A high-precision fluorogenic cholesteryl ester transfer protein assay compatible with animal serum and 3456-well assay technology

Eveland, Suzanne S.; Milot, Denise P.; Guo, Qiu; Chen, Ying; Hyland, Sheryl A.; Peterson, Laurence B.; Jezequel-Sur, Sylvie; O'Donnell, Gregory; Zuck, Paul; Ferrer, Marc; Strulovici, Berta; Wagner, John A.; Tanaka, Wesley; Hilliard, Deborah; Laterza, Omar; Wright, Samuel D.; Sparrow, Carl P.; Anderson, Matt S.

doi:10.1016/j.ab.2007.06.003

Cited by 23 publications

(19 citation statements)

References 21 publications

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“…5D ). Western-blot analysis of protein samples from wild-type and CETP Tg mice confi rmed that cynomolgus CETP migrates at an apparent molecular weight of 68-71 kDa ( 69 ), which was not detected in radioactive neutral lipid transfer assay ( 64,65 ). In agreement with previous reports ( 35, 50 ), we found torcetrapib was a potent inhibitor of both CE and TG transfer, whereas dalcetrapib was a signifi cantly weaker inhibitor ( Table 1, 2 ).…”

Section: Discussionmentioning

confidence: 99%

“…Assay conditions similar to the in vitro fl uorogenic CE transfer assay ( 64 ) were chosen to allow direct comparison of an inhibitor's binding and biochemical activity. Binding assays contained 1× CETP buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA) with 0.005% Tween20, and 20 nM purifi ed recombinant CETP in a 100 l fi nal volume.…”

Section: Cetp Inhibitor Binding Assaymentioning

confidence: 99%

“…For determination of in vitro CETP transfer activity, a continuous fl uorogenic assay described previously ( 64 ) was used. In brief, this assay measures the CE or TG transfer half-reaction using a synthetic donor particle similar in size and density to HDL-C, which bears a core of fl uorescent CE or TG.…”

Section: In Vitro Fl Uorogenic Assays Of Cetp-mediated Ce and Tg Tranmentioning

confidence: 99%

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Biochemical characterization of cholesteryl ester transfer protein inhibitors

Ranalletta

Bierilo

Chen

et al. 2010

Journal of Lipid Research

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Section: Discussionmentioning

confidence: 99%

Section: Cetp Inhibitor Binding Assaymentioning

confidence: 99%

Section: In Vitro Fl Uorogenic Assays Of Cetp-mediated Ce and Tg Tranmentioning

confidence: 99%

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Biochemical characterization of cholesteryl ester transfer protein inhibitors

Ranalletta

Bierilo

Chen

et al. 2010

Journal of Lipid Research

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“…However, in this study we synthesized 7KFAEs by the modification of a previous protocol for the synthesis of labeled triglycerides [23]. We have found that this method provides a higher yield and fewer side products.…”

Section: Methodsmentioning

confidence: 99%

Extra-hepatic metabolism of 7-ketocholesterol occurs by esterification to fatty acids via cPLA2α and SOAT1 followed by selective efflux to HDL

Lee

Huang

Rodríguez

2015

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

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Accumulation of 7-ketocholesterol (7KCh) in tissues has been previously associated with various chronic aging diseases. Orally ingested 7KCh is readily metabolized by the liver and does not pose a toxicity threat. However, 7KCh formed in situ, usually associated with lipoprotein deposits, can adversely affect surrounding tissues by causing inflammation and cytotoxicity. In this study we have investigated various mechanisms for extra-hepatic metabolism of 7KCh (e.g. hydroxylation, sulfation) and found only esterification to fatty acids. The esterification of 7KCh to fatty acids involves the combined action of cytosolic phospholipase A2 alpha (cPLA2α) and sterol O-acyltransferase (SOAT1). Inhibition of either one of these enzymes ablates 7KCh-fatty acid ester (7KFAE) formation. The 7KFAEs are not toxic and do not induce inflammatory responses. However, they can be unstable and re-release 7KCh. The higher the degree of unsaturation, the more unstable the 7KFAE (e.g. 18:0>18:1>18:2>18:3>>20:4). Biochemical inhibition and siRNA knockdown of SOAT1 and cPLA2α ablated the 7KFAE synthesis in cultured ARPE19 cells, but had little effect on the 7KCh-induced inflammatory response. Overexpression of SOAT1 reduced the 7KCh-induced inflammatory response and provided some protection from cell death. This effect is likely due to the increased conversion of 7KCh to 7KFAEs, which reduced the intracellular 7KCh levels. Addition of HDL selectively increased the efflux of 7KFAEs and enhanced the effect of SOAT1 overexpression. Our data suggests an additional function for HDL in aiding extra-hepatic tissues to eliminate 7KCh by returning 7KFAEs to the liver for bile acid formation.

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“…This investigation led to the discovery of compound 5, which displayed promising in vitro CETP inhibition and reasonable rat PK (Figure 2). 18 Interestingly, the optimal stereochemistry at C-2 of the tetrahydroquinoxaline is opposite to that found in tetrahydroquinoline 3.…”

mentioning

confidence: 97%

Discovery of Novel Indoline Cholesterol Ester Transfer Protein Inhibitors (CETP) through a Structure-Guided Approach

Wilson

Kurukulasuriya

Reibarkh

et al. 2016

ACS Med. Chem. Lett.

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Using the collective body of known (CETP) inhibitors as inspiration for design, a structurally novel series of tetrahydroquinoxaline CETP inhibitors were discovered. An exemplar from this series, compound 5, displayed potent in vitro CETP inhibition and was efficacious in a transgenic cynomologus-CETP mouse HDL PD (pharmacodynamic) assay. However, an undesirable metabolic profile and chemical instability hampered further development of the series. A three-dimensional structure of tetrahydroquinoxaline inhibitor 6 was proposed from 1 H NMR structural studies, and this model was then used in silico for the design of a new class of compounds based upon an indoline scaffold. This work resulted in the discovery of compound 7, which displayed potent in vitro CETP inhibition, a favorable PK−PD profile relative to tetrahydroquinoxaline 5, and dose-dependent efficacy in the transgenic cynomologus-CETP mouse HDL PD assay.

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A high-precision fluorogenic cholesteryl ester transfer protein assay compatible with animal serum and 3456-well assay technology

Cited by 23 publications

References 21 publications

Biochemical characterization of cholesteryl ester transfer protein inhibitors

Biochemical characterization of cholesteryl ester transfer protein inhibitors

Extra-hepatic metabolism of 7-ketocholesterol occurs by esterification to fatty acids via cPLA2α and SOAT1 followed by selective efflux to HDL

Discovery of Novel Indoline Cholesterol Ester Transfer Protein Inhibitors (CETP) through a Structure-Guided Approach

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