The fibroblast growth factor (FGF)/FGF receptor (FGFR) system plays a crucial role in cancer by affecting tumor growth, angiogenesis, drug resistance, and escape from anti-angiogenic anti-vascular endothelial growth factor therapy. The soluble pattern recognition receptor long-pentraxin 3 (PTX3) acts as a multi-FGF antagonist. Here we demonstrate that human PTX3 overexpression in transgenic mice driven by the Tie2 promoter inhibits tumor growth, angiogenesis, and metastasis in heterotopic, orthotopic, and autochthonous FGF-dependent tumor models. Using pharmacophore modeling of the interaction of a minimal PTX3-derived FGF-binding pentapeptide with FGF2, we identified a small-molecule chemical (NSC12) that acts as an extracellular FGF trap with significant implications in cancer therapy.

The discovery of methods suitable for the conversion in vitro of native proteins into amyloid fibrils has shed light on the molecular basis of amyloidosis and has provided fundamental tools for drug discovery. We have studied the capacity of a small library of tetracycline analogues to modulate the formation or destructuration of ␤2-microglobulin fibrils. The inhibition of fibrillogenesis of the wild type protein was first established in the presence of 20% trifluoroethanol and confirmed under a more physiologic environment including heparin and collagen. The latter conditions were also used to study the highly amyloidogenic variant, P32G. The NMR analysis showed that doxycycline inhibits ␤2-microglobulin self-association and stabilizes the native-like species through fast exchange interactions involving specific regions of the protein.Cell viability assays demonstrated that the drug abolishes the natural cytotoxic activity of soluble ␤2-microglobulin, further strengthening a possible in vivo therapeutic exploitation of this drug. Doxycycline can disassemble preformed fibrils, but the IC 50 is 5-fold higher than that necessary for the inhibition of fibrillogenesis. Fibril destructuration is a dynamic and timedependent process characterized by the early formation of cytotoxic protein aggregates that, in a few hours, convert into non-toxic insoluble material. The efficacy of doxycycline as a drug against dialysis-related amyloidosis would benefit from the ability of the drug to accumulate just in the skeletal system where amyloid is formed. In these tissues, the doxycycline concentration reaches values several folds higher than those resulting in inhibition of amyloidogenesis and amyloid destructuration in vitro.Amyloidosis associated with long term hemodialysis results from the deposition of full-length ␤2-microglobulin (␤2-m) 2 and its N-terminal truncated species ⌬N6␤2m in target tissues (1, 2). Although all peripheral organs (but not the brain) can be potentially affected (3), the muscle-skeletal tissues are the preferential target always involved in this type of amyloidosis. Despite significant progress achieved in the hemodialysis techniques, including the increased biocompatibility and the active removal of circulating ␤2-m, the onset of this amyloidosis can be delayed but not avoided in dialysis-related amyloidosis patients (4). New therapeutic approaches, targeting the process of protein aggregation and promoting fibril solubilization (5), are under investigation for the treatment of different types of amyloid diseases. Up until now, different classes of structurally unrelated compounds have been investigated for their ability to interfere with protein self-aggregation and to weaken the intermolecular interactions that stabilize the fibrillar structure of the aggregates (6). Over 10 years ago, iododoxorubicin was serendipitously discovered as the prototype of a class of compounds able to inhibit protein aggregation (7), but this compound was subsequently abandoned for its toxicity. The resemblance of the ...

Fibroblast growth factors (FGFs) are recognized targets for the development of therapies against angiogenesis-driven diseases, including cancer. The formation of a ternary complex with the transmembrane tyrosine kinase receptors (FGFRs), and heparan sulphate proteoglycans (HSPGs) is required for FGF2 pro-angiogenic activity. Here by using a combination of techniques including Nuclear Magnetic Resonance, Molecular Dynamics, Surface Plasmon Resonance and cell-based binding assays we clarify the molecular mechanism of inhibition of an angiostatic small molecule, sm27, mimicking the endogenous inhibitor of angiogenesis, thrombospondin-1. NMR and MD data demonstrate that sm27 engages the heparin-binding site of FGF2 and induces long-range dynamics perturbations along FGF2/FGFR1 interface regions. The functional consequence of the inhibitor binding is an impaired FGF2 interaction with both its receptors, as demonstrated by SPR and cell-based binding assays. We propose that sm27 antiangiogenic activity is based on a twofold–direct and allosteric–mechanism, inhibiting FGF2 binding to both its receptors.

