Characterizing the Oligomers Distribution along the Aggregation Pathway of Amyloid Aß1-40 by NMR.

This study delves into the early aggregation process of the Aß1-40 amyloid peptide, elucidating the associated oligomers distribution. Motivated by the acknowledged role of small oligomers in the neurotoxic damage linked to Alzheimer's disease, we present an experimental protocol for preparing 26-O-acyl isoAß1-40, a modified Aß1-40 peptide facilitating rapid isomerization to the native amide form at neutral pH. This ensures seed-free solutions, minimizing experimental variability. Additionally, we demonstrate the efficacy of coupling NMR diffusion ordered spectroscopy (DOSY) with the Inverse Laplace Transform (ILT) reconstruction method, for effective characterization of early aggregation processes. This innovative approach efficiently maps oligomers distributions across a wide spectrum of initial peptide concentrations offering unique insights into the evolution of oligomers relative populations. As a proof of concept, we demonstrate the efficacy of our approach assessing the impact of Epigallocathechin gallate, a known remodeling agent of amyloid fibrils, on the oligomeric distributions of aggregated Aß1-40. The DOSY-ILT proposed approach stands as a robust and discriminating asset, providing a powerful strategy for rapidly gaining insight into potential inhibitors' impact on the aggregation process.

Antibacterial Properties of Polyurethane Foams Additivated with Terpenes from a Bio-Based Polyol.

Water-blown polyurethane (PU) foams were prepared by bio-polyols from epoxidized linseed oils and caprylic acid in combination with toluene diisocianate (TDI). A series of terpenes (menthol, geraniol, terpineol, and borneol), natural compounds with recognized antibacterial properties, were included in the starting formulations to confer bactericidal properties to the final material. Foams additivated with Irgasan®, a broad-spectrum antimicrobial molecule, were prepared as reference. The bactericidal activity of foams against planktonic and sessile E. coli (ATCC 11229) and S. aureus (ATCC 6538) was evaluated following a modified AATCC 100-2012 static method. Menthol-additivated foams showed broad-spectrum antibacterial activity, reducing Gram+ and Gram- viability by more than 60%. Foams prepared with borneol and terpineol showed selective antibacterial activity against E. coli and S. aureus, respectively. NMR analysis of foams leaking in water supported a bactericidal mechanism mediated by contact killing rather than molecule release. The results represent the proof of concept of the possibility to develop bio-based PU foams with intrinsic bactericidal properties through a simple and innovative synthetic approach.

Molecular Basis of the Antiangiogenic Action of Rosmarinic Acid, a Natural Compound Targeting Fibroblast Growth Factor-2/FGFR Interactions.

Fibroblast growth factor (FGF2)/fibroblast growth factor receptor (FGFR) signalling plays a major role both in physiology and in several pathologies, including cancer development, metastasis formation and resistance to therapy. The development of small molecules, acting extracellularly to target FGF2/FGFR interactions, has the advantage of limiting the adverse effects associated with current intracellular FGFR inhibitors. Herein, we discuss the ability of the natural compound rosmarinic acid (RA) to induce FGF2/FGFR complex dissociation. The molecular-level description of the FGF2/FGFR/RA system, by NMR spectroscopy and docking, clearly demonstrates that RA binds to the FGFR-D2 domain and directly competes with FGF2 for the same binding site. Direct and allosteric perturbations combine to destabilise the complex. The proposed molecular mechanism is validated by cellular studies showing that RA inhibits FGF2-induced endothelial cell proliferation and FGFR activation. Our results can serve as the basis for the development of new extracellular inhibitors of the FGF/FGFR pathways.

The study of transient protein-nanoparticle interactions by solution NMR spectroscopy.

