Development of a Highly Selective Allosteric Antagonist Radioligand for the Type 1 Cholecystokinin Receptor and Elucidation of Its Molecular Basis of Binding

Dong, Maoqing; Vattelana, Ashton M.; Lam, Polo C.‐H.; Orry, Andrew; Abagyan, Ruben; Christopoulos, Arthur; Sexton, Patrick M.; Haines, David R.; Miller, Laurence J.

doi:10.1124/mol.114.095430

Cited by 11 publications

(10 citation statements)

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“…Synthetic CCK-(26 -33) (CCK8) was purchased from Bachem (Torrance, CA). The benzodiazepine CCK1R antagonist BDZ-1 was provided by Professor Phillip Portoghese (1), the CCK1R antagonist T-0632 was prepared as we described previously (16), and the 1,5-benzodiazepine agonist GI181771X (2) was provided by GlaxoSmithKline Research Laboratories (Research Triangle Park, NC). Tauroursodeoxycholic acid (TUDC), taurochenodeoxycholic acid (TCDC), and 25-hydroxycholesterol were purchased from Sigma-Aldrich (St. Louis, MO), Quest fluo 8-AM from AAT Bioquest (Sunnyvale, CA), soybean trypsin inhibitor from Gibco Life Technologies (Grand Island, NY), bovine serum albumin from Equitech-Bio (Kerrville, TX), and lipoprotein-deficient serum from Intracel Resources (Frederick, MD).…”

Section: Methodsmentioning

confidence: 99%

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Impact of ursodeoxycholic acid on a CCK1R cholesterol-binding site may contribute to its positive effects in digestive function

Desai

Dong

Harikumar

et al. 2015

American Journal of Physiology-Gastrointestinal and Liver Physi

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Dysfunction of the type 1 cholecystokinin (CCK) receptor (CCK1R) as a result of increased gallbladder muscularis membrane cholesterol has been implicated in the pathogenesis of cholesterol gallstones. Administration of ursodeoxycholic acid, which is structurally related to cholesterol, has been shown to have beneficial effects on gallstone formation. Our aims were to explore the possible direct effects and mechanism of action of bile acids on CCK receptor function. We studied the effects of structurally related hydrophobic chenodeoxycholic acid and hydrophilic ursodeoxycholic acid in vitro on CCK receptor function in the setting of normal and elevated membrane cholesterol. We also examined their effects on a cholesterol-insensitive CCK1R mutant (Y140A) disrupting a key site of cholesterol action. The results show that, similar to the impact of cholesterol on CCK receptors, bile acid effects were limited to CCK1R, with no effects on CCK2R. Chenodeoxycholic acid had a negative impact on CCK1R function, while ursodeoxycholic acid had no effect on CCK1R function in normal membranes but was protective against the negative impact of elevated cholesterol on this receptor. The cholesterol-insensitive CCK1R mutant Y140A was resistant to effects of both bile acids. These data suggest that bile acids compete with the action of cholesterol on CCK1R, probably by interacting at the same site, although the conformational impact of each bile acid appears to be different, with ursodeoxycholic acid capable of correcting the abnormal conformation of CCK1R in a high-cholesterol environment. This mechanism may contribute to the beneficial effect of ursodeoxycholic acid in reducing cholesterol gallstone formation.

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Section: Methodsmentioning

confidence: 99%

“…Nonspecific binding was determined in the presence of 1 M CCK and represented Ͻ10% of total binding. The whole cell binding assay was also used for CCK1R antagonist T-0632 binding using 125 I-T-0632, as we described previously (16).…”

Section: Methodsmentioning

confidence: 99%

Impact of ursodeoxycholic acid on a CCK1R cholesterol-binding site may contribute to its positive effects in digestive function

Desai

Dong

Harikumar

et al. 2015

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…Although, we observed some dose dependency, the levels of apparent increase did not reach statistical significance. At 10 pM, CCK is at the bottom end of the CCK-R A binding curve although this concentration is capable of inducing near maximal increase in intracellular Ca 2+ (Dong et al, 2014 ). Currently, the reasons why higher doses of CCK did not give statistically significant increases remain obscure.…”

Section: Discussionmentioning

confidence: 99%

Sulfated Cholecystokinin-8 Promotes CD36—Mediated Fatty Acid Uptake into Primary Mouse Duodenal Enterocytes

