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
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
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
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
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