Pyrazolones Activate the Proteasome by Gating Mechanisms and Protect Neuronal Cells from ß-Amyloid Toxicity.

Proteasome malfunction parallels abnormal amyloid accumulation in Alzheimer's Disease (AD). Here we scrutinize a small library of pyrazolones by assaying their ability to enhance proteasome activity and protect neuronal cells from amyloid toxicity. Tube tests evidenced that aminopyrine and nifenazone behave as 20S proteasome activators. Enzyme assays carried out on an "open gate" mutant (α3ΔN) proteasome demonstrated that aminopyrine activates proteasome through binding the α-ring surfaces and influencing gating dynamics. Docking studies coupled with STD-NMR experiments showed that H-bonds and π-π stacking interactions between pyrazolones and the enzyme play a key role in bridging α1 to α2 and, alternatively, α5 to α6 subunits of the outer α-ring. Aminopyrine and nifenazone exhibit neurotrophic properties and protect differentiated human neuroblastoma SH-SY5Y cells from ß-amyloid (Aß) toxicity. ESI-MS studies confirmed that aminopyrine enhances Aß degradation by proteasome in a dose-dependent manner. Our results suggest that some pyrazolones and, in particular, aminopyrine are promising compounds for the development of proteasome activators for AD treatment.

Receptor-mediated toxicity of human amylin fragment aggregated by short- and long-term incubations with copper ions.

Human amylin (hA1-37) is a polypeptide hormone secreted in conjunction with insulin from the pancreatic ß-cells involved in the pathogenesis of type 2 diabetes mellitus (T2DM). The shorter fragment hA17-29 than full-length peptide is capable to form amyloids "in vitro". Here, we monitored the time course of hA17-29 ß-amyloid fibril and oligomer formation [without and with copper(II)], cellular toxicity of different amyloid aggregates, and involvement of specific receptors (receptor for advanced glycation end-products, RAGE; low-affinity nerve growth factor receptor, p75-NGFR) in aggregate toxicity. Fibril and oligomer formation of hA17-29 incubated at 37 °C for 0, 48, and 120 h, without or with copper(II), were measured by the thioflavin T fluorescence assay and ELISA, respectively. Toxicity of hA17-29 aggregates and effects of anti-RAGE and anti-p75-NGFR antibodies were evaluated on neuroblastoma SH-SY5Y viability. Fluorescence assay of hA17-29 indicates an initial slow rate of soluble fibril formation (48 h), followed by a slower rate of insoluble aggregate formation (120 h). The highest quantity of oligomers was recorded when hA17-29 was pre-aggregated for 48 h in the presence of copper(II) showing also the maximal cell toxicity (-44% of cell viability, p < 0.01 compared to controls). Anti-RAGE or anti-p75-NGFR antibodies almost abolished cell toxicity of hA17-29 aggregates. These results indicate that copper(II) influences the aggregation process and hA17-29 toxicities are especially attributable to oligomeric aggregates. hA17-29 aggregate toxicity seems to be mediated by RAGE and p75-NGFR receptors which might be potential targets for new drugs in T2DM treatment.

Influence of the N-terminus acetylation of Semax, a synthetic analog of ACTH(4-10), on copper(II) and zinc(II) coordination and biological properties.

Semax is a heptapeptide (Met-Glu-His-Phe-Pro-Gly-Pro) that encompasses the sequence 4-7 of N-terminal domain of the adrenocorticotropic hormone and a C-terminal Pro-Gly-Pro tripeptide. N-terminal amino group acetylation (Ac-Semax) modulates the chemical and biological properties of parental peptide, modifying the ability of Semax to form complex species with Cu(II) ion. At physiological pH, the main complex species formed by Ac-Semax, [CuLH-2]2-, consists in a distorted CuN3O chromophore with a weak apical interaction of the methionine sulphur. Such a complex differs from the Cu(II)-Semax complex system, which exhibits a CuN4 chromophore. The reduced ligand field affects the [CuLH-2]2- formal redox potential, which is more positive than that of Cu(II)-Semax corresponding species. In the amino-free form, the resulting complex species is redox-stable and unreactive against ascorbic acid, unlike the acetylated form. Semax acetylation did not protect from Cu(II) induced toxicity on a SH-SY5Y neuroblastoma cell line, thus demonstrating the crucial role played by the free NH2 terminus in the cell protection. Since several brain diseases are associated either to Cu(II) or Zn(II) dyshomeostasis, here we characterized also the complex species formed by Zn(II) with Semax and Ac-Semax. Both peptides were able to form Zn(II) complex species with comparable strength. Confocal microscopy imaging confirmed that peptide group acetylation does not affect the Zn(II) influx in neuroblastoma cells. Moreover, a punctuate distribution of Zn(II) within the cells suggests a preferred subcellular localization that might explain the zinc toxic effect. A future perspective can be the use of Ac-Semax as ionophore in antibody drug conjugates to produce a dysmetallostasis in tumor cells.

A small linear peptide encompassing the NGF N-terminus partly mimics the biological activities of the entire neurotrophin in PC12 cells.

