Measuring H₂¹⁸O 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 H₂¹⁸O 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 H₂¹⁸O 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 H₂¹⁸O 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 H₂¹⁸O 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 H₂¹⁸O 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

A Maltose-Binding Protein Fusion Construct Yields a Robust Crystallography Platform for MCL1

<div><p>Crystallization of a maltose-binding protein MCL1 fusion has yielded a robust crystallography platform that generated the first apo MCL1 crystal structure, as well as five ligand-bound structures. The ability to obtain fragment-bound structures advances structure-based drug design efforts that, despite considerable effort, had previously been intractable by crystallography. In the ligand-independent crystal form we identify inhibitor binding modes not observed in earlier crystallographic systems. This MBP-MCL1 construct dramatically improves the structural understanding of well-validated MCL1 ligands, and will likely catalyze the structure-based optimization of high affinity MCL1 inhibitors.</p></div

The structure of Apo MBP-MCL1 determined at 1.90 Å.

<p>(A) The MBP domain (red) is connected by a short GS linker (orange) to MCL1 173–321 (blue). A portion of alpha helix four is not ordered in the structure (red dashed-line). Maltose ligand is shown in yellow. (B) The MCL1 domain is structurally very similar to the NMR structure of Apo-MCL1 (gray).</p

Comparison of PDB 4HW3 and MBP-MCL1 with fragment 4.

<p>The structure of MBP-MCL1 with fragment <b>4</b> (yellow) determined to 2.4 Å (blue) overlaid with the structure of MCL1 171–323 determined at 2.4 Å (PDB ID 4HW3, gray). The carboxylic acid of 4HW3 adopts multiple conformations depending on the chain; only chain A is shown for clarity.</p

Discovery of an Orally Bioavailable Benzimidazole Diacylglycerol Acyltransferase 1 (DGAT1) Inhibitor That Suppresses Body Weight Gain in Diet-Induced Obese Dogs and Postprandial Triglycerides in Humans

Modification of a gut restricted class of benzimidazole DGAT1 inhibitor <b>1</b> led to <b>9</b> with good oral bioavailability. The key structural changes to <b>1</b> include bioisosteric replacement of the amide with oxadiazole and α,α-dimethylation of the carboxylic acid, improving DGAT1 potency and gut permeability. Since DGAT1 is expressed in the small intestine, both <b>1</b> and <b>9</b> can suppress postprandial triglycerides during acute oral lipid challenges in rats and dogs. Interestingly, only <b>9</b> was found to be effective in suppressing body weight gain relative to control in a diet-induced obese dog model, suggesting the importance of systemic inhibition of DGAT1 for body weight control. <b>9</b> has advanced to clinical investigation and successfully suppressed postprandial triglycerides during an acute meal challenge in humans

Binding affinity (K_D) of ligands to MCL1 and MBP-MCL1.

<p>All experiments are n ≥ 3, and averaged values for K_D are reported.</p><p>Binding affinity (K_D) of ligands to MCL1 and MBP-MCL1.</p

The structure of MBP-MCL1 bound to fragment 5 determined at 1.9 Å.

<p>Fragment <b>5</b> (yellow) binds similarly in comparison to the elaborated ligand from PDB ID 4OQ6 (gray).</p

The conformational flexibility of the binding pocket of MCL1.

<p>Surface representations are shown as side views and ligands are shown as yellow sticks. (A and B) Fragment 4 maps onto L78 of NoxaB from PDB ID 2NLA, with only minor structural perturbation of the BH3-binding groove of MCL1. In contrast, binding of fragment 6 creates a significant pocket (C) which is further expanded upon binding of ligand 1 (D).</p

MCL1 ligands used in co-crystallization experiments.

<p>MCL1 ligands used in co-crystallization experiments.</p