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.

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.

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.

Photo-Induced Microfluidic Production of Ultrasmall Glyco Gold Nanoparticles.

Ultra-small gold nanoparticles (UAuNPs) are extremely interesting for applications in nanomedicine thanks to their good stability, biocompatibility, long circulation time and efficient clearance pathways. UAuNPs engineered with glycans (Glyco-UAuNPs) emerged as excellent platforms for many applications since the multiple copies of glycans can mimic the multivalent effect of glycoside clusters. Herein, we unravel a straightforward photo-induced synthesis of Glyco-UAuNPs based on a reliable and robust microfluidic approach. The synthesis occurs at room temperature avoiding the use of any further chemical reductant, templating agents or co-solvents. Exploiting 1 H NMR spectroscopy, we showed that the amount of thiol-ligand exposed on the UAuNPs is linearly correlated to the ligand concentration in the initial mixture. The results pave the way towards the development of a programmable synthetic approach, enabling an accurate design of the engineered UAuNPs or smart hybrid nano-systems.

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.

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.

Rhodamine binds to silk fibroin and inhibits its self-aggregation.

Amyloid structures are universal structures, widely diffuse in nature. Silk, capable of forming some of the strongest tensile materials on earth represents an important example of formation of functional amyloid fibrils, a process reminiscent of the oligomerization of peptides involved in neurodegenerative diseases. The stability of silk fibroin solutions in different conditions and its transition from α-helix/random coil to ß-sheet structures, at the basis of gelation processes and fibril formation, have been here investigated and monitored employing different biophysical approaches. Silk fibroin aggregation state as a function of concentration, pH and aging has been characterized employing NMR ordered diffusion spectroscopy. The change of silk fibroin diffusion coefficient over time, which reflects the progress of oligomerization, has been monitored for silk fibroin alone and in the presence of a polycondensed aromatic dye, namely rhodamine 6G. NMR, UV and DLS measurements indicated that rhodamine specifically binds to silk fibroin with a micromolar KD. The reported data reveal, for the first time, that RHD is capable of inhibiting fibroin self-association, thus controlling ß-conformational transition at the basis of fibril formation. The described approach could be extended to further protein systems, allowing better control of the oligomerisation process.

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.

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.

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.

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.

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.

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.

NMR unfolding studies on a liver bile acid binding protein reveal a global two-state unfolding and localized singular behaviors.

The folding properties of a bile acid binding protein, belonging to a subfamily of the fatty acid binding proteins, have been here investigated both by hydrogen exchange measurements, using the SOFAST NMR approach, and urea denaturation experiments. The urea unfolding profiles of individual residues, acting as single probes, were simultaneously analyzed through a global fit, according to a two-state unfolding model. The resulting conformational stability DeltaG(U)(H(2)O)=7.2+/-0.25kcal mol(-1) is in good agreement with hydrogen exchange stability DeltaG(op). While the majority of protein residues satisfy this model, few amino-acids display a singular behavior, not directly amenable to the presence of a folding intermediate, as reported for other fatty acid binding proteins. These residues are part of a protein patch characterized by enhanced plasticity. To explain this singular behavior a tentative model has been proposed which takes into account the interplay between the dynamic features and the formation of transient aggregates. A functional role for this plasticity, related to translocation across the nuclear membrane, is discussed.