A Kinetically Controlled Bioconjugation Method for the Synthesis of Radioimmunoconjugates and the Development of a Domain Mapping MS-Workflow for Its Characterization.

Immunoconjugates exploit the high affinity of monoclonal antibodies for a recognized antigen to selectively deliver a cytotoxic payload, such as drugs or radioactive nuclides, at the site of disease. Despite numerous techniques have been recently developed for site-selective bioconjugations of protein structures, reaction of ε-amine group of lysine residues with electrophilic reactants, such as activated esters (NHS), is the main method reported in the literature as it maintains proteins in their native conformation. Since antibodies hold a high number of lysine residues, a heterogeneous mixture of conjugates will be generated, which can result in decreased target affinity. Here, we report an intradomain regioselective bioconjugation between the monoclonal antibody Trastuzumab and the N-hydroxysuccinimide ester of the chelator 2,2',2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) by a kinetically controlled reaction adding substoichiometric quantities of the activated ester to the mAb working at slightly basic pH. Liquid chromatography-mass spectrometry (LC-MS) analyses were carried out to assess the chelator-antibody ratio (CAR) and the number of chelating moieties linked to the mAb chains. Proteolysis experiments showed four lysine residues mainly involved in bioconjugation (K188 for the light chain and K30, K293, and K417 for the heavy chain), each of which was located in a different domain. Since the displayed intradomain regioselectivity, a domain mapping MS-workflow, based on a selective domain denaturation, was developed to quantify the percentage of chelator linked to each mAb domain. The resulting immunoconjugate mixture showed an average CAR of 0.9. About a third of the heavy chains were found as monoconjugated, whereas conjugation of the chelator in the light chain was negligible. Domain mapping showed the CH3 domain bearing 13% of conjugated DOTA, followed by CH2 and VH respectively bearing 12.5 and 11% of bonded chelator. Bioconjugation was not found in the CH1 domain, whereas for the light chain, only the CL domain was conjugated (6%). Data analysis based on LC-MS quantification of different analytical levels (intact, reduced chains, and domains) provided the immunoconjugate formulation. A mixture of immunoconjugates restricted to 15 species was obtained, and the percentage of each one within the mixture was calculated. In particular, species bearing 1 DOTA with a relative abundance ranging from 4 to 20-fold, in comparison to species bearing 2DOTA, were observed. Pairing of bioconjugation under kinetic control with the developed domain mapping MS-workflow could raise the standard of chemical quality for immunoconjugates obtained with commercially available reactants.

Aß8-20 Fragment as an Anti-Fibrillogenic and Neuroprotective Agent: Advancing toward Efficient Alzheimer's Disease Treatment.

Alzheimer's disease (AD) is the most common cause of dementia, characterized by a spectrum of symptoms associated with memory loss and cognitive decline with deleterious consequences in everyday life. The lack of specific drugs for the treatment and/or prevention of this pathology makes AD an ever-increasing economic and social emergency. Oligomeric species of amyloid-beta (Aß) are recognized as the primary cause responsible for synaptic dysfunction and neuronal degeneration, playing a crucial role in the onset of the pathology. Several studies have been focusing on the use of small molecules and peptides targeting oligomeric species to prevent Aß aggregation and toxicity. Among them, peptide fragments derived from the primary sequence of Aß have also been used to exploit any eventual recognition abilities toward the full-length Aß parent peptide. Here, we test the Aß8-20 fragment which contains the self-recognizing Lys-Leu-Val-Phe-Phe sequence and lacks Arg 5 and Asp 7 and the main part of the C-terminus, key points involved in the aggregation pathway and stabilization of the fibrillary structure of Aß. In particular, by combining chemical and biological techniques, we show that Aß8-20 does not undergo random coil to ß sheet conformational transition, does not form amyloid fibrils by itself, and is not toxic for neuronal cells. Moreover, we demonstrate that Aß8-20 mainly interacts with the 4-11 region of Aß1-42 and inhibits the formation of toxic oligomeric species and Aß fibrils. Finally, our data show that Aß8-20 protects neuron-like cells from Aß1-42 oligomer toxicity. We propose Aß8-20 as a promising drug candidate for the treatment of AD.

