FATTY ACID BINDING PROTEINS (FABPs): SPECTROSCOPIC INTERACTION STUDIES WITH MEMBRANE MIMETICS AND LIPID-FUNCTIONALIZED DRUGS

The fatty acid binding proteins (FABPs) are a class of cytosolic proteins known to participate in lipid transport and homoeostasis. These relatively small biomolecules are responsible for the translocation of hydrophobic or amphiphilic molecules across the cell, delivering their cargo to the cell membrane or to other cellular components. Some members of the FABP family, called bile acid binding proteins (BABPs), are responsible for the translocation of bile acid (BA) in the enterocytes and in the hepatocytes. This transport is achieved mainly through a protein mediated mechanism that involves BABPs as intracellular carriers. Bile acids undergo a recycling pathway between the intestine and the liver, called “enterohepatic circulation”, which assure the recovery of these molecules and their subsequent reutilization. Alterations of intracellular BA transport are linked to cholestatic diseases and BA accumulation leads to liver damage and may promote the development of liver tumors. A complete understanding of the functional role of BABPs necessitates a detailed description of the mechanisms of ligand uptake from and release to the cell membranes. These mechanisms possibly involve a direct collision of the protein with the membrane, in analogy with other FABP proteins. However, the binding equilibria describing the partitioning of these proteins between free and membrane-bound states remain poorly characterized. In the first part of the project the details the mechanism of interaction of apo BABP with the membrane mimetic systems were investigated via NMR spectroscopy. Despite the system under study prevents a direct investigation of the lipid- bound protein state by solution NMR we applied advance NMR methods for the investigation on the so-called NMR invisible “dark state” of the liposome bound protein. An application of the acquired knowledge on FABPs/lipid interactions has been recently proposed for the development of hepatospecific contrast agents (CAs) for magnetic resonance imaging (MRI). About one-third of MRI clinical scans are carried out in the presence of gadolinium-agents because they add relevant physiological information to the superb anatomical resolution attainable with this technique. There is a growing interest in the development of CAs displaying high tissue specificity and high intrinsic relaxivity obtained with slowly-tumbling molecules binding. In the second part of the project two lipid-functionalized Gd-chelates (AAZTAC17 and B22626) were investigated here for targeting to human liver-fatty acid binding protein (hL-FABP) as a means to increase sensitivity and specificity of intracellular-directed MRI probes. The former consists of a long aliphatic chain bound to the AAZTA coordination cage, the latter has a bile acid-like body linked to the basic unit of DTPA which chelates Gd. The binding affinities of the two selected series of Gd(III) chelates molecules to a liver cytosolic fatty acid transporter, have been determined through relaxivity measurements while the interaction modes and localisation of binding site were characterized performing NMR experiments in solution exploiting the relaxation properties of Gd complexes. These preliminary studies will serve in a medicinal chemistry approach to the design of new Gd-based contrast agents for MRI.