The rapid development of novel nanoscale materials for applications in biomedicine urges an improved characterization of the nanobio interfaces. Nanoparticles exhibit unique structures and properties, often different from the corresponding bulk materials, and the nature of their interactions with biological systems remains poorly characterized. Solution NMR spectroscopy is a mature technique for the investigation of biomolecular structure, dynamics, and intermolecular associations, however its use in protein-nanoparticle interaction studies remains scarce and highly challenging, particularly due to unfavorable hydrodynamic properties of most nanoscale assemblies. Nonetheless, recent efforts demonstrated that a number of NMR observables, such as chemical shifts, signal intensities, amide exchange rates and relaxation parameters, together with newly designed saturation transfer experiments, could be successfully employed to characterize the orientation, structure and dynamics of proteins adsorbed onto nanoparticle surfaces. This review provides the first survey and critical assessment of the contributions from solution NMR spectroscopy to the study of transient interactions between proteins and both inorganic (gold, silver, and silica) and organic (polymer, carbon and lipid based) nanoparticles. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.

The role of dynamics in modulating ligand exchange in intracellular lipid binding proteins.

Lipids are essential for many biological processes and crucial in the pathogenesis of several diseases. Intracellular lipid-binding proteins (iLBPs) provide mobile hydrophobic binding sites that allow hydrophobic or amphipathic lipid molecules to penetrate into and across aqueous layers. Thus iLBPs mediate the lipid transport within the cell and participate to a spectrum of tissue-specific pathways involved in lipid homeostasis. Structural studies have shown that iLBPs' binding sites are inaccessible from the bulk, implying that substrate binding should involve a conformational change able to produce a ligand entry portal. Many studies have been reported in the last two decades on iLBPs indicating that their dynamics play a pivotal role in regulating ligand binding and targeted release. The ensemble of reported data has not been reviewed until today. This review is thus intended to summarize and possibly generalize the results up to now described, providing a picture which could help to identify the missing notions necessary to improve our understanding of the role of dynamics in iLBPs' molecular recognition. Such notions would clarify the chemistry of lipid binding to iLBPs and set the basis for the development of new drugs.

Structural dynamics of bacterial translation initiation factor IF2.

Bacterial translation initiation factor IF2 promotes ribosomal subunit association, recruitment, and binding of fMet-tRNA to the ribosomal P-site and initiation dipeptide formation. Here, we present the solution structures of GDP-bound and apo-IF2-G2 of Bacillus stearothermophilus and provide evidence that this isolated domain binds the 50 S ribosomal subunit and hydrolyzes GTP. Differences between the free and GDP-bound structures of IF2-G2 suggest that domain reorganization within the G2-G3-C1 regions underlies the different structural requirements of IF2 during the initiation process. However, these structural signals are unlikely forwarded from IF2-G2 to the C-terminal fMet-tRNA binding domain (IF2-C2) because the connected IF2-C1 and IF2-C2 modules show completely independent mobility, indicating that the bacterial interdomain connector lacks the rigidity that was found in the archaeal IF2 homolog aIF5B.

Encapsulation of a rhodamine dye within a bile acid binding protein: toward water processable functional bio host-guest materials.

New strategies are requested for the preparation of bioinspired host-guest complexes to be employed in technologically relevant applications, as sensors and optoelectronic devices. We report here a new approach employing a single monomeric protein as host for the strongly fluorescent rhodamine dye. The selected protein, belonging to the intracellular lipid binding protein family, fully encapsulates one rhodamine molecule inside its cavity forming a host-guest complex stabilized by H and π-hydrogen bonds, a salt bridge, and favorable hydrophobic contacts, as revealed by the NMR derived structural model. The protein-dye solutions are easily processable and form homogeneous thin films exhibiting excellent photophysical and morphological properties, as derived from photoluminescence and AFM data. The obtained results represent the proof of concept of the viability of this bio host-guest system for the development of bioinspired optoelectronic devices.

Flexible Polyurethane Foams from Bio-Based Polyols: Prepolymer Synthesis and Characterization.

Bio-polyols (BPOs), characterized by a hydroxyl number up to around 90 mg KOH/g, narrow polydispersity index and relatively low molecular mass up to 2000 g/mol, were synthetized from partially and completely epoxidized soybean and linseed oils and caprylic acid or 3-phenyl butyric acid. These BPOs were used in the presence of toluene diisocyanate to produce polyurethane (PU) foams by using a quasi-prepolymer method involving a two-step reaction. A detailed structural investigation of the prepolymers from toluene diisocyanate and both BPOs and polypropylene glycol was conducted by SEC and solution NMR. The apparent density of the foams was in the range of 40-90 kg/m3, with higher values for foams from the aromatic acid. All the foams showed an open-cell structure with uniform and regular shape and uniform size. The specific Young's moduli and compression deflection values suggest superior mechanical properties than the reference foams. The novel synthesized polyurethanes are excellent candidates to partially replace petroleum-based materials.