Aß and Tau Interact with Metal Ions, Lipid Membranes and Peptide-Based Amyloid Inhibitors: Are These Common Features Relevant in Alzheimer's Disease?

In the last two decades, the amyloid hypothesis, i.e., the abnormal accumulation of toxic Aß assemblies in the brain, has been considered the mainstream concept sustaining research in Alzheimer's Disease (AD). However, the course of cognitive decline and AD development better correlates with tau accumulation rather than amyloid peptide deposition. Moreover, all clinical trials of amyloid-targeting drug candidates have been unsuccessful, implicitly suggesting that the amyloid hypothesis needs significant amendments. Accumulating evidence supports the existence of a series of potentially dangerous relationships between Aß oligomeric species and tau protein in AD. However, the molecular determinants underlying pathogenic Aß/tau cross interactions are not fully understood. Here, we discuss the common features of Aß and tau molecules, with special emphasis on: (i) the critical role played by metal dyshomeostasis in promoting both Aß and tau aggregation and oxidative stress, in AD; (ii) the effects of lipid membranes on Aß and tau (co)-aggregation at the membrane interface; (iii) the potential of small peptide-based inhibitors of Aß and tau misfolding as therapeutic tools in AD. Although the molecular mechanism underlying the direct Aß/tau interaction remains largely unknown, the arguments discussed in this review may help reinforcing the current view of a synergistic Aß/tau molecular crosstalk in AD and stimulate further research to mechanism elucidation and next-generation AD therapeutics.

KLVFF oligopeptide-decorated amphiphilic cyclodextrin nanomagnets for selective amyloid beta recognition and fishing.

Recognition and capture of amyloid beta (Aß) is a challenging task for the early diagnosis of neurodegenerative disorders, such as Alzheimer's disease. Here, we report a novel KLVFF-modified nanomagnet based on magnetic nanoparticles (MNP) covered with a non-ionic amphiphilic ß-cyclodextrin (SC16OH) and decorated with KLVFF oligopeptide for the self-recognition of the homologous amino-acids sequence of Aß to collect Aß (1-42) peptide from aqueous samples. MNP@SC16OH and MNP@SC16OH/Ada-Pep nanoassemblies were fully characterized by complementary techniques both as solid powders and in aqueous dispersions. Single domain MNP@SC16OH/Ada-Pep nanomagnets of 20-40 nm were observed by TEM analysis. DLS and ζ-potential measurements revealed that MNP@SC16OH nanoassemblies owned in aqueous dispersion a hydrodynamic radius of about 150 nm, which was unaffected by Ada-Pep decoration, while the negative ζ-potential of MNP@SC16OH (-40 mV) became less negative (-30 mV) in MNP@SC16OH/Ada-Pep, confirming the exposition of positively charged KLVFF on nanomagnets surface. The ability of MNP@SC16OH/Ada-Pep to recruit Aß (1-42) in aqueous solution was evaluated by MALDI-TOF and compared with the ineffectiveness of undecorated MNP@SC16OH and VFLKF scrambled peptide-decorated nanoassemblies (MNP@SC16OH/Ada-scPep), pointing out the selectivity of KLVFF-decorated nanohybrid towards Aß (1-42). Finally, the property of nanomagnets to extract Aß in conditioned medium of cells over-producing Aß peptides was investigated as proof of concept of effectiveness of these nanomaterials as potential diagnostic tools.

Tau/Aß chimera peptides: A Thioflavin-T and MALDI-TOF study of Aß amyloidosis in the presence of Cu(II) or Zn(II) ions and total lipid brain extract (TLBE) vesicles.

