4 research outputs found

    Measuring H<sub>2</sub><sup>18</sup>O Tracer Incorporation on a QQQ-MS Platform Provides a Rapid, Transferable Screening Tool for Relative Protein Synthesis

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    Intracellular proteins are in a state of flux, continually being degraded into amino acids and resynthesized into new proteins. The rate of this biochemical recycling process varies across proteins and is emerging as an important consideration in drug discovery and development. Here, we developed a triple-stage quadrupole mass spectrometry assay based on product ion measurements at unit resolution and H<sub>2</sub><sup>18</sup>O stable tracer incorporation to measure relative protein synthesis rates. As proof of concept, we selected to measure the relative in vivo synthesis rate of ApoB100, an apolipoprotein where elevated levels are associated with an increased risk of coronary heart disease, in plasma-isolated very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in a mouse in vivo model. In addition, serial time points were acquired to measure the relative in vivo synthesis rate of mouse LDL ApoB100 in response to vehicle, microsomal triacylglycerol transfer protein (MTP) inhibitor, and site-1 protease inhibitor, two potential therapeutic targets to reduce plasma ApoB100 levels at 2 and 6 h post-tracer-injection. The combination of H<sub>2</sub><sup>18</sup>O tracer with the triple quadrupole mass spectrometry platform creates an assay that is relatively quick and inexpensive to transfer across different biological model systems, serving as an ideal rapid screening tool for relative protein synthesis in response to treatment

    Measuring H<sub>2</sub><sup>18</sup>O Tracer Incorporation on a QQQ-MS Platform Provides a Rapid, Transferable Screening Tool for Relative Protein Synthesis

    No full text
    Intracellular proteins are in a state of flux, continually being degraded into amino acids and resynthesized into new proteins. The rate of this biochemical recycling process varies across proteins and is emerging as an important consideration in drug discovery and development. Here, we developed a triple-stage quadrupole mass spectrometry assay based on product ion measurements at unit resolution and H<sub>2</sub><sup>18</sup>O stable tracer incorporation to measure relative protein synthesis rates. As proof of concept, we selected to measure the relative in vivo synthesis rate of ApoB100, an apolipoprotein where elevated levels are associated with an increased risk of coronary heart disease, in plasma-isolated very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in a mouse in vivo model. In addition, serial time points were acquired to measure the relative in vivo synthesis rate of mouse LDL ApoB100 in response to vehicle, microsomal triacylglycerol transfer protein (MTP) inhibitor, and site-1 protease inhibitor, two potential therapeutic targets to reduce plasma ApoB100 levels at 2 and 6 h post-tracer-injection. The combination of H<sub>2</sub><sup>18</sup>O tracer with the triple quadrupole mass spectrometry platform creates an assay that is relatively quick and inexpensive to transfer across different biological model systems, serving as an ideal rapid screening tool for relative protein synthesis in response to treatment

    Use of [<sup>13</sup>C<sub>18</sub>] Oleic Acid and Mass Isotopomer Distribution Analysis to Study Synthesis of Plasma Triglycerides In Vivo: Analytical and Experimental Considerations

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    We have previously reported on a liquid chromatography–mass spectrometry method to determine the disposition of [<sup>13</sup>C<sub>18</sub>]-oleic acid following intravenous and oral administration in vivo. This approach has enabled us to study a variety of aspects of lipid metabolism including a quantitative assessment of triglyceride synthesis. Here we present a more rigorous evaluation of the constraints imposed upon the analytical method in order to generate accurate data using this stable-isotope tracer approach along with more detail on relevant analytical figures of merit including limits of quantitation, precision, and accuracy. The use of mass isotopomer distribution analysis (MIDA) to quantify plasma triglyceride synthesis is specifically highlighted, and a re-evaluation of the underlying mathematics has enabled us to present a simplified series of equations. The derivation of this MIDA model and the significance of all underlying assumptions are explored in detail, and examples are given of how it can successfully be applied to detect differences in plasma triglyceride synthesis in lean and high-fat diet fed mouse models. More work is necessary to evaluate the applicability of this approach to triglyceride stores with slower rates of turnover such as in adipose or muscle tissue; however, the present report provides investigators with the tools necessary to conduct such studies

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

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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 <b>5</b>, 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 <b>6</b> was proposed from <sup>1</sup>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 <b>7</b>, which displayed potent in vitro CETP inhibition, a favorable PK–PD profile relative to tetrahydroquinoxaline <b>5</b>, and dose-dependent efficacy in the transgenic cynomologus-CETP mouse HDL PD assay
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