Bacteria as sensors: Real-time NMR analysis of extracellular metabolites detects sub-lethal amounts of bactericidal molecules released from functionalized materials.

BACKGROUND: Cells exposed to stress factors experience time-dependent variations of metabolite concentration, acting as reliable sensors of the effective concentration of drugs in solution. NMR can detect and quantify changes in metabolite concentration, thus providing an indirect estimate of drug concentration. The quantification of bactericidal molecules released from antimicrobial-treated biomedical materials is crucial to determine their biocompatibility and the potential onset of drug resistance. METHODS: Real-time NMR measurements of extracellular metabolites produced by bacteria grown in the presence of known concentrations of an antibacterial molecule (irgasan) are employed to quantify the bactericidal molecule released from antimicrobial-treated biomedical devices. Viability tests assess their activity against E. coli and S. aureus planktonic and sessile cells. AFM and contact angle measurements assisted in the determination of the mechanism of antibacterial action. RESULTS: NMR-derived concentration kinetics of metabolites produced by bacteria grown in contact with functionalized materials allows for indirectly evaluating the effective concentration of toxic substances released from the device, lowering the detection limit to the nanomolar range. NMR, AFM and contact angle measurements support a surface-killing mechanism of action against bacteria. CONCLUSIONS: The NMR based approach provides a reliable tool to estimate bactericidal molecule release from antimicrobial materials. GENERAL SIGNIFICANCE: The novelty of the proposed NMR-based strategy is that it i) exploits bacteria as sensors of the presence of bactericidal molecules in solution; ii) is independent of the chemo-physical properties of the analyte; iii) establishes the detection limit to nanomolar concentrations.

A disulfide bridge allows for site-selective binding in liver bile acid binding protein thereby stabilising the orientation of key amino acid side chains.

The presence of a disulfide bridge in liver bile acid binding protein (L-BABP/S-S) allows for site-selective binding of two bile acids, glycochenodeoxycholic (GCDA) and glycocholic acid (GCA), differing only in the presence of a hydroxyl group. The protein form devoid of the disulfide bridge (L-BABP) binds both bile salts without discriminating ability. We investigate the determinants of the molecular recognition process in the formation of the heterotypic L-BABP/S-S complex with GCDA [corrected] and GCA [corrected] located in the superficial and inner protein sites, respectively. The comparison of the NMR spectroscopy structure of heterotypic holo L-BABP/S-S, the first reported for this protein family, with that of the homotypic L-BABP complex demonstrates that the introduction of a S-S link between adjacent strands changes the conformation of three key residues, which function as hot-spot mediators of molecular discrimination. The favoured χ(1) rotameric states (t, g(+) and g(-) for E99, Q100 and E109 residues, respectively) allow the onset of an extended intramolecular hydrogen-bond network and the consequent stabilisation of the side-chain orientation of a buried histidine, which is capable of anchoring a specific ligand.

Non-peptidic thrombospondin-1 mimics as fibroblast growth factor-2 inhibitors: an integrated strategy for the development of new antiangiogenic compounds.