Currently, Alzheimer's Disease (AD) is a complex neurodegenerative condition, with limited therapeutic options. Several factors, like Amyloid ß (Aß) aggregation, tau protein hyperphosphorylation, bio-metals dyshomeostasis and oxidative stress contribute to AD pathogenesis. These pathogenic processes might occur in the aqueous phase but also on neuronal membranes. Thus, investigating the connection between Aß and biomembranes, becomes important for unveiling the molecular mechanism underlying Aß amyloidosis as a critical event in AD pathology. In this work, the interaction of two peptides, made up with hybrid sequences from Tau protein 9-16 (EVMEDHAG) or 26-33 (QGGYTMHQ) N-terminal domain and Aß16-20 (KLVFF) hydrophobic region, with full length Aß40 or Aß42 peptides is reported. The studied "chimera" peptides Ac-EVMEDHAGKLVFF-NH2 (τ9-16-KL) and Ac-QGGYTMHQKLVFF-NH2 (τ26-33-KL) are endowed with Aß recognition and metal ion interaction capabilities provided by the tau or Aß sequences, respectively. These peptides were characterized in previous study along with their metal dependent interaction and amyloidogenesis, either in the presence or absence of metal ion and artificial membranes made up with Total Lipid Brain Extract (TLBE) components, (Sciacca et al., 2020). In the present paper, the ability of the two peptides to inhibit Aß aggregation is studied using composite experimental conditions including aqueous solution, the presence of metal ions (Cu or Zn), the presence of lipid vesicles mimicking neuronal membranes as well as the co-presence of metals and TLBE artificial membranes. We used Thioflavine-T (ThT) fluorescence or MALDI-TOF spectrometry analysis of Aß limited proteolysis to respectively monitor the Aß aggregation kinetic or validation of the Aß interacting regions. We demonstrate that τ9-16-KL and τ26-33-KL peptides differently affect Aß aggregation kinetics, with the tau sequence playing a crucial role. The results are discussed in terms of chimera's peptides hydrophobicity and electrostatic driven interactions at the aqueous/membrane interface.

Novel Peptide-Calix[4]arene Conjugate Inhibits Aß Aggregation and Rescues Neurons from Aß's Oligomers Cytotoxicity In Vitro.

Alzheimer's disease (AD) is a progressive neurodegenerative condition affecting people in the elderly. Targeting aggregation of ß-amyloid peptides (Aß) is considered a promising approach for the therapeutic treatment of the disease. Peptide based inhibitors of ß-amyloid fibrillation are emerging as safe drug candidates as well as interesting compounds for early diagnosis of AD. Peptide conjugation via covalent bond with functional moieties enables the resultant hybrid system to acquire desired functions. Here we report the synthesis, the structural characterization, and the Aß42 interaction of a p-amino-calix[4]arene derivative bearing a GPGKLVFF peptide pendant at the lower rim. We demonstrate that the p-amino-calix[4]arene-GPGKLVFF conjugate alters the Aß42 aggregation pathways by preventing Aß42's conformational transition from random coil to ß-sheet with concomitant changes of the aggregation kinetic profile as evidenced by circular dichroism (CD), thioflavin T (ThT), and dynamic light scattering (DLS) measurements, respectively. High resolution mass spectrometry (HR-MS) confirmed a direct interaction of the p-amino-calix[4]arene-GPGKLVFF conjugate with Aß42 monomer which provided insight into a possible working mechanism, whereas the alteration of the Aß42's fibrillary architecture, by the calix-peptide conjugate, was further validated by atomic force microscopy (AFM) imaging. Finally, the herein proposed compound was shown to be effective against Aß42 oligomers' toxicity in differentiated neuroblastoma cells, SH-SY5Y.

Copper (II) binding properties of an octapeptide fragment from the R3 region of tau protein: A combined potentiometric, spectroscopic and mass spectrometric study.