2017

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Cholecystokinin (CCK) is an archetypal incretin hormone secreted by intestinal enteroendocrine cells (EEC) in response to ingested nutrients. The aim of this study was to determine whether CCK modulates enterocyte fatty acid uptake by primary mouse duodenal cells. Exposure of primary mouse duodenal cells to 10 pM sulfated CCK-8 caused a two fold increase in dodecanoic acid fatty acid (FA) uptake. The selective CCK A receptor antagonist loxiglumide (100 μM) completely abolished the CCK-8 induced FA uptake. The CD36 fatty acid translocase-specific inhibitor sulfo-N-succinimidyl oleate (1 μM) also completely inhibited CCK-8 induced FA uptake, as did treatment with 200 μM phloretin. Together these data show CCK induces FA uptake into duodenal enterocytes; this action involves the CCK-RA receptor and is carrier mediated by CD36.

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“…42,43 The allosteric nature of this small-molecule-binding site was further confirmed using pharmacologic analyses, including the kinetics of ligand dissociation and the impact of orthosteric and possible allosteric ligands on the functions of each other. [42][43][44] Radioiodinated small-molecule CCK1R ligands have been developed 45,46 that provide a means for direct displacement from this allosteric pocket to establish the molecular basis for the binding of small-molecule ligands. This was successfully applied to antagonist ligands to gain insights into the inactive conformation of this pocket.…”

Section: Characterization Of the Small-molecule Ligand-binding Pocketmentioning

confidence: 99%

Cholecystokinin-induced satiety, a key gut servomechanism that is affected by the membrane microenvironment of this receptor

et al. 2016

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The gastrointestinal (GI) tract has a central role in nutritional homeostasis, as location for food ingestion, digestion and absorption, with the gut endocrine system responding to and regulating these events, as well as influencing appetite. One key GI hormone with the full spectrum of these activities is cholecystokinin (CCK), a peptide released from neuroendocrine I cells scattered through the proximal intestine in response to fat and protein, with effects to stimulate gall bladder contraction and pancreatic exocrine secretion, to regulate gastric emptying and intestinal transit, and to induce satiety. There has been interest in targeting the type 1 CCK receptor (CCK1R) for drug development to provide non-caloric satiation as an aid to dieting and weight loss; however, there have been concerns about CCK1R agonists related to side effects and potential trophic impact on the pancreas. A positive allosteric modulator (PAM) of CCK action at this receptor without intrinsic agonist activity could provide a safer and more effective approach to long-term administration. In addition, CCK1R stimulus-activity coupling has been shown to be negatively affected by excess membrane cholesterol, a condition described in the metabolic syndrome, thereby potentially interfering with an important servomechanism regulating appetite. A PAM targeting this receptor could also potentially correct the negative impact of cholesterol on CCK1R function. We will review the molecular basis for binding natural peptide agonist, binding and action of small molecules within the allosteric pocket, and the impact of cholesterol. Novel strategies for taking advantage of this receptor for the prevention and management of obesity will be reviewed.International Journal of Obesity Supplements (2016) 6, S22-S27; doi:10.1038/ijosup.2016.5 INTRODUCTIONThe prevalence of obesity has been progressively increasing throughout the United States and around the world, contributing to a parallel increase in the prevalence of type 2 diabetes mellitus and its associated comorbidities. 1 These problems have been responsible for extraordinary morbidity, suffering, loss in productivity and premature mortality. In spite of major efforts to develop effective weight reduction diets, diet aids, medications, medical devices and surgical procedures, the trajectory for this continues in the wrong direction. Bariatric surgery has proven to be quite effective in patients with morbid obesity; 2 however, this is quite expensive and not scalable, certainly not keeping up with the increasing prevalence of the most severe end of this clinical continuum. The activity of acute dieting and exercise has been similarly effective in inducing weight loss; however, it has not been durable, with an extremely high incidence of regaining weight to or even beyond the starting point. After a long hiatus in approved drugs, three new diet medications have recently been approved, 3-5 but all carry concerns about safety, and there are requirements for registration and careful clinical follow-up, re...

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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

Cited by 11 publications

References 50 publications

Impact of ursodeoxycholic acid on a CCK1R cholesterol-binding site may contribute to its positive effects in digestive function

Impact of ursodeoxycholic acid on a CCK1R cholesterol-binding site may contribute to its positive effects in digestive function

Sulfated Cholecystokinin-8 Promotes CD36—Mediated Fatty Acid Uptake into Primary Mouse Duodenal Enterocytes

Cholecystokinin-induced satiety, a key gut servomechanism that is affected by the membrane microenvironment of this receptor

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