Endogenous inhibitors of angiogenesis, such as thrombospondin-1 (TSP-1), are promising sources of therapeutic agents to treat angiogenesis-driven diseases, including cancer. TSP-1 regulates angiogenesis through different mechanisms, including binding and sequestration of the angiogenic factor fibroblast growth factor-2 (FGF-2), through a site located in the calcium binding type III repeats. We hypothesized that the FGF-2 binding sequence of TSP-1 might serve as a template for the development of inhibitors of angiogenesis. Using a peptide array approach followed by binding assays with synthetic peptides and recombinant proteins, we identified a FGF-2 binding sequence of TSP-1 in the 15-mer sequence DDDDDNDKIPDDRDN. Molecular dynamics simulations, taking the full flexibility of the ligand and receptor into account, and nuclear magnetic resonance identified the relevant residues and conformational determinants for the peptide-FGF interaction. This information was translated into a pharmacophore model used to screen the NCI2003 small molecule databases, leading to the identification of three small molecules that bound FGF-2 with affinity in the submicromolar range. The lead compounds inhibited FGF-2-induced endothelial cell proliferation in vitro and affected angiogenesis induced by FGF-2 in the chicken chorioallantoic membrane assay. These small molecules, therefore, represent promising leads for the development of antiangiogenic agents. Altogether, this study demonstrates that new biological insights obtained by integrated multidisciplinary approaches can be used to develop small molecule mimics of endogenous proteins as therapeutic agents.

Fibroblast growth factor 2-antagonist activity of a long-pentraxin 3-derived anti-angiogenic pentapeptide.

Fibroblast growth factor-2 (FGF2) plays a major role in angiogenesis. The pattern recognition receptor long-pentraxin 3 (PTX3) inhibits the angiogenic activity of FGF2. To identify novel FGF2-antagonistic peptide(s), four acetylated (Ac) synthetic peptides overlapping the FGF2-binding region PTX3-(97-110) were assessed for their FGF2-binding capacity. Among them, the shortest pentapeptide Ac-ARPCA-NH(2) (PTX3-[100-104]) inhibits the interaction of FGF2 with PTX3 immobilized to a BIAcore sensorchip and suppresses FGF2-dependent proliferation in endothelial cells, without affecting the activity of unrelated mitogens. Also, Ac-ARPCA-NH(2) inhibits angiogenesis triggered by FGF2 or by tumorigenic FGF2-overexpressing murine endothelial cells in chick and zebrafish embryos, respectively. Accordingly, the peptide hampers the binding of FGF2 to Chinese Hamster ovary cells overexpressing the tyrosine-kinase FGF receptor-1 (FGFR1) and to recombinant FGFR1 immobilized to a BIAcore sensorchip without affecting heparin interaction. In all the assays the mutated Ac-ARPSA-NH(2) peptide was ineffective. In keeping with the observation that hydrophobic interactions dominate the interface between FGF2 and the FGF-binding domain of the Ig-like loop D2 of FGFR1, amino acid substitutions in Ac-ARPCA-NH(2) and saturation transfer difference-nuclear magnetic resonance analysis of its mode of interaction with FGF2 implicate the hydrophobic methyl groups of the pentapeptide in FGF2 binding. These results will provide the basis for the design of novel PTX3-derived anti-angiogenic FGF2 antagonists.

Site-specific investigation of the steady-state kinetics and dynamics of the multistep binding of bile acid molecules to a lipid carrier protein.

The investigation of multi-site ligand-protein binding and multi-step mechanisms is highly demanding. In this work, advanced NMR methodologies such as 2D (1)H-(15)N line-shape analysis, which allows a reliable investigation of ligand binding occurring on micro- to millisecond timescales, have been extended to model a two-step binding mechanism. The molecular recognition and complex uptake mechanism of two bile salt molecules by lipid carriers is an interesting example that shows that protein dynamics has the potential to modulate the macromolecule-ligand encounter. Kinetic analysis supports a conformational selection model as the initial recognition process in which the dynamics observed in the apo form is essential for ligand uptake, leading to conformations with improved access to the binding cavity. Subsequent multi-step events could be modelled, for several residues, with a two-step binding mechanism. The protein in the ligand-bound state still exhibits a conformational rearrangement that occurs on a very slow timescale, as observed for other proteins of the family. A global mechanism suggesting how bile acids access the macromolecular cavity is thus proposed.

Natural Compounds as Inhibitors of Aß Peptide Aggregation: Chemical Requirements and Molecular Mechanisms.