The copper(II) complexes of a peptide fragment of the R3 domain of tau protein (tau(326-333) Ac-GNIHHKPG-NH2) and its mutants (Ac-GNGHHKPG-NH2, Ac-GNIHHKAG-NH2, Ac-GNGAHKPG-NH2 and Ac-GNGHAKPG-NH2) have been studied by potentiometric and spectroscopic (UV-Vis, CD) methods. ESR spectroscopy and mass spectrometry were also used to prove the coordination mode of the mononuclear complexes and the formation of dinuclear species, respectively. It has been demonstrated that the (326-333) fragment of tau protein is a versatile and effective ligand for copper(II) coordination. The versatility of copper(II) binding is related to the presence of two adjacent histidyl residues in the sequence, which results in the coexistence of mononuclear, bis(ligand) and dinuclear complexes at different metal to ligand ratios. The 1:1 mononuclear complexes are, however, the dominant species with all peptides and the imidazole-N and one to three deprotonated amide nitrogen atoms towards the N-terminal side of the histidyl residue have been suggested as metal binding sites. This binding mode allows the formation of coordination isomers because any of the two histidine moieties can be the primary anchoring site. It is evident from the CD spectroscopic measurements that the isomers are present in almost equal concentration. The copper(II) binding affinity of the native fragment of tau protein is comparable to that of a similar 2-histidine fragment of amyloid-ß mutant, Ac-SGAEGHHQK-NH2 but the comparison with an independent histidyl residue (H32) from the N-terminal region of the protein reveals the predominance of H32 over the histidines in the R3 domain.

Interaction between Hemin and Prion Peptides: Binding, Oxidative Reactivity and Aggregation.

We investigate the interaction of hemin with four fragments of prion protein (PrP) containing from one to four histidines (PrP106-114, PrP95-114, PrP84-114, PrP76-114) for its potential relevance to prion diseases and possibly traumatic brain injury. The binding properties of hemin-PrP complexes have been evaluated by UV-visible spectrophotometric titration. PrP peptides form a 1:1 adduct with hemin with affinity that increases with the number of histidines and length of the peptide; the following log K1 binding constants have been calculated: 6.48 for PrP76-114, 6.1 for PrP84-114, 4.80 for PrP95-114, whereas for PrP106-114, the interaction is too weak to allow a reliable binding constant calculation. These constants are similar to that of amyloid-ß (Aß) for hemin, and similarly to hemin-Aß, PrP peptides tend to form a six-coordinated low-spin complex. However, the concomitant aggregation of PrP induced by hemin prevents calculation of the K2 binding constant. The turbidimetry analysis of [hemin-PrP76-114] shows that, once aggregated, this complex is scarcely soluble and undergoes precipitation. Finally, a detailed study of the peroxidase-like activity of [hemin-(PrP)] shows a moderate increase of the reactivity with respect to free hemin, but considering the activity over long time, as for neurodegenerative pathologies, it might contribute to neuronal oxidative stress.

Zinc Interactions with a Soluble Mutated Rat Amylin to Mimic Whole Human Amylin: An Experimental and Simulation Approach to Understand Stoichiometry, Speciation and Coordination of the Metal Complexes.

Islet amyloid polypeptide (IAPP) is a hormone co-secreted with insulin and zinc from pancreatic ß-cells. To overcome the low solubility of human IAPP, we characterized zinc complexes species formed with 1) a mutated form of rat-IAPP(1-37; R18 H) able to mimic the human IAPP, 2) the r-IAPP(1-37) and the IAPP(1-8) fragment. Stoichiometry, speciation and coordination features of zinc(II) complexes were unveiled by ESI-MS, potentiometry and NMR measurements combined with DFT and free-energy simulations. Mononuclear species start to form around pH 6; Zn2+ binds both His18 and N-amino terminus in rat-IAPP(1-37; R18 H). The in silico study allows us to assess not only a structured turn compact domain in r-IAPP(1-37) and r-IAPP(1-37; R18 H) featured by a different free energy barrier for the transition from the compact to elongated conformation upon the coordination of Zn2+ , but also to bring into light a coordination shell further stabilized by noncovalent interactions.