Ever since the discovery of its neurite growth promoting activity in sympathetic and sensory ganglia, nerve growth factor (NGF) became the prototype of the large family of neurotrophins. The use of primary cultures and clonal cell lines has revealed several distinct actions of NGF and other neurotrophins. Among several models of NGF activity, the clonal cell line PC12 is the most widely employed. Thus, in the presence of NGF, through the activation of the transmembrane protein TrkA, these cells undergo a progressive mitotic arrest and start to grow electrically excitable neuritis. A vast number of studies opened intriguing aspects of NGF mechanisms of action, its biological properties, and potential use as therapeutic agents. In this context, identifying and utilizing small portions of NGF is of great interest and involves several human diseases including Alzheimer's disease. Here we report the specific action of the peptide encompassing the 1-14 sequence of the human NGF (NGF(1-14)), identified on the basis of scattered indications present in literature. The biological activity of NGF(1-14) was tested on PC12 cells, and its binding with TrkA was predicted by means of a computational approach. NGF(1-14) does not elicit the neurite outgrowth promoting activity, typical of the whole protein, and it only has a moderate action on PC12 proliferation. However, this peptide exerts, in a dose and time dependent fashion, an effective and specific NGF-like action on some highly conserved and biologically crucial intermediates of its intracellular targets such as Akt and CREB. These findings indicate that not all TrkA pathways must be at all times operative, and open the possibility of testing each of them in relation with specific NGF needs, biological actions, and potential therapeutic use.

Zinc(II) interactions with brain-derived neurotrophic factor N-terminal peptide fragments: inorganic features and biological perspectives.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin essential for neuronal differentiation, growth, and survival; it is involved in memory formation and higher cognitive functions. The N-terminal domain of BDNF is crucial for the binding selectivity and activation of its specific TrkB receptor. Zn(2+) ion binding may influence BDNF activity. Zn(2+) complexes with the peptide fragment BDNF(1-12) encompassing the sequence 1-12 of the N-terminal domain of BDNF were studied by means of potentiometry, electrospray mass spectrometry, NMR, and density functional theory (DFT) approaches. The predominant Zn(2+) complex species, at physiological pH, is [ZnL] in which the metal ion is bound to an amino, an imidazole, and two water molecules (NH2, N(Im), and 2O(water)) in a tetrahedral environment. DFT-based geometry optimization of the zinc coordination environment showed a hydrogen bond between the carboxylate and a water molecule bound to zinc in [ZnL]. The coordination features of the acetylated form [AcBDNF(1-12)] and of a single mutated peptide [BDNF(1-12)D3N] were also characterized, highlighting the role of the imidazole side chain as the first anchoring site and ruling out the direct involvement of the aspartate residue in the metal binding. Zn(2+) addition to the cell culture medium induces an increase in the proliferative activity of the BDNF(1-12) peptide and of the whole protein on the SHSY5Y neuroblastoma cell line. The effect of Zn(2+) is opposite to that previously observed for Cu(2+) addition, which determines a decrease in the proliferative activity for both peptide and protein, suggesting that these metals might discriminate and modulate differently the activity of BDNF.

Oligonucleotides conjugated to natural lipids: synthesis of phosphatidyl-anchored antisense oligonucleotides.

Antisense oligonucleotides are promising therapeutic agents against a variety of diseases. Effective delivery of these molecules is critical in view of their clinical application. Despite the richness of synthetic strategies addressed to the lipophilic modification of oligodeoxynucleotides (ODNs) for enhancing their pharmacokinetic behavior and trans-membrane delivery, the phosphatidyl group (1,2-di-O-acyl-sn-glycero-3-phosphoryl) has been never used as the lipophilic moiety of lipid-ODN conjugates. The present paper reports a general procedure for synthesizing 5'-phosphatidyl-ODNs. By this procedure, phosphatidyl conjugates of a VEGF antisense-ODN have been prepared, which differ in the fatty acid composition of their phosphatidyl moiety. These new lipid-ODN conjugates, which have been characterized on the basis of their physicochemical properties, showed an improved resistance to exonucleases and were able to lower the VEGF-mRNA expression in human SH-SY5Y neuroblastoma cells more effectively than the relevant free antisense-ODN did.

The inorganic perspective of nerve growth factor: interactions of Cu2+ and Zn2+ with the N-terminus fragment of nerve growth factor encompassing the recognition domain of the TrkA receptor.

There is a significant overlap between brain areas with Zn(2+) and Cu(2+) pathological dys-homeostasis and those in which the nerve growth factor (NGF) performs its biological role. The protein NGF is necessary for the development and maintenance of the sympathetic and sensory nervous systems. Its flexible N-terminal region has been shown to be a critical domain for TrkA receptor binding and activation. Computational analyses show that Zn(2+) and Cu(2+) form pentacoordinate complexes involving both the His4 and His8 residues of the N-terminal domain of one monomeric unit and the His84 and Asp105 residues of the other monomeric unit of the NGF active dimer. To date, neither experimental data on the coordination features have been reported, nor has one of the hypotheses according to which Zn(2+) and Cu(2+) may have different binding environments or the Ser1 α-amino group could be involved in coordination been supported. The peptide fragment, encompassing the 1-14 sequence of the human NGF amino-terminal domain (NGF(1-14)), blocked at the C terminus, was synthesised and its Cu(2+) and Zn(2+) complexes characterized by means of potentiometric and spectroscopic (UV/Vis, CD, NMR, and EPR) techniques. The N-terminus-acetylated form of NGF(1-14) was also investigated to evaluate the involvement of the Ser1 α-amino group in metal-ion coordination. Our results demonstrate that the amino group is the first anchoring site for Cu(2+) and is involved in Zn(2+) coordination at physiological pH. Finally, a synergic proliferative activity of both NGF(1-14) and the whole protein on SHSY5Y neuroblastoma cell line was found after treatment in the presence of Cu(2+). This effect was not observed after treatment with the N-acetylated peptide fragment, demonstrating a functional involvement of the N-terminal amino group in metal binding and peptide activity.