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In this study natural-based complex polyphenols, obtained through a smart synthetic approach, have been evaluated for their ability to inhibit the formation of A? 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 A? 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 A? oligomeric species. Combined NMR and docking studies indicated that the A? 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 A? oligomer-induced acute mouse model highlighted that the most promising polyphenolic derivative (PP04) inhibits detrimental effects of A? 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 A? nucleation and growth rates, thus representing a promising candidate to be further developed into an effective drug against neurodegenerative diseases of the amyloid type.

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 DDDDDNDKIPD-DRDN. 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.

The use of spectroscopy in the study of fatty acids binding to bovine beta-lactoglobulin (BLG) appears to be a difficult task, as these acid compounds, assumed as the protein natural ligands, do not exhibit favorable optical response such as, for example, absorption or fluorescence. Therefore, the BLG fatty-acid equilibrium has been tackled by exploiting the competition between fatty acids and ANS, a widely used fluorescent hydrophobic probe, whose binding sites on the protein have been characterized recently. Two lifetime decays of the ANS-BLG complex have been found; the longer one has been attributed to the internal binding site and the shorter one to the external site. At increasing fatty acids concentration, the fractional weight associated with ANS bound to the internal site drops, in agreement with a model describing the competition of the dye with fatty acids, whereas the external site occupancy appears to be unaffected by the fatty acids binding to BLG. This model is supported by docking studies. An estimate of the acid-binding affinities for BLG has been obtained by implementing the fitting of the bound ANS intensities with a competitive binding model. A relevant dependence has been found upon the solution pH, in the range from 6 to 8, which correlates with the calyx accessibility modulated by the conformation of the EF loop. Fatty acids with longer aliphatic chains (palmitate and laurate) are found to display larger affinities for the protein and the interaction free energy nicely correlates with the number of contacts inside the protein calyx, in agreement with docking simulations.

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 GCA and GCDA 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 SS link between adjacent strands changes the conformation of three key residues, which function as hot-spot mediators of molecular discrimination. The favoured ?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

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 polar residues 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 K-d 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.

Bovine beta-lactoglobulin represents an interesting example of context-dependent secondary structure induction. In fact, secondary structure predictions indicated that this beta-barrel protein has a surprisingly high alpha-helical preference, which was retained for short fragments. Cooperative transitions from the native beta-sheet to alpha-helical structures were additionally induced by organic solvents, in particular trifluoroethanol. As a result of this high alpha-helical preference, it has been proposed that non-native alpha-helical intermediates could be formed in the unfolding pathway of this protein. In order to provide a better understanding of the processes that underlie conformational plasticity in this protein, CD measurements in the presence of increasing amounts of urea and in the presence of organic solvents were performed. Urea unfolding studies, performed at pH 2.1 and 37 degrees C, revealed an apparent two-state transition, and afforded no evidence of non native alpha-helical intermediates. The protein treated with up to 6M urea, refolded to the native structure, while treatment with higher molar concentration urea, lead to partial misfolding. A 29-mer peptide covering the region of strands a and b of the intact protein, characterized by the presence of 4/3 heptad repeats, was synthesized and studied by CD in the presence of different solvents. On the basis of the obtained results, a mechanism was proposed to explain the structural transition from the beta to alpha structure, provoked by organic solvents in the intact protein.