Tau/Aß chimera peptides: Evaluating the dual function of metal coordination and membrane interaction in one sequence.

Several abnormal events may concur as major risk factors for Alzheimer's disease (AD) pathogenesis. For instance, dysregulation of brain's metal homeostasis and amyloid-mediated membrane damage are established toxic mechanisms causing neuronal death. In this study, we assess the amyloidogenic propensity and membrane-damage effects, either in the presence or in absence of metal ions, of two newly synthesized bifunctional peptides. These were designed to comprise a metal chelating N-terminus region derived from Tau protein namely the Tau9-16 (EVMEDHAG) or Tau26-33 (QGGYTMHQ) sequences, merged with the C-terminal hydrophobic region analogous to the Amyloid beta (Aß) 16-20 aminoacid sequence KLVFF (KL). Comparative circular dichroism or fluorescence experiments were carried out to look at the peptide conformation, fibril formation and membrane affinity of Tau9-16KL and Tau26-33KL peptides. We found that Tau9-16KL and Tau26-33KL perturb the fibrillogenic process of Aß1-40. Furthermore Cu(II) and, to a lower extent, Zn(II) induced conformational changes Tau26-33KL both in water and in membrane-mimicking environment. By contrast, due to a different metal coordination mode we observed for Tau9-16KL an unstructured peptide conformation in all the experimental conditions. Unlike aqueous solution, a certain propensity to form amyloid structures at the lipid membrane interface clearly emerged for both the peptides. However, the two peptides exhibit a different capability to elicit membrane damage depending on the presence or absence of metal ions. Tau9-16KL and Tau26-33KL can be used as peptide-based molecular systems able to interfere with the metal dependent Aß/Tau cross-seeded generation of membrane active amyloid species.

Copper(II) Coordination Abilities of the Tau Protein's N-Terminus Peptide Fragments: A Combined Potentiometric, Spectroscopic and Mass Spectrometric Study.

Copper(II) complexes of the N-terminal peptide fragments of tau protein have been studied by potentiometric and various spectroscopic techniques (UV-vis, CD, ESR and ESI-MS). The octapeptide Tau(9-16) (Ac-EVMEDHAG-NH2 ) contains the H14 residue of the native protein, while Tau(26-33) (Ac-QGGYTMHQ-NH2 ) and its mutants Tau(Q26K-Q33K) (Ac-KGGYTMHK-NH2 ) and Tau(Q26K-Y29A-Q33K) (Ac-KGGATMHK-NH2 ) include the H32 residue. To compare the binding ability of H14 and H32 in a single molecule the decapeptide Ac-EDHAGTMHQD-NH2 (Tau(12-16)(30-34)) has also been synthesized and studied. The histidyl residue is the primary metal binding site for metal ions in all the peptide models studied. In the case of Tau(9-16) the side chain carboxylate functions enhance the stability of the M-Nim coordinated complexes compared to Tau(26-33) (logK(Cu-Nim )=5.04 and 3.78, respectively). Deprotonation and metal ion coordination of amide groups occur around the physiological pH range for copper(II). The formation of the imidazole- and amide-coordinated species changes the metal ion preference and the complexes formed with the peptides containing the H32 residue predominate over those of H14 at physiological pH values (90 %-10 %) and in alkaline samples (96 %-4 %).

Repurposing of Copper(II)-chelating Drugs for the Treatment of Neurodegenerative Diseases.