Alzheimer's disease (AD) is one of the most common neurodegenerative disorders, with no cure and preventive therapy. Misfolding and extracellular aggregation of Amyloid-ß (Aß) peptides are recognized as the main cause of AD progression, leading to the formation of toxic Aß oligomers and to the deposition of ß-amyloid plaques in the brain, representing the hallmarks of AD. Given the urgent need to provide alternative therapies, natural products serve as vital resources for novel drugs. In recent years, several natural compounds with different chemical structures, such as polyphenols, alkaloids, terpenes, flavonoids, tannins, saponins and vitamins from plants have received attention for their role against the neurodegenerative pathological processes. However, only for a small subset of them experimental evidences are provided on their mechanism of action. This review focuses on those natural compounds shown to interfere with Aß aggregation by direct interaction with Aß peptide and whose inhibitory mechanism has been investigated by means of biophysical and structural biology experimental approaches. In few cases, the combination of approaches offering a macroscopic characterization of the oligomers, such as TEM, AFM, fluorescence, together with high-resolution methods could shed light on the complex mechanism of inhibition. In particular, solution NMR spectroscopy, through peptide-based and ligand-based observation, was successfully employed to investigate the interactions of the natural compounds with both soluble NMR-visible (monomer and low molecular weight oligomers) and NMR-invisible (high molecular weight oligomers and protofibrils) species. The molecular determinants of the interaction of promising natural compounds are here compared to infer the chemical requirements of the inhibitors and the common mechanisms of inhibition. Most of the data converge to indicate that the Aß regions relevant to perturb the aggregation cascade and regulate the toxicity of the stabilized oligomers, are the N-term and ß1 region. The ability of the natural aggregation inhibitors to cross the brain blood barrier, together with the tactics to improve their low bioavailability are discussed. The analysis of the data ensemble can provide a rationale for the selection of natural compounds as molecular scaffolds for the design of new therapeutic strategies against the progression of early and late stages of AD.

New insights into the molecular interaction of the C-terminal sequence of CXCL4 with fibroblast growth factor-2.

Full-length CXCL4 chemokine and a peptide derived from its carboxyl-terminal domain exhibits significant antiangiogenic and anti-tumor activity in vivo and in vitro by interacting with fibroblast growth factor (FGF). In this study we used NMR spectroscopy to characterize at a molecular level the interactions between CXCL4 (47-70) and FGF-2 identifying the peptide residues mainly involved in the contact area with the growth factor. Altogether NMR data point to a major role of the hydrophobic contributions of the C-terminal region of CXCL4 (47-70) peptide in addition to specific contacts established by the N-terminal region through cysteine side chain. The proposed recognition mode constitutes a rationale for the observed effects of CXCL4 (47-70) on FGF-2 biological activity and lays the basis for developing novel inhibitors of angiogenesis.

Effects of Prion Protein on Aß42 and Pyroglutamate-Modified AßpΕ3-42 Oligomerization and Toxicity.

Soluble Aß oligomers are widely recognized as the toxic forms responsible for triggering AD, and Aß receptors are hypothesized to represent the first step in a neuronal cascade leading to dementia. Cellular prion protein (PrP) has been reported as a high-affinity binder of Aß oligomers. The interactions of PrP with both Aß42 and the highly toxic N-truncated pyroglutamylated species (AßpE3-42) are here investigated, at a molecular level, by means of ThT fluorescence, NMR and TEM. We demonstrate that soluble PrP binds both Aß42 and AßpE3-42, preferentially interacting with oligomeric species and delaying fibril formation. Residue level analysis of Aß42 oligomerization process reveals, for the first time, that PrP is able to differently interact with the forming oligomers, depending on the aggregation state of the starting Aß42 sample. A distinct behavior is observed for Aß42 1-30 region and C-terminal residues, suggesting that PrP protects Aß42 N-tail from entangling on the mature NMR-invisible fibril, consistent with the hypothesis that Aß42 N-tail is the locus of interaction with PrP. PrP/AßpE3-42 interactions are here reported for the first time. All interaction data are validated and complemented by cellular tests performed on Wt and PrP-silenced neuronal cell lines, clearly showing PrP dependent Aß oligomer cell internalization and toxicity. The ability of soluble PrP to compete with membrane-anchored PrP for binding to Aß oligomers bears relevance for studies of druggable pathways.