The structure of AcP from the hyperthermophilic archaeon Sulfolobus solfataricus has been determined by (1)H-NMR spectroscopy and X-ray crystallography. Solution and crystal structures (1.27 A resolution, R-factor 13.7%) were obtained on the full-length protein and on an N-truncated form lacking the first 12 residues, respectively. The overall Sso AcP fold, starting at residue 13, displays the same betaalphabetabetaalphabeta topology previously described for other members of the AcP family from mesophilic sources. The unstructured N-terminal tail may be crucial for the unusual aggregation mechanism of Sso AcP previously reported. Sso AcP catalytic activity is reduced at room temperature but rises at its working temperature to values comparable to those displayed by its mesophilic counterparts at 25-37 degrees C. Such a reduced activity can result from protein rigidity and from the active site stiffening due the presence of a salt bridge between the C-terminal carboxylate and the active site arginine. Sso AcP is characterized by a melting temperature, Tm, of 100.8 degrees C and an unfolding free energy, DeltaG(U-F)H2O, at 28 degrees C and 81 degrees C of 48.7 and 20.6 kJ mol(-1), respectively. The kinetic and structural data indicate that mesophilic and hyperthermophilic AcP's display similar enzymatic activities and conformational stabilities at their working conditions. Structural analysis of the factor responsible for Sso AcP thermostability with respect to mesophilic AcP's revealed the importance of a ion pair network stabilizing particularly the beta-sheet and the loop connecting the fourth and fifth strands, together with increased density packing, loop shortening and a higher alpha-helical propensity.

Alzheimer’s disease (AD) is one of the most common neurodegenerative disorders, with no cure and preventive therapy. Misfolding and extracellular aggregation of Amyloid-β (Aβ) peptides are recognized as the main cause of AD progression, leading to the formation of toxic Aβ oligomers and to the deposition of β-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 Aβ aggregation by direct interaction with Aβ 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 Aβ regions relevant to perturb the aggregation cascade and regulate the toxicity of the stabilized oligomers, are the N-term and β1 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.

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It has previously been shown that the acylphosphatase from Sulfolobus solfataricus is capable of forming amyloid-like aggregates under conditions in which the native structure is maintained and via the transient formation of native-like aggregates. Based on the previously determined NMR structure of the native protein, showing a ferredoxin-like fold and the peculiar presence of an unstructured N-terminal segment, we show here, at a molecular level using NMR spectroscopy, that indeed S. solfataricus acylphosphatase remains in a native-like conformation when placed in aggregating conditions and that such a nativelike structure persists when the protein forms the initial aggregates, at least within the low molecular weight species. The analysis carried out under different solution conditions, based on the measurement of the combined 1 H and 15 N chemical shifts and hydrogen/deuterium exchange rates, enabled the most significant conformational changes to be monitored upon transfer of the monomeric state into aggregating conditions and upon formation of the initial native-like aggregates. Important increases of the hydrogen/deuterium exchange rates throughout the native protein, accompanied by small and localized structural changes, in the monomeric protein were observed. The results also allow the identification of the intermolecular interaction regions within the native-like aggregates, that involve, in particular, the N-terminal unstructured segment, the apical region including strands S4 and S5 with the connecting loop, and the opposite active site.Proteins and peptides have a generic propensity to form well organized aggregates characterized by a fibrillar morphology and an extended cross-␤ structure, generally referred to as amyloid-like fibrils (1, 2). This process is important for a number of reasons. From a physicochemical perspective, it represents an essential feature of the behavior of polypeptide chains that need to be fully understood for a thorough characterization of the nature of proteins (3). From a more biological perspective, formation of amyloid fibrils, or intracellular inclusions with structurally related characteristics, is associated with over 40 pathological conditions in humans (4, 5). It is also a major problem in biotechnology because the large scale expression of proteins potentially interesting to the market often results in their self-assembly in inclusion bodies with amyloid-like structural features (4).