BACKGROUND: There is mounting urgency to find new drugs for the treatment of neurodegenerative disorders. A large number of reviews have exhaustively described either the molecular or clinical aspects of neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's (PD). Conversely, reports outlining how known drugs in use for other diseases can also be effective as therapeutic agents in neurodegenerative diseases are less reported. This review focuses on the current uses of some copper(II) chelating molecules as potential drug candidates in neurodegeneration. METHODS: Starting from the well-known harmful relationships existing between the dyshomeostasis and mis-management of metals and AD onset, we surveyed the experimental work reported in the literature, which deals with the repositioning of metal-chelating drugs in the field of neurodegenerative diseases. The reviewed papers were retrieved from common literature and their selection was limited to those describing the biomolecular aspects associated with neuroprotection. In particular, we emphasized the copper(II) coordination abilities of the selected drugs. RESULTS: Copper, together with zinc and iron, are known to play a key role in regulating neuronal functions. Changes in copper homeostasis are crucial for several neurodegenerative disorders. The studies included in this review may provide an overview on the current strategies aimed at repurposing copper (II) chelating drugs for the treatment of neurodegenerative disorders. Starting from the exemplary case of clioquinol repurposing, we discuss the challenge and the opportunities that repurposing of other metal-chelating drugs may provide (e.g. PBT-2, metformin and cyclodipeptides) in the treatment of neurodegenerative disease. CONCLUSIONS: In order to improve the success rate of drug repositioning, comprehensive studies on the molecular mechanism and therapeutic efficacy are still required. The present review upholds that drug repurposing makes significant advantages over drug discovery since repositioned drugs had already passed the safety and toxicity tests. Promising drug candidates in neurodegenerative diseases may be represented by copper chelating classes of drugs, provided that sufficient details on their mechanism of action are available to encourage further investigations and clinical trials.

Prion Peptides Are Extremely Sensitive to Copper Induced Oxidative Stress.

Copper(II) binding to prion peptides does not prevent Cu redox cycling and formation of reactive oxygen species (ROS) in the presence of reducing agents. The toxic effects of these species are exacerbated in the presence of catecholamines, indicating that dysfunction of catecholamine vesicular sequestration or recovery after synaptic release is a dangerous amplifier of Cu induced oxidative stress. Cu bound to prion peptides including the high affinity site involving histidines adjacent to the octarepeats exhibits marked catalytic activity toward dopamine and 4-methylcatechol. The resulting quinone oxidation products undergo parallel oligomerization and endogenous peptide modification yielding catechol adducts at the histidine binding ligands. These modifications add to the more common oxidation of Met and His residues produced by ROS. Derivatization of Cu-prion peptides is much faster than that undergone by Cu-ß-amyloid and Cu-α-synuclein complexes in the same conditions.

New comprehensive studies of a gold(III) Dithiocarbamate complex with proven anticancer properties: Aqueous dissolution with cyclodextrins, pharmacokinetics and upstream inhibition of the ubiquitin-proteasome pathway.

The gold(III)-dithiocarbamate complex AuL12 (dibromo [ethyl-N-(dithiocarboxy-kS,kS')-N-methylglycinate] gold(III)), is endowed with promising in vitro/in vivo antitumor activity and toxicological profile. Here, we report our recent strategies to improve its water solubility and stability under physiological conditions along with our efforts for unravelling its tangled mechanism of action. We used three types of α-cyclodextrins (CDs), namely ß-CD, Me-ß-CD and HP-ß-CD to prepare aqueous solutions of AuL12. The ability of these natural oligosaccharide carriers to enhance water solubility of hydrophobic compounds, allowed drug stability of AuL12 to be investigated. Moreover, pharmacokinetic experiments were first carried out for a gold(III) coordination compound, after i.v. injection of the nanoformulation AuL12/HP-ß-CD to female mice. The gold content in the blood samples was detected at scheduled times by AAS (atomic absorption spectrometry) analysis, highlighting a fast biodistribution with a tß1/2 of few minutes and a slow escretion (tα1/2 of 14.3 h). The in vitro cytotoxic activity of AuL12 was compared with the AuL12/HP-ß-CD mixture against a panel of three human tumor cell lines (i.e., HeLa, KB and MCF7). Concerning the mechanism of action, we previously reported the proteasome-inhibitory activity of some our gold(III)-based compounds. In this work, we moved from the proteasome target to upstream of the important ubiquitin-proteasome pathway, testing the effects of AuL12 on the polyubiquitination reactions involving the Ub-activating (E1) and -conjugating (E2) enzymes.