Biophysical and in Vivo Studies Identify a New Natural-Based Polyphenol, Counteracting Aß Oligomerization in Vitro and Aß Oligomer-Mediated Memory Impairment and Neuroinflammation in an Acute Mouse Model of Alzheimer's Disease.

In this study natural-based complex polyphenols, obtained through a smart synthetic approach, have been evaluated for their ability to inhibit the formation of Aß42 oligomers, the most toxic species causing synaptic dysfunction, neuroinflammation, and neuronal death leading to the onset and progression of Alzheimer's disease. In vitro neurotoxicity tests on primary hippocampal neurons have been employed to select nontoxic candidates. Solution NMR and molecular docking studies have been performed to clarify the interaction mechanism of Aß42 with the synthesized polyphenol derivatives, and highlight the sterical and chemical requirements important for their antiaggregating activity. NMR results indicated that the selected polyphenolic compounds target Aß42 oligomeric species. Combined NMR and docking studies indicated that the Aß42 central hydrophobic core, namely, the 17-31 region, is the main interaction site. The length of the peptidomimetic scaffold and the presence of a guaiacol moiety were identified as important requirements for the antiaggregating activity. In vivo experiments on an Aß42 oligomer-induced acute mouse model highlighted that the most promising polyphenolic derivative (PP04) inhibits detrimental effects of Aß42 oligomers on memory and glial cell activation. NMR kinetic studies showed that PP04 is endowed with the chemical features of true inhibitors, strongly affecting both the Aß42 nucleation and growth rates, thus representing a promising candidate to be further developed into an effective drug against neurodegenerative diseases of the amyloid type.

Conformational and dynamics changes induced by bile acids binding to chicken liver bile acid binding protein.

The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family.

NMR-based modeling and binding studies of a ternary complex between chicken liver bile acid binding protein and bile acids.

Chicken liver bile acid binding protein (cL-BABP) is involved in bile acid transport in the liver cytosol. A detailed study of the mechanism of binding and selectivity of bile acids binding proteins towards the physiological pool of bile salts is a key issue for the complete understanding of the role of these proteins and their involvement in cholesterol homeostasis. In the present study, we modeled the ternary complex of cL-BABP with two molecules of bile salts using the data driven docking program HADDOCK on the basis of NMR and mass spectrometry data. Docking resulted in good 3D models, satisfying the majority of experimental restraints. The docking procedure represents a necessary step to help in the structure determination and in functional analysis of such systems, in view of the high complexity of the 3D structure determination of a ternary complex with two identical ligands. HADDOCK models show that residues involved in binding are mainly located in the C-terminal end of the protein, with two loops, CD and EF, playing a major role in ligand binding. A spine, comprising polarresidues pointing toward the protein interior and involved in motion communication, has a prominent role in ligand interaction. The modeling approach has been complemented with NMR interaction and competition studies of cL-BABP with chenodeoxycholic and cholic acids. A higher affinity for chenodeoxycholic acid was observed and a Kd upper limit estimate was obtained. The binding is highly cooperative and no site selectivity was detected for the different bile salts, thus indicating that site selectivity and cooperativity are not correlated. Differences in physiological pathways and bile salt pools in different species is discussed in light of the binding results thus enlarging the body of knowledge of BABPs biological functions.

NMR structural studies of the supramolecular adducts between a liver cytosolic bile acid binding protein and gadolinium(III)-chelates bearing bile acids residues: molecular determinants of the binding of a hepatospecific magnetic resonance imaging contrast agent.

The binding affinities of a selected series of Gd(III) chelates bearing bile acid residues, potential hepatospecific MRI contrast agents, to a liver cytosolic bile acid transporter, have been determined through relaxivity measurements. The Ln(III) complexes of compound 1 were selected for further NMR structural analysis aimed at assessing the molecular determinants of binding. A number of NMR experiments have been carried out on the bile acid-like adduct, using both diamagnetic Y(III) and paramagnetic Gd(III) complexes, bound to a liver bile acid binding protein. The identified protein "hot spots" defined a single binding site located at the protein portal region. The presented findings will serve in a medicinal chemistry approach for the design of hepatocytes-selective gadolinium chelates for liver malignancies detection.