Identification of a novel extracellular inhibitor of FGF2/FGFR signaling axis by combined virtual screening and NMR spectroscopy approach.

The aberrant activation of the fibroblast growth factor 2 (FGF2)/fibroblast growth factor receptor (FGFR) signalling pathway drives severe pathologies, including cancer development and angiogenesis-driven pathologies. The perturbation of the FGF2/FGFR axis via extracellular allosteric small inhibitors is a promising strategy for developing FGFR inhibitors with improved safety and efficacy for cancer treatment. We have previously investigated the role of new extracellular inhibitors, such as rosmarinic acid (RA), which bind the FGFR-D2 domain and directly compete with FGF2 for the same binding site, enabling the disruption of the functional FGF2/FGFR interaction. To select ligands for the previously identified FGF2/FGFR RA binding site, NMR data-driven virtual screening has been performed on an in-house library of non-commercial small molecules and metabolites. A novel drug-like compound, a resorcinol derivative named RBA4 has been identified. NMR interaction studies demonstrate that RBA4 binds the FGF2/FGFR complex, in agreement with docking prediction. Residue-level NMR perturbations analysis highlights that the mode of action of RBA4 is similar to RA in terms of its ability to target the FGF2/FGFR-D2 complex, inducing perturbations on both proteins and triggering complex dissociation. Biological assays proved that RBA4 inhibited FGF2 proliferative activity at a level comparable to the previously reported natural product, RA. Identification of RBA4 chemical groups involved in direct interactions represents a starting point for further optimization of drug-like extracellular inhibitors with improved activity.

Inhibition of FGFR Signaling by Targeting FGF/FGFR Extracellular Interactions: Towards the Comprehension of the Molecular Mechanism through NMR Approaches.

NMR-based approaches play a pivotal role in providing insight into molecular recognition mechanisms, affording the required atomic-level description and enabling the identification of promising inhibitors of protein-protein interactions. The aberrant activation of the fibroblast growth factor 2 (FGF2)/fibroblast growth factor receptor (FGFR) signaling pathway drives several pathologies, including cancer development, metastasis formation, resistance to therapy, angiogenesis-driven pathologies, vascular diseases, and viral infections. Most FGFR inhibitors targeting the intracellular ATP binding pocket of FGFR have adverse effects, such as limited specificity and relevant toxicity. A viable alternative is represented by targeting the FGF/FGFR extracellular interactions. We previously identified a few small-molecule inhibitors acting extracellularly, targeting FGFR or FGF. We have now built a small library of natural and synthetic molecules that potentially act as inhibitors of FGF2/FGFR interactions to improve our understanding of the molecular mechanisms of inhibitory activity. Here, we provide a comparative analysis of the interaction mode of small molecules with the FGF2/FGFR complex and the single protein domains. DOSY and residue-level NMR analysis afforded insights into the capability of the potential inhibitors to destabilize complex formation, highlighting different mechanisms of inhibition of FGF2-induced cell proliferation.

Sodium fusidate prevents protein aggregation of silk fibroin and offers new perspectives for human lens material disaggregation.

Silk fibroin (SF) is a non-pathological amyloidogenic protein prone, in solution, to the formation of amyloid-like aggregated species, displaying similarities in fibrillation kinetics with pathological amyloids, as widely reported in the literature. We show here, on the basis of different biophysical approaches (turbidity, Congo Red assays, CD, DLS and fluorescence), that fusidic acid (FA), a well-known antibiotic, acts on SF as an anti-aggregating agent in a dose-dependent manner, being also able to revert SF aggregation. FA binds to SF inducing changes in the environment of SF aromatic residues. We further provide the proof of principle that FA, already approved as drug on humans and used in ophthalmic preparations, displays its anti-aggregation properties also on lens material derived from cataract surgery and is capable of reducing aggregation. Thus it is suggested that FA can be foreseen as a therapeutic treatment for cataract and other protein aggregation disorders.

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.

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.

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.

Unsaturated Long-Chain Fatty Acids Are Preferred Ferritin Ligands That Enhance Iron Biomineralization.

Ferritin is a ubiquitous nanocage protein, which can accommodate up to thousands of iron atoms inside its cavity. Aside from its iron storage function, a new role as a fatty acid binder has been proposed for this protein. The interaction of apo horse spleen ferritin (HoSF) with a variety of lipids has been here investigated through NMR spectroscopic ligand-based experiments, to provide new insights into the mechanism of ferritin-lipid interactions, and the link with iron mineralization. 1D 1 H, diffusion (DOSY) and saturation-transfer difference (STD) NMR experiments provided evidence for a stronger interaction of ferritin with unsaturated fatty acids compared to saturated fatty acids, detergents, and bile acids. Mineralization assays showed that oleate c aused the most efficient increase in the initial rate of iron oxidation, and the highest formation of ferric species in HoSF. The comprehension of the factors inducing a faster biomineralization is an issue of the utmost importance, given the association of ferritin levels with metabolic syndromes, such as insulin resistance and diabetes, characterized by fatty acid concentration dysregulation. The human ferritin H-chain homopolymer (HuHF), featuring ferroxidase activity, was also tested for its fatty acid binding capabilities. Assays show that oleate can bind with high affinity to HuHF, without altering the reaction rates at the ferroxidase site.

Searching for protein binding sites from Molecular Dynamics simulations and paramagnetic fragment-based NMR studies.

