Drugs Form Ternary Complexes with Human Liver Fatty Acid Binding Protein (FABP1) and FABP1 Binding Alters Drug Metabolism.

Liver fatty acid binding protein (FABP1) binds diverse endogenous lipids and is highly expressed in the human liver. Binding to FABP1 alters the metabolism and homeostasis of endogenous lipids in the liver. Drugs have also been shown to bind to rat FABP1, but limited data is available for human FABP1 (hFABP1). FABP1 has a large binding pocket and up to two fatty acids can bind to FABP1 simultaneously. We hypothesized that drug binding to hFABP1 results in formation of ternary complexes and that FABP1 binding alters drug metabolism. To test these hypotheses, native protein mass spectrometry (MS) and fluorescent 11-(dansylamino)undecanoic acid (DAUDA) displacement assays were used to characterize drug binding to hFABP1, and diclofenac oxidation by cytochrome P450 2C9 (CYP2C9) was studied in the presence and absence of hFABP1. DAUDA binding to hFABP1 involved high (Kd,1=0.2 µM) and low affinity (Kd,2 >10 µM) binding sites. Nine drugs bound to hFABP1 with Kd values ranging from 1 to 20 µM. None of the tested drugs completely displaced DAUDA from hFABP1 and fluorescence spectra showed evidence of ternary complex formation. Formation of DAUDA-hFABP1-diclofenac ternary complex was verified with native MS. Docking predicted diclofenac binding in the portal region of FABP1 with DAUDA in the binding cavity. The kcat of diclofenac hydroxylation by CYP2C9 was decreased by ~50% (p<0.01) in the presence of FABP1. Together, these results suggest that drugs form ternary complexes with hFABP1 and that hFABP1 binding in the liver will alter drug metabolism and clearance. Significance Statement Many commonly prescribed drugs bind FABP1 forming ternary complexes with FABP1 and the fluorescent fatty acid DAUDA. These findings suggests that drugs will bind to apo-FABP1 and fatty acid bound FABP1 in the human liver. The high expression of FABP1 in the liver, together with drug binding to FABP1 may alter drug disposition processes in vivo.

Drugs Form Ternary Complexes with Human Liver Fatty Acid Binding Protein (FABP1) and FABP1 Binding Alters Drug Metabolism.

Liver fatty acid binding protein (FABP1) binds diverse endogenous lipids and is highly expressed in the human liver. Binding to FABP1 alters the metabolism and homeostasis of endogenous lipids in the liver. Drugs have also been shown to bind to rat FABP1, but limited data is available for human FABP1 (hFABP1). FABP1 has a large binding pocket and multiple fatty acids can bind to FABP1 simultaneously. We hypothesized that drug binding to hFABP1 results in formation of ternary complexes and that FABP1 binding alters drug metabolism. To test these hypotheses native protein mass spectrometry (MS) and fluorescent 11-(dansylamino)undecanoic acid (DAUDA) displacement assays were used to characterize drug binding to hFABP1 and diclofenac oxidation by cytochrome P450 2C9 (CYP2C9) was studied in the presence and absence of hFABP1. DAUDA binding to hFABP1 involved high (Kd,1=0.2 µM) and low affinity (Kd,2 >10 µM) binding sites. Nine drugs bound to hFABP1 with Kd values ranging from 1 to 20 µM. None of the tested drugs completely displaced DAUDA from hFABP1 and fluorescence spectra showed evidence of ternary complex formation. Formation of DAUDA-diclofenac-hFABP1 ternary complex was verified with native MS. Docking placed diclofenac in the portal region of FABP1 with DAUDA in the binding cavity. Presence of hFABP1 decreased the kcat and Km,u of diclofenac with CYP2C9 by ~50% suggesting that hFABP1 binding in the liver will alter drug metabolism and clearance. Together, these results suggest that drugs form ternary complexes with hFABP1 and that hFABP1 interacts with CYP2C9.

Are the Gas-Phase Structures of Molecular Elephants Enduring or Ephemeral? Results from Time-Dependent, Tandem Ion Mobility.

The structural stability of biomolecules in the gas phase remains an important topic in mass spectrometry applications for structural biology. Here, we evaluate the kinetic stability of native-like protein ions using time-dependent, tandem ion mobility (IM). In these tandem IM experiments, ions of interest are mobility-selected after a first dimension of IM and trapped for up to ∼14 s. Time-dependent, collision cross section distributions are then determined from separations in a second dimension of IM. In these experiments, monomeric protein ions exhibited structural changes specific to both protein and charge state, whereas large protein complexes did not undergo resolvable structural changes on the timescales of these experiments. We also performed energy-dependent experiments, i.e., collision-induced unfolding, as a comparison for time-dependent experiments to understand the extent of unfolding. Collision cross section values observed in energy-dependent experiments using high collision energies were significantly larger than those observed in time-dependent experiments, indicating that the structures observed in time-dependent experiments remain kinetically trapped and retain some memory of their solution-phase structure. Although structural evolution should be considered for highly charged, monomeric protein ions, these experiments demonstrate that higher-mass protein ions can have remarkable kinetic stability in the gas phase.

A Flexible, Modular Platform for Multidimensional Ion Mobility of Native-like Ions.

Native ion mobility (IM) mass spectrometry (MS) is used to probe the size, shape, and assembly of biomolecular complexes. IM-IM-MS can increase the amount of information available in structural studies by isolating subpopulations of structures for further analysis. Previously, IM-IM-MS has been implemented using the Structures for Lossless Ion Manipulations (SLIM) architecture to probe the structural stability of gas-phase protein ions. Here, a new multidimensional IM instrument constructed from SLIM devices is characterized using multiple operational modes. In this new design, modular devices are used to perform all ion manipulations, including initial accumulation, injection, separation, selection, and trapping. Using single-dimension IM, collision cross section (Ω) values are determined for a set of native-like ions. These Ω values are within 3% of those reported previously based on measurements using RF-confining drift cells. Tandem IM experiments are performed on a sample of ubiquitin ions that contains both compact and partially unfolded structures, demonstrating that this platform can isolate subpopulations of structures. Finally, additional modes of analysis, including multiplexed IM and inverse IM, are demonstrated using this platform. The ability of this platform to quickly switch between different modes of IM analysis makes it a highly flexible tool for studying protein structures and dynamics.

Enhancing the Depth of Analyses with Next-Generation Ion Mobility Experiments.

Recent developments in ion mobility (IM) technology have expanded the capability to separate and characterize gas-phase ions of biomolecules, especially when paired with mass spectrometry. This next generation of IM technology has been ushered in by creative innovation focused on both instrument architectures and how electric fields are applied. In this review, we focus on the application of high-resolution and multidimensional IM to biomolecular analyses, encompassing the fields of glycomics, lipidomics, peptidomics, and proteomics. We highlight selected research that demonstrates the application of the new IM toolkit to challenging biomolecular systems. Through our review of recently published literature, we outline the current strengths of respective technologies and perspectives for future applications.