Ubiquitin Associates with the N-Terminal Domain of Nerve Growth Factor: The Role of Copper(II) Ions.

Many biochemical pathways involving nerve growth factor (NGF), a neurotrophin with copper(II) binding abilities, are regulated by the ubiquitin (Ub) proteasome system. However, whether NGF binds Ub and the role played by copper(II) ions in modulating their interactions have not yet been investigated. Herein NMR spectroscopy, circular dichroism, ESI-MS, and titration calorimetry are employed to characterize the interactions of NGF with Ub. NGF1-14 , which is a short model peptide encompassing the first 14 N-terminal residues of NGF, binds the copper-binding regions of Ub (KD =8.6 10-5 m). Moreover, the peptide undergoes a random coil-polyproline type II helix structural conversion upon binding to Ub. Notably, copper(II) ions inhibit NGF1-14 /Ub interactions. Further experiments performed with the full-length NGF confirmed the existence of a copper(II)-dependent association between Ub and NGF and indicated that the N-terminal domain of NGF was a valuable paradigm that recapitulated many traits of the full-length protein.

Cross-talk between the octarepeat domain and the fifth binding site of prion protein driven by the interaction of copper(II) with the N-terminus.

Prion diseases are a group of neurodegenerative diseases based on the conformational conversion of the normal form of the prion protein (PrP(C)) to the disease-related scrapie isoform (PrP(Sc)). Copper(II) coordination to PrP(C) has attracted considerable interest for almost 20 years, mainly due to the possibility that such an interaction would be an important event for the physiological function of PrP(C). In this work, we report the copper(II) coordination features of the peptide fragment Ac(PEG11)3PrP(60-114) [Ac = acetyl] as a model for the whole N-terminus of the PrP(C) metal-binding domain. We studied the complexation properties of the peptide by means of potentiometric, UV/Vis, circular dichroism and electrospray ionisation mass spectrometry techniques. The results revealed that the preferred histidyl binding sites largely depend on the pH and copper(II)/peptide ratio. Formation of macrochelate species occurs up to a 2:1 metal/peptide ratio in the physiological pH range and simultaneously involves the histidyl residues present both inside and outside the octarepeat domain. However, at increased copper(II)/peptide ratios amide-bound species form, especially within the octarepeat domain. On the contrary, at basic pH the amide-bound species predominate at any copper/peptide ratio and are formed preferably with the binding sites of His96 and His111, which is similar to the metal-binding-affinity order observed in our previous studies.

Neoadjuvant strategies for pancreatic cancer.

Pancreatic cancer (PC) is the fourth cause of cancer death in Western countries, the only chance for long term survival is an R0 surgical resection that is feasible in about 10%-20% of all cases. Five years cumulative survival is less than 5% and rises to 25% for radically resected patients. About 40% has locally advanced in PC either borderline resectable (BRPC) or unresectable locally advanced (LAPC). Since LAPC and BRPC have been recognized as a particular form of PC neoadjuvant therapy (NT) has increasingly became a valid treatment option. The aim of NT is to reach local control of disease but, also, it is recognized to convert about 40% of LAPC patients to R0 resectability, thus providing a significant improvement of prognosis for responding patients. Once R0 resection is achieved, survival is comparable to that of early stage PCs treated by upfront surgery. Thus it is crucial to look for a proper patient selection. Neoadjuvant strategies are multiples and include neoadjuvant chemotherapy (nCT), and the association of nCT with radiotherapy (nCRT) given as either a combination of a radio sensitizing drug as gemcitabine or capecitabine or and concomitant irradiation or as upfront nCT followed by nRT associated to a radio sensitizing drug. This latter seem to be most promising as it may select patients who do not go on disease progression during initial treatment and seem to have a better prognosis. The clinical relevance of nCRT may be enhanced by the application of higher active protocols as FOLFIRINOX.

Affinity, speciation, and molecular features of copper(II) complexes with a prion tetraoctarepeat domain in aqueous solution: insights into old and new results.