Hotspot delineation on protein surfaces represents a fundamental step for targeting protein-protein interfaces. Disruptors of protein-protein interactions can be designed provided that the sterical features of binding pockets, including the transient ones, can be defined. Molecular Dynamics, MD, simulations have been used as a reliable framework for identifying transient pocket openings on the protein surface. Accessible surface area and intramolecular H-bond involvement of protein backbone amides are proposed as descriptors for characterizing binding pocket occurrence and evolution along MD trajectories. TEMPOL induced paramagnetic perturbations on (1)H-(15)N HSQC signals of protein backbone amides have been analyzed as a fragment-based search for surface hotspots, in order to validate MD predicted pockets. This procedure has been applied to CXCL12, a small chemokine responsible for tumor progression and proliferation. From combined analysis of MD data and paramagnetic profiles, two CXCL12 sites suitable for the binding of small molecules were identified. One of these sites is the already well characterized CXCL12 region involved in the binding to CXCR4 receptor. The other one is a transient pocket predicted by Molecular Dynamics simulations, which could not be observed from static analysis of CXCL12 PDB structures. The present results indicate how TEMPOL, instrumental in identifying this transient pocket, can be a powerful tool to delineate minor conformations which can be highly relevant in dynamic discovery of antitumoral drugs.

Urine metabolic signature of pancreatic ductal adenocarcinoma by (1)h nuclear magnetic resonance: identification, mapping, and evolution.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis and is highly chemoresistant. Early detection is the only means to impact long-term survival, but screening methods are lacking. Given the complex and heterogeneous nature of pancreatic cancer, unbiased analytical methods such as metabolomics by nuclear magnetic resonance (NMR) spectroscopy show promise to identify disease-specific molecular fingerprints. NMR profiles constitute a fingerprint of the biofluid, reporting quantitatively on all detectable small biomolecules. NMR spectroscopy was applied to investigate the urine metabolome of PDAC patients (n = 33) and to detect altered metabolic profiles in comparison with healthy matched controls (n = 54). The spectral data were analyzed using multivariate statistical techniques. Statistically significant differences were found between urine metabolomic profiles of PDAC and control individuals (p < 10(-5)). Group discrimination was possible due to average concentration differences of several metabolite signals, pointing to a multimolecular signature of the disease. The robustness of the determined statistical model is confirmed by its predictive performance (sensitivity = 75.8%, specificity = 90.7%). Additionally, the method allowed for a neat separation between spectral profiles of individuals with intermediate and advanced pathologic staging, as well as for the discrimination of samples based on tumor localization. NMR spectroscopy analysis of urinary metabolic profiles proved successful in identifying a complex molecular signature of PDAC. Furthermore, results of a descriptive-level analysis show the possibility to follow disease evolution and to carry out tumor site mapping. Given the high reproducibility and the noninvasive nature of the analytical procedure, the described method bears potential to impact large-scale screening programs.

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.

NMR solution structure of viscotoxin C1 from Viscum album species Coloratum ohwi: toward a structure-function analysis of viscotoxins.

The high resolution three-dimensional structure of the newly discovered plant viscotoxin C1, from the Asiatic Viscum album ssp. Coloratum ohwi, has been determined in solution by (1)H NMR spectroscopy at pH 3.6 and 285 K. The viscotoxin C1-fold, consisting of a helix-turn-helix motif and a short stretch of an antiparralel beta-sheet is very similar to that found for the highly similar viscotoxins A2 and A3 and for other related thionins. Different functional properties of members of the thionin family are discussed here in light of the structural and electrostatic properties. Among the very homologous family of alpha- and beta-thionins, known for their antimicrobial activity, the viscotoxin subfamily differs from the other members because of its high toxicity against tumoral cells. Key residues for the modulation of viscotoxin cytotoxicity have been identified on the basis of sequence and structural alignment.

Solution structure of chicken liver basic fatty acid binding protein.

Chicken liver basic fatty acid binding protein (Lb-FABP) belongs to the basic-type fatty acid binding proteins, a novel group of proteins isolated from liver of different non mammalian species whose structure is not known. The structure of Lb-FABP has been solved by (1)H NMR. The overall fold of Lb-FABP, common to the other proteins of the family, consists of ten antiparallel beta-strands organised in two nearly ortogonal beta-sheets with two alpha helices closing the protein cavity where small hydrophobic ligands can be bound. The binding specificity of the protein is not known, however, based on the high sequence and structural similarity with an orthologous protein, ileal lipid binding protein, it is suggested that bile acids may be the putative ligands.

Peptide models of folding initiation sites of bovine beta-lactoglobulin: identification of nativelike hydrophobic interactions involving G and H strands.

In an attempt to characterize the early folding events in bovine beta-lactoglobulin (BLG), a set of peptides, covering the flexible N-terminal region and the stable C-terminus beta-core, was synthesized and analyzed by circular dichroism and by nuclear magnetic resonance in water, trifluoroethanol (TFE), and sodium dodecyl sulfate (SDS) below and above the critical micellar concentration. The role of local and long-range hydrophobic interactions in guiding the folding has been investigated. For the peptide fragment covering the more flexible N-terminal region of BLG (beta-strands A, B), where both theoretical predictions and kinetic refolding experiments suggested the formation of non-native alpha-helix, no native long-range contacts were identified, and a helical secondary structure was stabilized only in the presence of 25 mM SDS. At variance, in 50% (v/v) TFE, native, long-range hydrophobic interactions were observed in the peptide covering the core region comprising G and H beta-strands. The side chains involved in these interactions form a nativelike hydrophobic cluster, thus suggesting that the GH region may act as the folding initiation site for BLG. This result is reinforced by the identification, in the urea denaturated BLG, of residual structure located at the level of the GH interface, as evidenced by NMR analysis. These results, in excellent agreement with kinetic, thermodynamic, and cold denaturation folding data, once more underline the utmost importance of the GH region for the stability and folding of BLG. Severe aggregation effects prevented the structural analysis of the peptide covering the EFGH region, indicating that this larger segment does not represent an independent folding domain and that the terminal alpha-helix is necessary for stabilizing the BLG folding core.