreatitis
Digestive Diseases and Sciences, 2020Co-Authors: Sandeep Nadella, Robin D Tucker, Hong Cao, Victor Ciofoaia, Bhaskar Kallakury, Jill P SmithAbstract:Background and Aims Chronic pancreatitis is associated with recurrent inflammation, pain, fibrosis, and loss of exocrine and endocrine pancreatic function and risk of cancer. We hypothesized that activation of the CCK Receptor contributes to pancreatitis and blockade of this pathway would improve chronic pancreatitis.
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a Cholecystokinin Receptor antagonist halts nonalcoholic steatohepatitis and prevents hepatocellular carcinoma
Digestive Diseases and Sciences, 2020Co-Authors: Robin D Tucker, Sandeep Nadella, Narayan Shivapurkar, Hong Cao, Victor Ciofoaia, Bhaskar Kallakury, Matthew Huber, Anita Safronenka, Annie J Kruger, Alexander KroemerAbstract:Nonalcoholic steatohepatitis (NASH) is a common inflammatory liver condition that may lead to cirrhosis and hepatocellular carcinoma (HCC). Risk factors for NASH include a saturated fat diet, altered lipid metabolism, and genetic and epigenetic factors, including microRNAs. Serum levels of Cholecystokinin (CCK) are elevated in mice and humans that consume a high-saturated fat diet. CCK Receptors (CCK-Rs) have been reported on fibroblasts which when activated can induce fibrosis; however, their role in hepatic fibrosis remains unknown. We hypothesized that elevated levels of CCK acting on the CCK-Rs play a role in the development of NASH and in NASH-associated HCC. We performed a NASH Prevention study and Reversal study in mice fed a saturated fat 75% choline-deficient–ethionine-supplemented (CDE) diet for 12 or 18 weeks. In each study, half of the mice received untreated drinking water, while the other half received water supplemented with the CCK-R antagonist proglumide. CCK-R expression was evaluated in mouse liver and murine HCC cells. CCK Receptor antagonist treatment not only prevented NASH but also reversed hepatic inflammation, fibrosis, and steatosis and normalized hepatic transaminases after NASH was established. Thirty-five percent of the mice on the CDE diet developed HCC compared with none in the proglumide-treated group. We found that CCK-BR expression was markedly upregulated in mouse CDE liver and HCC cells compared with normal hepatic parenchymal cells, and this expression was epigenetically regulated by microRNA-148a. These results support the novel role of CCK Receptors in the pathogenesis of NASH and HCC.
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Cholecystokinin Receptor Antagonist Therapy Decreases Inflammation and Fibrosis in Chronic Pancreatitis
Digestive Diseases and Sciences, 2019Co-Authors: Sandeep Nadella, Robin D Tucker, Hong Cao, Victor Ciofoaia, Bhaskar Kallakury, Jill P SmithAbstract:Background and Aims Chronic pancreatitis is associated with recurrent inflammation, pain, fibrosis, and loss of exocrine and endocrine pancreatic function and risk of cancer. We hypothesized that activation of the CCK Receptor contributes to pancreatitis and blockade of this pathway would improve chronic pancreatitis. Methods Two murine models were used to determine whether CCK Receptor blockade with proglumide could prevent and reverse histologic and biochemical features of chronic pancreatitis: the 6-week repetitive chronic cerulein injection model and the modified 75% choline-deficient ethionine (CDE) diet. In the CDE-fed model, half the mice received water supplemented with proglumide, for 18 weeks. After chronic pancreatitis was established in the cerulein model, half the mice were treated with proglumide and half with water. Histology was scored in a blinded fashion for inflammation, fibrosis and acinar ductal metaplasia (ADM) and serum lipase levels were measured. RNA was extracted and examined for differentially expressed fibrosis genes. Results Proglumide therapy decreased pancreatic weight in the CDE diet study and the cerulein-induced chronic pancreatitis model. Fibrosis, inflammation, and ADM scores were significantly reduced in both models. Lipase values improved with proglumide but not in controls in both models. Proglumide decreased pancreas mRNA expression of amylase, collagen-4, and TGFβR2 gene expression by 44, 38, and 25%, respectively, compared to control mice. Conclusion New strategies are needed to decreased inflammation and reduce fibrosis in chronic pancreatitis. CCK Receptor antagonist therapy may improve chronic pancreatitis by reversing fibrosis and inflammation. The decrease in ADM may reduce the risk of the development of pancreatic cancer.
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Cholecystokinin Receptor targeted polyplex nanoparticle inhibits growth and metastasis of pancreatic cancer
Cellular and molecular gastroenterology and hepatology, 2018Co-Authors: Julian Burks, Sandeep Nadella, Abdullah Mahmud, Charoen Mankongpaisarnrung, Juan Wang, Jongin Hahm, Robin D Tucker, Narayan Shivapurkar, Stephan T Stern, Jill P SmithAbstract:Background & Aims Pancreatic ductal adenocarcinoma (PDAC) remains the most aggressive malignancy with the lowest 5-year survival rate of all cancers in part owing to the lack of tumor-specific therapy and the rapid metastatic nature of this cancer. The gastrointestinal peptide gastrin is a trophic peptide that stimulates growth of PDAC in an autocrine fashion by interaction with the Cholecystokinin Receptor that is overexpressed in this malignancy. Methods We developed a therapeutic novel polyplex nanoparticle (NP) that selectively targets the Cholecystokinin Receptor on PDAC. The NP was characterized in vitro and stability testing was performed in human blood. The effects of the target-specific NP loaded with gastrin small interfering RNA (siRNA) was compared with an untargeted NP and with an NP loaded with a scrambled siRNA in vitro and in 2 orthotopic models of PDAC. A polymerase chain reaction metastasis array examined differentially expressed genes from control tumors compared with tumors of mice treated with the targeted polyplex NP. Results The polyplex NP forms a micelle that safely delivers specific gastrin siRNA to the tumor without off-target toxicity. Consistent with these findings, cellular uptake was confirmed only with the targeted fluorescently labeled NP by confocal microscopy in vitro and by IVIS fluorescent based imaging in mice bearing orthotopic pancreatic cancers but not found with untargeted NPs. Tumor uptake and release of the gastrin siRNA NP was verified by decreased cellular gastrin gene expression by quantitative reverse-transcription polymerase chain reaction and peptide expression by immunohistochemistry. Growth of PDAC was inhibited in a dose-related fashion in cell culture and in vivo. The targeted NP therapy completely blocked tumor metastasis and altered tumor-specific genes. Conclusions Our polyplex nanoparticle platform establishes both a strong foundation for the development of Receptor-targeted therapeutics and a unique approach for the delivery of siRNA in vivo, thus warranting further exploration of this approach in other types of cancers.