Characterization of the copper(II) complexes formed with the tetraoctarepeat peptide at low and high metal-to-ligand ratios and in a large pH range, would provide a breakthrough in the interpretation of biological relevance of the different metal complexes of copper(II)-tetraoctarepeat system. In the present work, the potentiometric, UV/Vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) studies were carried out on copper(II) complexes with a PEG-ylated derivative of the tetraoctarepeats peptide sequence (Ac-PEG27 -(PHGGGWGQ)4 -NH2 ) and the peptide Ac-(PHGGGWGQ)2 -NH2 . Conjugation of tetraoctarepeat peptide sequence with polyethyleneglycol improved the solubility of the copper(II) complexes. The results enable a straightforward explanation of the conflicting results originated from the underestimation of all metal-ligand equilibria and the ensuing speciation. A complete and reliable speciation is therefore obtained with the released affinity and binding details of the main complexes species formed in aqueous solution. The results contribute to clarify the discrepancies of several studies in which the authors ascribe the redox activity of copper(II)-tetraoctarepeat system considering only the average effects of several coexisting species with very different stoichiometries and binding modes.

Antioxidants counteract lipopolysaccharide-triggered alterations of human colonic smooth muscle cells.

Gut dysmotility develops in individuals during and after recovering from infective acute gastroenteritis and it is apparently due to a direct effect of circulating lipopolysaccharides (LPS). This is an endotoxin with a prooxidant activity derived from gram-negative bacteria. Due to the lack of human models available so far, the mechanisms underlying LPS-induced gut dysmotility are, however, poorly investigated. In the present work long-term effects of LPS and their reversibility have been assessed by means of different analytical cytology methods on pure primary cultures of human colonic smooth muscle cells. We found that LPS triggered the following alterations: (i) a redox imbalance with profound changes of contractile microfilament network, and (ii) the induction of cell cycle progression with dedifferentiation from a contractile to a synthetic phenotype. These alterations persisted also after LPS removal. Importantly, two unrelated antioxidants, alpha-tocopherol and N-acetylcysteine, were able to reverse the cytopathic effects of LPS and to restore normal muscle cell function. The present data indicate that LPS is capable of triggering a persistent and long-term response that could contribute to muscle dysfunction occurring after an infective and related inflammatory burst and suggest a reappraisal of antioxidants in the management of postinfective motor disorders of the gut.

Mixed metal copper(II)-nickel(II) and copper(II)-zinc(II) complexes of multihistidine peptide fragments of human prion protein.

Mixed metal copper(II)-nickel(II) and copper(II)-zinc(II) complexes of four peptide fragments of human prion protein have been studied by potentiometric, UV-vis and circular dichroism spectroscopic techniques. One peptide contained three histidyl residues: HuPrP(84-114) with H85 inside and H96, H111 outside the octarepeat domain. The other three peptides contained two histidyl residues; H96 and H111 for HuPrP(91-115) and HuPrP(84-114)H85A while HuPrP(84-114)H96A contained the histidyl residues at positions 85 and 111. It was found that both histidines of the latter peptides can simultaneously bind copper(II) and nickel(II) ions and dinuclear mixed metal complexes can exist in slightly alkaline solution. One molecule of the peptide with three histidyl residues can bind two copper(II) and one nickel(II) ions. H85 and H111 were identified as the major copper(II) and H96 as the preferred nickel(II) binding sites in mixed metal species. The studies on the zinc(II)-PrP peptide binary systems revealed that zinc(II) ions can coordinate to the 31-mer PrP peptide fragments in the form of macrochelates with two or three coordinated imidazol-nitrogens but the low stability of these complexes cannot prevent the hydrolysis of the metal ion in slightly alkaline solution. These data provide further support for the outstanding affinity of copper(II) ions towards the peptide fragments of prion protein but the binding of nickel(II) can significantly modify the distribution of copper(II) among the available metal binding sites.