Characterization of Two Distinct Modes of Drug Binding to Human Intestinal Fatty Acid Binding Protein

Abstract

The aqueous cytoplasm of cells poses a potentially significant barrier for many lipophilic drugs to reach their sites of action. Fatty acid binding proteins (FABPs) bind to poorly water-soluble fatty acids (FAs) and lipophilic compounds and facilitate their intracellular transport. Several structures of FA in complex with FABPs have been described, but data describing the binding sites of other lipophilic ligands including drugs are limited. Here the environmentally sensitive fluorophores, 1-anilinonapthalene 8-sulfonic acid (ANS), and 11-dansylamino undecanoic acid (DAUDA) were used to investigate drug binding to human intestinal FABP (hIFABP). Most drugs that bound hIFABP were able to displace both ANS and DAUDA. A notable exception was ketorolac, a non-steroidal anti-inflammatory drug that bound to hIFABP and displaced DAUDA but failed to displace ANS. Isothermal titration calorimetry revealed that for the majority of ligands including FA, ANS, and DAUDA, binding to hIFABP was exothermic. In contrast, ketorolac binding to hIFABP was endothermic and entropy-driven. The X-ray crystal structure of DAUDA-hIFABP revealed a FA-like binding mode where the carboxylate of DAUDA formed a network of hydrogen bonds with residues at the bottom of the binding cavity and the dansyl group interacted with residues in the portal region. In contrast, NMR chemical shift perturbation (CSP) data suggested that ANS bound only toward the bottom of the hIFABP cavity, whereas ketorolac occupied only the portal region. The CSP data further suggested that ANS and ketorolac were able to bind simultaneously to hIFABP, consistent with the lack of displacement of ANS observed by fluorescence and supported by a model of the ternary complex. The NMR solution structure of the ketorolac-hIFABP complex therefore describes a newly characterized, hydrophobic ligand binding site in the portal region of hIFABP.

Fatty Acid-binding Proteins 1 and 2 Differentially Modulate the Activation of Peroxisome Proliferator-activated Receptor α in a Ligand-selective Manner

“…This construct produced recombinant FABP1 with a noncleavable hexahistidine tag at the N terminus to aid purification. Conditions for expression, purification, and delipidation of FABP1 were identical to those reported previously for human FABP2 (23). The purity of the protein preparation was assessed by SDS-PAGE, and protein concentration was measured by UV-visible spectrometry by recording absorbance at 280 nm (A 280 ) using an extinction coefficient of 1490 M Ϫ1 cm…”

“…FABP2 was expressed and purified as described previously (23). Isothermal Titration Calorimetry (ITC) Data-ITC experiments were carried out using an iTC 200 microcalorimeter (MicroCal, Malvern UK) with coin shaped sample cell (200 l) at 37°C with stirring at 1000 rpm.…”

Fatty acid binding proteins have the potential to channel dietary fatty acids into enterocyte nuclei

“…BODIPY [boron dipyrromethene (4,4-difl uoro-5,7-dimethyl-4-bora-3a,4a-diaza-s -indacene)] and BODIPY-FL analogs (Life Technologies) were resuspended in methanol (1 µg/µl) and mixed well with a CEY emulsion (10 µl/ml), prepared from dried CEY mixed in water using an IKA Ultra-Turrax Homogenizer. The BODIPY 493/503 , BODIPY-FLC 5 (D3834), or BODIPY-FLC 12 (D3822)-CEY emulsion was maintained at 37°C for 10 min for the solvent to evaporate. BODIPY 493/503 (D3922) was used as a negative control.…”

“…Fluorescence emission spectra of BODIPY-FLC 5 or BODIPY-FLC 12 in the presence and absence of Fabp1b.1 were determined at 25°C using a Cary Eclipse fl uorescence spectrophotometer (Agilent Technologies, Santa Clara). BODIPY-FLC 5 or BODIPY-FLC 12 2 mM stock solutions in ethanol were diluted in PBS prior to mixing and incubating for 2 min with purifi ed FABP1b.1.…”

“…BODIPY-FLC 5 or BODIPY-FLC 12 2 mM stock solutions in ethanol were diluted in PBS prior to mixing and incubating for 2 min with purifi ed FABP1b.1. The protein sample was previously dissolved with 30 mM Tris HCl, pH 8.3, buffer.…”